Quercetin attenuates toosendanin-induced hepatotoxicity through inducing the Nrf2/GCL/GSH antioxidant signaling pathway.

PubMed

Jin, Yao; Huang, Zhen-Lin; Li, Li; Yang, Yang; Wang, Chang-Hong; Wang, Zheng-Tao; Ji, Li-Li

2018-06-19

Toosendanin (TSN) is the main active compound in Toosendan Fructus and Meliae Cortex, two commonly used traditional Chinese medicines. TSN has been reported to induce hepatotoxicity, but its mechanism remains unclear. In this study, we demonstrated the critical role of nuclear factor erythroid 2-related factor 2 (Nrf2) in protecting against TSN-induced hepatotoxicity in mice and human normal liver L-02 cells. In mice, administration of TSN (10 mg/kg)-induced acute liver injury evidenced by increased serum alanine/aspartate aminotransferase (ALT/AST) and alkaline phosphatase (ALP) activities, and total bilirubin (TBiL) content as well as the histological changes. Furthermore, TSN markedly increased liver reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased liver glutathione (GSH) content and Nrf2 expression. In L-02 cells, TSN (2 μM) time-dependently reduced glutamate-cysteine ligase (GCL) activity and cellular expression of the catalytic/modify subunit of GCL (GCLC/GCLM). Moreover, TSN reduced cellular GSH content and the increased ROS formation, and time-dependently decreased Nrf2 expression and increased the expression of the Nrf2 inhibitor protein kelch-like ECH-associated protein-1 (Keap1). Pre-administration of quercetin (40, 80 mg/kg) effectively inhibited TSN-induced liver oxidative injury and reversed the decreased expression of Nrf2 and GCLC/GCLM in vivo and in vitro. In addition, the quercetin-provided protection against TSN-induced hepatotoxicity was diminished in Nrf2 knock-out mice. In conclusion, TSN decreases cellular GSH content by reducing Nrf2-mediated GCLC/GCLM expression via decreasing Nrf2 expression. Quercetin attenuates TSN-induced hepatotoxicity by inducing the Nrf2/GCL/GSH antioxidant signaling pathway. This study implies that inducing Nrf2 activation may be an effective strategy to prevent TSN-induced hepatotoxicity.

Paeonol Ameliorates Diabetic Renal Fibrosis Through Promoting the Activation of the Nrf2/ARE Pathway via Up-Regulating Sirt1.

PubMed

Zhang, Lei; Chen, Zhiquan; Gong, Wenyan; Zou, Yezi; Xu, Futian; Chen, Lihao; Huang, Heqing

2018-01-01

Diabetic nephropathy (DN) is rapidly becoming the leading cause of end-stage renal disease worldwide and a major cause of morbidity and mortality in patients of diabetes. The main pathological change of DN is renal fibrosis. Paeonol (PA), a single phenolic compound extracted from the root bark of Cortex Moutan, has been demonstrated to have many potential pharmacological activities. However, the effects of PA on DN have not been fully elucidated. In this study, high glucose (HG)-treated glomerular mesangial cells (GMCs) and streptozotocin (STZ)-induced diabetic mice were analyzed in exploring the potential mechanisms of PA on DN. Results in vitro showed that: (1) PA inhibited HG-induced fibronectin (FN) and ICAM-1 overexpressions; (2) PA exerted renoprotective effect through activating the Nrf2/ARE pathway; (3) Sirt1 mediated the effects of PA on the activation of Nrf2/ARE pathway. What is more, in accordance with the in vitro results, significant elevated levels of Sirt1, Nrf2 and downstream proteins related to Nrf2 were observed in the kidneys of PA treatment group compared with model group. Taken together, our study shows that PA delays the progression of diabetic renal fibrosis, and the underlying mechanism is probably associated with regulating the Nrf2 pathway. The effect of PA on Nrf2 is at least partially dependent on Sirt1 activation.

Keap1 redox-dependent regulation of doxorubicin-induced oxidative stress response in cardiac myoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nordgren, Kendra K.S., E-mail: knordgre@d.umn.edu; Wallace, Kendall B., E-mail: kwallace@d.umn.edu

Doxorubicin (DOX) is a widely prescribed treatment for a broad scope of cancers, but clinical utility is limited by the cumulative, dose-dependent cardiomyopathy that occurs with repeated administration. DOX-induced cardiotoxicity is associated with the production of reactive oxygen species (ROS) and oxidation of lipids, DNA and proteins. A major cellular defense mechanism against such oxidative stress is activation of the Keap1/Nrf2-antioxidant response element (ARE) signaling pathway, which transcriptionally regulates expression of antioxidant genes such as Nqo1 and Gstp1. In the present study, we address the hypothesis that an initial event associated with DOX-induced oxidative stress is activation of the Keap1/Nrf2-dependentmore » expression of antioxidant genes and that this is regulated through drug-induced changes in redox status of the Keap1 protein. Incubation of H9c2 rat cardiac myoblasts with DOX resulted in a time- and dose-dependent decrease in non-protein sulfhydryl groups. Associated with this was a near 2-fold increase in Nrf2 protein content and enhanced transcription of several of the Nrf2-regulated down-stream genes, including Gstp1, Ugt1a1, and Nqo1; the expression of Nfe2l2 (Nrf2) itself was unaltered. Furthermore, both the redox status and the total amount of Keap1 protein were significantly decreased by DOX, with the loss of Keap1 being due to both inhibited gene expression and increased autophagic, but not proteasomal, degradation. These findings identify the Keap1/Nrf2 pathway as a potentially important initial response to acute DOX-induced oxidative injury, with the primary regulatory events being the oxidation and autophagic degradation of the redox sensor Keap1 protein. - Highlights: • DOX caused a ∼2-fold increase in Nrf2 protein content. • DOX enhanced transcription of several Nrf2-regulated down-stream genes. • Redox status and total amount of Keap1 protein were significantly decreased by DOX. • Loss of Keap1 protein was due to inhibited gene expression and increased autophagy. • Keap1/Nrf2 pathway is an important initial response to DOX-induced oxidative injury.« less

Naringenin protects against 6-OHDA-induced neurotoxicity via activation of the Nrf2/ARE signaling pathway.

PubMed

Lou, Haiyan; Jing, Xu; Wei, Xinbing; Shi, Huanying; Ren, Dongmei; Zhang, Xiumei

2014-04-01

There is increasing evidence that oxidative stress is critically involved in the pathogenesis of Parkinson's disease (PD), suggesting that pharmacological targeting of the antioxidant machinery may have therapeutic value. Naringenin, a natural flavonoid compound, has been reported to possess neuroprotective effect against PD related pathology; however the mechanisms underlying its beneficial effects are poorly defined. Thus, the purpose of the present study was to investigate the potential neuroprotective role of naringenin and to delineate its mechanism of action against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in models of PD both in vitro and in vivo. Naringenin treatment resulted in an increase in nuclear factor E2-related factor 2 (Nrf2) protein levels and subsequent activation of antioxidant response element (ARE) pathway genes in SH-SY5Y cells and in mice. Exposure of SH-SY5Y cells to naringenin provided protection against 6-OHDA-induced oxidative insults that was dependent on Nrf2, since treatment with Nrf2 siRNA failed to block against 6-OHDA neurotoxicity or induce Nrf2-dependent cytoprotective genes in SH-SY5Y cells. In mice, oral administration of naringenin resulted in significant protection against 6-OHDA-induced nigrostriatal dopaminergic neurodegeneration and oxidative damage. Our results indicate that activation of Nrf2/ARE signaling by naringenin is strongly associated with its neuroprotective effects against 6-OHDA neurotoxicity and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in PD. Copyright © 2013 Elsevier Ltd. All rights reserved.

The hypertension drug, verapamil, activates Nrf2 by promoting p62-dependent autophagic Keap1 degradation and prevents acetaminophen-induced cytotoxicity.

PubMed

Lee, Da Hyun; Park, Jeong Su; Lee, Yu Seol; Sung, Su Haeng; Lee, Yong-Ho; Bae, Soo Han

2017-02-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) provides a cellular defense against oxidative stress by inducing the expression of antioxidant and detoxification enzymes. The calcium antagonist, verapamil, is an FDA-approved drug prescribed for the treatment of hypertension. Here, we show that verapamil acts as a potent Nrf2 activator without causing cytotoxicity, through degradation of Kelch-like ECH-associated protein 1 (Keap1), a Nrf2 repressor. Furthermore, verapamilinduced Keap1 degradation is prominently mediated by a p62-dependent autophagic pathway. Correspondingly, verapamil protects cells from acetaminophen-induced oxidative damage through Nrf2 activation. These results demonstrated the underlying mechanisms for the protective role of verapamil against acetaminophen-induced cytotoxicity. [BMB Reports 2017; 50(2): 91-96].

Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: Biochemical, pharmacological and toxicological implications

PubMed Central

Walsh, Joanne; Jenkins, Rosalind E.; Wong, Michael; Olayanju, Adedamola; Powell, Helen; Copple, Ian; O’Neill, Paul M.; Goldring, Christopher E.P.; Kitteringham, Neil R.; Park, B. Kevin

2014-01-01

The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2(−/−)) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2(−/−) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR < 1%). One hundred sixty-one were significantly different (P < 0.05) between WT and Nrf2(−/−) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3 mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24 h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2(−/−) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway. Biological significance The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2(−/−) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers. PMID:24859727

Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: biochemical, pharmacological and toxicological implications.

PubMed

Walsh, Joanne; Jenkins, Rosalind E; Wong, Michael; Olayanju, Adedamola; Powell, Helen; Copple, Ian; O'Neill, Paul M; Goldring, Christopher E P; Kitteringham, Neil R; Park, B Kevin

2014-08-28

The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2((-/-))) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2((-/-)) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR <1%). One hundred sixty-one were significantly different (P<0.05) between WT and Nrf2((-/-)) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2((-/-)) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway. The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2((-/-)) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers. Copyright © 2014. Published by Elsevier B.V.

Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mussotter, Franz, E-mail: franz.mussotter@bfr.bund

Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2{sup +/+}) and Nrf2 knockout (nrf2{sup −/−}) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gelmore » electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100 μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100 μM CA, respectively. For 5 and 10 μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2{sup −/−} BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. - Highlights: • Contact allergens induce proteins involved in DC maturation Nrf2-dependently. • Induction of these proteins points to a functional role of Nrf2 in DC maturation. • Evidence that metabolic reprogramming enables DC activation by contact allergens. • Identification of biomarker candidates for development of in vitro testing methods.« less

Glutathione S-transferase pi expression regulates the Nrf2-dependent response to hormetic diselenides.

PubMed

Bartolini, D; Commodi, J; Piroddi, M; Incipini, L; Sancineto, L; Santi, C; Galli, F

2015-11-01

Glutathione S-transferase pi (GSTP), a phase II gene downstream of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant-responsive element (ARE)/electrophile response element (EpRE) transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of diphenyl diselenide [(PhSe)2]. These diselenides, and particularly 2,2'-diselenyl dibenzoic acid (DSBA), behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce submaximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and then the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and stress activated protein kinase (SAPK)/JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of an Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA-treated HepG2 cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of Nrf2 transcription factor in viral infection.

PubMed

Ramezani, Ali; Nahad, Mehdi Parsa; Faghihloo, Ebrahim

2018-05-08

The nuclear factor erythroid 2 related factor 2 (Nrf2) is a major regulator of intracellular inducible defense systems against harmful endogenous and exogenous substances in the body. Under normal conditions Nrf2 is mainly binds to keap1 and located in the cytoplasm. However, in response to oxidative and electrophile stress, Nrf2 translocated to the nucleus and link to anti-oxidant response elements to induce the transcription of cytoprotective genes. Most viruses cause oxidative stress and increase the activity of radicals and reactive oxygen species (ROS), subsequently, the cellular protection system activates the Nrf2 and increases the expression of cytoprotective genes. However, in some cases, the activation of Nrf2 is not ROS-dependent, and is carried out directly via the ROS-independent pathway. Many viruses cause the activation of Nrf2, which is involved in the pathogenesis and the progression of the virus infection and even in its chronic form. However, some viruses inhibit the activation of Nrf2, in which case the virus also benefits of this mechanism to maintain the homeostasis of the cell. However, the challenge between the Nrf2/ARE signaling pathway of and viral infections is unknown in some cases, and in order to know more details in this regard, a more detailed seems necessary. © 2018 Wiley Periodicals, Inc.

CDDO-Im protects from acetaminophen hepatotoxicity through induction of Nrf2-dependent genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reisman, Scott A.; Buckley, David B.; Tanaka, Yuji

CDDO-Im is a synthetic triterpenoid recently shown to induce cytoprotective genes through the Nrf2-Keap1 pathway, an important mechanism for the induction of cytoprotective genes in response to oxidative stress. Upon oxidative or electrophilic insult, the transcription factor Nrf2 translocates to the nucleus, heterodimerizes with small Maf proteins, and binds to antioxidant response elements (AREs) in the upstream promoter regions of various cytoprotective genes. To further elucidate the hepatoprotective effects of CDDO-Im, wild-type and Nrf2-null mice were pretreated with CDDO-Im (1 mg/kg, i.p.) or vehicle (DMSO), and then administered acetaminophen (500 mg/kg, i.p.). Pretreatment of wild-type mice with CDDO-Im reduced livermore » injury caused by acetaminophen. In contrast, hepatoprotection by CDDO-Im was not observed in Nrf2-null mice. CDDO-Im increased Nrf2 protein expression and Nrf2-ARE binding in wild-type, but not Nrf2-null mice. Furthermore, CDDO-Im increased the mRNA expression of the Nrf2 target genes NAD(P)H: quinone oxidoreductase-1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); and heme-oxygenase-1 (Ho-1), in both a dose- and time-dependent manner. Conversely, CDDO-Im did not induce Nqo1, Gclc, and Ho-1 mRNA expression in Nrf2-null mice. Collectively, the present study shows that CDDO-Im pretreatment induces Nrf2-dependent cytoprotective genes and protects the liver from acetaminophen-induced hepatic injury.« less

Britanin Ameliorates Cerebral Ischemia-Reperfusion Injury by Inducing the Nrf2 Protective Pathway.

PubMed

Wu, Guozhen; Zhu, Lili; Yuan, Xing; Chen, Hao; Xiong, Rui; Zhang, Shoude; Cheng, Hao; Shen, Yunheng; An, Huazhang; Li, Tiejun; Li, Honglin; Zhang, Weidong

2017-10-10

Oxidative stress is considered the major cause of tissue injury after cerebral ischemia. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important defensive mechanisms against oxidative stresses and has been confirmed as a target for stroke treatment. Thus, we desired to find new Nrf2 activators and test their neuronal protective activity both in vivo and in vitro. The herb-derived compound, Britanin, is a potent inducer of the Nrf2 system. Britanin can induce the expression of protective enzymes and reverse oxygen-glucose deprivation, followed by reperfusion (OGD-R)-induced neuronal injury in primary cortical neurons in vitro. Furthermore, the administration of Britanin significantly ameliorated middle cerebral artery occlusion-reperfusion (MCAO-R) insult in vivo. We report here the crystal structure of the complex of Britanin and the BTB domain of Keap1. Britanin selectively binds to a conserved cysteine residue, cysteine 151, of Keap1 and inhibits Keap1-mediated ubiquitination of Nrf2, leading to induction of the Nrf2 pathway. Britanin is a potent inducer of Nrf2. The complex crystal structure of Britanin and the BTB domain of Keap1 help clarify the mechanism of Nrf2 induction. Britanin was proven to protect primary cortical neurons against OGD-R-induced injury in an Nrf2-dependant way. Additionally, Britanin had excellent cerebroprotective effect in an MCAO-R model. Our results demonstrate that the natural product Britanin with potent Nrf2-activating and neural protective activities both in vitro and in vivo could be developed into a cerebroprotective therapeutic agent. Antioxid. Redox Signal. 27, 754-768.

Nrf2 Expression Modifies Influenza A Entry and Replication inNasal Epithelial Cells

EPA Science Inventory

Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could playa role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene exp...

PM2.5 induces Nrf2-mediated defense mechanisms against oxidative stress by activating PIK3/AKT signaling pathway in human lung alveolar epithelial A549 cells.

PubMed

Deng, Xiaobei; Rui, Wei; Zhang, Fang; Ding, Wenjun

2013-06-01

It has been well documented in in vitro studies that ambient airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM(2.5)) is capable of inducing oxidative stress, which plays a key role in PM(2.5)-mediated cytotoxicity. Although nuclear factor erythroid-2-related factor 2 (Nrf2) has been shown to regulate the intracellular defense mechanisms against oxidative stress, a potential of the Nrf2-mediated cellular defense against oxidative stress induced by PM(2.5) remains to be determined. This study was aimed to explore the potential signaling pathway of Nrf2-mediated defense mechanisms against PM(2.5)-induced oxidative stress in human type II alveolar epithelial A549 cells. We exposed A549 cells to PM(2.5) particles collected from Beijing at a concentration of 16 μg/cm(2). We observed that PM(2.5) triggered an increase of intracellular reactive oxygen species (ROS) in a time-dependent manner during a period of 2 h exposure. We also found that Nrf2 overexpression suppressed and Nrf2 knockdown increased PM(2.5)-induced ROS generation. Using Western blot and confocal microscopy, we found that PM(2.5) exposure triggered significant translocation of Nrf2 into nucleus, resulting in AKT phosphorylation and significant transcription of ARE-driven phases II enzyme genes, such as NAD(P)H:quinone oxidoreductase (NQO-1), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic subunit (GCLC) in A549 cells. Evaluation of signaling pathways showed that a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not an ERK 1/2 inhibitor (PD98059) or a p38 MAPK (SB203580), significantly down-regulated PM(2.5)-induced Nrf2 nuclear translocation and HO-1 mRNA expression, indicating PI3K/AKT is involved in the signaling pathway leads to the PM(2.5)-induced nuclear translocation of Nrf2 and subsequent Nrf2-mediated HO-1 transcription. Taken together, our results suggest that PM(2.5)-induced ROS may function as signaling molecules to activate Nrf2-mediated defenses, such as HO-1 expression, against oxidative stress induced by PM(2.5) through the PI3K/AKT signaling pathway.

Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimoto, Shoichi; Suzuki, Toshihiro; Koike, Shin

Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress. Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date. This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased inmore » acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system. Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO. - Highlights: • Nrf2 activation attenuates the effect of arsenic trioxide to acute promyelocytic leukemia cells. • The sensitivity of arsenic trioxide to NB4 cells was dependent on efflux rate of arsenic. • Activation of the GSH biosynthesis is essential in Nrf2-regulated responses for arsenic efflux.« less

Magnolia officinalis (Hou Po) bark extract stimulates the Nrf2-pathway in hepatocytes and protects against oxidative stress.

PubMed

Rajgopal, Arun; Missler, Stephen R; Scholten, Jeffery D

2016-12-04

The highly aromatic bark of Magnolia officinalis Rehder and EH Wilson, (magnolia bark) has been widely used in traditional Chinese medicine where it is known as Hou Po. Historically the bark of the tree has been used for treating variety of disorders the most common use of magnolia bark in traditional prescription has been to treat stress and anxiety disorders. Till date it is not clear regarding the fundamental cellular pathway it modulates. NRF2 signaling has emerged as the central pathway that protects cells from variety of stressors this led us to hypothesize that basis for magnolia bark's effects could be via activating NRF2 pathway. We utilized variety of biochemical procedures like luciferase reporter assay, enzyme induction, gene expression to determine NRF2 inducing activity by magnolia bark extract and its significance. Further we identified the phytochemicals inducing this activity using bio-directed fractionation procedure. In this study, we demonstrate that magnolia bark extract activates Nrf2-dependent gene expression and protects against hydrogen peroxide mediated oxidative stress in hepatocytes. We further identified through HPLC fractionation and mass spectroscopy that magnolol, 4-methoxy honokiol and honokiol are the active phytochemicals inducing the Nrf2-mediated activity. This could be the molecular basis for its numerous beneficial activity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms.

PubMed

Palliyaguru, Dushani L; Chartoumpekis, Dionysios V; Wakabayashi, Nobunao; Skoko, John J; Yagishita, Yoko; Singh, Shivendra V; Kensler, Thomas W

2016-12-01

Small molecules of plant origin offer presumptively safe opportunities to prevent carcinogenesis, mutagenesis and other forms of toxicity in humans. However, the mechanisms of action of such plant-based agents remain largely unknown. In recent years the stress responsive transcription factor Nrf2 has been validated as a target for disease chemoprevention. Withania somnifera (WS) is a herb used in Ayurveda (an ancient form of medicine in South Asia). In the recent past, withanolides isolated from WS, such as Withaferin A (WA) have been demonstrated to be preventive and therapeutic against multiple diseases in experimental models. The goals of this study are to evaluate withanolides such as WA as well as Withania somnifera root extract as inducers of Nrf2 signaling, to probe the underlying signaling mechanism of WA and to determine whether prevention of acetaminophen (APAP)-induced hepatic toxicity in mice by WA occurs in an Nrf2-dependent manner. We observed that WA profoundly protects wild-type mice but not Nrf2-disrupted mice against APAP hepatotoxicity. WA is a potent inducer of Nrf2-dependent cytoprotective enzyme expression both in vivo and in vitro. Unexpectedly, WA induces Nrf2 signaling at least in part, in a Keap1-independent, Pten/Pi3k/Akt-dependent manner in comparison to prototypical Nrf2 inducers, sulforaphane and CDDO-Im. The identification of WA as an Nrf2 inducer that can signal through a non-canonical, Keap1-independent pathway provides an opportunity to evaluate the role of other regulatory partners of Nrf2 in the dietary and pharmacological induction of Nrf2-mediated cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

Metformin Sensitizes Non-small Cell Lung Cancer Cells to an Epigallocatechin-3-Gallate (EGCG) Treatment by Suppressing the Nrf2/HO-1 Signaling Pathway.

PubMed

Yu, Chenxiao; Jiao, Yang; Xue, Jiao; Zhang, Qi; Yang, Hongying; Xing, Ligang; Chen, Guangxia; Wu, Jinchang; Zhang, Shuyu; Zhu, Wei; Cao, Jianping

2017-01-01

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, is widely studied as a cancer chemopreventive agent with potential anti-cancer effects. The NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway is considered to mediate cellular resistance to EGCG. Metformin, a classical antidiabetic drug, has been shown to prevent cancer progression. Researchers have not reported whether metformin potentiates the anti-cancer efficacy of EGCG. In this study, metformin inhibited HO-1 expression and augmented the anti-tumor effect of EGCG. Metformin also enhanced ROS (reactive oxygen species) generation induced by EGCG (100 μM), subsequently resulting in apoptosis. Based on the results of the in vivo study, size of xenografts treated with the combination of metformin and EGCG was smaller than other groups. Mechanistically, metformin modulated the EGCG-activated Nrf2/HO-1 pathway through Sirtuin 1 (SIRT1)-dependent deacetylation of Nrf2. Moreover, metformin upregulated SIRT1 expression partially through the NF-kB pathway. Comparatively, the combination of EGCG and metformin showed little impact on normal lung epithelial BEAS-2B cells. Based on our findings, metformin sensitized NSCLC cells to the EGCG treatment by suppressing the Nrf2/HO-1 signaling pathway.

Genetic silencing of Nrf2 enhances X-ROS in dysferlin-deficient muscle

PubMed Central

Kombairaju, Ponvijay; Kerr, Jaclyn P.; Roche, Joseph A.; Pratt, Stephen J. P.; Lovering, Richard M.; Sussan, Thomas E.; Kim, Jung-Hyun; Shi, Guoli; Biswal, Shyam; Ward, Christopher W.

2014-01-01

Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy—a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression. PMID:24600403

Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng

2013-04-19

Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal andmore » glial cultures, and protects neurons against glutamate-induced excitotoxicity.« less

Protective Effects of Maillard Reaction Products of Whey Protein Concentrate against Oxidative Stress through an Nrf2-Dependent Pathway in HepG2 Cells.

PubMed

Pyo, Min Cheol; Yang, Sung-Yong; Chun, Su-Hyun; Oh, Nam Su; Lee, Kwang-Won

2016-09-01

Whey protein concentrate (WPC), which contains α-lactalbumin and β-lactoglobulin, is utilized widely in the food industry. The Maillard reaction is a complex reaction that produces Maillard reaction products (MRPs), which are associated with the formation of antioxidant compounds. In this study, the hepatoprotection activity of MRPs of WPC against oxidative stress through the nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant pathway in HepG2 cells was examined. Glucose-whey protein concentrate conjugate (Glc-WPC) was obtained from Maillard reaction between WPC and glucose. The fluorescence intensity of Glc-WPC increased after 7 d compared to native WPC, and resulted in loss of 48% of the free amino groups of WPC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of Glc-WPC showed the presence of a high-molecular-weight portion. Treatment of HepG2 cells with Glc-WPC increased cell viability in the presence of oxidative stress, inhibited the generation of intracellular reactive oxygen species by tert-butyl hydroperoxide (t-BHP), and increased the glutathione level. Nrf2 translocation and Nrf2, reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-quinone oxidoreductase 1 (NOQ1), heme oxygenase-1 (HO-1), glutamate-L-cysteine ligase (GCL)M and GCLC mRNA levels were increased by Glc-WPC. Also, Glc-WPC increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK). The results of this study demonstrate that Glc-WPC activates the Nrf2-dependent pathway through the phosphorylation of ERK1/2 and JNK in HepG2 cells, and induces production of antioxidant enzymes and phase II enzymes.

Targeting the Nrf2/Amyloid-Beta Liaison in Alzheimer's Disease: A Rational Approach.

PubMed

Simoni, Elena; Serafini, Melania M; Caporaso, Roberta; Marchetti, Chiara; Racchi, Marco; Minarini, Anna; Bartolini, Manuela; Lanni, Cristina; Rosini, Michela

2017-07-19

Amyloid is a prominent feature of Alzheimer's disease (AD). Yet, a linear linkage between amyloid-β peptide (Aβ) and the disease onset and progression has recently been questioned. In this context, the crucial partnership between Aβ and Nrf2 pathways is acquiring paramount importance, offering prospects for deciphering the Aβ-centered disease network. Here, we report on a new class of antiaggregating agents rationally designed to simultaneously activate transcription-based antioxidant responses, whose lead 1 showed interesting properties in a preliminary investigation. Relying on the requirements of Aβ recognition, we identified the catechol derivative 12. In SH-SY5Y neuroblastoma cells, 12 combined remarkable free radical scavenger properties to the ability to trigger the Nrf2 pathway and induce the Nrf2-dependent defensive gene NQO1 by means of electrophilic activation of the transcriptional response. Moreover, 12 prevented the formation of cytotoxic stable oligomeric intermediates, being significantly more effective, and per se less toxic, than prototype 1. More importantly, as different chemical features were exploited to regulate Nrf2 and Aβ activities, the two pathways could be tuned independently. These findings point to compound 12 and its derivatives as promising tools for investigating the therapeutic potential of the Nrf2/Aβ cellular network, laying foundation for generating new drug leads to confront AD.

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells.

PubMed

Chiu, Po Yee; Chen, Na; Leong, Po Kuan; Leung, Hoi Yan; Ko, Kam Ming

2011-04-01

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

PubMed

Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2014-02-23

Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

PubMed Central

2014-01-01

Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway. PMID:24559113

The Crosstalk Between Nrf2 and AMPK Signal Pathways Is Important for the Anti-Inflammatory Effect of Berberine in LPS-Stimulated Macrophages and Endotoxin-Shocked Mice

PubMed Central

Mo, Chunfen; Wang, Ling; Zhang, Jie; Numazawa, Satoshi; Tang, Hong; Tang, Xiaoqiang; Han, XiaoJuan; Li, Junhong; Yang, Ming; Wang, Zhe; Wei, Dandan

2014-01-01

Abstract Aims: The response of AMP-activated protein kinase (AMPK) to oxidative stress has been recently reported but the downstream signals of this response are largely unknown. Meanwhile, the upstream events for the activation of nuclear factor erythroid-2-related factor-2 (Nrf2), a critical transcriptional activator for antioxidative responses, remain unclear. In the present study, we investigated the relationship between AMPK and Nrf2 signal pathways in lipopolysaccharide (LPS)-triggered inflammatory system, in which berberine (BBR), a known AMPK activator, was used for inflammation suppression. Results and Innovation: In inflammatory macrophages, BBR attenuated LPS-induced expression of inflammatory genes (inducible nitric oxide synthase [iNOS], cyclooxygenase-2 [COX2], interleukin [IL]-6), and the generation of nitric oxide and reactive oxygen species, but increased the transcription of Nrf2-targeted antioxidative genes (NADPH quinone oxidoreductase-1 [NQO-1], heme oxygenase-1 [HO-1]), as well as the nuclear localization and phosphorylation of Nrf2 protein. Importantly, we found BBR-induced activation of Nrf2 is AMPK-dependent, as either pharmacologically or genetically inactivating AMPK blocked the activation of Nrf2. Consistent with in vitro experiments, BBR down-regulated the expression of proinflammatory genes but upregulated those of Nrf2-targeted genes in lungs of LPS-injected mice, and these effects were attenuated in Nrf2-deficient mice. Moreover, the effect of BBR on survival time extension and plasma redox regulation in endotoxin-shocked mice was largely weakened when Nrf2-depleted. Conclusions: Our results demonstrate convergence between AMPK and Nrf2 pathways and this intersection is essential for anti-inflammatory effect of BBR in LPS-stimulated macrophages and endotoxin-shocked mice. Uncovering this intersection is significant for understanding the relationship between energy homeostasis and antioxidative responses and may be beneficial for developing new therapeutic strategies against inflammatory diseases. Antioxid. Redox Signal. 20, 574–588. PMID:23875776

Induction of the pi class of glutathione S-transferase by carnosic acid in rat Clone 9 cells via the p38/Nrf2 pathway.

PubMed

Lin, Chia-Yuan; Wu, Chi-Rei; Chang, Shu-Wei; Wang, Yu-Jung; Wu, Jia-Jiuan; Tsai, Chia-Wen

2015-06-01

Induction of phase II enzymes is important in cancer chemoprevention. We compared the effect of rosemary diterpenes on the expression of the pi class of glutathione S-transferase (GSTP) in rat liver Clone 9 cells and the signaling pathways involved. Culturing cells with 1, 5, 10, or 20 μM carnosic acid (CA) or carnosol (CS) for 24 h in a dose-dependent manner increased the GSTP expression. CA was more potent than CS. The RNA level and the enzyme activity of GSTP were also enhanced by CA treatment. Treatment with 10 μM CA highly induced the reporter activity of the enhancer element GPEI. Furthermore, CA markedly increased the translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) from the cytosol to the nucleus after 30 to 60 min. CA the stimulated the protein induction of p38, nuclear Nrf2, and GSTP was diminished in the presence of SB203580 (a p38 inhibitor). In addition, SB203580 pretreatment or silencing of Nrf2 by siRNA suppressed the CA-induced GPEI-DNA binding activity and GSTP protein expression. Knockdown of p38 or Nrf2 by siRNA abolished the activation of p38 and Nrf2 as well as the protein induction and enzyme activity of GSTP by CA. These results suggest that CA up-regulates the expression and enzyme activity of GSTP via the p38/Nrf2/GPEI pathway.

Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway.

PubMed

Lin, Wen; Wu, Rachel T; Wu, Tienyuan; Khor, Tin-Oo; Wang, Hu; Kong, Ah-Ng

2008-10-15

Sulforaphane (SFN) is a natural isothiocyanate that is present in cruciferous vegetables such as broccoli and cabbage. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents, which is related in part to its potent anti-inflammation properties. In the present study, we compared the anti-inflammatory effect of SFN on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 (-/-) mice. Pretreatment of SFN in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNF-alpha, IL-1beta, COX-2 and iNOS. HO-1 expression was significantly augmented in LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by SFN. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (-/-) primary peritoneal macrophages. We concluded that SFN exerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages.

Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5–Nrf2 pathway

PubMed Central

Jimenez-Blasco, D; Santofimia-Castaño, P; Gonzalez, A; Almeida, A; Bolaños, J P

2015-01-01

Neurotransmission unavoidably increases mitochondrial reactive oxygen species. However, the intrinsic antioxidant defense of neurons is weak and hence the mechanism whereby these cells are physiologically protected against oxidative damage is unknown. Here we found that the antioxidant defense of neurons is repressed owing to the continuous protein destabilization of the master antioxidant transcriptional activator, nuclear factor-erythroid 2-related factor-2 (Nrf2). By contrast, Nrf2 is highly stable in neighbor astrocytes explaining their robust antioxidant defense and resistance against oxidative stress. We also show that subtle and persistent stimulation of N-methyl-d-aspartate receptors (NMDAR) in astrocytes, through a mechanism not requiring extracellular Ca2+ influx, upregulates a signal transduction pathway involving phospholipase C-mediated endoplasmic reticulum release of Ca2+ and protein kinase Cδ activation. Active protein kinase Cδ promotes, by phosphorylation, the stabilization of p35, a cyclin-dependent kinase-5 (Cdk5) cofactor. Active p35/Cdk5 complex in the cytosol phosphorylates Nrf2 at Thr395, Ser433 and Thr439 that is sufficient to promote Nrf2 translocation to the nucleus and induce the expression of antioxidant genes. Furthermore, this Cdk5–Nrf2 transduction pathway boosts glutathione metabolism in astrocytes efficiently protecting closely spaced neurons against oxidative damage. Thus, intercellular communication through NMDAR couples neurotransmission with neuronal survival. PMID:25909891

Akt and ERK/Nrf2 activation by PUFA oxidation-derived aldehydes upregulates FABP4 expression in human macrophages.

PubMed

Lázaro, Iolanda; Ferré, Raimon; Masana, Lluís; Cabré, Anna

2013-10-01

In macrophages, adipocyte fatty acid-binding protein (FABP4) coordinates key events in oxidized LDL-induced foam cell formation, such as cholesterol trafficking and inflammatory responses. Nrf2 is a redox-sensitive transcription factor with antioxidant and anti-inflammatory properties. We investigated the involvement of the Nrf2 signaling pathway in FABP4-upregulation in response to aldehydes that are derived from polyunsaturated fatty acid (PUFA) oxidation. Using RT-PCR and western blotting, we found that the aldehyde 2,4-decadienal (2,4-DDE) produced a marked increase in FABP4 mRNA and protein levels. 2,4-DDE acts at the transcriptional level of FABP4 by promoting mRNA synthesis and prolonging the half-life of the de novo synthesized mRNA. 2,4-DDE consistently enhanced nuclear translocation of phosphorylated Nrf2, which was mediated by the activation of the Akt and ERK signaling pathways. A chromatin immunoprecipitation assay showed the in vivo binding of activated Nrf2 to a newly identified ARE site in the human FABP4 promoter. We propose an Akt and ERK/Nrf2-dependent FABP4 upregulation pathway in response to PUFA oxidation end-products in human macrophages. These results open a new avenue for putative therapeutic targets addressed to control atherogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Suppression of antioxidant Nrf-2 and downstream pathway in H9c2 cells by advanced glycation end products (AGEs) via ERK phosphorylation.

PubMed

Ko, Shun-Yao; Chang, Shu-Shing; Lin, I-Hsuan; Chen, Hong-I

2015-11-01

Diabetic cardiomyopathy is related to oxidative stress and correlated with the presence of advanced glycation end products (AGEs). In a clinical setting, AGEs can be detected in patients presenting diabetic cardiomyopathy; however, the underlying mechanism has yet to be elucidated. In our previous study, AGEs increase cell hypertrophy via ERK phosphorylation in a process closely related to ROS production. Thus, we propose that AGEs regulate the antioxidant gene nuclear factor-erythroid 2-related factor (Nrf-2). In H9c2 cells treated with AGEs, the expression of Nrf-2 was reduced; however, ERK phosphorylation was shown to increase. Treatment with H2O2 was also shown to increase Nrf-2 and ERK phosphorylation. In cells pretreatment with ROS scavenger NAC, the effects of H2O2 were reduced; however, the effects of the AGEs remained largely unchanged. Conversely, when cells were pretreated with PD98059 (ERK inhibitor), the expression of Nrf-2 was recovered following treatment with AGEs. Our results suggest that AGEs inhibit Nrf-2 via the ERK pathway; however, this influence is partly associated with ROS. Our finding further indicated that AGEs possess both ROS-dependent and ROS-independent pathways, resulting in a reduction in Nrf-2. This report reveals an important mechanism underlying the regulation of diabetic cardiomyopathy progression by AGEs. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway

PubMed Central

Ye, Fang; Li, Xiaoyi; Li, Lili; Yuan, Jing; Chen, Jun

2016-01-01

The neurotoxicity of lead has been well established, and oxidative stress is strongly associated with lead-induced neurotoxicity. Nrf2 is important for protection against oxidative stress in many disease models. We applied t-BHQ, which is an Nrf2 activator, to investigate the possible role of Nrf2 in the protection against lead neurotoxicity. t-BHQ significantly attenuated the oxidative stress in developmental rats by decreasing MDA level, as well as by increasing SOD activity and GSH content, in the hippocampus and frontal cortex. Furthermore, neuronal apoptosis was detected by Nissl staining, and Bax expression was inhibited in the t-BHQ-treated group. Results showed that t-BHQ suppressed ROS production and caspase 3/7 activity but increased intracellular GSH content, in SH-SY5Y cells under lead exposure. Moreover, in vivo and in vitro, t-BHQ enhanced the nuclear translocation of Nrf2 and binding to ARE areas but did not induce Nrf2 transcription. These phenomena were confirmed using RT-PCR, EMSA, Western blot, and immunofluorescence analyses. Subsequent upregulation of the expression of HO-1, NQO1, and GCLC was observed. However, knockdown of Nrf2 or HO-1 adversely affected the protective effects of t-BHQ against lead toxicity in SH-SY5Y cells. Thus, t-BHQ can protect against lead neurotoxicity, depending on the Nrf2/HO-1 pathway. PMID:26798413

Dendritic cells' death induced by contact sensitizers is controlled by Nrf2 and depends on glutathione levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Ali, Zeina

Dendritic cells (DC) are known to play a major role during contact allergy induced by contact sensitizers (CS). Our previous studies showed that Nrf2 was induced in DC and controlled allergic skin inflammation in mice in response to chemicals. In this work, we raised the question of the role of Nrf2 in response to a stress provoked by chemical sensitizers in DC. We used two well-described chemical sensitizers, dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA), known to have different chemical reactivity and mechanism of action. First, we performed a RT-qPCR array showing that CinA was a higher inducer of immune and detoxificationmore » genes compared to DNCB. Interestingly, in the absence of Nrf2, gene expression was dramatically affected in response to DNCB but was slightly affected in response to CinA. These observations prompted us to study DC's cell death in response to both chemicals. DNCB and CinA increased apoptotic cells and decreased living cells in the absence of Nrf2. The characterization of DC apoptosis induced by both CS involved the mitochondrial-dependent caspase pathway and was regulated via Nrf2 in response to both chemicals. Oxidative stress induced by DNCB, and leading to cell death, was regulated by Nrf2. Unlike CinA, DNCB treatment provoked a significant reduction of intracellular GSH levels and up-regulated bcl-2 gene expression, under the control of Nrf2. This work underlies that chemical reactivity may control Nrf2-dependent gene expression leading to different cytoprotective mechanisms in DC. - Highlights: • Nrf2 controls cell death induced by contact sensitizers in dendritic cells. • DNCB reduced GSH levels and up-regulated bcl-2 gene expression unlike CinA. • Chemical reactivity controls Nrf2-dependent genes having protective effect in DC.« less

Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lu; Gallagher, Evan P., E-mail: evang3@uw.edu

2013-01-15

Exposure to trace metals can disrupt olfactory function in fish leading to a loss of behaviors critical to survival. Cadmium (Cd) is an olfactory toxicant that elicits cellular oxidative stress as a mechanism of toxicity while also inducing protective cellular antioxidant genes via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the molecular mechanisms of Cd-induced olfactory injury have not been characterized. In the present study, we investigated the role of the Nrf2-mediated antioxidant defense pathway in protecting against Cd-induced olfactory injury in zebrafish. A dose-dependent induction of Nrf2-regulated antioxidant genes associated with cellular responses to oxidativemore » stress was observed in the olfactory system of adult zebrafish following 24 h Cd exposure. Zebrafish larvae exposed to Cd for 3 h showed increased glutathione S-transferase pi (gst pi), glutamate–cysteine ligase catalytic subunit (gclc), heme oxygenase 1 (hmox1) and peroxiredoxin 1 (prdx1) mRNA levels indicative of Nrf2 activation, and which were blocked by morpholino-mediated Nrf2 knockdown. The inhibition of antioxidant gene induction in Cd-exposed Nrf2 morphants was associated with disruption of olfactory driven behaviors, increased cell death and loss of olfactory sensory neurons (OSNs). Nrf2 morphants also exhibited a downregulation of OSN-specific genes after Cd exposure. Pre-incubation of embryos with sulforaphane (SFN) partially protected against Cd-induced olfactory tissue damage. Collectively, our results indicate that oxidative stress is an important mechanism of Cd-mediated injury in the zebrafish olfactory system. Moreover, the Nrf2 pathway plays a protective role against cellular oxidative damage and is important in maintaining zebrafish olfactory function. -- Highlights: ► Oxidative stress is an important mechanism of Cd-mediated olfactory injury. ► Cd induces antioxidant gene expression in the zebrafish olfactory system. ► The olfactory antioxidant response is blocked by Nrf2 knockdown. ► Disruption of olfactory neurobehaviors is associated with Nrf2 knockdown. ► Nrf2 morphants show increased cell death and olfactory sensory neuron loss.« less

Luteolin inhibits the Nrf2 signaling pathway and tumor growth in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chian, Song; Thapa, Ruby; Chi, Zhexu

Highlights: • Luteolin inhibits the Nrf2 pathway in mouse liver and in xenografted tumors. • Luteolin markedly inhibits the growth of xenograft tumors. • Luteolin enhances the anti-cancer effect of cisplatin in mice in vivo. • Luteolin could serve as an adjuvant in the chemotherapy of NSCLC. - Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) is over-expressed in many types of tumor, promotes tumor growth, and confers resistance to anticancer therapy. Hence, Nrf2 is regarded as a novel therapeutic target in cancer. Previously, we reported that luteolin is a strong inhibitor of Nrf2 in vitro. Here, we showed thatmore » luteolin reduced the constitutive expression of NAD(P)H quinone oxidoreductase 1 in mouse liver in a time- and dose-dependent manner. Further, luteolin inhibited the expression of antioxidant enzymes and glutathione transferases, decreasing the reduced glutathione in the liver of wild-type mice under both constitutive and butylated hydroxyanisole-induced conditions. In contrast, such distinct responses were not detected in Nrf2{sup −/−} mice. In addition, oral administration of luteolin, either alone or combined with intraperitoneal injection of the cytotoxic drug cisplatin, greatly inhibited the growth of xenograft tumors from non-small-cell lung cancer (NSCLC) cell line A549 cells grown subcutaneously in athymic nude mice. Cell proliferation, the expression of Nrf2, and antioxidant enzymes were all reduced in tumor xenograft tissues. Furthermore, luteolin enhanced the anti-cancer effect of cisplatin. Together, our findings demonstrated that luteolin inhibits the Nrf2 pathway in vivo and can serve as an adjuvant in the chemotherapy of NSCLC.« less

Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; College of Pharmacy, Chosun University, Gwangju; Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.k

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gbeta1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation,more » which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gbeta1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.« less

Cerebroprotection of Flavanol (−)-Epicatechin after Traumatic Brain Injury via Nrf2-dependent and –independent Pathways

PubMed Central

Cheng, Tian; Wang, Wenzhu; Li, Qian; Han, Xiaoning; Xing, Jing; Qi, Cunfang; Lan, Xi; Wan, Jieru; Potts, Alexa; Guan, Fangxia; Wang, Jian

2016-01-01

Traumatic brain injury (TBI), which leads to disability, dysfunction, and even death, is a prominent health problem worldwide with no effective treatment. A brain-permeable flavonoid named (−)-epicatechin (EC) modulates redox/oxidative stress and has been shown to be beneficial for vascular and cognitive function in humans and for ischemic and hemorrhagic stroke in rodents. Here we examined whether EC is able to protect the brain against TBI-induced brain injury in mice and if so, whether it exerts neuroprotection by modulating the NF-E2-related factor (Nrf2) pathway. We used the controlled cortical impact model to mimic TBI. EC was administered orally at 3 h after TBI and then every 24 h for either 3 or 7 days. We evaluated lesion volume, brain edema, white matter injury, neurologic deficits, cognitive performance and emotion-like behaviors, neutrophil infiltration, reactive oxygen species (ROS), and a variety of injury-related protein markers. Nrf2 knockout mice were used to determine the role of the Nrf2 signaling pathway after EC treatment. In wild-type mice, EC significantly reduced lesion volume, edema, and cell death and improved neurologic function on days 3 and 28; cognitive performance and depression-like behaviors were also improved with EC administration. In addition, EC reduced white matter injury, heme oxygenase-1 expression, and ferric iron deposition after TBI. These changes were accompanied by attenuation of neutrophil infiltration and oxidative insults, reduced activity of matrix metalloproteinase 9, decreased Keap 1 expression, increased Nrf2 nuclear accumulation, and increased expression of superoxide dismutase 1 and quinone 1. However, EC did not significantly reduce lesion volume or improve neurologic deficits in Nrf2 knockout mice after TBI. Our results show that EC protects the TBI brain by activating the Nrf2 pathway, inhibiting heme oxygenase-1 protein expression, and reducing iron deposition. The latter two effects could represent an Nrf2-independent mechanism in this model of TBI. PMID:26724590

Curcumin plays neuroprotective roles against traumatic brain injury partly via Nrf2 signaling.

PubMed

Dong, Wenwen; Yang, Bei; Wang, Linlin; Li, Bingxuan; Guo, Xiangshen; Zhang, Miao; Jiang, Zhenfei; Fu, Jingqi; Pi, Jingbo; Guan, Dawei; Zhao, Rui

2018-05-01

Traumatic brain injury (TBI), which leads to high mortality and morbidity, is a prominent public health problem worldwide with no effective treatment. Curcumin has been shown to be beneficial for neuroprotection in vivo and in vitro, but the underlying mechanism remains unclear. This study determined whether the neuroprotective role of curcumin in mouse TBI is dependent on the NF-E2-related factor (Nrf2) pathway. The Feeney weight-drop contusion model was used to mimic TBI. Curcumin was administered intraperitoneally 15 min after TBI induction, and brains were collected at 24 h after TBI. The levels of Nrf2 and its downstream genes (Hmox-1, Nqo1, Gclm, and Gclc) were detected by Western blot and qRT-PCR at 24 h after TBI. In addition, edema, oxidative damage, cell apoptosis and inflammatory reactions were evaluated in wild type (WT) and Nrf2-knockout (Nrf2-KO) mice to explore the role of Nrf2 signaling after curcumin treatment. In wild type mice, curcumin treatment resulted in reduced ipsilateral cortex injury, neutrophil infiltration, and microglia activation, improving neuron survival against TBI-induced apoptosis and degeneration. These effects were accompanied by increased expression and nuclear translocation of Nrf2, and enhanced expression of antioxidant enzymes. However, Nrf2 deletion attenuated the neuroprotective effects of curcumin in Nrf2-KO mice after TBI. These findings demonstrated that curcumin effects on TBI are associated with the activation the Nrf2 pathway, providing novel insights into the neuroprotective role of Nrf2 and the potential therapeutic use of curcumin for TBI. Copyright © 2018. Published by Elsevier Inc.

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients

PubMed Central

Kanamori, Masayuki; Higa, Tsuyoshi; Sonoda, Yukihiko; Murakami, Shohei; Dodo, Mina; Kitamura, Hiroshi; Taguchi, Keiko; Shibata, Tatsuhiro; Watanabe, Mika; Suzuki, Hiroyoshi; Shibahara, Ichiyo; Saito, Ryuta; Yamashita, Yoji; Kumabe, Toshihiro; Yamamoto, Masayuki; Motohashi, Hozumi; Tominaga, Teiji

2015-01-01

Background Nuclear factor erythroid 2–related factor 2 (NRF2) plays pivotal roles in cytoprotection. We aimed at clarifying the contribution of the NRF2 pathway to malignant glioma pathology. Methods NRF2 target gene expression and its association with prognosis were examined in 95 anaplastic gliomas with or without isocitrate dehydrogenase (IDH) 1/2 gene mutations and 52 glioblastomas. To explore mechanisms for the altered activity of the NRF2 pathway, we examined somatic mutations and expressions of the NRF2 gene and those encoding NRF2 regulators, Kelch-like ECH-associated protein 1 (KEAP1) and p62/SQSTSM. To clarify the functional interaction between IDH1 mutations and the NRF2 pathway, we introduced a mutant IDH1 to T98 glioblastoma-derived cells and examined the NRF2 activity in these cells. Results NRF2 target genes were elevated in 13.7% and 32.7% of anaplastic gliomas and glioblastomas, respectively. Upregulation of NRF2 target genes correlated with poor prognosis in anaplastic gliomas but not in glioblastomas. Neither somatic mutations of NRF2/KEAP1 nor dysregulated expression of KEAP1/p62 explained the increased expression of NRF2 target genes. In most cases of anaplastic glioma with mutated IDH1/2, NRF2 and its target genes were downregulated. This was reproducible in IDH1 R132H–expressing T98 cells. In minor cases of IDH1/2-mutant anaplastic gliomas with increased expression of NRF2 target genes, the clinical outcomes were significantly poor. Conclusions The NRF2 activity is increased in a significant proportion of malignant gliomas in general but decreased in the majority of IDH1/2-mutant anaplastic gliomas. It is plausible that the NRF2 pathway plays an important role in tumor progression of anaplastic gliomas with IDH1/2 mutations. PMID:25304134

Sulforaphane activates the cerebral vascular Nrf2-ARE pathway and suppresses inflammation to attenuate cerebral vasospasm in rat with subarachnoid hemorrhage.

PubMed

Zhao, Xudong; Wen, Liting; Dong, Min; Lu, Xiaojie

2016-12-15

Nrf2-ARE pathway reportedly plays a protective role in several central nervous system diseases. No study has explored the role of the Nrf2-ARE pathway in cerebral vasospasm(CVS) after subarachnoid hemorrhage(SAH). The purpose of the present study was to investigate the activation of the cerebral vascular Nrf2-ARE pathway and to determine the potential role of this pathway in the development of CVS following SAH. We investigated whether the administration of sulforaphane (SFN, a specific Nrf2 activator) modulated vascular caliber, Nrf2-ARE pathway activity, proinflammatory cytokine expression, and clinical behavior in a rat model of SAH. A two-hemorrhage protocol was used to generate an animal model of SAH in male Sprague-Dawley rats. Administration of SFN to these rats following SAH enhanced the activity of the Nrf2-ARE pathway and suppressed the release of proinflammatory cytokines. Vasospasm was markedly attenuated in the basilar arteries after SFN therapy. Additionally, SFN administration significantly ameliorated two behavioral functions disrupted by SAH. These results suggest that SFN has a therapeutic benefit in post-SAH, and this may be due to elevated Nrf2-ARE pathway activity and inhibition of cerebral vascular proinflammatory cytokine expression. Copyright © 2016. Published by Elsevier B.V.

Activation of p62-keap1-Nrf2 antioxidant pathway in the early stage of acetaminophen-induced acute liver injury in mice.

PubMed

Shen, Zhenyu; Wang, Yu; Su, Zhenhui; Kou, Ruirui; Xie, Keqin; Song, Fuyong

2018-02-25

Acetaminophen (APAP) overdose can cause severe liver failure even death. Nearly half of drug-induced liver injury is attributed to APAP in the US and many European countries. Oxidative stress has been validated as a critical event involved in APAP-induced liver failure. p62/SQSTM1, a selective autophagy adaptor protein, is reported to regulate Nrf2-ARE antioxidant pathway in response to oxidative stress. However, the exact role of p62-keap1-Nrf2 antioxidant pathway in APAP-induced hepatotoxicity remains unknown. In the present study, the dose-response and time-course model in C57/BL6 mice were established by intraperitoneal injection of APAP. The results of serum alanine/aspartate aminotransferases (ALT/AST) and histological examination demonstrated that APAP overdose resulted in the severe liver injury. In the meantime, the levels of p62, phospho-p62 and nuclear Nrf2 were significantly increased by APAP in mice liver, suggesting an activation of p62-keap1-Nrf2 pathway. In addition, the expression of GSTA1 mRNA was increased in a dose-dependent manner, while the mRNA levels of HO-1 and GCLC were decreased with the increase of APAP dose. Our further investigation found that expression of HO-1 and GCLC peaked at 3 h∼6 h, and then were decreased gradually. Taken together, these results indicated that p62-keap1-Nrf2 antioxidant pathway was primarily activated in the early stage of APAP hepatotoxicity, which might play a protective role in the process of APAP-induced acute liver injury. Copyright © 2018 Elsevier B.V. All rights reserved.

Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

PubMed Central

Bailey-Downs, Lora C.; Mitschelen, Matthew; Sosnowska, Danuta; Toth, Peter; Pinto, John T.; Ballabh, Praveen; Valcarcel-Ares, M.Noa; Farley, Julie; Koller, Akos; Henthorn, Jim C.; Bass, Caroline; Sonntag, William E.; Csiszar, Anna

2012-01-01

Recent studies demonstrate that age-related dysfunction of NF-E2–related factor-2 (Nrf2)–driven pathways impairs cellular redox homeostasis, exacerbating age-related cellular oxidative stress and increasing sensitivity of aged vessels to oxidative stress–induced cellular damage. Circulating levels of insulin-like growth factor (IGF)-1 decline during aging, which significantly increases the risk for cardiovascular diseases in humans. To test the hypothesis that adult-onset IGF-1 deficiency impairs Nrf2-driven pathways in the vasculature, we utilized a novel mouse model with a liver-specific adeno-associated viral knockdown of the Igf1 gene using Cre-lox technology (Igf1f/f + MUP-iCre-AAV8), which exhibits a significant decrease in circulating IGF-1 levels (∼50%). In the aortas of IGF-1–deficient mice, there was a trend for decreased expression of Nrf2 and the Nrf2 target genes GCLC, NQO1 and HMOX1. In cultured aorta segments of IGF-1–deficient mice treated with oxidative stressors (high glucose, oxidized low-density lipoprotein, and H2O2), induction of Nrf2-driven genes was significantly attenuated as compared with control vessels, which was associated with an exacerbation of endothelial dysfunction, increased oxidative stress, and apoptosis, mimicking the aging phenotype. In conclusion, endocrine IGF-1 deficiency is associated with dysregulation of Nrf2-dependent antioxidant responses in the vasculature, which likely promotes an adverse vascular phenotype under pathophysiological conditions associated with oxidative stress (eg, diabetes mellitus, hypertension) and results in accelerated vascular impairments in aging. PMID:22021391

Nrf2 protects against oxidative stress induced by SiO2 nanoparticles.

PubMed

Liu, Wei; Hu, Tao; Zhou, Li; Wu, Desheng; Huang, Xinfeng; Ren, Xiaohu; Lv, Yuan; Hong, Wenxu; Huang, Guanqin; Lin, Zequn; Liu, Jianjun

2017-10-01

The aim of our study was to explore the role of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) on the exposure of SiO 2 nanoparticles (NPs) and its influence. To understand the mechanism of NP-induced oxidative stress, the involvement of oxidative-stress-responding transcription factors and the Nrf2/antioxidant reactive element (ARE) signaling pathway in the toxicity of SiO 2 NPs' exposure was investigated via in vivo and in vitro models. A549 cells showed a significant cytotoxic effect while A549-shNrf2 cells showed decreased cell viability after nm-SiO 2 exposure. SiO 2 NPs' exposure activated the Nrf2/ARE signaling pathway. Nrf2 -/- exposed mice showed increased reactive oxygen species, 8-hydroxyl deoxyguanosine level and decreased total antioxidant capacity. Nrf2/ARE signaling pathway activation disrupted, leading inhibition of heme oxygenase-1 and upregulation of PKR-like endoplasmic-reticulum-regulated kinase. Our findings suggested that Nrf2 could protect against oxidative stress induced by SiO 2 NPs, and the Nrf2/ARE pathway might be involved in mild-to-moderate SiO 2 NP-induced oxidative stress that was evident from dampened activity of Nrf2.

Temporal activation of Nrf2 in the penumbra and Nrf2 activator-mediated neuroprotection in ischemia-reperfusion injury.

PubMed

Takagi, Toshinori; Kitashoji, Akira; Iwawaki, Takao; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

2014-07-01

Oxidative stress plays a critical role in mediating tissue injury and neuron death during ischemia-reperfusion injury (IRI). The Keap1-Nrf2 defense pathway serves as a master regulator of endogenous antioxidant defense, and Nrf2 has been attracting attention as a target for the treatment of IRI. In this study, we evaluated Nrf2 expression in IRI using OKD (Keap1-dependent oxidative stress detector) mice and investigated the neuroprotective ability of an Nrf2 activator. We demonstrated temporal changes in Nrf2 expression in the same mice with luciferase assays and an Nrf2 activity time course using Western blotting. We also visualized Nrf2 expression in the ischemic penumbra and investigated Nrf2 expression in mice and humans using immunohistochemistry. Endogenous Nrf2 upregulation was not detected early in IRI, but expression peaked 24h after ischemia. Nrf2 expression was mainly detected in the penumbra, and it was found in neurons and astrocytes in both mice and humans. Intravenous administration of the Nrf2 activator bardoxolone methyl (BARD) resulted in earlier upregulation of Nrf2 and heme oxygenase-1. Furthermore, BARD decreased infarction volume and improved neurological symptoms after IRI. These findings indicate that earlier Nrf2 activation protects neurons, possibly via effects on astrocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; Kim, Hyung Gyun; Han, Eun Hee

2008-09-15

The zinc-binding protein metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. In this study, we demonstrate that MT-III prevents the accumulation of reactive oxygen species (ROS) in dopaminergic SH-SY5Y cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) and ERK kinase/NF-E2-related factor 2 (Nrf2) dependent induction of the stress response protein heme oxygenase-1 (HO-1). Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, MT-III up-regulates HO-1 expression and this expression confers neuroprotectionmore » against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that the PI3K and ERK/Nrf2 signaling pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.« less

Natural product-derived pharmacological modulators of Nrf2/ARE pathway for chronic diseases.

PubMed

Kumar, Hemant; Kim, In-Su; More, Sandeep Vasant; Kim, Byung-Wook; Choi, Dong-Kug

2014-01-01

Covering: 2000 to 2013. Oxidative stress is the central component of chronic diseases. The nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway is vital in the up-regulation of cytoprotective genes and enzymes in response to oxidative stress and treatment with certain dietary phytochemicals. Herein, we classify bioactive compounds derived from natural products that are Nrf2/ARE pathway activators and recapitulate the molecular mechanisms for inducing Nrf2 to provide favorable effects in experimental models of chronic diseases. Moreover, pharmacological inhibition of Nrf2 signalling has emerged as promising strategy against multi-drug resistance thereby improving the treatment efficacy. We have also enlisted natural product-derived inhibitors of Nrf2/ARE pathway.

Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway

PubMed Central

Lin, Wen; Wu, Rachel T; Wu, Tienyuan; Khor, Tin-Oo; Wang, Hu; Kong, Ah-Ng

2008-01-01

Sulforaphane (SFN) is a natural isothiocyanate that is present in cruciferous vegetables such as broccoli and cabbage. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents, which is related in part to its potent anti-inflammation properties. In the present study, we compared the anti-inflammatory effect of SFN on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 mice. Pretreatment of SFN in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNFα, IL-1β, Cox-2 and iNOS. HO-1 expression, which is significantly augmented in LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by SFN. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (−/−) primary peritoneal macrophages. We concluded that SFN exerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages. PMID:18755157

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

PubMed

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S; Franklin, Christopher C; McCord, Joe M; Day, Brian J

2011-09-15

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization of the Antioxidant Effects of γ-Oryzanol: Involvement of the Nrf2 Pathway

PubMed Central

Rungratanawanich, W.; Serafini, M. M.; Guarienti, M.; Catanzaro, M.; Marziano, M.; Memo, M.; Lanni, C.

2018-01-01

γ-Oryzanol (ORY) is well known for its antioxidant potential. However, the mechanism by which ORY exerts its antioxidant effect is still unclear. In this paper, the antioxidant properties of ORY were investigated for its potential effects as a reactive oxygen and nitrogen species (ROS/RNS) scavenger and in activating antioxidant-promoting intracellular pathways utilizing the human embryonic kidney cells (HEK-293). The 24 h ORY exposure significantly prevented hydrogen peroxide- (H2O2-) induced ROS/RNS production at 3 h, and this effect was sustained for at least 24 h. ORY pretreatment also enhanced the activity of antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidase (GPX). Interestingly, ORY induced the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation and upregulation of Nrf2-dependent defensive genes such as NAD(P)H quinone reductase (NQO1), heme oxygenase-1 (HO-1), and glutathione synthetase (GSS) at mRNA and protein levels in both basal condition and after H2O2 insult. Thus, this study suggested an intriguing effect of ORY in modulating the Nrf2 pathway, which is also involved in regulating longevity as well as age-related diseases. PMID:29725495

Characterization of the Antioxidant Effects of γ-Oryzanol: Involvement of the Nrf2 Pathway.

PubMed

Rungratanawanich, W; Abate, G; Serafini, M M; Guarienti, M; Catanzaro, M; Marziano, M; Memo, M; Lanni, C; Uberti, D

2018-01-01

γ -Oryzanol (ORY) is well known for its antioxidant potential. However, the mechanism by which ORY exerts its antioxidant effect is still unclear. In this paper, the antioxidant properties of ORY were investigated for its potential effects as a reactive oxygen and nitrogen species (ROS/RNS) scavenger and in activating antioxidant-promoting intracellular pathways utilizing the human embryonic kidney cells (HEK-293). The 24 h ORY exposure significantly prevented hydrogen peroxide- (H 2 O 2 -) induced ROS/RNS production at 3 h, and this effect was sustained for at least 24 h. ORY pretreatment also enhanced the activity of antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidase (GPX). Interestingly, ORY induced the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation and upregulation of Nrf2-dependent defensive genes such as NAD(P)H quinone reductase (NQO1), heme oxygenase-1 (HO-1), and glutathione synthetase (GSS) at mRNA and protein levels in both basal condition and after H 2 O 2 insult. Thus, this study suggested an intriguing effect of ORY in modulating the Nrf2 pathway, which is also involved in regulating longevity as well as age-related diseases.

Ezetimibe, an NPC1L1 inhibitor, is a potent Nrf2 activator that protects mice from diet-induced nonalcoholic steatohepatitis.

PubMed

Lee, Da Hyun; Han, Dai Hoon; Nam, Ki Taek; Park, Jeong Su; Kim, Soo Hyun; Lee, Milim; Kim, Gyuri; Min, Byung Soh; Cha, Bong-Soo; Lee, Yu Seol; Sung, Su Haeng; Jeong, Haengdueng; Ji, Hye Won; Lee, Moon Joo; Lee, Jae Sung; Lee, Hui-Young; Chun, Yoomi; Kim, Joungmok; Komatsu, Masaaki; Lee, Yong-Ho; Bae, Soo Han

2016-10-01

Oxidative stress is important for the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a chronic disease that ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The nuclear factor erythroid 2-related factor 2-Kelch-like ECH associated protein 1 (Nrf2-Keap1) pathway is essential for cytoprotection against oxidative stress. In this study, we found that oxidative stress or inflammatory biomarkers and TUNEL positive cells were markedly increased in NASH patients compared to normal or simple steatosis. In addition, we identified that the hepatic mRNA levels of Nrf2 target genes such as Nqo-1 and GSTA-1 were significantly increased in NASH patients. Ezetimibe, a drug approved by the Food and Drug Administration for the treatment of hypercholesterolemia, improves NAFLD and alleviates oxidative stress. However, the precise mechanism of its antioxidant function remains largely unknown. We now demonstrate that ezetimibe activates Nrf2-Keap1 pathway which was dependent of autophagy adaptor protein p62, without causing cytotoxicity. Ezetimibe activates AMP-activated protein kinase (AMPK), which in turn phosphorylates p62 (p-S351) via their direct interaction. Correspondingly, Ezetimibe protected liver cells from saturated fatty acid-induced apoptotic cell death through p62-dependent Nrf2 activation. Furthermore, its role as an Nrf2 activator was supported by methione- and choline- deficient (MCD) diet-induced NASH mouse model, showing that ezetimibe decreased the susceptibility of the liver to oxidative injury. These data demonstrate that the molecular mechanisms underlying ezetimibe's antioxidant role in the pathogenesis of NASH. Copyright © 2016 Elsevier Inc. All rights reserved.

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant response promotes melanocyte viability and reduces toxicity of the vitiligo-inducing phenol monobenzone.

PubMed

Arowojolu, Omotayo A; Orlow, Seth J; Elbuluk, Nada; Manga, Prashiela

2017-07-01

Vitiligo, characterised by progressive melanocyte death, can be initiated by exposure to vitiligo-inducing phenols (VIPs). VIPs generate oxidative stress in melanocytes and activate the master antioxidant regulator NRF2. While NRF2-regulated antioxidants are reported to protect melanocytes from oxidative stress, the role of NRF2 in the melanocyte response to monobenzone, a clinically relevant VIP, has not been characterised. We hypothesised that activation of NRF2 may protect melanocytes from monobenzone-induced toxicity. We observed that knockdown of NRF2 or NRF2-regulated antioxidants NQO1 and PRDX6 reduced melanocyte viability, but not viability of keratinocytes and fibroblasts, suggesting that melanocytes were preferentially dependent upon NRF2 activity for growth compared to other cutaneous cells. Furthermore, melanocytes activated the NRF2 response following monobenzone exposure and constitutive NRF2 activation reduced monobenzone toxicity, supporting NRF2's role in the melanocyte stress response. In contrast, melanocytes from individuals with vitiligo (vitiligo melanocytes) did not activate the NRF2 response as efficiently. Dimethyl fumarate-mediated NRF2 activation protected normal and vitiligo melanocytes against monobenzone-induced toxicity. Given the contribution of oxidant-antioxidant imbalance in vitiligo, modulation of this pathway may be of therapeutic interest. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiple regulations of Keap1/Nrf2 system by dietary phytochemicals.

PubMed

Qin, Si; Hou, De-Xing

2016-08-01

Keap1/Nrf2 system plays a critical role on cellular protection by regulating many antioxidant and detoxification enzyme genes through the antioxidant response element (ARE). Thus, it must work constantly to prevent the accumulation of reactive oxygen species (ROS) because excess ROS are associated with many diseases such as cancer, cardiovascular complications, inflammation, and neurodegeneration. Dietary phytochemicals widely distributing in fruits and vegetables have been considered to possess cancer chemopreventive potential through the induction of Keap1/Nrf2 system-mediated antioxidant and detoxification enzymes in a variety of manners. The data are extensive and are not well classified on the molecular mechanisms. In this review, we first briefly introduce the current knowledge on Keap1/Nrf2 system regulation including Keap1-dependent and Keap1-independent cascades, and epigenetic pathway. Then, we summarize the molecular targets of Keap1/Nrf2 system by dietary phytochemicals, and finally review the crosstalk between Keap1/Nrf2 system and other cellular signaling pathways to regulate diverse chronic diseases by dietary phytochemicals. These comprehensive data will help us to understand the potential effects of dietary phytochemicals on the prevention of chronic diseases and maintenance of human health. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

PubMed

Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

2014-04-01

Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. Copyright © 2013 John Wiley & Sons, Ltd.

Sulforaphane enhances the activity of the Nrf2-ARE pathway and attenuates inflammation in OxyHb-induced rat vascular smooth muscle cells.

PubMed

Zhao, X-D; Zhou, Y-T; Lu, X-J

2013-09-01

A growing body of evidence indicates that the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a protective role in many physiological stress processes such as inflammatory damage, oxidative stress, and the accumulation of toxic metabolites, which are all involved in the cerebral vasospasm following subarachnoid hemorrhage (SAH). We hypothesized that the Nrf2-ARE pathway might have a protective role in cerebral vasospasm following SAH. In our study, we investigate whether the oxyhemoglobin (OxyHb) can induce the activation of the Nrf2-ARE pathway in vascular smooth muscle cells (VSMCs), and evaluate the modulatory effects of sulforaphane (SUL) on OxyHb-induced inflammation in VSMCs. As a result, both the protein level and the mRNA level of the nuclear Nrf2 were significantly increased, while the mRNA levels of two Nrf2-regulated gene products, both heme oxygenase-1 and NAD(P)H: quinone oxidoreductase-1, were also up-regulated in VSMCs induced with OxyHb. A marked increase of inflammatory cytokines such as IL-1β, IL-6 and TNF-α release was observed at 48 h after cells were treated with OxyHb. SUL enhanced the activity of the Nrf2-ARE pathway and suppressed cytokine release. Our results indicate that the Nrf2-ARE pathway was activated in OxyHb-induced VSMCs. SUL suppressed cytokine release via the activation of the Nrf2-ARE pathway in OxyHb-induced VSMCs.

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jing, Xu; Ren, Dongmei; Wei, Xinbing

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependentmore » genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke. - Highlights: • E7G activates Nrf2 in astrocytes. • E7G stimulates expression of Nrf2-mediated cytoprotective proteins in astrocytes. • E7G protects astrocytes against OGD-induced cell death and apoptosis. • The neuroprotective effect of E7G involves the Nrf2/ARE pathway. • E7G protects rats against cerebral ischemic injury.« less

Protective effect of ganodermanondiol isolated from the Lingzhi mushroom against tert-butyl hydroperoxide-induced hepatotoxicity through Nrf2-mediated antioxidant enzymes.

PubMed

Li, Bin; Lee, Dong-Sung; Kang, Yue; Yao, Nai-Qi; An, Ren-Bo; Kim, Youn-Chul

2013-03-01

Ganodermanondiol, a biologically active compound, was isolated from the Lingzhi mushroom (Ganoderma lucidum). The present study examined the protective effects of ganodermanondiol against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity. Ganodermanondiol protected human liver-derived HepG2 cells through nuclear factor-E2-related factor 2 (Nrf2) pathway-dependent heme oxygenase-1 expressions. Moreover, ganodermanondiol increased cellular glutathione levels and the expression of the glutamine-cysteine ligase gene in a dose-dependent manner. Furthermore, ganodermanondiol exposure enhanced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, LKB1 and Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII). This study indicates that ganodermanondiol exhibits potent cytoprotective effects on t-BHP-induced hepatotoxicity in human liver-derived HepG2 cells, presumably through Nrf2-mediated antioxidant enzymes and AMPK. Copyright © 2012 Elsevier Ltd. All rights reserved.

Hyperoside attenuates hydrogen peroxide-induced L02 cell damage via MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Hai-Yan; Liu, Yao; Chen, Jian-Hong

Highlights: {yields} Hyperoside attenuated H{sub 2}O{sub 2}-induced L02 cell damage. {yields} Hyperoside up-regulated HO-1 expression at both mRNA and protein levels. {yields} Hyperoside activated both Nrf{sub 2} nuclear translocation and gene expression. {yields} Hyperoside may inhibit Keap{sub 1} mRNA translation or protein degradation. {yields} Phosphorylation of ERK and p38 is involved in hyperoside-mediated Nrf{sub 2} activation. -- Abstract: The flavonoid hyperoside has been reported to elicit cytoprotection against oxidative stress partly by increasing the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase. However, the cellular and molecular mechanisms underlying this effect remain unclear. Here, hepatic L02more » cells exposed to H{sub 2}O{sub 2} (100 {mu}M) were used to demonstrate that hyperoside protected cells by significantly inhibiting overproduction of intracellular ROS, depletion of the mitochondrial membrane potential and leakage of lactate dehydrogenase. Hyperoside further enhanced the cellular antioxidant defense system through increasing the activity of heme oxygenase-1 (HO-1), and by up-regulating HO-1 expression. Meanwhile, real time PCR, western blot and immunofluorescence studies revealed that hyperoside stimulated nuclear translocation of the Nrf{sub 2} transcription factor in a dose-dependent manner, and this effect was significantly suppressed by pharmacological inhibition of the mitogen-activated protein kinases (MAPK) p38 and ERK. Collectively, our data provide the first description of the mechanism underlying hyperoside's ability to attenuate H{sub 2}O{sub 2}-induced cell damage, namely this compound interacts with the MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway to up-regulate HO-1 expression and enhance intracellular antioxidant activity.« less

Synthesis, evaluation, and metabolism of novel [6]-shogaol derivatives as potent Nrf2 activators.

PubMed

Zhu, Yingdong; Wang, Pei; Zhao, Yantao; Yang, Chun; Clark, Anderson; Leung, TinChung; Chen, Xiaoxin; Sang, Shengmin

2016-06-01

Oxidative stress is a central component of many chronic diseases. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 p45-related factor 2 (Nrf2) system is a major regulatory pathway of cytoprotective genes against oxidative and electrophilic stress. Activation of the Nrf2 pathway plays crucial roles in the chemopreventive effects of various inducers. In this study, we developed a novel class of potent Nrf2 activators derived from ginger compound, [6]-shogaol (6S), using the Tg[glutathione S-transferase pi 1 (gstp1):green fluorescent protein (GFP)] transgenic zebrafish model. Investigation of structure-activity relationships of 6S derivatives indicates that the combination of an α,β-unsaturated carbonyl entity and a catechol moiety in one compound enhances the Tg(gstp1:GFP) fluorescence signal in zebrafish embryos. Chemical reaction and in vivo metabolism studies of the four most potent 6S derivatives showed that both α,β-unsaturated carbonyl entity and catechol moiety act as major active groups for conjugation with the sulfhydryl groups of the cysteine residues. In addition, we further demonstrated that 6S derivatives increased the expression of Nrf2 downstream target, heme oxygenase-1, in both a dose- and time-dependent manner. These results suggest that α,β-unsaturated carbonyl entity and catechol moiety of 6S derivatives may react with the cysteine residues of Keap1, disrupting the Keap1-Nrf2 complex, thereby liberating and activating Nrf2. Our findings of natural product-derived Nrf2 activators lead to design options of potent Nrf2 activators for further optimization. Copyright © 2016 Elsevier Inc. All rights reserved.

The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, Raymond; Matthews, Jason, E-mail: jason.matthews@utoronto.ca

2013-07-15

Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 andmore » HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ERα, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ERα repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ERα-dependent repression of HMOX1 and NQO1.« less

Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

PubMed Central

Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

2014-01-01

Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

PubMed Central

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

2011-01-01

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells.

PubMed

Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

2018-02-06

KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF.

KEAP1-NRF2 COMPLEX IN ISCHEMIA-INDUCED HEPATOCELLULAR DAMAGE OF MOUSE LIVER TRANSPLANTS

PubMed Central

Ke, Bibo; Shen, Xiu-Da; Zhang, Yu; Ji, Haofeng; Gao, Feng; Yue, Shi; Kamo, Naoko; Zhai, Yuan; Yamamoto, Masayuki; Busuttil, Ronald W.; Kupiec-Weglinski, Jerzy W.

2015-01-01

Background The Keap1-Nrf2 signaling pathway regulates host cell defense responses against oxidative stress and maintains the cellular redox balance. Aims&Methods: We investigated the function/molecular mechanisms by which Keap1-Nrf2 complex may influence liver ischemia/reperfusion injury (IRI) in a mouse model of hepatic cold storage (20h at 4 C) followed by orthotopic liver transplantation (OLT). Results The Keap1 hepatocyte-specific knock-out (HKO) in the donor liver ameliorated post-transplant IRI, evidenced by improved hepatocellular function and OLT outcomes (Keap1HKO Keap1HKO; 100% survival), as compared with controls (WT WT; 50% survival; p<0.01). In contrast, donor liver Nrf2 deficiency exacerbated IRI in transplant recipients (Nrf2KO Nrf2KO; 40% survival). Ablation of Keap1 signaling reduced macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while simultaneously promoting anti-apoptotic functions in OLTs. At the molecular level, Keap1HKO increased Nrf2 levels, stimulated Akt phosphorylation, and enhanced expression of anti-oxidant Trx1, HIF-1 , and HO-1. Pretreatment of liver donors with PI3K inhibitor (LY294002) disrupted Akt/HIF-1 signaling and recreated hepatocellular damage in otherwise IR-resistant Keap1HKO transplants. In parallel in vitro studies, hydrogen peroxide-stressed Keap1-deficient hepatocytes were characterized by enhanced expression of Nrf2, Trx1, and Akt phosphorylation, in association with decreased release of lactate dehydrogenase (LDH) in cell culture supernatants. Conclusions Keap1-Nrf2 complex prevents oxidative injury in IR-stressed OLTs through Keap1 signaling, which negatively regulates Nrf2 pathway. Activation of Nrf2 induces Trx1 and promotes PI3K/Akt, crucial for HIF-1 activity. HIF-1 -mediated overexpression of HO-1/CyclinD1 facilitates cytoprotection by limiting hepatic inflammatory responses, and hepatocellular necrosis/apoptosis in PI3K-dependent manner. PMID:23867319

Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK-3β

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Jae-Sook; Kim, Eun-Kyung; Choi, Yong-Won

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level.more » Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3β through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3β inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3β signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant-negative MEK1 or wild-type GSK-3β. Finally, our data demonstrate that nectandrin B has the ability to protect hepatocytes against oxidative injury through the activation of Nrf2/ARE pathway mediated by ERK phosphorylation and AMPK-dependent inactivation of GSK-3β. - Highlight: • Nectandrin B, a nutmeg lignan protects hepatocytes against oxidative damage. • Nectandrin B exerts an indirect antioxidative effect via the activation of Nrf2. • Both ERK and GSK-3β pathways contribute to the activation of Nrf2 and hepatocyte-protection by nectandrin B. • Nectandrin B can inactivate GSK-3β through the activation of AMPK in HepG2 cells.« less

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Bin; Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208; Abdalrahman, Akram

2014-02-21

Highlights: • Dh404 suppresses the expression of a selected set of pro-inflammatory cytokines in inflamed macrophages via activating Nrf2. • Dh404 activates Nrf2 while keeping Keap1 function intact in macrophages. • Dh404 minimally regulates NF-κB pathway in macrophages. - Abstract: Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promisesmore » in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation of Nrf2 independently of Keap1 and NF-κB, suggesting a unique therapeutic potential of dh404 for specific targeting a Nrf2-mediated resolution of inflammation.« less

Intracellular degradation of functionalized carbon nanotube/iron oxide hybrids is modulated by iron via Nrf2 pathway

PubMed Central

Elgrabli, Dan; Dachraoui, Walid; Marmier, Hélène de; Ménard-Moyon, Cécilia; Bégin, Dominique; Bégin-Colin, Sylvie; Bianco, Alberto; Alloyeau, Damien; Gazeau, Florence

2017-01-01

The in vivo fate and biodegradability of carbon nanotubes is still a matter of debate despite tremendous applications. In this paper we describe a molecular pathway by which macrophages degrade functionalized multi-walled carbon nanotubes (CNTs) designed for biomedical applications and containing, or not, iron oxide nanoparticles in their inner cavity. Electron microscopy and Raman spectroscopy show that intracellularly-induced structural damages appear more rapidly for iron-free CNTs in comparison to iron-loaded ones, suggesting a role of iron in the degradation mechanism. By comparing the molecular responses of macrophages derived from THP1 monocytes to both types of CNTs, we highlight a molecular mechanism regulated by Nrf2/Bach1 signaling pathways to induce CNT degradation via NOX2 complex activation and O2•−, H2O2 and OH• production. CNT exposure activates an oxidative stress-dependent production of iron via Nrf2 nuclear translocation, Ferritin H and Heme oxygenase 1 translation. Conversely, Bach1 was translocated to the nucleus of cells exposed to iron-loaded CNTs to recycle embedded iron. Our results provide new information on the role of oxidative stress, iron metabolism and Nrf2-mediated host defence for regulating CNT fate in macrophages. PMID:28120861

Inorganic Arsenic Induces NRF2-Regulated Antioxidant Defenses in Both Cerebral Cortex and Hippocampus in Vivo.

PubMed

Zhang, Yang; Duan, Xiaoxu; Li, Jinlong; Zhao, Shuo; Li, Wei; Zhao, Lu; Li, Wei; Nie, Huifang; Sun, Guifang; Li, Bing

2016-08-01

Inorganic arsenic is reported to induce the reactive oxygen species-mediated oxidative stress, which is supposed to be one of the main mechanisms of arsenic-related neurological diseases. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant defense systems, up-regulates the expression of target genes to fight against oxidative damages caused by harmful substances, including metals. In the present study, mice were used as a model to investigate the oxidative stress levels and the expressions of NRF2-regulated antioxidant substances in both cerebral cortex and hippocampus with 5, 10 and 20 mg/kg NaAsO2 exposure intra-gastrically. Our results showed that acute NaAsO2 treatment resulted in decreased total anti-oxidative capacity (T-AOC) and increased maleic dialdehyde production in the nervous system. We also detected rapidly elevation of NRF2 protein levels by enhancement of Nrf2 transcription, especially at 20 mg/kg NaAsO2 exposure group. In the meantime, mRNA and protein levels of Nrf2 encoding antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) were consistently elevated time- and dose-dependently both in the cerebral cortex and hippocampus. Taken together, the presence study demonstrated the activation of NRF2 pathway, an early antioxidant defensive response, in both cerebral cortex and hippocampus upon inorganic arsenic (iAs) exposure in vivo. A better knowledge on the roles of NRF2 pathway in maintaining cellular redox homeostasis would be helpful for the strategies on improvement of neurotoxicity related to this metalloid.

Mechanism of phytoestrogen puerarin-mediated cytoprotection following oxidative injury: Estrogen receptor-dependent up-regulation of PI3K/Akt and HO-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; Jeong, Hye Gwang

2008-12-15

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. The phytoestrogen puerarin, the main isoflavone glycoside found in the root of Pueraria lobata, has been used for various medicinal purposes in traditional Chinese medicines for thousands of years. Recent studies have indicated that the estrogen receptor (ER), through interaction with p85, regulates phosphoinositide 3-kinase (PI3K) activity, revealing a physiologic, non-nuclear function of ER that may be relevant in cytoprotection. In this study, we demonstrate that the phytoestrogen puerarin inhibits tert-butyl hydroperoxide (t-BHP)-induced oxidative injury via an ER-dependent G{beta}1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of Hepa1c1c7 and HepG2 cellsmore » with puerarin significantly reduced t-BHP-induced caspase-3 activation and subsequent cell death. Also, puerarin up-regulated HO-1 expression and this expression conferred cytoprotection against oxidative injury induced by t-BHP. Moreover, puerarin induced Nrf2 nuclear translocation, which is upstream of puerarin-induced HO-1 expression, and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Puerarin-induced up-regulation of HO-1 and cytoprotection against t-BHP were abolished by silencing Nrf2 expression with specific siRNA. Also, puerarin-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that puerarin augments cellular antioxidant defense capacity through ER-dependent HO-1 induction via the G{beta}1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress.« less

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming.

PubMed

Saito, Tetsuya; Ichimura, Yoshinobu; Taguchi, Keiko; Suzuki, Takafumi; Mizushima, Tsunehiro; Takagi, Kenji; Hirose, Yuki; Nagahashi, Masayuki; Iso, Tetsuro; Fukutomi, Toshiaki; Ohishi, Maki; Endo, Keiko; Uemura, Takefumi; Nishito, Yasumasa; Okuda, Shujiro; Obata, Miki; Kouno, Tsuguka; Imamura, Riyo; Tada, Yukio; Obata, Rika; Yasuda, Daisuke; Takahashi, Kyoko; Fujimura, Tsutomu; Pi, Jingbo; Lee, Myung-Shik; Ueno, Takashi; Ohe, Tomoyuki; Mashino, Tadahiko; Wakai, Toshifumi; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Motohashi, Hozumi; Waguri, Satoshi; Soga, Tomoyoshi; Yamamoto, Masayuki; Tanaka, Keiji; Komatsu, Masaaki

2016-06-27

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sanghwan; Hur, Eu-gene; Ryoo, In-geun

2012-11-01

The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2‐related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma β-cell line, βTC-6. shRNA-mediated silencing of Nrf2 expression in βTC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ERmore » stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in βTC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated‐like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic β-cells by enhancing proteasome-mediated ERAD. -- Highlights: ► Nrf2 silencing in pancreatic β-cells enhanced tunicamycin-mediated ER stress. ► Expression of the proteasome was inducible by Nrf2 signaling. ► Nrf2 activator D3T protected β-cells from tunicamycin-mediated ER stress. ► Protective effect of D3T was associated with Nrf2-dependent proteasome induction.« less

The role of Nrf2 in oxidative stress-induced endothelial injuries.

PubMed

Chen, Bo; Lu, Yanrong; Chen, Younan; Cheng, Jingqiu

2015-06-01

Endothelial dysfunction is an important risk factor for cardiovascular disease, and it represents the initial step in the pathogenesis of atherosclerosis. Failure to protect against oxidative stress-induced cellular damage accounts for endothelial dysfunction in the majority of pathophysiological conditions. Numerous antioxidant pathways are involved in cellular redox homeostasis, among which the nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is perhaps the most prominent. Nrf2, a transcription factor with a high sensitivity to oxidative stress, binds to AREs in the nucleus and promotes the transcription of a wide variety of antioxidant genes. Nrf2 is located in the cytoskeleton, adjacent to Keap1. Keap1 acts as an adapter for cullin 3/ring-box 1-mediated ubiquitination and degradation of Nrf2, which decreases the activity of Nrf2 under physiological conditions. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which results in its binding to ARE and the transcription of downstream target genes. Experimental evidence has established that Nrf2-driven free radical detoxification pathways are important endogenous homeostatic mechanisms that are associated with vasoprotection in the setting of aging, atherosclerosis, hypertension, ischemia, and cardiovascular diseases. The aim of the present review is to briefly summarize the mechanisms that regulate the Nrf2/Keap1-ARE signaling pathway and the latest advances in understanding how Nrf2 protects against oxidative stress-induced endothelial injuries. Further studies regarding the precise mechanisms by which Nrf2-regulated endothelial protection occurs are necessary for determining whether Nrf2 can serve as a therapeutic target in the treatment of cardiovascular diseases. © 2015 Society for Endocrinology.

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductasemore » 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. - Highlights: • We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum. • 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress. • KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression. • KetoC promoted the expression of antioxidative enzymes in mice organs. • The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.« less

Quercetin protects rat dorsal root ganglion neurons against high glucose-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition.

PubMed

Shi, Yue; Liang, Xiao-chun; Zhang, Hong; Wu, Qun-li; Qu, Ling; Sun, Qing

2013-09-01

To examine the effects of quercetin, a natural antioxidant, on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons of rats. DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of NF-кB, IкBα, phosphorylated IкBα and Nrf2 was examined using RT PCR and Western blot assay. The expression of hemeoxygenase-1 (HO-1), IL-6, TNF-α, iNOS, COX-2, and caspase-3 were also examined. HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway. Co-treatment with quercetin (2.5, 5, and 10 mmol/L) dose-dependently decreased HG-induced caspase-3 activation and apoptosis. Quercetin could directly scavenge ROS and significantly increased the expression of Nrf-2 and HO-1 in DRG neurons. Quercetin also dose-dependently inhibited the NF-κB signaling pathway and suppressed the expression of iNOS, COX-2, and proinflammatory cytokines IL-6 and TNF-α. Quercetin protects rat DRG neurons against HG-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition, thus may be beneficial for the treatment of diabetic neuropathy.

l-Arginine induces antioxidant response to prevent oxidative stress via stimulation of glutathione synthesis and activation of Nrf2 pathway.

PubMed

Liang, Mingcai; Wang, Zhengxuan; Li, Hui; Cai, Liang; Pan, Jianghao; He, Hongjuan; Wu, Qiong; Tang, Yinzhao; Ma, Jiapei; Yang, Lin

2018-05-01

Arginine is a conditionally essential amino acid. To elucidate the influence of l-arginine on the activation of endogenous antioxidant defence, male Wistar rats were orally administered daily with l-arginine at different levels of 25, 50, 100 mg/100 g body weight. After 7 and 14 days feeding, the antioxidative capacities and glutathione (GSH) contents in the plasma and in the liver were uniformly enhanced with the increasing consumption of l-arginine, whereas the oxidative stress was effectively suppressed by l-arginine treatment. After 14 days feeding, the mRNA levels and protein expressions of Keap1 and Cul3 were gradually reduced by increasing l-arginine intake, resulting that the nuclear factor Nrf2 was activated. Upon activation of Nrf2, the expressions of antioxidant responsive element (ARE)-dependent genes and proteins (GCLC, GCLM, GS, GR, GST, GPx, CAT, SOD, NQO1, HO-1) were up-regulated by l-arginine feeding, indicating an upward trend in antioxidant capacity uniformly with the increasing consumption of l-arginine. The present study demonstrates that the supplementation of l-arginine stimulates GSH synthesis and activates Nrf2 pathway, leading to the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway. Results suggest the availability of l-arginine is a critical factor to suppress oxidative stress and induce an endogenous antioxidant response. Copyright © 2018 Elsevier Ltd. All rights reserved.

Berberine, a natural antidiabetes drug, attenuates glucose neurotoxicity and promotes Nrf2-related neurite outgrowth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Ya-Yun; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; Tseng, Yu-Ting

Reactive oxygen intermediates production and apoptotic damage induced by high glucose are major causes of neuronal damage in diabetic neuropathy. Berberine (BBR), a natural antidiabetes drug with PI3K-activating activity, holds promise for diabetes because of its dual antioxidant and anti-apoptotic activities. We have previously reported that BBR attenuated H{sub 2}O{sub 2} neurotoxicity via activating the PI3K/Akt/Nrf2-dependent pathway. In this study, we further explored the novel protective mechanism of BBR on high glucose-induced apoptotic death and neurite damage of SH-SY5Y cells. Results indicated BBR (0.1–10 nM) significantly attenuated reactive oxygen species (ROS) production, nucleus condensation, and apoptotic death in high glucose-treatedmore » cells. However, AG1024, an inhibitor of insulin growth factor-1 (IGF-1) receptor, significantly abolished BBR protection against high glucose-induced neuronal death. BBR also increased Bcl-2 expression and decreased cytochrome c release. High glucose down-regulated IGF-1 receptor and phosphorylation of Akt and GSK-3β, the effects of which were attenuated by BBR treatment. BBR also activated nuclear erythroid 2-related factor 2 (Nrf2), the key antioxidative transcription factor, which is accompanied with up-regulation of hemeoxygenase-1 (HO-1). Furthermore, BBR markedly enhanced nerve growth factor (NGF) expression and promoted neurite outgrowth in high glucose-treated cells. To further determine the role of the Nrf2 in BBR neuroprotection, RNA interference directed against Nrf2 was used. Results indicated Nrf2 siRNA abolished BBR-induced HO-1, NGF, neurite outgrowth and ROS decrease. In conclusion, BBR attenuated high glucose-induced neurotoxicity, and we are the first to reveal this novel mechanism of BBR as an Nrf2 activator against glucose neurotoxicity, providing another potential therapeutic use of BBR on the treatment of diabetic complications. - Highlights: • BBR attenuates high glucose-induced ROS production and neuronal cell death. • BBR activates IGF-1/Akt/GSK-3β signaling under normal and high glucose conditions. • BBR enhances HO-1 and NGF expression through stimulating Nrf2 translocation. • BBR promotes neurite outgrowth through Nrf2-dependent pathway.« less

Apigenin sensitizes BEL-7402/ADM cells to doxorubicin through inhibiting miR-101/Nrf2 pathway

PubMed Central

Gao, Ai-Mei; Zhang, Xiao-Yu; Ke, Zun-Ping

2017-01-01

Chemo-resistance is one of the main obstacle in hepatocellular carcinoma therapy. Apigenin as a natural bioflavonoid has been exhibited anti-cancer properties in various malignant cancers. The aim of this study is to evaluate the potential chemo-sensitization effect of apigenin in doxorubicin-resistant hepatocellular carcinoma cell line BEL-7402/ADM and to investigate its possible mechanism. We found that apigenin significantly reversed doxorubicin sensitivity and induced caspase-dependent apoptosis in BEL-7402/ADM cells. Furthermore, apigenin induced miR-101 expression, and overexpression of miR-101 mimicked the doxorubicin-sensitizing effect of apigenin. Importantly, we showed that miR-101 was able to target the 3′-UTR of Nrf2. The results suggested that apigenin sensitizes BEL-7402/ADM cells to doxorubicin through miR-101/Nrf2 pathway, which furtherly supports apigenin as a potential chemo-sensitizer for hepatocellular carcinoma. PMID:29137246

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway.

PubMed

Lee, Hyeong-Seon; Lee, Gyeong-Seon; Kim, Seon-Hee; Kim, Hyun-Kyung; Suk, Dong-Hee; Lee, Dong-Seok

2014-02-01

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway.

NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

PubMed

Türei, Dénes; Papp, Diána; Fazekas, Dávid; Földvári-Nagy, László; Módos, Dezső; Lenti, Katalin; Csermely, Péter; Korcsmáros, Tamás

2013-01-01

NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Xiaojun; Sun Zheng; Chen Weimin

2007-12-01

Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using a UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2more » pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III) and MMA(III). Furthermore, the wild-type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF, whereas neither tBHQ nor SF conferred protection in the Nrf2{sup -/-}MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.« less

Disruption of the Transcription Factor Nrf2 Promotes Pro-Oxidative Dendritic Cells That Stimulate Th2-Like Immunoresponsiveness upon Activation by Ambient Particulate Matter

PubMed Central

Williams, Marc A.; Rangasamy, Tirumalai; Bauer, Stephen M.; Killedar, Smruti; Karp, Matthew; Kensler, Thomas W.; Yamamoto, Masayuki; Breysse, Patrick; Biswal, Shyam; Georas, Steve N.

2011-01-01

Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2−/− DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2−/− DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2+/+ DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2+/+ DCs, coincubation of Nrf2−/− DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs. PMID:18802057

Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - Implication in modification of radiation damage

PubMed Central

Anuranjani; Bala, Madhu

2014-01-01

Whole body exposure to low linear energy transfer (LET) ionizing radiations (IRs) damages vital intracellular bio-molecules leading to multiple cellular and tissue injuries as well as pathophysiologies such as inflammation, immunosuppression etc. Nearly 70% of damage is caused indirectly by radiolysis of intracellular water leading to formation of reactive oxygen species (ROS) and free radicals and producing a state of oxidative stress. The damage is also caused by direct ionization of biomolecules. The type of radiation injuries is dependent on the absorbed radiation dose. Sub-lethal IR dose produces more of DNA base damages, whereas higher doses produce more DNA single strand break (SSBs), and double strand breaks (DSBs). The Nrf2-ARE pathway is an important oxidative stress regulating pathway. The DNA DSBs repair regulated by MRN complex, immunomodulation and inflammation regulated by HMGB1 and various types of cytokines are some of the key pathways which interact with each other in a complex manner and modify the radiation response. Because the majority of radiation damage is via oxidative stress, it is essential to gain in depth understanding of the mechanisms of Nrf2-ARE pathway and understand its interactions with MRN complex, HMGB1 and cytokines to increase our understanding on the radiation responses. Such information is of tremendous help in development of medical radiation countermeasures, radioprotective drugs and therapeutics. Till date no approved and safe countermeasure is available for human use. This study reviews the Nrf2-ARE pathway and its crosstalk with MRN-complex, HMGB1 and cytokines (TNF-a, IL-6, IFN-? etc.). An attempt is also made to review the modification of some of these pathways in presence of selected antioxidant radioprotective compounds or herbal extracts. PMID:25009785

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress.

PubMed

Yonchuk, John G; Foley, Joseph P; Bolognese, Brian J; Logan, Gregory; Wixted, William E; Kou, Jen-Pyng; Chalupowicz, Diana G; Feldser, Heidi G; Sanchez, Yolanda; Nie, Hong; Callahan, James F; Kerns, Jeffrey K; Podolin, Patricia L

2017-10-01

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2 H -chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant ( tert -butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1 β -induced nuclear factor- κ B translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence.

PubMed

Zhou, Tengfei; Zhang, Mengqian; Zhao, Liang; Li, Aiqin; Qin, Xiaomei

2016-10-01

Oxidative stress and impaired antioxidant defense are believed to be contributors to the cardiovascular aging process. The transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis. Our previous study showed that Exendin-4, a glucagon-like peptide-1 analog, alleviates angiotensin II (ANG II)-induced vascular smooth muscle cell (VSMC) senescence by inhibiting Rac1 activation via cAMP/PKA (Zhao L, Li AQ, Zhou TF, Zhang MQ, Qin XM. Am J Physiol Cell Physiol 307: C1130-C1141, 2014). The objective of this study is to investigate if Nrf2 mediates the antisenescent effect of Exendin-4 in ANG II-induced VSMCs. Here we report that Exendin-4 triggered Nrf2 nuclear translocation, a downstream target of cAMP-responsive element-binding protein (CREB) and expressions of antioxidant genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1) in a dose- and time-dependent manner. In addition, knock-down of Nrf2 attenuated the inhibitory effects of Exendin-4 on ANG II-induced superoxidant generation and VSMC senescence. PKA/CREB pathway participated in the upregulations of HO-1 and NQO-1 induced by Exendin-4. Notably, our study revealed that Exendin-4 dose-dependently increased the acetylation of Nrf2 and the recruitment of transcriptional coactivator CREB binding protein (CBP) to Nrf2. The Exendin-4-induced Nrf2 transactivation was diminished in the presence of CBP small interfering RNA. Microscope imaging of Nrf2, as well as immunoblotting for Nrf2, showed that the Exendin-4-evoked Nrf2 acetylation favored its nuclear retention. Importantly, CBP silencing attenuated the suppressing effects of Exendin-4 on ANG II-induced VSMC senescence and superoxidant production. In conclusion, these results provide a mechanistic insight into how Nrf2 signaling mediates the antisenescent and antioxidative effects induced by Exendin-4 in VSMCs. Copyright © 2016 the American Physiological Society.

Systems-Level Feedbacks of NRF2 Controlling Autophagy upon Oxidative Stress Response

PubMed Central

Kapuy, Orsolya; Papp, Diána; Bánhegyi, Gábor

2018-01-01

Although the primary role of autophagy-dependent cellular self-eating is cytoprotective upon various stress events (such as starvation, oxidative stress, and high temperatures), sustained autophagy might lead to cell death. A transcription factor called NRF2 (nuclear factor erythroid-related factor 2) seems to be essential in maintaining cellular homeostasis in the presence of either reactive oxygen or nitrogen species generated by internal metabolism or external exposure. Accumulating experimental evidence reveals that oxidative stress also influences the balance of the 5′ AMP-activated protein kinase (AMPK)/rapamycin (mammalian kinase target of rapamycin or mTOR) signaling pathway, thereby inducing autophagy. Based on computational modeling here we propose that the regulatory triangle of AMPK, NRF2 and mTOR guaranties a precise oxidative stress response mechanism comprising of autophagy. We suggest that under conditions of oxidative stress, AMPK is crucial for autophagy induction via mTOR down-regulation, while NRF2 fine-tunes the process of autophagy according to the level of oxidative stress. We claim that the cellular oxidative stress response mechanism achieves an incoherently amplified negative feedback loop involving NRF2, mTOR and AMPK. The mTOR-NRF2 double negative feedback generates bistability, supporting the proper separation of two alternative steady states, called autophagy-dependent survival (at low stress) and cell death (at high stress). In addition, an AMPK-mTOR-NRF2 negative feedback loop suggests an oscillatory characteristic of autophagy upon prolonged intermediate levels of oxidative stress, resulting in new rounds of autophagy stimulation until the stress events cannot be dissolved. Our results indicate that AMPK-, NRF2- and mTOR-controlled autophagy induction provides a dynamic adaptation to altering environmental conditions, assuming their new frontier in biomedicine. PMID:29510589

Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-β1 in rat glomerular messangial cells.

PubMed

Huang, Kaipeng; Chen, Cheng; Hao, Jie; Huang, Junying; Wang, Shaogui; Liu, Peiqing; Huang, Heqing

2015-01-05

Sirt1 and nuclear factor-E2 related factor 2 (Nrf2)-anti-oxidant response element (ARE) anti-oxidative pathway play important regulatory roles in the pathological progression of diabetic nephropathy (DN) induced by advanced glycation-end products (AGEs). Polydatin (PD), a glucoside of resveratrol, has been shown to possess strong anti-oxidative bioactivity. Our previous study demonstrated that PD markedly resists the progression of diabetic renal fibrosis and thus, inhibits the development of DN. Whereas, whether PD could resist DN through regulating Sirt1 and consequently promoting Nrf2-ARE pathway needs further investigation. Here, we found that concomitant with decreasing RAGE (the specific receptor for AGEs) expression, PD significantly reversed the downregulation of Sirt1 in terms of protein expression and deacetylase activity and attenuated FN and TGF-β1 expression in GMCs exposed to AGEs. Under AGEs-treatment condition, PD could decrease Keap1 expression and promote the nuclear content, ARE-binding ability, and transcriptional activity of Nrf2. In addition, PD increased the protein levels of heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1), two target genes of Nrf2. The activation of Nrf2-ARE pathway by PD eventually led to the quenching of ROS overproduction sharply boosted by AGEs. Depletion of Sirt1 blocked Nrf2-ARE pathway activation and reversed FN and TGF-β1 downregulation induced by PD in GMCs challenged with AGEs. Along with reducing HO-1 and SOD1 expression, silencing of Nrf2 increased FN and TGF-β1 levels. PD treatment elevated Sirt1 and Nrf2 levels in the kidney tissues of diabetic rats, then improved the anti-oxidative capacity and renal dysfunction of diabetic models, and finally reversed the upregulation of FN and TGF-β1. Taken together, the resistance of PD on upregulated FN and TGF-β1 induced by AGEs via oxidative stress in GMCs is closely associated with its activation of Sirt1-Nrf2-ARE pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

PubMed

Chatterjee, Anwesha; Ronghe, Amruta; Singh, Bhupendra; Bhat, Nimee K; Chen, Jie; Bhat, Hari K

2014-12-01

The objective of the present study was to characterize the role of resveratrol (Res) and vitamin C (VC) in prevention of estrogen-induced breast cancer through regulation of cap "n"collar (CNC) b-zip transcription factors. Human breast epithelial cell line MCF-10A was treated with 17β-estradiol (E2) and VC or Res with or without E2. mRNA and protein expression levels of CNC b-zip transcription factors nuclear factor erythroid 2-related factor 1 (Nrf1), nuclear factor erythroid 2 related factor 2 (Nrf2), nuclear factor erythroid 2 related factor 3 (Nrf3), and Nrf2-regulated antioxidant enzymes superoxide dismutase 3 (SOD3) and quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2-mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF-10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17β-Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen-induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf-dependent pathways. © 2014 Wiley Periodicals, Inc.

Role of miR-155 in fluorooctane sulfonate-induced oxidative hepatic damage via the Nrf2-dependent pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Chong; Han, Rui; Liu, Limin

Studies demonstrated that perfluorooctane sulfonate (PFOS) tends to accumulate in the liver and is capable to cause hepatomegaly. In the present study, we investigated the roles of miR-155 in PFOS-induced hepatotoxicity in SD rats and HepG2 cells. Male SD rats were orally administrated with PFOS at 1 or 10 mg/kg/day for 28 days while HepG2 cells were treated with 0–50 μM of PFOS for 24 h or 50 μM of PFOS for 1, 3, 6, 12 or 24 h, respectively. We found that PFOS significantly increased the liver weight and serum alanine transaminase (ALT) and aspartate amino transferase (AST) levelsmore » in rats. Morphologically, PFOS caused actin filament remodeling and endothelial permeability changes in the liver. Moreover, PFOS triggered reactive oxygen species (ROS) generation and induced apoptosis in both in vivo and in vitro assays. Immunoblotting data showed that NF-E2-related factor-2 (Nrf2) expression and activation and its target genes were all suppressed by PFOS in the liver and HepG2 cells. However, PFOS significantly increased miR-155 expression. Further studies showed that pretreatment of HepG2 cells with catalase significantly decreased miR-155 expression and substantially increased Nrf2 expression and activation, resulting in reduction of PFOS-induced cytotoxicity and oxidative stress. Taken together, these results indicated that miR-155 plays an important role in the PFOS-induced hepatotoxicity by disrupting Nrf2/ARE signaling pathway. - Highlights: • PFOS is capable to cause hepatotoxicity. • PFOS triggers ROS generation and induces apoptosis both in vivo and in vitro assays. • PFOS-induced ROS inhibits Nrf2 expression and its transactivation function. • PFOS promotes miR155 expression in liver and HepG2 cells. • miR-155 is involved in PFOS-induced hepatotoxicity by disrupting Nrf2/ARE pathway.« less

Alkylating agent induced NRF2 blocks endoplasmic reticulum stress-mediated apoptosis via control of glutathione pools and protein thiol homeostasis

PubMed Central

Zanotto-Filho, Alfeu; Masamsetti, V. Pragathi; Loranc, Eva; Tonapi, Sonal S.; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C. F.; Chen, Yidong; Bishop, Alexander J. R.

2016-01-01

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival; an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine (BSO) caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine (NAC) or glutathione-ethyl-ester (GSH-E), decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 targets overexpression and poor survival. In KEAP1 mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. PMID:27638861

Sirt1 activation prevents anti-Thy 1.1 mesangial proliferative glomerulonephritis in the rat through the Nrf2/ARE pathway.

PubMed

Huang, Kaipeng; Li, Ruiming; Wei, Wentao

2018-08-05

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by glomerular mesangial cells proliferation and extracellular matrix deposition in mesangial area, which develop into glomerulosclerosis. Both silent information regulator 2-related protein 1 (Sirt1) and nuclear factor erythroid 2-related factor 2/anti-oxidant response element (Nrf2/ARE) pathway had remarkable renoprotective effects. However, whether Sirt1 and Nrf2/ARE pathway can regulate the pathological process of MsPGN remains unknown. Here, we found that Sirt1 activation by SRT1720 decreased mesangial hypercellularity and mesangial matrix areas, reduced renal Col4 and α-SMA expressions, lowered 24 h proteinuria, and eventually reduced FN and TGF-β1 expressions in rats received anti-Thy 1.1 IgG. Further study showed that SRT1720 markedly enhanced the activity of Nrf2/ARE pathway including promoting the nuclear content and ARE-binding ability of Nrf2, elevating the protein levels of HO-1 and SOD1, two target genes of Nrf2, which eventually increased total SOD activity and decreased malondialdehyde level in the kidney tissues of experimental anti-Thy 1.1 MsPGN rats. Taken together, Sirt1 prevented the pathological process of experimental anti-Thy 1.1 MsPGN through promoting the activation of Nrf2/ARE pathway, which warrants further elucidation. Sirt1 might be a potential therapeutic target for treating MsPGN. Copyright © 2018 Elsevier B.V. All rights reserved.

Brain ischemic preconditioning protects against ischemic injury and preserves the blood-brain barrier via oxidative signaling and Nrf2 activation.

PubMed

Yang, Tuo; Sun, Yang; Mao, Leilei; Zhang, Meijuan; Li, Qianqian; Zhang, Lili; Shi, Yejie; Leak, Rehana K; Chen, Jun; Zhang, Feng

2018-05-06

Brain ischemic preconditioning (IPC) with mild ischemic episodes is well known to protect the brain against subsequent ischemic challenges. However, the underlying mechanisms are poorly understood. Here we demonstrate the critical role of the master redox transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), in IPC-mediated neuroprotection and blood-brain barrier (BBB) preservation. We report that IPC causes generation of endogenous lipid electrophiles, including 4-hydroxy-2-nonenal (4-HNE), which release Nrf2 from inhibition by Keap1 (via Keap1-C288) and inhibition by glycogen synthase kinase 3β (via GSK3β-C199). Nrf2 then induces expression of its target genes, including a new target, cadherin 5, a key component of adherens junctions of the BBB. These effects culminate in mitigation of BBB leakage and of neurological deficits after stroke. Collectively, these studies are the first to demonstrate that IPC protects the BBB against ischemic injury by generation of endogenous electrophiles and activation of the Nrf2 pathway through inhibition of Keap1- and GSK3β-dependent Nrf2 degradation. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Anti-inflammatory effects of novel polygonum multiflorum compound via inhibiting NF-κB/MAPK and upregulating the Nrf2 pathways in LPS-stimulated microglia.

PubMed

Park, Sun Young; Jin, Mei Ling; Kang, Nam Jun; Park, Geuntae; Choi, Young-Whan

2017-06-09

The incorporation of Polygonum multiflorum into the diet can result in anti-aging effects owing to its wide range of biological and pharmaceutical properties. We investigated the anti-neuroinflammatory properties of CRPE56IGIH isolated from P. multiflorum by focusing on its role in the induction of phase II antioxidant enzymes and the modulation of upstream signaling pathways. In microglia, CRPE56IGIH significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide and prostaglandin E 2 production with nonspecific cytotoxicity. CRPE56IGIH also markedly inhibited LPS-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 protein and mRNA expression in the same manner as it inhibited nitric oxide and prostaglandin E 2 production. In the control cells, NF-κB transactivation and nuclear translocation occurred at a baseline level, which was significantly increased in response to LPS. However, pretreatment with CRPE56IGIH concentration-dependently inhibited the LPS-induced NF-κB transactivation and nuclear translocation. The phosphorylation of Janus kinase-signal transducers and activators of transcription and mitogen-activated protein kinases was markedly upregulated by LPS, but considerably and dose-dependently inhibited by pretreatment with CRPE56IGIH. Furthermore, CRPE56IGIH induced the expression of phase II antioxidant enzymes, including heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone-1 (NQO-1). The activation of upstream signaling pathways, such as the Nrf2 pathway, was significantly increased following CRPE56IGIH treatment. Furthermore, the anti-neuroinflammatory effect of CRPE56IGIH was reversed by transfection of Nrf2, HO-1, and NQO-1 siRNA. Our results indicated that CRPE56IGIH isolated from P. multiflorum could be used as a natural anti-neuroinflammatory agent that induces phase II antioxidant enzymes via Nrf2 signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic inactivation of Nrf2 prevents clonal expansion of initiated cells in a nutritional model of rat hepatocarcinogenesis.

PubMed

Orrù, Claudia; Szydlowska, Marta; Taguchi, Keiko; Zavattari, Patrizia; Perra, Andrea; Yamamoto, Masayuki; Columbano, Amedeo

2018-06-21

Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in cancer development. Herein, we examined whether Nrf2 (Nfe2l2) activation occurs at early steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the onset of hepatocellular carcinoma (HCC). We used wild-type (WT) and Nrf2 knockout (Nrf2KO) rats treated with a single injection of diethylnitrosamine (DENA) followed by choline-devoid methionine-deficient (CMD) diet. This experimental model causes massive fatty liver and steatohepatitis with fibrosis and enables identification of early stages of hepatocarcinogenesis. We found that Nrf2 activation takes place in early preneoplastic lesions identified by the marker glutathione S-transferase placental form (GSTP). Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to increased liver injury and chronic compensatory hepatocyte regeneration when rats were fed a CMD diet. However, in spite of such a permissive environment, the livers of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. These results demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of initiated cells, indicating that Nrf2 is critical in the onset of HCC. Dysregulation of the Keap1-Nrf2 molecular pathway has been observed in human tumors. In a nutritional model of hepatocarcinogenesis, the protein Nrf2 is frequently mutated/activated at early steps of the tumorigenic process. Herein, we show that Nrf2 is mandatory for the development of preneoplastic lesions. These results suggest that Nrf2 has a critical role in the onset of hepatocellular carcinoma. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Epigenetics Reactivation of Nrf2 in Prostate TRAMP C1 Cells by Curcumin Analogue FN1.

PubMed

Li, Wenji; Pung, Doug; Su, Zheng-Yuan; Guo, Yue; Zhang, Chengyue; Yang, Anne Yuqing; Zheng, Xi; Du, Zhi-Yun; Zhang, Kun; Kong, Ah-Ng

2016-04-18

It has previously been shown that curcumin can effectively inhibit prostate cancer proliferation and progression in TRAMP mice, potentially acting through the hypomethylation of the Nrf2 gene promoter and hence activation of the Nrf2 pathway to enhance cell antioxidative defense. FN1 is a synthetic curcumin analogue that shows stronger anticancer activity than curcumin in other reports. We aimed to explore the epigenetic modification of FN1 that restores Nrf2 expression in TRAMP-C1 cells. Stably transfected HepG2-C8 cells were used to investigate the effect of FN1 on the Nrf2- antioxidant response element (ARE) pathway. Real-time quantitative PCR and Western blotting were applied to study the influence of FN1 on endogenous Nrf2 and its downstream genes. Bisulfite genomic sequencing (BGS) and methylated DNA immunoprecipitation (MeDIP) were then performed to examine the methylation profile of the Nrf2 promoter. An anchorage-independent colony-formation analysis was conducted to examine the tumor inhibition activity of FN1. Epigenetic modification enzymes, including DNMTs and HDACs, were investigated by Western blotting. The luciferase reporter assay indicated that FN1 was more potent than curcumin in activating the Nrf2-ARE pathway. FN1 increased the expression of Nrf2 and its downstream detoxifying enzymes. FN1 significantly inhibited the colony formation of TRAMP-C1 cells. BGS and MeDIP assays revealed that FN1 treatment (250 nM for 3 days) reduced the percentage of CpG methylation of the Nrf2 promoter. FN1 also downregulated epigenetic modification enzymes. In conclusion, our results suggest that FN1 is a novel anticancer agent for prostate cancer. In the TRAMP-C1 cell line, FN1 can increase the level of Nrf2 and downstream genes via activating the Nrf2-ARE pathway and inhibit the colony formation potentially through the decreased expression of keap1 coupled with CpG demethylation of the Nrf2 promoter. This CpG demethylation effect may come from decreased epigenetic modification enzymes, such as DNMT1, DNMT3a, DNMT3b, and HDAC4.

Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway: the involvement of endoplasmic reticulum stress and the Nrf2 signaling pathway.

PubMed

Jo, Guk Heui; Kim, Gi-Young; Kim, Wun-Jae; Park, Kun Young; Choi, Yung Hyun

2014-10-01

Sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, has received a great deal of attention because of its ability to inhibit cell proliferation and induce apoptosis in cancer cells. In this study, we investigated the anticancer activity of sulforaphane in the T24 human bladder cancer line, and explored its molecular mechanism of action. Our results showed that treatment with sulforaphane inhibited cell viability and induced apoptosis in T24 cells in a concentration-dependent manner. Sulforaphane-induced apoptosis was associated with mitochondria dysfunction, cytochrome c release and Bcl-2/Bax dysregulation. Furthermore, the increased activity of caspase-9 and -3, but not caspase-8, was accompanied by the cleavage of poly ADP-ribose polymerase, indicating the involvement of the mitochondria-mediated intrinsic apoptotic pathway. Concomitant with these changes, sulforaphane triggered reactive oxygen species (ROS) generation, which, along with the blockage of sulforaphane-induced loss of mitochondrial membrane potential and apoptosis, was strongly attenuated by the ROS scavenger N-acetyl-L-cysteine. Furthermore, sulforaphane was observed to activate endoplasmic reticulum (ER) stress and the nuclear factor-E2-related factor-2 (Nrf2) signaling pathway, as demonstrated by the upregulation of ER stress‑related proteins, including glucose-regulated protein 78 and C/EBP-homologous protein, and the accumulation of phosphorylated Nrf2 proteins in the nucleus and induction of heme oxygenase-1 expression, respectively. Taken together, these results demonstrate that sulforaphane has antitumor effects against bladder cancer cells through an ROS-mediated intrinsic apoptotic pathway, and suggest that ER stress and Nrf2 may represent strategic targets for sulforaphane-induced apoptosis.

Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway.

PubMed

Hong, Bo; Su, Zhengyuan; Zhang, Chengyue; Yang, Yuqing; Guo, Yue; Li, Wenjing; Kong, Ah-Ng Tony

2016-05-01

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a crucial transcription factor that regulates the expression of defensive antioxidants and detoxification enzymes in cells. In a previous study, we showed that expression of the Nrf2 gene is regulated by an epigenetic modification. Rauvolfia verticillata, a traditional Chinese herbal medicine widely used in China, possesses anticancer and antioxidant effects. In this study, we investigated how Nrf2 is epigenetically regulated by reserpine, the main active component in R. verticillata, in mouse skin epidermal JB6 P+ cells. Reserpine induced ARE (antioxidant response element)-luciferase activity in HepG2-C8 cells. Accordingly, in JB6 P+ cells, it upregulated the mRNA and protein levels of Nrf2 and its downstream target genes heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), while it only increased the protein level of UDP-glucuronosyltransferase 1A1 (UGT1A1). Furthermore, reserpine decreased the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced colony formation of JB6 cells in a dose-dependent manner. DNA sequencing and methylated DNA immunoprecipitation further demonstrated the demethylation effect of reserpine on the first 15 CpGs of the Nrf2 promoter in JB6 P+ cells. Reserpine also reduced the mRNA and protein expression of DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferases 3a), and DNMT3b (DNA methyltransferases 3b). Moreover, reserpine induced Nrf2 expression via an epigenetic pathway in skin epidermal JB6 P+ cells, enhancing the protective antioxidant activity and decreasing TPA-induced cell transformation. These results suggest that reserpine exhibits a cancer preventive effect by reactivating Nrf2 and inducing the expression of target genes involved in cellular protection, potentially providing new insight into the chemoprevention of skin cancer using reserpine.

Benzo[a]pyrene affects Jurkat T cells in the activated state via the antioxidant response element dependent Nrf2 pathway leading to decreased IL-2 secretion and redirecting glutamine metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murugaiyan, Jayaseelan; Rockstroh, Maxie; Wagner, Juliane

2013-06-15

There is a clear evidence that environmental pollutants, such as benzo[a]pyrene (B[a]P), can have detrimental effects on the immune system, whereas the underlying mechanisms still remain elusive. Jurkat T cells share many properties with native T lymphocytes and therefore are an appropriate model to analyze the effects of environmental pollutants on T cells and their activation. Since environmental compounds frequently occur at low, not acute toxic concentrations, we analyzed the effects of two subtoxic concentrations, 50 nM and 5 μM, on non- and activated cells. B[a]P interferes directly with the stimulation process as proven by an altered IL-2 secretion. Furthermore,more » B[a]P exposure results in significant proteomic changes as shown by DIGE analysis. Pathway analysis revealed an involvement of the AhR independent Nrf2 pathway in the altered processes observed in unstimulated and stimulated cells. A participation of the Nrf2 pathway in the change of IL-2 secretion was confirmed by exposing cells to the Nrf2 activator tBHQ. tBHQ and 5 μM B[a]P caused similar alterations of IL-2 secretion and glutamine/glutamate metabolism. Moreover, the proteome changes in unstimulated cells point towards a modified regulation of the cytoskeleton and cellular stress response, which was proven by western blotting. Additionally, there is a strong evidence for alterations in metabolic pathways caused by B[a]P exposure in stimulated cells. Especially the glutamine/glutamate metabolism was indicated by proteome pathway analysis and validated by metabolite measurements. The detrimental effects were slightly enhanced in stimulated cells, suggesting that stimulated cells are more vulnerable to the environmental pollutant model compound B[a]P. - Highlights: • B[a]P affects the proteome of Jurkat T cells also at low concentrations. • Exposure to B[a]P (50 nM, 5 μM) did not change Jurkat T cell viability. • Both B[a]P concentrations altered the IL-2 secretion of stimulated cells. • 608 different protein spots of Jurkat T cells were quantified using 2-DE-DIGE. • Pathway analysis identified Nrf2 and AhR pathway as regulated.« less

Lycium barbarum polysaccharide protects human keratinocytes against UVB-induced photo-damage.

PubMed

Li, Huaping; Li, Zhenjie; Peng, Liqian; Jiang, Na; Liu, Qing; Zhang, Erting; Liang, Bihua; Li, Runxiang; Zhu, Huilan

2017-02-01

Ultraviolet B (UVB) irradiation plays a key role in skin damage, which induces oxidative and inflammatory damages, thereby causing photoaging or photocarcinogenesis. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses significant antioxidative and anti-inflammatory effects on multiple tissues. In the present study, the photoprotective effects and potential underlying molecular mechanisms of LBP against UVB-induced photo-damage were investigated in immortalized human keratinocytes (HaCaT cells). The data indicated that pretreatment with LBP significantly attenuated UVB-induced decrease in cell viability, increase in ROS production and DNA damage. LBP also significantly suppressed UVB-induced p38 MAPK activation, and subsequently reversed caspase-3 activation and MMP-9 expression. Notably, LBP was found to induce Nrf2 nuclear translocation and increase the expression of Nrf2-dependent ARE target genes. Furthermore, the protective effects of LBP were abolished by siRNA-mediated Nrf2 silencing. These results showed that the antioxidant LBP could partially protect against UVB irradiation-induced photo-damage through activation of Nrf2/ARE pathway, thereby scavenging ROS and reducing DNA damage, and subsequently suppressing UVB-induced p38 MAP pathway. Thus, LBP can be potentially used for skincare against oxidative damage from environmental insults.

Transcript Profiling Identifies Dynamic Gene Expression Patterns and an Important Role for Nrf2/Keap1 Pathway in the Developing Mouse Esophagus

PubMed Central

Li, Haiyan; Hu, Yuhui; Tevebaugh, Whitney; Yamamoto, Masayuki; Que, Jianwen; Chen, Xiaoxin

2012-01-01

Background and Aims Morphological changes during human and mouse esophageal development have been well characterized. However, changes at the molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how the Nrf2/Keap1 pathway regulates the morphogenesis of the esophageal epithelium. Methods Gene expression microarrays were used to survey gene expression in the esophagus at three critical phases: specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2−/−, Keap1−/−, or Nrf2−/−Keap1−/− embryos or young adult mice. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, Pparβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium. Results Dynamic gene expression patterns accompanied the morphological changes of the developing esophagus at critical phases. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia phase. In addition, Keap1−/− mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray and immunostaining data also suggested that esophageal hyperkeratosis in the Keap1−/− mice was due to activation of Pparβ/δ and the PI3K/Akt pathway. Conclusions Morphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. Nrf2/Keap1 pathway activity is required for maturation of mouse esophageal epithelium. PMID:22567161

Sulforaphane Ameliorates Okadaic Acid-Induced Memory Impairment in Rats by Activating the Nrf2/HO-1 Antioxidant Pathway.

PubMed

Dwivedi, Subhash; Rajasekar, N; Hanif, Kashif; Nath, Chandishwar; Shukla, Rakesh

2016-10-01

Okadaic acid (OKA) causes memory impairment and attenuates nuclear factor erythroid 2-related factor 2 (Nrf2) along with oxidative stress and neuroinflammation in rats. Sulforaphane (dietary isothiocyanate compound), an activator of Nrf2 signaling, exhibits neuroprotective effects. However, the protective effect of sulforaphane in OKA-induced neurotoxicity remains uninvestigated. Therefore, in the present study, the role of sulforaphane in OKA-induced memory impairment in rats was explored. A significant increased Nrf2 expression in the hippocampus and cerebral cortex was observed in trained (Morris water maze) rats, and a significant decreased Nrf2 expression in memory-impaired (OKA, 200 ng icv) rats indicated its involvement in memory function. Sulforaphane administration (5 and 10 mg/kg, ip, days 1 and 2) ameliorates OKA-induced memory impairment in rats. The treatment also restored Nrf2 and its downstream antioxidant protein expression (GCLC, HO-1) and attenuated oxidative stress (ROS, nitrite, GSH), neuroinflammation (NF-κB, TNF-α, IL-10), and neuronal apoptosis in the cerebral cortex and hippocampus of OKA-treated rats. Further, to determine whether modulation of Nrf2 signaling is responsible for the protective effect of sulforaphane, in vitro, Nrf2 siRNA and its downstream HO-1 inhibition studies were carried out in a rat astrocytoma cell line (C6). The protective effects of sulforaphane were abolished with Nrf2 siRNA and HO-1 inhibition in astrocytes. The results suggest that Nrf2-dependent activation of cellular antioxidant machinery results in sulforaphane-mediated protection against OKA-induced memory impairment in rats. Graphical Abstract ᅟ.

Activation of anti-oxidant Nrf2 signaling by enone analogues of curcumin.

PubMed

Deck, Lorraine M; Hunsaker, Lucy A; Vander Jagt, Thomas A; Whalen, Lisa J; Royer, Robert E; Vander Jagt, David L

2018-01-01

Inflammation and oxidative stress are common in many chronic diseases. Targeting signaling pathways that contribute to these conditions may have therapeutic potential. The transcription factor Nrf2 is a major regulator of phase II detoxification and anti-oxidant genes as well as anti-inflammatory and neuroprotective genes. Nrf2 is widespread in the CNS and is recognized as an important regulator of brain inflammation. The natural product curcumin exhibits numerous biological activities including ability to induce the expression of Nrf2-dependent phase II and anti-oxidant enzymes. Curcumin has been examined in a number of clinical studies with limited success, mainly owing to limited bioavailability and rapid metabolism. Enone analogues of curcumin were examined with an Nrf2 reporter assay to identify Nrf2 activators. Analogues were separated into groups with a 7-carbon dienone spacer, as found in curcumin; a 5-carbon enone spacer with and without a ring; and a 3-carbon enone spacer. Activators of Nrf2 were found in all three groups, many of which were more active than curcumin. Dose-response studies demonstrated that a range of substituents on the aromatic rings of these enones influenced not only the sensitivity to activation, reflected in EC 50 values, but also the extent of activation, which suggests that multiple mechanisms are involved in the activation of Nrf2 by these analogues. Copyright © 2017. Published by Elsevier Masson SAS.

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

PubMed

Heyninck, Karen; Sabbe, Linde; Chirumamilla, Chandra Sekhar; Szarc Vel Szic, Katarzyna; Vander Veken, Pieter; Lemmens, Kristien J A; Lahtela-Kakkonen, Maija; Naulaerts, Stefan; Op de Beeck, Ken; Laukens, Kris; Van Camp, Guy; Weseler, Antje R; Bast, Aalt; Haenen, Guido R M M; Haegeman, Guy; Vanden Berghe, Wim

2016-06-01

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA. Copyright © 2016 Elsevier Inc. All rights reserved.

Nrf2-p62 autophagy pathway and its response to oxidative stress in hepatocellular carcinoma.

PubMed

Bartolini, Desirée; Dallaglio, Katiuscia; Torquato, Pierangelo; Piroddi, Marta; Galli, Francesco

2018-03-01

Deregulation of autophagy is proposed to play a key pathogenic role in hepatocellular carcinoma (HCC), the most common primary malignancy of the liver and the third leading cause of cancer death. Autophagy is an evolutionarily conserved catabolic process activated to degrade and recycle cell's components. Under stress conditions, such as oxidative stress and nutrient deprivation, autophagy is an essential survival pathway that operates in harmony with other stress response pathways. These include the redox-sensitive transcription complex Nrf2-Keap1 that controls groups of genes with roles in detoxification and antioxidant processes, intermediary metabolism, and cell cycle regulation. Recently, a functional association between a dysfunctional autophagy and Nrf2 pathway activation has been identified in HCC. This appears to occur through the physical interaction of the autophagy adaptor p62 with the Nrf2 inhibitor Keap1, thus leading to increased stabilization and transcriptional activity of Nrf2, a key event in reprogramming metabolic and stress response pathways of proliferating hepatocarcinoma cells. These emerging molecular mechanisms and the therapeutic perspective of targeting Nrf2-p62 interaction in HCC are discussed in this paper along with the prognostic value of autophagy in this type of cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Specific bioactive compounds in ginger and apple alleviate hyperglycemia in mice with high fat diet-induced obesity via Nrf2 mediated pathway.

PubMed

Sampath, Chethan; Rashid, Muhammed Raihan; Sang, Shengmin; Ahmedna, Mohamed

2017-07-01

Prolonged hyperglycemia activates the formation of advanced glycation end-products (AGEs). Major dicarbonyl compounds such as methylglyoxal or glyoxal are found to be the main precursors of AGEs and N(ε)-(carboxymethyl)lysine (CML) found to be predominantly higher in the diabetic population. We hypothesized that phloretin from apple and [6]-gingerol from ginger inhibit formation of AGEs and suppress the receptor for advanced glycation end products (RAGE) via nuclear factor erythroid-2-related-factor-2 (Nrf2)-dependent pathway. Phloretin and [6]-gingerol were supplemented at two different doses to C57BL/6 mice on high fat diet or standard diet for a period of 17weeks. Phloretin or [6]-gingerol supplementation significantly reduced plasma glucose, alanine aminotransferase, aspartate aminotransferase, AGEs and insulin levels. Phloretin and [6]-gingerol also decreased the levels of AGEs and CML levels, via Nrf2 pathway, enhancing GSH/GSSG ratio, heme oxygenase-1 and glyoxalase 1 in liver tissue. These results suggest that phloretin and [6]-gingerol are potential dietary compounds that can alleviate diabetes-induced complications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anti-apoptotic effect of phloretin on cisplatin-induced apoptosis in HEI-OC1 auditory cells.

PubMed

Choi, Byung-Min; Chen, Xiao Yan; Gao, Shang Shang; Zhu, Rizhe; Kim, Bok-Ryang

2011-01-01

Cisplatin is a highly effective chemotherapeutic agent, but it has significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to cisplatin. The present study examined the effects of phloretin, a natural polyphenolic compound found in apples and pears, on cisplatin-induced apoptosis. We found that phloretin induced the expression of heme oxygenase-1 (HO-1) protein in a concentration- and time-dependent manner. Phloretin induced nuclear factor-E2-related factor 2 (Nrf2) nuclear translocation, and dominant-negative Nrf2 attenuated phloretin-induced expression of HO-1. Phloretin activated the JNK, ERK and p38 mitogen-activated protein kinase (MAPK) pathways, and the JNK pathway played an important role in phloretin-induced HO-1 expression. Phloretin protected the cells against cisplatin-induced apoptosis. The protective effect of phloretin was abrogated by zinc protoporphyrin IX (ZnPP IX), a HO inhibitor. Furthermore, phloretin pretreatment inhibited mitochondrial dysfunction and the activation of caspases. These results demonstrate that the expression of HO-1 induced by phloretin is mediated by both the JNK pathway and Nrf2; the expression inhibits cisplatin-induced apoptosis in HEI-OC1 cells.

Nrf2 regulates cellular behaviors and Notch signaling in oral squamous cell carcinoma cells.

PubMed

Fan, Hong; Paiboonrungruan, Chorlada; Zhang, Xinyan; Prigge, Justin R; Schmidt, Edward E; Sun, Zheng; Chen, Xiaoxin

2017-11-04

Oxidative stress is known to play a pivotal role in the development of oral squamous cell carcinoma (OSCC). We have demonstrated that activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway has chemopreventive effects against oxidative stress-associated OSCC. However, Nrf2 have dual roles in cancer development; while it prevents carcinogenesis of normal cells, hyperactive Nrf2 also promotes the survival of cancer cells. This study is aimed to understand the function of Nrf2 in regulating cellular behaviors of OSCC cells, and the potential mechanisms through which Nrf2 facilitates OSCC. We established the Nrf2-overexpressing and Nrf2-knockdown OSCC cell lines, and examined the function of Nrf2 in regulating cell proliferation, migration, invasion, cell cycle and colony formation. Our data showed that Nrf2 overexpression promoted cancer phenotypes in OSCC cells, whereas Nrf2 silencing inhibited these phenotypes. In addition, Nrf2 positively regulated Notch signaling pathway in OSCC cells in vitro. Consistent with this observation, Nrf2 activation in Keap1 -/- mice resulted in not only hyperproliferation of squamous epithelial cells in mouse tongue as evidenced by increased expression of PCNA, but also activation of Notch signaling in these cells as evidenced by increased expression of NICD1 and Hes1. In conclusion, Nrf2 regulates cancer behaviors and Notch signaling in OSCC cells. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

PubMed

Trio, Phoebe Zapanta; Kawahara, Atsuyoshi; Tanigawa, Shunsuke; Sakao, Kozue; Hou, De-Xing

2017-01-01

6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

Early Induction of NRF2 Antioxidant Pathway by RHBDF2 Mediates Rapid Cutaneous Wound Healing

PubMed Central

Hosur, Vishnu; Burzenski, Lisa M.; Stearns, Timothy M.; Farley, Michelle L.; Sundberg, John P.; Wiles, Michael V.; Shultz, Leonard D.

2017-01-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15 min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15 min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. PMID:28268192

Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing.

PubMed

Hosur, Vishnu; Burzenski, Lisa M; Stearns, Timothy M; Farley, Michelle L; Sundberg, John P; Wiles, Michael V; Shultz, Leonard D

2017-04-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2 cub/cub ) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2 cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2 cub/cub and Rhbdf2 +/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2 cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Taurine protects against As2O3-induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway.

PubMed

Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Qiu, Tianming; Liu, Shuang; Qi, Baoxu; Zheng, Yue; Kong, Yuan; Yang, Guang; Chen, Min; Liu, Xiaofang; Sun, Xiance

2016-04-01

Arsenic was increasingly to blame as a risk factor for type 2 diabetes mellitus. In our previous study, we had found iAs stimulated autophagic flux and caused autophagic cell death through ROS pathway in INS-1 cells. Since NF-E2-related factor 2 (Nrf2) and the thioredoxin (Trx) system was a crucial line of defense against ROS, we investigated whether Nrf2/Trx pathway contributed to As2O3-stimulated autophagy and the role of taurine in this study. After treatment with 2 mg/kg BW-8 mg/kg BW As2O3 for 57 d, the expression of Nrf2 protein was decreased significantly in offsprings' pancreas. The expression of Trx gene was decreased significantly in pancreas subsequently. Finally, the generation of reactive oxygen species stimulated autophagy in arsenic-treated pancreas. Taurine could reverse arsenic-inhibited Nrf2 and Trx and inhibit autophagy. In short, inhibition of Nrf2/Trx pathway might play an important role in the pathogenesis of arsenic-related diabetes. Taurine could serve as nutrition supplementation against arsenic-related diabetes in high arsenic exposure area. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Xiangbao; Wang, Min; Sun, Guibo, E-mail: sunguibo@126.com

Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio.more » Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or gypenosides. - Highlights: • GP-17 showed protection against Aβ{sub 25–35}-induced neurotoxicity. • The neuroprotective effects of GP-17 are dependent on estrogen receptors. • GP-17 activates Nrf2/ARE/HO-1 pathways. • GP-17 activates PI3K/Akt/GSK-3β pathways.« less

The angiogenic related functions of bone marrow mesenchymal stem cells are promoted by CBDL rat serum via the Akt/Nrf2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Cheng-Cheng; Chen, Bing; Gu, Jian-Teng

Hepatopulmonary syndrome (HPS) is a complication of severe liver disease. It is characterized by an arterial oxygenation defect. Recent studies have demonstrated that pulmonary angiogenesis contributes to the abnormal gas exchange found in HPS. Additionally, mesenchymal stem cells (MSCs) are considered the stable source of VEGF-producing cells and have the potential to differentiate into multiple cell types. However, it has not been determined whether bone marrow mesenchymal stem cells (BM-MSCs) are mobilized and involved in the pulmonary angiogenesis in HPS. In this study, a CFU-F assay showed that the number of peripheral blood MSCs was increased in common bile ductmore » ligation (CBDL) rats; however, there was no significant difference found in the number of BM-MSCs. In vitro, CBDL rat serum induced the overexpression of CXCR4 and PCNA in BM-MSCs. Consistently, the directional migration as well as the proliferation ability of BM-MSCs were enhanced by CBDL rat serum, as determined by a transwell migration and MTT assays. Moreover, the secretion of VEGF by BM-MSCs increased after treatment with CBDL rat serum. We also found that the expression of phospho-Akt, phospho-ERK, and Nrf2 in BM-MSCs was significantly up-regulated by CBDL rat serum in a time dependent manner, and the blockage of the Akt/Nrf2 signalling pathway with an Akt Inhibitor or Nrf2 siRNA, instead of an ERK inhibitor, attenuated the migration, proliferation and paracrine capacity of BM-MSCs. In conclusion, these findings indicated that the number of MSCs increased in the peripheral blood of CBDL rats, and the Akt/Nrf2 pathway plays a vital role in promoting the angiogenic related functions of BM-MSCs, which could be a potent contributor to pulmonary angiogenesis in HPS. - Highlights: • Peripheral blood MSCs was increased in CBDL rats; however, the difference found for the number of BM-MSCs was not significant. • The directional migration, proliferation and ability to secrete VEGF of BM-MSCs were enhanced by CBDL rat serum. • The Akt/Nrf2 instead of ERK/Nrf2 pathway regulates the angiogenic related functions of BM-MSCs.« less

4-hydroxy-2-nonenal protects against cardiac ischemia-reperfusion injury via the Nrf2-dependent pathway.

PubMed

Zhang, Yan; Sano, Motoaki; Shinmura, Ken; Tamaki, Kayoko; Katsumata, Yoshinori; Matsuhashi, Tomohiro; Morizane, Shintaro; Ito, Hideyuki; Hishiki, Takako; Endo, Jin; Zhou, Heping; Yuasa, Shinsuke; Kaneda, Ruri; Suematsu, Makoto; Fukuda, Keiichi

2010-10-01

Reactive oxygen species (ROS) attack polyunsaturated fatty acids of the membrane and trigger lipid peroxidation, which results in the generation of alpha,beta-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). There is compelling evidence that high concentrations of aldehydes are responsible for much of the damage elicited by cardiac ischemia-reperfusion injury, while sublethal concentrations of aldehydes stimulate stress resistance pathways, to achieve cardioprotection. We investigated the mechanism of cardioprotection mediated by 4-HNE. For cultured cardiomyocytes, 4-HNE was cytotoxic at higher concentrations (>or=20 microM) but had no appreciable cytotoxicity at lower concentrations. Notably, a sublethal concentration (5muM) of 4-HNE primed cardiomyocytes to become resistant to cytotoxic concentrations of 4-HNE. 4-HNE induced nuclear translocation of transcription factor NF-E2-related factor 2 (Nrf2), and enhanced the expression of gamma-glutamylcysteine ligase (GCL) and the core subunit of the Xc(-) high-affinity cystine transporter (xCT), thereby increasing 1.45-fold the intracellular GSH levels. Cardiomyocytes treated with either Nrf2-specific siRNA or the GCL inhibitor l-buthionine sulfoximine (BSO) were less tolerant to 4-HNE. Moreover, the cardioprotective effect of 4-HNE pretreatment against subsequent glucose-free anoxia followed by reoxygenation was completely abolished in these cells. Intravenous administration of 4-HNE (4 mg/kg) activated Nrf2 in the heart and increased the intramyocardial GSH content, and consequently improved the functional recovery of the left ventricle following ischemia-reperfusion in Langendorff-perfused hearts. This cardioprotective effect of 4-HNE was not observed for Nrf2-knockout mice. In summary, 4-HNE activates Nrf2-mediated gene expression and stimulates GSH biosynthesis, thereby conferring on cardiomyocytes protection against ischemia-reperfusion injury. Copyright 2010 Elsevier Ltd. All rights reserved.

Protective effects of curcumin against mercury-induced hepatic injuries in rats, involvement of oxidative stress antagonism, and Nrf2-ARE pathway activation.

PubMed

Liu, W; Xu, Z; Li, H; Guo, M; Yang, T; Feng, S; Xu, B; Deng, Yu

2017-09-01

Mercury (Hg) represents a ubiquitous environmental heavy metal that could lead to severe toxic effects in a variety of organs usually at a low level. The present study focused on the liver oxidative stress, one of the most important roles playing in Hg hepatotoxicity, by evaluation of different concentrations of mercuric chloride (HgCl 2 ) administration. Moreover, the protective potential of curcumin against Hg hepatotoxic effects was also investigated. Eighty-four rats were randomly divided into six groups for a three-days experiment: control, dimethyl sulfoxide control, HgCl 2 treatment (0.6, 1.2, and 2.4 mg kg -1 day -1 ), and curcumin pretreatment (100 mg kg -1 day -1 ) groups. Exposure of HgCl 2 resulted in acute dose-dependent hepatotoxic effects. Administration of 2.4 mg kg -1 HgCl 2 significantly elevated total Hg, nonprotein sulfhydryl, reactive oxygen species formation, malondialdehyde, apoptosis levels, serum lactate dehydrogenase, and alanine transaminase activities, with an impairment of superoxide dismutase and glutathione peroxidase in the liver. Moreover, HgCl 2 treatment activated nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway in further investigation, with a significant upregulation of Nrf2, heme oxygenase-1, and γ-glutamylcysteine synthetase heavy subunit expression, relative to control. Pretreatment with curcumin obviously prevented HgCl 2 -induced liver oxidative stress, which may be due to its free radical scavenging or Nrf2-ARE pathway-inducing properties. Taking together these data suggest that curcumin counteracts HgCl 2 hepatotoxicity through antagonizing liver oxidative stress.

Omeprazole induces heme oxygenase-1 in fetal human pulmonary microvascular endothelial cells via hydrogen peroxide-independent Nrf2 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, Ananddeep; Zhang, Shaojie; Shrestha, Amrit

Omeprazole (OM) is an aryl hydrocarbon receptor (AhR) agonist and a proton pump inhibitor that is used to treat humans with gastric acid related disorders. Recently, we showed that OM induces NAD (P) H quinone oxidoreductase-1 (NQO1) via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent mechanism. Heme oxygenase-1 (HO-1) is another cytoprotective and antioxidant enzyme that is regulated by Nrf2. Whether OM induces HO-1 in fetal human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce HO-1 expression via Nrf2 in HPMEC. OM induced HO-1 mRNA and protein expression in a dose-dependent manner.more » siRNA-mediated knockdown of AhR failed to abrogate, whereas knockdown of Nrf2 abrogated HO-1 induction by OM. To identify the underlying molecular mechanisms, we determined the effects of OM on cellular hydrogen peroxide (H{sub 2}O{sub 2}) levels since oxidative stress mediated by the latter is known to activate Nrf2. Interestingly, the concentration at which OM induced HO-1 also increased H{sub 2}O{sub 2} levels. Furthermore, H{sub 2}O{sub 2} independently augmented HO-1 expression. Although N-acetyl cysteine (NAC) significantly decreased H{sub 2}O{sub 2} levels in OM-treated cells, we observed that OM further increased HO-1 mRNA and protein expression in NAC-pretreated compared to vehicle-pretreated cells, suggesting that OM induces HO-1 via H{sub 2}O{sub 2}-independent mechanisms. In conclusion, we provide evidence that OM transcriptionally induces HO-1 via AhR - and H{sub 2}O{sub 2} - independent, but Nrf2 - dependent mechanisms. These results have important implications for human disorders where Nrf2 and HO-1 play a beneficial role. - Highlights: • Omeprazole induces HO-1 in human fetal lung cells. • AhR deficiency fails to abrogate omeprazole-mediated induction of HO-1. • Nrf2 knockdown abrogates omeprazole-mediated HO-1 induction in human lung cells. • Hydrogen peroxide depletion augments omeprazole-mediated induction of HO-1.« less

Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo

PubMed Central

Kawamura, Tomohiro; Wakabayashi, Nobunao; Shigemura, Norihisa; Huang, Chien-Sheng; Masutani, Kosuke; Tanaka, Yugo; Noda, Kentaro; Peng, Ximei; Takahashi, Toru; Billiar, Timothy R.; Okumura, Meinoshin; Toyoda, Yoshiya; Kensler, Thomas W.

2013-01-01

Hyperoxic lung injury is a major concern in critically ill patients who receive high concentrations of oxygen to treat lung diseases. Successful abrogation of hyperoxic lung injury would have a huge impact on respiratory and critical care medicine. Hydrogen can be administered as a therapeutic medical gas. We recently demonstrated that inhaled hydrogen reduced transplant-induced lung injury and induced heme oxygenase (HO)-1. To determine whether hydrogen could reduce hyperoxic lung injury and investigate the underlying mechanisms, we randomly assigned rats to four experimental groups and administered the following gas mixtures for 60 h: 98% oxygen (hyperoxia), 2% nitrogen; 98% oxygen (hyperoxia), 2% hydrogen; 98% balanced air (normoxia), 2% nitrogen; and 98% balanced air (normoxia), 2% hydrogen. We examined lung function by blood gas analysis, extent of lung injury, and expression of HO-1. We also investigated the role of NF-E2-related factor (Nrf) 2, which regulates HO-1 expression, by examining the expression of Nrf2-dependent genes and the ability of hydrogen to reduce hyperoxic lung injury in Nrf2-deficient mice. Hydrogen treatment during exposure to hyperoxia significantly improved blood oxygenation, reduced inflammatory events, and induced HO-1 expression. Hydrogen did not mitigate hyperoxic lung injury or induce HO-1 in Nrf2-deficient mice. These findings indicate that hydrogen gas can ameliorate hyperoxic lung injury through induction of Nrf2-dependent genes, such as HO-1. The findings suggest a potentially novel and applicable solution to hyperoxic lung injury and provide new insight into the molecular mechanisms and actions of hydrogen. PMID:23475767

Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwak, Mi-Kyoung; Kensler, Thomas W.

The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this genemore » was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.« less

Visualization of Oxidative Stress Induced by Experimental Periodontitis in Keap1-Dependent Oxidative Stress Detector-Luciferase Mice.

PubMed

Kataoka, Kota; Ekuni, Daisuke; Tomofuji, Takaaki; Irie, Koichiro; Kunitomo, Muneyoshi; Uchida, Yoko; Fukuhara, Daiki; Morita, Manabu

2016-11-16

The aim of this study was to investigate whether a Keap1-dependent oxidative stress detector-luciferase (OKD-LUC) mouse model would be useful for the visualization of oxidative stress induced by experimental periodontitis. A ligature was placed around the mandibular first molars for seven days to induce periodontitis. Luciferase activity was measured with an intraperitoneal injection of d-luciferin on days 0, 1, and 7. The luciferase activity in the periodontitis group was significantly greater than that in the control group at seven days. The expressions of heme oxygenase-1 (HO-1) and malondialdehyde in periodontal tissue were significantly higher in the periodontitis group than in the control group. Immunofluorescent analysis confirmed that the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) occurred more frequently in the periodontitis group than in the control group. This study found that under oxidative stress induced by experimental periodontitis, the Nrf2/antioxidant defense pathway was activated and could be visualized from the luciferase activity in the OKD-LUC model. Thus, the OKD-LUC mouse model may be useful for exploring the mechanism underlying the relationship between the Nrf2/antioxidant defense pathway and periodontitis by enabling the visualization of oxidative stress over time.

Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity.

PubMed

Williamson, Tracy P; Johnson, Delinda A; Johnson, Jeffrey A

2012-06-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease. Copyright © 2012 Elsevier Inc. All rights reserved.

Astaxanthin Induces the Nrf2/HO-1 Antioxidant Pathway in Human Umbilical Vein Endothelial Cells by Generating Trace Amounts of ROS.

PubMed

Niu, Tingting; Xuan, Rongrong; Jiang, Ligang; Wu, Wei; Zhen, Zhanghe; Song, Yuling; Hong, Lili; Zheng, Kaiqin; Zhang, Jiaxing; Xu, Qingshan; Tan, Yinghong; Yan, Xiaojun; Chen, Haimin

2018-02-14

Astaxanthin is a powerful antioxidant that possesses potent protective effects against various human diseases and physiological disorders. However, the mechanisms underlying its antioxidant functions in cells are not fully understood. In the present study, the effects of astaxanthin on reactive oxygen species (ROS) production and antioxidant enzyme activity, as well as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt, and the nuclear factor erythroid 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathways in human umbilical vein endothelial cells (HUVECs), were examined. It was shown that astaxanthin (0.1, 1, and 10 μM) induced ROS production by 9.35%, 14.8%, and 18.06% compared to control, respectively, in HUVECs. In addition, astaxanthin increased the mRNA levels of phase II enzymes HO-1 and also promoted GSH-Px enzyme activity. Furthermore, we observed ERK phosphorylation, nuclear translocation of Nrf-2, and activation of antioxidant response element-driven luciferase activity upon astaxanthin treatment. Knockdown of Nrf-2 by small interfering RNA inhibited HO-1 mRNA expression by 60%, indicating that the Nrf-2/ARE signaling pathway is activated by astaxanthin. Our results suggest that astaxanthin activates the Nrf-2/HO-1 antioxidant pathway by generating small amounts of ROS.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

PubMed

Mascuch, Samantha J; Boudreau, Paul D; Carland, Tristan M; Pierce, N Tessa; Olson, Joshua; Hensler, Mary E; Choi, Hyukjae; Campanale, Joseph; Hamdoun, Amro; Nizet, Victor; Gerwick, William H; Gaasterland, Teresa; Gerwick, Lena

2018-03-23

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses

PubMed Central

Chaiprasongsuk, Anyamanee; Onkoksoong, Tasanee; Pluemsamran, Thanyawan; Limsaengurai, Saowalak; Panich, Uraiwan

2015-01-01

Dietary phenolics may play a protective role in UV-mediated skin pigmentation through their antioxidant and UV-absorbing actions. In this study, we investigated whether genetic silencing of Nrf2, regulating the transcription of antioxidant genes, affected melanogenesis in primary human epidermal melanocytes (HEMn) and B16F10 melanoma cells subjected to UVA (8 J/cm2) exposure. Then, we explored the antimelanogenic actions of phenolics; caffeic acid (CA) and ferulic acid (FA) providing partial UVA protection; quercetin (QU) and rutin (RU) providing strong UVA protection and; avobenzone (AV), an efficient UVA filter, in association with modulation of Nrf2-mediated antioxidant defenses in response to UVA insults in B16F10 cells. Upon oxidative insults, Nrf2 silencing promoted melanogenesis in both HEMn and B16F10 cells irradiated with UVA. Stimulation of melanogenesis by UVA correlated with increased ROS and oxidative DNA damage (8-OHdG), GSH depletion as well as a transient downregulation of Nrf2 nuclear translocation and of Nrf2-ARE signaling in B16F10 cells. All test compounds exerted antimelanogenic effects with respect to their abilities to reverse UVA-mediated oxidative damage as well as downregulation of Nrf2 activity and its target antioxidants (GCLC, GST and NQO1) in B16F10 cells. In conclusion, defective Nrf2 may promote melanogenesis under UVA irradiation through oxidative stress mechanisms. Compounds with antioxidant and/or UVA absorption properties could protect against UVA-induced melanogenesis through indirect regulatory effect on Nrf2-ARE pathway. PMID:26765101

Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway.

PubMed

Narayanan, Srinivasan V; Dave, Kunjan R; Perez-Pinzon, Miguel A

2018-04-01

Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2 -/- mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.IPC-induced cytoprotection in wild-type, but not Nrf2 -/- mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress.

PubMed

Yang, Ya-Chen; Lii, Chong-Kuei; Lin, Ai-Hsuan; Yeh, Yu-Wen; Yao, Hsien-Tsung; Li, Chien-Chun; Liu, Kai-Li; Chen, Haw-Wen

2011-12-01

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

Green tea polyphenol (−)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: Responses of major antioxidant enzymes and the Nrf2 rescue pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Dongxu; Wang, Yijun; Wan, Xiaochun

(−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been suggested to have numerous health-promoting effects. On the other hand, high-dose EGCG is able to evoke hepatotoxicity. In the present study, we elucidated the responses of hepatic major antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2) rescue pathway to high-dose levels of EGCG in Kunming mice. At a non-lethal toxic dose (75 mg/kg, i.p.), repeated EGCG treatments markedly decreased the levels of superoxide dismutase, catalase, and glutathione peroxidase. As a rescue response, the nuclear distribution of Nrf2 was significantly increased; a battery of Nrf2-target genes, including heme oxygenase 1more » (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and those involved in glutathione and thioredoxin systems, were all up-regulated. At the maximum tolerated dose (45 mg/kg, i.p.), repeated EGCG treatments did not disturb the major antioxidant defense. Among the above-mentioned genes, only HO1, NQO1, and GST genes were significantly but modestly up-regulated, suggesting a comprehensive and extensive activation of Nrf2-target genes principally occurs at toxic levels of EGCG. At a lethal dose (200 mg/kg, i.p.), a single EGCG treatment dramatically decreased not only the major antioxidant defense but also the Nrf2-target genes, demonstrating that toxic levels of EGCG are able to cause a biphasic response of Nrf2. Overall, the mechanism of EGCG-triggered hepatotoxicity involves suppression of major antioxidant enzymes, and the Nrf2 rescue pathway plays a vital role for counteracting EGCG toxicity. - Highlights: • EGCG at maximum tolerated dose does not disturb hepatic major antioxidant defense. • EGCG at maximum tolerated dose modestly upregulates hepatic Nrf2 target genes. • EGCG at toxic dose suppresses hepatic major antioxidant enzymes. • EGCG at non-lethal toxic dose pronouncedly activates hepatic Nrf2 rescue response. • EGCG at lethal dose substantially suppresses hepatic Nrf2 pathway.« less

Shikonin inhibits oxidized LDL-induced monocyte adhesion by suppressing NFκB activation via up-regulation of PI3K/Akt/Nrf2-dependent antioxidation in EA.hy926 endothelial cells.

PubMed

Huang, Chin-Shiu; Lin, Ai-Hsuan; Yang, Ting-Chun; Liu, Kai-Li; Chen, Haw-Wen; Lii, Chong-Kuei

2015-02-01

Oxidized low-density lipoprotein (oxLDL) is a key contributor to atherogenesis through multiple mechanisms, including the reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NFκB) signaling pathway. Although shikonin, one of the main active components isolated from the Chinese herb Lithospermum erythrorhizon, has been shown to possess cardioprotective, antioxidative, and anti-inflammatory effects, the mechanisms underlying these actions are not well understood. In this study, we used EA.hy926 endothelial-like cells to examine the anti-atherogenic activity of shikonin. Shikonin (0-1 μM) concentration-dependently induced heme oxygenase-1, glutamate cysteine ligase modifier subunit, catalase, superoxide dismutase 1, glutathione peroxidase 1, and glutathione reductase protein and mRNA expression and glutathione content via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/Nrf2 signaling pathway. In the presence of oxLDL (40 μg/ml), shikonin pretreatment reversed oxLDL-induced ROS production, antioxidant response element reporter activity, NFκB nuclear translocation, and intercellular adhesion molecule (ICAM)-1 and E-selectin expression and suppressed the increase of monocyte adhesion to endothelial cells. Nrf2 knockdown by using RNA interference attenuated the ability of shikonin to inhibit oxLDL-induced NFκB DNA binding activity, adhesion molecule expression, and monocyte adhesion. Taken together, these results suggest that shikonin protects against oxLDL-induced endothelial damage by suppressing ROS/NFκB-mediated ICAM-1 and E-selectin expression via up-regulation of PI3K/Akt/Nrf2-dependent antioxidant enzyme expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

PubMed

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2–Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells

PubMed Central

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state. PMID:29410668

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis.

PubMed

Niso-Santano, Mireia; González-Polo, Rosa A; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Lastres-Becker, Isabel; Ortiz-Ortiz, Miguel A; Soler, Germán; Morán, José M; Cuadrado, Antonio; Fuentes, José M

2010-05-15

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases. Copyright 2010 Elsevier Inc. All rights reserved.

Anti-Inflammatory Mechanism Involved in Pomegranate-Mediated Prevention of Breast Cancer: the Role of NF-κB and Nrf2 Signaling Pathways

PubMed Central

Mandal, Animesh; Bhatia, Deepak; Bishayee, Anupam

2017-01-01

Pomegranate (Punica granatum L.), a nutrient-rich unique fruit, has been used for centuries for the prevention and treatment of various inflammation-driven diseases. Based on our previous study, a characterized pomegranate emulsion (PE) exhibited a striking inhibition of dimethylbenz(a)anthracene (DMBA)-initiated rat mammary tumorigenesis via antiproliferative and apoptosis-inducing mechanisms. The objective of the present work is to investigate the anti-inflammatory mechanism of action of PE during DMBA rat mammary carcinogenesis by evaluating the expression of cyclooxygenase-2 (COX-2), heat shock protein 90 (HSP90), nuclear factor-κB (NF-κB) and nuclear factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2). Mammary tumor samples were harvested from our previous chemopreventive study in which PE (0.2–5.0 g/kg) was found to reduce mammary tumorigenesis in a dose-dependent manner. The expressions of COX-2, HSP90, NF-κB, inhibitory κBα (IκBα) and Nrf2 were detected by immunohistochemical techniques. PE decreased the expression of COX-2 and HSP90, prevented the degradation of IκBα, hindered the translocation of NF-κB from cytosol to nucleus and increased the expression and nuclear translocation of Nrf2 during DMBA-induced mammary tumorigenesis. These findings, together with our previous results, indicate that PE-mediated prevention of DMBA-evoked mammary carcinogenesis may involve anti-inflammatory mechanisms through concurrent but differential regulation of two interrelated molecular pathways, namely NF-κB and Nrf2 signaling. PMID:28452959

Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

PubMed

Zanotto-Filho, Alfeu; Masamsetti, V Pragathi; Loranc, Eva; Tonapi, Sonal S; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C F; Chen, Yidong; Bishop, Alexander J R

2016-12-01

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR. ©2016 American Association for Cancer Research.

PPARβ/δ ameliorates fructose-induced insulin resistance in adipocytes by preventing Nrf2 activation.

PubMed

Barroso, Emma; Rodríguez-Rodríguez, Rosalía; Chacón, Matilde R; Maymó-Masip, Elsa; Ferrer, Laura; Salvadó, Laia; Salmerón, Emilio; Wabistch, Martin; Palomer, Xavier; Vendrell, Joan; Wahli, Walter; Vázquez-Carrera, Manuel

2015-05-01

We studied whether PPARβ/δ deficiency modifies the effects of high fructose intake (30% fructose in drinking water) on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhances adipose tissue inflammation and impairs insulin signaling. Fructose intake for 8 weeks significantly increased body and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-type mice. Feeding PPARβ/δ-deficient mice with fructose exacerbated glucose intolerance and led to macrophage infiltration, inflammation, enhanced mRNA and protein levels of CD36, and activation of the JNK pathway in white adipose tissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhibited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interestingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36 expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubation with GW501516 abolished this increase. In agreement with Nrf2 playing a role in the fructose-induced CD36 protein level increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulated AKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target gene quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting that PPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficient mice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36 protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue. Copyright © 2015 Elsevier B.V. All rights reserved.

Toll-Like Receptor Signaling Induces Nrf2 Pathway Activation through p62-Triggered Keap1 Degradation.

PubMed

Yin, Shasha; Cao, Wangsen

2015-08-01

Toll-like receptors (TLRs) induce inflammation and tissue repair through multiple signaling pathways. The Nrf2 pathway plays a key role in defending against the tissue damage incurred by microbial infection or inflammation-associated diseases. The critical event that mediates TLR-induced Nrf2 activation is still poorly understood. In this study, we found that lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists activate Nrf2 signaling and the activation is due to the reduction of Keap1, the key Nrf2 inhibitor. TLR signaling-induced Keap1 reduction promoted Nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. TLR agonists modulated Keap1 at the protein posttranslation level through autophagy. TLR signaling increased the expression of autophagy protein p62 and LC3-II and induced their association with Keap1 in the autophagosome-like structures. We also characterized the interaction between p62 and Keap1 and found that p62 is indispensable for TLR-mediated Keap1 reduction: TLR signaling had no effect on Keap1 if cells lacked p62 or if cells expressed a mutant Keap1 that could not interact with p62. Our study indicates that p62-mediated Keap1 degradation through autophagy represents a critical linkage for TLR signaling regulation of the major defense network, the Nrf2 signaling pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The involvement of Nrf2 in the protective effects of (-)-Epigallocatechin-3-gallate (EGCG) on NaAsO2-induced hepatotoxicity.

PubMed

Han, Xiao-Dong; Zhang, Yan-Yan; Wang, Ke-Lei; Huang, Yong-Pan; Yang, Zhong-Bao; Liu, Zhi

2017-09-12

Arsenic exposure produces hepatotoxicity. The common mechanism determining its toxicity is the generation of oxidative stress. Oxidative stress induced by arsenic leads to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. (-)-Epigallocatechin-3-gallate (EGCG) possesses a potent antioxidant capacity and exhibits extensive pharmacological activities. This study aims to evaluate effects of EGCG on arsenic-induced hepatotoxicity and activation of Nrf2 pathway. Plasma activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase were measured; Histological analyses were conducted to observe morphological changes; Biochemical indexes such as oxidative stress (Catalase (CAT), malonyldialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS)), Nrf2 signaling related genes ( Nrf2, Nqo1, and Ho-1 ) were assessed. The results showed that EGCG inhibited arsenic-induced hepatic pathological damage, liver ROS level and MDA level. Arsenic decreases the antioxidant enzymes SOD, GPX, and CAT activity and the decrease was inhibited by treatment of EGCG. Furthermore, EGCG attenuated the retention of arsenic in liver tissues and improved the expressions of Nrf2 signaling related genes ( Nrf2, Nqo1, and Ho-1 ). These findings provide evidences that EGCG may be useful for reducing hepatotoxicity associated with oxidative stress by the activation of Nrf2 signaling pathway. Our findings suggest a possible mechanism of antioxidant EGCG in preventing hepatotoxicity, which implicate that EGCG may be a potential treatment for arsenicosis therapy.

α-Lipoic acid protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells through regeneration of glutathione by glutathione reductase via Nrf2/ARE signaling pathway.

PubMed

Shi, Chunli; Zhou, Xue; Zhang, Jiayu; Wang, Jiachun; Xie, Hong; Wu, Zhigang

2016-07-01

α-Lipoic acid (α-LA) is a potent natural antioxidant, which is capable of regenerating glutathione (GSH). However, the mechanisms by which α-LA regenerates reduced glutathione (rGSH) via the reduction of oxidized glutathione (GSSG) by glutathione reductase (GR) are still not well understood. In the present study, we investigated if α-LA replenished rGSH by GR via Nrf2/ARE signaling pathway in cadmium-treated HepG2 cells. We found that α-LA antagonized the oxidative damage and alleviated the cytotoxicity in cadmium-induced HepG2 cells by regeneration of rGSH. α-LA regenerated rGSH by activating Nrf2 signaling pathway via promoting the nuclear translocation of Nrf2, which upregulates the transcription of GR, and thus increased the activity of GR. Our results indicated that α-LA was an effective agent to antagonize the oxidative stress and alleviate the cytotoxicity in cadmium-treated HepG2 cells by regenerating rGSH through activating Nrf2 signaling pathway. Copyright © 2016. Published by Elsevier B.V.

NRF2 Mediates Neuroblastoma Proliferation and Resistance to Retinoic Acid Cytotoxicity in a Model of In Vitro Neuronal Differentiation.

PubMed

de Miranda Ramos, Vitor; Zanotto-Filho, Alfeu; de Bittencourt Pasquali, Matheus Augusto; Klafke, Karina; Gasparotto, Juciano; Dunkley, Peter; Gelain, Daniel Pens; Moreira, José Cláudio Fonseca

2016-11-01

Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox ® as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.

Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.

PubMed

Lin, Ai-Hsuan; Chen, Haw-Wen; Liu, Cheng-Tze; Tsai, Chia-Wen; Lii, Chong-Kuei

2012-07-04

Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

β-TrCP1 Is a Vacillatory Regulator of Wnt Signaling.

PubMed

Long, Marcus John; Lin, Hong-Yu; Parvez, Saba; Zhao, Yi; Poganik, Jesse Richard; Huang, Paul; Aye, Yimon

2017-08-17

Simultaneous hyperactivation of Wnt and antioxidant response (AR) are often observed during oncogenesis. However, it remains unclear how the β-catenin-driven Wnt and the Nrf2-driven AR mutually regulate each other. The situation is compounded because many players in these two pathways are redox sensors, rendering bolus redox signal-dosing methods uninformative. Herein we examine the ramifications of single-protein target-specific AR upregulation in various knockdown lines. Our data document that Nrf2/AR strongly inhibits β-catenin/Wnt. The magnitude and mechanism of this negative regulation are dependent on the direct interaction between β-catenin N terminus and β-TrCP1 (an antagonist of both Nrf2 and β-catenin), and independent of binding between Nrf2 and β-TrCP1. Intriguingly, β-catenin positively regulates AR. Because AR is a negative regulator of Wnt regardless of β-catenin N terminus, this switch of function is likely sufficient to establish a new Wnt/AR equilibrium during tumorigenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

PubMed Central

Lu, Yongke; Zhang, Xu Hannah

2012-01-01

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 –/–) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 –/– mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 –/– KI) mice but not in CYP2E1 knockout (Cyp2e1 –/–) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 –/– KI mice but not in Cyp2e1 –/– mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 –/–) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 –/– mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 –/– KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis. PMID:22552773

Novel antioxidants' synthesis and their anti-oxidative activity through activating Nrf2 signaling pathway.

PubMed

Wu, Jianzhang; Ren, Jiye; Yao, Song; Wang, Jiabing; Huang, Lili; Zhou, Peng; Yun, Di; Xu, Qing; Wu, Shoubiao; Wang, Zhankun; Qiu, Peihong

2017-04-01

Novel structure compounds (WS) containing 3,4,5-trimethoxyphenyl and acyl pyrazole were designed and synthesized based combination principles. Among them, WS13 was screened out to possess desirable anti-oxidative activity in vitro. Cell survival assay and apoptosis experiment in H 2 O 2 induced PC12 cells injury model all showed that its cytoprotection exhibited a concentration-effect manner. WS13 at 10μM could remove ROS with equal effiency to edaravone. Further, it clearly activated Nrf2 nuclear translocation and upregulated GCLC mRNA transcription and protein expression in dose-dependent manner, and its cytoprotection was reversed by GCLC protein inhibitor. In total, WS13 with further promotion can serve as Nrf2-GCLC activator in anti-oxidative therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative Stress Signaling in Mouse Prostate Cancer TRAMP C1 Cells.

PubMed

Li, Wenji; Su, Zheng-Yuan; Guo, Yue; Zhang, Chengyue; Wu, Renyi; Gao, Linbo; Zheng, Xi; Du, Zhi-Yun; Zhang, Kun; Kong, Ah-Ng

2018-02-19

The carcinogenesis of prostate cancer (PCa) in TRAMP model is highly correlated with hypermethylation in the promoter region of Nrf2 and the accompanying reduced transcription of Nrf2 and its regulated detoxifying genes. We aimed to investigate the effects of (3E,5E)-3,5-bis-(3,4,5-trimethoxybenzylidene)-tetrahydro-thiopyran-4-one (F10) and (3E,5E)-3,5-bis-(3,4,5-trimethoxy-benzylidene)-tetrahydropyran-4-one (E10), two synthetic curcumin derivatives, on restoring Nrf2 activity in TRAMP C1 cells. HepG2-C8 cells transfected with an antioxidant-response element (ARE)-luciferase vector were treated with F10, E10, curcumin, and sulforaphane (SFN) to compare their effects on Nrf2-ARE pathways. We performed real-time quantitative PCR and Western blotting to investigate the effects of F10 and E10 on Nrf2, correlated phase II detoxification genes. We also measured expression and activity of DNMTand HDAC enzymes. Enrichment of H3K27me3 on the promoter region of Nrf2 was explored with a chromatin immunoprecipitation (ChIP) assay. Methylation of the CpG region in Nrf2 promoter was doubly examined by bisulfite genomic sequencing (BGS) and methylation DNA immunoprecipitation (MeDIP). Compared with curcumin and SFN, F10 is more potent in activating Nrf2-ARE pathways. Both F10 and E10 enhanced level of Nrf2 and the correlated phase II detoxifying genes. BGS and MeDIP assays indicated that F10 but not E10 hypomethylated the Nrf2 promoter. F10 also downregulated the protein level of DNMT1, DNMT3a, DNMT3b, HDAC1, HDAC4, and HDAC7 and the activity of DNMTs and HDACs. F10 but not E10 effectively reduced the accumulation of H3k27me3 on the promoter of Nrf2. F10 and E10 can activate the Nrf2-ARE pathway and increase the level of Nrf2 and correlated phase II detoxification genes. The reactivation effect on Nrf2 by F10 in TRAMP C1 may come from demethylation, decrease of HDACs, and inhibition of H3k27me3 accumulation.

Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Juanjuan, E-mail: jwu32@emory.edu; Williams, Devin; Walter, Grant A.

The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activitymore » in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer cells and suggest that patients' hormonal status through this activity may play a significant role in some therapeutic outcomes. - Highlights: • Estrogen (E2) increases Nrf2 activity in E2-responsive breast cancer cells. • ERα, but not ERβ is essential for E2-mediated stimulation of Nrf2 in MCF7 cells. • The PI3K/Akt/GSK3β pathway is involved in E2 stimulation of Nrf2. • E2 stimulates phosphorylation of Akt and GSK3β.« less

The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway

NASA Astrophysics Data System (ADS)

Goldstein, Alona; Soroka, Yoram; Frušić-Zlotkin, Marina; Lewis, Aaron; Kohen, Ron

2016-06-01

Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.

Targeting of the Glutathione, Thioredoxin, and Nrf2 Antioxidant Systems in Head and Neck Cancer.

PubMed

Roh, Jong-Lyel; Jang, Hyejin; Kim, Eun Hye; Shin, Daiha

2017-07-10

The glutathione (GSH), thioredoxin (Trx), and Nrf2 systems represent a major defense against reactive oxygen species (ROS), the cellular imbalance of which in cancer promotes growth and therapeutic resistance. This study investigated whether targeting the GSH, Trx, and Nrf2 antioxidant systems effectively eliminated head and neck cancer (HNC). At high concentrations, auranofin, but not buthionine sulfoximine (BSO) alone, decreased the viability of HNC, whereas even at low concentrations, auranofin plus BSO synergized to kill HNC cells. Dual silencing of the genes for GCLM and TrxR1 induced GSH depletion, Trx activity inhibition, and ROS accumulation, synergistically killing HNC cells. Inhibition of the GSH and Trx systems resulted in activation of the Nrf2-antioxidant response element (ARE) pathway, which may result in suboptimal GSH and Trx inhibition where HNC is resistant. Genetic inhibition of Nrf2 and/or HO-1 or trigonelline enhanced growth suppression, ROS accumulation, and cell death from GSH and Trx inhibition. The in vivo effects of GSH, Trx, and Nrf2 system inhibition were confirmed in a mouse HNC xenograft model by achieving growth inhibition >60% compared with those of control. Innovations: This study is the first to show that triple inhibition of GSH, Trx, and Nrf2 pathways could be an effective method to overcome the resistance of HNC. Inhibition of the Nrf2-ARE pathway in addition to dual inhibition of the GSH and Trx antioxidant systems can effectively eliminate resistant HNC. Antioxid. Redox Signal. 27, 106-114.

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibuya, Akiko; Onda, Kenji, E-mail: knjond@toyaku.ac.jp; Kawahara, Hirofumi

2010-07-30

Research highlights: {yields} Sofalcone increases HO-1 in gastric epithelial cells. {yields} The induction of HO-1 by sofalcone treatment follows the activation of Nrf2. {yields} The production of VEGF by sofalcone treatment is mediated by HO-1 induction. -- Abstract: Sofalcone, 2'-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction inmore » gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.« less

Apigenin exhibits protective effects in a mouse model of d-galactose-induced aging via activating the Nrf2 pathway.

PubMed

Sang, Ying; Zhang, Fan; Wang, Heng; Yao, Jianqiao; Chen, Ruichuan; Zhou, Zhengdao; Yang, Kun; Xie, Yan; Wan, Tianfeng; Ding, Hong

2017-06-21

The aim of the present research was to study the protective effects and underlying mechanisms of apigenin on d-galactose-induced aging mice. Firstly, apigenin exhibited a potent antioxidant activity in vitro. Secondly, d-galactose was administered by subcutaneous injection once daily for 8 weeks to establish an aging mouse model to investigate the protective effect of apigenin. We found that apigenin supplementation significantly ameliorated aging-related changes such as behavioral impairment, decreased organic index, histopathological injury, increased senescence-associated β-galactosidase (SAβ-gal) activity and advanced glycation end product (AGE) level. Further data showed that apigenin facilitated Nrf2 nuclear translocation both in aging mice and normal young mice, and the Nrf2 expression of normal young mice was higher than that of natural senile mice. In addition, the expressions of Nrf2 downstream gene targets, including HO-1 and NQO1, were also promoted by apigenin administration. Moreover, apigenin also decreased the MDA level and elevated SOD and CAT activities. In conclusion, focusing on the Nrf2 pathway is a suitable strategy to delay the aging process, and apigenin may exert an anti-senescent effect process via activating the Nrf2 pathway.

Structure activity relationship of phenolic diterpenes from Salvia officinalis as activators of the nuclear factor E2-related factor 2 pathway

PubMed Central

Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; Johnson, Jeffrey A.

2013-01-01

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate cytoprotective genes which may be useful in the treatment of neurodegenerative disease. In order to better understand the structure activity relationship of phenolic diterpenes from Salvia officinalis L., we isolated carnosic acid, carnosol, epirosmanol, rosmanol, 12-methoxy-carnosic acid, sageone, and carnosaldehyde using polyamide column, centrifugal partition chromatography, and semi-preparative high performance liquid chromatography. Isolated compounds were screened in-vitro for their ability to active the Nrf2 and general cellular toxicity using mouse primary cortical cultures. All compounds except 12-methoxy-carnosic acid were able to activate the antioxidant response element. Furthermore both carnosol and carnoasldehyde were able to induce Nrf2-dependent gene expression as well as protect mouse primary cortical neuronal cultures from H2O2 induced cell death. PMID:23507152

Activation of the Nrf2-ARE Pathway in Hepatocytes Protects Against Steatosis in Nutritionally Induced Non-alcoholic Steatohepatitis in Mice

PubMed Central

Lee, Lung-Yi; Köhler, Ulrike A.; Zhang, Li; Roenneburg, Drew; Werner, Sabine; Johnson, Jeffrey A.; Foley, David P.

2014-01-01

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2− animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and β-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2− and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model. PMID:25294219

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

PubMed

Best, Sarah A; De Souza, David P; Kersbergen, Ariena; Policheni, Antonia N; Dayalan, Saravanan; Tull, Dedreia; Rathi, Vivek; Gray, Daniel H; Ritchie, Matthew E; McConville, Malcolm J; Sutherland, Kate D

2018-04-03

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1 f/f /Pten f/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations. Copyright © 2018 Elsevier Inc. All rights reserved.

MicroRNA-140-5p attenuated oxidative stress in Cisplatin induced acute kidney injury by activating Nrf2/ARE pathway through a Keap1-independent mechanism.

PubMed

Liao, Weitang; Fu, Zongjie; Zou, Yanfang; Wen, Dan; Ma, Hongkun; Zhou, Fangfang; Chen, Yongxi; Zhang, Mingjun; Zhang, Wen

2017-11-15

Oxidative stress was predominantly involved in the pathogenesis of acute kidney injury (AKI). Recent studies had reported the protective role of specific microRNAs (miRNAs) against oxidative stress. Hence, we investigated the levels of miR140-5p and its functional role in the pathogenesis of Cisplatin induced AKI. A mice Cisplatin induced-AKI model was established. We found that miR-140-5p expression was markedly increased in mice kidney. Bioinformatics analysis revealed nuclear factor erythroid 2-related factor (Nrf2) was a potential target of miR-140-5p, We demonstrated that miR-140-5p did not affect Kelch-like ECH-associated protein 1 (Keap1) level but directly targeted the 3'-UTR of Nrf2 mRNA and played a positive role in the regulation of Nrf2 expression which was confirmed by luciferase activity assay and western blot. What was more, consistent with miR140-5p expression, the mRNA and protein levels of Nrf2, as well as antioxidant response element (ARE)-driven genes Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1) were significantly increased in mice kidney tissues. In vitro study, Enforced expression of miR-140-5p in HK2 cells significantly attenuated oxidative stress by decreasing ROS level and increasing the expression of manganese superoxide dismutase (MnSOD). Simultaneously, miR-140-5p decreased lactate dehydrogenase (LDH) leakage and improved cell vitality in HK2 cells under Cisplatin-induced oxidative stress. However, HK2 cells transfected with a siRNA targeting Nrf2 abrogated the protective effects of miR-140-5p against oxidative stress. These results indicated that miR-140-5p might exert its anti-oxidative stress function via targeting Nrf2. Our findings showed the novel transcriptional role of miR140-5p in the expression of Nrf2 and miR-140-5p protected against Cisplatin induced oxidative stress by activating Nrf2-dependent antioxidant pathway, providing a potentially therapeutic target in acute kidney injury. Copyright © 2017. Published by Elsevier Inc.

Gemfibrozil pretreatment affecting antioxidant defense system and inflammatory, but not Nrf-2 signaling pathways resulted in female neuroprotection and male neurotoxicity in the rat models of global cerebral ischemia-reperfusion.

PubMed

Mohagheghi, Fatemeh; Khalaj, Leila; Ahmadiani, Abolhassan; Rahmani, Behrouz

2013-04-01

Two important pathophysiological mechanisms involved during cerebral ischemia are oxidative stress and inflammation. In pathological conditions such as brain ischemia the ability of free radicals production is greater than that of elimination by endogenous antioxidative systems, so brain is highly injured due to oxidation and neuroinflammation. Fibrates as peroxisome proliferator-activated receptor (PPAR)-α ligands, are reported to have antioxidant and anti-inflammatory actions. In this study, gemfibrozil, a fibrate is investigated for its therapeutic potential against global cerebral ischemia-reperfusion (I/R) injury of male and female rats. This study particularly has focused on inflammatory and antioxidant signaling pathways, such as nuclear factor erythroid-related factor (Nrf)-2, as well as the activity of some endogenous antioxidant agents. It was found that pretreatment of animals with gemfibrozil prior to I/R resulted in a sexually dimorphic outcome. Within females it proved to be protective, modulating inflammatory factors and inducing antioxidant defense system including superoxide dismutase (SOD), catalase, as well as glutathione level. However, Nrf-2 signaling pathway was not affected. It also decreased malondialdehyde level as an index of lipid peroxidation. In contrast, gemfibrozil pretreatment was toxic to males, enhancing the expression of inflammatory factors such as tumor necrosis factor-α, nuclear factor-κB, and cyclooxygenase-2, and decreasing Nrf-2 expression and SOD activity, leading to hippocampal neurodegeneration. Considering that gemfibrozil is a commonly used anti-hyperlipidemic agent in clinic, undoubtedly more investigations are crucial to exactly unravel its sex-dependent neuroprotective/neurodegenerative potential.

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

PubMed

Wang, Rong; Yu, Zhen; Sunchu, Bharath; Shoaf, James; Dang, Ivana; Zhao, Stephanie; Caples, Kelsey; Bradley, Lynda; Beaver, Laura M; Ho, Emily; Löhr, Christiane V; Perez, Viviana I

2017-06-01

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Neuroprotection against 6-OHDA toxicity in PC12 cells and mice through the Nrf2 pathway by a sesquiterpenoid from Tussilago farfara.

PubMed

Lee, Joohee; Song, Kwangho; Huh, Eugene; Oh, Myung Sook; Kim, Yeong Shik

2018-06-01

Oxidative stress plays a key role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Therefore, the nuclear factor-E2-related factor 2 (Nrf2), a key regulator of the antioxidative response, is considered to be important as a therapeutic target for neurodegenerative diseases. We investigated the underlying mechanism of Nrf2-mediated neuroprotective effects against oxidative stress in the PC12 cell line by 7β-(3-ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), one of the sesquiterpenoids in Farfarae Flos. Pretreatment of PC12 cells with ECN had a protective effect against hydrogen peroxide (H 2 O 2 )- or 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. ECN upregulated the ARE-luciferase activity and induced the mRNA expression of Nrf2 and antioxidant enzyme heme oxygenase-1 (HO-1). Knockdown of Nrf2 by small, interfering RNA (siRNA) abrogated the upregulation of HO-1, indicating that ECN had induced HO-1 via the Nrf2 pathway. Pretreatment with the thiol reducing agents, N-acetylcysteine (NAC) or dithiothreitol (DTT), attenuated Nrf2 activation and HO-1 expression. However, the non-thiol reducing antioxidant, Trolox, failed to inhibit HO-1 induction by ECN. These results suggest that ECN may directly interact with Kelch-like ECH-associated protein 1 (Keap1) and modify critical cysteine thiols present in the proteins responsible for Nrf2-mediated upregulation of HO-1. In a 6-OHDA-induced mouse model of PD, administration of ECN ameliorated motor impairments and dopaminergic neuronal damage. Taken together, ECN exerts neuroprotective effects by activating the Nrf2/HO-1 signaling pathway in both PC12 cells and mice. Thus, ECN, as an Nrf2 activator, could be an attractive therapeutic candidate for the neuroprotection or treatment of neurodegenerative diseases. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Adipose-derived mesenchymal stem cells promote the survival of fat grafts via crosstalk between the Nrf2 and TLR4 pathways

PubMed Central

Chen, Xiaosong; Yan, Liu; Guo, Zhihui; Chen, Zhaohong; Chen, Ying; Li, Ming; Huang, Chushan; Zhang, Xiaoping; Chen, Liangwan

2016-01-01

Autologous fat grafting is an effective reconstructive surgery technique; however, its success is limited by inconsistent graft retention and an environment characterized by high oxidative stress and inflammation. Adipose-derived stem cells (ADSCs) increase the survival of fat grafts, although the underlying mechanisms remain unclear. Here, TLR4−/− and Nrf2−/− mice were used to explore the effects of oxidative stress and inflammation on the viability and function of ADSCs in vitro and in vivo. Enrichment of fat grafts with ADSCs inhibited inflammatory cytokine production, enhanced growth factor levels, increased fat graft survival, downregulated NADPH oxidase (NOX)1 and 4 expression, increased vascularization and reduced ROS production in a manner dependent on toll-like receptor (TLR)-4 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Immunohistochemical analysis showed that exposure to hypoxia enhanced ADSC growth and promoted the differentiation of ADSCs into vascular endothelial cells. Hypoxia-induced inflammatory cytokine, growth factor and NOX1/4 upregulation, as well as increased ROS production and apoptosis in ADSCs were dependent on TLR4 and Nrf2, which also modulated the effect of ADSCs on promoting endothelial progenitor cell migration and angiogenesis. Western blot analyses showed that the effects of hypoxia on ADSCs were regulated by crosstalk between Nrf2 antioxidant responses and NF-κB- and TLR4-mediated inflammatory responses. Taken together, our results indicate that ADSCs can increase the survival of fat transplants through the modulation of inflammatory and oxidative responses via Nrf2 and TLR4, suggesting potential strategies to improve the use of ADSCs for cell therapy. PMID:27607584

Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway1[S

PubMed Central

Latham Birt, Sally H.; Purcell, Robert; Botham, Kathleen M.; Wheeler-Jones, Caroline P. D.

2016-01-01

Circulating levels of chylomicron remnants (CMRs) increase postprandially and their composition directly reflects dietary lipid intake. These TG-rich lipoproteins likely contribute to the development of endothelial dysfunction, albeit via unknown mechanisms. Here, we investigated how the FA composition of CMRs influences their actions on human aortic endothelial cells (HAECs) by comparing the effects of model CMRs—artificial TG-rich CMR-like particles (A-CRLPs)—containing TGs extracted from fish, DHA-rich algal, corn, or palm oils. HAECs responded with distinct transcriptional programs according to A-CRLP TG content and oxidation status, with genes involved in antioxidant defense and cytoprotection most prominently affected by n-3 PUFA-containing A-CRLPs. These particles were significantly more efficacious inducers of heme oxygenase-1 (HO-1) than n-6 PUFA corn or saturated FA-rich palm CRLPs. Mechanistically, HO-1 induction by all CRLPs requires NADPH oxidase 4, with PUFA-containing particles additionally dependent upon mitochondrial reactive oxygen species. Activation of both p38 MAPK and PPARβ/δ culminates in increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression/nuclear translocation and HO-1 induction. These studies define new molecular pathways coupling endothelial cell activation by model CMRs with adaptive regulation of Nrf2-dependent HO-1 expression and may represent key mechanisms through which dietary FAs differentially impact progression of endothelial dysfunction. PMID:27185859

miR-214 protects erythroid cells against oxidative stress by targeting ATF4 and EZH2.

PubMed

Gao, Ming; Liu, Yun; Chen, Yue; Yin, Chunyang; Chen, Jane-Jane; Liu, Sijin

2016-03-01

Nuclear factor (erythroid-derived 2) like 2 (Nrf2) is a key regulator in protecting cells against stress by targeting many anti-stress response genes. Recent evidence also reveals that Nrf2 functions partially by targeting mircroRNAs (miRNAs). However, the understanding of Nrf2-mediated cytoprotection through miRNA-dependent mechanisms is largely unknown. In the current study, we identified a direct Nrf2 targeting miRNA, miR-214, and demonstrated a protective role of miR-214 in erythroid cells against oxidative stresses generated by radiation, excess iron and arsenic (As) exposure. miR-214 expression was transcriptionally repressed by Nrf2 through a canonical antioxidant response element (ARE) within its promoter region, and this repression is ROS-dependence. The suppression of miR-214 by Nrf2 could antagonize oxidative stress-induced cell death in erythroid cells by two ways. First, miR-214 directly targeted ATF4, a crucial transcriptional factor involved in anti-stress responses, down regulation of miR-214 releases the repression of ATF4 translation and leads to increased ATF4 protein content. Second, miR-214 was able to prevent cell death by targeting EZH2, the catalytic core component of PRC2 complex that is responsible for tri-methylation reaction at lysine 27 (K27) of histone 3 (H3) (H3K27me3), by which As-induced miR-214 reduction resulted in an increased global H3K27me3 level and a compromised overexpression of a pro-apoptotic gene Bim. These two pathways downstream of miR-214 synergistically cooperated to antagonize erythroid cell death upon oxidative stress. Our combined data revealed a protective role of miR-214 signaling in erythroid cells against oxidative stress, and also shed new light on Nrf2-mediated cytoprotective machinery. Copyright © 2016 Elsevier Inc. All rights reserved.

DL-3-n-butylphthalide alleviates vascular cognitive impairment induced by chronic cerebral hypoperfusion by activating the Akt/Nrf2 signaling pathway in the hippocampus of rats.

PubMed

Qi, Qianqian; Xu, Jing; Lv, Peiyuan; Dong, Yanhong; Liu, Zhijuan; Hu, Ming; Xiao, Yining; Jia, Yanqiu; Jin, Wei; Fan, Mingyue; Zhang, Dandan; Meng, Nan

2018-04-13

Oxidative stress induced by chronic cerebral hypoperfusion (CCH) plays an important role in the pathogenesis of vascular cognitive impairment (VCI). The Akt/Nrf2 signaling pathway is one of the most important antioxidative stress pathways. To explore whether NBP (DL-3-n-butylphthalide) could alleviate VCI induced by CCH via activating the Akt/Nrf2 signaling pathway and modifying the levels of apoptosis-related proteins, adult male Sprague-Dawley rats were subjected to permanent occlusion of bilateral common carotid arteries (BCCAO) and treated either with vehicle or NBP (applied in two doses, 40 mg/kg and 80 mg/kg) while sham operated animals were treated with vehicle. Treatments were administered daily for 28 days. The obtained results indicate that both administrated doses of NBP significantly ameliorated the spatial learning and memory impairments as indicated by the Morris water maze test while Hematoxylin-Eosin staining revealed that morphological defects in the CA1 area of hippocampus were improved. Moreover, NBP reversed the BCCAO-induced downregulation of investigated oxidative stress-related proteins (p-Akt, t-Nrf2, n-Nrf2 and HO-1) along with the upregulation of pro-apoptotic molecule, Bax and reduction of the expression of anti-apoptotic protein, Bcl-2. According to presented results, NBP may have a protective effect against cognitive and morphological impairments induced by CCH via activation of Akt/Nrf2 signaling pathway and inhibition of apoptotic cascade. Copyright © 2017. Published by Elsevier B.V.

Prx I Suppresses K-ras-Driven Lung Tumorigenesis by Opposing Redox-Sensitive ERK/Cyclin D1 Pathway

PubMed Central

Park, Young-Ho; Kim, Sun-Uk; Lee, Bo-Kyoung; Kim, Hyun-Sun; Song, In-Sung; Shin, Hye-Jun; Han, Ying-Hao; Chang, Kyu-Tae; Kim, Jin-Man; Lee, Dong-Seok; Kim, Yeul-Hong; Choi, Chang-Min; Kim, Bo-Yeon

2013-01-01

Abstract Aims: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non–small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-rasG12D-mediated lung adenocarcinogenesis. Results: Using human-lung adenocarcinoma tissues and lung-specific K-rasG12D-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-rasG12D-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. Innovation: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. Conclusion: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis. Antioxid. Redox Signal. 19, 482–496. PMID:23186333

Electrophilic nitro-fatty acids prevent astrocyte-mediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation.

PubMed

Diaz-Amarilla, Pablo; Miquel, Ernesto; Trostchansky, Andrés; Trias, Emiliano; Ferreira, Ana M; Freeman, Bruce A; Cassina, Patricia; Barbeito, Luis; Vargas, Marcelo R; Rubbo, Homero

2016-06-01

Nitro-fatty acids (NO2-FA) are electrophilic signaling mediators formed in tissues during inflammation, which are able to induce pleiotropic cytoprotective and antioxidant pathways including up regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) responsive genes. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons associated to an inflammatory process that usually aggravates the disease progression. In ALS animal models, the activation of the transcription factor Nrf2 in astrocytes confers protection to neighboring neurons. It is currently unknown whether NO2-FA can exert protective activity in ALS through Nrf2 activation. Herein we demonstrate that nitro-arachidonic acid (NO2-AA) or nitro-oleic acid (NO2-OA) administrated to astrocytes expressing the ALS-linked hSOD1(G93A) induce antioxidant phase II enzyme expression through Nrf2 activation concomitant with increasing intracellular glutathione levels. Furthermore, treatment of hSOD1(G93A)-expressing astrocytes with NO2-FA prevented their toxicity to motor neurons. Transfection of siRNA targeted to Nrf2 mRNA supported the involvement of Nrf2 activation in NO2-FA-mediated protective effects. Our results show for the first time that NO2-FA induce a potent Nrf2-dependent antioxidant response in astrocytes capable of preventing motor neurons death in a culture model of ALS. Copyright © 2016 Elsevier Inc. All rights reserved.

HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway.

PubMed

Ade, Nadège; Leon, Fanny; Pallardy, Marc; Peiffer, Jean-Luc; Kerdine-Romer, Saadia; Tissier, Marie-Hélène; Bonnet, Pierre-Antoine; Fabre, Isabelle; Ourlin, Jean-Claude

2009-02-01

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

Structural and functional characterization of Nrf2 degradation by glycogen synthase kinase 3/β-TrCP.

PubMed

Cuadrado, Antonio

2015-11-01

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular homeostasis that controls the expression of more than 1% of human genes related to biotransformation reactions, redox homeostasis, energetic metabolism, DNA repair, and proteostasis. Its activity has a tremendous impact on physiology and pathology and therefore it is very tightly regulated, mainly at the level of protein stability. In addition to the very well established regulation by the ubiquitin E3 ligase adapter Keap1, recent advances have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinse-3 (GSK-3). This kinase phosphorylates specific serine residues in the Neh6 domain of Nrf2 to create a degradation domain that is then recognized by the ubiquitin ligase adapter β-TrCP and tagged for proteasome degradation by a Cullin1/Rbx1 complex. Here we review the mechanistic elements and the signaling pathways that participate in this regulation by GSK-3/β-TrCP. These pathways include those activated by ligands of tyrosine kinase, G protein-coupled, metabotropic, and ionotropic receptors that activate phosphatidyl inositol 3-kinase (PI3K)/ATK and by the canonical WNT signaling pathway, where a fraction of Nrf2 interacts with Axin1/GSK-3. Considering that free Nrf2 protein is localized in the nucleus, we propose a model termed "double flux controller" to explain how Keap1 and β-TrCP coordinate the stability of Nrf2 in several scenarios. The GSK-3/β-TrCP axis provides a novel therapeutic strategy to modulate Nrf2 activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages

PubMed Central

Chai, Jianshen; Luo, Li; Hou, Fengyan; Fan, Xia; Yu, Jing; Ma, Wei; Tang, Wangqi; Yang, Xue; Zhu, Junyu; Kang, Wenyuan; Yan, Jun; Liang, Huaping

2016-01-01

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. PMID:27685463

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

PubMed

Ahuja, Manuj; Ammal Kaidery, Navneet; Yang, Lichuan; Calingasan, Noel; Smirnova, Natalya; Gaisin, Arsen; Gaisina, Irina N; Gazaryan, Irina; Hushpulian, Dmitry M; Kaddour-Djebbar, Ismail; Bollag, Wendy B; Morgan, John C; Ratan, Rajiv R; Starkov, Anatoly A; Beal, M Flint; Thomas, Bobby

2016-06-08

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects. Copyright © 2016 the authors 0270-6474/16/366333-20$15.00/0.

Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu-Kun Jennifer; Yeager, Ronnie L.; Tanaka, Yuji

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione andmore » exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gst{alpha}1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.« less

Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet.

PubMed

Zhang, Yu-Kun Jennifer; Yeager, Ronnie L; Tanaka, Yuji; Klaassen, Curtis D

2010-06-15

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstalpha1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities. Copyright 2010. Published by Elsevier Inc.

Licochalcone E activates Nrf2/antioxidant response element signaling pathway in both neuronal and microglial cells: therapeutic relevance to neurodegenerative disease.

PubMed

Kim, Sa Suk; Lim, Juhee; Bang, Yeojin; Gal, Jiyeong; Lee, Sang-Uk; Cho, Young-Chang; Yoon, Goo; Kang, Bok Yun; Cheon, Seung Hoon; Choi, Hyun Jin

2012-10-01

Oxidative stress and neuroinflammation are hallmarks of neurodegenerative diseases, which do not play independently but work synergistically through complex interactions exacerbating neurodegeneration. Therefore, the mechanism that is directly implicated in controlling oxidative stress and inflammatory response could be an attractive strategy to prevent the onset and/or delay the progression of neurodegenerative diseases. The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is the guardian of redox homeostasis by regulating a battery of antioxidant and phase II detoxification genes, which are relevant to defense mechanism against oxidative stress and inflammatory responses. In this study, we show that a recently identified Glycyrrhiza-inflata-derived chalcone, licochalcone E (Lico-E), attenuates lipopolysaccharide-induced inflammatory responses in microglial BV2 cells and protects dopaminergic SH-SY5Y cells from 6-hydroxydopamine cytotoxicity. Lico-E activates Nrf2-antioxidant response element (ARE) system and up-regulates downstream NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Anti-inflammatory and cytoprotective effects of Lico-E are attenuated in siRNA-mediated Nrf2-silencing cells as well as in the presence with specific inhibitor of HO-1 or NQO1, respectively. Lico-E also has neuroprotective effect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal dopaminergic neurodegeneration in mice, with up-regulation of HO-1 and NQO1 in the substantia nigra of the brain. This study demonstrates that Lico-E is a potential activator of the Nrf2/ARE-dependent pathway and is therapeutically relevant not only to oxidative-stress-related neurodegeneration but also inflammatory responses of microglial cells both in vitro and in vivo. Copyright © 2012 Elsevier Inc. All rights reserved.

Flavonoids as Putative Inducers of the Transcription Factors Nrf2, FoxO, and PPARγ

PubMed Central

Duckstein, Nils; Hasler, Mario; Rimbach, Gerald

2017-01-01

Dietary flavonoids have been shown to extend the lifespan of some model organisms and may delay the onset of chronic ageing-related diseases. Mechanistically, the effects could be explained by the compounds scavenging free radicals or modulating signalling pathways. Transcription factors Nrf2, FoxO, and PPARγ possibly affect ageing by regulating stress response, adipogenesis, and insulin sensitivity. Using Hek-293 cells transfected with luciferase reporter constructs, we tested the potency of flavonoids from different subclasses (flavonols, flavones, flavanols, and isoflavones) to activate these transcription factors. Under cell-free conditions (ABTS and FRAP assays), we tested their free radical scavenging activities and used α-tocopherol and ascorbic acid as positive controls. Most of the tested flavonoids, but not the antioxidant vitamins, stimulated Nrf2-, FoxO-, and PPARγ-dependent promoter activities. Flavonoids activating Nrf2 also tended to induce a FoxO and PPARγ response. Interestingly, activation patterns of cellular stress response by flavonoids were not mirrored by their activities in ABTS and FRAP assays, which depended mostly on hydroxylation in the flavonoid B ring and, in some cases, extended that of the vitamins. In conclusion, the free radical scavenging properties of flavonoids do not predict whether these molecules can stimulate a cellular response linked to activation of longevity-associated transcription factors. PMID:28761622

Nrf2 and regulation of the antioxidant system in the Antarctic silverfish, Pleuragramma antarctica: Adaptation to environmental changes of pro-oxidant pressure.

PubMed

Giuliani, Maria Elisa; Benedetti, Maura; Nigro, Marco; Regoli, Francesco

2017-08-01

Despite the key importance of Nrf2-Keap1 in regulating antioxidant system in vertebrates, this system is still poorly investigated in marine species. The present study focused on the Antarctic silverfish Pleuragramma antarctica which, during the final phases of embryo development in platelet ice, is challenged by a sudden enhancement of environmental oxidative conditions associated to ice melting. Partial coding sequences were identified for Nrf2, its repressor Keap1 and for typical Nrf2-target antioxidant genes, like catalase, glutathione peroxidase isoform 1 and Cu/Zn-dependent superoxide dismutase. Compared to temperate homologues, the protein sequences showed an elevated conservation of amino acids essential for catalytic functions, while a few specific substitutions in non-essential regions may represent a molecular adaptation to improve flexibility and accessibility to active site at cold temperatures. The role of the Nrf2-Keap1 pathway in modulating the activation of antioxidant defences was demonstrated at both transcriptional and functional levels with a clear temporal increase of antioxidant protection in embryos before the hatching. Such findings confirm the importance of Nrf2 and highlight regulation of antioxidants as an adaptive strategy in P. antarctica to protect the early life stages toward the environmental changes of pro-oxidant pressure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Hong Shik; Hong, Eun-Hee; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791

2013-09-27

Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotypemore » of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.« less

Dietary phytochemicals and cancer prevention: Nrf2 signaling, epigenetics, and cell death mechanisms in blocking cancer initiation and progression.

PubMed

Lee, Jong Hun; Khor, Tin Oo; Shu, Limin; Su, Zheng-Yuan; Fuentes, Francisco; Kong, Ah-Ng Tony

2013-02-01

Reactive metabolites from carcinogens and oxidative stress can drive genetic mutations, genomic instability, neoplastic transformation, and ultimately carcinogenesis. Numerous dietary phytochemicals in vegetables/fruits have been shown to possess cancer chemopreventive effects in both preclinical animal models and human epidemiological studies. These phytochemicals could prevent the initiation of carcinogenesis via either direct scavenging of reactive oxygen species/reactive nitrogen species (ROS/RNS) or, more importantly, the induction of cellular defense detoxifying/antioxidant enzymes. These defense enzymes mediated by Nrf2-antioxidative stress and anti-inflammatory signaling pathways can contribute to cellular protection against ROS/RNS and reactive metabolites of carcinogens. In addition, these compounds would kill initiated/transformed cancer cells in vitro and in in vivo xenografts via diverse anti-cancer mechanisms. These mechanisms include the activation of signaling kinases (e.g., JNK), caspases and the mitochondria damage/cytochrome c pathways. Phytochemicals may also have anti-cancer effects by inhibiting the IKK/NF-κB pathway, inhibiting STAT3, and causing cell cycle arrest. In addition, other mechanisms may include epigenetic alterations (e.g., inhibition of HDACs, miRNAs, and the modification of the CpG methylation of cancer-related genes). In this review, we will discuss: the current advances in the study of Nrf2 signaling; Nrf2-deficient tumor mouse models; the epigenetic control of Nrf2 in tumorigenesis and chemoprevention; Nrf2-mediated cancer chemoprevention by naturally occurring dietary phytochemicals; and the mutation or hyper-expression of the Nrf2-Keap1 signaling pathway in advanced tumor cells. The future development of dietary phytochemicals for chemoprevention must integrate in vitro signaling mechanisms, relevant biomarkers of human diseases, and combinations of different phytochemicals and/or non-toxic therapeutic drugs, including NSAIDs. Copyright © 2012 Elsevier Inc. All rights reserved.

Ulinastatin alleviates neurological deficiencies evoked by transient cerebral ischemia via improving autophagy, Nrf-2-ARE and apoptosis signals in hippocampus.

PubMed

Jiang, Xiao-Ming; Hu, Jing-Hai; Wang, Lu-Lu; Ma, Chi; Wang, Xu; Liu, Xiao-Liang

2018-05-10

Ulinastatin [or called as urinary trypsin inhibitor (UTI)] plays a role in regulating neurological deficits evoked by transient cerebral ischemia. However, the underlying mechanisms still need to be determined. The present study was to examine the effects of UTI on autophagy, Nrf2-ARE and apoptosis signal pathway in the hippocampus in the process of neurological functions after cerebral ischemia using a rat model of cardiac arrest (CA). CA was induced by asphyxia followed by cardiopulmonary resuscitation (CPR) in rats. Western Blot analysis was employed to determine the expression of representative autophagy (namely, Atg5, LC3, Beclin 1), p62 protein (a maker of autophagic flux), and Nrf2-ARE pathways. Neuronal apoptosis was assessed by determining expression levels of Caspase-3 and Caspase-9, and by examining terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). The modified neurological severity score (mNSS) and spatial working memory performance were used to assess neurological deficiencies in CA rats. Our results show that CA amplified autophagy and apoptotic Caspase-3/Caspase-9, and downregulated Nrf2-ARE pathway in the hippocampus CA1 region. Systemic administration of UTI attenuated autophagy and apoptosis, and largely restored Nrf2-ARE signal pathway following cerebral ischemia and thereby alleviated neurological deficits with increasing survival of CA rats. Our data suggest that UTI improves the worsened protein expression of autophagy and apoptosis, and restores Nrf2-ARE signals in the hippocampus and this is linked to inhibition of neurological deficiencies in transient cerebral ischemia. UTI plays a beneficial role in modulating neurological deficits induced by transient cerebral ischemia via central autophagy, apoptosis and Nrf2-ARE mechanisms.

Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuse, Yuji; Nguyen, Vu Thanh; Kobayashi, Makoto, E

Transcription factor Nrf2 induces a number of detoxifying enzymes and antioxidant proteins to confer protection against the toxic effects of a diverse range of chemicals including inorganic arsenicals. Although a number of studies using cultured cells have demonstrated that Nrf2 has a cell-protective function against acute and high-dose arsenic toxicity, there is no clear in vivo evidence of this effect. In the present study, we genetically investigated the protective role of Nrf2 against acute sodium arsenite toxicity using the zebrafish Nrf2 mutant, nrf2a{sup fh318}. After treatment with 1 mM sodium arsenite, the survival of nrf2a{sup fh318} larvae was significantly shortermore » than that of wild-type siblings, suggesting that Nrf2 protected the zebrafish larvae against high-dose arsenite exposure. To understand the molecular basis of the Nrf2-dependent protection, we analyzed the gene expression profiles after arsenite exposure, and found that the genes involved in the antioxidative function (prdx1 and gclc), arsenic metabolism (gstp1) and xenobiotic elimination (abcc2) were induced in an Nrf2-dependent manner. Furthermore, pre-treatment with sulforaphane, a well-known Nrf2 activator improved the survival of zebrafish larvae after arsenic exposure. Based on these results, we concluded that Nrf2 plays a fundamental and conserved role in protection against acute sodium arsenite toxicity. - Highlights: • The role of Nrf2 under arsenite exposure was valuated using zebrafish. • Nrf2 mutant zebrafish was highly sensitive to acute arsenic toxicity. • Nrf2 induced anti-arsenic genes in response to arsenite. • Sulforaphane attenuated arsenic toxicity through Nrf2 activation. • Nrf2 system plays an important role in the defense against acute arsenic toxicity.« less

Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

PubMed Central

Gong, Li-rong; Dong, Shu-an; Cao, Xin-shun; Wu, Li-li; Wu, Li-na

2015-01-01

Electroacupuncture at select acupoints have been verified to protect against organ dysfunctions during endotoxic shock. And, heme oxygenase (HO)-1 as a phase II enzyme and antioxidant contributed to the protection of kidney in septic shock rats. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway mediated the activation of NF-E2 related factor-2 (Nrf2), which was involved in HO-1 induction. To understand the efficacy of electroacupuncture stimulation in ameliorating acute kidney injury (AKI) through the PI3K/Akt/Nrf2 pathway and subsequent HO-1 upregulation, a dose of LPS 5mg/kg was administered intravenously to replicate the rabbit model of AKI induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Neiguan acupoints for five consecutive days while sham electroacupuncture at non-acupoints as control. Results displayed that electroacupuncture stimulation significantly alleviated the morphologic renal damage, attenuated renal tubular apoptosis, suppressed the elevated biochemical indicators of AKI caused by LPS, enhanced the expressions of phospho-Akt, HO-1protein, Nrf2 total and nucleoprotein, and highlighted the proportions of Nrf2 nucleoprotein as a parallel. Furthermore, partial protective effects of elecroacupuncture were counteracted by preconditioning with wortmannin (the selective PI3K inhibitor), indicating a direct involvement of PI3K/Akt pathway. Inconsistently, wortmannin pretreatment made little difference to the expressions of HO-1, Nrf2 nucleoprotein and total protein, which indicated that PI3K/Akt may be not the only pathway responsible for electroacupuncture-afforded protection against LPS-induced AKI. These findings provide new insights into the potential future clinical applications of electroacupuncture for AKI induced by endotoxic shock instead of traditional remedies. PMID:26524181

Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways.

PubMed

Yu, Jian-Bo; Shi, Jia; Zhang, Yuan; Gong, Li-Rong; Dong, Shu-An; Cao, Xin-Shun; Wu, Li-Li; Wu, Li-Na

2015-01-01

Electroacupuncture at select acupoints have been verified to protect against organ dysfunctions during endotoxic shock. And, heme oxygenase (HO)-1 as a phase II enzyme and antioxidant contributed to the protection of kidney in septic shock rats. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway mediated the activation of NF-E2 related factor-2 (Nrf2), which was involved in HO-1 induction. To understand the efficacy of electroacupuncture stimulation in ameliorating acute kidney injury (AKI) through the PI3K/Akt/Nrf2 pathway and subsequent HO-1 upregulation, a dose of LPS 5mg/kg was administered intravenously to replicate the rabbit model of AKI induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Neiguan acupoints for five consecutive days while sham electroacupuncture at non-acupoints as control. Results displayed that electroacupuncture stimulation significantly alleviated the morphologic renal damage, attenuated renal tubular apoptosis, suppressed the elevated biochemical indicators of AKI caused by LPS, enhanced the expressions of phospho-Akt, HO-1protein, Nrf2 total and nucleoprotein, and highlighted the proportions of Nrf2 nucleoprotein as a parallel. Furthermore, partial protective effects of elecroacupuncture were counteracted by preconditioning with wortmannin (the selective PI3K inhibitor), indicating a direct involvement of PI3K/Akt pathway. Inconsistently, wortmannin pretreatment made little difference to the expressions of HO-1, Nrf2 nucleoprotein and total protein, which indicated that PI3K/Akt may be not the only pathway responsible for electroacupuncture-afforded protection against LPS-induced AKI. These findings provide new insights into the potential future clinical applications of electroacupuncture for AKI induced by endotoxic shock instead of traditional remedies.

The Roles of 4β-Hydroxywithanolide E from Physalis peruviana on the Nrf2-Anti-Oxidant System and the Cell Cycle in Breast Cancer Cells.

PubMed

Peng, Chieh Yu; You, Bang Jau; Lee, Chia Lin; Wu, Yang Chang; Lin, Wen Hsin; Lu, Te Ling; Chang, Fei-Ching; Lee, Hong Zin

2016-01-01

4[Formula: see text]-Hydroxywithanolide E is an active component of the extract of Physalis peruviana that has been reported to exhibit antitumor effects. Although the involvement of reactive oxygen species (ROS) production and the ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway in 4[Formula: see text]-hydroxywithanolide E-induced apoptosis of breast cancer MCF-7 cells was demonstrated in our previous study, the relationship between ROS production and the cellular defense system response in 4[Formula: see text]-hydroxywithanolide E-induced cell death requires further verification. The present study suggests that ROS play an important role in 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in which anti-oxidants, such as glutathione or N-acetylcysteine, can resist the 4[Formula: see text]-hydroxywithanolide E-induced accumulation of ROS and cell death. Furthermore, N-acetylcysteine or glutathione can reverse the 4[Formula: see text]-hydroxywithanolide E-induced changes in the cell cycle distribution and the expression of cell cycle regulators. We found that the 4[Formula: see text]-hydroxywithanolide E-induced ROS accumulation was correlated with the upregulation of Nrf2 and Nrf2-downstream genes, such as antioxidative defense enzymes. In general, the activity of Nrf2 is regulated by the Ras signalling pathway. However, we demonstrated that Nrf2 was activated during 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in spite of the 4[Formula: see text]-hydroxywithanolide E-induced inhibition of the Ras/Raf/ERK pathway. The activity and protein expression of superoxide dismutase and catalase were involved in the 4[Formula: see text]-hydroxywithanolide E-induced ROS production in MCF-7 cells. Furthermore, 4[Formula: see text]-hydroxywithanolide E was demonstrated to significantly reduce the sizes of the tumor nodules in the human breast cancer MDA-MB231 xenograft tumor model.

Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells.

PubMed

Zagoura, Dimitra; Canovas-Jorda, David; Pistollato, Francesca; Bremer-Hoffmann, Susanne; Bal-Price, Anna

2017-06-01

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH + ) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Pharmacokinetics of dietary cancer chemopreventive compound dibenzoylmethane in rats and the impact of nanoemulsion and genetic knockout of Nrf2 on its disposition.

PubMed

Lin, Wen; Hong, Jin-Liern; Shen, Guoxiang; Wu, Rachel T; Wang, Yuwen; Huang, Mou-Tuan; Newmark, Harold L; Huang, Qingrong; Khor, Tin Oo; Heimbach, Tycho; Kong, Ah-Ng

2011-03-01

The pharmacokinetic disposition of a dietary cancer chemopreventive compound dibenzoylmethane (DBM) was studied in male Sprague-Dawley rats after intravenous (i.v.) and oral (p.o.) administrations. Following a single i.v. bolus dose, the mean plasma clearance (CL) of DBM was low compared with the hepatic blood flow. DBM displayed a high volume of distribution (Vss). The elimination terminal t1/2 was long. The mean CL, Vss and AUC0-∞/dose were similar between the i.v. 10 and 10 mg/kg doses. After single oral doses (10, 50 and 250 mg/kg), the absolute oral bioavailability (F*) of DBM was 7.4%-13.6%. The increase in AUC was not proportional to the oral doses, suggesting non-linearity. In silico prediction of oral absorption also demonstrated low DBM absorption in vivo. An oil-in-water nanoemulsion containing DBM was formulated to potentially overcome the low F* due to poor water solubility of DBM, with enhanced oral absorption. Finally, to examine the role of Nrf2 on the pharmacokinetics of DBM, since DBM activates the Nrf2-dependent detoxification pathways, Nrf2 wild-type (+/+) mice and Nrf2 knockout (-/-) mice were utilized. There was an increased systemic plasma exposure of DBM in Nrf2 (-/-) mice, suggesting that the Nrf2 genotype could also play a role in the pharmacokinetic disposition of DBM. Taken together, the results show that DBM has low oral bioavailability which could be due in part to poor water solubility and this could be overcome by a nanotechnology-based drug delivery system and furthermore the Nrf2 genotype could also play a role in the pharmacokinetics of DBM. Copyright © 2010 John Wiley & Sons, Ltd.

A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway.

PubMed

Morimitsu, Yasujiro; Nakagawa, Yoko; Hayashi, Kazuhiro; Fujii, Hiroyuki; Kumagai, Takeshi; Nakamura, Yoshimasa; Osawa, Toshihiko; Horio, Fumihiko; Itoh, Ken; Iida, Katsuyuki; Yamamoto, Masayuki; Uchida, Koji

2002-02-01

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of the protective phase II detoxification enzymes, such as glutathione S-transferase (GST). We have recently developed a cell culture system, using rat liver epithelial RL 34 cells, that potently responds to the phenolic antioxidants resulting in the induction of GST activity (Kawamoto, Y., Nakamura, Y., Naito, Y., Torii, Y., Kumagai, T., Osawa, T., Ohigashi, H., Satoh, K., Imagawa, M., and Uchida, K. (2000) J. Biol. Chem. 275, 11291-11299.) In the present study, we investigated the phase II-inducing potency of an isothiocyanate compound in vitro and in vivo and examined a possible induction mechanism. Based on an extensive screening of vegetable extracts for GST inducer activity in RL34 cells, we found Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane (4-methylsulfinylbutyl isothiocyanate) isolated from broccoli, as the major GST inducer in wasabi. 6-HITC potently induced both class alpha GSTA1 and class pi GSTP1 isozymes in RL34 cells. In animal experiments, we found that 6-MSHI was rapidly absorbed into the body and induced hepatic phase II detoxification enzymes more potently than sulforaphane. The observations that (i) 6-HITC activated the antioxidant response element (ARE), (ii) 6-HITC induced nuclear localization of the transcription factor Nrf2 that binds to ARE, and (iii) the induction of phase II enzyme genes by 6-HITC was completely abrogated in the nrf2-deficient mice, suggest that 6-HITC is a potential activator of the Nrf2/ARE-dependent detoxification pathway.

Expression of Nrf2 Promotes Schwann Cell-Mediated Sciatic Nerve Recovery in Diabetic Peripheral Neuropathy.

PubMed

Tang, Wei; Chen, Xiangfang; Liu, Haoqi; Lv, Qian; Zou, Junjie; Shi, Yongquan; Liu, Zhimin

2018-04-26

High glucose-induced oxidative stress and inflammatory responses play an important role in painful diabetic neuropathy by activating the TLR4/NFκB signal pathway. Schwann cells (SCs) are integral to peripheral nerve biology, contributing to saltatory conduction along axons, nerve and axon development, and axonal regeneration. SCs provide a microenvironment favoring vascular regeneration but their low survival ratio in hyperglycemic conditions suppress the function to promote nerve growth. Nuclear factor erythroid 2-related factor 2 (Nrf2) promotes remyelination after peripheral nerve injury. The aim of this study was to identify the role of Nrf2 in SC-mediated functional recovery after sciatic nerve injury. We compared plasma inflammatory factors in diabetic patients (DN) with/without diabetic peripheral neuropathy (DPN) and assessed whether Nrf2 expression in SCs could repair peripheral nerve injury in a rat model. Nrf2, TLR4/NFκB signal pathway and apoptosis relative protein expression were detected by western blot. Apoptosis and angiogenesis were determined by immunofluorescence and tubule formation assay, respectively. Regenerated nerves were determined by transmission electron microscope. Higher levels of inflammatory factors and VEGF expression were found in DPN patients. Cellular experiments indicate that Nrf2 expression inhibits hyperglycemia-induced apoptosis and promotes angiogenesis by regulating the TLR4/NFκB signal pathway. Animal experiments show that nerve conduction velocity, myelin sheath thickness, and sciatic vasa nervorum are restored with transplantation of SCs overexpressing Nrf2. Taken together, the high survival ratio of SCs in a DPN rat model indicates that overexpression of Nrf2 restores nerve injury. © 2018 The Author(s). Published by S. Karger AG, Basel.

Rosmarinic acid counteracts activation of hepatic stellate cells via inhibiting the ROS-dependent MMP-2 activity: Involvement of Nrf2 antioxidant system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Changfang; Zou, Yu; Liu, Yuzhang

Recently, oxidative stress is involved in hepatofibrogenesis. Matrix metalloproteinase-2 (MMP-2) is required for activation of hepatic stellate cells (HSCs) in response to reactive oxygen species (ROS). This study was designed to explore the hypothesis that the inhibitory effect of rosmarinic acid (RA) on HSCs activation might mainly result from its antioxidant capability by increasing the synthesis of glutathione (GSH) involved in nuclear factor kappa B (NF-κB)-dependent inhibition of MMP-2 activity. Here, we demonstrate that RA reverses activated HSCs to quiescent cells. Concomitantly, RA inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished down-regulation of MMP-2 by RA. RA-mediated inactivation ofmore » NF-κB could be blocked by the diphenyleneiodonium chloride (DPI; a ROS inhibitor). Conversely, transfection of dominant-negative (DN) mutant of extracellular signal-regulated kinases 2 (ERK2), c-Jun N-terminal kinase 1 (JNK1), or p38α kinase had no such effect. Simultaneously, RA suppresses ROS generation and lipid peroxidation (LPO) whereas increases cellular GSH in HSC-T6 cells. Furthermore, RA significantly increased antioxidant response element (ARE)-mediated luciferase activity, nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and catalytic subunits from glutamate cysteine ligase (GCLc) expression, but not modulatory subunits from GCL (GCLm). RA-mediated up-regulation of GClc is inhibited by the shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 or buthionine sulfoximine (a GCL inhibitor) abolished RA-mediated inhibition of ROS. Collectively, these results provide novel insights into the mechanisms of RA as an antifibrogenic candidate in the prevention and treatment of liver fibrosis. - Highlights: • RA reverses activated HSCs to quiescent cells. • RA suppresses MMP-2 activity through a NF-κB-dependent pathway. • Inhibition of oxidative stress by RA is dependent on nuclear translocation of Nrf2. • RA-mediated down-regulation of MMP-2 was ROS-dependent.« less

Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

PubMed

Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

2012-05-25

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

NRF2, cancer and calorie restriction.

PubMed

Martín-Montalvo, A; Villalba, J M; Navas, P; de Cabo, R

2011-02-03

The transcription factor NF-E2-related factor (NRF2) is a key regulator of several enzymatic pathways, including cytoprotective enzymes in highly metabolic organs. In this review, we summarize the ongoing research related to NRF2 activity in cancer development, focusing on in vivo studies using NRF2 knockout (KO) mice, which have helped in defining the crucial role of NRF2 in chemoprevention. The lower cancer protection observed in NRF2 KO mice under calorie restriction (CR) suggests that most of the beneficial effects of CR on the carcinogenesis process are likely mediated by NRF2. We propose that future interventions in cancer treatment would be carried out through the activation of NRF2 in somatic cells, which will lead to a delay or prevention of the onset of some forms of human cancers, and subsequently an extension of health- and lifespan.

Nrf2: bane or blessing in cancer?

PubMed

Xiang, MingJun; Namani, Akhileshwar; Wu, ShiJun; Wang, XiaoLi

2014-08-01

The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor-E2-related factor 2 (Nrf2)-antioxidant response element pathway serves a major function in endogenous cytoprotection in normal cells. Nrf2 is a transcription factor that mainly regulates the expression of a wide array of genes that produce the antioxidants and other proteins responsible for the detoxification of xenobiotics and reactive oxygen species. Nrf2 mediates the chemoprevention of cancer in normal cells. Growing body of evidence suggests that Nrf2 is not only involved in the chemoprevention of normal cells but also promotes the growth of cancer cells. However, the mechanism underlying the function of Nrf2 in oncogenesis and tumor protection in cancer cells remains unclear and thus requires further study. This review aims to rationalize the existing functions of Nrf2 in chemoprevention and tumorigenesis, as well as the somatic mutations of Nrf2 and Keap1 in cancer and Nrf2 cross talk with miRNAs. This review also discusses the future challenges in Nrf2 research.

Mangiferin Mitigates Gastric Ulcer in Ischemia/ Reperfused Rats: Involvement of PPAR-γ, NF-κB and Nrf2/HO-1 Signaling Pathways

PubMed Central

Mahmoud-Awny, Magdy; Attia, Ahmed S.; Abd-Ellah, Mohamed F.; El-Abhar, Hanan Salah

2015-01-01

Mangiferin (MF), a xanthonoid from Mangifera indica, has been proved to have antisecretory and antioxidant gastroprotective effects against different gastric ulcer models; however, its molecular mechanism has not been previously elucidated. Therefore, the aim of this study was to test its modulatory effect on several signaling pathways using the ischemia/reperfusion model for the first time. Animals were treated with MF, omeprazole (OMP), and the vehicle. The mechanistic studies revealed that MF mediated its gastroprotective effect partly via inducing the expression of Nrf2, HO-1 and PPAR-γ along with downregulating that of NF-κB. Surprisingly, the effect of MF, especially the high dose, exceeded that mediated by OMP except for Nrf2. The molecular results were reflected on the biomarkers measured, where the antioxidant effect of MF was manifested by increasing total antioxidant capacity and glutathione, besides normalizing malondialdehyde level. Additionally, MF decreased the I/R-induced nitric oxide elevation, an effect that was better than that of OMP. In the serum, MF, dose dependently, enhanced endothelial nitric oxide synthase, while reduced the inducible isoform. Regarding the anti-inflammatory effect of MF, it reduced serum level of IL-1β and sE-selectin, effects that were mirrored on the tissue level of myeloperoxidase, the neutrophil infiltration marker. In addition, MF possessed an antiapoptotic character evidenced by elevating Bcl-2 level and reducing that of caspase-3 in a dose related order. As a conclusion, the intimated gastroprotective mechanisms of MF are mediated, partially, by modulation of oxidative stress, inflammation and apoptosis possibly via the Nrf2/HO-1, PPAR-γ/NF-κB signaling pathways. PMID:26196679

Induction of Lung GSH and Glutamate Cysteine Ligase by 1,4-phenylenebis(methylene)selenocyanate and its Glutathione Conjugate: Role of Nuclear factor-erythroid 2-Related Factor 2

PubMed Central

Emmert, Sans W.; El-Bayoumy, Karam; Das, Arunangshu; Sun, Yuan-Wan; Amin, Shantu; Desai, Dhimant; Aliaga, Cesar; Richie, John P.

2012-01-01

The synthetic organoselenium agent 1,4- phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG), are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamate cysteine ligase (GCL), through activation of antioxidant response elements (ARE) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular related kinase (ERK) (220%) and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), while the naturally-occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE reporter cells up to 3-fold for p-XSC and ≥5-fold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2 (siNrf2). The dependence of GCL induction on Nrf2 was confirmed in Nrf2 deficient mouse embryonic fibroblasts (MEF) where p-XSeSG induced GCL subunits in wildtype, but not Nrf2 deficient cells (p<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo, and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious inducer of GSH. PMID:22542796

Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.

PubMed

Zagorski, Joseph W; Maser, Tyler P; Liby, Karen T; Rockwell, Cheryl E

2017-05-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert -butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NF κ B activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NF κ B activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NF κ B activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NF κ B and Nrf2. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Regeneration of glutathione by α-lipoic acid via Nrf2/ARE signaling pathway alleviates cadmium-induced HepG2 cell toxicity.

PubMed

Zhang, Jiayu; Zhou, Xue; Wu, Wenbo; Wang, Jiachun; Xie, Hong; Wu, Zhigang

2017-04-01

Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). However, the underlying molecular mechanism by which α-LA regenerates GSH remains poorly understood. The current study aimed to investigate whether α-LA regenerates GSH by activation of Nrf2 to alleviate cadmium-induced cytotoxicity in HepG2 cells. In the present study, we found that cadmium induced cell death by depletion of GSH through inactivation of Nrf2. Addition of α-LA to cadmium-treated cells reactivated Nrf2 and regenerated GSH through elevating the Nrf2-downstream genes γ-glutamate-cysteine ligase (γ-GCL) and GR, both of which are key enzymes for GSH synthesis. However, blocking Nrf2 with brusatol in the cells co-treated with α-LA and cadmium reduced the mRNA and the protein levels of γ-GCL and GR, thus suppressed GSH regeneration by α-LA. Our results indicated that α-LA activated Nrf2 signaling pathway, which upregulated the transcription of the enzymes for GSH synthesis and therefore GSH contents to alleviate cadmium-induced cytotoxicity in HepG2 cells. Copyright © 2017. Published by Elsevier B.V.

Hydrogen peroxide prevents vascular calcification induced ROS production by regulating Nrf-2 pathway.

PubMed

Zhang, Wensong; Li, Yi; Ding, Hanlu; Du, Yaqin; Wang, Li

2016-08-01

Although vascular calcification in end-stage renal disease (ESRD) represents a ubiquitous human health problem, effective therapies with limited side effects are still lacking, and the precise mechanisms are not fully understood. The Nrf-2/ARE pathway is a pivotal to regulate anti-oxidative responses in vascular calcification upon ESRD. Although Nrf-2 plays a crucial role in atherosclerosis, pulmonary fibrosis, and brain ischemia, the effect of Nrf-2 and oxidative stress on vascular calcification in ESRD patients is still unclear. The aim of this research was to study the protective role of hydrogen peroxide in vascular calcification and the mechanism of Nrf-2 and oxidative stress on vascular calcification. Here we used the rat vascular smooth muscle cell model of β-glycerophosphate-induced calcification resembling vascular calcification in ESRD to investigate the therapeutic effect of 0.01 mM hydrogen peroxide on vascular calcification and further explores the possible underlying mechanisms. Our current report shows the in vitro role of 0.01 mM hydrogen peroxide in protecting against intracellular ROS accumulation upon vascular calcification. Both hydrogen peroxide and sulforaphane pretreatment reduced ROS production, increased the expression of Nrf-2, and decreased the expression of Runx2 following calcification. Our study demonstrates that 0.01 mM hydrogen peroxide can effectively protect rat aortic vascular smooth muscle cells against oxidative stress by preventing vascular calcification induced ROS production through Nrf-2 pathway. These data might define an antioxidant role of hydrogen peroxide in vascular calcification upon ESRD.

Mangiferin alleviates lipopolysaccharide and D-galactosamine-induced acute liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

PubMed

Pan, Chen-wei; Pan, Zhen-zhen; Hu, Jian-jian; Chen, Wei-lai; Zhou, Guang-yao; Lin, Wei; Jin, Ling-xiang; Xu, Chang-long

2016-01-05

Mangiferin, a glucosylxanthone from Mangifera indica, has been reported to have anti-inflammatory effects. However, the protective effects and mechanisms of mangiferin on liver injury remain unclear. This study aimed to determine the protective effects and mechanisms of mangiferin on lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute liver injury. Mangiferin was given 1h after LPS and D-GalN treatment. The results showed that mangiferin inhibited the levels of serum ALT, AST, IL-1β, TNF-α, MCP-1, and RANTES, as well as hepatic malondialdehyde (MDA) and ROS levels. Moreover, mangiferin significantly inhibited IL-1β and TNF-α production in LPS-stimulated primary hepatocytes. Mangiferin was found to up-regulate the expression of Nrf2 and HO-1 in a dose-dependent manner. Furthermore, mangiferin inhibited LPS/d-GalN-induced hepatic NLRP3, ASC, caspase-1, IL-1β and TNF-α expression. In conclusion, mangiferin protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. Copyright © 2015 Elsevier B.V. All rights reserved.

A prenylated flavonoid, 10-oxomornigrol F, exhibits anti-inflammatory effects by activating the Nrf2/heme oxygenase-1 pathway in macrophage cells.

PubMed

Tran, Phi-Long; Tran, Phuong Thao; Tran, Huynh Nguyen Khanh; Lee, Suhyun; Kim, Okwha; Min, Buyng-Sun; Lee, Jeong-Hyung

2018-02-01

Prenylated flavonoids are a unique class of naturally occurring flavonoids that have various pharmacological activities. In the present study, we investigated the anti-inflammatory effect in murine macrophages of a prenylated flavonoid, 10-oxomornigrol F (OMF), which was isolated from the twigs of Morus alba (Moraceae). OMF inhibited the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in RAW264.7 cells, as well as in mouse bone marrow-derived macrophages (BMMs). OMF also rescued LPS-induced septic mortality in ICR mice. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 was also significantly suppressed by OMF treatment in RAW264.7 cells. Treatment of RAW264.7 cells with OMF induced heme oxygenase (HO)-1 mRNA and protein expression and increased the nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1). Treatment of RAW264.7 cells with OMF increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK); co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked this OMF-induced p38 MAPK phosphorylation. Moreover, NAC, or SB203580 (a p38 MAPK inhibitor), blocked the OMF-induced nuclear translocation of Nrf2 and HO-1 expression, suggesting that OMF induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of OMF in LPS-stimulated RAW264.7 cells. Taken together, these findings suggest that OMF exerts its anti-inflammatory effect by activating the Nrf2/HO-1 pathway, and may be a potential Nrf2 activator to prevent or treat inflammatory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of Mastin® on expression of Nrf2 in the rat heart with subtotally nephrectomy chronic Kidney disease model

NASA Astrophysics Data System (ADS)

Nathania, J.; Soetikno, V.

2017-08-01

Chronic kidney disease (CKD) is increasingly prevalent in Indonesia and worldwide. One of the major causes of morbidity and mortality in CKD is the complication of cardiovascular disease. Mastin® is a supplement that is locally produced in Indonesia and is made from extract of mangosteen pericarp, which is reported to have antioxidative, anti-inflammatory, and antitumor properties. The present study aimed to investigate whether Mastin® could improve antioxidant responses in the rat heart during CKD by measuring the expression of nuclear factor erythroid-2-related factor (Nrf)2, a master regulator of antioxidant response elements. RNA was extracted from the heart tissue of three groups of rats: a normal group, a nephrectomy group, and a nephrectomy with Mastin® group. Two-step real-time RT-PCR was then conducted to calculate the relative expression of the Nrf2 gene. Nrf2 expression was markedly decreased in the nephrectomy group vs the normal group, but slightly increas ed in the nephrectomy with Mastin® group vs the nephrectomy group. CKD resulted in impaired activation of the Nrf2 pathway in the rat heart. Although the administration of Mastin® slightly increased Nrf2 expression, it was not enough to confer cardioprotective effects through the Nrf2 pathway.

Sulforaphane Ameliorates Bladder Dysfunction through Activation of the Nrf2-ARE Pathway in a Rat Model of Partial Bladder Outlet Obstruction

PubMed Central

Liu, Chong; Xu, Huan; Fu, Shi; Chen, Yanbo; Chen, Qi; Cai, Zhikang; Zhou, Juan; Wang, Zhong

2016-01-01

Purpose. We evaluated the effect of sulforaphane (SFN) treatment on the function and changes of expression of Nrf2-ARE pathway in the bladder of rats with bladder outlet obstruction (BOO). Materials and Methods. A total of 18 male Sprague-Dawley rats at age of 8 weeks were divided into 3 groups (6 of each): the sham operated group, the BOO group, and the BOO+SFN group. We examined histological alterations and the changes of oxidative stress markers and the protein expression of the Nrf2-ARE pathway. Results. We found that SFN treatment could prolong micturition interval and increase bladder capacity and bladder compliance. However, the peak voiding pressure was lower than BOO group. SFN treatment can ameliorate the increase of collagen fibers induced by obstruction. SFN treatment also increased the activity of SOD, GSH-Px, and CAT compared to the other groups. The level of bladder cell apoptosis was decreased in BOO rats with SFN treatment. Moreover, SFN could reduce the ratio of Bax/Bcl-2 expression. Furthermore, SFN could activate the Nrf2 expression with elevation of its target antioxidant proteins. Conclusions. The sulforaphane-mediated decrease of oxidative stress and activation of the Nrf2-ARE pathway may ameliorate bladder dysfunction caused by bladder outlet obstruction. PMID:27433291

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

PubMed

Zakharova, E T; Sokolov, A V; Pavlichenko, N N; Kostevich, V A; Abdurasulova, I N; Chechushkov, A V; Voynova, I V; Elizarova, A Yu; Kolmakov, N N; Bass, M G; Semak, I V; Budevich, A I; Kozhin, P M; Zenkov, N K; Klimenko, V M; Kirik, O V; Korzhevskii, D E; Menshchikova, E B; Vasilyev, V B

2018-05-10

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

Hydrogen sulphide induces HIF-1α and Nrf2 in THP-1 macrophages.

PubMed

Lohninger, Lilian; Tomasova, Lenka; Praschberger, Monika; Hintersteininger, Michael; Erker, Thomas; Gmeiner, Bernhard M K; Laggner, Hilde

2015-05-01

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Serum Oxidative Stress-Induced Repression of Nrf2 and GSH Depletion: A Mechanism Potentially Involved in Endothelial Dysfunction of Young Smokers

PubMed Central

Fratta Pasini, Anna; Albiero, Anna; Stranieri, Chiara; Cominacini, Mattia; Pasini, Andrea; Mozzini, Chiara; Vallerio, Paola; Cominacini, Luciano; Garbin, Ulisse

2012-01-01

Background Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs). Methods and Results 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers' serum but not to non-smokers' serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamate-cysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers' serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs. Conclusions In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion. PMID:22272327

Protective effect of forsythiaside A on OVA-induced asthma in mice.

PubMed

Qian, Jin; Ma, Xiaorong; Xun, Yali; Pan, Lei

2017-10-05

Forsythiaside A (FSA), an active constituent isolated from the Chinese medicinal herb Forsythia suspensa, has been known to have anti-inflammatory effect. However, the effect of FSA on allergic airway inflammation remains unclear. The aim of this study was to investigate the effect of FSA on OVA-induced asthma in mice. Mice model of asthma was induced by OVA. OVA-induced airway hyperresponsiveness (AHR) and inflammatory cells in BALF were detected. The production of IgE, IL-4, IL-5, IFN-γ, and IL-13 were detected by ELISA. The effects of FSA on Nrf2 and NF-κB signaling pathways were detected by western blot analysis. The results showed that treatment of FSA significantly attenuated OVA-induced lung histopathological changes. FSA inhibited OVA-induced AHR and inflammatory cells in BALF. OVA-induced IgE, IL-4, IL-5, and IL-13 production were also inhibited by FSA. Western blot analysis showed that treatment of FSA inhibited OVA-induced NF-κB activation. Treatment of FSA dose-dependently up-regulated the expression of Nrf2 and HO-1. In addition, we found that FSA up-regulated the expression of Nrf2 and HO-1 both in A549 cells and MS-H cells. Taken together, FSA suppressed inflammatory responses in OVA-induced asthma through activating Nrf2/HO-1 signaling pathway. FSA may be a promising potential preventive agent for asthma treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrogen Peroxide responsive miR153 targets Nrf2/ARE cytoprotection in paraquat induced dopaminergic neurotoxicitya

PubMed Central

Narasimhan, Madhusudhanan; Riar, Amanjot Kaur; Rathinam, Mary Latha; Vedpathak, Dhanashree; Henderson, George; Mahimainathan, Lenin

2014-01-01

Epidemiological and animal studies suggest that environmental toxins including paraquat (PQ) increase the risk of developing Parkinson's disease (PD) by damaging nigrostriatal dopaminergic neurons. We previously showed that overexpression of a group of microRNAs (miRs) affects the antioxidant promoting factor, Nrf2 and related glutathione-redox homeostasis in SH-SY5Y dopaminergic neurons. Although, dysregulation of redox balance by PQ is well documented, the role for miRs and their impact have not been elucidated. In the current study we investigated whether PQ impairs Nrf2 and its related cytoprotective machinery by misexpression of specific fine tune miRs in SH-SY5Y neurons. Real time PCR analysis revealed that PQ significantly (p<0.05) increased the expression of brain enriched miR153 with an associated decrease in Nrf2 and its function as revealed by decrease in 4× ARE activity and expression of GCLC and NQO1. Also, PQ and H2O2-induced decrease in Nrf2 3′ UTR activity was restored on miR153 site mutation suggesting a 3′ UTR interacting role. Overexpression of either anti-miR153 or Nrf2 cDNA devoid of 3′ UTR prevented PQ and H2O2-induced loss in Nrf2 activity confirming that PQ could cause miR153 to bind to and target Nrf2 3′ UTR thereby weakening the cellular antioxidant defense. Adenovirus mediated overexpression of cytoplasmic catalase (Ad cCAT) confirmed that PQ induced miR153 is hydrogen peroxide (H2O2) dependent. In addition, Ad cCAT significantly (p<0.05) negated the PQ induced dysregulation of Nrf2 and function along with minimizing ROS, caspase 3/7 activation and neuronal death. Altogether, these results suggest a critical role for oxidant mediated miR153-Nrf2/ARE pathway interaction in paraquat neurotoxicity. This novel finding facilitates the understanding of molecular mechanisms and to develop appropriate management alternatives to counteract PQ-induced neuronal pathogenesis. PMID:24866057

Sulforaphane induces neurovascular protection against a systemic inflammatory challenge via both Nrf2-dependent and independent pathways.

PubMed

Holloway, Paul M; Gillespie, Scarlett; Becker, Felix; Vital, Shantel A; Nguyen, Victoria; Alexander, J Steven; Evans, Paul C; Gavins, Felicity N E

2016-10-01

Sepsis is often characterized by an acute brain inflammation and dysfunction, which is associated with increased morbidity and mortality worldwide. Preventing cerebral leukocyte recruitment may provide the key to halt progression of systemic inflammation to the brain. Here we investigated the influence of the anti-inflammatory and anti-oxidant compound, sulforaphane (SFN) on lipopolysaccharide (LPS)-induced cellular interactions in the brain. The inflammatory response elicited by LPS was blunted by SFN administration (5 and 50mg/kg i.p.) 24h prior to LPS treatment in WT animals, as visualized and quantified using intravital microscopy. This protective effect of SFN was lost in Nrf2-KO mice at the lower dose tested, however 50mg/kg SFN revealed a partial effect, suggesting SFN works in part independently of Nrf2 activity. In vitro, SFN reduced neutrophil recruitment to human brain endothelial cells via a down regulation of E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Our data confirm a fundamental dose-dependent role of SFN in limiting cerebral inflammation. Furthermore, our data demonstrate that not only is Nrf2 in part essential in mediating these neuroprotective effects, but they occur via down-regulation of E-selectin and VCAM-1. In conclusion, SFN may provide a useful therapeutic drug to reduce cerebral inflammation in sepsis. Copyright © 2016 Elsevier Inc. All rights reserved.

Schisantherin A Attenuates Neuroinflammation in Activated Microglia: Role of Nrf2 Activation Through ERK Phosphorylation.

PubMed

Li, Chuwen; Chen, Tongkai; Zhou, Hefeng; Zhang, Chao; Feng, Yu; Tang, Fan; Hoi, Maggie Pui-Man; He, Chengwei; Zheng, Ying; Lee, Simon Ming-Yuen

2018-06-28

In the present study, we investigated whether schisantherin A (StA) had anti-inflammatory effects under neuroinflammatory conditions. The effects of StA and its underlying mechanisms were examined in lipopolysaccharide (LPS)-activated BV-2 microglial cells by ELISA, qPCR, EMSA, Western blot, and IHC. Firstly, we found that StA inhibited the inflammatory response in LPS-activated BV-2 microglia. Secondly, we found that StA suppressed LPS-induced activation of NF-κB via interfering with degradation of IκB and phosphorylation of IκB, IKK, PI3K/Akt, JNK, and p38 MAPK. Thirdly, StA conferred indirect antioxidative effects via quenching ROS and promoted expression of antioxidant enzymes, including HO-1 and NQO-1, via stimulating activation of Nrf2 pathways. Finally, we demonstrated that anti-neuroinflammatory actions of StA were dependent on ERK phosphorylation-mediated Nrf2 activation. StA induced ERK phosphorylation-mediated Nrf2 activation, which contributed to its anti-inflammation and anti-oxidation. The anti-neuroinflammatory and anti-oxidative effects of StA may show preventive therapeutic potential for various neuroinflammatory disorders. © 2018 The Author(s). Published by S. Karger AG, Basel.

Taxifolin Activates the Nrf2 Anti-Oxidative Stress Pathway in Mouse Skin Epidermal JB6 P+ Cells through Epigenetic Modifications

PubMed Central

Kuang, Haixue; Tang, Zhenqiu; Zhang, Chengyue; Wang, Zhibin; Li, Wenji; Yang, Chunjuan; Wang, Qiuhong; Yang, Bingyou; Kong, Ah-Ng

2017-01-01

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a vital transcription factor that regulates the anti-oxidative defense system. Previous reports suggested that the expression of the Nrf2 gene can be regulated by epigenetic modifications. The potential epigenetic effect of taxifolin (TAX), a potent cancer chemopreventive agent, in skin cancer chemoprotection is unknown. In this study, we investigated how Nrf2 is epigenetically regulated by TAX in JB6 P+ cells. TAX was found to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced colony formation of JB6 P+ cells. TAX induced antioxidant response element (ARE)-luciferase activity in HepG2-C8 cells and up-regulated mRNA and protein levels of Nrf2 and its downstream genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), in JB6 P+ cells. Furthermore, bisulfite genomic sequencing revealed that TAX treatment reduces the methylation level of the first 15 CpGs sites in the Nrf2 promoter. Western blotting showed that TAX inhibits the expression levels of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) proteins. In summary, our results revealed that TAX can induce expression of Nrf2 and its downstream target genes in JB6 P+ cells by CpG demethylation. These finding suggest that TAX may exhibit a skin cancer preventive effect by activating Nrf2 via an epigenetic pathway. PMID:28714938

Curcumin ameliorates dopaminergic neuronal oxidative damage via activation of the Akt/Nrf2 pathway.

PubMed

Cui, Qunli; Li, Xin; Zhu, Hongcan

2016-02-01

Parkinson's disease (PD) is an age-related complex neurodegenerative disease that affects ≤ 80% of dopaminergic neurons in the substantia nigra pars compacta (SNpc). It has previously been suggested that mitochondrial dysfunction, oxidative stress and oxidative damage underlie the pathogenesis of PD. Curcumin, which is a major active polyphenol component extracted from the rhizomes of Curcuma longa (Zingiberaceae), has been reported to exert neuroprotective effects on an experimental model of PD. The present study conducted a series of in vivo experiments, in order to investigate the effects of curcumin on behavioral deficits, oxidative damage and related mechanisms. The results demonstrated that curcumin was able to significantly alleviate motor dysfunction and increase suppressed tyrosine hydroxylase (TH) activity in the SNpc of rotenone (ROT)-injured rats. Biochemical measurements indicated that rats pretreated with curcumin exhibited increased glutathione (GSH) levels, and reduced reactive oxygen species activity and malondialdehyde content. Mechanistic studies demonstrated that curcumin significantly restored the expression levels of heme oxygenase-1 and quinone oxidoreductase 1, thus ameliorating ROT-induced damage in vivo, via the phosphorylation of Akt and nuclear factor erythroid 2-related factor 2 (Nrf2). Further studies indicated that the Akt/Nrf2 signaling pathway was associated with the protective role of curcumin in ROT-treated rats. Inhibiting the Akt/Nrf2 pathway using a lentiviral vector containing Nrf2-specific short hairpin RNA, or the phosphoinositide 3-kinase inhibitor LY294002, markedly reduced the expression levels of TH and GSH, ultimately attenuating the neuroprotective effects of curcumin against oxidative damage. These results indicated that curcumin was able to significantly ameliorate ROT-induced dopaminergic neuronal oxidative damage in the SNpc of rats via activation of the Akt/Nrf2 signaling pathway.

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusunoki, Chisato, E-mail: yosizaki@belle.shiga-med.ac.jp; Yang, Liu; Yoshizaki, Takeshi

Highlights: Black-Right-Pointing-Pointer Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. Black-Right-Pointing-Pointer EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. Black-Right-Pointing-Pointer Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid ({omega}3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of {omega}3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treatedmore » with {omega}3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). {omega}3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with {omega}3-PUFA prevented H{sub 2}O{sub 2}-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.« less

Emodin Inhibition of Influenza A Virus Replication and Influenza Viral Pneumonia via the Nrf2, TLR4, p38/JNK and NF-kappaB Pathways.

PubMed

Dai, Jian-Ping; Wang, Qian-Wen; Su, Yun; Gu, Li-Ming; Zhao, Ying; Chen, Xiao-Xua; Chen, Cheng; Li, Wei-Zhong; Wang, Ge-Fei; Li, Kang-Sheng

2017-10-18

Lasting activations of toll-like receptors (TLRs), MAPK and NF-κB pathways can support influenza A virus (IAV) infection and promote pneumonia. In this study, we have investigated the effect and mechanism of action of emodin on IAV infection using qRT-PCR, western blotting, ELISA, Nrf2 luciferase reporter, siRNA and plaque inhibition assays. The results showed that emodin could significantly inhibit IAV (ST169, H1N1) replication, reduce IAV-induced expressions of TLR2/3/4/7, MyD88 and TRAF6, decrease IAV-induced phosphorylations of p38/JNK MAPK and nuclear translocation of NF-κB p65. Emodin also activated the Nrf2 pathway, decreased ROS levels, increased GSH levelss and GSH/GSSG ratio, and upregulated the activities of SOD, GR, CAT and GSH-Px after IAV infection. Suppression of Nrf2 via siRNA markedly blocked the inhibitory effects of emodin on IAV-induced activations of TLR4, p38/JNK, and NF-κB pathways and on IAV-induced production of IL-1β, IL-6 and expression of IAV M2 protein. Emodin also dramatically increased the survival rate of mice, reduced lung edema, pulmonary viral titer and inflammatory cytokines, and improved lung histopathological changes. In conclusion, emodin can inhibit IAV replication and influenza viral pneumonia, at least in part, by activating Nrf2 signaling and inhibiting IAV-induced activations of the TLR4, p38/JNK MAPK and NF-κB pathways.

5-HMF attenuates striatum oxidative damage via Nrf2/ARE signaling pathway following transient global cerebral ischemia.

PubMed

Ya, Bai-Liu; Li, Hong-Fang; Wang, Hai-Ying; Wu, Fei; Xin, Qing; Cheng, Hong-Ju; Li, Wen-Juan; Lin, Na; Ba, Zai-Hua; Zhang, Ru-Juan; Liu, Qian; Li, Ya-Nan; Bai, Bo; Ge, Feng

2017-01-01

Recent studies have shown 5-hydroxymethyl-2-furfural (5-HMF) has favorable biological effects, and its neuroprotection in a variety of neurological diseases has been noted. Our previous study showed that treatment of 5-HMF led to protection against permanent global cerebral ischemia. However, the underlying mechanisms in cerebral ischemic injury are not fully understood. This study was conducted to investigate the neuroprotective effect of 5-HMF and elucidate the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway mechanism in the striatum after transient global cerebral ischemia. C57BL/6 mice were subjected to bilateral common carotid artery occlusion for 20 min and sacrificed 24 h after reperfusion. 5-HMF (12 mg/kg) or an equal volume of vehicle was intraperitoneally injected 30 min before ischemia and 5 min after the onset of reperfusion. At 24 h after reperfusion, neurological function was evaluated by neurological disability status scale, locomotor activity test and inclined beam walking test. Histological injury of the striatum was observed by cresyl violet staining and terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) staining. Oxidative stress was evaluated by the carbonyl groups introduced into proteins, and malondialdehyde (MDA) levels. An enzyme-linked immunosorbent assay (ELISA)-based measurement was used to detect Nrf2 DNA binding activity. Nrf2 and its downstream ARE pathway protein expression such as heme oxygenase-1, NAD (P)H:quinone oxidoreductase 1, glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modulatory subunit were detected by western blot. Our results showed that 5-HMF treatment significantly ameliorated neurological deficits, reduced brain water content, attenuated striatum neuronal damage, decreased the carbonyl groups and MDA levels, and activated Nrf2/ARE signaling pathway. Taken together, these results demonstrated that 5-HMF exerted significant antioxidant and neuroprotective effects following transient cerebral ischemia, possibly through the activation of the Nrf2/ARE signaling pathway.

Keap1-knockdown decreases fasting-induced fatty liver via altered lipid metabolism and decreased fatty acid mobilization from adipose tissue.

PubMed

Xu, Jialin; Donepudi, Ajay C; Moscovitz, Jamie E; Slitt, Angela L

2013-01-01

The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting). Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters--CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle. Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.

Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

PubMed Central

Xu, Jialin; Donepudi, Ajay C.; Moscovitz, Jamie E.; Slitt, Angela L.

2013-01-01

Aims The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting). Methods and Results Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle. Conclusion Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation. PMID:24224011

Ginsenoside Rb1 Treatment Attenuates Pulmonary Inflammatory Cytokine Release and Tissue Injury following Intestinal Ischemia Reperfusion Injury in Mice

PubMed Central

Zhou, Zhen; Meng, Qing-tao; Sun, Qian; Su, Wating; Xia, Zhengyuan; Xia, Zhong-yuan

2015-01-01

Objective. Intestinal ischemia reperfusion (II/R) injury plays a critical role in remote organ dysfunction, such as lung injury, which is associated with nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. In the present study, we tested whether ginsenoside Rb1 attenuated II/R induced lung injury by Nrf2/HO-1 pathway. Methods. II/R injury was induced in male C57BL/6J mice by 45 min of superior mesenteric artery (SMA) occlusion followed by 2 hours of reperfusion. Ginsenoside Rb1 was administrated prior to reperfusion with or without ATRA (all-transretinoic acid, the inhibitor of Nrf2/ARE signaling pathway) administration before II/R. Results. II/R induced lung histological injury, which is accompanied with increased levels of malondialdehyde (MDA), interleukin- (IL-) 6, and tumor necrosis factor- (TNF-) α but decreased levels of superoxide dismutase (SOD) and IL-10 in the lung tissues. Ginsenoside Rb1 reduced lung histological injury and the levels of TNF-α and MDA, as well as wet/dry weight ratio. Interestingly, the increased Nrf2 and HO-1 expression induced by II/R in the lung tissues was promoted by ginsenoside Rb1 treatment. All these changes could be inhibited or prevented by ATRA. Conclusion. Ginsenoside Rb1 is capable of ameliorating II/R induced lung injuries by activating Nrf2/HO-1 pathway. PMID:26161243

Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster.

PubMed

Nagy, Péter; Varga, Agnes; Pircs, Karolina; Hegedűs, Krisztina; Juhász, Gábor

2013-01-01

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.

Nrf2-dependent suppression of azoxymethane/dextran sulfate sodium-induced colon carcinogenesis by the cinnamon-derived dietary factor cinnamaldehyde.

PubMed

Long, Min; Tao, Shasha; Rojo de la Vega, Montserrat; Jiang, Tao; Wen, Qing; Park, Sophia L; Zhang, Donna D; Wondrak, Georg T

2015-05-01

The progressive nature of colorectal cancer and poor prognosis associated with the metastatic phase of the disease create an urgent need for the development of more efficacious strategies targeting colorectal carcinogenesis. Cumulative evidence suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defence, represents a promising molecular target for colorectal cancer chemoprevention. Recently, we have identified cinnamon, the ground bark of Cinnamomum aromaticum (cassia cinnamon) and Cinnamomum verum (Ceylon cinnamon), as a rich dietary source of the Nrf2 inducer cinnamaldehyde (CA) eliciting the Nrf2-regulated antioxidant response in human epithelial colon cells, conferring cytoprotection against electrophilic and genotoxic insult. Here, we have explored the molecular mechanism underlying CA-induced Nrf2 activation in colorectal epithelial cells and have examined the chemopreventive potential of CA in a murine colorectal cancer model comparing Nrf2(+/+) with Nrf2(-/-) mice. In HCT116 cells, CA caused a Keap1-C151-dependent increase in Nrf2 protein half-life via blockage of ubiquitination with upregulation of cytoprotective Nrf2 target genes and elevation of cellular glutathione. After optimizing colorectal Nrf2 activation and target gene expression by dietary CA-supplementation regimens, we demonstrated that CA suppresses AOM/DSS-induced inflammatory colon carcinogenesis with modulation of molecular markers of colorectal carcinogenesis. Dietary suppression of colorectal cancer using CA supplementation was achieved in Nrf2(+/+) but not in Nrf2(-/-) mice confirming the Nrf2 dependence of CA-induced chemopreventive effects. Taken together, our data suggest feasibility of colorectal cancer suppression by dietary CA, an FDA-approved food additive derived from the third most consumed spice in the world. ©2015 American Association for Cancer Research.

Systemic administration of the apocarotenoid bixin protects skin against solar UV-induced damage through activation of NRF2.

PubMed

Tao, Shasha; Park, Sophia L; Rojo de la Vega, Montserrat; Zhang, Donna D; Wondrak, Georg T

2015-12-01

Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and an urgent need exists for improved molecular photoprotective strategies different from (or synergistic with) photon absorption. Recent studies suggest a photoprotective role of cutaneous gene expression orchestrated by the transcription factor NRF2 (nuclear factor-E2-related factor 2). Here we have explored the molecular mechanism underlying carotenoid-based systemic skin photoprotection in SKH-1 mice and provide genetic evidence that photoprotection achieved by the FDA-approved apocarotenoid and food additive bixin depends on NRF2 activation. Bixin activates NRF2 through the critical Cys-151 sensor residue in KEAP1, orchestrating a broad cytoprotective response in cultured human keratinocytes as revealed by antioxidant gene expression array analysis. Following dose optimization studies for cutaneous NRF2 activation by systemic administration of bixin, feasibility of bixin-based suppression of acute cutaneous photodamage from solar UV exposure was investigated in Nrf2(+/+) versus Nrf2(-/-) SKH-1 mice. Systemic administration of bixin suppressed skin photodamage, attenuating epidermal oxidative DNA damage and inflammatory responses in Nrf2(+/+) but not in Nrf2(-/-) mice, confirming the NRF2-dependence of bixin-based cytoprotection. Taken together, these data demonstrate feasibility of achieving NRF2-dependent cutaneous photoprotection by systemic administration of the apocarotenoid bixin, a natural food additive consumed worldwide. Copyright © 2015 Elsevier Inc. All rights reserved.

Piper betle Induced Cytoprotective Genes and Proteins via the Nrf2/ARE Pathway in Aging Mice.

PubMed

Aliahmat, Nor Syahida; Abdul Sani, Nur Fathiah; Wan Hasan, Wan Nuraini; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2016-01-01

The objective of this study was to elucidate the underlying antioxidant mechanism of aqueous extract of Piper betle (PB) in aging rats. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE pathway involving phase II detoxifying and antioxidant enzymes plays an important role in the antioxidant system by reducing electrophiles and reactive oxygen species through induction of phase II enzymes and proteins. Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis. PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes. PB was testified for the first time to induce cytoprotective genes through the Nrf2/ARE signaling pathway, thus unraveling the antioxidant mechanism of PB during the aging process. © 2016 S. Karger AG, Basel.

Sargassum fusiforme polysaccharides activate antioxidant defense by promoting Nrf2-dependent cytoprotection and ameliorate stress insult during aging.

PubMed

Chen, Peichao; He, Dan; Zhang, Ya; Yang, Shanshan; Chen, Liujun; Wang, Shengqin; Zou, Huixi; Liao, Zhiyong; Zhang, Xu; Wu, Mingjiang

2016-11-09

Aging is a complex issue, which results in a progressive decline process in cellular protection and physiological functions. Illustrating the causes of aging and pharmaceutical interference with the aging process has been a pivotal issue for thousands of years. Sargassum fusiforme (S. fusiforme), a kind of brown alga, is also named the "longevity vegetable" as it is not only a kind of food, but also used as an herb in traditional Chinese Medicine for maintaining health and treatment of thyroid disease, cardiovascular disease and so on. But how S. fusiforme promotes longevity is vastly equivocal. We got clues from S. fusiforme polysaccharides, which exhibited antioxidant activity, but the underlying mechanisms remained unclear. In this study, we evaluated the antioxidant effect and the possible mechanisms that S. fusiforme polysaccharides have against d-galactose-induced aging and chronic aging. We selected the SFPS as the candidate for antioxidant defense evaluation, which is a type of S. fusiforme polysaccharide with strong free radical scavenging activity and non-toxicity. It revealed that the antioxidant defense of the d-galactose-induced mice was markedly recovered when they were intragastrically administrated with the SFPS. However, oxidative damage may not be the only cause of aging. We further evaluated the function of the SFPS in the chronic aging mice. Intriguingly, we even found an obvious aging phenotype in the middle aged male ICR mice, which showed a significant decline in Nrf2-dependent cytoprotection. When 9-month old male ICR mice were treated with the SFPS for 2 months or even 11 months to their mean survival age, experimental measurements showed that the SFPS significantly promoted the antioxidant defense and mitochondrial integrity during aging. Furthermore, we suggest that the SFPS promotes Nrf2-dependent cytoprotection by upregulating the nuclear Nrf2 translocation, which may be mediated by p21 and JNK dependent pathways. These results suggest that the SFPS may decelerate the aging process by enhancing Nrf2-dependent cytoprotection, especially antioxidant defense.

Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

PubMed Central

Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

2015-01-01

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. PMID:25589789

Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes

PubMed Central

Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R.; Scott, Melanie J.

2014-01-01

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NFκB transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress. PMID:24683544

Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.

PubMed

Chen, Christine; Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R; Scott, Melanie J

2014-01-01

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

Sulforaphane Attenuated the Pro-Inflammatory State Induced by Hydrogen Peroxide in SH-SY5Y Cells Through the Nrf2/HO-1 Signaling Pathway.

PubMed

de Oliveira, Marcos Roberto; Brasil, Flávia Bittencourt; Fürstenau, Cristina Ribas

2018-02-23

Sulforaphane (SFN), an isothiocyanate obtained from cruciferous vegetables, exerts antioxidant, antiapoptotic, and antitumor activities in different cell types. Moreover, SFN has been viewed as an anti-inflammatory agent. Nonetheless, the mechanism underlying the ability of SFN in modulating the immune response in mammalian cells is not completely understood yet. Therefore, we investigated here whether and how SFN would be effective in preventing inflammation induced by a pro-oxidant agent (hydrogen peroxide, H 2 O 2 ) in the human neuroblastoma SH-SY5Y cells. The cells were treated with SFN at 5 μM for 30 min before a challenge with H 2 O 2 for an additional 24 h. Pretreatment with SFN reduced the secretion of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as decreased the levels of cyclooxygenase-2 (COX-2) in H 2 O 2 -treated cells. SFN also decreased the activity of the transcription factor nuclear factor-κB (NF-κB) and the immunocontent of the p65 NF-κB subunit in the cell nucleus. The inhibition of heme oxygenase-1 (HO-1) by ZnPP-IX at 10 μM or the silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor by small interfering RNA targeting Nrf2 attenuated the anti-inflammatory and cytoprotective effects induced by SFN. Therefore, SFN exerted an anti-inflammatory effect in H 2 O 2 -challenged SH-SY5Y cells by a mechanism dependent on the Nrf2/HO-1 signaling pathway.

Interplay between VEGF and Nrf2 regulates angiogenesis due to intracranial venous hypertension.

PubMed

Li, Liwen; Pan, Hao; Wang, Handong; Li, Xiang; Bu, Xiaomin; Wang, Qiang; Gao, Yongyue; Wen, Guodao; Zhou, Yali; Cong, Zixiang; Yang, Youqing; Tang, Chao; Liu, Zhengwei

2016-11-21

Venous hypertension(VH) plays an important role in the pathogenesis of cerebral arteriovenous malformations (AVMs) and is closely associated with the HIF-1α/VEGF signaling pathway. Nuclear factor erythroid 2-related factor 2(Nrf2) significantly influences angiogenesis; however, the interplay between Nrf2 and VEGF under VH in brain AVMs remains unclear. Therefore, our study aimed to investigate the interplay between Nrf2 and VEGF due to VH in brain AVMs. Immunohistochemistry indicated that Nrf2 and VEGF were highly expressed in human brain AVM tissues. In vivo, we established a VH model in both wild-type (WT) and siRNA-mediated Nrf2 knockdown rats. VH significantly increased the expression of Nrf2 and VEGF. Loss of Nrf2 markedly inhibited the upregulation of VEGF, as determined by Western blot analysis and qRT-PCR. In vitro, primary brain microvascular endothelial cells (BMECs) were isolated from WT and Nrf2 -/- mice, and a VEGF-Nrf2 positive feed-back loop was observed in BMECs. By trans well assay and angiogenesis assay, Nrf2 knockout significantly inhibited the migration and vascular tube formation of BMECs. These findings suggest that the interplay between Nrf2 and VEGF can contribute to VH-induced angiogenesis in brain AVMs pathogenesis.

Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr-Nrf2 pathway and MAPK signaling.

PubMed

Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Moley, Kelle H

2015-02-01

Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate (CSC) causes growth arrest of a mouse spermatocyte cell line [GC-2spd(ts)] through activation of the AHR-NRF2 pathway. The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts) via arylhydrocarbon receptor (AHR). Pharmacological inhibition by using AHR-antagonist, or p38 MAPK and ERK (MEK1) inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation. QRT-PCR, western blot, and immunofluorescence of Ahr-target of Nrf2, and stress-inducible growth suppressive Atf3 and E2f4 following treatments indicate a crosstalk among these pathways. Regulation of Atf3 by Nrf2 and Ahr through RNA interference suggests the existence of a cross-regulatory loop between the targets. CSC induction of E2f4 via Atf3 and its regulation by pharmacological inhibitors reveal a possible regulatory mechanism of growth inhibitory CSC. SiRNA silencing of Ahr, Nrf2, Atf3, and E2f4 genes and downregulation of cyclins by CSC corroborate the growth inhibitory effect of cigarette smoke. Thus, the data obtained suggest that the CSC-mediated MAPKs and AHR-NRF2 crosstalks lay the molecular basis for the growth arrest and cell death of spermatocytes. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Tanshindiol C inhibits oxidized low-density lipoprotein induced macrophage foam cell formation via a peroxiredoxin 1 dependent pathway.

PubMed

Yang, Yuyu; Li, Xueyan; Peng, Liying; An, Lin; Sun, Ningyuan; Hu, Xuewen; Zhou, Ping; Xu, Yong; Li, Ping; Chen, Jun

2018-03-01

NF-E2-related factor 2 (Nrf2) has been shown to be protective in atherosclerosis. The loss of Nrf2 in macrophages enhances foam cell formation and promotes early atherogenesis. Tanshindiol C (Tan C) is isolated from the root of Salvia miltiorrhiza Bge., a traditional Chinese medicine that has been used for the treatment of several cardiovascular diseases for many years. This study was aimed to test the potential role of Tan C against macrophage foam cell formation and to explore the underlying mechanism. Firstly, we observed that Tan C markedly suppressed oxidized low-density lipoprotein (oxLDL) induced macrophage foam cell formation. Then, we found that Tan C was an activator of both Nrf2 and Sirtuin 1 (Sirt1) in macrophages. Nrf2 and Sirt1 synergistically activated the transcription of anti-oxidant peroxiredoxin 1 (Prdx1) after Tan C treatment. More important, we demonstrated that silencing of Prdx1 promoted oxLDL-induced macrophage foam cell formation. Prdx1 upregulated adenosine triphosphate-binding cassette (ABC) transporter A1 (ABCA1) expression and decreased intracellular lipid accumulation. Furthermore, Tan C ameliorated oxLDL induced macrophage foam cell formation in a Prdx1-dependent manner. These observations suggest that Tan C protects macrophages from oxLDL induced foam cell formation via activation of Prdx1/ABCA1 signaling and that Prdx1 may be a novel target for therapeutic intervention of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of Nrf2/ARE Pathway in Protective Effect of Electroacupuncture against Endotoxic Shock-Induced Acute Lung Injury in Rabbits

PubMed Central

Yu, Jian-bo; Shi, Jia; Gong, Li-rong; Dong, Shu-an; Xu, Yan; Zhang, Yuan; Cao, Xin-shun; Wu, Li-li

2014-01-01

NF-E2 related factor 2 (Nrf2) is a major transcription factor and acts as a key regulator of antioxidant genes to exogenous stimulations. The aim of current study was to determine whether Nrf2/ARE pathway is involved in the protective effect of electroacupuncture on the injured lung in a rabbit model of endotoxic shock. A dose of lipopolysaccharide (LPS) 5 mg/kg was administered intravenously to replicate the model of acute lung injury induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Feishu acupoints for five consecutive days while sham electroacupuncture punctured at non-acupoints. Fourty anesthetized New England male rabbits were randomized into normal control group (group C), LPS group (group L), electroacupuncture + LPS group (group EL) and sham electroacupuncture + LPS (group SEL). At 6 h after LPS administration, the animals were sacrificed and the blood samples were collected for biochemical measurements. The lungs were removed for calculation of wet-to-dry weight ratios (W/D), histopathologic examination, determination of heme oxygenase (HO)-1 protein and mRNA, Nrf2 total and nucleoprotein, as well as Nrf2 mRNA expression, and evaluation of the intracellular distribution of Nrf2 nucleoprotein. LPS caused extensive morphologic lung damage, which was lessened by electroacupuncture treatment. Besides, lung W/D ratios were significantly decreased, the level of malondialdehyde was inhibited, plasma levels of TNF-α and interleukin-6 were decreased, while the activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced in the electroacupucnture treated animals. In addition, electroacupuncture stimulation distinctly increased the expressions of HO-1 and Nrf2 protein including Nrf2 total protein and nucleoprotein as well as mRNA in lung tissue, while these effects were blunted in the sham electroacupuncture group. We concluded that electroacupuncture treatment at ST36 and BL13 effectively attenuates lung injury in a rabbit model of endotoxic shock through activation of Nrf2/ARE pathway and following up-regulation of HO-1 expression. PMID:25115759

Role of Nrf2/ARE pathway in protective effect of electroacupuncture against endotoxic shock-induced acute lung injury in rabbits.

PubMed

Yu, Jian-bo; Shi, Jia; Gong, Li-rong; Dong, Shu-an; Xu, Yan; Zhang, Yuan; Cao, Xin-shun; Wu, Li-li

2014-01-01

NF-E2 related factor 2 (Nrf2) is a major transcription factor and acts as a key regulator of antioxidant genes to exogenous stimulations. The aim of current study was to determine whether Nrf2/ARE pathway is involved in the protective effect of electroacupuncture on the injured lung in a rabbit model of endotoxic shock. A dose of lipopolysaccharide (LPS) 5 mg/kg was administered intravenously to replicate the model of acute lung injury induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Feishu acupoints for five consecutive days while sham electroacupuncture punctured at non-acupoints. Fourty anesthetized New England male rabbits were randomized into normal control group (group C), LPS group (group L), electroacupuncture + LPS group (group EL) and sham electroacupuncture + LPS (group SEL). At 6 h after LPS administration, the animals were sacrificed and the blood samples were collected for biochemical measurements. The lungs were removed for calculation of wet-to-dry weight ratios (W/D), histopathologic examination, determination of heme oxygenase (HO)-1 protein and mRNA, Nrf2 total and nucleoprotein, as well as Nrf2 mRNA expression, and evaluation of the intracellular distribution of Nrf2 nucleoprotein. LPS caused extensive morphologic lung damage, which was lessened by electroacupuncture treatment. Besides, lung W/D ratios were significantly decreased, the level of malondialdehyde was inhibited, plasma levels of TNF-α and interleukin-6 were decreased, while the activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced in the electroacupucnture treated animals. In addition, electroacupuncture stimulation distinctly increased the expressions of HO-1 and Nrf2 protein including Nrf2 total protein and nucleoprotein as well as mRNA in lung tissue, while these effects were blunted in the sham electroacupuncture group. We concluded that electroacupuncture treatment at ST36 and BL13 effectively attenuates lung injury in a rabbit model of endotoxic shock through activation of Nrf2/ARE pathway and following up-regulation of HO-1 expression.

Investigation of the effect of a panel of model hepatotoxins on the Nrf2-Keap1 defence response pathway in CD-1 mice.

PubMed

Randle, Laura E; Goldring, Chris E P; Benson, Craig A; Metcalfe, Peter N; Kitteringham, Neil R; Park, B Kevin; Williams, Dominic P

2008-01-20

The Keap1-Nrf2-ARE signalling pathway has emerged as an important regulator of the mammalian defence system to enable detoxification and clearance of foreign chemicals. Recent studies by our group using paracetamol (APAP), diethylmaleate and buthionine sulphoximine have shown that for a given xenobiotic molecule, Nrf2 induction in the murine liver is associated with protein reactivity and glutathione depletion. Here, we have investigated, in vivo, whether the ability of four murine hepatotoxins, paracetamol, bromobenzene (BB), carbon tetrachloride (CCl4) and furosemide (FS) to deplete hepatic glutathione (GSH) is related to induction of hepatic Nrf2 nuclear translocation and Nrf2-dependent gene expression. Additionally, we studied whether hepatic Nrf2 nuclear translocation is a general response during the early stages of acute hepatic chemical stress in vivo. Male CD-1 mice were administered APAP (3.5 mmol/kg), FS (1.21 mmol/kg), BB (4.8 mmol/kg) and CCl4 (1 mmol/kg) for 1, 5 and 24h. Each compound elicited significant serum ALT increases after 24h (ALT U/L: APAP, 3036+/-1462; BB, 5308+/-2210; CCl4, 5089+/-1665; FS, 2301+/-1053), accompanied by centrilobular damage as assessed by histopathology. Treatment with APAP also elicited toxicity at a much earlier time point (5h) than the other hepatotoxins (ALT U/L: APAP, 1780+/-661; BB, 161+/-15; CCl4, 90+/-23; FS, 136+/-27). Significant GSH depletion was seen with APAP (9.6+/-1.7% of control levels) and BB (52.8+/-6.2% of control levels) 1h after administration, but not with FS and CCl4. Western Blot analysis revealed an increase in nuclear Nrf2, 1h after administration of BB (209+/-10% control), CCl4 (146+/-3% control) and FS (254+/-41% control), however this was significantly lower than the levels observed in the APAP-treated mice (462+/-36% control). The levels of Nrf2-dependent gene induction were also analysed by quantitative real-time PCR and Western blotting. Treatment with APAP for 1h caused a significant increase in the levels of haem oxygenase-1 (HO-1; 2.85-fold) and glutamate cysteine ligase (GCLC; 1.62-fold) mRNA. BB and FS did not affect the mRNA levels of either gene after 1h of treatment; however CCl4 significantly increased HO-1 mRNA at this time point. After 24h treatment with the hepatotoxins, there was evidence for the initiation of a late defence response. BB significantly increased both HO-1 and GCLC protein at this time point, CCl4 increased GCLC protein alone, although FS did not alter either of these proteins. In summary, we have demonstrated that the hepatotoxins BB, CCl4 and FS can induce a small but significant increase in Nrf2 accumulation in hepatic nuclei. However, this was associated with modest changes in hepatic GSH, a delayed development of toxicity and was insufficient to activate an early functional adaptive response to these hepatotoxins.

Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse.

PubMed

Liu, Qianying; Lei, Zhixin; Guo, Jingchao; Liu, Aimei; Lu, Qirong; Fatima, Zainab; Khaliq, Haseeb; Shabbir, Muhammad A B; Maan, Muhammad Kashif; Wu, Qinghua; Dai, Menghong; Wang, Xu; Pan, Yuanhu; Yuan, Zonghui

2018-01-01

Mequindox (MEQ), belonging to quinoxaline-di- N -oxides (QdNOs), is a synthetic antimicrobial agent widely used in China. Previous studies found that the kidney was one of the main toxic target organs of the QdNOs. However, the mechanisms underlying the kidney toxicity caused by QdNOs in vivo still remains unclear. The present study aimed to explore the molecular mechanism of kidney toxicity in mice after chronic exposure to MEQ. MEQ led to the oxidative stress, apoptosis, and mitochondrial damage in the kidney of mice. Meanwhile, MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. The oxidative stress mediated by MEQ might led to mitochondria damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, upregulation of the Nrf2-Keap1 signaling pathway was also observed. Our findings revealed that the oxidative stress, mitochondrial dysfunction, and the Nrf2-Keap1 signaling pathway were associated with the kidney apoptosis induced by MEQ in vivo .

Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse

PubMed Central

Liu, Qianying; Lei, Zhixin; Guo, Jingchao; Liu, Aimei; Lu, Qirong; Fatima, Zainab; Khaliq, Haseeb; Shabbir, Muhammad A. B.; Maan, Muhammad Kashif; Wu, Qinghua; Dai, Menghong; Wang, Xu; Pan, Yuanhu; Yuan, Zonghui

2018-01-01

Mequindox (MEQ), belonging to quinoxaline-di-N-oxides (QdNOs), is a synthetic antimicrobial agent widely used in China. Previous studies found that the kidney was one of the main toxic target organs of the QdNOs. However, the mechanisms underlying the kidney toxicity caused by QdNOs in vivo still remains unclear. The present study aimed to explore the molecular mechanism of kidney toxicity in mice after chronic exposure to MEQ. MEQ led to the oxidative stress, apoptosis, and mitochondrial damage in the kidney of mice. Meanwhile, MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. The oxidative stress mediated by MEQ might led to mitochondria damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, upregulation of the Nrf2-Keap1 signaling pathway was also observed. Our findings revealed that the oxidative stress, mitochondrial dysfunction, and the Nrf2-Keap1 signaling pathway were associated with the kidney apoptosis induced by MEQ in vivo. PMID:29765325

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791; Baek, Jeong-Hwa

2014-07-11

Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates themore » radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.« less

Gene-expression signature regulated by the KEAP1-NRF2-CUL3 axis is associated with a poor prognosis in head and neck squamous cell cancer.

PubMed

Namani, Akhileshwar; Matiur Rahaman, Md; Chen, Ming; Tang, Xiuwen

2018-01-06

NRF2 is the key regulator of oxidative stress in normal cells and aberrant expression of the NRF2 pathway due to genetic alterations in the KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor erythroid 2 like 2)-CUL3 (cullin 3) axis leads to tumorigenesis and drug resistance in many cancers including head and neck squamous cell cancer (HNSCC). The main goal of this study was to identify specific genes regulated by the KEAP1-NRF2-CUL3 axis in HNSCC patients, to assess the prognostic value of this gene signature in different cohorts, and to reveal potential biomarkers. RNA-Seq V2 level 3 data from 279 tumor samples along with 37 adjacent normal samples from patients enrolled in the The Cancer Genome Atlas (TCGA)-HNSCC study were used to identify upregulated genes using two methods (altered KEAP1-NRF2-CUL3 versus normal, and altered KEAP1-NRF2-CUL3 versus wild-type). We then used a new approach to identify the combined gene signature by integrating both datasets and subsequently tested this signature in 4 independent HNSCC datasets to assess its prognostic value. In addition, functional annotation using the DAVID v6.8 database and protein-protein interaction (PPI) analysis using the STRING v10 database were performed on the signature. A signature composed of a subset of 17 genes regulated by the KEAP1-NRF2-CUL3 axis was identified by overlapping both the upregulated genes of altered versus normal (251 genes) and altered versus wild-type (25 genes) datasets. We showed that increased expression was significantly associated with poor survival in 4 independent HNSCC datasets, including the TCGA-HNSCC dataset. Furthermore, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and PPI analysis revealed that most of the genes in this signature are associated with drug metabolism and glutathione metabolic pathways. Altogether, our study emphasizes the discovery of a gene signature regulated by the KEAP1-NRF2-CUL3 axis which is strongly associated with tumorigenesis and drug resistance in HNSCC. This 17-gene signature provides potential biomarkers and therapeutic targets for HNSCC cases in which the NRF2 pathway is activated.

Alkaloids from Piper nigrum Exhibit Antiinflammatory Activity via Activating the Nrf2/HO-1 Pathway.

PubMed

Ngo, Quynh Mai Thi; Tran, Phuong Thao; Tran, Manh Hung; Kim, Jeong Ah; Rho, Seong Soo; Lim, Chi-Hwan; Kim, Jin-Cheol; Woo, Mi Hee; Choi, Jae Sui; Lee, Jeong-Hyung; Min, Byung Sun

2017-04-01

In the present study, ten alkaloids, namely chabamide (1), pellitorine (2), retrofractamide A (3), pyrroperine (4), isopiperolein B (5), piperamide C9:1 (8E) (6), 6,7-dehydrobrachyamide B (7), 4,5-dihydropiperine (8), dehydropipernonaline (9), and piperine (10), were isolated from the fruits of Piper nigrum. Among these, chabamide (1), pellitorine (2), retrofractamide A (3), isopiperolein B (5), and 6,7-dehydrobrachyamide B (7) exhibited significant inhibitory activity on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells, with IC 50 values of 6.8, 14.5, 30.2, 23.7, and 38.5 μM, respectively. Furthermore, compound 1 inhibited lipopolysaccharide-induced NO production in bone marrow-derived macrophages with IC 50 value of 9.5 μM. Consistent with NO inhibition, treatment of RAW264.7 cells with chabamide (1), pellitorine (2), and 6,7-dehydrobrachyamide B (7) suppressed expression of inducible NO synthase and cyclooxygenase-2. Chabamide (1), pellitorine (2), and 6,7-dehydrobrachyamide B (7) induced heme-oxygenase-1 expression at the transcriptional level. In addition, compound 1 induced the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and upregulated the expression of Nrf2 target genes, NAD(P)H:quinone oxidoreductase 1 and γ-glutamyl cysteine synthetase catalytic subunit, in a concentration-dependent manner in RAW264.7 cells. These findings suggest that chabamide (1) from P. nigrum exert antiinflammatory effects via the activation of the Nrf2/heme-oxygenase-1 pathway; hence, it might be a promising candidate for the treatment of inflammatory diseases. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Hui; Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing; Wang, Huihui

2016-02-01

Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As{sub 2}O{sub 3}), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide,more » that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As{sub 2}O{sub 3}-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As{sub 2}O{sub 3}-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As{sub 2}O{sub 3}-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents. - Highlights: • Identification of novel inhibitors of ARE-dependent transcription • Suppression of NRF2–ARE sensitizes THP-1 cells to chemotherapy. • Ethionamide suppresses ARE-dependent transcriptional activity. • Ethionamide and isoniazid increase the cytotoxicity of As{sub 2}O{sub 3} in AML cells. • Sensitization of THP-1 cells to As{sub 2}O{sub 3} toxicity by ethionamide is NRF2-dependent.« less

Curcumin analog 1, 5-bis (2-trifluoromethylphenyl)-1, 4-pentadien-3-one exhibits enhanced ability on Nrf2 activation and protection against acrolein-induced ARPE-19 cell toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yuan; Zou, Xuan; Cao, Ke

2013-11-01

Curcumin, a phytochemical agent in the spice turmeric, has received increasing attention for its anticancer, anti-inflammatory and antioxidant properties. However, application of curcumin has been limited due to its insolubility in water and poor bioavailability both clinically and experimentally. In addition, the protective effects and mechanisms of curcumin in eye diseases have been poorly studied. In the present study, we synthesized a curcumin analog, 1, 5-bis (2-trifluoromethylphenyl)-1, 4-pentadien-3-one (C3), which displayed improved protective effect against acrolein-induced toxicity in a human retinal pigment epithelial cell line (ARPE-19). At 5 μM, curcumin completely protected against acrolein-induced cell oxidative damage and preserved GSHmore » levels and mitochondrial function. Surprisingly, C3 displayed a complete protective effect at 0.5 μM, which was much more efficient than curcumin. Both 0.5 μM C3 and 5 μM curcumin induced Nrf2 nuclear translocation and Nrf2 target genes transcription similarly. Experiments using Nrf2 siRNA showed that the protective effects of curcumin and C3 were eliminated by Nrf2 knockdown. Additionally, both curcumin and C3 activated the PI3/Akt pathway, however, Nrf2 activation was independent of this pathway, and therefore, we hypothesized that both curcumin and C3 activated phase II enzymes via directly disrupting the Nrf2/Keap1 complex and promoting Nrf2's nuclear translocation. Since acrolein challenge of ARPE-19 cells has been used as a model of smoking and age-related macular degeneration (AMD), we concluded that the curcumin analog, C3, may be a more promising drug candidate for its potential application for the prevention and treatment of eye diseases, such as AMD. - Highlights: • We examine toxicity effects of cigarette smoking component acrolein in retina cells. • We report a more efficient curcumin analog (C3) protecting cellular function. • Mitochondrial function and phase II enzyme activation are the major targets of C3. • C3 is ten-fold more potent than curcumin on activating Nrf2 nuclear translocation. • Nrf2 translocation and Phase II enzyme induction are independent of PI3K/Akt pathway.« less

Protective Effect of Decursin Extracted from Angelica gigas in Male Infertility via Nrf2/HO-1 Signaling Pathway

PubMed Central

Bae, Woong Jin; Ha, U. Syn; Choi, Jin Bong; Kim, Kang Sup; Kim, Su Jin; Cho, Hyuk Jin; Hong, Sung Hoo; Lee, Ji Youl; Wang, Zhiping; Hwang, Sung Yeoun; Kim, Sae Woong

2016-01-01

Higher testicular temperature results in altered spermatogenesis due to heat-related oxidative stress. We examined the effects of decursin extracted from Angelica gigas Nakai on antioxidant activity in vitro and in a cryptorchidism-induced infertility rat model. TM3 Leydig cell viability was measured based on oxidative stress according to treatment. Either distilled water or AG 400 mg/kg of A. gigas extract was administered orally for 4 weeks after unilateral cryptorchidism was induced. After 1, 2, and 4 weeks, six rats from the control group and six rats from treatment group were sacrificed. Testicular weight, semen quality, antioxidant activities, nuclear factor erythroid 2-related factor 2 (Nrf2) protein, and mRNA expression of Nrf2-regulated genes were analyzed. Treatment with A. gigas extract (1) protected TM3 cells against oxidative stress in a dose-dependent manner, (2) improved the mean weight of the cryptorchid testis, (3) maintained sperm counts, motility, and spermatogenic cell density, (4) decreased levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and increased levels of superoxide dismutase (SOD), (5) significantly increased Nrf2 and heme oxygenase-1 (HO-1), and (6) significantly decreased apoptosis. This study suggests that decursin extracted from A. gigas is a supplemental agent that can reduce oxidative stress by Nrf2-mediated upregulation of HO-1 in rat experimentally induced unilateral cryptorchidism and may improve cryptorchidism-induced infertility. PMID:27034737

Protective Effect of Decursin Extracted from Angelica gigas in Male Infertility via Nrf2/HO-1 Signaling Pathway.

PubMed

Bae, Woong Jin; Ha, U Syn; Choi, Jin Bong; Kim, Kang Sup; Kim, Su Jin; Cho, Hyuk Jin; Hong, Sung Hoo; Lee, Ji Youl; Wang, Zhiping; Hwang, Sung Yeoun; Kim, Sae Woong

2016-01-01

Higher testicular temperature results in altered spermatogenesis due to heat-related oxidative stress. We examined the effects of decursin extracted from Angelica gigas Nakai on antioxidant activity in vitro and in a cryptorchidism-induced infertility rat model. TM3 Leydig cell viability was measured based on oxidative stress according to treatment. Either distilled water or AG 400 mg/kg of A. gigas extract was administered orally for 4 weeks after unilateral cryptorchidism was induced. After 1, 2, and 4 weeks, six rats from the control group and six rats from treatment group were sacrificed. Testicular weight, semen quality, antioxidant activities, nuclear factor erythroid 2-related factor 2 (Nrf2) protein, and mRNA expression of Nrf2-regulated genes were analyzed. Treatment with A. gigas extract (1) protected TM3 cells against oxidative stress in a dose-dependent manner, (2) improved the mean weight of the cryptorchid testis, (3) maintained sperm counts, motility, and spermatogenic cell density, (4) decreased levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and increased levels of superoxide dismutase (SOD), (5) significantly increased Nrf2 and heme oxygenase-1 (HO-1), and (6) significantly decreased apoptosis. This study suggests that decursin extracted from A. gigas is a supplemental agent that can reduce oxidative stress by Nrf2-mediated upregulation of HO-1 in rat experimentally induced unilateral cryptorchidism and may improve cryptorchidism-induced infertility.

Protective properties of Huperzine A through activation Nrf2/ARE-mediated transcriptional response in X-rays radiation-induced NIH3T3 cells.

PubMed

Zhu, Huan-Feng; Yan, Peng-Wei; Wang, Li-Jun; Liu, Ya-Tian; Wen, Jing; Zhang, Qian; Fan, Yan-Xin; Luo, Yan-Hong

2018-06-22

Huperzine A (HupA), derived from Huperzia Serrata, has exhibited a variety of biological actions, in particular neuroprotective effect. However, the protective activities of HupA on murine embryonic fibroblast NIH3T3 cells after X-rays radiation have not been fully elucidated. Herein, HupA treatment dramatically promoted cell viability, abated a G0/G1 peak accumulation, and ameliorated increase of cell apoptosis in NIH3T3 cells after X-rays radiation. Simultaneously, HupA notably enhanced activities of anti-oxidant enzymes, inhibited activity of lipid peroxide, and efficiently eliminated production of reactive oxygen species in NIH3T3 cells after X-rays radiation. Dose-dependent increase of antioxidant genes by HupA were associated with up-regulated Nrf2 and down-regulated Keap-1 expression, which was confirmed by increasing nuclear accumulation, and inhibiting of degradation of Nrf2. Notably, augmented luciferase activity of ARE may explained Nrf2/ARE-mediated signaling pathways behind HupA protective properties. Moreover, expression of Nrf2 HupA-mediated was significant attenuated by AKT inhibitor (LY294002), p38 MAPK inhibitor (SB202190) and ERK inhibitor (PD98059). Besides, HupA-mediated cell viability, and ROS production were dramatically bated by LY294002, SB202190, and PD98059. Taken together, HupA effectively ameliorated X-rays radiation-induced damage Nrf2-ARE-mediated transcriptional response via activation AKT, p38, and ERK signaling in NIH3T3 cells. © 2018 Wiley Periodicals, Inc.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5.

PubMed

Ashino, Takashi; Ohkubo-Morita, Haruyo; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

2014-01-01

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient (Nrf2(-⧸-)) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2(-⧸-) mouse livers were lower than that in wild-type mouse livers. Nrf2(-⧸-) mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5☆

PubMed Central

Ashino, Takashi; Ohkubo-Morita, Haruyo; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

2014-01-01

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)–deficient (Nrf2−⧸−) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2−⧸− mouse livers were lower than that in wild-type mouse livers. Nrf2−⧸− mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression. PMID:24494203

Curcumin mitigates axonal injury and neuronal cell apoptosis through the PERK/Nrf2 signaling pathway following diffuse axonal injury.

PubMed

Huang, Tingqin; Zhao, Junjie; Guo, Dan; Pang, Honggang; Zhao, Yonglin; Song, Jinning

2018-05-23

Diffuse axonal injury (DAI) accounts for more than 50% of all traumatic brain injury. In response to the mechanical damage associated with DAI, the abnormal proteins produced in the neurons and axons, namely, β-APP and p-tau, induce endoplasmic reticulum (ER) stress. Curcumin, a major component extracted from the rhizome of Curcuma longa, has shown potent anti-inflammatory, antioxidant, anti-infection, and antitumor activity in previous studies. Moreover, curcumin is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2) and promotes its nuclear translocation. In this study, we evaluated the therapeutic potential of curcumin for the treatment of DAI and investigated the mechanisms underlying the protective effects of curcumin against neural cell death and axonal injury after DAI. Rats subjected to a model of DAI by head rotational acceleration were treated with vehicle or curcumin to evaluate the effect of curcumin on neuronal and axonal injury. We observed that curcumin (20 mg/kg intraperitoneal) administered 1 h after DAI induction alleviated the aggregation of p-tau and β-APP in neurons, reduced ER-stress-related cell apoptosis, and ameliorated neurological deficits. Further investigation showed that the protective effect of curcumin in DAI was mediated by the PERK/Nrf2 pathway. Curcumin promoted PERK phosphorylation, and then Nrf2 dissociated from Keap1 and was translocated to the nucleus, which activated ATF4, an important bZIP transcription factor that maintains intracellular homeostasis, but inhibited the CHOP, a hallmark of ER stress and ER-associated programmed cell death. In summary, we demonstrate for the first time that curcumin confers protection against abnormal proteins and neuronal apoptosis after DAI, that the process is mediated by strengthening of the unfolded protein response to overcome ER stress, and that the protective effect of curcumin against DAI is dependent on the activation of Nrf2.

Withanolide A Prevents Neurodegeneration by Modulating Hippocampal Glutathione Biosynthesis during Hypoxia

PubMed Central

Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

2014-01-01

Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner. PMID:25310001

Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia.

PubMed

Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

2014-01-01

Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.

Mechanisms of activation of the transcription factor Nrf2 by redox stressors, nutrient cues, and energy status and the pathways through which it attenuates degenerative disease.

PubMed

Tebay, Lauren E; Robertson, Holly; Durant, Stephen T; Vitale, Steven R; Penning, Trevor M; Dinkova-Kostova, Albena T; Hayes, John D

2015-11-01

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and stress-inducible expression of a battery of genes encoding key components of the glutathione-based and thioredoxin-based antioxidant systems, as well as aldo-keto reductase, glutathione S-transferase, and quinone oxidoreductase-1 drug-metabolizing isoenzymes along with multidrug-resistance-associated efflux pumps. It therefore plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS) and xenobiotics. Activation of Nrf2 can, however, serve as a double-edged sword because some of the genes it induces may contribute to chemical carcinogenesis by promoting futile redox cycling of polycyclic aromatic hydrocarbon metabolites or confer resistance to chemotherapeutic drugs by increasing the expression of efflux pumps, suggesting its cytoprotective effects will vary in a context-specific fashion. In addition to cytoprotection, Nrf2 also controls genes involved in intermediary metabolism, positively regulating those involved in NADPH generation, purine biosynthesis, and the β-oxidation of fatty acids, while suppressing those involved in lipogenesis and gluconeogenesis. Nrf2 is subject to regulation at multiple levels. Its ability to orchestrate adaptation to oxidants and electrophiles is due principally to stress-stimulated modification of thiols within one of its repressors, the Kelch-like ECH-associated protein 1 (Keap1), which is present in the cullin-3 RING ubiquitin ligase (CRL) complex CRLKeap1. Thus modification of Cys residues in Keap1 blocks CRLKeap1 activity, allowing newly translated Nrf2 to accumulate rapidly and induce its target genes. The ability of Keap1 to repress Nrf2 can be attenuated by p62/sequestosome-1 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent manner, thereby allowing refeeding after fasting to increase Nrf2-target gene expression. In parallel with repression by Keap1, Nrf2 is also repressed by β-transducin repeat-containing protein (β-TrCP), present in the Skp1-cullin-1-F-box protein (SCF) ubiquitin ligase complex SCFβ-TrCP. The ability of SCFβ-TrCP to suppress Nrf2 activity is itself enhanced by prior phosphorylation of the transcription factor by glycogen synthase kinase-3 (GSK-3) through formation of a DSGIS-containing phosphodegron. However, formation of the phosphodegron in Nrf2 by GSK-3 is inhibited by stimuli that activate protein kinase B (PKB)/Akt. In particular, PKB/Akt activity can be increased by phosphoinositide 3-kinase and mTORC2, thereby providing an explanation of why antioxidant-responsive element-driven genes are induced by growth factors and nutrients. Thus Nrf2 activity is tightly controlled via CRLKeap1 and SCFβ-TrCP by oxidative stress and energy-based signals, allowing it to mediate adaptive responses that restore redox homeostasis and modulate intermediary metabolism. Based on the fact that Nrf2 influences multiple biochemical pathways in both positive and negative ways, it is likely its dose-response curve, in terms of susceptibility to certain degenerative disease, is U-shaped. Specifically, too little Nrf2 activity will lead to loss of cytoprotection, diminished antioxidant capacity, and lowered β-oxidation of fatty acids, while conversely also exhibiting heightened sensitivity to ROS-based signaling that involves receptor tyrosine kinases and apoptosis signal-regulating kinase-1. By contrast, too much Nrf2 activity disturbs the homeostatic balance in favor of reduction, and so may have deleterious consequences including overproduction of reduced glutathione and NADPH, the blunting of ROS-based signal transduction, epithelial cell hyperplasia, and failure of certain cell types to differentiate correctly. We discuss the basis of a putative U-shaped Nrf2 dose-response curve in terms of potentially competing processes relevant to different stages of tumorigenesis. Copyright © 2015. Published by Elsevier Inc.

Mechanisms of activation of the transcription factor Nrf2 by redox stressors, nutrient cues, and energy status and the pathways through which it attenuates degenerative disease

PubMed Central

Tebay, Lauren E.; Robertson, Holly; Durant, Stephen T.; Vitale, Steven R.; Penning, Trevor M.; Dinkova-Kostova, Albena T.; Hayes, John D.

2015-01-01

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and stress-inducible expression of a battery of genes encoding key components of the glutathione-based and thioredoxin-based anti-oxidant systems, as well as aldo-keto reductase, glutathione S-transferase, and NAD(P)H:quinone oxi-doreductase-1 drug-metabolizing isoenzymes along with multidrug-resistance-associated efflux pumps. It therefore plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS) and xenobiotics. Activation of Nrf2 can, however, serve as a double-edged sword because some of the genes it induces may contribute to chemical carcinogenesis by promoting futile redox cycling of polycyclic aromatic hydrocarbon metabolites or confer resistance to chemotherapeutic drugs by increasing the expression of efflux pumps, suggesting its cytoprotective effects will vary in a context-specific fashion. In addition to cytoprotection, Nrf2 also controls genes involved in intermediary metabolism, positively regulating those involved in NADPH generation, purine biosynthesis, and the β-oxidation of fatty acids, while suppressing those involved in lipogenesis and gluconeogenesis. Nrf2 is subject to regulation at multiple levels. Its ability to orchestrate adaptation to oxidants and electrophiles is due principally to stress-stimulated modification of thiols within one of its repressors, the Kelch-like ECH-associated protein 1 (Keap1), which is present in the cullin-3 RING ubiquitin ligase (CRL) complex CRLKeap1. Thus modification of Cys residues in Keap1 blocks CRLKeap1 activity, allowing newly translated Nrf2 to accumulate rapidly and induce its target genes. The ability of Keap1 to repress Nrf2 can be attenuated by p62/sequestosome-1 in a mechanistic target of rapamycin complex 1 (mTORC1)-depen-dent manner, thereby allowing refeeding after fasting to increase Nrf2-target gene expression. In parallel with repression by Keap1, Nrf2 is also repressed by β-transducin repeat-containing protein (β-TrCP), present in the Skp1–cullin-1–F-box protein (SCF) ubiquitin ligase complex SCFβ-TrCP. The ability of SCFβ-TrCP to suppress Nrf2 activity is itself enhanced by prior phosphorylation of the transcription factor by glycogen synthase kinase-3 (GSK-3) through formation of a DSGIS-containing phosphodegron. However, formation of the phosphodegron in Nrf2 by GSK-3 is inhibited by stimuli that activate protein kinase B (PKB)/Akt. In particular, PKB/Akt activity can be increased by phosphoinositide 3-kinase and mTORC2, thereby providing an explanation of why antioxidant-responsive element-driven genes are induced by growth factors and nutrients. Thus Nrf2 activity is tightly controlled via CRLKeap1 and SCFβ-TrCP by oxidative stress and energy-based signals, allowing it to mediate adaptive responses that restore redox homeostasis and modulate intermediary metabolism. Based on the fact that Nrf2 influences multiple biochemical pathways in both positive and negative ways, it is likely its dose–response curve, in terms of susceptibility to certain degenerative disease, is U-shaped. Specifically, too little Nrf2 activity will lead to loss of cytoprotection, diminished antioxidant capacity, and lowered β-oxidation of fatty acids, while conversely also exhibiting heightened sensitivity to ROS-based signaling that involves receptor tyrosine kinases and apoptosis signal-regulating kinase-1. By contrast, too much Nrf2 activity disturbs the homeostatic balance in favor of reduction, and so may have deleterious consequences including overproduction of reduced glutathione and NADPH, the blunting of ROS-based signal transduction, epithelial cell hyperplasia, and failure of certain cell types to differentiate correctly. We discuss the basis of a putative U-shaped Nrf2 dose–response curve in terms of potentially competing processes relevant to different stages of tumorigenesis. PMID:26122708

Cardiac and renal upregulation of Nox2 and NF-κB and repression of Nox4 and Nrf2 in season- and diabetes-mediated models of vascular oxidative stress in guinea-pig and rat.

PubMed

Gajos-Draus, Anna; Duda, Monika; Beręsewicz, Andrzej

2017-11-01

The superoxide-forming NADPH oxidase homologues, Nox1, Nox2, and Nox5, seem to mediate the pro-atherosclerotic vascular phenotype. The hydrogen peroxide-forming Nox4 afforded vascular protection, likely via NF-E2-related factor-2 (Nrf2) activation and/or Nox2 downregulation in transgenic mice. We hypothesized that oxidative stress in the intact vasculature involves, aside from the upregulation of the superoxide-forming Noxs, the downregulation of the Nox4/Nrf2 pathway. Guinea-pigs and rats were studied either in winter or in summer, and the streptozotocin diabetic rats in winter. Plasma nitrite, and superoxide production by isolated hearts were measured, while frozen tissues served in biochemical analyses. Summer in both species and diabetes in rats downregulated myocardial Nox4 while reciprocally upregulating Nox2 and Nox5 in guinea-pigs, and Nox2 in rats. Simultaneously, myocardial Nrf2 activity and the expression of the Nrf2-directed heme oxygenase-1 and endothelial NO synthase were reduced while activity of the nuclear factor κ B (NF- κ B) and the expression of NF- κ B-directed inducible NO synthase and the vascular cell adhesion molecule-1 were increased. Cardiac superoxide production was increased while plasma nitrite was decreased reciprocally. Analogous disregulation of Noxs, Nrf2, and NF- κ B, occurred in diabetic rat kidneys. Given the diversity of the experimental settings and the uniform pattern of the responses, we speculate that: (1) chronic vascular oxidative stress is a nonspecific (model-, species-, organ-independent) response involving the induction of Nox2 (and Nox5 in guinea-pigs) and the NF- κ B pathway, and the repression of Nox4 and the Nrf2 pathway; and (2) the systems Nox2-NF- κ B and Nox4-Nrf2 regulate each other negatively. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Nrf2 target genes are induced under marginal selenium-deficiency

PubMed Central

Müller, Mike; Banning, Antje; Brigelius-Flohé, Regina

2010-01-01

A suboptimal selenium supply appears to prevail in Europe. The current study, therefore, was focused on the changes in gene expression under a suboptimal selenium intake. Previous microarray analyses in the colon of mice fed either a selenium-adequate or a moderately deficient diet revealed a change in genes of several pathways. Severe selenium-deficiency has been found previously to influence Nrf2-regulated genes of the adaptive response. Since the previous pathway analyses were done with a program not searching for Nrf2 target genes, respective genes were manually selected and confirmed by qPCR. qPCR revealed an induction of phase II (Nqo1, Gsts, Sult1b1 and Ugt1a6) and antioxidant enzymes (Hmox1, Mt2, Prdx1, Srxn1, Sod1 and Gclc) under the selenium-poor diet, which is considered to compensate for the loss of selenoproteins. The strongest effects were observed in the duodenum where preferentially genes for antioxidant enzymes were up-regulated. These also include the mRNA of the selenoproteins TrxR1 and GPx2 that would enable their immediate translation upon selenium refeeding. The down-regulation of Gsk3β in moderate selenium-deficiency observed in the previous paper provides a possible explanation for the activation of the Nrf2 pathway, because inhibition of GSK3β results in the nuclear accumulation of Nrf2. PMID:21189866

Fisetin alleviates oxidative stress after traumatic brain injury via the Nrf2-ARE pathway.

PubMed

Zhang, Li; Wang, Handong; Zhou, Yali; Zhu, Yihao; Fei, Maoxin

2018-05-22

Fisetin, a natural flavonoid, has neuroprotection properties in many brain injury models. However, its role in traumatic brain injury (TBI) has not been fully explained. In the present study, we aimed to explore the neuroprotective effects of fisetin in a mouse model of TBI. We found that fisetin improved neurological function, reduced cerebral edema, attenuated brain lesion and ameliorated blood-brain barrier (BBB) disruption after TBI. Moreover, the up-regulation of malondialdehyde (MDA) and the activity of glutathione peroxidase (GPx) were reversed by fisetin treatment. Furthermore, administration of fisetin suppressed neuron cell death and apoptosis, increased the expression of B-cell lymphoma 2 (Bcl-2), while decreased the expression of Bcl-2-associated X protein (Bax) and caspase-3 after TBI. In addition, fisetin activated the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway following TBI. However, fisetin only failed to suppress oxidative stress in Nrf2 -/- mice. In conclusion, our data provided the first evidence that fisetin played a critical role in neuroprotection after TBI partly through the activation of the Nrf2-ARE pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

PubMed Central

Wu, Shouhai; Zhang, Tianpeng; Du, Jingsheng

2016-01-01

Background Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin. Materials and methods Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis. Results The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 μg/mL and 2.25 μg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells. Conclusion The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway. PMID:27822011

Oncogenic mutations in KEAP1 disturbing inhibitory Nrf2-Keap1 interaction: Activation of antioxidative pathway in papillary thyroid carcinoma.

PubMed

Danilovic, Debora Lucia Seguro; de Mello, Evandro Sobroza; Frazzato, Eliana Salgado Turri; Wakamatsu, Alda; de Lima Jorge, Alexander Augusto; Hoff, Ana Oliveira; Marui, Suemi

2018-06-01

Nuclear factor erythroid 2-like 2 (NFE2L2) encodes Nrf2, transcription factor of antioxidative genes. In the presence of reactive oxygen species, Keap1 (Kelch-ECH-associating protein-1) inhibitor complex undergoes conformational changes disrupting Keap1-Nrf2 binding and Nrf2 translocates into nucleus. We evaluated the presence of mutations in NFE2L2 and KEAP1 in papillary thyroid carcinomas (PTCs) and correlated them with clinical presentation. Coding regions of NFE2L2 and KEAP1 were sequenced in 131 patients with PTC. Clinical and histopathological features were analyzed. Immunohistochemical analysis of Nrf2 expression was performed in mutated carcinomas. Although no mutations were found in NFE2L2, missense mutations in KEAP1 were observed in 6 patients with PTC (4.6%). Immunohistochemistry showed increased Nrf2 expression in nuclei of all mutated carcinomas, which presented poor prognostic features in histopathology. We identified mutations in KEAP1 associated with Nrf2 overexpression in PTC. Mutations favored disruption of inhibitory interaction Nrf2-Keap1 to enable increased antioxidant Nrf2 activity, possibly with prognostic consequences. © 2018 Wiley Periodicals, Inc.

Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

PubMed

Korenori, Yoshimi; Tanigawa, Shunsuke; Kumamoto, Takuma; Qin, Si; Daikoku, Yosuke; Miyamori, Koji; Nagai, Masashi; Hou, De-Xing

2013-05-01

6-Methylthiohexyl isothiocyanate (6-MTITC), one of the major bioactive ingredients in Japanese Wasabi, has revealed cytoprotective and cancer chemopreventive effects. This study aims to clarify the molecular mechanisms how 6-MTITC modulates nuclear factor E2-related factor 2 (Nrf2)/Kelchlike ECH-associating protein 1 (Keap1) system in antioxidant-responsive element (ARE)-mediated nicotinamide adenine dinucleotide phosphate (NADP): quinone oxidoreductase 1 (NQO1) expression. HepG2 cells were treated with 6-MTITC with varying time and dose. NQO1, Nrf2, and Keap1 proteins were detected by Western blotting. ARE transactivation was detected by electrophilic mobility shift assay and reporter gene assay. Nuclear localization of Nrf2 was determined by immunocytochemistry assay. Ubiquitination of Nrf2 and Keap1 was detected using immunoprecipitation after treatment with MG132. Small interfering RNA was used to knockdown Nrf2 or Keap1. The results revealed that 6-MTITC modulated Nrf2/ARE pathway by stimulating Keap1 modification, and inhibiting Nrf2 ubiquitination and protein turnover. These actions finally increased nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by 6-MTITC. 6-MTITC modulated ARE-driven NQO1 expression by stabilizing Nrf2 with enhanced Keap1 modification and decreased Nrf2 degradation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear factor E2-related factor-2 has a differential impact on MCT1 and MCT4 lactate carrier expression in colonic epithelial cells: a condition favoring metabolic symbiosis between colorectal cancer and stromal cells.

PubMed

Diehl, K; Dinges, L-A; Helm, O; Ammar, N; Plundrich, D; Arlt, A; Röcken, C; Sebens, S; Schäfer, H

2018-01-04

Malignant tumors, such as colorectal cancer (CRC), are heterogeneous diseases characterized by distinct metabolic phenotypes. These include Warburg- and reverse Warburg phenotypes depending on differential distribution of the lactate carrier proteins monocarboxylate transporter-4 and -1 (MCT4 and MCT1). Here, we elucidated the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) as the key regulator of cellular adaptation to inflammatory/environmental stress in shaping the metabolism toward a reverse Warburg phenotype in malignant and premalignant colonic epithelial cells. Immunohistochemistry of human CRC tissues revealed reciprocal expression of MCT1 and MCT4 in carcinoma and stroma cells, respectively, accompanied by strong epithelial Nrf2 activation. In colorectal tissue from inflammatory bowel disease patients, MCT1 and Nrf2 were coexpressed as well, relating to CD68+inflammatory infiltrates. Indirect coculture of human NCM460 colonocytes with M1- but not M2 macrophages induces MCT1 as well as G6PD, LDHB and TALDO expression, whereas MCT4 expression was decreased. Nrf2 knockdown or reactive oxygen species (ROS) scavenging blocked these coculture effects in NCM460 cells. Likewise, Nrf2 knockdown inhibited similar effects of tBHQ-mediated Nrf2 activation on NCM460 and HCT15 CRC cells. M1 coculture or Nrf2 activation/overexpression greatly altered the lactate uptake but not glucose uptake and mitochondrial activities in these cells, reflecting the reverse Warburg phenotype. Depending on MCT1-mediated lactate uptake, Nrf2 conferred protection from TRAIL-induced apoptosis in NCM460 and HCT15 cells. Moreover, metabolism-dependent clonal growth of HCT15 cells was induced by Nrf2-dependent activation of MCT1-driven lactate exchange. These findings indicate that Nrf2 has an impact on the metabolism already in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations. Favoring the reverse Warburg effect, these Nrf2-dependent alterations add to malignant transformation of the colonic epithelium.

Eriodictyol protects against H(2)O(2)-induced neuron-like PC12 cell death through activation of Nrf2/ARE signaling pathway.

PubMed

Lou, Haiyan; Jing, Xu; Ren, Dongmei; Wei, Xinbing; Zhang, Xiumei

2012-07-01

Eriodictyol, a flavonoid isolated from the Chinese herb Dracocephalum rupestre has long been established as an antioxidant. The present study was designed to explore the protective effects of eriodictyol against hydrogen peroxide (H(2)O(2))-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, differentiated PC12 cells were cultured and exposed to 200 μM H(2)O(2) in the absence or presence of eriodictyol (20, 40 and 80 μM). In addition, the potential contribution of the Nrf2/ARE neuroprotective pathway in eriodictyol-mediated protection against H(2)O(2)-induced neurotoxicity was also investigated. The results showed that H(2)O(2)-induced cell death can be inhibited in the presence of eriodictyol as measured by assays for MTT and apoptosis. Further study revealed that eriodictyol induced the nuclear translocation of Nrf2, enhanced the expression of heme oxygenase (HO-1) and γ-glutamylcysteine synthetase (γ-GCS), and increased the levels of intracellular glutathione. Treatment of PC12 cells with Nrf2 small interference RNA abolished eriodictyol-induced HO-1 and γ-GCS expression and its protective effects. In conclusion, these results suggest that eriodictyol upregulates HO-1 and γ-GCS expression through the activation of Nrf2/ARE pathway and protects PC12 cells against H(2)O(2)-induced oxidative stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis

PubMed Central

Malhotra, Deepti; Portales-Casamar, Elodie; Singh, Anju; Srivastava, Siddhartha; Arenillas, David; Happel, Christine; Shyr, Casper; Wakabayashi, Nobunao; Kensler, Thomas W.; Wasserman, Wyeth W.; Biswal, Shyam

2010-01-01

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1−/−) or depletion (Nrf2−/−) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response. PMID:20460467

Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet

PubMed Central

Senger, Donald R.; Li, Dan; Jaminet, Shou-Ching; Cao, Shugeng

2016-01-01

The Nrf2 (NFE2L2) cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both in vitro and in vivo. Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The absence of alkyl catechols from the modern Western diet suggests serious negative consequences for Nrf2 cell defense, resulting in reduced protection against multiple chronic diseases associated with oxidative stress. PMID:26885667

Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

PubMed

Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

2015-03-13

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jingyun; Wei, Xing; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai

Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressedmore » MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line. - Highlights: • Chaetominine enhanced chemosensitivity of ADR against K562/Adr cells. • Chaetominine increased intracellular ADR levels via inhibiting MRP1. • Chaetominine induced apoptosis of K562/Adr cells through upregulation of ROS and modulation of Bax/Bcl-2. • Inhibition of MRP1 and Nrf2 by chaetominine treatment was correlative with blockade of PI3K/Akt signaling.« less

Inflammation and airway hyperresponsiveness after chlorine exposure are prolonged by Nrf2 deficiency in mice.

PubMed

Ano, Satoshi; Panariti, Alice; Allard, Benoit; O'Sullivan, Michael; McGovern, Toby K; Hamamoto, Yoichiro; Ishii, Yukio; Yamamoto, Masayuki; Powell, William S; Martin, James G

2017-01-01

Chlorine gas (Cl 2 ) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2-related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl 2 , using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48h after a 5-min nose-only exposure to 100ppm Cl 2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. Cl 2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24h after Cl 2 exposure, were significantly greater at 48h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl 2 exposure. BSO reduced GSH levels and promoted Cl 2 -induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl 2 -induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48h post Cl 2 exposure. Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl 2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes

PubMed Central

Stepp, Wesley; Sjeklocha, Lucas; Long, Clayton; Riley, Caitlin; Callahan, James; Sanchez, Yolanda; Gough, Peter; Knowlin, Laquanda; van Duin, David; Ortiz-Pujols, Shiara; Jones, Samuel; Maile, Robert; Hong, Zhi; Berger, Scott; Cairns, Bruce

2017-01-01

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0–48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells. PMID:28886135

Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes.

PubMed

Eitas, Timothy K; Stepp, Wesley H; Sjeklocha, Lucas; Long, Clayton V; Riley, Caitlin; Callahan, James; Sanchez, Yolanda; Gough, Peter; Knowlin, Laquanda; van Duin, David; Ortiz-Pujols, Shiara; Jones, Samuel W; Maile, Robert; Hong, Zhi; Berger, Scott; Cairns, Bruce A

2017-01-01

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0-48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells.

Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway.

PubMed

Wu, Juan; Liu, Xinhui; Fan, Jinjin; Chen, Wenfang; Wang, Juan; Zeng, Youjia; Feng, Xiaorang; Yu, Xueqing; Yang, Xiao

2014-04-06

Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling

PubMed Central

Del Vecchio, Catherine A.; Feng, Yuxiong; Sokol, Ethan S.; Tillman, Erik J.; Sanduja, Sandhya; Reinhardt, Ferenc; Gupta, Piyush B.

2014-01-01

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)–scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. PMID:25203443

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

PubMed Central

McNeill, Eileen; Crabtree, Mark J.; Sahgal, Natasha; Patel, Jyoti; Chuaiphichai, Surawee; Iqbal, Asif J.; Hale, Ashley B.; Greaves, David R.; Channon, Keith M.

2015-01-01

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation. PMID:25451639

Tartary buckwheat flavonoids ameliorate high fructose-induced insulin resistance and oxidative stress associated with the insulin signaling and Nrf2/HO-1 pathways in mice.

PubMed

Hu, Yuanyuan; Hou, Zuoxu; Yi, Ruokun; Wang, Zhongming; Sun, Peng; Li, Guijie; Zhao, Xin; Wang, Qiang

2017-08-01

The present study was conducted to explore the effects of a purified tartary buckwheat flavonoid fraction (TBF) on insulin resistance and hepatic oxidative stress in mice fed high fructose in drinking water (20%) for 8 weeks. The results indicated that continuous administration of TBF dose-dependently improved the insulin sensitivity and glucose intolerance in high fructose-fed mice. TBF treatment also reversed the reduced level of insulin action on the phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (Akt) and phosphatidylinositol 3-kinase (PI3K), as well as the translocation of glucose transporter type 4 (GLUT4) in the insulin-resistant liver. Furthermore, TBF was found to exert high antioxidant capacity as it acts as a shield against oxidative stress induced by high fructose by restoring the antioxidant status, and modulating nuclear factor E2 related factor 2 (Nrf2) translocation to the nucleus with subsequently up-regulated antioxidative enzyme protein expression. Histopathological examinations revealed that impaired pancreatic/hepatic tissues were effectively restored in high fructose-fed mice following TBF treatment. Our results show that TBF intake is effective in preventing the conversion of high fructose-induced insulin resistance and hepatic oxidative stress in mice by improving the insulin signaling molecules and the Nrf2 signal pathway in the liver.

Sulforaphane suppresses LPS-induced inflammation in primary rat microglia.

PubMed

Brandenburg, Lars-Ove; Kipp, Markus; Lucius, Ralph; Pufe, Thomas; Wruck, Christoph J

2010-06-01

The aim of this study was to investigate the signal transduction pathways involved in sulforaphane (SF) mediated inhibition of the inflammatory response to lipopolysaccharide (LPS). Additionally, we investigated the effects of SF and LPS on the activity of Nrf2. Primary rat microglia and the murine microglia cell line BV2 were used. Cells were treated with LPS with or without SF. Cell viability was measured via WST-assay. Real-time RT-PCR was performed to analyze cytokine mRNA levels. The nitric oxide (NO) release was measured in LPS-stimulated microglia. The induction of various signal transduction pathways and Nrf2 was determined by Western blotting. NF-kappaB and AP-1 activation was measured by dual luciferase assay. We showed that SF attenuates the LPS-induced increase of IL-1beta, IL-6, and TNF-alpha expression in microglia. In addition, SF significantly decreases the NO in a concentration-dependent manner. SF inhibits LPS-stimulated ERK1/2 and JNK phosphorylation and thereby inhibits the LPS-induced activation of NF-kappaB- and activator protein-1 (AP-1). Moreover, SF and LPS together are able to induce Nrf2 activation. We showed that SF, and also LPS by itself, are able to activate the cell's defence against oxidative and electrophilic stress. We conclude that SF could be a candidate agent for anti-inflammatory treatment of the central nervous system.

4-Nitrophenol induces activation of Nrf2 antioxidant pathway and apoptosis of the germ cells in rat testes.

PubMed

Zhang, Yonghui; Cao, Yun; Wang, Fei; Song, Meiyan; Rui, Xiaoli; Li, Yansen; Li, ChunMei

2016-07-01

The potential of 4-nitrophenol (PNP) to affect testicular function of rats was assessed by intratesticular injection (IT). The protective effects of phytosterin (PS) on PNP-induced injury were assessed. Rats were sacrificed on days 1, 3, and 7 after IT of PNP (0.1 M, 50 μl). PNP induced hemorrhage in intertubular areas and denudation of germinal epithelium. The expression of caspase-3 and sperm abnormalities were significantly increased (P < 0.05). The concentrations of testosterone in serum were significantly increased (P < 0.05) on the 1st and 3rd day. PNP induced oxidative stress in testes, which manifested increased SOD, CAT, GSH-Px activities, and increases in MDA, GSH, H2O2 concentrations (P < 0.05). The Nrf2 antioxidant pathway was activated as indicated by increased expression of Nrf2, HO-1, and GCLC mRNA (P < 0.05). Moreover, supplementation with PS resulted in an amelioration of PNP-induced oxidative damage. These results suggest that PNP induced activation of Nrf2 antioxidant pathway and apoptosis of the germ cells.

The effects of chromium picolinate and chromium histidinate administration on NF-κB and Nrf2/HO-1 pathway in the brain of diabetic rats.

PubMed

Sahin, Kazim; Tuzcu, Mehmet; Orhan, Cemal; Gencoglu, Hasan; Ulas, Mustafa; Atalay, Mustafa; Sahin, Nurhan; Hayirli, Armagan; Komorowski, James R

2012-12-01

The objective of this experiment was to investigate the effects of supplemental chromium picolinate (CrPic) and chromium histidinate (CrHis) on nuclear factor-kappa B (NF-κB p65) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway in diabetic rat brain. Nondiabetic (n = 45) and diabetic (n = 45) male Wistar rats were either not supplemented or supplemented with CrPic or CrHis via drinking water to consume 8 μg elemental chromium (Cr) per day for 12 weeks. Diabetes was induced by streptozotocin injection (40 mg/kg i.p., for 2 weeks) and maintained by high-fat feeding (40 %). Diabetes was associated with increases in cerebral NF-κB and 4-hydroxynonenal (4-HNE) protein adducts and decreased in cerebral nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα) and Nrf2 levels. Both Cr chelates were effective to decrease levels of NF-κB and 4-HNE protein adducts and to increase levels of IκBα and Nrf2 in the brain of diabetic rats. However, responses of these increases and decreases were more notable when Cr was supplemented as CrHis than as CrPic. In conclusion, Cr may play a protective role in cerebral antioxidant defense system in diabetic subjects via the Nrf2 pathway by reducing inflammation through NF-κB p65 inhibition. Histidinate form of Cr was superior to picolinate form of Cr in reducing NF-κB expression and increasing Nrf2 expression in the brain of diabetic rats.

Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases.

PubMed

Preciados, Mark; Yoo, Changwon; Roy, Deodutta

2016-12-13

During the development of an individual from a single cell to prenatal stages to adolescence to adulthood and through the complete life span, humans are exposed to countless environmental and stochastic factors, including estrogenic endocrine disrupting chemicals. Brain cells and neural circuits are likely to be influenced by estrogenic endocrine disruptors (EEDs) because they strongly dependent on estrogens. In this review, we discuss both environmental, epidemiological, and experimental evidence on brain health with exposure to oral contraceptives, hormonal therapy, and EEDs such as bisphenol-A (BPA), polychlorinated biphenyls (PCBs), phthalates, and metalloestrogens, such as, arsenic, cadmium, and manganese. Also we discuss the brain health effects associated from exposure to EEDs including the promotion of neurodegeneration, protection against neurodegeneration, and involvement in various neurological deficits; changes in rearing behavior, locomotion, anxiety, learning difficulties, memory issues, and neuronal abnormalities. The effects of EEDs on the brain are varied during the entire life span and far-reaching with many different mechanisms. To understand endocrine disrupting chemicals mechanisms, we use bioinformatics, molecular, and epidemiologic approaches. Through those approaches, we learn how the effects of EEDs on the brain go beyond known mechanism to disrupt the circulatory and neural estrogen function and estrogen-mediated signaling. Effects on EEDs-modified estrogen and nuclear respiratory factor 1 (NRF1) signaling genes with exposure to natural estrogen, pharmacological estrogen-ethinyl estradiol, PCBs, phthalates, BPA, and metalloestrogens are presented here. Bioinformatics analysis of gene-EEDs interactions and brain disease associations identified hundreds of genes that were altered by exposure to estrogen, phthalate, PCBs, BPA or metalloestrogens. Many genes modified by EEDs are common targets of both 17 β-estradiol (E2) and NRF1. Some of these genes are involved with brain diseases, such as Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis, Autism Spectrum Disorder, and Brain Neoplasms. For example, the search of enriched pathways showed that top ten E2 interacting genes in AD- APOE , APP , ATP5A1 , CALM1 , CASP3 , GSK3B , IL1B , MAPT , PSEN2 and TNF- underlie the enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) AD pathway. With AD, the six E2-responsive genes are NRF1 target genes: APBB2 , DPYSL2 , EIF2S1 , ENO1 , MAPT , and PAXIP1 . These genes are also responsive to the following EEDs: ethinyl estradiol ( APBB2 , DPYSL2 , EIF2S1 , ENO1 , MAPT , and PAXIP1 ), BPA ( APBB2 , EIF2S1 , ENO1 , MAPT , and PAXIP1 ), dibutyl phthalate (DPYSL2, EIF2S1, and ENO1), diethylhexyl phthalate ( DPYSL2 and MAPT ). To validate findings from Comparative Toxicogenomics Database (CTD) curated data, we used Bayesian network (BN) analysis on microarray data of AD patients. We observed that both gender and NRF1 were associated with AD. The female NRF1 gene network is completely different from male human AD patients. AD-associated NRF1 target genes- APLP1 , APP , GRIN1 , GRIN2B , MAPT , PSEN2 , PEN2 , and IDE -are also regulated by E2. NRF1 regulates targets genes with diverse functions, including cell growth, apoptosis/autophagy, mitochondrial biogenesis, genomic instability, neurogenesis, neuroplasticity, synaptogenesis, and senescence. By activating or repressing the genes involved in cell proliferation, growth suppression, DNA damage/repair, apoptosis/autophagy, angiogenesis, estrogen signaling, neurogenesis, synaptogenesis, and senescence, and inducing a wide range of DNA damage, genomic instability and DNA methylation and transcriptional repression, NRF1 may act as a major regulator of EEDs-induced brain health deficits. In summary, estrogenic endocrine disrupting chemicals-modified genes in brain health deficits are part of both estrogen and NRF1 signaling pathways. Our findings suggest that in addition to estrogen signaling, EEDs influencing NRF1 regulated communities of genes across genomic and epigenomic multiple networks may contribute in the development of complex chronic human brain health disorders.

[Synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells and its related mechanism].

PubMed

Zhang, Shao-nan; Yong, Qun; Wu, Xin-li; Liu, Xiao-ping

2014-11-01

To investigate the synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells and the down-regulating effect of curcumin on the Keapl-Nrf2 pathway, a well recognized anti-drug pathway in almost drugged tumor cells. T24 cells were cultured and treated with increasing concentrations of curcumin(5 ,10 and 20 µmol/mL) combined with cisplatin(30 µg/mL) for 24 hours. The inhibitory effects on T24 cells were tested with MTI colorimetric assay. Nuclear Nrf2 and Keapl , cytoplasmic Keapl and two typical phase II enzymes (GSTP1 and NQOl) were checked with Western blotting. The proliferation of T24 cells was significantly inhibited by different concentrations of curcumin combined with cisplatin. After the treatment with different concentrations of curcumin, Nuclear Nrf2 was decreased but Keapl was increased, and GSTP1 and NQO1 were decreased. Synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells is observed in this research. The Keapl-Nrf2 pathway in T24 cells is down-regulated by curcumin. The expression of typical phase I enzymes (GSTP1 and NQO1) mediated by Nrf2 are decreased by curcumin. The sensitivity of tumor cells to chemotherapeutic drugs is then enhanced. These may be the mechanism of synergism effect of curcumin combined with cisplatin.

Nrf2 Is an Attractive Therapeutic Target for Retinal Diseases

PubMed Central

2016-01-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many species. The retina is constantly exposed to reactive oxygen species, and oxidative stress is a major contributor to age-related macular diseases. Moreover, the resulting inflammation and neuronal degeneration are also related to other retinal diseases. The well-known Nrf2 activators, bardoxolone methyl and its derivatives, have been the subject of a number of clinical trials, including those aimed at treating chronic kidney disease, pulmonary arterial hypertension, and mitochondrial myopathies. Recent studies suggest that Nrf2 activation protects the retina from retinal diseases. In particular, this is supported by the finding that Nrf2 knockout mice display age-related retinal degeneration. Moreover, the concept has been validated by the efficacy of Nrf2 activators in a number of retinal pathological models. We have also recently succeeded in generating a novel Nrf2 activator, RS9, using a biotransformation technique. This review discusses current links between retinal diseases and Nrf2 and the possibility of treating retinal diseases by activating the Nrf2 signaling pathway. PMID:27818722

Carnosic Acid Induces Anti-Inflammatory Effects in Paraquat-Treated SH-SY5Y Cells Through a Mechanism Involving a Crosstalk Between the Nrf2/HO-1 Axis and NF-κB.

PubMed

de Oliveira, Marcos Roberto; de Souza, Izabel Cristina Custódio; Fürstenau, Cristina Ribas

2018-01-01

Carnosic acid (CA) is a phenolic diterpene obtained from Rosmarinus officinalis L. and has demonstrated cytoprotective properties in several experimental models. CA exerts antioxidant effects by upregulating the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which controls the expression of antioxidant and phase II detoxification enzymes. Heme oxygenase-1 (HO-1) expression is modulated by Nrf2 and has been demonstrated as part of the mechanism underlying the CA-induced cytoprotection. Nonetheless, it remains to be studied whether and how HO-1 would mediate CA-elicited anti-inflammatory effects. Therefore, we have investigated here whether and how CA would prevent paraquat (PQ)-induced inflammation-related alterations in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated for 12 h with CA at 1 μM before exposure to PQ for further 24 h. CA suppressed the PQ-induced alterations on the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) through a mechanism involving the activation of the Nrf2/HO-1 axis. Furthermore, we observed a crosstalk between the Nrf2/HO-1 signaling pathway and the activation of the nuclear factor-κB (NF-κB) transcription factor, since administration of ZnPP IX (specific inhibitor of HO-1) or Nrf2 knockdown using small interfering RNA (siRNA) abolished the anti-inflammatory effects induced by CA. Moreover, administration of SN50 (specific inhibitor of NF-κB) inhibited the PQ-induced inflammation-related effects in SH-SY5Y cells. Therefore, CA exerted anti-inflammatory effects in SH-SY5Y cells through an Nrf2/HO-1 axis-dependent manner associated with downregulation of NF-κB.

Prediction of Binding Energy of Keap1 Interaction Motifs in the Nrf2 Antioxidant Pathway and Design of Potential High-Affinity Peptides.

PubMed

Karttunen, Mikko; Choy, Wing-Yiu; Cino, Elio A

2018-06-07

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor and principal regulator of the antioxidant pathway. The Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) binds to motifs in the N-terminal region of Nrf2, promoting its degradation. There is interest in developing ligands that can compete with Nrf2 for binding to Kelch, thereby activating its transcriptional activities and increasing antioxidant levels. Using experimental Δ G bind values of Kelch-binding motifs determined previously, a revised hydrophobicity-based model was developed for estimating Δ G bind from amino acid sequence and applied to rank potential uncharacterized Kelch-binding motifs identified from interaction databases and BLAST searches. Model predictions and molecular dynamics (MD) simulations suggested that full-length MAD2A binds Kelch more favorably than a high-affinity 20-mer Nrf2 E78P peptide, but that the motif in isolation is not a particularly strong binder. Endeavoring to develop shorter peptides for activating Nrf2, new designs were created based on the E78P peptide, some of which showed considerable propensity to form binding-competent structures in MD, and were predicted to interact with Kelch more favorably than the E78P peptide. The peptides could be promising new ligands for enhancing the oxidative stress response.

Nrf2 pathway modulates Substance P-induced human mast cell activation and degranulation in the hair follicle.

PubMed

Jadkauskaite, Laura; Bahri, Rajia; Farjo, Nilofer; Farjo, Bessam; Jenkins, Gail; Bhogal, Ranjit; Haslam, Iain; Bulfone-Paus, Silvia; Paus, Ralf

2018-05-30

Activation of Nrf2 in primary human mast cells exposed to oxidative stress induced by substance P suppresses pro-inflammatory gene transcription, activation and degranulation. Copyright © 2018. Published by Elsevier Inc.

Neochlorogenic acid inhibits against LPS-activated inflammatory responses through up-regulation of Nrf2/HO-1 and involving AMPK pathway.

PubMed

Park, Sun Young; Jin, Mei Ling; Yi, Eun Hye; Kim, Yoon; Park, Geuntae

2018-06-08

Acute and chronic inflammatory diseases are associated with excessive inflammation due to the accumulation of pro-inflammatory mediators and cytokines produced by macrophages. In the present study, we investigated the anti-inflammatory properties of neochlorogenic acid (nCGA) from Lonicera japonica on lipopolysaccharide (LPS)-activated inflammation in macrophages and participation of the AMPK/Nrf2 pathway. nCGA pretreatment significantly reduced the production of nitric oxide, prostaglandin E 2 , TNF-α, reactive oxygen species, IL-1β, and IL-6 by LPS-activated macrophages. Moreover, both transcript and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 were reduced by nCGA in LPS-activated macrophages. nCGA inhibited NF-κB activation by attenuating IKKα/β and IκBα phosphorylation in LPS-stimulated macrophages. Moreover, nCGA attenuated LPS-elevated JAK-1, STAT-1, and MAPK phosphorylation. We further evaluated the possible role of nCGA in the induction of AMPK/Nrf2 signal pathways required for the protein expression of HO-1 and NQO-1. nCGA induced AMPK activation via phosphorylation of LKB1 and CaMKII and by the inhibitory phosphorylation of GSK3β. It stimulated the overexpression of Nrf2/ARE-regulated downstream proteins, such as NQO-1 and HO-1. Furthermore, the anti-inflammatory effects of nCGA were attenuated in macrophages subjected to siRNAs specific for HO-1, NQO-1, Nrf2, and AMPK. Accordingly, these results indicate that nCGA, as an AMPK/Nrf2 signal activator, prevents excessive macrophage-mediated responses associated with acute and chronic inflammatory disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington's disease knock-in striatal cells.

PubMed

Ribeiro, Márcio; Rosenstock, Tatiana R; Oliveira, Ana M; Oliveira, Catarina R; Rego, A Cristina

2014-09-01

Oxidative stress and mitochondrial dysfunction have been described in Huntington's disease, a disorder caused by expression of mutant huntingtin (mHtt). IGF-1 was previously shown to protect HD cells, whereas insulin prevented neuronal oxidative stress. In this work we analyzed the role of insulin and IGF-1 in striatal cells derived from HD knock-in mice on mitochondrial production of reactive oxygen species (ROS) and related antioxidant and signaling pathways influencing mitochondrial function. Insulin and IGF-1 decreased mitochondrial ROS induced by mHtt and normalized mitochondrial SOD activity, without affecting intracellular glutathione levels. IGF-1 and insulin promoted Akt phosphorylation without changing the nuclear levels of phosphorylated Nrf2 or Nrf2/ARE activity. Insulin and IGF-1 treatment also decreased mitochondrial Drp1 phosphorylation, suggesting reduced mitochondrial fragmentation, and ameliorated mitochondrial function in HD cells in a PI-3K/Akt-dependent manner. This was accompanied by increased total and phosphorylated Akt, Tfam, and mitochondrial-encoded cytochrome c oxidase II, as well as Tom20 and Tom40 in mitochondria of insulin- and IGF-1-treated mutant striatal cells. Concomitantly, insulin/IGF-1-treated mutant cells showed reduced apoptotic features. Hence, insulin and IGF-1 improve mitochondrial function and reduce mitochondrial ROS caused by mHtt by activating the PI-3K/Akt signaling pathway, in a process independent of Nrf2 transcriptional activity, but involving enhanced mitochondrial levels of Akt and mitochondrial-encoded complex IV subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

Bardoxolone brings Nrf2-based therapies to light.

PubMed

Zhang, Donna D

2013-08-10

The targeted activation of nuclear factor erythroid-derived-2-like 2 (Nrf2) to alleviate symptoms of chronic kidney disease has recently garnered much attention. Unfortunately, the greatest clinical success to date, bardoxolone, failed in phase III clinical trial for unspecified safety reasons. The present letter to the editor discusses the clinical development of bardoxolone and explores potential reasons for the ultimate withdrawal from clinical trials. In particular, was the correct clinical indication pursued and would improved specificity have mitigated the safety concerns? Ultimately, it is concluded that the right clinical indication and heightened specificity will lead to successful Nrf2-based therapies. Therefore, the bardoxolone clinical results do not dampen enthusiasm for Nrf2-based therapies; rather it illuminates the clinical potential of the Nrf2 pathway as a drug target.

SPBP Is a Sulforaphane Induced Transcriptional Coactivator of NRF2 Regulating Expression of the Autophagy Receptor p62/SQSTM1

PubMed Central

Darvekar, Sagar Ramesh; Elvenes, Julianne; Brenne, Hanne Britt; Johansen, Terje; Sjøttem, Eva

2014-01-01

Organisms exposed to oxidative stress respond by orchestrating a stress response to prevent further damage. Intracellular levels of antioxidant agents increase, and damaged components are removed by autophagy induction. The KEAP1-NRF2 signaling pathway is the main pathway responsible for cell defense against oxidative stress and for maintaining the cellular redox balance at physiological levels. Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is a potent inducer of KEAP1-NRF2 signaling and antioxidant response element driven gene expression. In this study, we show that sulforaphane enhances the expression of the transcriptional coregulator SPBP. The expression curve peaks 6–8 hours post stimulation, and parallels the sulforaphane-induced expression of NRF2 and the autophagy receptor protein p62/SQSTM1. Reporter gene assays show that SPBP stimulates the expression of p62/SQSTM1 via ARE elements in the promoter region, and siRNA mediated knock down of SPBP significantly decreases the expression of p62/SQSTM1 and the formation of p62/SQSTM1 bodies in HeLa cells. Furthermore, SPBP siRNA reduces the sulforaphane induced expression of NRF2, and the expression of the autophagy marker protein LC3B. Both these proteins contain ARE-like elements in their promoter regions. Over-expressed SPBP and NRF2 acts synergistically on the p62/SQSTM1 promoter and colocalize in nuclear speckles in HeLa cells. Collectively, these results suggest that SPBP is a coactivator of NRF2, and hence may be important for securing enhanced and sustained expression of NRF2 induced genes such as proteins involved in selective autophagy. PMID:24416372

Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes

PubMed Central

Ajit, Deepa; Simonyi, Agnes; Li, Runting; Chen, Zihong; Hannink, Mark; Fritsche, Kevin L.; Mossine, Valeri V.; Smith, Robert E.; Dobbs, Thomas K.; Luo, Rensheng; Folk, William R.; Gu, Zezong; Lubahn, Dennis B.; Weisman, Gary A.; Sun, Grace Y.

2016-01-01

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemiscals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promotor activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells. PMID:27166148

The neuroprotective effects of α-iso-cubebene on dopaminergic cell death: involvement of CREB/Nrf2 signaling.

PubMed

Park, Sun Young; Son, Beung Gu; Park, Young Hoon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

2014-09-01

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.

Dextran loading protects macrophages from lipid peroxidation and induces a Keap1/Nrf2/ARE-dependent antioxidant response.

PubMed

Chechushkov, Anton; Zaitseva, Natalia; Vorontsova, Elena; Kozhin, Petr; Menshchikova, Elena; Shkurupiy, Vyacheslav

2016-12-01

Linear dextrans are often proposed as drug delivery systems with milder adverse effects and lower effective drug concentrations. Linear dextrans are polysaccharides that can potentially be used to load macrophages with drugs to transport them to a site of inflammation. Recently, it was reported that dextrans may exert a protective effect vis-à-vis drug cytotoxicity and during wound healing. The aim of the current work was to evaluate molecular mechanisms of action of dextrans that may be relevant to the cytoprotective effects. We determined the effect of treatment with 40- or 70-kDa dextran on production of reactive oxygen species, lipid peroxidation, and lysosomal pH in the J774 macrophage cell line. In addition, induction of Keap1/Nrf2/ARE and autophagic activity were evaluated. Dextrans of both molecular weights protected the cells from oxidative stress induced by cumene hydroperoxide and from lysosomal stress induced by ammonium chloride. The effect was associated with induction of the Keap1/Nrf2/ARE signaling pathway. Furthermore, dextran stimulated autophagy in a dose-dependent manner but inhibited the autophagosome-lysosome fusion in a time-dependent manner. This study shows possible cytoprotective effects of dextran under oxidative stress, and these findings may be used for the development of novel (dextran-based) drug delivery approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

The NRF2-KEAP1 Pathway Is an Early Responsive Gene Network in Arsenic Exposed Lymphoblastoid Cells

PubMed Central

Córdova, Emilio J.; Martínez-Hernández, Angélica; Uribe-Figueroa, Laura; Centeno, Federico; Morales-Marín, Mirna; Koneru, Harsha; Coleman, Matthew A.; Orozco, Lorena

2014-01-01

Inorganic arsenic (iAs), a major environmental contaminant, has risen as an important health problem worldwide. More detailed identification of the molecular mechanisms associated with iAs exposure would help to establish better strategies for prevention and treatment. Although chronic iAs exposures have been previously studied there is little to no information regarding the early events of exposure to iAs. To better characterize the early mechanisms of iAs exposure we conducted gene expression studies using sublethal doses of iAs at two different time-points. The major transcripts differentially regulated at 2 hrs of iAs exposure included antioxidants, detoxificants and chaperones. Moreover, after 12 hrs of exposure many of the down-regulated genes were associated with DNA replication and S phase cell cycle progression. Interestingly, the most affected biological pathway by both 2 or 12 hrs of iAs exposure were the Nrf2-Keap1 pathway, represented by the highly up-regulated HMOX1 transcript, which is transcriptionally regulated by the transcription factor Nrf2. Additional Nrf2 targets included SQSTM1 and ABCB6, which were not previously associated with acute iAs exposure. Signalling pathways such as interferon, B cell receptor and AhR route were also responsive to acute iAs exposure. Since HMOX1 expression increased early (20 min) and was responsive to low iAs concentrations (0.1 µM), this gene could be a suitable early biomarker for iAs exposure. In addition, the novel Nrf2 targets SQSTM1 and ABCB6 could play an important and previously unrecognized role in cellular protection against iAs. PMID:24516582

Z-ligustilide ameliorated ultraviolet B-induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO-1 and suppression of NF-κB pathway.

PubMed

Wu, Zhouwei; Uchi, Hiroshi; Morino-Koga, Saori; Shi, Weimin; Furue, Masutaka

2015-09-01

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z-ligustilide (Z-lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB-induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z-lig significantly rescued UVB-induced NHEKs damage in a dosage-dependent manner. Pretreatment of NHEKs with Z-lig inhibited UVB-induced ROS production in NHEKs. Both silencing the nuclear factor E2-related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase-1 (HO-1) inhibitor, cancelled the inhibitory effect of Z-lig on UVB-induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z-lig reduced UVB-induced nuclear factor kappa B (NF-κB)-dependent inflammatory mediators (IL-6, IL-8 and MCP-1) production at both mRNA and protein level. In the presence of Z-lig, UVB-induced NF-κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z-lig can suppress UVB-induced ROS generation through Nrf2/HO-1 upregulation and inflammation by suppressing the NF-κB pathway, suggesting that Z-lig may be beneficial in protecting skin from UVB exposure. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

UVA Irradiation Enhances Brusatol-Mediated Inhibition of Melanoma Growth by Downregulation of the Nrf2-Mediated Antioxidant Response

PubMed Central

Wang, Mei; Shi, Guangwei; Bian, Chunxiang; Nisar, Muhammad Farrukh; Guo, Yingying; Wu, Yan; Li, Wei; Huang, Xiao; Jiang, Xuemei; Bartsch, Jörg W.

2018-01-01

Brusatol (BR) is a potent inhibitor of Nrf2, a transcription factor that is highly expressed in cancer tissues and confers chemoresistance. UVA-generated reactive oxygen species (ROS) can damage both normal and cancer cells and may be of potential use in phototherapy. In order to provide an alternative method to treat the aggressive melanoma, we sought to investigate whether low-dose UVA with BR is more effective in eliminating melanoma cells than the respective single treatments. We found that BR combined with UVA led to inhibition of A375 melanoma cell proliferation by cell cycle arrest in the G1 phase and triggers cell apoptosis. Furthermore, inhibition of Nrf2 expression attenuated colony formation and tumor development from A375 cells in heterotopic mouse models. In addition, cotreatment of UVA and BR partially suppressed Nrf2 and its downstream target genes such as HO-1 along with the PI3K/AKT pathway. We propose that cotreatment increased ROS-induced cell cycle arrest and cellular apoptosis and inhibits melanoma growth by regulating the AKT-Nrf2 pathway in A375 cells which offers a possible therapeutic intervention strategy for the treatment of human melanoma. PMID:29670684

Cell‑specific and roasting‑dependent regulation of the Keap1/Nrf2 pathway by coffee extracts.

PubMed

Priftis, Alexandros; Angeli-Terzidou, Antonia-Eugenia; Veskoukis, Aristidis S; Spandidos, Demetrios A; Kouretas, Dimitrios

2018-06-01

Coffee is a popular beverage that contains various bioactive compounds. However, its molecular mechanism of action is not fully elucidated. In this context, two previously characterized coffee extracts, a lightly roasted and the corresponding green one, were investigated for their effect on nuclear factor erythroid 2‑related factor 2 (Nrf2) target gene expression in myoblasts and endothelial cells using quantitative PCR. The tested concentrations were non‑cytotoxic and led to improved redox cell status, as was evident by increased reduced glutathione (GSH) levels. In both cell lines, the roasted extract upregulated gene expression more readily than its green counterpart leading to increased NAD(P)H quinone dehydrogenase 1 and γ‑glutamyl cysteine ligase catalytic subunit, among others. The green extract had a mixed effect on the endothelial cells, while, as regards the myoblasts it caused the downregulation of some Nrf‑target genes. Therefore, a potential dose‑ and roasting‑dependent mechanism is proposed in the current study, accounting for coffee's antioxidant activity.

Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2.

PubMed

Sun, Chengcao; Yang, Cuili; Xue, Ruilin; Li, Shujun; Zhang, Ting; Pan, Lei; Ma, Xuejiao; Wang, Liang; Li, Dejia

2015-01-15

Sulforaphane (SFN), one of the most important isothiocyanates in the human diet, is known to have chemo-preventive and antioxidant activities in different tissues via activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of SFN on Duchenne muscular dystrophy. Four-week-old mdx mice were treated with SFN by gavage (2 mg·kg body wt(-1)·day(-1) for 8 wk), and our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1 with a Nrf2-dependent manner. SFN significantly increased skeletal muscle mass, muscle force (∼30%), running distance (∼20%), and GSH-to-GSSG ratio (∼3.2-fold) of mdx mice and decreased the activities of plasma creatine phosphokinase (∼45%) and lactate dehydrogenase (∼40%), gastrocnemius hypertrophy (∼25%), myocardial hypertrophy (∼20%), and malondialdehyde levels (∼60%). Furthermore, SFN treatment also reduced the central nucleation (∼40%), fiber size variability, and inflammation and improved the sarcolemmal integrity of mdx mice. Collectively, these results show that SFN can improve muscle function and pathology and protect dystrophic muscle from oxidative damage in mdx mice associated with Nrf2 signaling pathway, which indicate Nrf2 may have clinical implications for the treatment of patients with muscular dystrophy. Copyright © 2015 the American Physiological Society.

Nrf2 protects against airway disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Hye-Youn, E-mail: cho2@niehs.nih.go; Kleeberger, Steven R.

Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master transcription factor that regulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. In the unstressed condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and drives its proteasomal degradation. Nrf2 can be activated by diverse stimuli including oxidants, pro-oxidants, antioxidants, and chemopreventive agents. Nrf2 induces cellular rescue pathways against oxidative injury, abnormal inflammatory and immune responses, apoptosis, and carcinogenesis. Application of Nrf2 germ-line mutant mice has identified an extensive range of protective roles for Nrf2 in experimental models of human disorders in the liver,more » gastrointestinal tract, airway, kidney, brain, circulation, and immune or nerve system. In the lung, lack of Nrf2 exacerbated toxicity caused by multiple oxidative insults including supplemental respiratory therapy (e.g., hyperoxia, mechanical ventilation), cigarette smoke, allergen, virus, bacterial endotoxin and other inflammatory agents (e.g., carrageenin), environmental pollution (e.g., particles), and a fibrotic agent bleomycin. Microarray analyses and bioinformatic studies elucidated functional AREs and Nrf2-directed genes that are critical components of signaling mechanisms in pulmonary protection by Nrf2. Association of loss of function with promoter polymorphisms in NRF2 or somatic and epigenetic mutations in KEAP1 and NRF2 has been found in cohorts of patients with acute lung injury/acute respiratory distress syndrome or lung cancer, which further supports the role for NRF2 in these lung diseases. In the current review, we address the role of Nrf2 in airways based on emerging evidence from experimental oxidative disease models and human studies.« less

Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

PubMed Central

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

2014-01-01

Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

PubMed

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

2013-10-10

Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

The Nrf2/Keap1/ARE Pathway and Oxidative Stress as a Therapeutic Target in Type II Diabetes Mellitus

PubMed Central

2017-01-01

Despite improvements in awareness and treatment of type II diabetes mellitus (TIIDM), this disease remains a major source of morbidity and mortality worldwide, and prevalence continues to rise. Oxidative damage caused by free radicals has long been known to contribute to the pathogenesis and progression of TIIDM and its complications. Only recently, however, has the role of the Nrf2/Keap1/ARE master antioxidant pathway in diabetic dysfunction begun to be elucidated. There is accumulating evidence that this pathway is implicated in diabetic damage to the pancreas, heart, and skin, among other cell types and tissues. Animal studies and clinical trials have shown promising results suggesting that activation of this pathway can delay or reverse some of these impairments in TIIDM. In this review, we outline the role of oxidative damage and the Nrf2/Keap1/ARE pathway in TIIDM, focusing on current and future efforts to utilize this relationship as a therapeutic target for prevention, prognosis, and treatment of TIID. PMID:28913364

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus.

PubMed

Burroughs, Andrea Flores; Eluhu, Sylvia; Whalen, Diva; Goodwin, J Shawn; Sakwe, Amos M; Arinze, Ifeanyi J

2018-05-22

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

The Keap1/Nrf2 pathway in health and disease: from the bench to the clinic.

PubMed

O'Connell, Maria A; Hayes, John D

2015-08-01

The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, with gene called NFE2L2) is a master regulator of the antioxidant response. In the last decade, interest has intensified in this research area as its importance in several physiological and pathological processes has become widely recognized; these include redox signalling and redox homoeostasis, drug metabolism and disposition, intermediary metabolism, cellular adaptation to stress, chemoprevention and chemoresistance, toxicity, inflammation, neurodegeneration, lipogenesis and aging. Regulation of Nrf2 is complex and although much attention has focussed on its repression by Kelch-like ECH-associated protein-1 (Keap1), recently it has become increasingly apparent that it is also controlled by cross-talk with other signalling pathways including the glycogen synthase kinase-3 (GSK-3)-β-transducin repeat-containing protein (β-TrCP) axis, ERAD (endoplasmic reticulum-associated degradation)-associated E3 ubiquitin-protein ligase (Hrd1, also called synoviolin), nuclear factor-kappa B (NF-κB), Notch and AMP kinase. Due to its beneficial role in several diseases, Nrf2 has become a major therapeutic target, with novel natural, synthetic and targeted small molecules currently under investigation to modulate the pathway and in clinical trials. © 2015 Authors; published by Portland Press Limited.

Effect of natural and semi-synthetic cadinanes from Heterotheca inuloides on NF-κB, Nrf2 and STAT3 signaling pathways and evaluation of their in vitro cytotoxicity in human cancer cell lines.

PubMed

Egas, Verónica; Millán, Estrella; Collado, Juan A; Ramírez-Apan, Teresa; Méndez-Cuesta, Carlos A; Muñoz, Eduardo; Delgado, Guillermo

2017-06-15

The effects of ten natural cadinane sesquiterpenoids isolated from Heterotheca inuloides on the pathways of the NF-κB, Nrf2 and STAT3 transcription factors were studied for the first time. The main constituent in this species, 7-hydroxy-3,4-dihydrocadalene (1), showed anti-NF-κB activity and activated the antioxidant Nrf2 pathway, which may explain the properties reported for the traditional use of the plant. In addition to the main metabolite, a structurally similar compound, 7-hydroxy-cadalene (2), also displayed anti-NF-κB activity. Thus, both natural compounds were used as templates for the preparation of a novel semi-synthetic derivative set, including esters and carbamates, which were evaluated for their potential in vitro antiproliferative activities against six human cancer cell lines. Carbamate derivatives 32 and 33 were found to exhibit potent activity against human colorectal adenocarcinoma and showed important selectivity in cancer cells. Among ester derivatives, compound 13 was determined to be a more potent NF-κB inhibitor and Nrf2 activator than its parent, 7-hydroxy-3,4-dihydrocadalene (1). Furthermore, this compound decreases levels of phospho-IκBα, a protein complex involved in the NF-κB activation pathway. Molecular simulations suggest that all active compounds interact with the activation loop of the IKKβ subunit in the IKK complex, which is the responsible of IκBα phosphorylation. Thus, we identified two natural, and one semi-synthetic, NF-κB and Nrf2 modulators and two new promising cytotoxic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways

PubMed Central

Singh, Bhupendra; Shoulson, Rivka; Chatterjee, Anwesha; Ronghe, Amruta; Bhat, Nimee K.; Dim, Daniel C.; Bhat, Hari K.

2014-01-01

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17β-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways. PMID:24894866

Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia.

PubMed

Ungvari, Zoltan; Bailey-Downs, Lora; Gautam, Tripti; Jimenez, Rosario; Losonczy, Gyorgy; Zhang, Cuihua; Ballabh, Praveen; Recchia, Fabio A; Wilkerson, Donald C; Sonntag, William E; Pearson, Kevin; de Cabo, Rafael; Csiszar, Anna

2011-04-01

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10-30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H(2)O(2) mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2(+/+) and Nrf2(-/-) mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2(+/+) mice, but not Nrf2(-/-) mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2(-/-) than Nrf2(+/+) mice. HFD-induced endothelial dysfunction was more severe in Nrf2(-/-) mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2(+/+) mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

Canonical and non-canonical mechanisms of Nrf2 activation.

PubMed

Silva-Islas, Carlos Alfredo; Maldonado, Perla D

2018-06-15

Nuclear Factor Erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of genes involved in the metabolism, immune response, cellular proliferation, and other processes; however, the attention has been focused on the study of its ability to induce the expression of proteins involved in the antioxidant defense. Nrf2 is mainly regulated by Kelch-like ECH-associated protein 1 (Keap1), an adapter substrate of Cullin 3 (Cul3) ubiquitin E3 ligase complex. Keap1 represses Nrf2 activity in the cytoplasm by its sequestering, ubiquitination and proteosomal degradation. Nrf2 activation, through the canonical mechanism, is carried out by electrophilic compounds and oxidative stress where some cysteine residues in Keap1 are oxidized, resulting in a decrease in Nrf2 ubiquitination and an increase in its nuclear translocation and activation. In the nucleus, Nrf2 induces a variety of genes involved in the antioxidant defense. Recently a new mechanism of Nrf2 activation has been described, called the non-canonical pathway, where proteins such as p62, p21, dipeptidyl peptidase III (DPP3), wilms tumor gene on X chromosome (WTX) and others are able to disrupt the Nrf2-Keap1 complex, by direct interaction with Keap1 decreasing Nrf2 ubiquitination and increasing its nuclear translocation and activation. In this review, the regulatory mechanisms involved in both canonical and non-canonical Nrf2 activation are discussed. Copyright © 2018. Published by Elsevier Ltd.

An overview of the molecular mechanisms and novel roles of Nrf2 in neurodegenerative disorders.

PubMed

Yang, Yang; Jiang, Shuai; Yan, Juanjuan; Li, Yue; Xin, Zhenlong; Lin, Yan; Qu, Yan

2015-02-01

Recently, growing evidence has demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of endogenous defense systems that function via the activation of a set of protective genes, and this is particularly clear in the central nervous system (CNS). Therefore, it is highly useful to summarize the current literature on the molecular mechanisms and role of Nrf2 in the CNS. In this review, we first briefly introduce the molecular features of Nrf2. We then discuss the regulation, cerebral actions, upstream modulators and downstream targets of Nrf2 pathway. Following this background, we expand our discussion to the role of Nrf2 in several major neurodegenerative disorders (NDDs) such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis. Lastly, we discuss some potential future directions. The information reviewed here may be significant in the design of further experimental research and increase the potential of Nrf2 as a therapeutic target in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bardoxolone Brings Nrf2-Based Therapies to Light

PubMed Central

2013-01-01

Abstract The targeted activation of nuclear factor erythroid-derived-2-like 2 (Nrf2) to alleviate symptoms of chronic kidney disease has recently garnered much attention. Unfortunately, the greatest clinical success to date, bardoxolone, failed in phase III clinical trial for unspecified safety reasons. The present letter to the editor discusses the clinical development of bardoxolone and explores potential reasons for the ultimate withdrawal from clinical trials. In particular, was the correct clinical indication pursued and would improved specificity have mitigated the safety concerns? Ultimately, it is concluded that the right clinical indication and heightened specificity will lead to successful Nrf2-based therapies. Therefore, the bardoxolone clinical results do not dampen enthusiasm for Nrf2-based therapies; rather it illuminates the clinical potential of the Nrf2 pathway as a drug target. Antioxid. Redox Signal. 19, 517–518. PMID:23227819

Targeting the Transcription Factor Nrf2 to Ameliorate Oxidative Stress and Inflammation in Chronic Kidney Disease

PubMed Central

Ruiz, Stacey; Pergola, Pablo E.; Zager, Richard A.; Vaziri, Nosratola D.

2012-01-01

Oxidative stress and inflammation are mediators in the development and progression of chronic kidney disease (CKD) and its complications, and they are inseparably linked as each begets and amplifies the other. CKD-associated oxidative stress is due to increased production of reactive oxygen species (ROS) and diminished antioxidant capacity. The latter is largely caused by impaired activation of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules. Protective effects of Nrf2 are evidenced by amelioration of oxidative stress, inflammation, and kidney disease in response to natural Nrf2 activators in animal models, while Nrf2 deletion amplifies these pathogenic pathways and leads to autoimmune nephritis. Given the role of impaired Nrf2 activity in CKD-induced oxidative stress and inflammation, interventions aimed at restoring Nrf2 may be effective in retarding CKD progression. Clinical trials of the potent Nrf2 activator bardoxolone methyl showed significant improvement in renal function in CKD patients with type 2 diabetes. Results of the ongoing BEACON trial investigating the effect of this drug on time to end-stage renal disease or cardiovascular death will help further characterize the efficacy of Nrf2 pharmacological modulation in CKD. This article provides an overview of the role of impaired Nrf2 activity in the pathogenesis of CKD-associated oxidative stress and inflammation and the potential utility of targeting Nrf2 in the treatment of CKD. PMID:23325084

EGCG evokes Nrf2 nuclear translocation and dampens PTP1B expression to ameliorate metabolic misalignment under insulin resistance condition.

PubMed

Mi, Yashi; Zhang, Wentong; Tian, Haoyu; Li, Runnan; Huang, Shuxian; Li, Xingyu; Qi, Guoyuan; Liu, Xuebo

2018-03-01

As a major nutraceutical component of green tea (-)-epigallocatechin-3-gallate (EGCG) has attracted interest from scientists due to its well-documented antioxidant and antiobesity bioactivities. In the current study, we aimed to investigate the protective effect of EGCG on metabolic misalignment and in balancing the redox status in mice liver and HepG2 cells under insulin resistance condition. Our results indicated that EGCG accelerates the glucose uptake and evokes IRS-1/Akt/GLUT2 signaling pathway via dampening the expression of protein tyrosine phosphatase 1B (PTP1B). Consistently, ectopic expression of PTP1B by Ad-PTP1B substantially impaired EGCG-elicited IRS-1/Akt/GLUT2 signaling pathway. Moreover, EGCG co-treatment stimulated nuclear translocation of Nrf2 by provoking P13K/AKT signaling pathway and thus modulated the downstream expressions of antioxidant enzymes such as HO-1 and NQO-1 in HepG2 cells. Furthermore, knockdown Nrf2 by small interfering RNA (siRNA) notably enhanced the expression of PTP1B and blunt EGCG-stimulated glucose uptake. Consistent with these results, in vivo study revealed that EGCG supplement significantly ameliorated high-fat and high-fructose diet (HFFD)-triggered insulin resistance and oxidative stress by up-regulating the IRS-1/AKT and Keap1/Nrf2 transcriptional pathways. Administration of an appropriate chemopreventive agent, such as EGCG, could potentially serve as an additional therapeutic intervention in the arsenal against obesity.

Fimasartan, a Novel Angiotensin-Receptor Blocker, Protects against Renal Inflammation and Fibrosis in Mice with Unilateral Ureteral Obstruction: the Possible Role of Nrf2

PubMed Central

Kim, Soojeong; Kim, Sung Jun; Yoon, Hye Eun; Chung, Sungjin; Choi, Bum Soon; Park, Cheol Whee; Shin, Seok Joon

2015-01-01

Objectives: A newly developed angiotensin II receptor blocker, fimasartan, is effective in lowering blood pressure through its action on the renin-angiotensin system. Renal interstitial fibrosis, believed to be due to oxidative injury, is an end-stage process in the progression of chronic kidney disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to regulate cellular oxidative stress and induce expression of antioxidant genes. In this study we investigated the role of Nrf2 in fimasartan-mediated antioxidant effects in mice with renal fibrosis induced by unilateral ureteral obstruction (UUO). Materials and Methods: UUO was induced surgically in mice, followed by either no treatment with fimasartan or the intraperitoneal administration of fimasartan (3 mg/kg/day). On day 7, we evaluated the changes in the renin-angiotensin system (RAS) and the expression of Nrf2 and its downstream antioxidant genes, as well as renal inflammation, apoptosis, and fibrosis in the obstructed kidneys. The effect of fimasartan on the Nrf2 pathway was also investigated in HK-2 cells stimulated by tumor necrosis factor-α. Results: The mice with surgically induced UUO showed increased renal inflammation and fibrosis as evidenced by histopathologic findings and total collagen content in the kidney. These effects were attenuated in the obstructed kidneys of the fimasartan-treated mice. Fimasartan treatment inhibited RAS activation and the expression of Nox1, Nox2, and Nox4. In contrast, fimasartan upregulated the renal expression of Nrf2 and its downstream signaling molecules (such as NQO1; HO-1; GSTa2 and GSTm3). Furthermore, it increased the expression of antioxidant enzymes, including CuSOD, MnSOD, and catalase. The fimasartan-treated mice had significantly less apoptosis on TUNEL staining, with decreased levels of pro-apoptotic protein and increased levels of anti-apoptotic protein. In the HK-2 cells, fimasartan treatment inhibited RAS activation, decreased expression of mitogen-activated protein kinases (MAPKs), and upregulated the Nrf2 pathway. Conclusions: These results suggest that fimasartan has beneficial effects in reducing renal oxidative stress, inflammation, and fibrosis. Possible mechanisms to explain these effects are inhibition of RAS and MAPKs and upregulation of Nrf2 signaling, with subsequent induction of antioxidant pathways. PMID:26640409

Simvastatin Treatment Upregulates HO-1 in Patients with Abdominal Aortic Aneurysm but Independently of Nrf2

PubMed Central

Kopacz, Aleksandra; Kloska, Damian; Zagrapan, Branislav; Neumayer, Christoph; Grochot-Przeczek, Anna; Huk, Ihor; Brostjan, Christine; Dulak, Jozef

2018-01-01

Heme oxygenase-1 (HO-1), encoded by HMOX1 gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin (N = 28) or without statins (N = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes HMOX1, NQO1, and GCLM expression remained unchanged in AAA. In vitro studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although HMOX1 expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs in vitro is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including HMOX1, in AAA tissue denotes Nrf2 independency. PMID:29743974

The Nrf2-antioxidant response element pathway: a target for regulating energy metabolism

USDA-ARS?s Scientific Manuscript database

The nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that responds to oxidative stress by binding to the antioxidant response element (ARE) in the promoter of genes coding for antioxidant enzymes like NAD(P)H:quinone oxidoreductase 1 (NQO1) and proteins for glutathione synthesis. ...

Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators

PubMed Central

Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.

2011-01-01

SUMMARY The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin—induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism. PMID:21700211

Palmitate-induced endothelial dysfunction is attenuated by cyanidin-3-O-glucoside through modulation of Nrf2/Bach1 and NF-κB pathways.

PubMed

Fratantonio, D; Speciale, A; Ferrari, D; Cristani, M; Saija, A; Cimino, F

2015-12-15

Free fatty acids (FFA), commonly elevated in diabetes and obesity, have been shown to impair endothelial functions and cause oxidative stress, inflammation, and insulin resistance. Anthocyanins represent one of the most important and interesting classes of flavonoids and seem to play a role in preventing cardiovascular diseases. Herein, we investigated the in vitro protective effects of cyanidin-3-O-glucoside (C3G) on cell signaling pathways in human umbilical vein endothelial cells (HUVECs) exposed to palmitic acid (PA), the most prevalent saturated FFA in circulation. Our data reported a significant augmentation of free radicals and oxidative stress in HUVECs exposed to PA for 3h, while C3G pretreatment improved intracellular redox status altered by FFA. Moreover, C3G significantly inhibited NF-κB proinflammatory pathway and adhesion molecules induced by PA, and these effects were attributed to the activation of Nrf2/EpRE pathway. In fact, C3G induced Nrf2 nuclear localization and activation of cellular antioxidant and cytoprotective genes at baseline and after PA exposure in endothelial cells. Our data confirm the hypothesis that natural Nrf2 inducers, such as C3G, might be a potential therapeutic strategy to protect vascular system against various stressors preventing several pathological conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Hydrogen-Rich Water Intake Accelerates Oral Palatal Wound Healing via Activation of the Nrf2/Antioxidant Defense Pathways in a Rat Model

PubMed Central

Orihuela-Campos, Rita Cristina; Fukui, Makoto; Ito, Hiro-O

2016-01-01

The wound healing process attempts to restore the integrity and function of the injured tissue. Additionally, proinflammatory cytokines, growth factors, and oxidative stress play important roles in wound healing. The aim of this study was to determine whether hydrogen-rich water intake induces the activation of the Nrf2/antioxidant defense pathway in rat palatal tissue, thereby reducing systemic oxidative stress and proinflammatory cytokine levels and promoting healing-associated genes. A circular excisional wound was created in the oral palatal region, and the wound healing process was observed. The rats were divided into two experimental groups in which either hydrogen-rich water or distilled water was consumed. In the drinking hydrogen-rich water, the palatal wound healing process was accelerated compared to that in the control group. As molecular hydrogen upregulated the Nrf2 pathway, systemic oxidative stresses were decreased by the activation of antioxidant activity. Furthermore, hydrogen-rich water intake reduced proinflammatory cytokine levels and promoted the expression of healing-associated factors in rat palatal tissue. In conclusion, hydrogen-rich water intake exhibited multiple beneficial effects through activation of the Nrf2/antioxidant defense pathway. The results of this study support the hypothesis that oral administration of hydrogen-rich water benefits the wound healing process by decreasing oxidative stress and inflammatory responses. PMID:26798423

Hydrogen-Rich Water Intake Accelerates Oral Palatal Wound Healing via Activation of the Nrf2/Antioxidant Defense Pathways in a Rat Model.

PubMed

Tamaki, Naofumi; Orihuela-Campos, Rita Cristina; Fukui, Makoto; Ito, Hiro-O

2016-01-01

The wound healing process attempts to restore the integrity and function of the injured tissue. Additionally, proinflammatory cytokines, growth factors, and oxidative stress play important roles in wound healing. The aim of this study was to determine whether hydrogen-rich water intake induces the activation of the Nrf2/antioxidant defense pathway in rat palatal tissue, thereby reducing systemic oxidative stress and proinflammatory cytokine levels and promoting healing-associated genes. A circular excisional wound was created in the oral palatal region, and the wound healing process was observed. The rats were divided into two experimental groups in which either hydrogen-rich water or distilled water was consumed. In the drinking hydrogen-rich water, the palatal wound healing process was accelerated compared to that in the control group. As molecular hydrogen upregulated the Nrf2 pathway, systemic oxidative stresses were decreased by the activation of antioxidant activity. Furthermore, hydrogen-rich water intake reduced proinflammatory cytokine levels and promoted the expression of healing-associated factors in rat palatal tissue. In conclusion, hydrogen-rich water intake exhibited multiple beneficial effects through activation of the Nrf2/antioxidant defense pathway. The results of this study support the hypothesis that oral administration of hydrogen-rich water benefits the wound healing process by decreasing oxidative stress and inflammatory responses.

Discovery of the first dual GSK3β inhibitor/Nrf2 inducer. A new multitarget therapeutic strategy for Alzheimer’s disease

NASA Astrophysics Data System (ADS)

Gameiro, Isabel; Michalska, Patrycja; Tenti, Giammarco; Cores, Ángel; Buendia, Izaskun; Rojo, Ana I.; Georgakopoulos, Nikolaos D.; Hernández-Guijo, Jesús M.; Teresa Ramos, María; Wells, Geoffrey; López, Manuela G.; Cuadrado, Antonio; Menéndez, J. Carlos; León, Rafael

2017-03-01

The formation of neurofibrillary tangles (NFTs), oxidative stress and neuroinflammation have emerged as key targets for the treatment of Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder. These pathological hallmarks are closely related to the over-activity of the enzyme GSK3β and the downregulation of the defense pathway Nrf2-EpRE observed in AD patients. Herein, we report the synthesis and pharmacological evaluation of a new family of multitarget 2,4-dihydropyrano[2,3-c]pyrazoles as dual GSK3β inhibitors and Nrf2 inducers. These compounds are able to inhibit GSK3β and induce the Nrf2 phase II antioxidant and anti-inflammatory pathway at micromolar concentrations, showing interesting structure-activity relationships. The association of both activities has resulted in a remarkable anti-inflammatory ability with an interesting neuroprotective profile on in vitro models of neuronal death induced by oxidative stress and energy depletion and AD. Furthermore, none of the compounds exhibited in vitro neurotoxicity or hepatotoxicity and hence they had improved safety profiles compared to the known electrophilic Nrf2 inducers. In conclusion, the combination of both activities in this family of multitarget compounds confers them a notable interest for the development of lead compounds for the treatment of AD.

Discovery of the first dual GSK3β inhibitor/Nrf2 inducer. A new multitarget therapeutic strategy for Alzheimer’s disease

PubMed Central

Gameiro, Isabel; Michalska, Patrycja; Tenti, Giammarco; Cores, Ángel; Buendia, Izaskun; Rojo, Ana I.; Georgakopoulos, Nikolaos D.; Hernández-Guijo, Jesús M.; Teresa Ramos, María; Wells, Geoffrey; López, Manuela G.; Cuadrado, Antonio; Menéndez, J. Carlos; León, Rafael

2017-01-01

The formation of neurofibrillary tangles (NFTs), oxidative stress and neuroinflammation have emerged as key targets for the treatment of Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder. These pathological hallmarks are closely related to the over-activity of the enzyme GSK3β and the downregulation of the defense pathway Nrf2-EpRE observed in AD patients. Herein, we report the synthesis and pharmacological evaluation of a new family of multitarget 2,4-dihydropyrano[2,3-c]pyrazoles as dual GSK3β inhibitors and Nrf2 inducers. These compounds are able to inhibit GSK3β and induce the Nrf2 phase II antioxidant and anti-inflammatory pathway at micromolar concentrations, showing interesting structure-activity relationships. The association of both activities has resulted in a remarkable anti-inflammatory ability with an interesting neuroprotective profile on in vitro models of neuronal death induced by oxidative stress and energy depletion and AD. Furthermore, none of the compounds exhibited in vitro neurotoxicity or hepatotoxicity and hence they had improved safety profiles compared to the known electrophilic Nrf2 inducers. In conclusion, the combination of both activities in this family of multitarget compounds confers them a notable interest for the development of lead compounds for the treatment of AD. PMID:28361919

Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es; Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es; Berzal, Sergio, E-mail: sberzal@fjd.es

The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed thatmore » in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory events in mice and human.« less

Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

PubMed Central

Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R.; Paules, Richard S.; Xia, Menghang; Auerbach, Scott S.

2017-01-01

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals. PMID:28531190

Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats.

PubMed

Di, Wei; Shi, Xiaolei; Lv, Hua; Liu, Jun; Zhang, Hong; Li, Zhiwei; Fang, Yannan

2016-12-01

Antioxidants have been proven to weaken hyperalgesia in neuropathic pain. Endogenous antioxidant defense system may have a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in regulating the activation of the trigeminovascular system (TGVS) and hypersensitivity in nitroglycerin (NTG)-induced hyperalgesia rats. The expression levels of Nrf2, HO, HO1, and NQO1 in the trigeminal nucleus caudalis (TNC) were detected by western blot. Immunofluorescence was used to demonstrate the cell-specific localization of Nrf2 in TNC. Sulforaphane, a Nrf2 activator, was administered to NTG-induced rats. Then, the number of c-Fos- and nNOS-immunoreactive neurons in TNC was evaluated using immunofluorescence, and c-Fos and nNOS protein levels were quantified using western blot. Von Frey hair testing was used to evaluate the tactile thresholds of rats at different time points in different groups. Total cellular and nuclear levels of the proteins Nrf2, HO1, and NQO1 were elevated in TNC after NTG injection, and Nrf2 was found to be located in the nucleus and cytoplasm of the neurons. Sulforaphane pretreatment significantly increased the nuclear Nrf2, HO1, and NQO1 levels in TNC. In addition, sulforaphane exposure effectively inhibited the expression of nNOS and c-Fos, reduced the number of nNOS and c-Fos immunoreactive neurons in TNC, and attenuated the tactile thresholds induced by NTG injection. Oxidative stress was involved in nitroglycerin-induced hyperalgesia. Activation of the Nrf2/ARE pathway inhibited the activation of TGVS and prevented the induction of hyperalgesia. Sulforaphane might therefore be an effective agent for hyperalgesia. Further studies are needed to discover the underlying mechanisms of the process.

The Anti-Aging Effect of Erythropoietin via the ERK/Nrf2-ARE Pathway in Aging Rats.

PubMed

Wu, Haiqin; Zhao, Jiaxin; Chen, Mengyi; Wang, Huqing; Yao, Qingling; Fan, Jiaxin; Zhang, Meng

2017-03-01

Erythropoietin (EPO) has a neuroprotective effect and can resist aging, which most likely occur through EPO increasing the activity of antioxidant enzymes and scavenging free radicals. In this study, we verified the anti-aging function of EPO and discussed the mechanism occurring through the extracellular signal-regulated kinase (ERK)/NF-E2-related factor 2 (Nrf2)-ARE pathway. A rat model of aging was induced by the continuous subcutaneous injection of 5 % D-galactose for 6 weeks. At the beginning of the sixth week, physiological saline or EPO was administered twice per day through a lateral ventricle system for a total of 7 days. In one group, 2 μl PD98059 was administered 30 min before EPO. Learning and memory ability were analyzed with the Morris water maze system. HE staining was used to observe the morphological changes in the neurons in the hippocampus, and immunohistochemical staining as well as Western blots were carried out to detect the expression of ERK for each group of rats and the expression of phosphorylated-ERK (P-ERK), Nrf2, and superoxide dismutase (SOD). Real-Time PCR was carried out to detect the amount of Nrf2 mRNA and the KEAP1 mRNA expression. EPO can significantly improve learning and memory ability in aging rats and can provide protection against aging by improving the hippocampus morphology. Immunohistochemical staining and Western blots showed P-ERK, Nrf2, and Cu-Zn SOD decreases in aging rats compared to the normal group, while the expression for those proteins increased after EPO intervention. PD98059 inhibited the enhanced expression of P-ERK, Nrf2, and Cu-Zn SOD induced by EPO. Real-Time PCR results suggested that the trend of Nrf2mRNA expression was the same as that for the proteins, which confirmed that the enhancement occurred at the gene level. As such, EPO can significantly resist or delay aging and protect the brain by reducing oxidative stress. The most likely mechanism is that EPO can promote the ERK/Nrf2-ARE pathway in aging rats and that PD98059 can inhibit that process. These findings may facilitate further studies on the mechanism of aging and applications for the neuroprotective properties of EPO for clinical treatments.

Signal transduction involved in lipoxin A4-induced protection of tubular epithelial cells against hypoxia/reoxygenation injury

PubMed Central

Wu, Sheng-Hua; Wang, Ming-Jie; Lü, Jing; Chen, Xiao-Qing

2017-01-01

Previous studies have reported that lipoxin A4 (LXA4) may exert a renoprotective effect on ischemia/reperfusion injury in various animal models. The underlying mechanism of LXA4-induced renoprotection during ischemia/reperfusion injury remains to be elucidated. The present study investigated LXA4-induced protection on renal tubular cells subjected to hypoxia/reoxygenation (H/R) injury, and determined the effects of peroxisome proliferator-activated receptor-γ (PPARγ) and heme oxygenase-1 (HO-1) on LXA4 treatment. HK-2 human tubular epithelial cells exposed to H/R injury were pretreated with LXA4, signal molecule inhibitors or the HO-1 inhibitor zinc protoporphyrin-IX, or were transfected with PPARγ small interfering RNA (siRNA) or nuclear factor E2-related factor 2 (Nrf2) siRNA. The protein and mRNA expression levels of PPARγ and HO-1 were analyzed using western blotting and reverse transcription-quantitative polymerase chain reaction. Binding activity of Nrf2 to the HO-1 E1 enhancer was determined using chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) was assessed using electrophoretic mobility shift assay. Preincubation of cells with LXA4 exposed to H/R injury led to a decreased production of inducible nitrogen oxide synthase, malondialdehyde, γ-glutamyl transpeptidase, leucine aminopeptidase and N-acetyl-β-glucosaminidase. In addition, LXA4 pretreatment increased cell viability, protein and mRNA expression levels of PPARγ and HO-1 and PPARγ and HO-1 promoter activity. SB20358 is a p38 mitogen-activated protein kinase (p38 MAPK) pathway inhibitor, which reduced LXA4-induced PPARγ expression levels. LXA4 treatment upregulated p38 MAPK activation, Nrf2 nuclear translocation and increased binding activity of Nrf2 to HO-1 ARE and E1 enhancer in cells exposed to H/R injury. Transfection of the cells with PPARγ siRNA reduced the LXA4-induced Nrf2 translocation. Transfection of the cells with PPARγ siRNA or Nrf2 siRNA also reduced the LXA4-induced increase in HO-1 expression. In conclusion, LXA4-induced protection of renal tubular cells against H/R injury was associated with the induction of PPARγ and HO-1, via activation of the p38 MAPK pathway, as well as Nrf2 nuclear translocation and binding to HO-1 ARE and E1 enhancer. Therefore, LXA4-induced renoprotection is associated with activation of the p38 MAPK/PPARγ/Nrf2-ARE/HO-1 pathway. PMID:28259922

Insulin is a potential antioxidant for diabetes-associated cognitive decline via regulating Nrf2 dependent antioxidant enzymes.

PubMed

Song, Ying; Ding, Wei; Bei, Yun; Xiao, Yan; Tong, Hai-Da; Wang, Li-Bo; Ai, Li-Yao

2018-05-21

To investigate the neuroprotective effects of insulin on diabetic encephalopathy and its mechanism. The diabetic model was established by injection of streptozotocin. Behavior examinations were conducted by the Morris water maze. Histopathological alterations were detected by HE staining. ROS, CAT levels and SOD activity were measured using a microplate reader. In vitro, the viability of wild type and knock-down PC12 cells was detected by MTT assay, the morphology of cells was monitored under a microscope. The subcellular distribution of Nrf2 was observed by western blotting and immunohistochemistry. Evident oxidative stress injury was observed in diabetic rats and H 2 O 2 -induced PC12 cells. Insulin not only protect diabetic rat from oxidative stress injury but also significantly inhibited H 2 O 2 -induced apoptosis and intracellular ROS in cells. In addition, the level of malondialdehyde was reduced, and the activities of superoxide dismutase, catalase and glutathione peroxidase were augmented in both diabetic rats and PC12 cells. Interestingly, insulin promoted the translocation of Nrf2 into the nucleus and activation of downstream antioxidant protein expression. Further, the Nrf2 knockdown cells suffered more serious H 2 O 2 -induced damage than the wild PC12 cells. Moreover, insulin had no significant protective effect on knockdown cells with H 2 O 2 -damage. Collectively, our results suggested that insulin significantly inhibited neuronal damage through the Nrf2 signaling pathway, which regulates endogenous oxidant-antioxidant balance, therefore, insulin may be a potential protective agent for the treatment of oxidative stress-induced diabetic encephalopathy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Minocycline attenuates sevoflurane-induced cell injury via activation of Nrf2

PubMed Central

Tian, Yue; Wu, Xiuying; Guo, Shanbin; Ma, Ling; Huang, Wei; Zhao, Xiaochun

2017-01-01

Minocycline has been demonstrated to exert neuroprotective effects in various experimental models. In the present study, we investigated the mechanisms underlying the protective effects of minocycline on cell injury induced by the inhalation of the anesthetic, sevoflurane. In our in vivo experiments using rats, minocycline attenuated sevoflurane-induced neuronal degeneration and apoptosis in the rat hippocampus, and this effect was associated with the minocycline-mediated suppression of oxidative stress in the hippocampus. In in vitro experiments, minocycline inhibited sevoflurane-induced apoptosis and the production of reactive oxygen species (ROS) in H4 human neuroglioma cells. In addition, minocycline suppressed the sevoflurane-induced upregulation of interleukin (IL)-6 and the activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells. Furthermore, we found that nuclear factor E2-related factor 2 (Nrf2), an activator of the stress response, was upregulated and activated upon sevoflurane treatment both in the rat hippocampus and in H4 cells. In addition, minocycline further augmented the upregulation and activation of Nrf2 when used in conjunction with sevoflurane. Moreover, the knockdown of Nrf2 in H4 cells by small interfering RNA (siRNA) diminished the cytoprotective effect of minocycline, and attenuated the inhibitory effect of minocycline on ROS production, IL-6 upregulation and the activation of the NF-κB signaling pathway. On the whole, our findings indicate that minocycline may exert protective effects against sevoflurane-induced cell injury via the Nrf2-modulated antioxidant response and the inhibition of the activation of the NF-κB signaling pathway. PMID:28260081

Dual regulation of skin sensitizer-induced HMOX1 expression by Bach1 and Nrf2: Comparison to regulation of the AKR1C2-ARE element in the KeratinoSens cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emter, Roger; Natsch, Andreas, E-mail: andreas.natsch@givaudan.com

2015-11-01

Heme oxygenase (decycling) 1 (HMOX1) is the most consistently found genetic marker induced by skin sensitizers. HMOX1 is often referred to as typical gene regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), however, it is also regulated by other DNA-binding factors, including BTB and CNC homolog 1 (Bach1). The KeratinoSens™ assay is the first validated in vitro assay for sensitizers that measures gene induction. It is based on luciferase expression regulated by the antioxidant response element (ARE) of the aldoketoreductase 1C2 (AKR1C2) gene. Luciferase upregulation is dependent on Nrf2, while HMOX1 upregulation is only partially Nrf2-dependent. Thus, sensitizer-dependent activationmore » of HMOX1 may integrate multiple signals thereby providing additional information. We constructed reporter cell lines containing the full HMOX1 regulatory region or the HMOX1-ARE sequence and compared them with the construct containing the AKR1C2-ARE sequence. Induction of the AKR1C2-ARE depends on Nrf2, but not on the repressor Bach1. Results obtained with HMOX1-ARE and the full HMOX1 promoter indicate that, within the HMOX1 promoter, the HMOX1-ARE is sufficient to explain the induction by sensitizers and that (i) inhibiting Bach1 leads to strong basal expression, (ii) fold-induction by sensitizers above this level is reduced in the absence of Bach1 and (iii) these constructs are less dependent on Nrf2 as compared to the AKR1C2-ARE. Nevertheless, congruent dose response curves for luciferase activity were obtained with all constructs. Thus, while sensitizer-induced HMOX1 activation is dependent on Nrf2 and Bach1, all constructs give identical information for the in vitro prediction of the sensitization potential. - Highlights: • HMOX1 is a key genetic marker up-regulated by skin sensitizers. • HMOX1-, but not AKR1C2-upregulation, is dependent on both Nrf2 and Bach1. • AKR1C2 and HMOX1-dependent reporter constructs yield congruent dose response curves. • Combining both constructs offers no advantage over either construct used alone.« less

An Essential Role of NRF2 in Diabetic Wound Healing

PubMed Central

Long, Min; Rojo de la Vega, Montserrat; Wen, Qing; Bharara, Manish; Jiang, Tao; Zhang, Rui; Zhou, Shiwen; Wong, Pak K.

2016-01-01

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2–related factor 2 (NRF2)–mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2−/− mice have delayed wound closure rates compared with Nrf2+/+ mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers. PMID:26718502

Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

PubMed

Zhang, Xiuli; Liang, Dan; Lian, Xu; Jiang, Yan; He, Hui; Liang, Wei; Zhao, Yue; Chi, Zhi-Hong

2016-06-01

Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Berberine (BBR) is identified as a potential anti-diabetic herbal medicine due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. In this study, the underlying mechanisms involved in the protective effects of BBR on high glucose-induced apoptosis were explored using cultured renal tubular epithelial cells (NRK-52E cells) and human kidney proximal tubular cell line (HK-2 cells). We identified the pivotal role of phosphatidylinositol 3-kinase (PI3K)/Akt in BBR cellular defense mechanisms and revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2) and heme oxygenase (HO)-1 in NRK-52E and HK-2 cells. BBR attenuated reactive oxygen species production, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (Nrf2 and HO-1), which also were blocked by LY294002 (an inhibitor of PI3K) in HG-treated NRK-52E and HK-2 cells. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential. BBR-induced anti-apoptotic function was demonstrated by decreasing apoptotic proteins (cytochrome c, Bax, caspase3 and caspase9). All these findings suggest that BBR exerts the anti-apoptosis effects through activation of PI3K/Akt signal pathways and leads to activation of Nrf2 and induction of Nrf2 target genes, and consequently protecting the renal tubular epithelial cells from HG-induced apoptosis.

Identification of an Unfavorable Immune Signature in Advanced Lung Tumors from Nrf2-Deficient Mice.

PubMed

Zhang, Di; Rennhack, Jonathan; Andrechek, Eran R; Rockwell, Cheryl E; Liby, Karen T

2018-04-16

Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in normal cells inhibits carcinogenesis, whereas constitutive activation of Nrf2 in cancer cells promotes tumor growth and chemoresistance. However, the effects of Nrf2 activation in immune cells during lung carcinogenesis are poorly defined and could either promote or inhibit cancer growth. Our studies were designed to evaluate tumor burden and identify immune cell populations in the lungs of Nrf2 knockout (KO) versus wild-type (WT) mice challenged with vinyl carbamate. Nrf2 KO mice developed lung tumors earlier than the WT mice and exhibited more and larger tumors over time, even at late stages. T cell populations were lower in the lungs of Nrf2 KO mice, whereas tumor-promoting macrophages and myeloid-derived suppressor cells were elevated in the lungs and spleen, respectively, of Nrf2 KO mice relative to WT mice. Moreover, 34 immune response genes were significantly upregulated in tumors from Nrf2 KO mice, especially a series of cytokines (Cxcl1, Csf1, Ccl9, Cxcl12, etc.) and major histocompatibility complex antigens that promote tumor growth. Our studies discovered a novel immune signature, characterized by the infiltration of tumor-promoting immune cells, elevated cytokines, and increased expression of immune response genes in the lungs and tumors of Nrf2 KO mice. A complementary profile was also found in lung cancer patients, supporting the clinical significance of our findings. Overall, our results confirmed a protective role for Nrf2 in late-stage carcinogenesis and, unexpectedly, suggest that activation of Nrf2 in immune cells may be advantageous for preventing or treating lung cancer. Antioxid. Redox Signal. 00, 000-000.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney

PubMed Central

Walsh, Joanne; Jenkins, Rosalind E.; Wong, Michael H. L.; Rowe, Cliff; Ricci, Emanuele; Ressel, Lorenzo; Fang, Yongxiang; Demougin, Philippe; Vukojevic, Vanja; O’Neill, Paul M.; Goldring, Christopher E.; Kitteringham, Neil R.; Park, B. Kevin; Odermatt, Alex; Copple, Ian M.

2015-01-01

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 hours, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared to wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared to vehicle control. In light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target. PMID:26422507

Sulforaphane Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity by Activating the Keap1-Nrf2-ARE Pathway and Inhibiting the MAPKs and NF-κB Pathways.

PubMed

Jang, Minhee; Cho, Ik-Hyun

2016-05-01

The potential neuroprotective value of sulforaphane (SFN) in Huntington's disease (HD) has not been established yet. We investigated whether SFN prevents and improves the neurological impairment and striatal cell death in a 3-nitropropionic acid (3-NP)-induced mouse model of HD. SFN (2.5 and 5.0 mg/kg/day, i.p.) was given daily 30 min before 3-NP treatment (pretreatment) and from onset/progression/peak points of the neurological scores. Pretreatment with SFN (5.0 mg/kg/day) produced the best neuroprotective effect with respect to the neurological scores and lethality among other conditions. The protective effects due to pretreatment with SFN were associated with the following: suppression of the formation of a lesion area, neuronal death, succinate dehydrogenase activity, apoptosis, microglial activation, and mRNA or protein expression of inflammatory mediators, including tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the striatum after 3-NP treatment. Also, pretreatment with SFN activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and inhibited the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways in the striatum after 3-NP treatment. As expected, the pretreatment with activators (dimethyl fumarate and antioxidant response element inducer-3) of the Keap1-Nrf2-ARE pathway decreased the neurological impairment and lethality after 3-NP treatment. Our findings suggest that SFN may effectively attenuate 3-NP-induced striatal toxicity by activating the Keap1-Nrf2-ARE pathway and inhibiting the MAPKs and NF-κB pathways and that SFN has a wide therapeutic time-window for HD-like symptoms.

NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

USDA-ARS?s Scientific Manuscript database

The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of the expression of several antioxidant genes. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. However little is kno...

Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells.

PubMed

Kanlaya, Rattiyaporn; Khamchun, Supaporn; Kapincharanon, Chompunoot; Thongboonkerd, Visith

2016-07-25

This study evaluated effect of oxalate on epithelial mesenchymal transition (EMT) and potential anti-fibrotic property of epigallocatechin-3-gallate (EGCG). MDCK renal tubular cells were incubated with 0.5 mM sodium oxalate for 24-h with/without 1-h pretreatment with 25 μM EGCG. Microscopic examination, immunoblotting and immunofluorescence staining revealed that oxalate-treated cells gained mesenchymal phenotypes by fibroblast-like morphological change and increasing expression of vimentin and fibronectin, while levels of epithelial markers (E-cadherin, occludin, cytokeratin and ZO-1) were decreased. EGCG pretreatment could prevent all these changes and molecular mechanisms underlying the prevention by EGCG were most likely due to reduced production of intracellular ROS through activation of Nrf2 signaling and increased catalase anti-oxidant enzyme. Knockdown of Nrf2 by small interfering RNA (siRNA) abrogated all the effects of EGCG, confirming that the EGCG protection against oxalate-induced EMT was mediated via Nrf2. Taken together, our data indicate that oxalate turned on EMT of renal tubular cells that could be prevented by EGCG via Nrf2 pathway. These findings also shed light onto development of novel therapeutics or preventive strategies of renal fibrosis in the future.

Mangiferin Upregulates Glyoxalase 1 Through Activation of Nrf2/ARE Signaling in Central Neurons Cultured with High Glucose.

PubMed

Liu, Yao-Wu; Cheng, Ya-Qin; Liu, Xiao-Li; Hao, Yun-Chao; Li, Yu; Zhu, Xia; Zhang, Fan; Yin, Xiao-Xing

2017-08-01

Mangiferin, a natural C-glucoside xanthone, has anti-inflammatory, anti-oxidative, neuroprotective actions. Our previous study showed that mangiferin could attenuate diabetes-associated cognitive impairment of rats by enhancing the function of glyoxalase 1 (Glo-1) in brain. The aim of this study was to investigate whether Glo-1 upregulation by mangiferin in central neurons exposed to chronic high glucose may be related to activation of Nrf2/ARE pathway. Compared with normal glucose (25 mmol/L) culture, Glo-1 protein, mRNA, and activity levels were markedly decreased in primary hippocampal and cerebral cortical neurons cultured with high glucose (50 mmol/L) for 72 h, accompanied by the declined Nrf2 nuclear translocation and protein expression of Nrf2 in cell nucleus, as well as protein expression and mRNA level of γ-glutamylcysteine synthetase (γ-GCS) and superoxide dismutase activity, target genes of Nrf2/ARE signaling. Nonetheless, high glucose cotreating with mangiferin or sulforaphane, a typical inducer of Nrf2 activation, attenuated the above changes in both central neurons. In addition, mangiferin and sulforaphane significantly prevented the formation of advanced glycation end-products (AGEs) reflecting Glo-1 activity, while elevated the level of glutathione, a cofactor of Glo-1 activity and production of γ-GCS, in high glucose cultured central neurons. These findings demonstrated that Glo-1 was greatly downregulated in central neurons exposed to chronic high glucose, which is expected to lead the formation of AGEs and oxidative stress damages. We also proved that mangiferin enhanced the function of Glo-1 under high glucose condition by inducing activation of Nrf2/ARE signaling pathway.

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

PubMed Central

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects. PMID:27547033

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells.

PubMed

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

A novel role of topical iodine in skin: Activation of the Nrf2 pathway.

PubMed

Ben-Yehuda Greenwald, Maya; Frušić-Zlotkin, Marina; Soroka, Yoram; Ben-Sasson, Shmuel; Bianco-Peled, Havazelet; Kohen, Ron

2017-03-01

For a long time iodine has been used as an active dermal agent in the treatment of inflammatory, immune-mediated and infectious diseases. Moreover, topical iodine application has been reported to provide protection against sulfur-mustard-induced skin lesions, heat-induced and acid-induced skin burns in both haired guinea-pigs and mouse ear swelling models. However, the exact mechanism of action underlying these benefits of iodine has not yet been elucidated. In the current study, a novel mechanism of action by which iodine provides skin protection and relief, based on its electrophilic nature, is suggested. This study demonstrates that both iodine and iodide are capable of activating the Nrf2 pathway in human skin. As a result, skin protection against UVB-induced damage was acquired and the secretion of pro-inflammatory cytokines (IL-6, IL-8) from LPS-challenged skin was reduced. Iodide role in the enhanced activation of this pathway is demonstrated. The mode of action by which iodine and iodide activate the Nrf2 pathway is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuroprotective effects of α-iso-cubebene against glutamate-induced damage in the HT22 hippocampal neuronal cell line.

PubMed

Park, Sun Young; Jung, Won Jung; Kang, Jum Soon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

2015-02-01

Since oxidative stress is critically involved in excitotoxic damage, we sought to determine whether the activation of the transcription factors, cAMP-responsive element binding protein (CREB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2, also known as NFE2L2), by α-iso-cubebene is involved in its protective effects against glutamate-induced neuronal cell death. Pre-treatment with α-iso-cubebene significantly attenuated glutamate-induced cytotoxicity in mouse hippocampus-derived neuronal cells. α-iso-cubebene also reduced the glutamate-induced generation of reactive oxygen species and calcium influx, thus preventing apoptotic cell death. α-iso-cubebene inhibited glutamate-induced mitochondrial membrane depolarization and, consequently, inhibited the release of the apoptosis-inducing factor from the mitochondria. Immunoblot anlaysis revealed that the phosphorylation of extracellular signal-regulated kinase (ERK) by glutamate was reduced in the presence of α-iso-cubebene. α-iso-cubebene activated protein kinase A (PKA), CREB and Nrf2, which mediate the expression of the antioxidant enzymes, heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1), involved in neuroprotection. In addition, α-iso-cubebene induced the expression of antioxidant responsive element and CRE transcriptional activity, thus conferring neuroprotection against glutamate-induced oxidative injury. α-iso-cubebene also induced the expression of Nrf2-dependent genes encoding HO-1 and NQO1. Furthermore, the knockdown of CREB and Nrf2 by small interfering RNA attenuated the neuroprotective effects of α-iso-cubebene. Taken together, our results indicate that α-iso-cubebene protects HT22 cells from glutamate-induced oxidative damage through the activation of Nrf2/HO-1/NQO-1, as well as through the PKA and CREB signaling pathways.

PCB 126 toxicity is modulated by cross-talk between caveolae and Nrf2 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petriello, Michael C.; University of Kentucky Superfund Research Center, Lexington, KY 40536; Han, Sung Gu

2014-06-01

Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alters PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehiclemore » and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1 −/− mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1 −/− endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1 −/− endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation. - Highlights: • Reduction of caveolin-1 protein protects against polychlorinated biphenyl toxicity. • Decreasing caveolin-1 levels increases the Nrf2 antioxidant response. • Reducing caveolin-1 levels decreases expression of Nrf2 inhibitory proteins. • Caveolin-1/Nrf2 cross-talk is evident in mouse, human, and porcine endothelial cells.« less

5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO-1 signaling cascade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Mingyi; Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou; Department of Pediatrics, The Third Xiangya Hospital, Central South University, Changsha

Background: Hypoxia causes cardiac disease via oxidative stress and mitochondrial dysfunction. 5-Aminolevulinic acid in combination with sodium ferrous citrate (ALA/SFC) has been shown to up-regulate heme oxygenase-1 (HO-1) and decrease macrophage infiltration and renal cell apoptosis in renal ischemia injury mice. However, its underlying mechanism remains largely unknown. The aim of this study was to investigate whether ALA/SFC could protect cardiomyocytes from hypoxia-induced apoptosis by autophagy via HO-1 signaling. Materials & methods: Murine atrial cardiomyocyte HL-1 cells were pretreated with ALA/SFC and then exposed to hypoxia. Results: ALA/SFC pretreatment significantly attenuated hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury,more » while it increased cell viability and autophagy levels. HO-1 expression by ALA/SFC was associated with up-regulation and nuclear translocation of Nrf-2, whereas Nrf-2 siRNA dramatically reduced HO-1 expression. ERK1/2, p38, and SAPK/JNK pathways were activated by ALA/SFC and their specific inhibitors significantly reduced ALA/SFC-mediated HO-1 upregulation. Silencing of either Nrf-2 or HO-1and LY294002, inhibitor of autophagy, abolished the protective ability of ALA/AFC against hypoxia-induced injury and reduced ALA/SFC-induced autophagy. Conclusion: Taken together, our data suggest that ALA/SFC induces autophagy via activation of MAPK/Nrf-2/HO-1 signaling pathway to protect cardiomyocytes from hypoxia-induced apoptosis. - Highlights: • ALA/SFC attenuates hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury. • ALA/SFC increases the heme oxygenase-1 expression via Nrf-2 and ERK1/2, p38, and SAPK/JNK pathways. • ALA/SFC induces autophagy and inhibition of autophagy prevent ALA/SFC-mediated suppression of hypoxia-induced injury.« less

Sulforaphane protects rodent retinas against ischemia-reperfusion injury through the activation of the Nrf2/HO-1 antioxidant pathway.

PubMed

Pan, Hong; He, Meihua; Liu, Ruixing; Brecha, Nicholas C; Yu, Albert Cheung Hoi; Pu, Mingliang

2014-01-01

Retinal ischemia-reperfusion (I/R) injury induces oxidative stress, leukocyte infiltration, and neuronal cell death. Sulforaphane (SF), which can be obtained in cruciferous vegetables such as broccoli, exerts protective effects in response to oxidative stress in various tissues. These effects can be initiated through nuclear factor E2-related factor 2 (Nrf2)-mediated induction of heme oxygenase-1 (HO-1). This investigation was designed to elucidate the neural protective mechanisms of SF in the retinal I/R rat model. Animals were intraperitoneally (i.p.) injected with SF (12.5 mg/kg) or vehicle (corn oil) once a day for 7 consecutive days. Then, retinal I/R was made by elevating the intraocular pressure (IOP) to 130 mmHg for 1 h. To determine if HO-1 was involved in the Nrf2 antioxidant pathway, rats were subjected to protoporphyrin IX zinc (II) (ZnPP, 30 mg/kg, i.p.) treatments at 24 h before retinal ischemia. The neuroprotective effects of SF were assessed by determining the morphology of the retina, counting the infiltrating inflammatory cells and the surviving retinal ganglion cells (RGCs) and amacrine cells, and measuring apoptosis in the retinal layers. The expression of Nrf2 and HO-1 was studied by immunofluorescence analysis and western blotting. I/R induced a marked increase of ROS generation, caused pronounced inflammation, increased the apoptosis of RGCs and amacrine cells and caused the thinning of the inner retinal layer (IRL), and these effects were diminished or abolished by SF pretreatment. Meanwhile, SF pretreatment significantly elevated the nuclear accumulation of Nrf2 and the level of HO-1 expression in the I/R retinas; however, ZnPP reversed the protective effects of SF on I/R retinas. Together, we offer direct evidence that SF had protective effects on I/R retinas, which could be attributed, at least in part, to the activation of the Nrf2/HO-1 antioxidant pathway.

Sulforaphane Protects Rodent Retinas against Ischemia-Reperfusion Injury through the Activation of the Nrf2/HO-1 Antioxidant Pathway

PubMed Central

Liu, Ruixing; Brecha, Nicholas C.; Yu, Albert Cheung Hoi; Pu, Mingliang

2014-01-01

Retinal ischemia-reperfusion (I/R) injury induces oxidative stress, leukocyte infiltration, and neuronal cell death. Sulforaphane (SF), which can be obtained in cruciferous vegetables such as broccoli, exerts protective effects in response to oxidative stress in various tissues. These effects can be initiated through nuclear factor E2-related factor 2 (Nrf2)-mediated induction of heme oxygenase-1 (HO-1). This investigation was designed to elucidate the neural protective mechanisms of SF in the retinal I/R rat model. Animals were intraperitoneally (i.p.) injected with SF (12.5 mg/kg) or vehicle (corn oil) once a day for 7 consecutive days. Then, retinal I/R was made by elevating the intraocular pressure (IOP) to 130 mmHg for 1 h. To determine if HO-1 was involved in the Nrf2 antioxidant pathway, rats were subjected to protoporphyrin IX zinc (II) (ZnPP, 30 mg/kg, i.p.) treatments at 24 h before retinal ischemia. The neuroprotective effects of SF were assessed by determining the morphology of the retina, counting the infiltrating inflammatory cells and the surviving retinal ganglion cells (RGCs) and amacrine cells, and measuring apoptosis in the retinal layers. The expression of Nrf2 and HO-1 was studied by immunofluorescence analysis and western blotting. I/R induced a marked increase of ROS generation, caused pronounced inflammation, increased the apoptosis of RGCs and amacrine cells and caused the thinning of the inner retinal layer (IRL), and these effects were diminished or abolished by SF pretreatment. Meanwhile, SF pretreatment significantly elevated the nuclear accumulation of Nrf2 and the level of HO-1 expression in the I/R retinas; however, ZnPP reversed the protective effects of SF on I/R retinas. Together, we offer direct evidence that SF had protective effects on I/R retinas, which could be attributed, at least in part, to the activation of the Nrf2/HO-1 antioxidant pathway. PMID:25470382

Nrf2 Activation Induced by Sirt1 Ameliorates Acute Lung Injury After Intestinal Ischemia/Reperfusion Through NOX4-Mediated Gene Regulation.

PubMed

Chai, DongDong; Zhang, Lei; Xi, SiWei; Cheng, YanYong; Jiang, Hong; Hu, Rong

2018-01-01

Nuclear erythroid 2-related factor-2 (Nrf2) is a major stress-response transcription factor that has been implicated in regulating ischemic angiogenesis. We investigated the effects of Nrf2 in regulating revascularization and modulating acute lung injury. The expression of Nrf2 and sirtuin1 (Sirt1) was assessed in lung tissue by western blotting and immunofluorescence staining after intestinal ischemia/reperfusion (IIR) in Nrf2-/- and wild-type (WT) mice. The involvement of Nrf2 in angiogenesis, cell viability, and migration was investigated in human pulmonary microvascular endothelial cells (PMVECs). Additionally, the influence of Nrf2 expression on NOX pathway activation was measured in PMVECs after oxygen-glucose deprivation/reoxygenation. We found activation and nuclear accumulation of Nrf2 in lung tissue after IIR. Compared to IIR in WT mice, IIR in Nrf2-/- mice significantly enhanced leukocyte infiltration and collagen deposit, and inhibited endothelial cell marker CD31 expression. Nrf2 upregulation and translocation into the nucleus stimulated by Sirt1 overexpression exhibited remission of histopathologic changes and enhanced CD31 expression. Nrf2 knockdown repressed non-phagocytic cell oxidase 4 (NOX4), hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) expression after IIR. Nrf2 upregulation by Sirt1 enhances NOX4, HIF-1α and VEGF expression after IIR in WT mice. Furthermore, Nrf2 knockdown suppressed cell viability, capillary tube formation and cell migration in PMVECs after oxygen-glucose deprivation/reoxygenation and also inhibited NOX4, HIF-1 and VEGF expression. Moreover, NOX4 knockdown in PMVECs decreased the levels of VEGF, HIF-1α and angiogenesis. Nrf2 stimulation by Sirt1 plays an important role in sustaining angiogenic potential through NOX4-mediated gene regulation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Curcumin attenuates quinocetone induced apoptosis and inflammation via the opposite modulation of Nrf2/HO-1 and NF-kB pathway in human hepatocyte L02 cells.

PubMed

Dai, Chongshan; Li, Bin; Zhou, Yan; Li, Daowen; Zhang, Shen; Li, Hui; Xiao, Xilong; Tang, Shusheng

2016-09-01

The potential toxicity of quinocetone (QCT) has raised widely concern, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on QCT induced apoptosis and the underlying mechanism in human hepatocyte L02 cells. The results showed that QCT treatment significantly decreased the cell viability of L02 cell and increased the release of lactate dehydrogenase (LDH), which was attenuated by curcumin pre-treatment at 1.25, 2.5 and 5 μM. Compared to the QCT alone group, curcumin pre-treatment significantly attenuated QCT induced oxidative stress, mitochondrial dysfunction and apoptosis. In addition, curcumin pretreatment markedly attenuated QCT-induced increase of iNOS activity and NO production in a dose-dependent manner. Meanwhile, curcumin pretreatment markedly down-regulated the expression of nuclear factor -kB (NF-kB) and iNOS mRNAs, but up-regulated the expressions of Nrf2 and HO-1 mRNAs, compared to the QCT alone group. Zinc protoporphyrin IX, a HO-1 inhibitor, markedly partly abolished the cytoprotective effect of curcumin against QCT-induced caspase activation, NF-kB mRNA expression. These results indicate that curcumin could effectively inhibit QCT induced apoptosis and inflammatory response in L02 cells, which may involve the activation of Nrf2/HO-1 and inhibition of NF-kB pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

Upregulation of transcription factor NRF2-mediated oxidative stress response pathway in rat brain under short-term chronic hypobaric hypoxia.

PubMed

Sethy, Niroj Kumar; Singh, Manjulata; Kumar, Rajesh; Ilavazhagan, Govindasamy; Bhargava, Kalpana

2011-03-01

Exposure to high altitude (and thus hypobaric hypoxia) induces electrophysiological, metabolic, and morphological modifications in the brain leading to several neurological clinical syndromes. Despite the known fact that hypoxia episodes in brain are a common factor for many neuropathologies, limited information is available on the underlying cellular and molecular mechanisms. In this study, we investigated the temporal effect of short-term (0-12 h) chronic hypobaric hypoxia on global gene expression of rat brain followed by detailed canonical pathway analysis and regulatory network identification. Our analysis revealed significant alteration of 33, 17, 53, 81, and 296 genes (p < 0.05, <1.5-fold) after 0.5, 1, 3, 6, and 12 h of hypoxia, respectively. Biological processes like regulation, metabolic, and transport pathways are temporally activated along with anti- and proinflammatory signaling networks like PI3K/AKT, NF-κB, ERK/MAPK, IL-6 and IL-8 signaling. Irrespective of exposure durations, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress response pathway and genes were detected at all time points suggesting activation of NRF2-ARE antioxidant defense system. The results were further validated by assessing the expression levels of selected genes in temporal as well as brain regions with quantitative RT-PCR and western blot. In conclusion, our whole brain approach with temporal monitoring of gene expression patterns during hypobaric hypoxia has resulted in (1) deciphering sequence of pathways and signaling networks activated during onset of hypoxia, and (2) elucidation of NRF2-orchestrated antioxidant response as a major intrinsic defense mechanism. The results of this study will aid in better understanding and management of hypoxia-induced brain pathologies.

Co-regulation of nuclear respiratory factor-1 by NFκB and CREB links LPS-induced inflammation to mitochondrial biogenesis

PubMed Central

Suliman, Hagir B.; Sweeney, Timothy E.; Withers, Crystal M.; Piantadosi, Claude A.

2010-01-01

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H2O2. These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses. PMID:20587593

Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2.

PubMed

Zhu, Lingxiang; Barrett, Erika C; Barret, Erika C; Xu, Yuxue; Liu, Zuguo; Manoharan, Aditya; Chen, Yin

2013-01-01

Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome.

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension.

PubMed

Hood, Katie Y; Mair, Kirsty M; Harvey, Adam P; Montezano, Augusto C; Touyz, Rhian M; MacLean, Margaret R

2017-07-01

Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1 -/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT 1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT 1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT 1B receptor signaling and Nox1, confirmed in PASMCs from Nox1 -/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT 1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT 1B receptors contribute to experimental pulmonary hypertension by inducing lung ROS production. Our results suggest that 5-HT 1B receptor-dependent cellular Src-related kinase-Nox1-pathways contribute to vascular remodeling in PAH. © 2017 The Authors.

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension

PubMed Central

Hood, Katie Y.; Mair, Kirsty M.; Harvey, Adam P.; Montezano, Augusto C.; Touyz, Rhian M.

2017-01-01

Objective— Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase–derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. Approach and Results— HPASMCs from controls and PAH patients, and PASMCs from Nox1−/− mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1−/− mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Conclusions— Serotonin can induce cellular Src-related kinase–regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT1B receptors contribute to experimental pulmonary hypertension by inducing lung ROS production. Our results suggest that 5-HT1B receptor–dependent cellular Src-related kinase-Nox1-pathways contribute to vascular remodeling in PAH. PMID:28473438

Reversal of cigarette smoke extract-induced sinonasal epithelial cell barrier dysfunction through Nrf2 Activation.

PubMed

Tharakan, Anuj; Halderman, Ashleigh A; Lane, Andrew P; Biswal, Shyam; Ramanathan, Murugappan

2016-11-01

Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke-induced sinonasal epithelial cell (SNEC) barrier dysfunction. Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE. CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels. This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke-associated sinonasal inflammation. © 2016 ARS-AAOA, LLC.

Advanced glycation end products influence oral cancer cell survival via Bcl-xl and Nrf-2 regulation in vitro.

PubMed

Ko, Shun-Yao; Ko, Hshin-An; Shieh, Tzong-Ming; Chi, Tzong-Cherng; Chen, Hong-I; Chen, Yi-Ting; Yu, Ya-Hui; Yang, Shu-Han; Chang, Shu-Shing

2017-05-01

An irreversible non-enzymatic reaction between carbohydrates and proteins results in the formation of advanced glycation end products (AGEs). AGEs have been demonstrated to be a risk factor of complications in patients with diabetes mellitus (DM). Previous studies have suggested that patients with DM exhibit a higher rate of metastasis of oral cancer and a lower cancer-associated survival rate. The receptor for AGEs (RAGE) has been associated with angiogenesis and an increase in cancer malignancy. Previous studies have suggested that AGE-RAGE regulates cell migration via extracellular signal-regulated kinase (ERK) phosphorylation. Nuclear factor-erythroid 2-related factor 2 (Nrf-2) is associated with the regulation of tumor protein p53 (p53) and the apoptotic response of oral cancer cells. AGEs are associated with oral cancer; however, the mechanism underlying this association remains to be elucidated. The present study hypothesized that AGEs regulate Nrf-2 and downstream pathways through ERK phosphorylation. The results of the current study demonstrated that AGEs inhibit the expression of Nrf-2, p53 and Bcl-2 associated × apoptosis regulator, and increase the expression of apoptosis regulator Bcl-x protein. The effect of AGEs was inhibited through the use of the PD98059. The present study demonstrated that AGEs regulate the downstream pathways Nrf-2 and Bcl-xl via ERK phosphorylation. It is suggested that AGEs regulate the survival of oral cancer cells via Nrf-2 and Bcl-xl through p53 regulation, which explains the poor prognosis of patients with DM who have oral cancer.

Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu-Kun Jennifer; Wu, Kai Connie; Liu, Jie

Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat Western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulatingmore » glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling. -- Highlights: ► Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet. ► The anti-diabetic hormone, Fgf21, is highly expressed in livers of Nrf2-null mice. ► The absence of Nrf2 increases the insulin-regulated Igfbp-1 mRNA in liver. ► Nrf2 deficiency improves glucose tolerance by influencing Fgf21 and insulin signaling.« less

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes

PubMed Central

Kerns, Michelle L.; Hakim, Jill M.C.; Lu, Rosemary G.; Guo, Yajuan; Berroth, Andreas; Kaspar, Roger L.

2016-01-01

Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16–/– mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid–derived 2 related factor 2–dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16–/– mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16–/– footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16–/– mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment. PMID:27183391

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus

PubMed Central

Sutter, Carrie Hayes; Olesen, Kristin M; Kensler, Thomas W

2018-01-01

Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of Nrf2 function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells. Consistent with this concept, activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude. In hepatocytes, activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2, increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription. Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping. PMID:29481323

Introducing the "TCDD-inducible AhR-Nrf2 gene battery".

PubMed

Yeager, Ronnie L; Reisman, Scott A; Aleksunes, Lauren M; Klaassen, Curtis D

2009-10-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 mug/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid-synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

PubMed

Nath, Samir R; Yu, Zhigang; Gipson, Theresa A; Marsh, Gregory B; Yoshidome, Eriko; Robins, Diane M; Todi, Sokol V; Housman, David E; Lieberman, Andrew P

2018-05-29

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-Seq to identify pathways that are disrupted in diseased muscle using AR113Q knock-in mice. This analysis unexpectedly identified significantly diminished expression of numerous ubiquitin-proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age-, hormone- and glutamine length-dependent and arose due to a toxic gain-of-function conferred by the mutation. Moreover, altered gene expression was associated with decreased level of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.

Characterization of docosahexaenoic acid (DHA)-induced heme oxygenase-1 (HO-1) expression in human cancer cells: the importance of enhanced BTB and CNC homology 1 (Bach1) degradation.

PubMed

Wang, Shuai; Hannafon, Bethany N; Wolf, Roman F; Zhou, Jundong; Avery, Jori E; Wu, Jinchang; Lind, Stuart E; Ding, Wei-Qun

2014-05-01

The effect of docosahexaenoic acid (DHA) on heme oxygenase-1 (HO-1) expression in cancer cells has never been characterized. This study examines DHA-induced HO-1 expression in human cancer cell model systems. DHA enhanced HO-1 gene expression in a time- and concentration-dependent manner, with maximal induction at 21 h of treatment. This induction of HO-1 expression was confirmed in vivo using a xenograft nude mouse model fed a fish-oil-enriched diet. The increase in HO-1 gene transcription induced by DHA was significantly attenuated by the antioxidant N-acetyl cysteine, suggesting the involvement of oxidative stress. This was supported by direct measurement of lipid peroxide levels after DHA treatment. Using a human HO-1 gene promoter reporter construct, we identified two antioxidant response elements (AREs) that mediate the DHA-induced increase in HO-1 gene transcription. Knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression compromised the DHA-induced increase in HO-1 gene transcription, indicating the importance of the Nrf2 pathway in this event. However, the nuclear protein levels of Nrf2 remained unchanged upon DHA treatment. Further studies demonstrated that DHA reduces nuclear Bach1 protein expression by promoting its degradation and attenuates Bach1 binding to the AREs in the HO-1 gene promoter. In contrast, DHA enhanced Nrf2 binding to the AREs without affecting nuclear Nrf2 expression levels, indicating a new cellular mechanism that mediates DHA's induction of HO-1 gene transcription. To our knowledge, this is the first characterization of DHA-induced HO-1 expression in human malignant cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Compound C Stimulates Heme Oxygenase-1 Gene Expression via the Nrf2-ARE Pathway to Preserve Human Endothelial Cell Survival

PubMed Central

Liu, Xiao-ming; Peyton, Kelly J.; Shebib, Ahmad R.; Wang, Hong; Durante, William

2011-01-01

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor-2 (Nrf2). Compound C also stimulated Nrf2 expression and this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-L-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions. PMID:21635873

Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases

PubMed Central

Preciados, Mark; Yoo, Changwon; Roy, Deodutta

2016-01-01

During the development of an individual from a single cell to prenatal stages to adolescence to adulthood and through the complete life span, humans are exposed to countless environmental and stochastic factors, including estrogenic endocrine disrupting chemicals. Brain cells and neural circuits are likely to be influenced by estrogenic endocrine disruptors (EEDs) because they strongly dependent on estrogens. In this review, we discuss both environmental, epidemiological, and experimental evidence on brain health with exposure to oral contraceptives, hormonal therapy, and EEDs such as bisphenol-A (BPA), polychlorinated biphenyls (PCBs), phthalates, and metalloestrogens, such as, arsenic, cadmium, and manganese. Also we discuss the brain health effects associated from exposure to EEDs including the promotion of neurodegeneration, protection against neurodegeneration, and involvement in various neurological deficits; changes in rearing behavior, locomotion, anxiety, learning difficulties, memory issues, and neuronal abnormalities. The effects of EEDs on the brain are varied during the entire life span and far-reaching with many different mechanisms. To understand endocrine disrupting chemicals mechanisms, we use bioinformatics, molecular, and epidemiologic approaches. Through those approaches, we learn how the effects of EEDs on the brain go beyond known mechanism to disrupt the circulatory and neural estrogen function and estrogen-mediated signaling. Effects on EEDs-modified estrogen and nuclear respiratory factor 1 (NRF1) signaling genes with exposure to natural estrogen, pharmacological estrogen-ethinyl estradiol, PCBs, phthalates, BPA, and metalloestrogens are presented here. Bioinformatics analysis of gene-EEDs interactions and brain disease associations identified hundreds of genes that were altered by exposure to estrogen, phthalate, PCBs, BPA or metalloestrogens. Many genes modified by EEDs are common targets of both 17 β-estradiol (E2) and NRF1. Some of these genes are involved with brain diseases, such as Alzheimer’s Disease (AD), Parkinson’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis, Autism Spectrum Disorder, and Brain Neoplasms. For example, the search of enriched pathways showed that top ten E2 interacting genes in AD—APOE, APP, ATP5A1, CALM1, CASP3, GSK3B, IL1B, MAPT, PSEN2 and TNF—underlie the enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) AD pathway. With AD, the six E2-responsive genes are NRF1 target genes: APBB2, DPYSL2, EIF2S1, ENO1, MAPT, and PAXIP1. These genes are also responsive to the following EEDs: ethinyl estradiol (APBB2, DPYSL2, EIF2S1, ENO1, MAPT, and PAXIP1), BPA (APBB2, EIF2S1, ENO1, MAPT, and PAXIP1), dibutyl phthalate (DPYSL2, EIF2S1, and ENO1), diethylhexyl phthalate (DPYSL2 and MAPT). To validate findings from Comparative Toxicogenomics Database (CTD) curated data, we used Bayesian network (BN) analysis on microarray data of AD patients. We observed that both gender and NRF1 were associated with AD. The female NRF1 gene network is completely different from male human AD patients. AD-associated NRF1 target genes—APLP1, APP, GRIN1, GRIN2B, MAPT, PSEN2, PEN2, and IDE—are also regulated by E2. NRF1 regulates targets genes with diverse functions, including cell growth, apoptosis/autophagy, mitochondrial biogenesis, genomic instability, neurogenesis, neuroplasticity, synaptogenesis, and senescence. By activating or repressing the genes involved in cell proliferation, growth suppression, DNA damage/repair, apoptosis/autophagy, angiogenesis, estrogen signaling, neurogenesis, synaptogenesis, and senescence, and inducing a wide range of DNA damage, genomic instability and DNA methylation and transcriptional repression, NRF1 may act as a major regulator of EEDs-induced brain health deficits. In summary, estrogenic endocrine disrupting chemicals-modified genes in brain health deficits are part of both estrogen and NRF1 signaling pathways. Our findings suggest that in addition to estrogen signaling, EEDs influencing NRF1 regulated communities of genes across genomic and epigenomic multiple networks may contribute in the development of complex chronic human brain health disorders. PMID:27983596

Activation of Nrf2/HO-1 Pathway by Glycogen Synthase Kinase-3β Inhibition Attenuates Renal Ischemia/Reperfusion Injury in Diabetic Rats.

PubMed

Shen, Xiaohua; Hu, Bo; Xu, Guangtao; Chen, Fengjuan; Ma, Ruifen; Zhang, Nenghua; Liu, Jie; Ma, Xiaoqin; Zhu, Jia; Wu, Yuhong; Shen, Ruilin

2017-01-01

Diabetes mellitus can exacerbate renal ischemia-reperfusion (I/R) injury (RI/RI). The aim of the present study was to evaluate the protective effect of GSK-3β inhibition (TDZD-8) on I/R-induced renal injury through the Nrf2/HO-1 pathway in a streptozocin (STZ)-induced diabetic rat model. STZ-induced diabetic rats preconditioned with TDZD-8 and ZnPP were subjected to renal I/R. The extent of renal morphologic lesions. Renal function was assessed from blood urea nitrogen (BUN) and serum creatinine (Scr), as determined utlizing commercial kits. Oxidative stress and inflammatory activity in the kidney tissue was estimated from levels of malondialdehyde (MDA), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), as well as the activities of superoxide dismutase (SOD) and glutathione (GSH) using qRT-PCR and ELISA. The expressions of Nrf2, HO-1, Bcl-2 and NF-κB in the renal tissue were measured by qRT-PCR and western blotting. I/R-induced renal inflammation was reduced significantly by TDZD-8 pretreatment. Preconditioning with TDZD-8 suppressed NF-κB expression and enhanced Bcl-2 expression in the renal tissue. The upregulated level of malondialdehyde (MDA), and reduced activities of superoxide dismutase (SOD) and glutathione (GSH) in I/R-shocked rats were markedly restored by TDZD-8 pretreatment. Furthermore, pretreatment with TDZD-8 enhanced activation of the Nrf2/HO-1 pathway in the renal tissue of diabetic RI/RI rats. These findings suggest that preconditioning with TDZD-8 may protect the kidney from I/R-induced damage via the activation of the Nrf2/HO-1 pathway in STZ-induced diabetic rats. Further detailed studies are needed to further clarify the underlying mechanisms. © 2017 The Author(s). Published by S. Karger AG, Basel.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

PubMed Central

Rada, Patricia; Rojo, Ana I.; Evrard-Todeschi, Nathalie; Innamorato, Nadia G.; Cotte, Axelle; Jaworski, Tomasz; Tobón-Velasco, Julio C.; Devijver, Herman; García-Mayoral, María Flor; Van Leuven, Fred; Hayes, John D.

2012-01-01

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3. PMID:22751928

3H-1,2-Dithiole-3-thione as a novel therapeutic agent for the treatment of ischemic stroke through Nrf2 defense pathway.

PubMed

Kuo, Ping-Chang; Yu, I-Chen; Scofield, Barbara A; Brown, Dennis A; Curfman, Eric T; Paraiso, Hallel C; Chang, Fen-Lei; Yen, Jui-Hung

2017-05-01

Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in brain tissue induce oxidative stress and inflammatory responses. D3T, the simplest compound of the cyclic, sulfur-containing dithiolethiones, is found in cruciferous vegetables and has been reported to induce antioxidant genes and glutathione biosynthesis through activation of Nrf2. In addition to antioxidant activity, D3T was also reported to possess anti-inflammatory effects. In this study, we evaluated the therapeutic potential of D3T for the treatment of ischemic stroke and investigated the mechanisms underlying the protective effects of D3T in ischemic stroke. Mice subjected to transient middle cerebral artery occlusion/reperfusion (tMCAO/R) were administered with vehicle or D3T to evaluate the effect of D3T in cerebral brain injury. We observed D3T reduced infarct size, decreased brain edema, lessened blood-brain barrier disruption, and ameliorated neurological deficits. Further investigation revealed D3T suppressed microglia (MG) activation and inhibited peripheral inflammatory immune cell infiltration of CNS in the ischemic brain. The protective effect of D3T in ischemic stroke is mediated through Nrf2 induction as D3T-attenuated brain injury was abolished in Nrf2 deficient mice subjected to tMCAO/R. In addition, in vitro results indicate the induction of Nrf2 by D3T is required for its suppressive effect on MG activation and cytokine production. In summary, we demonstrate for the first time that D3T confers protection against ischemic stroke, which is mediated through suppression of MG activation and inhibition of CNS peripheral cell infiltration, and that the protective effect of D3T in ischemic stroke is dependent on the activation of Nrf2. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel Nrf2 activators from microbial transformation products inhibit blood-retinal barrier permeability in rabbits.

PubMed

Nakagami, Yasuhiro; Masuda, Kayoko; Hatano, Emiko; Inoue, Tatsuya; Matsuyama, Takuya; Iizuka, Mayumi; Ono, Yasunori; Ohnuki, Takashi; Murakami, Yoko; Iwasaki, Masaru; Yoshida, Kazuhiro; Kasuya, Yuji; Komoriya, Satoshi

2015-03-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood-retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease. © 2014 The British Pharmacological Society.

Novel Nrf2 activators from microbial transformation products inhibit blood–retinal barrier permeability in rabbits

PubMed Central

Nakagami, Yasuhiro; Masuda, Kayoko; Hatano, Emiko; Inoue, Tatsuya; Matsuyama, Takuya; Iizuka, Mayumi; Ono, Yasunori; Ohnuki, Takashi; Murakami, Yoko; Iwasaki, Masaru; Yoshida, Kazuhiro; Kasuya, Yuji; Komoriya, Satoshi

2015-01-01

Background and Purpose Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. Experimental Approach The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood–retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. Key Results Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. Conclusion and Implications Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease. PMID:25363737

Targeting NRF2 signaling for cancer chemoprevention

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwak, Mi-Kyoung, E-mail: mkwak@ynu.ac.k; Kensler, Thomas W.

2010-04-01

Modulation of the metabolism and disposition of carcinogens through induction of cytoprotective enzymes is one of several promising strategies to prevent cancer. Chemopreventive efficacies of inducers such as dithiolethiones and sulforaphane have been extensively studied in animals as well as in humans. The KEAP1-NRF2 system is a key, but not unilateral, molecular target for these chemopreventive agents. The transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of the expression of a subset of genes, which produce proteins responsible for the detoxication of electrophiles and reactive oxygen species as well as the removal or repair of some of theirmore » damage products. It is believed that chemopreventive enzyme inducers affect the interaction between KEAP1 and NRF2 through either mediating conformational changes of the KEAP1 protein or activating phosphorylation cascades targeting the KEAP1-NRF2 complex. These events in turn affect NRF2 stability and trafficking. Recent advances elucidating the underlying structural biology of KEAP1-NRF2 signaling and identification of the gene clusters under the transcriptional control of NRF2 are facilitating understanding of the potential pleiotropic effects of NRF2 activators and discovery of novel classes of potent chemopreventive agents such as the triterpenoids. Although there is appropriately a concern regarding a deleterious role of the KEAP1-NRF2 system in cancer cell biology, especially as the pathway affects cell survival and drug resistance, the development and the use of NRF2 activators as chemopreventive agents still holds a great promise for protection of normal cells from a diversity of environmental stresses that contribute to the burden of cancer and other chronic, degenerative diseases.« less

Pyrrolidine dithiocarbamate activates the Nrf2 pathway in astrocytes.

PubMed

Liddell, Jeffrey R; Lehtonen, Sarka; Duncan, Clare; Keksa-Goldsteine, Velta; Levonen, Anna-Liisa; Goldsteins, Gundars; Malm, Tarja; White, Anthony R; Koistinaho, Jari; Kanninen, Katja M

2016-02-26

Endogenous defense against oxidative stress is controlled by nuclear factor erythroid 2-related factor 2 (Nrf2). The normal compensatory mechanisms to combat oxidative stress appear to be insufficient to protect against the prolonged exposure to reactive oxygen species during disease. Counterbalancing the effects of oxidative stress by up-regulation of Nrf2 signaling has been shown to be effective in various disease models where oxidative stress is implicated, including Alzheimer's disease. Stimulation of Nrf2 signaling by small-molecule activators is an appealing strategy to up-regulate the endogenous defense mechanisms of cells. Here, we investigate Nrf2 induction by the metal chelator and known nuclear factor-κB inhibitor pyrrolidine dithiocarbamate (PDTC) in cultured astrocytes and neurons, and mouse brain. Nrf2 induction is further examined in cultures co-treated with PDTC and kinase inhibitors or amyloid-beta, and in Nrf2-deficient cultures. We show that PDTC is a potent inducer of Nrf2 signaling specifically in astrocytes and demonstrate the critical role of Nrf2 in PDTC-mediated protection against oxidative stress. This induction appears to be regulated by both Keap1 and glycogen synthase kinase 3β. Furthermore, the presence of amyloid-beta magnifies PDTC-mediated induction of endogenous protective mechanisms, therefore suggesting that PDTC may be an effective Nrf2 inducer in the context of Alzheimer's disease. Finally, we show that PDTC increases brain copper content and glial expression of heme oxygenase-1, and decreases lipid peroxidation in vivo, promoting a more antioxidative environment. PDTC activates Nrf2 and its antioxidative targets in astrocytes but not neurons. These effects may contribute to the neuroprotection observed for PDTC in models of Alzheimer's disease.

The effect of natural antioxidants in cyclophosphamide-induced hepatotoxicity: Role of Nrf2/HO-1 pathway.

PubMed

Sherif, Iman O

2018-05-22

Hepatotoxicity induced by cyclophosphamide (Cyclo) is a major concern in clinical practice. This study was designed to investigate the possible cytoprotective effect of natural antioxidants as oleuropein and quercetin against Cyclo induced hepatotoxicity via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Male Wistar rats were randomly divided into six groups and treated for 10 days as follow: Group I (Normal control) received saline, group II (Oleu control): received orally oleuropein 30 mg/kg/day, group III (Quer control): administered orally quercetin 50 mg/kg/day, group IV (Cyclo): received saline and injected with single intraperitoneal (i.p) dose of Cyclo 200 mg/kg at day 5, group V (Oleu ttt): treated with oleuropein plus Cyclo i.p. injection at day 5, and group VI (Quer ttt): treated with quercetin plus Cyclo i.p. injection at day 5. Injection of Cyclo showed marked increase in serum transaminases and alkaline phosphatase, hepatic malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-⍺) levels along with significant reduction in hepatic reduced glutathione (GSH), superoxide dismutase (SOD), and catalase levels in addition to downregulation of hepatic Nrf2 and HO-1 expressions and reduction in hepatic nuclear Nrf2 binding activity when compared with normal group. Histopathological examination of Cyclo treated rats revealed hepatic damage. Both oleuropein and quercetin exhibited an improvement in the biochemical and histopathological findings. In conclusion, the natural antioxidants oleuropein and quercetin counteract the Cyclo induced hepatotoxicity through activation of Nrf2/HO-1 signaling pathway with subsequent suppression of oxidative stress and inflammation. Copyright © 2018 Elsevier B.V. All rights reserved.

Minocycline attenuates sevoflurane-induced cell injury via activation of Nrf2.

PubMed

Tian, Yue; Wu, Xiuying; Guo, Shanbin; Ma, Ling; Huang, Wei; Zhao, Xiaochun

2017-04-01

Minocycline has been demonstrated to exert neuroprotective effects in various experimental models. In the present study, we investigated the mechanisms underlying the protective effects of minocycline on cell injury induced by the inhalation of the anesthetic, sevoflurane. In our in vivo experiments using rats, minocycline attenuated sevoflurane-induced neuronal degeneration and apoptosis in the rat hippocampus, and this effect was associated with the minocycline-mediated suppression of oxidative stress in the hippocampus. In in vitro experiments, minocycline inhibited sevoflurane-induced apoptosis and the production of reactive oxygen species (ROS) in H4 human neuroglioma cells. In addition, minocycline suppressed the sevoflurane-induced upregulation of interleukin (IL)-6 and the activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells. Furthermore, we found that nuclear factor E2-related factor 2 (Nrf2), an activator of the stress response, was upregulated and activated upon sevoflurane treatment both in the rat hippocampus and in H4 cells. In addition, minocycline further augmented the upregulation and activation of Nrf2 when used in conjunction with sevoflurane. Moreover, the knockdown of Nrf2 in H4 cells by small interfering RNA (siRNA) diminished the cytoprotective effect of minocycline, and attenuated the inhibitory effect of minocycline on ROS production, IL-6 upregulation and the activation of the NF-κB signaling pathway. On the whole, our findings indicate that minocycline may exert protective effects against sevoflurane-induced cell injury via the Nrf2-modulated antioxidant response and the inhibition of the activation of the NF-κB signaling pathway.

Nrf2 inhibits oxaliplatin-induced peripheral neuropathy via protection of mitochondrial function.

PubMed

Yang, Yang; Luo, Lan; Cai, Xueting; Fang, Yuan; Wang, Jiaqi; Chen, Gang; Yang, Jie; Zhou, Qian; Sun, Xiaoyan; Cheng, Xiaolan; Yan, Huaijiang; Lu, Wuguang; Hu, Chunping; Cao, Peng

2018-05-20

Oxaliplatin-induced peripheral neuropathy (OIPN) is a severe, dose-limiting toxicity associated with cancer chemotherapy. The efficacy of antioxidant administration in OIPN is debatable, as the promising preliminary results obtained with a number of antioxidants have not been confirmed in larger clinical trials. Besides its antioxidant activity, the transcription factor, nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2) plays a crucial role in the maintenance of mitochondrial homeostasis, and mitochondrial dysfunction is a key contributor to OIPN. Here, we have investigated the protective properties of Nrf2 in OIPN. Nrf2 -/- mice displayed severe mechanical allodynia and cold sensitivity and thus experienced increased peripheral nervous system injury compared to Nrf2 +/+ mice. Furthermore, Nrf2 knockout aggravated oxaliplatin-induced reactive oxygen species production, decreased the mitochondrial membrane potential, led to abnormal intracellular calcium levels, and induced cytochrome c-related apoptosis and overexpression of the TRP protein family. Sulforaphane-induced activation of the Nrf2 signaling pathway alleviated morphological alterations, mitochondrial dysfunction in dorsal root ganglion neurons, and nociceptive sensations in mice. Our findings reveal that Nrf2 may play a critical role in ameliorating OIPN, through protection of mitochondrial function by alleviating oxidative stress and inhibiting TRP protein family expression. This suggests that pharmacological or therapeutic activation of Nrf2 may be used to prevent or slow down the progression of OIPN. Copyright © 2018 Elsevier Inc. All rights reserved.

Potential Ameliorative Effects of Qing Ye Dan Against Cadmium Induced Prostatic Deficits via Regulating Nrf-2/HO-1 and TGF-β1/Smad Pathways.

PubMed

Du, Lifen; Lei, Yongfang; Chen, Jinglou; Song, Hongping; Wu, Xinying

2017-01-01

Cadmium (Cd) is an environmental pollutant with reproductive toxicity. Swertia mileensis is used in Chinese medicine for the treatment of prostatic deficits and named as Qing Ye Dan (QYD). This study was undertaken to investigate the potential protective effects of QYD against Cd-induced prostatic deficits. Rat model of prostatic deficits was induced by 0.2 mg/kg/d CdCl2 subcutaneous injection for 15 days. The prostatic oxidative stress was evaluated by detecting the levels of malondialdehyde, nitric oxide, reduced/ oxidized glutathione, total sulfhydryl groups and enzymatic antioxidant status. The prostatic inflammation was estimated by testing the levels of pro-inflammatory cytokines. The levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, fibronectin, vimentin and α-smooth muscle actin were measured by qPCR analysis. Additionally, the prostatic expressions of transforming growth factor-β1 (TGF-β1), type I TGF-β receptor (TGF-βRI), Smad2, phosphorylation-Smad2 (p-Smad2), Smad3, p-Smad3, Smad7, nuclear related factor-2 (Nrf-2), heme oxygenase-1 (HO-1), B-cell CLL/lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax) were measured by western blot assay. It was found that QYD ameliorated the Cd-induced prostatic oxidative stress and inflammation, attenuated prostatic EMT, inhibited the TGF-β1/Smad pathway, increased Bcl-2/Bax ratio and enhanced the activity of Nrf-2/HO-1 pathway. These results showed that QYD could ameliorate Cd-induced prostatic deficits via modulating Nrf-2/HO-1 and TGF-β1/Smad pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Ebselen attenuates cisplatin-induced ROS generation through Nrf2 activation in auditory cells.

PubMed

Kim, Se-Jin; Park, Channy; Han, A Lum; Youn, Myung-Ja; Lee, Jeong-Han; Kim, Yunha; Kim, Eun-Sook; Kim, Hyung-Jin; Kim, Jin-Kyung; Lee, Ho-Kyun; Chung, Sang-Young; So, Hongseob; Park, Raekil

2009-05-01

Ebselen, an organoselenium compound that acts as a glutathione peroxidase mimetic, has been demonstrated to possess antioxidant and anti-inflammatory activities. However, the molecular mechanism underlying this effect is not fully understood in auditory cells. The purpose of the present study is to investigate the protective effect of ebselen against cisplatin-induced toxicity in HEI-OC1 auditory cells, organotypic cultures of cochlear explants from two-day postnatal rats (P(2)) and adult Balb/C mice. Pretreatment with ebselen ameliorated apoptotic death induced by cisplatin in HEI-OC1 cells and organotypic cultures of Corti's organ. Ebselen pretreatment also significantly suppressed cisplatin-induced increases in intracellular reactive oxygen species (ROS), intracellular reactive nitrogen species (RNS) and lipid peroxidation levels. Ebselen dose-dependently increased the expression level of an antioxidant response element (ARE)-luciferase reporter in HEI-OC1 cells through the translocation of Nrf2 into the nucleus. Furthermore, we found that pretreatment with ebselen significantly restored Nrf2 function, whereas it ameliorated the cytotoxicity of cisplatin in cells transfectants with either a pcDNA3.1 (control) or a DN-Nrf2 (dominant-negative) plasmid. We also observed that Nrf2 activation by ebselen increased the expression of phase II antioxidant genes, including heme oxygenase (HO-1), NAD(P)H:quinine oxidoreductase, and gamma-glutamylcysteine synthetase (gamma-GCS). Treatment with ebselen resulted in an increased expression of HO-1 and intranuclear Nrf2 in hair cells of organotypic cultured cochlea. After intraperitoneal injection with cisplatin, auditory brainstem responses (ABRs) threshold was measured on 8th day in Balb/C mice. ABR threshold shift was marked occurred in mice injected with cisplatin (16 mg/kg, n=5; Click and 8-kHz stimuli, p<0.05; 4, 16 and 32 kHz, p<0.01), whereas that of animal group which was treated with cisplatin and ebselen was not significantly changed. These results suggest that ebselen activates the Nrf2-ARE signaling pathway, which ultimately prevents free radical stresses from cisplatin and further contributes to protect auditory sensory hair cells from free radicals produced by cisplatin.

Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury

PubMed Central

Kim, Eun Nim; Lim, Ji Hee; Kim, Min Young; Ban, Tae Hyun; Jang, In-Ae; Yoon, Hye Eun; Park, Cheol Whee; Chang, Yoon Sik

2018-01-01

Background. Two important issues in the aging kidney are mitochondrial dysfunction and oxidative stress. An Nrf2 activator, resveratrol, is known to have various effects. Resveratrol may prevent inflammation and oxidative stress by activating Nrf2 and SIRT1 signaling. We examined whether resveratrol could potentially ameliorate the cellular condition, such as renal injury due to cellular oxidative stress and mitochondrial dysfunction caused by aging. Methods. Male 18-month-old C57BL/6 mice were used. Resveratrol (40 mg/kg) was administered to aged mice for 6 months. We compared histological changes, oxidative stress, and aging-related protein expression in the kidney between the resveratrol-treated group (RSV) and the control group (cont). We performed experiments using small-interfering RNAs (siRNAs) for Nrf2 and SIRT1 in cultured HK2 cells. Results. Resveratrol improved renal function, proteinuria, histological changes and inflammation in aging mice. Also, expression of Nrf2-HO-1-NOQ-1 signaling and SIRT1-AMPK-PGC-1α signaling was increased in the RSV group. Transfection with Nrf2 and SIRT1 siRNA prevented resveratrol-induced anti-oxidative effect in HK2 cells in media treated with H2O2. Conclusions. Activation of the Nrf2 and SIRT1 signaling pathways ameliorated oxidative stress and mitochondrial dysfunction. Pharmacological targeting of Nrf2 signaling molecules may reduce the pathologic changes of aging in the kidney. PMID:29326403

Lithospermic acid B protects beta-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Byung-Wan; Chun, Sung Wan; Kim, Soo Hyun

2011-04-01

Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo. Pretreatment with 50 {mu}M LAB significantly reduced the 1000 U/mL INF-{gamma} and 100 U/mL IL-1{beta}-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against themore » cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxigenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo. Our results suggest that the cytoprotective effects of LAB on pancreatic {beta}-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.« less

Zinc L-carnosine suppresses inflammatory responses in lipopolysaccharide-induced RAW 264.7 murine macrophages cell line via activation of Nrf2/HO-1 signaling pathway.

PubMed

Ooi, Theng Choon; Chan, Kok Meng; Sharif, Razinah

2017-10-01

Zinc L-carnosine (ZnC) is a chelate of Zn and L-carnosine and is used clinically in the treatment of peptic ulcer. In this study, we aim to investigate the involvement of heme oxygenase-1 (HO-1) in the anti-inflammatory effects of ZnC in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages. We used immunoblotting analysis to evaluate the involvement of HO-1 in the anti-inflammatory effects of ZnC and the signaling pathway involved was measured using Dual luciferase reporter assay. Results from immunoblotting analysis demonstrated that pretreatment of cells with ZnC enhanced the expression of HO-1 in RAW 264.7 cells. Pretreatment of cells with HO-1 inhibitor (tin protoporphyrin IX dichloride) significantly attenuated the inhibitory effects of ZnC on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and NF-κB activation in LPS-induced RAW 264.7 cells, suggesting that HO-1 play an important role in the suppression of inflammatory responses induced by ZnC. Furthermore, results from co-immunoprecipitation of Nrf2 and Keap1 and dual luciferase reporter assay showed that pretreatment of ZnC was able to activate the Nrf2 signaling pathway. Treatment of cells with p38 inhibitor (SB203580), c-Jun N-terminal kinase inhibitor (SP600125), and MEK 1/2 inhibitor (U0126) did not significantly suppress the induction of HO-1 by ZnC. Moreover, our present findings suggest that the effects of ZnC on NO production, HO-1 expression, and Nrf2 activation were attributed to its Zn subcomponent, but not l-carnosine. Pretreatment with ZnC was able to activate Nrf2/HO-1 signaling pathway, thus suppressing the expression of inflammatory mediators, such as NO and iNOS in LPS-induced RAW 264.7 cells.

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells.

PubMed

Zhang, Benping; Zhao, Jie; Li, Shanshan; Zeng, Linglan; Chen, Yan; Fang, Jun

2015-04-01

Mangiferin (2-C-β-d-gluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) is a well-known natural antioxidant distributed in various plants of the Anacardiaceae and Gentianaceae families. Mangiferin can inhibit carcinogen-induced lung or colon tumor formation in experimental animals. However, the molecular mechanisms of its chemopreventive activity remain unexplored. This study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-ARE signaling in hematopoietic cells. Mononuclear cells (MNCs) were isolated from human umbilical cord blood (hUCB). DNA damage was evaluated by comet and micronucleus assays. The expression of Nrf2 and NQO1 was examined by immunofluorescence and western blotting. An electrophoretic mobility shift assay (EMSA) was used to detect the binding activity of Nrf2 with NQO1-ARE sequences. We found that mangiferin treatment significantly reduced DNA damage in etoposide-treated MNCs, which was verified by decreased olive tail moment (OTM) and micronucleus (MN) frequency. Mangiferin treatment significantly promoted Nrf2 translocation into the nucleus and increased nuclear Nrf2 expression. Moreover, NQO1, an Nrf2 signaling target, was significantly upregulated by mangiferin treatment, and the binding activity of Nrf2 with NQO1-ARE sequences was elevated after mangiferin treatment. Mangiferin activated Nrf2 signaling, upregulated NQO1 expression, and significantly reduced etoposide-induced DNA damage. Thus, mangiferin is a potential cytoprotective agent for hematopoietic cells.

KEAP1 and Done? Targeting the NRF2 Pathway with Sulforaphane.

PubMed

Dinkova-Kostova, Albena T; Fahey, Jed W; Kostov, Rumen V; Kensler, Thomas W

2017-11-01

Since the re-discovery of sulforaphane in 1992 and the recognition of the bioactivity of this phytochemical, many studies have examined its mode of action in cells, animals and humans. Broccoli, especially as young sprouts, is a rich source of sulforaphane and broccoli-based preparations are now used in clinical studies probing efficacy in health preservation and disease mitigation. Many putative cellular targets are affected by sulforaphane although only one, KEAP1-NRF2 signaling, can be considered a validated target at this time. The transcription factor NRF2 is a master regulator of cell survival responses to endogenous and exogenous stressors. This review summarizes the chemical biology of sulforaphane as an inducer of NRF2 signaling and efficacy as an inhibitor of carcinogenesis. It also provides a summary of the current findings from clinical trials using a suite of broccoli sprout preparations on a series of short-term endpoints reflecting a diversity of molecular actions. Sulforaphane, as a pure chemical, protects against chemical-induced skin, oral, stomach, colon, lung and bladder carcinogenesis and in genetic models of colon and prostate carcinogenesis. In many of these settings the antitumorigenic efficacy of sulforaphane is dampened in Nrf2 -disrupted animals. Broccoli preparations rich in glucoraphanin or sulforaphane exert demonstrable pharmacodynamic action in over a score of clinical trials. Measures of NRF2 pathway response and function are serving as guideposts for the optimization of dose, schedule and formulation as clinical trials with broccoli-based preparations become more commonplace and more rigorous in design and implementation.

Abrupt salinity stress induces oxidative stress via the Nrf2-Keap1 signaling pathway in large yellow croaker Pseudosciaena crocea.

PubMed

Zeng, Lin; Ai, Chun-Xiang; Wang, Yong-Hong; Zhang, Jian-She; Wu, Chang-Wen

2017-08-01

The aim of the present study was to evaluate the effects of abrupt salinity stress (12, 26 (control), and 40) on lipid peroxidation, activities and mRNA levels of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx, and GR), and gene expression of the Nrf2-Keap1 signaling molecules at different times (6, 12, 24, and 48 h) in the liver of large yellow croaker Pseudosciaena crocea. The results showed that lipid peroxidation was sharply reduced at 6 h and increased at 12 h before returning to control levels in the hypo-salinity group. Similarly, lipid peroxidation was significantly decreased at 6 h followed by a sharp increase towards the end of the exposure in the hyper-salinity group. Negative relationships between lipid peroxidation and antioxidant enzyme activities and positive relationships between activities and gene expression of antioxidant enzymes were observed, suggesting that the changes at molecular levels and enzyme activity levels may provide protective roles against damage from salinity stress. Obtained results also showed a coordinated transcriptional regulation of antioxidant genes, suggesting that Nrf2 is required for regulating these genes. Furthermore, there was a positive relationship between the mRNA levels of Nrf2 and Keap1, indicating that Keap1 plays an important role in switching off the Nrf2 response. In conclusion, this is the first study to elucidate effects of salinity stress on antioxidant responses in large yellow croaker through the Keap1-Nrf2 pathway.

Sinomenine attenuates renal fibrosis through Nrf2-mediated inhibition of oxidative stress and TGFβ signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Tian

Renal fibrosis is the common feature of chronic kidney disease and mainly mediated by TGFβ-associated pro-fibrogenic signaling, which causes excessive extracellular matrix accumulation and successive loss of kidney functions. Sinomenine (SIN), an alkaloid derived from medicinal herb extensively used in treatment of rheumatoid arthritis and various inflammatory disorders, displays renal protective properties in experimental animals; however its pharmacological potency against renal fibrosis is not explored. In this study we report that SIN possesses strong anti-renal fibrosis functions in kidney cell and in mouse fibrotic kidney. SIN beneficially modulated the pro-fibrogenic protein expression in TGFβ-treated kidney cells and attenuated the renalmore » fibrotic pathogenesis incurred by unilateral ureteral obstruction (UUO), which correlated with its activation of Nrf2 signaling - the key defender against oxidative stress with anti-fibrotic potentials. Further investigation on its regulation of Nrf2 downstream events revealed that SIN significantly balanced oxidative stress via improving the expression and activity of anti-oxidant and detoxifying enzymes, and interrupted the pro-fibrogenic signaling of TGFβ/Smad and Wnt/β-catenin. Even more impressively SIN achieved its anti-fibrotic activities in an Nrf2-dependent manner, suggesting that SIN regulation of Nrf2-associated anti-fibrotic activities constitutes a critical component of SIN's renoprotective functions. Collectively our studies have demonstrated a novel anti-fibrotic property of SIN and its upstream events and provided a molecular basis for SIN's potential applications in treatment of renal fibrosis-associated kidney disorders. - Highlights: • Sinomenine has strong potency of inhibiting renal fibrosis in UUO mouse kidney. • Sinomenine attenuates the expression of profibrogenic proteins. • Sinomenine balances renal fibrosis-associated oxidative stress. • Sinomenine mitigates profibrogenic signaling of TGBβ/Smad and Wnt/β-catenin. • Sinomenine exerts anti-renal fibrotic functions mainly via activating Nrf2 pathways.« less

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

PubMed

Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

2005-12-01

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions.

Sulforaphane induces Nrf2 target genes and attenuates inflammatory gene expression in microglia from brain of young adult and aged mice.

PubMed

Townsend, Brigitte E; Johnson, Rodney W

2016-01-01

Increased neuroinflammation and oxidative stress resulting from heightened microglial activation are associated with age-related cognitive impairment. The objectives of this study were to examine the effects of the bioactive sulforaphane (SFN) on the nuclear factor E2-related factor 2 (Nrf2) pathway in BV2 microglia and primary microglia, and to evaluate proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated primary microglia from adult and aged mice. BV2 microglia and primary microglia isolated from young adult and aged mice were treated with SFN and LPS. Changes in Nrf2 activity, expression of Nrf2 target genes, and levels of proinflammatory markers were assessed by quantitative PCR and immunoassay. SFN increased Nrf2 DNA-binding activity and upregulated Nrf2 target genes in BV2 microglia, while reducing LPS-induced interleukin (IL-)1β, IL-6, and inducible nitric oxide synthase (iNOS). In primary microglia from adult and aged mice, SFN increased expression of Nrf2 target genes and attenuated IL-1β, IL-6, and iNOS induced by LPS. These data indicate that SFN is a potential beneficial supplement that may be useful for reducing microglial mediated neuroinflammation and oxidative stress associated with aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, Sherin T.; Bergström, Petra; Hammarsten, Ola, E-mail: ola.hammarsten@clinchem.gu.se

2014-05-01

Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA andmore » reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2′-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. - Highlights: • Repeated treatment with sulforaphane protects fibroblasts from ionizing radiation • Repeated sulforaphane treatment attenuates radiation induced ROS and DNA damage • Sulforaphane mediated protection is Nrf2 dependent.« less

Dose and Time Dependencies in Stress Pathway Responses during Chemical Exposure: Novel Insights from Gene Regulatory Networks.

PubMed

Souza, Terezinha M; Kleinjans, Jos C S; Jennen, Danyel G J

2017-01-01

Perturbation of biological networks is often observed during exposure to xenobiotics, and the identification of disturbed processes, their dynamic traits, and dose-response relationships are some of the current challenges for elucidating the mechanisms determining adverse outcomes. In this scenario, reverse engineering of gene regulatory networks (GRNs) from expression data may provide a system-level snapshot embedded within accurate molecular events. Here, we investigate the composition of GRNs inferred from groups of chemicals with two distinct outcomes, namely carcinogenicity [azathioprine (AZA) and cyclophosphamide (CYC)] and drug-induced liver injury (DILI; diclofenac, nitrofurantoin, and propylthiouracil), and a non-carcinogenic/non-DILI group (aspirin, diazepam, and omeprazole). For this, we analyzed publicly available exposed in vitro human data, taking into account dose and time dependencies. Dose-Time Network Identification (DTNI) was applied to gene sets from exposed primary human hepatocytes using four stress pathways, namely endoplasmic reticulum (ER), NF-κB, NRF2, and TP53. Inferred GRNs suggested case specificity, varying in interactions, starting nodes, and target genes across groups. DILI and carcinogenic compounds were shown to directly affect all pathway-based GRNs, while non-DILI/non-carcinogenic chemicals only affected NF-κB. NF-κB-based GRNs clearly illustrated group-specific disturbances, with the cancer-related casein kinase CSNK2A1 being a target gene only in the carcinogenic group, and opposite regulation of NF-κB subunits being observed in DILI and non-DILI/non-carcinogenic groups. Target genes in NRF2-based GRNs shared by DILI and carcinogenic compounds suggested markers of hepatotoxicity. Finally, we indicate several of these group-specific interactions as potentially novel. In summary, our reversed-engineered GRNs are capable of revealing dose dependent, chemical-specific mechanisms of action in stress-related biological networks.

Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

NASA Astrophysics Data System (ADS)

Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

2014-03-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

Elevated Hepatic Iron Activates NF-E2–Related Factor 2–Regulated Pathway in a Dietary Iron Overload Mouse Model

PubMed Central

Isom, Harriet C.

2012-01-01

Hepatic iron overload has been associated classically with the genetic disorder hereditary hemochromatosis. More recently, it has become apparent that mild-to-moderate degrees of elevated hepatic iron stores observed in other liver diseases also have clinical relevance. The goal was to use a mouse model of dietary hepatic iron overload and isobaric tag for relative and absolute quantitation proteomics to identify, at a global level, differentially expressed proteins in livers from mice fed a control or 3,5,5-trimethyl-hexanoyl-ferrocene (TMHF) supplemented diet for 4 weeks. The expression of 74 proteins was altered by ≥ ±1.5-fold, showing that the effects of iron on the liver proteome were extensive. The top canonical pathway altered by TMHF treatment was the NF-E2–related factor 2 (NRF2–)–mediated oxidative stress response. Because of the long-standing association of elevated hepatic iron with oxidative stress, the remainder of the study was focused on NRF2. TMHF treatment upregulated 25 phase I/II and antioxidant proteins previously categorized as NRF2 target gene products. Immunoblot analyses showed that TMHF treatment increased the levels of glutathione S-transferase (GST) M1, GSTM4, glutamate-cysteine ligase (GCL) catalytic subunit, GCL modifier subunit, glutathione synthetase, glutathione reductase, heme oxygenase 1, epoxide hydrolase 1, and NAD(P)H dehydrogenase quinone 1. Immunofluorescence, carried out to determine the cellular localization of NRF2, showed that NRF2 was detected in the nucleus of hepatocytes from TMHF-treated mice and not from control mice. We conclude that elevated hepatic iron in a mouse model activates NRF2, a key regulator of the cellular response to oxidative stress. PMID:22649188

Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

PubMed Central

He, Jin-Lian; Zhou, Zhi-Wei; Yin, Juan-Juan; He, Chang-Qiang; Zhou, Shu-Feng; Yu, Yang

2015-01-01

Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to the nuclei. Additionally, SCE significantly suppressed the expression of cytosolic Kelch-like ECH-associated protein 1 (the repressor of Nrf2) and remarkably increased Nrf2 stability in HepG2 cells. Taken together, our findings suggest that the hepatoprotective effects of SCE may be partially ascribed to the modulation of DMEs and drug transporters via Nrf2-mediated signaling pathway. SCE may alter the pharmacokinetics of other coadministered drugs that are substrates of these DMEs and transporters and thus cause unfavorable herb–drug interactions. PMID:25552902

Hydrogen sulfide (H2S) attenuates uranium-induced acute nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways.

PubMed

Zheng, Jifang; Zhao, Tingting; Yuan, Yan; Hu, Nan; Tang, Xiaoqing

2015-12-05

As an endogenous gaseous mediator, H2S exerts anti-oxidative, anti-inflammatory and cytoprotective effects in kidneys. This study was designed to investigate the protective effect of H2S against uranium-induced nephrotoxicity in adult SD male rats after in vivo effect of uranium on endogenous H2S formation was explored in kidneys. The levels of endogenous H2S and H2S-producing enzymes (CBS and CSE) were measured in renal homogenates from rats intoxicated by an intraperitoneally (i.p.) injection of uranyl acetate at a single dose of 2.5, 5 or 10 mg/kg. In rats injected i.p. with uranyl acetate (5 mg/kg) or NaHS (an H2S donor, 28 or 56 μmol/kg) alone or in combination, we determined biochemical parameters and histopathological alteration to assess kidney function, examined oxidative stress markers, and investigated Nrf2 and NF-κB pathways in kidney homogenates. The results suggest that uranium intoxication in rats decreased endogenous H2S generation as well as CBS and CSE protein expression. NaHS administration in uranium-intoxicated rats ameliorated the renal biochemical indices and histopathological effects, lowered MDA accumulation, and restored GSH level and anti-oxidative enzymes activities like SOD, CAT, GPx and GST. NaHS treatment in uranium-intoxicated rats activated uranium-inhibited protein expression and nuclear translocation of transcription factor Nrf2, which increased protein expression of downstream target-Nrf2 genes HO-1, NQO-1, GCLC, and TXNRD-1. NaHS administration in uranium-intoxicated rats inhibited uranium-induced nuclear translocation and phosphorylation of transcription factor κB/p65, which decreased protein expression of target-p65 inflammatory genes TNF-α, iNOS, and COX-2. Taken together, these data implicate that H2S can afford protection to rat kidneys against uranium-induced adverse effects through induction of antioxidant defense by activating Nrf2 pathway and reduction of inflammatory response by suppressing NF-κB pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Clinically significant association of elevated expression of nuclear factor E2-related factor 2 expression with higher glucose uptake and progression of upper urinary tract cancer.

PubMed

Nukui, Akinori; Narimatsu, Takahiro; Kambara, Tsunehito; Abe, Hideyuki; Sakamoto, Setsu; Yoshida, Ken-Ichiro; Kamai, Takao

2018-05-02

There is growing evidence that the transcription factor nuclear factor E2-related factor 2 (Nrf2) is the major participant in regulating antioxidants and pathways for detoxifying reactive oxygen species (ROS), as well as having a vital role in tumor proliferation, invasion, and chemoresistance. It was also recently reported that Nrf2 supports cell proliferation by promoting metabolic activity. Thus, Nrf2 is involved in progression of cancer. Upper urinary tract urothelial carcinoma (UTUC) is a biologically aggressive tumor with high rates of recurrence and progression, resulting in a poor prognosis. However, the role of Nrf2 in UTUC is largely unknown. In order to study the role of Nrf2 in UTUC from the metabolic perspective, we retrospectively assessed Nrf2 expression in the surgical specimen and the preoperative maximum standard glucose uptake (SUVmax) on [ 18 F]fluorodeoxy-glucose positron emission tomography ( 18 F-FDG-PET) of 107 patients with UTUC who underwent radical nephroureterectomy. Increased expression of Nrf2 in the primary lesion was correlated with less differentiated histology, local invasion, and lymph node metastasis, and was also an independent indicator of shorter overall survival according to multivariate analysis. Furthermore, increased expression of Nrf2 was associated with higher preoperative SUVmax by the primary tumor on 18 F-FDG-PET, while Nrf2 expression and SUVmax were also significantly correlated in the metastatic lymph nodes. Among the 18 patients with lymph node metastasis at nephroureterectomy who underwent retroperitoneal lymph node dissection and received adjuvant chemotherapy, the patients with higher Nrf2 expression in the primary tumor had worse recurrence-free survival. These results suggest that constitutive activation of Nrf2 might be linked with tumor aerobic glycolysis and progression of UTUC, indicating that Nrf2 signaling in the tumor microenvironment promotes progression of UTUC.

A biomarker-based screen of a gene expression compendium ...

EPA Pesticide Factsheets

Computational approaches were developed to identify factors that regulate Nrf2 in a large gene expression compendium of microarray profiles including >2000 comparisons which queried the effects of chemicals, genes, diets, and infectious agents on gene expression in the mouse liver. A gene expression biomarker of 48 genes which accurately predicted Nrf2 activation was used to identify factors which resulted in a gene expression profile with significant correlation to the biomarker. A number of novel insights were made. Chemicals that activated the xenosensor constitutive activated receptor (CAR) consistently activated Nrf2 across hundreds of profiles, possibly downstream of Cyp-induced increases in oxidative stress. Nrf2 activation was also found to be negatively regulated by the growth hormone (GH)- and androgen-regulated transcription factor STAT5b, a transcription factor suppressed by CAR. Nrf2 was activated when STAT5b was suppressed in female mice vs. male mice, after exposure to estrogens, or in genetic mutants in which GH signaling was disrupted. A subset of the mutants that show STAT5b suppression and Nrf2 activation result in increased resistance to environmental stressors and increased longevity. This study describes a novel approach for understanding the network of factors that regulate the Nrf2 pathway and highlights novel interactions between Nrf2, CAR and STAT5b transcription factors. (This abstract does not represent EPA policy.) Computational appr

Agmatine induces Nrf2 and protects against corticosterone effects in hippocampal neuronal cell line.

PubMed

Freitas, Andiara E; Egea, Javier; Buendía, Izaskun; Navarro, Elisa; Rada, Patricia; Cuadrado, Antonio; Rodrigues, Ana Lúcia S; López, Manuela G

2015-01-01

Hyperactivation of the hypothalamic-pituitary-adrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration- and time-dependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α2-adrenoceptor antagonist), ketanserin (5-HT2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α2-adrenergic and 5-HT2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis

PubMed Central

Jiang, Ming; Ku, Wei-Yao; Zhou, Zhongren; Dellon, Evan S.; Falk, Gary W.; Nakagawa, Hiroshi; Wang, Mei-Lun; Liu, Kuancan; Wang, Jun; Katzka, David A.; Peters, Jeffrey H.; Lan, Xiaopeng; Que, Jianwen

2015-01-01

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett’s intestinal differentiation; however, in mice, basal progenitor cell–specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE. PMID:25774506

Intranasal Pretreatment with Z-Ligustilide, the Main Volatile Component of Rhizoma Chuanxiong, Confers Prophylaxis against Cerebral Ischemia via Nrf2 and HSP70 Signaling Pathways.

PubMed

Li, Juan; Yu, Jie; Ma, Hui; Yang, Na; Li, Li; Zheng, Ding-Ding; Wu, Ming-Xia; Zhao, Zhi-Long; Qi, Hong-Yi

2017-03-01

Z-Ligustilide (Z-LIG) is a major component in Rhizoma Chuanxiong, which has been traditionally used as a health food supplement for the prevention of cerebrovascular disease in China. This study investigates the ability of intranasal Z-LIG pretreatment to enhance protection against neuronal damage in rats with middle cerebral artery occlusion (MCAO) and the role of cellular stress response mechanisms Nrf2 and HSP70. Z-LIG significantly mitigated infarct volume, neurological dysfunction, blood-brain barrier disruption, and brain edema (p < 0.01). Moreover, Z-LIG prevented the loss of collagen IV, occludin, and ZO-1 (p < 0.05) and decreased MMP-2 and -9 levels (p < 0.01). Meanwhile, Z-LIG up-regulated NQO1 and HSP70. Notably, blockage of Nrf2-driven transcription or down-regulation of HSP70 remarkably attenuated the preventive effect of Z-LIG (p < 0.05). Together, intranasal delivery of Z-LIG enhanced protection against ischemic injury via Nrf2 and HSP70 signaling pathways and has prophylactic potential in the population at high risk of stroke.

4-Methoxychalcone Enhances Cisplatin-Induced Oxidative Stress and Cytotoxicity by Inhibiting the Nrf2/ARE-Mediated Defense Mechanism in A549 Lung Cancer Cells

PubMed Central

Lim, Juhee; Lee, Sung Ho; Cho, Sera; Lee, Ik-Soo; Kang, Bok Yun; Choi, Hyun Jin

2013-01-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for the protection of cells against oxidative and xenobiotic stresses. Recent studies have demonstrated that high constitutive expression of Nrf2 is observed in many types of cancer cells showing resistance to anti-cancer drugs, suggesting that the suppression of overexpressed Nrf2 could be an attractive therapeutic strategy to overcome cancer drug resistance. In the present study, we aimed to find small molecule compounds that enhance the sensitivity of tumor cells to cisplatin induced cytotoxicity by suppressing Nrf2-mediated defense mechanism. A549 lung cancer cells were shown to be more resistant to the anti-cancer drug cisplatin than HEK293 cells, with higher Nrf2 signaling activity; constitutively high amounts of Nrf2-downstream target proteins were observed in A549 cells. Among the three chalcone derivatives 4-methoxy-chalcone (4-MC), hesperidin methylchalcone, and neohesperidin dihydrochalcone, 4-MC was found to suppress transcriptional activity of Nrf2 in A549 cells but to activate it in HEK293 cells. 4-MC was also shown to down-regulate expression of Nrf2 and the downstream phase II detoxifying enzyme NQO1 in A549 cells. The PI3K/Akt pathway was found to be involved in the 4-MC-induced inhibition of Nrf2/ARE activity in A549 cells. This inhibition of Nrf2 signaling results in the accelerated generation of reactive oxygen species and exacerbation of cytotoxicity in cisplatin-treated A549 cells. Taken together, these results suggest that the small molecule compound 4-MC could be used to enhance the sensitivity of tumor cells to the therapeutic effect of cisplatin through the regulation of Nrf2/ARE signaling. PMID:24046186

4-methoxychalcone enhances cisplatin-induced oxidative stress and cytotoxicity by inhibiting the Nrf2/ARE-mediated defense mechanism in A549 lung cancer cells.

PubMed

Lim, Juhee; Lee, Sung Ho; Cho, Sera; Lee, Ik-Soo; Kang, Bok Yun; Choi, Hyun Jin

2013-10-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for the protection of cells against oxidative and xenobiotic stresses. Recent studies have demonstrated that high constitutive expression of Nrf2 is observed in many types of cancer cells showing resistance to anti-cancer drugs, suggesting that the suppression of overexpressed Nrf2 could be an attractive therapeutic strategy to overcome cancer drug resistance. In the present study, we aimed to find small molecule compounds that enhance the sensitivity of tumor cells to cisplatin induced cytotoxicity by suppressing Nrf2-mediated defense mechanism. A549 lung cancer cells were shown to be more resistant to the anti-cancer drug cisplatin than HEK293 cells, with higher Nrf2 signaling activity; constitutively high amounts of Nrf2-downstream target proteins were observed in A549 cells. Among the three chalcone derivatives 4-methoxy-chalcone (4-MC), hesperidin methylchalcone, and neohesperidin dihydrochalcone, 4-MC was found to suppress transcriptional activity of Nrf2 in A549 cells but to activate it in HEK293 cells. 4-MC was also shown to down-regulate expression of Nrf2 and the downstream phase II detoxifying enzyme NQO1 in A549 cells. The PI3K/Akt pathway was found to be involved in the 4-MC-induced inhibition of Nrf2/ARE activity in A549 cells. This inhibition of Nrf2 signaling results in the accelerated generation of reactive oxygen species and exacerbation of cytotoxicity in cisplatin-treated A549 cells. Taken together, these results suggest that the small molecule compound 4-MC could be used to enhance the sensitivity of tumor cells to the therapeutic effect of cisplatin through the regulation of Nrf2/ARE signaling.

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

PubMed Central

Chen, Xiao-Qing; Wu, Sheng-Hua; Zhou, Yu; Tang, Yan-Rong

2013-01-01

Objective To investigate whether lipoxin A4 (LXA4) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA4-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA4-induced HO-1 induction. Methods Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA4 or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay. Results Pretreatment of the cells undergoing H/R lesion with LXA4 significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA4 on the cells undergoing H/R lesion. LXA4 increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure. Conclusion The protection role of LXA4 against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway. PMID:23826208

Effects of trigonelline inhibition of the Nrf2 transcription factor in vitro on Echinococcus granulosus.

PubMed

Qin, Wenjuan; Guan, Dongfang; Ma, Rongji; Yang, Rentan; Xing, Guoqiang; Shi, Hongjuan; Tang, Guangyao; Li, Jiajie; Lv, Hailong; Jiang, Yufeng

2017-08-01

The aim of this study was to investigate the impact of trigonelline (TRG) on Echinococcus granulosus, and to explore the inhibition impact of nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway on E. granulosus protoscoleces. Echinococcus granulosus protoscoleces were incubated with various concentrations of TRG, and then Nrf2 protein expression and its localization in protoscoleces were detected by western blot analysis and immunofluorescence assay, respectively. Reactive oxygen species (ROS) level in protoscoleces was measured using ROS detection kit. Caspase-3 activity was measured using a caspase-3 activity assay kit, and NAD(P)H quinone oxidoreductase (NQO)-1 and heme oxygenase (HO)-1 activities in protoscoleces were measured by ELISA. The effect of TRG on protoscoleces viability was investigated using 0.1% eosin staining, and ultrastructural alterations in protoscoleces were examined by scanning electron microscopy (SEM). Immunolocalization experiment clearly showed that Nrf2 protein was predominantly present in cells of protoscoleces. TRG treatment reduced NQO-1 and HO-1 activities in protoscoleces, but could increase ROS level at early time. Protoscoleces could not survive when treated with 250 μM TRG for 12 days. SEM results showed that TRG-treated protoscoleces presented damage in the protoscoleces region, including hook deformation, lesions, and digitiform protuberance. Nrf2 protein expression was significantly decreased and caspase-3 activity was clearly increased in protoscoleces treated with TRG for 24 and 48 h, respectively, when compared with that in controls (P < 0.05). Our results demonstrated that TRG had scolicidal activity against E. granulosus protoscoleces. Nrf2 protein was mainly expressed in the cells and TRG could efficiently inhibit the Nrf2 signaling pathway in E. granulosus. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ganglioside GM1 protects against high altitude cerebral edema in rats by suppressing the oxidative stress and inflammatory response via the PI3K/AKT-Nrf2 pathway.

PubMed

Gong, Gu; Yin, Liang; Yuan, Libang; Sui, Daming; Sun, Yangyang; Fu, Haiyu; Chen, Liang; Wang, Xiaowu

2018-03-01

High altitude cerebral edema (HACE) is a severe type of acute mountain sickness (AMS) that occurs in response to a high altitude hypobaric hypoxic (HH) environment. GM1 monosialoganglioside can alleviate brain injury under adverse conditions including amyloid-β-peptide, ischemia and trauma. However, its role in HACE-induced brain damage remains poorly elucidated. In this study, GM1 supplementation dose-dependently attenuated increase in rat brain water content (BWC) induced by hypobaric chamber (7600 m) exposurefor 24 h. Compared with the HH-treated group, rats injected with GM1 exhibited less brain vascular leakage, lower aquaporin-4 and higher occludin expression, but they also showed increase in Na+/K+-ATPase pump activities. Importantly, HH-incurred consciousness impairment and coordination loss also were ameliorated following GM1 administration. Furthermore, the increased oxidative stress and decrease in anti-oxidant stress system under the HH condition were also reversely abrogated by GM1 treatment via suppressing accumulation of ROS, MDA and elevating the levels of SOD and GSH. Simultaneously, GM1 administration also counteracted the enhanced inflammation in HH-exposed rats by muting pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 levels in serum and brain tissues. Subsequently, GM1 potentiated the activation of the PI3K/AKT-Nrf2 pathway. Cessation of this pathway by LY294002 reversed GM1-mediated inhibitory effects on oxidative stress and inflammation, and ultimately abrogated the protective role of GM1 in abating brain edema, cognitive and motor dysfunction. Overall, GM1 may afford a protective intervention in HACE by suppressing oxidative stress and inflammatory response via activating the PI3K/AKT-Nrf2 pathway, implying a promising agent for the treatment of HACE. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood-brain barrier disruption and neurological deficits in stroke.

PubMed

Alfieri, Alessio; Srivastava, Salil; Siow, Richard C M; Cash, Diana; Modo, Michel; Duchen, Michael R; Fraser, Paul A; Williams, Steven C R; Mann, Giovanni E

2013-12-01

Disruption of the blood-brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70 min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72 h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4-72 h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Ginsenoside Rb1 improves postoperative fatigue syndrome by reducing skeletal muscle oxidative stress through activation of the PI3K/Akt/Nrf2 pathway in aged rats.

PubMed

Zhuang, Cheng-Le; Mao, Xiang-Yu; Liu, Shu; Chen, Wei-Zhe; Huang, Dong-Dong; Zhang, Chang-Jing; Chen, Bi-Cheng; Shen, Xian; Yu, Zhen

2014-10-05

Ginsenoside Rb1 is reported to possess anti-fatigue activity, but the mechanisms remain unknown. The aim of this study was to investigate the molecular mechanisms responsible for the anti-fatigue effect of ginsenoside Rb1 on postoperative fatigue syndrome induced by major small intestinal resection (MSIR) in aged rat. Aged rats with MSIR were administrated with ginsenoside Rb1 (15 mg/kg) once a day from 3 days before surgery to the day of sacrifice, or with saline as corresponding controls. Rats without MSIR but going through the same surgery procedure were administrated with saline as blank controls. Anti-fatigue effect was assessed by an open field test; superoxide dismutase, reactive oxygen species and malondialdehyde in skeletal muscle were determined. The mRNA levels of Akt2 and Nrf2 in skeletal muscle were measured by real-time quantitative PCR. The activation of Akt and Nrf2 was examined by western blot and immunohistofluorescence. Our results revealed that ginsenoside Rb1 significantly increased the journey and the rearing frequency, decreased the time of rest in aged rats with MSIR. In addition, ginsenoside Rb1 significantly reduced reactive oxygen species and malondialdehyde release and increased the superoxide dismutase activity of skeletal muscle in aged rats with MSIR. Ginsenoside Rb1 also increased the expression of Akt2 and Nrf2 mRNA, up-regulated Akt phosphorylation and Nrf2 nuclear translocation. These findings indicate that ginsenoside Rb1 has an anti-fatigue effect on postoperative fatigue syndrome in aged rat, and the mechanism possibly involves activation of the PI3K/Akt pathway with subsequent Nrf2 nuclear translocation and induction of antioxidant enzymes. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibition of curcumin on influenza A virus infection and influenzal pneumonia via oxidative stress, TLR2/4, p38/JNK MAPK and NF-κB pathways.

PubMed

Dai, Jianping; Gu, Liming; Su, Yun; Wang, Qianwen; Zhao, Ying; Chen, Xiaoxua; Deng, Huixiong; Li, Weizhong; Wang, Gefei; Li, Kangsheng

2018-01-01

Oxidative stress, Nrf2-HO-1 and TLR-MAPK/NF-κB signaling pathways have been proved to be involved in influenza A virus (IAV) replication and influenzal pneumonia. In the previous studies, we have performed several high-throughput drug screenings based on the TLR pathways. In the present study, through plaque inhibition test, luciferase reporter assay, TCID 50 , qRT-PCR, western blotting, ELISA and siRNA assays, we investigated the effect and mechanism of action of curcumin against IAV infection in vitro and in vivo. The results showed that curcumin could directly inactivate IAV, blocked IAV adsorption and inhibited IAV proliferation. As for the underlying mechanisms, we found that curcumin could significantly inhibit IAV-induced oxidative stress, increased Nrf2, HO-1, NQO1, GSTA3 and IFN-β production, and suppressed IAV-induced activation of TLR2/4/7, Akt, p38/JNK MAPK and NF-κB pathways. Suppression of Nrf2 via siRNA significantly abolished the stimulatory effect of curcumin on HO-1, NQO1, GSTA3 and IFN-β production and meanwhile blocked the inhibitory effect of curcumin on IAV M2 production. Oxidant H 2 O 2 and TLR2/4, p38/JNK and NF-κB agonists could significantly antagonize the anti-IAV activity of curcumin in vitro. Additionally, curcumin significantly increased the survival rate of mice, reduced lung index, inflammatory cytokines and lung IAV titer, and finally improved pulmonary histopathological changes after IAV infection. In conclusion, curcumin can directly inactivate IAV, inhibits IAV adsorption and replication; and its inhibition on IAV replication may be via activating Nrf2 signal and inhibiting IAV-induced activation of TLR2/4, p38/JNK MAPK and NF-κB pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased Alzheimer's disease-like pathology in the APP/ PS1ΔE9 mouse model lacking Nrf2 through modulation of autophagy.

PubMed

Joshi, Gururaj; Gan, Kok Ann; Johnson, Delinda A; Johnson, Jeffrey A

2015-02-01

The presence of senile plaques is one of the major pathologic hallmarks of the brain with Alzheimer's disease (AD). The plaques predominantly contain insoluble amyloid β-peptide, a cleavage product of the larger amyloid precursor protein (APP). Two enzymes, named β and γ secretase, generate the neurotoxic amyloid-β peptide from APP. Mature APP is also turned over endogenously by autophagy, more specifically by the endosomal-lysosomal pathway. A defective lysosomal system is known to be pathogenic in AD. Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders, and Nrf2 has become a potential therapeutic target for various neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis. In the current study, we explored the effect of genetic ablation of Nrf2 on APP/Aβ processing and/or aggregation as well as changes in autophagic dysfunction in APP/PS1 mice. There was a significant increase in inflammatory response in APP/PS1 mice lacking Nrf2. This was accompanied by increased intracellular levels of APP, Aβ (1-42), and Aβ (1-40), without a change total full-length APP. There was a shift of APP and Aβ into the insoluble fraction, as well as increased poly-ubiquitin conjugated proteins in mice lacking Nrf2. APP/PS1-mediated autophagic dysfunction is also enhanced in Nrf2-deficient mice. Finally, neurons in the APP/PS1/Nrf2-/- mice had increased accumulation of multivesicular bodies, endosomes, and lysosomes. These outcomes provide a better understanding of the role of Nrf2 in modulating autophagy in an AD mouse model and may help design better Nrf2 targeted therapeutics that could be efficacious in the treatment of AD. Published by Elsevier Inc.

Pomegranate protects against arsenic-induced p53-dependent ROS-mediated inflammation and apoptosis in liver cells.

PubMed

Choudhury, Sreetama; Ghosh, Sayan; Mukherjee, Sudeshna; Gupta, Payal; Bhattacharya, Saurav; Adhikary, Arghya; Chattopadhyay, Sreya

2016-12-01

Molecular mechanisms involved in arsenic-induced toxicity are complex and elusive. Liver is one of the most favored organs for arsenic toxicity as methylation of arsenic occurs mostly in the liver. In this study, we have selected a range of environmentally relevant doses of arsenic to examine the basis of arsenic toxicity and the role of pomegranate fruit extract (PFE) in combating it. Male Swiss albino mice exposed to different doses of arsenic presented marked hepatic injury as evident from histological and electron microscopic studies. Increased activities of enzymes alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase corroborated extensive liver damage. It was further noted that arsenic exposure initiated reactive oxygen species (ROS)-dependent apoptosis in the hepatocytes involving loss of mitochondrial membrane potential. Arsenic significantly increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-κB (NF-κB), coupled with increase in phosphorylated Iκ-B, possibly as adaptive cellular survival strategies. Arsenic-induced oxidative DNA damage to liver cells culminated in p53 activation and increased expression of p53 targets like miR-34a and Bax. Pomegranate polyphenols are known to possess remarkable antioxidant properties and are capable of protecting normal cells from various stimuli-induced oxidative stress and toxicities. We explored the protective role of PFE in ameliorating arsenic-induced hepatic damage. PFE was shown to reduce ROS generation in hepatocytes, thereby reducing arsenic-induced Nrf2 activation. PFE also inhibited arsenic-induced NF-κB-inflammatory pathway. Data revealed that PFE reversed arsenic-induced hepatotoxicity and apoptosis by modulating the ROS/Nrf2/p53-miR-34a axis. For the first time, we have mapped the possible signaling pathways associated with arsenic-induced hepatotoxicity and its rescue by pomegranate polyphenols. Copyright © 2016 Elsevier Inc. All rights reserved.

Nrf2 promotes neuronal cell differentiation.

PubMed

Zhao, Fei; Wu, Tongde; Lau, Alexandria; Jiang, Tao; Huang, Zheping; Wang, Xiao-Jun; Chen, Weimin; Wong, Pak Kin; Zhang, Donna D

2009-09-15

The transcription factor Nrf2 has emerged as a master regulator of the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate, two well-studied inducers of neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 in a dose- and time-dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M and microtubule-associated protein 2. Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation, whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to those from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents.

PubMed

Abed, Dhulfiqar Ali; Goldstein, Melanie; Albanyan, Haifa; Jin, Huijuan; Hu, Longqin

2015-07-01

The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.

Hydrogen gas protects against serum and glucose deprivation‑induced myocardial injury in H9c2 cells through activation of the NF‑E2‑related factor 2/heme oxygenase 1 signaling pathway.

PubMed

Xie, Qiang; Li, Xue-Xiang; Zhang, Peng; Li, Jin-Cao; Cheng, Ying; Feng, Yan-Ling; Huang, Bing-Sheng; Zhuo, Yu-Feng; Xu, Guo-Hua

2014-08-01

Ischemia or hypoxia‑induced myocardial injury is closely associated with oxidative stress. Scavenging free radicals and/or enhancing endogenous antioxidative defense systems may be beneficial for the impediment of myocardial ischemic injury. Hydrogen (H2) gas, as a water‑ and lipid‑soluble small molecule, is not only able to selectively eliminate hydroxyl (·OH) free radicals, but also to enhance endogenous antioxidative defense systems in rat lungs and arabidopsis plants. However, thus far, it has remained elusive whether H2 gas protects cardiomyocytes through enhancement of endogenous antioxidative defense systems. In the present study, the cardioprotective effect of H2 gas against ischemic or hypoxic injury was investigated, along with the underlying molecular mechanisms. H9c2 cardiomyoblasts (H9c2 cells) were treated in vitro with a chemical hypoxia inducer, cobalt chloride (CoCl2), to imitate hypoxia, or by serum and glucose deprivation (SGD) to imitate ischemia. Cell viability and intracellular ·OH free radicals were assessed. The role of an endogenous antioxidative defense system, the NF‑E2‑related factor 2 (Nrf2)/heme oxygenase 1 (HO‑1) signaling pathway, was evaluated. The findings revealed that treatment with CoCl2 or SGD markedly reduced cell viability in H9c2 cells. H2 gas‑rich medium protected against cell injury induced by SGD, but not that induced by CoCl2. When the cells were exposed to SGD, levels of intracellular ·OH free radicals were markedly increased; this was mitigated by H2 gas‑rich medium. Exposure of the cells to SGD also resulted in significant increases in HO‑1 expression and nuclear Nrf2 levels, and the HO‑1 inhibitor ZnPP IX and the Nrf2 inhibitor brusatol aggravated SGD‑induced cellular injury. H2 gas‑rich medium enhanced SGD‑induced upregulation of HO‑1 and Nrf2, and the HO‑1 or Nrf2 inhibition partially suppressed H2 gas‑induced cardioprotection. Furthermore, following genetic silencing of Nrf2 by RNA interference, the effects of H2 gas on the induction of HO‑1 and cardioprotection were markedly reduced. In conclusion, H2 gas protected cardiomyocytes from ischemia‑induced myocardial injury through elimination of ·OH free radicals and also through activation of the Nrf2/HO‑1 signaling pathway.

Estrogen-Dependent Nrf2 Expression Protects Against Reflux-Induced Esophagitis.

PubMed

Torihata, Yudai; Asanuma, Kiyotaka; Iijima, Katsunori; Mikami, Tetsuhiko; Hamada, Shin; Asano, Naoki; Koike, Tomoyuki; Imatani, Akira; Masamune, Atsushi; Shimosegawa, Tooru

2018-02-01

Gastroesophageal reflux disease is more common in males than in females. The enhanced antioxidative capacity of estrogen in females might account for the gender difference. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the host defense mechanism against oxidative stress. This study aimed to clarify the role of Nrf2 in reflux-induced esophageal inflammation, focusing on the gender difference and nitric oxide. Gastroesophageal reflux was surgically induced in male and female rats. Nitrite and ascorbic acid were administered for 1 week to provoke nitric oxide in the esophageal lumen. Male rats with gastroesophageal reflux were supplemented with 17β-estradiol or tert-butylhydroquinone, an Nrf2-inducing reagent. Esophageal squamous cell carcinoma KYSE30 cells were treated with 17β-estradiol. Nrf2 expression was examined by Western blotting and quantitative real-time PCR. Antioxidant gene expression profiles were examined by a PCR array. In the presence of nitric oxide, reflux-induced esophageal damage was less evident, whereas esophageal expression of Nrf2 and its target genes such as Nqo1 was more evident in female or male rats supplemented with 17β-estradiol than in male rats. 17β-Estradiol increased nuclear Nrf2 expression in KYSE30 cells. tert-Butylhydroquinone increased tissue Nqo1 mRNA expression, leading to a reduction in reflux-induced esophageal damage. Estrogen-dependent Nrf2 expression might contribute to protection against the development of gastroesophageal reflux disease in females.

An Essential Role of NRF2 in Diabetic Wound Healing.

PubMed

Long, Min; Rojo de la Vega, Montserrat; Wen, Qing; Bharara, Manish; Jiang, Tao; Zhang, Rui; Zhou, Shiwen; Wong, Pak K; Wondrak, Georg T; Zheng, Hongting; Zhang, Donna D

2016-03-01

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2-related factor 2 (NRF2)-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2(-/-) mice have delayed wound closure rates compared with Nrf2(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.

PubMed

Li, Jinqing; Ichikawa, Tomonaga; Jin, Yu; Hofseth, Lorne J; Nagarkatti, Prakash; Nagarkatti, Mitzi; Windust, Anthony; Cui, Taixing

2010-07-20

Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes. A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFalpha) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H(2)O(2)). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral over-expression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart. American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFalpha-induced free radical formation and H(2)O(2)-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral administration of American ginseng markedly increased Nrf2 activity in murine hearts. These results demonstrate that American ginseng suppresses oxidative stress and oxidative stress-induced cell death in cardiomyocytes through activating the Nrf2 pathway, thereby providing cardioprotection against pathological cardiac remodeling. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

3H-1,2-dithiole-3-thione protects retinal pigment epithelium cells against Ultra-violet radiation via activation of Akt-mTORC1-dependent Nrf2-HO-1 signaling.

PubMed

Li, Ke-Ran; Yang, Su-Qing; Gong, Yi-Qing; Yang, Hong; Li, Xiu-Miao; Zhao, Yu-Xia; Yao, Jin; Jiang, Qin; Cao, Cong

2016-05-06

Excessive UV radiation and reactive oxygen species (ROS) cause retinal pigment epithelium (RPE) cell injuries. Nrf2 regulates transcriptional activation of many anti-oxidant genes. Here, we tested the potential role of 3H-1,2-dithiole-3-thione (D3T) against UV or ROS damages in cultured RPE cells (both primary cells and ARPE-19 line). We showed that D3T significantly inhibited UV-/H2O2-induced RPE cell death and apoptosis. UV-stimulated ROS production was dramatically inhibited by D3T pretreatment. D3T induced Nrf2 phosphorylation in cultured RPE cells, causing Nrf2 disassociation with KEAP1 and its subsequent nuclear accumulation. This led to expression of antioxidant response elements (ARE)-dependent gene heme oxygenase-1 (HO-1). Nrf2-HO-1 activation was required for D3T-mediated cytoprotective effect. Nrf2 shRNA knockdown or S40T dominant negative mutation as well as the HO-1 inhibitor Zinc protoporphyrin (ZnPP) largely inhibited D3T's RPE cytoprotective effects against UV radiation. Yet, exogenous overexpression Nrf2 enhanced D3T's activity in RPE cells. Further studies showed that D3T activated Akt/mTORC1 in cultured RPE cells. Akt-mTORC1 inhibitors, or Akt1 knockdown by shRNA, not only inhibited D3T-induced Nrf2-HO-1 activation, but also abolished the RPE cytoprotective effects. In vivo, D3T intravitreal injection protected from light-induced retinal dysfunctions in mice. Thus, D3T protects RPE cells from UV-induced damages via activation of Akt-mTORC1-Nrf2-HO-1 signaling axis.

Maresin 1 Ameliorates Lung Ischemia/Reperfusion Injury by Suppressing Oxidative Stress via Activation of the Nrf-2-Mediated HO-1 Signaling Pathway

PubMed Central

Wu, You; Zhao, Feng

2017-01-01

Lung ischemia/reperfusion (I/R) injury occurs in various clinical conditions and heavily damaged lung function. Oxidative stress reaction and antioxidant enzymes play a pivotal role in the etiopathogenesis of lung I/R injury. In the current study, we investigated the impact of Maresin 1 on lung I/R injury and explored the possible mechanism involved in this process. MaR 1 ameliorated I/R-induced lung injury score, wet/dry weight ratio, myeloperoxidase, tumor necrosis factor, bronchoalveolar lavage fluid (BALF) leukocyte count, BALF neutrophil ratio, and pulmonary permeability index levels in lung tissue. MaR 1 significantly reduced ROS, methane dicarboxylic aldehyde, and 15-F2t-isoprostane generation and restored antioxidative enzyme (superoxide dismutase, glutathione peroxidase, and catalase) activities. Administration of MaR 1 improved the expression of nuclear Nrf-2 and cytosolic HO-1 in I/R-treated lung tissue. Furthermore, we also found that the protective effects of MaR 1 on lung tissue injury and oxidative stress were reversed by HO-1 activity inhibitor, Znpp-IX. Nrf-2 transcription factor inhibitor, brusatol, significantly decreased MaR 1-induced nuclear Nrf-2 and cytosolic HO-1 expression. In conclusion, these results indicate that MaR 1 protects against lung I/R injury through suppressing oxidative stress. The mechanism is partially explained by activation of the Nrf-2-mediated HO-1 signaling pathway. PMID:28751936

Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte Necroptosis.

PubMed

Lu, Chunfeng; Xu, Wenxuan; Zhang, Feng; Shao, Jiangjuan; Zheng, Shizhong

2016-12-05

It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD). Our previous study has identified that the beneficial therapeutic effect of curcumin on alcohol-caused liver injury might be attributed to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), whereas the role of curcumin in regulating necroptosis and the underlying mechanism remain to be determined. We first found that chronic alcohol consumption triggered obvious hepatocyte necroptosis, leading to increased expression of receptor-interacting protein 1, receptor-interacting protein 3, high-mobility group box 1, and phosphorylated mixed lineage kinase domain-like in murine livers. Curcumin dose-dependently ameliorated hepatocyte necroptosis and alleviated alcohol-caused decrease in hepatic Nrf2 expression in alcoholic mice. Then Nrf2 shRNA lentivirus was introduced to generate Nrf2-knockdown mice. Our results indicated that Nrf2 knockdown aggravated the effects of alcohol on liver injury and necroptosis and even abrogated the inhibitory effect of curcumin on necroptosis. Further, activated Nrf2 by curcumin inhibited p53 expression in both livers and cultured hepatocytes under alcohol stimulation. The next in vitro experiments, similar to in vivo ones, revealed that although Nrf2 knockdown abolished the suppression of curcumin on necroptosis of hepatocytes exposed to ethanol, p53 siRNA could clearly rescued the relative effect of curcumin. In summary, for the first time, we concluded that curcumin attenuated alcohol-induced hepatocyte necroptosis in a Nrf2/p53-dependent mechanism. These findings make curcumin an excellent candidate for ALD treatment and advance the understanding of ALD mechanisms associated with hepatocyte necroptosis.

Targeting Nrf2 Signaling Improves Bacterial Clearance by Alveolar Macrophages in Patients with COPD and in a Mouse Model

PubMed Central

Harvey, Christopher J.; Thimmulappa, Rajesh K.; Sethi, Sanjay; Kong, Xiaoni; Yarmus, Lonny; Brown, Robert H.; David, Feller-Kopman; Wise, Robert; Biswal, Shyam

2016-01-01

Patients with chronic obstructive pulmonary disease (COPD) have innate immune dysfunction in the lung largely due to defective macrophage phagocytosis. This deficiency results in periodic bacterial infections that cause acute exacerbations of COPD, a major source of morbidity and mortality. Recent studies indicate that a decrease in Nrf2 (nuclear erythroid–related factor 2) signaling in patients with COPD may hamper their ability to defend against oxidative stress, although the role of Nrf2 in COPD exacerbations has not been determined. Here, we test whether activation of Nrf2 by the phytochemical sulforaphane restores phagocytosis of clinical isolates of nontypeable Haemophilus influenza (NTHI) and Pseudomonas aeruginosa (PA) by alveolar macrophages from patients with COPD. Sulforaphane treatment restored bacteria recognition and phagocytosis in alveolar macrophages from COPD patients. Furthermore, sulforaphane treatment enhanced pulmonary bacterial clearance by alveolar macrophages and reduced inflammation in wild-typemice but not in Nrf2-deficientmice exposed to cigarette smoke for 6 months. Gene expression and promoter analysis revealed that Nrf2 increased phagocytic ability of macrophages by direct transcriptional up-regulation of the scavenger receptor MARCO. Disruption of Nrf2 or MARCO abrogated sulforaphane-mediated bacterial phagocytosis by COPD alveolar macrophages. Our findings demonstrate the importance of Nrf2 and its downstream target MARCO in improving antibacterial defenses and provide a rationale for targeting this pathway, via pharmacological agents such as sulforaphane, to prevent exacerbations of COPD caused by bacterial infection. PMID:21490276

Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism.

PubMed

Loboda, Agnieszka; Damulewicz, Milena; Pyza, Elzbieta; Jozkowicz, Alicja; Dulak, Jozef

2016-09-01

The multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) is considered not only as a cytoprotective factor regulating the expression of genes coding for anti-oxidant, anti-inflammatory and detoxifying proteins, but it is also a powerful modulator of species longevity. The vertebrate Nrf2 belongs to Cap 'n' Collar (Cnc) bZIP family of transcription factors and shares a high homology with SKN-1 from Caenorhabditis elegans or CncC found in Drosophila melanogaster. The major characteristics of Nrf2 are to some extent mimicked by Nrf2-dependent genes and their proteins including heme oxygenase-1 (HO-1), which besides removing toxic heme, produces biliverdin, iron ions and carbon monoxide. HO-1 and their products exert beneficial effects through the protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis. On the other hand, the disturbances in the proper HO-1 level are associated with the pathogenesis of some age-dependent disorders, including neurodegeneration, cancer or macular degeneration. This review summarizes our knowledge about Nrf2 and HO-1 across different phyla suggesting their conservative role as stress-protective and anti-aging factors.

Leptin induces SIRT1 expression through activation of NF-E2-related factor 2: Implications for obesity-associated colon carcinogenesis.

PubMed

Song, Na-Young; Lee, Yeon-Hwa; Na, Hye-Kyung; Baek, Jeong-Heum; Surh, Young-Joon

2018-07-01

Leptin, a representative adipokine secreted from the white adipose tissue, is considered as a potential linker between obesity and cancer. SIRT1 is an NAD + -dependent histone/protein deacetylase speculated to function as an oncogene. In the present study, we found that leptin signaling-defective ob/ob and db/db mice had lower colonic expression of SIRT1 compared with leptin signaling-intact C57BL/6J mice, implying that leptin signaling is crucial for SIRT1 expression in vivo. Moreover, leptin induced up-regulation of SIRT1 in human colon cancer (HCT-116) cells. Leptin stimulated migration and invasion of cultured HCT-116 cells and tumor growth in the xenograft assay, and these effects were abrogated by a SIRT1 inhibitor sirtinol, suggesting that SIRT1 plays a role in leptin-induced colon carcinogenesis. Leptin-induced SIRT1 expression was regulated by the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2). Leptin stimulated nuclear accumulation of Nrf2 as well as its binding to the antioxidant response elements located in the SIRT1 promoter. Moreover, siRNA knockdown of Nrf2 abrogated the leptin-induced SIRT1 expression. Notably, SIRT1 was significantly reduced in colon tissues of Nrf2-null mice, lending further support to Nrf2-dependent SIRT1 expression. Expression of leptin, Nrf2 and SIRT1 was coordinately increased in human colon tumor tissues. In conclusion, leptin might play a role in colon carcinogenesis by inducing Nrf2-dependent SIRT1 overexpression. Copyright © 2018 Elsevier Inc. All rights reserved.

Procyanidin B2 ameliorates carrageenan-induced chronic nonbacterial prostatitis in rats via anti-inflammatory and activation of the Nrf2 pathway.

PubMed

Wang, Wei; Chen, Renzong; Wang, Jiye

2017-11-04

Prostatitis is one of the most prevalent problems in andriatry and urinary surgery. In the present study, we evaluated the effect of procyanidin B2 (PB) on carrageenan-induced chronic nonbacterial prostatitis in rats. Results showed that PB significantly decreased the prostatic index and enhanced the body weight inhibited by carrageenan. Biochemical results revealed that PB significantly lowered the prostatic specific antigen (PSA) and alleviated oxidative stress in serum. The levels of TNF-α, IL-6, and IL-10 in prostatic homogenate were also significantly decreased after PB treatment. We also found evidence that PB treatment reversed the suppression of Nrf2 nuclear translocation, and increased the expressions of NQO1 and HO-1 in the prostate glands. In conclusion, treatment with PB attenuates carrageenan-induced chronic nonbacterial prostatitis via anti-inflammatory and activation of the Nrf2 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction☆

PubMed Central

Chepelev, Nikolai L.; Zhang, Hongqiao; Liu, Honglei; McBride, Skye; Seal, Andrew J.; Morgan, Todd E.; Finch, Caleb E.; Willmore, William G.; Davies, Kelvin J.A.; Forman, Henry Jay

2013-01-01

Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals. PMID:24024152

Alleviation of Acute Lung Injury in Rats with Sepsis by Resveratrol via the Phosphatidylinositol 3-Kinase/Nuclear Factor-Erythroid 2 Related Factor 2/Heme Oxygenase-1 (PI3K/Nrf2/HO-1) Pathway.

PubMed

Wang, Yu; Wang, Xiaofeng; Zhang, Lichun; Zhang, Rong

2018-05-30

BACKGROUND Resveratrol (Res) is a type of polyphenol found in many plants, which can protect important organs from the damage induced by sepsis. However, the exact mechanism of its protective effect has not been established. This study investigated the effect of Res on the PI3K/Nrf2/HO-1 signaling pathway in rats with sepsis-induced acute lung injury (ALI). MATERIAL AND METHODS Male Wistar rats were treated with 30 mg/kg Res by intraperitoneal administration for 1 hour immediately after cecal ligation and puncture. Levels of MIP-2, IL-18, and IL-10 in bronchoalveolar lavage fluid (BALF) were determined. Lung tissues were collected to measure the wet-to-dry (W/D) ratios, oxidative stress index, and lung injury scores. Expression levels of Akt, p-Akt, HO-1, Nrf-2, and active caspase-3 proteins were determined by western blotting; expression of HO-1 mRNA was determined by RT-PCR. RESULTS Treatment with Res significantly decreased the levels of MIP-2 and IL-18 and increased IL-10 in the BALF of rats with sepsis-induced ALI. In addition, Res also effectively reduced the W/D lung weight ratio, lung injury score, and the levels of MDA (malondialdehyde) and 8-OHdG. Conversely, Res increased SOD (superoxide dismutase) activity in the lung tissue. Moreover, Res significantly induced higher HO-1 mRNA expression, upregulated HO-1 and Nrf-2 protein expression, and the phosphorylation of Akt in the lung tissue. In contrast, the levels of activated caspase-3 protein were decreased in Res-treated rats (P<0.05). CONCLUSIONS Res could inhibit inflammation, oxidative stress, and cell apoptosis to alleviate ALI in septic rats through the inhibition of the PI3K/Nrf2/HO-1 signaling pathway.

Taurine Protects Mouse Spermatocytes from Ionizing Radiation-Induced Damage Through Activation of Nrf2/HO-1 Signaling.

PubMed

Yang, Wenjun; Huang, Jinfeng; Xiao, Bang; Liu, Yan; Zhu, Yiqing; Wang, Fang; Sun, Shuhan

2017-01-01

The increasing prevalence of ionizing radiation exposure has inevitably raised public concern over the potential detrimental effects of ionizing radiation on male reproductive system function. The detection of drug candidates to prevent reproductive system from damage caused by ionizing radiation is urgent. We aimed to investigate the protective role of taurine on the injury of mouse spermatocyte-derived cells (GC-2) subjected to ionizing radiation. mouse spermatocytes (GC-2 cells) were exposed to ionizing radiation with or without treatment of Taurine. The effect of ionizing radiation and Taurine treatment on GC-2 cells were evaluated by cell viability assay (CCK8), cell cycle and apoptosis. The relative protein abundance change was determined by Western blotting. The siRNA was used to explore whether Nrf2 signaling was involved in the cytoprotection of Taurine. Taurine significantly inhibited the decrease of cell viability, percentage of apoptotic cells and cell cycle arrest induced by ionizing radiation. Western blot analysis showed that taurine significantly limited the ionizing radiation-induced down-regulation of CyclinB1 and CDK1, and suppressed activation of Fas/FasL system pathway. In addition, taurine treatment significantly increased the expression of Nrf2 and HO-1 in GC-2 cells exposed to ionizing radiation, two components in antioxidant pathway. The above cytoprotection of Taurine was blocked by siNrf2. Our results demonstrate that taurine has the potential to effectively protect GC-2 cells from ionizing radiation- triggered damage via upregulation of Nrf2/HO-1 signaling. © 2017 The Author(s). Published by S. Karger AG, Basel.

Rapamycin protects against paraquat-induced pulmonary fibrosis: Activation of Nrf2 signaling pathway.

PubMed

Xu, Yiheng; Tai, Wenlin; Qu, Xiaoyuan; Wu, Wenjuan; Li, ZhenKun; Deng, Shuhao; Vongphouttha, Chanthasone; Dong, Zhaoxing

2017-08-19

Paraquat (PQ) is a widely used herbicide indeveloping countries worldwide, and pulmonary fibrosis is one of the most typical features of PQ poisoning. The molecular mechanism of PQ toxicity especially how to treat PQ-induced pulmonary fibrosis is still largely unknown. In animal model of pulmonary fibrosis, we used HE staining, western blotting assay and Real-time PCR assay to analyze the effects of rapamycin on the PQ-induced epithelial mesenchymal transition (EMT). We found that PQ induced the pulmonary fibrosis using HE staining and Masson's staining, and up-regulated the activity of HYP and the mRNA expressions of Collagen I and III (COL-1and COL-3) in pulmonary tissues. We also found that rapamycin down-regulated the mesenchymal cell marker Vimentin and up-regulated the epithelial cell marker E-cadherin both in mRNA and protein levels compared with PQ group. And the EMT associated transcription factor Snail was decreased by rapamycin treatment compared with PQ group. And PQ decreased the Nrf2 expression both in mRNA and protein levels, and rapamycin inhibited these effects of PQ. SFN, a activator of Nrf2, could inhibit the EMT and the expression of Snail. And knockdowon of Nrf2 could abolish the inhibitory effects of rapamycin of PQ-induced EMT. In conclusion, rapamycin protects against paraquat-induced pulmonary fibrosis by activation of Nrf2 signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioenergetic Approaches and Inflammation of MPTP Toxicity

DTIC Science & Technology

2011-09-01

out a large number of studies, which showed that there were indeed significant protective effects. In particular, we were able to show that CoQ ...the Nrf2/ARE pathway. We demonstrated that these compounds can induce antioxidant enzymes in normal fibroblasts, however, the ability to induce thee...2 enzymes glutathione·c transferase, NADPH quinone oxidoreductase and heme·oxygenase was blocked in fibroblasts knocked out for Nrf2/ARE. We

Denitrification versus respiratory ammonification: environmental controls of two competing dissimilatory NO3−/NO2− reduction pathways in Shewanella loihica strain PV-4

PubMed Central

Yoon, Sukhwan; Cruz-García, Claribel; Sanford, Robert; Ritalahti, Kirsti M; Löffler, Frank E

2015-01-01

Denitrification and respiratory ammonification are two competing, energy-conserving NO3−/NO2− reduction pathways that have major biogeochemical consequences for N retention, plant growth and climate. Batch and continuous culture experiments using Shewanella loihica strain PV-4, a bacterium possessing both the denitrification and respiratory ammonification pathways, revealed factors that determine NO3−/NO2− fate. Denitrification dominated at low carbon-to-nitrogen (C/N) ratios (that is, electron donor-limiting growth conditions), whereas ammonium was the predominant product at high C/N ratios (that is, electron acceptor-limiting growth conditions). pH and temperature also affected NO3−/NO2− fate, and incubation above pH 7.0 and temperatures of 30 °C favored ammonium formation. Reverse-transcriptase real-time quantitative PCR analyses correlated the phenotypic observations with nirK and nosZ transcript abundances that decreased up to 1600-fold and 27-fold, respectively, under conditions favoring respiratory ammonification. Of the two nrfA genes encoded on the strain PV-4 genome, nrfA0844 transcription decreased only when the chemostat reactor received medium with the lowest C/N ratio of 1.5, whereas nrfA0505 transcription occurred at low levels (≤3.4 × 10−2 transcripts per cell) under all growth conditions. At intermediate C/N ratios, denitrification and respiratory ammonification occurred concomitantly, and both nrfA0844 (5.5 transcripts per cell) and nirK (0.88 transcripts per cell) were transcribed. Recent findings suggest that organisms with both the denitrification and respiratory ammonification pathways are not uncommon in soil and sediment ecosystems, and strain PV-4 offers a tractable experimental system to explore regulation of dissimilatory NO3−/NO2− reduction pathways. PMID:25350157

The redox-sensing gene Nrf2 affects intestinal homeostasis, insecticide resistance, and Zika virus susceptibility in the mosquito Aedes aegypti.

PubMed

Bottino-Rojas, Vanessa; Talyuli, Octavio A C; Carrara, Luana; Martins, Ademir J; James, Anthony A; Oliveira, Pedro L; Paiva-Silva, Gabriela O

2018-06-08

Production and degradation of reactive oxygen species (ROS) are extensively regulated to ensure proper cellular responses to various environmental stimuli and stresses. Moreover, physiologically generated ROS function as secondary messengers that can influence tissue homeostasis. The cap'n'collar transcription factor known as nuclear factor erythroid-derived factor 2 (Nrf2) coordinates an evolutionarily conserved transcriptional activation pathway that mediates antioxidant and detoxification responses in many animal species, including insects and mammals. Here, we show that Nrf2-mediated signaling affects embryo survival, midgut homeostasis, and redox biology in Aedes aegypti , a mosquito species vector of dengue, Zika, and other disease-causing viruses. We observed that AeNrf2 silencing increases ROS levels and stimulates intestinal stem cell proliferation. Because ROS production is a major aspect of innate immunity in mosquito gut, we found that a decrease in Nrf2 signaling results in reduced microbiota growth and Zika virus infection. Moreover, we provide evidence that AeNrf2 signaling also controls transcriptional adaptation of A. aegypti to insecticide challenge. Therefore, we conclude that Nrf2-mediated response regulates assorted gene clusters in A. aegypti that determine cellular and midgut redox balance, affecting overall xenobiotic resistance and vectorial adaptation of the mosquito. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Commiphora molmol Modulates Glutamate-Nitric Oxide-cGMP and Nrf2/ARE/HO-1 Pathways and Attenuates Oxidative Stress and Hematological Alterations in Hyperammonemic Rats

PubMed Central

Alqahtani, Sultan; Othman, Sarah I.; Germoush, Mousa O.; Hussein, Omnia E.; Al-Basher, Gadh; Khim, Jong Seong; Al-Qaraawi, Maha A.; Al-Harbi, Hanan M.; Fadel, Abdulmannan; Allam, Ahmed A.

2017-01-01

Hyperammonemia is a serious complication of liver disease and may lead to encephalopathy and death. This study investigated the effects of Commiphora molmol resin on oxidative stress, inflammation, and hematological alterations in ammonium chloride- (NH4Cl-) induced hyperammonemic rats, with an emphasis on the glutamate-NO-cGMP and Nrf2/ARE/HO-1 signaling pathways. Rats received NH4Cl and C. molmol for 8 weeks. NH4Cl-induced rats showed significant increase in blood ammonia, liver function markers, and tumor necrosis factor-alpha (TNF-α). Concurrent supplementation of C. molmol significantly decreased circulating ammonia, liver function markers, and TNF-α in hyperammonemic rats. C. molmol suppressed lipid peroxidation and nitric oxide and enhanced the antioxidant defenses in the liver, kidney, and cerebrum of hyperammonemic rats. C. molmol significantly upregulated Nrf2 and HO-1 and decreased glutamine and nitric oxide synthase, soluble guanylate cyclase, and Na+/K+-ATPase expression in the cerebrum of NH4Cl-induced hyperammonemic rats. Hyperammonemia was also associated with hematological and coagulation system alterations. These alterations were reversed by C. molmol. Our findings demonstrated that C. molmol attenuates ammonia-induced liver injury, oxidative stress, inflammation, and hematological alterations. This study points to the modulatory effect of C. molmol on glutamate-NO-cGMP and Nrf2/ARE/HO-1 pathways in hyperammonemia. Therefore, C. molmol might be a promising protective agent against hyperammonemia. PMID:28744340

Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation.

PubMed

Mathew, Sherin T; Bergström, Petra; Hammarsten, Ola

2014-05-01

Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA and reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2'-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. Copyright © 2014 Elsevier Inc. All rights reserved.

EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi-Hwi; Kim, Eung-Hwi

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H{sub 2}O{sub 2}. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factormore » erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is mediated by Nrf2. • EX4 increases Nrf2 level by stabilizing Nrf2 protein. • EX4 stabilizes Nrf2 by activation of PKCδ.« less

Combination of quercetin, cinnamaldehyde and hirudin protects rat dorsal root ganglion neurons against high glucose-induced injury through Nrf-2/HO-1 activation and NF-κB inhibition.

PubMed

Shi, Yue; Liang, Xiao-Chun; Zhang, Hong; Sun, Qing; Wu, Qun-Li; Qu, Ling

2017-09-01

To examine the effects of the combination of quercetin (Q), cinnamaldehyde (C) and hirudin (H), a Chinese medicine formula on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons. DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of nuclear factor of Kappa B (NF-κB), inhibitory kappa Bα(IκBα), phosphorylated IκBα and Nf-E2 related factor 2 (Nrf2) were examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. The expression of hemeoxygenase-1 (HO-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and caspase-3 were also examined by RT-PCR and Western blot assay. HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway (P<0.05). Co-treatment with Q, C, H and their combination decreased HG-induced caspase-3 activation and apoptosis (P<0.05 or P<0.01). The expressions of NF-κB, IL-6 and TNF-α were down-regulated, and Nrf2/HO-1 expression was up-regulated (P<0.05 or P<0.01). QCH has better effect in scavenging ROS, activating Nrf-2/HO-1, and down-regulating the NF-κB pathway than other treatment group. DRG neurons' apoptosis was increased in diabetic conditions, which was reduced by QCH formula treatment. The possible reason could be activating Nrf-2/HO-1 pathway, scavenging ROS, and inhibition of NF-κB activation. The effect of QCH combination was better than each monomer or the combination of the two monomers.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.

PubMed

Davidson, Rose K; Jupp, Orla; de Ferrars, Rachel; Kay, Colin D; Culley, Kirsty L; Norton, Rosemary; Driscoll, Clare; Vincent, Tonia L; Donell, Simon T; Bao, Yongping; Clark, Ian M

2013-12-01

Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis. Gene expression, histone acetylation, and signaling of the transcription factors NF-E2-related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels. SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB-dependent genes. Compared with cytokines alone, SFN (10 μM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 μmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes. SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo. © The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

Neuroprotective efficacy of naringin on 3-nitropropionic acid-induced mitochondrial dysfunction through the modulation of Nrf2 signaling pathway in PC12 cells.

PubMed

Kulasekaran, Gopinath; Ganapasam, Sudhandiran

2015-11-01

Oxidative stress and mitochondrial dysfunction are implicated in neuronal apoptosis associated with Huntington's disease. Naringin is the flavanone present in grapefruit and related citrus species possess diverse pharmacological and therapeutic properties including antioxidant, anti-apoptotic, and neuroprotective properties. The aim of this study was to investigate the protective effect of naringin on 3-nitropropionic acid (3-NP)-induced neurotoxicity in pheochromocytoma cells (PC12) cells and to explore its mechanism of action. Naringin protects PC12 cells from 3-NP neurotoxicity, as evaluated the by cell viability assays. The lactate dehydrogenase release was decreased upon naringin treatment in 3-NP-induced PC12 cells. Naringin treatment enhances the antioxidant defense by increasing the activities of enzymatic antioxidants and the level of reduced glutathione. The increase in levels of reactive oxygen species and lipid peroxidation induced by 3-NP were significantly decreased by naringin. PC12 cells induced with 3-NP showed decrease in the mitochondrial membrane potential and mitochondrial respiratory complex enzymes, succinate dehydrogenase and cytochrome c oxidase activities, and it was significantly altered to near normal upon naringin treatment. Naringin reduced the 3-NP-induced apoptosis through the modulation in expressions of B-cell lymphoma 2 and Bcl-2-associated X protein. Further, naringin enhances the nuclear translocation of Nrf2 and induces the quinone oxidoreductase-1 and Heme oxygenase-1 expressions through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. Taken together, the above findings suggest that naringin augments cellular antioxidant defense capacity and reduces the 3-NP-induced neurotoxicity in PC12 cells through the PI-3K/Akt-dependent Nrf2 activation in PC12 cells.

Procyanidin B2 induces Nrf2 translocation and glutathione S-transferase P1 expression via ERKs and p38-MAPK pathways and protect human colonic cells against oxidative stress.

PubMed

Rodríguez-Ramiro, Ildefonso; Ramos, Sonia; Bravo, Laura; Goya, Luis; Martín, Maria Ángeles

2012-10-01

Procyanidin B2 (PB2) is a naturally occurring flavonoid widely found in cocoa, red wine and grape juice. Recent studies have suggested that PB2 could protect against oxidative stress- and chemical-induced injury in colonic cells by modulating the endogenous cellular defence. However, the precise mechanism for this protection is not fully understood. Herein, we examined the effect of PB2 on the expression of one of the major antioxidant/detoxificant enzymes related to intestinal protection, the glutathione S-transferase P1 (GSTP1), and the molecular mechanisms involved. Human colonic Caco-2 cells were treated with PB2 at different times and enzymatic activity, and mRNA and protein levels of GSTP1 were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation states of specific proteins central to intracellular signalling cascades were also investigated. PB2 induced the expression and activity of GSTP1 and the nuclear translocation of Nrf2. Interestingly, two important signalling proteins involved in Nrf2 translocation, the extracellular signal-regulated protein kinases (ERKs) and the p38 mitogen-activated protein kinase (MAPK) were also activated. Further experiments with specific inhibitors of both pathways confirmed their critical role in the beneficial effects induced by PB2. The present results show that PB2 protects against oxidative injury in colonic cells and up-regulate the expression of GSTP1 via a mechanism that involves ERK and p38 MAPK activation and Nrf2 translocation. These results provide a molecular basis for the potential contribution of PB2 in the prevention of oxidative stress-related intestinal injury and gut pathologies.

Hepatoprotective effect of 2'-O-galloylhyperin against oxidative stress-induced liver damage through induction of Nrf2/ARE-mediated antioxidant pathway.

PubMed

Wang, Peng; Gao, Yi-Meng; Sun, Xing; Guo, Na; Li, Ji; Wang, Wei; Yao, Li-Ping; Fu, Yu-Jie

2017-04-01

2'-O-galloylhyperin (2'-O-GH), an active compound isolated from Pyrola calliantha, possesses remarkable antioxidant activity. The aims of this study were to investigate the hepatoprotective effect of 2'-O-GH against oxidative stress and elucidate the underlying mechanistic signaling pathways in HepG2 cells as well as in an animal model. Results showed that 2'-O-GH significantly inhibited hydrogen peroxide (H 2 O 2 )-induced HepG2 cell death in a dose dependent manner. The mitogen-activated protein kinase activation, ROS production, mitochondrial membrane potential, intracellular calcium level and subsequent apoptotic protein activation in H 2 O 2 -stimulated HepG2 cells were remarkably inhibited by 2'-O-GH. Furthermore, 2'-O-GH stimulation resulted in a fast and dramatic activation of Akt and nuclear translocation of the NF-E2-related factor 2 (Nrf2), along with the increased expression of heme oxygenase-1 (HO-1) and levels of glutathione (GSH). Meanwhile, histopathological evaluation of the liver also revealed that 2'-O-GH effectively ameliorated CCl 4 -induced the hepatic damage by reducing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Therefore, these results suggested the hepatoprotective effect of 2'-O-GH might be correlated with its antioxidant and free radical scavenger effect. Copyright © 2017. Published by Elsevier Ltd.

α-Lipoic Acid Promotes Neurological Recovery After Ischemic Stroke by Activating the Nrf2/HO-1 Pathway to Attenuate Oxidative Damage.

PubMed

Lv, Chengmei; Maharjan, Surendra; Wang, Qingqing; Sun, Yongxin; Han, Xu; Wang, Shan; Mao, Zhengchun; Xin, Yanming; Zhang, Bing

2017-01-01

Alpha-lipoic acid (α-LA) has been demonstrated to be protective against cerebral ischemia injury. Herein, we investigate the neuroprotective effect and underlying mechanisms of α-LA. In vivo study, α-LA was administered intravenously upon reperfusion of transient middle cerebral artery occlusion. Garcia score was used to evaluate neurologic recovery. Infarct volume was examined by TTC staining, and oxidative damage was evaluated by ELISA assay. In an in vitro study, neurons were pretreated with α-LA at different doses and then subjected to OGD. Lentiviral vectors were applied to knockdown nuclear factor-erythroid 2-related factor-2 (Nrf2) or heme oxygenase-1 (HO-1). Cell viability was measured using CCK8. Protein expression was evaluated using western blot, and immunofluorescence staining was assessed. α-LA significantly reduced the infarct volume, brain edema, and oxidative damage and promoted neurologic recovery in rats. Pretreatment of α-LA caused an obvious increase in cell viability and a decrease in intracellular reactive oxygen species. Western blot analyses and immunofluorescence staining demonstrated a distinct increase in Nrf2 and HO-1 protein expression. Conversely, knockdown of Nrf2 or HO-1 resulted in the down-regulation of HO-1 protein and inhibited the neuroprotective effect of α-LA. α-LA treatment is neuroprotective and promotes functional recovery after ischemic stroke by attenuating oxidative damage, which is partially mediated by the Nrf2/HO-1 pathway. © 2017 The Author(s). Published by S. Karger AG, Basel.

Glycosylation enables aesculin to activate Nrf2.

PubMed

Kim, Kyun Ha; Park, Hyunsu; Park, Hee Jin; Choi, Kyoung-Hwa; Sadikot, Ruxana T; Cha, Jaeho; Joo, Myungsoo

2016-07-15

Since aesculin, 6,7-dihydroxycoumarin-6-O-β-glucopyranoside, suppresses inflammation, we asked whether its anti-inflammatory activity is associated with the activation of nuclear factor-E2-related factor 2 (Nrf2), a key anti-inflammatory factor. Our results, however, show that aesculin marginally activated Nrf2. Since glycosylation can enhance the function of a compound, we then asked whether adding a glucose makes aesculin activate Nrf2. Our results show that the glycosylated aesculin, 3-O-β-d-glycosyl aesculin, robustly activated Nrf2, inducing the expression of Nrf2-dependent genes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase 1 in macrophages. Mechanistically, 3-O-β-d-glycosyl aesculin suppressed ubiquitination of Nrf2, retarding degradation of Nrf2. Unlike aesculin, 3-O-β-d-glycosyl aesculin significantly suppressed neutrophilic lung inflammation, a hallmark of acute lung injury (ALI), in mice, which was not recapitulated in Nrf2 knockout mice, suggesting that the anti-inflammatory function of the compound largely acts through Nrf2. In a mouse model of sepsis, a major cause of ALI, 3-O-β-d-glycosyl aesculin significantly enhanced the survival of mice, compared with aesculin. Together, these results show that glycosylation could confer the ability to activate Nrf2 on aesculin, enhancing the anti-inflammatory function of aesculin. These results suggest that glycosylation can be a way to improve or alter the function of aesculin.

6-Shogaol, an active compound of ginger, alleviates allergic dermatitis-like skin lesions via cytokine inhibition by activating the Nrf2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Gunhyuk, E-mail: uranos5@kiom.re.kr

Allergic dermatitis (AD) clinically presents with skin erythematous plaques, eruption, and elevated serum IgE, and T helper cell type 2 and 1 (Th2 and Th1) cytokine levels. 6-Shogaol [1-(4-hydroxy-methoxyphenyl)-4-decen-one], a pungent compound isolated from ginger, has shown anti-inflammatory effects, but its inhibitory effects on AD are unknown. The aim of this study was to examine whether 6-shogaol inhibits AD-like skin lesions and their underlying mechanism in vivo and in vitro. An AD-like response was induced by tumor necrosis factor-α (TNF-α) + IFN-γ in human keratinocytes or by 2,4-dinitrochlorobenzene (DNCB) in mice. In vivo, 6-shogaol inhibited the development of DNCB-induced AD-likemore » skin lesions and scratching behavior, and showed significant reduction in Th2/1-mediated inflammatory cytokines, IgE, TNF-α, IFN-γ, thymus and activation-regulated chemokine, IL-1, 4, 12, and 13, cyclooxygenase-2, and nitric oxide synthase levels. In vitro, 6-shogaol inhibited reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) signaling, and increased the levels of total glutathione, heme oxygenase-1, and quinone 1 via nuclear factor erythroid 2 related factor 2 (Nrf2) activation. 6-Shogaol can alleviate AD-like skin lesions by inhibiting immune mediators via regulating the ROS/MAPKs/Nrf2 signaling pathway, and may be an effective alternative therapy for AD. - Highlights: • 6-Shogaol inhibited Th2/1-mediated inflammatory mediators in vitro and in vivo. • 6-Shogaol regulated ROS/MAPKs/Nrf2 signaling pathway. • 6-Shogaol can protect against the development of AD-like skin lesions.« less

Translational control of Nrf2 within the open reading frame

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Leal, Oscar, E-mail: operez@temple.edu; Barrero, Carlos A.; Merali, Salim, E-mail: smerali@temple.edu

2013-07-19

Highlights: •Identification of a novel Nrf2 translational repression mechanism. •The repressor is within the 3′ portion of the Nrf2 ORF. •The translation of Nrf2 or eGFP is reduced by the regulatory element. •The translational repression can be reversed with synonymous codon substitutions. •The molecular mechanism requires the mRNA sequence, but not the encoded amino acids. -- Abstract: Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) is a transcription factor that is essential for the regulation of an effective antioxidant and detoxifying response. The regulation of its activity can occur at transcription, translation and post-translational levels. Evidence suggests that under environmental stressmore » conditions, new synthesis of Nrf2 is required – a process that is regulated by translational control and is not fully understood. Here we described the identification of a novel molecular process that under basal conditions strongly represses the translation of Nrf2 within the open reading frame (ORF). This mechanism is dependent on the mRNA sequence within the 3′ portion of the ORF of Nrf2 but not in the encoded amino acid sequence. The Nrf2 translational repression can be reversed with the use of synonymous codon substitutions. This discovery suggests an additional layer of control to explain the reason for the low Nrf2 concentration under quiescent state.« less

Sulforaphane reduction of testicular apoptotic cell death in diabetic mice is associated with the upregulation of Nrf2 expression and function.

PubMed

Wang, Yonggang; Zhang, Zhiguo; Guo, Weiying; Sun, Weixia; Miao, Xiao; Wu, Hao; Cong, Xianling; Wintergerst, Kupper A; Kong, Xiangbo; Cai, Lu

2014-07-01

Diabetes-induced testicular cell death is due predominantly to oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor in controlling the antioxidative system and is inducible by sulforaphane (SFN). To test whether SFN prevents diabetes-induced testicular cell death, an insulin-defective stage of type 2 diabetes (IDS-T2DM) was induced in mice. This was accomplished by feeding them a high-fat diet (HFD) for 3 mo to induce insulin resistance and then giving one intraperitoneal injection of streptozotocin to induce hyperglycemia while age-matched control mice were fed a normal diet (ND). IDS-T2DM and ND-fed control mice were then further subdivided into those with or without 4-mo SFN treatment. IDS-T2DM induced significant increases in testicular cell death presumably through receptor and mitochondrial pathways, shown by increased ratio of Bax/Bcl2 expression and cleavage of caspase-3 and caspase-8 without significant change of endoplasmic reticulum stress. Diabetes also significantly increased testicular oxidative damage and inflammation. All of these diabetic effects were significantly prevented by SFN treatment with upregulated Nrf2 expression. These results suggest that IDS-T2DM induces testicular cell death presumably through caspase-8 activation and mitochondria-mediated cell death pathways and also by significantly downregulating testicular Nrf2 expression and function. SFN upregulates testicular Nrf2 expression and its target antioxidant expression, which was associated with significant protection of the testis from IDS-T2DM-induced germ cell death. Copyright © 2014 the American Physiological Society.

Effect of cellular ubiquitin levels on the regulation of oxidative stress response and proteasome function via Nrf1.

PubMed

Lee, Donghee; Ryu, Kwon-Yul

2017-04-01

The polyubiquitin genes Ubb and Ubc are upregulated under oxidative stress induced by arsenite [As(III)]. However, the role of ubiquitin (Ub) under As(III) exposure is not known in detail. In a previous study, we showed that the reduced viability observed in Ubc -/- mouse embryonic fibroblasts under As(III) exposure was not due to dysregulation of the Nrf2-Keap1 pathway, which prompted us to investigate another NFE2 family protein, nuclear factor erythroid 2-related factor 1 (Nrf1). In this study, we found that Ub deficiency due to Ubc knockdown in N2a cells reduced cell viability and proteasome activity under As(III) exposure. Furthermore, mRNA levels of the proteasome subunit Psma1 were also reduced. In addition, Ub deficiency led to the nuclear accumulation of the p65 isoform of Nrf1 under As(III) exposure. Interestingly, the overexpression of p65-Nrf1 recapitulated the phenotypes of Ub-deficient N2a cells under As(III) exposure. On the other hand, Nrf1 knockdown suppressed the death of Ub-deficient N2a cells upon exposure to As(III). Therefore, the levels of p65-Nrf1 may play an important role in the maintenance of cell viability under oxidative stress induced by As(III). Copyright © 2017 Elsevier Inc. All rights reserved.

Dl-3-n-Butylphthalide Inhibits NLRP3 Inflammasome and Mitigates Alzheimer's-Like Pathology via Nrf2-TXNIP-TrX Axis.

PubMed

Wang, Chun-Yan; Xu, Ye; Wang, Xu; Guo, Chuang; Wang, Tao; Wang, Zhan-You

2018-04-25

Oxidative stress and neuroinflammation play important roles in the pathology of Alzheimer's disease (AD). Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin, is suspected to be an important modulator of oxidative stress and inflammation. However, the underlying mechanism involved in the abnormal homeostasis of TXNIP-thioredoxin (TrX) in AD pathogenesis remains unclear. Using the Swedish mutant form of APP (APPswe)/PSEN1dE9 transgenic mouse (APP/PS1) and human-derived neuronal cells as model systems, we disclosed the impairment of the nuclear factor erythroid 2-related factor 2 (Nrf2)-TXNIP-TrX signaling in Alzheimer's-like pathology. We observed that the immune staining of TXNIP was increased in postmortem AD brain. The chronic accumulation of inflammatory mediator in neuronal cells facilitates interactions of TXNIP-nucleotide binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and NLRP3-ASC, which increases β-amyloid (Aβ) secretion. The antioxidant Dl-3-n-butylphthalide (Dl-NBP) is commonly used for cerebral ischemia treatment. In our study, we elucidated for new mechanisms by which Dl-NBP enhanced TrX activity, suppressed TXNIP, and ameliorated neuronal apoptosis in the APP/PS1 mouse brains. In human glioblastoma A172 cells and neuroblastoma SH-SY5Y cells, we delineated the Dl-NBP-mediated signaling pathways by which Dl-NBP-dependent upregulation of Nrf2 mediated the reciprocal regulation of reducing proinflammatory cytokine and inhibiting Aβ production in the glial and neuronal cells overexpressing APPswe. Our data provide a novel insight into the molecular mechanism that impairments of Nrf2-TXNIP-TrX system may be involved in the imbalance of cellular redox homeostasis and inflammatory damage in the AD brain. Dl-NBP treatment could suppress TXNIP-NLRP3 interaction and inhibit NLRP3 inflammasome activation via upregulating Nrf2. These findings may provide an instrumental therapeutic approach for AD. Antioxid. Redox Signal. 00, 000-000.

Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

PubMed

Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

2016-04-01

Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could be a therapeutic agent for treating ischemic stroke or neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice

PubMed Central

Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

2014-01-01

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

Nitroxide delivery system for Nrf2 activation and skin protection.

PubMed

Ben Yehuda Greenwald, Maya; Frušić-Zlotkin, Marina; Soroka, Yoram; Sasson, Shmuel Ben; Bianco-Peled, Havazelet; Bitton, Ronit; Kohen, Ron

2015-08-01

Cyclic nitroxides are a large group of compounds composed of diverse stable radicals also known as synthetic antioxidants. Although nitroxides are valuable for use in several skin conditions, in in vivo conditions they have several drawbacks, such as nonspecific dispersion in normal tissue, preferential renal clearance and rapid reduction of the nitroxide to the corresponding hydroxylamine. However, these drawbacks can be easily addressed by encapsulating the nitroxides within microemulsions. This approach would allow nitroxide activity and therefore their valuable effects (e.g. activation of the Keap1-Nrf2-EpRE pathway) to continue. In this work, nitroxides were encapsulated in a microemulsion composed of biocompatible ingredients. The nanometric size and shape of the vehicle microemulsion and nitroxide microemulsion displayed high similarity, indicating that the stability of the microemulsions was preserved. Our studies demonstrated that nitroxide microemulsions were more potent inducers of the Keap1-Nrf2-EpRE pathway than the free nitroxides, causing the activation of phase II enzymes. Moreover, microemulsions containing nitroxides significantly reduced UVB-induced cytotoxicity in the skin. Understanding the mechanism of this improved activity may expand the usage of many other Nrf2 modulating molecules in encapsulated form, as a skin protection strategy against oxidative stress-related conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Induction of activation of the antioxidant response element and stabilization of Nrf2 by 3-(3-pyridylmethylidene)-2-indolinone (PMID) confers protection against oxidative stress-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jia-Wei; Beijing Institute of Radiation Medicine, Beijing 100850; Liu, Jing

2012-03-01

The antioxidant response elements (ARE) are a cis-acting enhancer sequence located in regulatory regions of antioxidant and detoxifying genes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a member of the Cap ‘n’ Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes. Under oxidative stress, Nrf2/ARE induction is fundamental to defense against reactive oxygen species (ROS) and serves as a key factor in the protection against toxic xenobiotics. 3-(3-Pyridylmethylidene)-2-Indolinone (PMID) is a derivative of 2-indolinone compounds which act as protein kinase inhibitors and show anti-tumor activity. However, the role of PMID inmore » the oxidative stress remains unknown. In the present study, we showed that PMID induced the activation of ARE-mediated transcription, increased the DNA-binding activity of Nrf2 and then up-regulated the expression of antioxidant genes such as HO-1, SOD, and NQO1. The level of Nrf2 protein was increased in cells treated with PMID by a post-transcriptional mechanism. Under CHX treatment, the stability of Nrf2 protein was enhanced by PMID with decreased turnover rate. We showed that PMID reduced the ubiquitination of Nrf2 and disrupted the Cullin3 (Cul3)-Keap1 interaction. Furthermore, cells treated with PMID showed resistance to cytotoxicity by H{sub 2}O{sub 2} and pro-oxidant 6-OHDA. PMID also up-regulated the antioxidant level in BALB/c mice. Taken together, the compound PMID induces the ARE-mediated gene expression through stabilization of Nrf2 protein and activation of Nrf2/ARE pathway and protects against oxidative stress-mediated cell death. -- Highlights: ► PMID up-regulates ARE-mediated antioxidant gene expression in vitro and in vivo. ► PMID enhances the stabilization of Nrf2 protein, decreasing Nrf2 turnover rate. ► PMID disrupted the Cullin3 (Cul3)-Keap1 interaction. ► PMID protects against cell death induced by H{sub 2}O{sub 2} and pro-oxidant 6-OHDA.« less

Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

PubMed

Wu, Muzo; Gibbons, John G; DeLoid, Glen M; Bedugnis, Alice S; Thimmulappa, Rajesh K; Biswal, Shyam; Kobzik, Lester

2017-07-01

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia. Copyright © 2017 the American Physiological Society.

Small Maf proteins (MafF, MafG, MafK): History, structure and function.

PubMed

Katsuoka, Fumiki; Yamamoto, Masayuki

2016-07-25

The small Maf proteins (sMafs) are basic region leucine zipper (bZIP)-type transcription factors. The basic region of the Maf family is unique among the bZIP factors, and it contributes to the distinct DNA-binding mode of this class of proteins. MafF, MafG and MafK are the three vertebrate sMafs, and no functional differences have been observed among them in terms of their bZIP structures. sMafs form homodimers by themselves, and they form heterodimers with cap 'n' collar (CNC) proteins (p45 NF-E2, Nrf1, Nrf2, and Nrf3) and also with Bach proteins (Bach1 and Bach2). Because CNC and Bach proteins cannot bind to DNA as monomers, sMafs are indispensable partners that are required by CNC and Bach proteins to exert their functions. sMafs lack the transcriptional activation domain; hence, their homodimers act as transcriptional repressors. In contrast, sMafs participate in transcriptional activation or repression depending on their heterodimeric partner molecules and context. Mouse genetic analyses have revealed that various biological pathways are under the regulation of CNC-sMaf heterodimers. In this review, we summarize the history and current progress of sMaf studies in relation to their partners. Copyright © 2016 Elsevier B.V. All rights reserved.

Gemfibrozil pretreatment resulted in a sexually dimorphic outcome in the rat models of global cerebral ischemia-reperfusion via modulation of mitochondrial pro-survival and apoptotic cell death factors as well as MAPKs.

PubMed

Mohagheghi, Fatemeh; Ahmadiani, Abolhassan; Rahmani, Behrouz; Moradi, Fatemeh; Romond, Nathalie; Khalaj, Leila

2013-07-01

Inducers of mitochondrial biogenesis are widely under investigation for use in a novel therapeutic approach in neurodegenerative disorders. The ability of Gemfibrozil, a fibrate, is investigated for the first time to modulate mitochondrial pro-survival factors involved in the mitochondrial biogenesis signaling pathway, including peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A (TFAM) in the brain. Gemfibozil is clinically administered to control hyperlipidemia. It secondarily prevents cardiovascular events such as cardiac arrest in susceptible patients. In this study, pretreatment of animals with gemfibrozil prior to ischemia-reperfusion (I/R) resulted in a sexually dimorphic outcome. While the expression of NRF-1 and TFAM were induced in gemfibrozil-pretreated met-estrous females, they were suppressed in males. Gemfibrozil also proved to be neuroprotective in met-estrous females, as it inhibited caspase-dependent apoptosis while in males it led to hippocampal neurodegeneration via activation of both the caspase-dependent and caspase-independent apoptosis. In the mitogen-activated protein kinase (MAPKs) pathway, gemfibrozil pretreatment induced the expression of extracellular signal-regulated kinases (ERK1/2) in met-estrous females and reduced it in males. These findings correlatively point to the sexual-dimorphic effects of gemfibrozil in global cerebral I/R context by affecting important factors involved in the mitochondrial biogenesis, MAPKs, and apoptotic cell death pathways.

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet.

PubMed

Hu, Haitao; Hao, Lanxiang; Tang, Chunzhou; Zhu, Yunxi; Jiang, Qin; Yao, Jin

2018-01-15

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19 cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation. Copyright © 2017 Elsevier Inc. All rights reserved.

Antioxidant response elements: Discovery, classes, regulation and potential applications.

PubMed

Raghunath, Azhwar; Sundarraj, Kiruthika; Nagarajan, Raju; Arfuso, Frank; Bian, Jinsong; Kumar, Alan P; Sethi, Gautam; Perumal, Ekambaram

2018-07-01

Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Nrf2-driven CD36 and HO-1 gene expression in circulating monocytes correlates with favourable clinical outcome in pregnancy-associated malaria.

PubMed

Aubouy, Agnès; Olagnier, David; Bertin, Gwladys; Ezinmegnon, Sem; Majorel, Clarisse; Mimar, Saliha; Massougbodji, Achille; Deloron, Philippe; Pipy, Bernard; Coste, Agnès

2015-09-18

Pregnancy-associated malaria (PAM) constitutes one of the most severe forms of malaria infection leading to fetal growth restriction and high risk of infant death. The severity of the pathology is largely attributed to the recruitment of monocytes and macrophages in the placenta which is evidenced by dysregulated inflammation found in placental blood. Importantly, CD36(+) monocytes/macrophages are also thought to participate in the tight control of the pro- and anti-inflammatory responses following Plasmodium detection through elimination of apoptotic cells and malaria-infected erythrocytes, internalization and recycling of oxidized forms of low-density lipoprotein and collaboration with TLR2 in pro-inflammatory response. Interestingly, previous work demonstrated that CD36 expression was upregulated on inflammatory macrophages following stimulation of the Nrf2 transcription factor, whilst the PPARγ pathway was inhibited and non-functional in the same inflammatory conditions. This current study examined the possible role of Nrf2-driven gene expression, CD36 and Haem-Oxygenase-1 (HO-1), in PAM clinical outcomes. Clinical data and biological samples including peripheral blood mononuclear cells were collected from 27 women presenting PAM. Polychromatic flow cytometry was used to characterize innate immune cell subpopulations and quantify CD36 protein expression level on monocytes. mRNA levels of CD36, PPARγ, Nrf2 and HO-1 were determined by qPCR and related to clinical outcomes. Finally, the capacity of monocytes to modulate CD36 expression upon rosiglitazone or sulforaphane treatment, two respective PPARγ or Nrf2 activators, was also investigated. The CD36 receptor, mostly expressed by CD14(+) circulating monocytes, statistically correlated with increased infant birth weights. Interestingly, mRNA levels of the transcription factor Nrf2 and the enzyme HO-1 also correlated with lower parasitaemia and increased infant birth weight, while PPARγ mRNA levels did not. Finally, monocytes isolated from low infant birth weight pregnant women were capable of up-regulating CD36 via the Nrf2 pathway ex vivo. Altogether these results suggest that Nrf2-driven CD36 and HO-1 expression on innate immune cells could contribute to a protective and detoxifying mechanism during PAM. More powered and mechanistical studies are however needed to strengthen the conclusions of this study.

Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

PubMed Central

Dias, Irundika H. K.; Chapple, Ian L. C.; Milward, Mike; Grant, Melissa M.; Hill, Eric; Brown, James; Griffiths, Helen R.

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients’ neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. PMID:23826097

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

PubMed

Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

Fluoxetine coupled with zinc in a chronic mild stress model of depression: Providing a reservoir for optimum zinc signaling and neuronal remodeling.

PubMed

Omar, Nesreen Nabil; Tash, Reham Fathy

2017-09-01

Recently, depression has been envisioned as more than an alteration in neurotransmitters centered around receptor signaling pathways. Consequently, the precise mechanisms of selective serotonin reuptake inhibitor (SSRI) antidepressant drugs such as fluoxetine are being revisited. Zinc is a trace element that has been long implicated in the psychopathology and therapy of depression. Zinc has been found to be sequestered and dispensed during stress and inflammation through a family of proteins called metallothioneins (MTs). In addition, MTs are well known for their antioxidant and therefore cytoprotective action. Changes in MTs, their upstream regulators and downstream effectors in response to fluoxetine have not been yet studied. The aim of the present study is to examine whether depression-induced changes in protein levels and mRNA levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), MTs, antioxidant defensive enzyme heme oxygenase (HO-1), zinc-specific receptor GPR39 and brain derived neurotrophic factor (BDNF) in the hippocampus can be reversed by fluoxetine treatment, zinc supplementation or a combination of the two. The present study investigated the effect of chronic (4weeks) combined treatment with zinc hydroaspartate (15mg/kg) and fluoxetine (10mg/kg) on a chronic mild stress model (CMS) in male Sprague-Dawley rats. Hippocampal mRNA and protein levels of Nrf2, HO-1, MTs, GPR39 (protein level only) and BDNF were significantly higher in response to a combined therapy of fluoxetine and zinc than to either monotherapy. Additionally, HO-1 and MTs gene expression was correlated with that of Nrf2 in the FLX-only group. Fluoxetine therapy activated the expression of MTs and HO-1 through an Nrf2-dependent pathway. When FLX was escorted by zinc, activated MTs had a positive impact on BDNF through the zinc signaling receptor GPR39, resulting in general improvement in neuronal plasticity as well as reduction of neuronal atrophy and neuronal cell loss. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting NRF2 for Improved Skin Barrier Function and Photoprotection: Focus on the Achiote-Derived Apocarotenoid Bixin

PubMed Central

Rojo de la Vega, Montserrat; Krajisnik, Andrea; Zhang, Donna D.; Wondrak, Georg T.

2017-01-01

The transcription factor NRF2 (nuclear factor-E2-related factor 2) orchestrates major cellular defense mechanisms including phase-II detoxification, inflammatory signaling, DNA repair, and antioxidant response. Recent studies strongly suggest a protective role of NRF2-mediated gene expression in the suppression of cutaneous photodamage induced by solar UV (ultraviolet) radiation. The apocarotenoid bixin, a Food and Drug Administration (FDA)-approved natural food colorant (referred to as ‘annatto’) originates from the seeds of the achiote tree native to tropical America, consumed by humans since ancient times. Use of achiote preparations for skin protection against environmental insult and for enhanced wound healing has long been documented. We have recently reported that (i) bixin is a potent canonical activator of the NRF2-dependent cytoprotective response in human skin keratinocytes; that (ii) systemic administration of bixin activates NRF2 with protective effects against solar UV-induced skin damage; and that (iii) bixin-induced suppression of photodamage is observable in Nrf2+/+ but not in Nrf2−/− SKH-1 mice confirming the NRF2-dependence of bixin-induced antioxidant and anti-inflammatory effects. In addition, bixin displays molecular activities as sacrificial antioxidant, excited state quencher, PPAR (peroxisome proliferator-activated receptor) α/γ agonist, and TLR (Toll-like receptor) 4/NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) antagonist, all of which might be relevant to the enhancement of skin barrier function and environmental stress protection. Potential skin photoprotection and photochemoprevention benefits provided by topical application or dietary consumption of this ethno-pharmacologically validated phytochemical originating from the Americas deserves further preclinical and clinical examination. PMID:29258247

NRF2-regulated metabolic gene signature as a prognostic biomarker in non-small cell lung cancer

PubMed Central

Namani, Akhileshwar; Cui, Qin Qin; Wu, Yihe; Wang, Hongyan; Wang, Xiu Jun; Tang, Xiuwen

2017-01-01

Mutations in Kelch-like ECH-associated protein 1 (KEAP1) cause the aberrant activation of nuclear factor erythroid-derived 2-like 2 (NRF2), which leads to oncogenesis and drug resistance in lung cancer cells. Our study was designed to identify the genes involved in lung cancer progression targeted by NRF2. A series of microarray experiments in normal and cancer cells, as well as in animal models, have revealed regulatory genes downstream of NRF2 that are involved in wide variety of pathways. Specifically, we carried out individual and combinatorial microarray analysis of KEAP1 overexpression and NRF2 siRNA-knockdown in a KEAP1 mutant-A549 non-small cell lung cancer (NSCLC) cell line. As a result, we identified a list of genes which were mainly involved in metabolic functions in NSCLC by using functional annotation analysis. In addition, we carried out in silico analysis to characterize the antioxidant responsive element sequences in the promoter regions of known and putative NRF2-regulated metabolic genes. We further identified an NRF2-regulated metabolic gene signature (NRMGS) by correlating the microarray data with lung adenocarcinoma RNA-Seq gene expression data from The Cancer Genome Atlas followed by qRT-PCR validation, and finally showed that higher expression of the signature conferred a poor prognosis in 8 independent NSCLC cohorts. Our findings provide novel prognostic biomarkers for NSCLC. PMID:29050246

Nrf2/ARE-Mediated Antioxidant Actions of Pro-Electrophilic Drugs

PubMed Central

Satoh, Takumi; McKercher, Scott R.; Lipton, Stuart A.

2013-01-01

Living cells maintain a balance between oxidation and reduction, and perturbations of this redox balance are thought to contribute to various diseases. Recent attempts to regulate redox state have focused on electrophiles (EPs), which activate potent cellular defense systems against oxidative stress. One example of this approach is exemplified by carnosic acid (CA) and carnosol (CS), compounds that are found in the herb rosemary (Rosmarinus officinalis). Importantly, CA and CS themselves are not electrophilic, but in response to oxidation, become electrophilic, and then activate the Keap1/Nrf2/ARE (antioxidant response element) transcription pathway to synthesize endogenous anti-oxidant ‘phase 2’ enzymes. As a result of our efforts to develop these compounds as therapeutics for brain health, we have formulated two innovative criteria for drug development: the first concept is the use of Pro-Electrophilic Drugs (PEDs) that are innocuous in and of themselves; and the second concept involves the use of compounds that are Pathologically-Activated Therapeutics (PATs), i.e., these small molecules are chemically converted to their active form by the very oxidative stress that they are designed to then combat. The chemical basis for PED and PAT drugs is embodied in the ortho- and para-hydroquinone electrophilic cores of the molecules, which are oxidized by the Cu2+/Cu+ cycling system (or potentially by other transition metals). Importantly, this cycling pathway is under stringent regulation by the cell redox state. We propose that redox-dependent quinone-formation is the predominant mechanism for formation of PED and PAT drugs from their precursor compounds. In fact, redox-dependent generation of the active form of drug from the “pro-form” distinguishes this therapeutic approach from traditional EPs such as curcumin, and results in a decrease in clinical side effects at therapeutic concentrations, e.g., lack of reaction with other thiols such as glutathione (GSH), which can result in lowering GSH and inducing oxidative stress in normal cells. We consider this pro-drug quality of PED/PAT compounds to be a key factor for generating drugs to be used to combat neurodegenerative diseases that will be clinically tolerated. Given the contribution of oxidative stress to the pathology of multiple neurodegenerative diseases, the Keap1/Nrf2/ARE pathway represents a promising drug target for these PED/PAT agents. PMID:23892355

Baicalin Ameliorates H2O2 Induced Cytotoxicity in HK-2 Cells through the Inhibition of ER Stress and the Activation of Nrf2 Signaling

PubMed Central

Lin, Miao; Li, Long; Zhang, Yi; Zheng, Long; Xu, Ming; Rong, Ruiming; Zhu, Tongyu

2014-01-01

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H2O2 with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H2O2 group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H2O2-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H2O2-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved. PMID:25029541

Activation of Nrf2 by cadmium and its role in protection against cadmium-induced apoptosis in rat kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Jun; Shaikh, Zahir A., E-mail: ZShaikh@uri.ed

Kidney is the primary target organ in chronic cadmium (Cd) toxicity, and oxidative stress plays an important role in this process. The nuclear transcription factor Nrf2 binds to antioxidant response elements (AREs) and regulates genes involved in protecting cells from oxidative damage. Whether kidney cells respond to Cd by activating Nrf2 is unknown. This study was designed to examine the Cd-induced activation of Nrf2 transcriptional activity in a stable rat kidney cell line, NRK-52E, and to investigate the protection this might offer against apoptosis. The cells were treated with 5-20 muM CdCl{sub 2} for 5 h, followed by a recoverymore » period of up to 24 h. A concentration-dependent increase (up to 2.9-fold) in the level of reactive oxygen species was noted upon termination of 5-h Cd treatment. The Nrf2-ARE binding activity also increased and peaked (6.1-fold) at 10 muM Cd concentration. Time-course study revealed that the binding activity increased at 1 h of Cd treatment and peaked 2 h post Cd treatment. Apoptosis was detected 6 h post treatment with Cd and a concentration- and time-dependent increase in the apoptotic cell population occurred during the next 18 h. Over-expression of Nrf2 by transient transfection conferred resistance against Cd-induced apoptosis. Conversely, suppression of Nrf2 expression by specific siRNA resulted in greater sensitivity of the cells to Cd by decreasing the levels of two antioxidant enzymes, hemeoxygenase-1 and glutamate-cysteine ligase. Taken together, these results suggest that in kidney cells the activation of Nrf2 is an adaptive intracellular response to Cd-induced oxidative stress, and that Nrf2 is protective against Cd-induced apoptosis.« less

Type I interferon enhances necroptosis of Salmonella Typhimurium–infected macrophages by impairing antioxidative stress responses

PubMed Central

Hos, Nina Judith; Hos, Deniz; Klimek, Jennifer; Abdullah, Zeinab; Krönke, Martin

2017-01-01

Salmonella enterica serovar Typhimurium exploits the host’s type I interferon (IFN-I) response to induce receptor-interacting protein (RIP) kinase–mediated necroptosis in macrophages. However, the events that drive necroptosis execution downstream of IFN-I and RIP signaling remain elusive. In this study, we demonstrate that S. Typhimurium infection causes IFN-I–mediated up-regulation of the mitochondrial phosphatase Pgam5 through RIP3. Pgam5 subsequently interacts with Nrf2, which sequesters Nrf2 in the cytosol, thereby repressing the transcription of Nrf2-dependent antioxidative genes. The impaired ability to respond to S. Typhimurium–induced oxidative stress results in reactive oxygen species–mediated mitochondrial damage, energy depletion, transient induction of autophagy, and autophagic degradation of p62. Reduced p62 levels impair interaction of p62 with Keap1, which further decreases Nrf2 function and antioxidative responses to S. Typhimurium infection, eventually leading to cell death. Collectively, we identify impaired Nrf2-dependent redox homeostasis as an important mechanism that promotes cell death downstream of IFN-I and RIP3 signaling in S. Typhimurium–infected macrophages. PMID:29055012

Regulation of mitochondrial biogenesis and its intersection with inflammatory responses.

PubMed

Cherry, Anne D; Piantadosi, Claude A

2015-04-20

Mitochondria play a vital role in cellular homeostasis and are susceptible to damage from inflammatory mediators released by the host defense. Cellular recovery depends, in part, on mitochondrial quality control programs, including mitochondrial biogenesis. Early-phase inflammatory mediator proteins interact with PRRs to activate NF-κB-, MAPK-, and PKB/Akt-dependent pathways, resulting in increased expression or activity of coactivators and transcription factors (e.g., PGC-1α, NRF-1, NRF-2, and Nfe2l2) that regulate mitochondrial biogenesis. Inflammatory upregulation of NOS2-induced NO causes mitochondrial dysfunction, but NO is also a signaling molecule upregulating mitochondrial biogenesis via PGC-1α, participating in Nfe2l2-mediated antioxidant gene expression and modulating inflammation. NO and reactive oxygen species generated by the host inflammatory response induce the redox-sensitive HO-1/CO system, causing simultaneous induction of mitochondrial biogenesis and antioxidant gene expression. Recent evidence suggests that mitochondrial biogenesis and mitophagy are coupled through redox pathways; for instance, parkin, which regulates mitophagy in chronic inflammation, may also modulate mitochondrial biogenesis and is upregulated through NF-κB. Further research on parkin in acute inflammation is ongoing. This highlights certain common features of the host response to acute and chronic inflammation, but caution is warranted in extrapolating findings across inflammatory conditions. Inflammatory mitochondrial dysfunction and oxidative stress initiate further inflammatory responses through DAMP/PRR interactions and by inflammasome activation, stimulating mitophagy. A deeper understanding of mitochondrial quality control programs' impact on intracellular inflammatory signaling will improve our approach to the restoration of mitochondrial homeostasis in the resolution of acute inflammation.

Neuroprotection by Curcumin in Ischemic Brain Injury Involves the Akt/Nrf2 Pathway

PubMed Central

Wu, Jingxian; Li, Qiong; Wang, Xiaoyan; Yu, Shanshan; Li, Lan; Wu, Xuemei; Chen, Yanlin; Zhao, Jing; Zhao, Yong

2013-01-01

Oxidative damage plays a critical role in many diseases of the central nervous system. This study was conducted to determine the molecular mechanisms involved in the putative anti-oxidative effects of curcumin against experimental stroke. Oxygen and glucose deprivation/reoxygenation (OGD/R) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular expression of NAD(P)H: quinone oxidoreductase1 (NQO1) induced by OGD was counteracted by curcumin post-treatment, which paralleled attenuated cell injury. The reduction of phosphorylation Akt induced by OGD was restored by curcumin. Consequently, NQO1 expression and the binding activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) to antioxidant response element (ARE) were increased. LY294002 blocked the increase in phospho-Akt evoked by curcumin and abolished the associated protective effect. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 60 minutes. Curcumin administration significantly reduced infarct size. Curcumin also markedly reduced oxidative stress levels in middle cerebral artery occlusion (MCAO) rats; hence, these effects were all suppressed by LY294002. Taken together, these findings provide evidence that curcumin protects neurons against ischemic injury, and this neuroprotective effect involves the Akt/Nrf2 pathway. In addition, Nrf2 is involved in the neuroprotective effects of curcumin against oxidative damage. PMID:23555802

Molecular targets for anticancer redox chemotherapy and cisplatin-induced ototoxicity: the role of curcumin on pSTAT3 and Nrf-2 signalling.

PubMed

Fetoni, A R; Paciello, F; Mezzogori, D; Rolesi, R; Eramo, S L M; Paludetti, G; Troiani, D

2015-11-17

In oncology, an emerging paradigm emphasises molecularly targeted approaches for cancer prevention and therapy and the use of adjuvant chemotherapeutics to overcome cisplatin limitations. Owing to their safe use, some polyphenols, such as curcumin, modulate important pathways or molecular targets in cancers. This paper focuses on curcumin as an adjuvant molecule to cisplatin by analysing its potential implications on the molecular targets, signal transducer and activator of transcription 3 (STAT3) and NF-E2 p45-related factor 2 (Nrf-2), in tumour progression and cisplatin resistance in vitro and the adverse effect ototoxicity in vivo. The effects of curcumin and/or cisplatin treatment have been evaluated in head and neck squamous cell carcinoma as well as in a rat model of cisplatin-induced ototoxicity by using immunofluorescence, western blot, and functional and morphological analysis. This study demonstrates that curcumin attenuates all stages of tumour progression (survival, proliferation) and, by targeting pSTAT3 and Nrf-2 signalling pathways, provides chemosensitisation to cisplatin in vitro and protection from its ototoxic adverse effects in vivo. These results indicate that curcumin can be used as an efficient adjuvant to cisplatin cancer therapy. This treatment strategy in head and neck cancer could mediate cisplatin chemoresistance by modulating therapeutic targets (STAT3 and Nrf2) and, at the same time, reduce cisplatin-related ototoxic adverse effects.

Plumbagin ameliorates memory dysfunction in streptozotocin induced Alzheimer's disease via activation of Nrf2/ARE pathway and inhibition of β-secretase.

PubMed

Nakhate, Kartik T; Bharne, Ashish P; Verma, Vinay Sagar; Aru, Deepali N; Kokare, Dadasaheb M

2018-05-01

Although plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) protects against cerebral ischemia and spinal cord injury-induced oxidative stress and inflammation by activating the nuclear factor-erythroid 2-related factor-2 /antioxidant response element (Nrf2/ARE) pathway, its role in the amelioration of neurodegenerative diseases remains unexplored. In the present study, we investigated the effect of plumbagin on Alzheimer's disease (AD)-like condition in mice. The animals were treated intracerebroventricularly with streptozotocin (STZ; 3 mg/kg) twice, on day 1 and 3, to induce AD-like condition, and the symptoms were evaluated after 14 days. While the loss of learning and memory performance was evident in the mice subjected to Morris water maze (MWM), there was a striking increase in the population of astrocytes labelled with glial fibrillary acidic protein (GFAP) in the hippocampus. Daily intraperitoneal (i.p.) treatment with plumbagin (0.5 and 1 mg/kg), starting from 1 h prior to first dose of STZ, significantly prevented the cognitive deficits in MWM. On the other hand, administration of Nrf2/ARE pathway inhibitor, trigonelline (10 and 15 mg/kg, i.p.) enhanced the effects of STZ. Pre-treatment with subeffective dose of trigonelline (5 mg/kg) significantly attenuated the effects of plumbagin in MWM. While plumbagin prevented the STZ induced GFAP expression, this effect of plumbagin was attenuated by trigonelline. Moreover, the in silico docking study revealed potent inhibitory effect of plumbagin on β-secretase enzyme. The results of the present study suggest that plumbagin improves cognitive function in STZ induced mouse model of AD possibly via Nrf2/ARE mediated suppression of astrogliosis and inhibition of β-secretase enzyme. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A systematic molecular dynamics approach to the study of peptide Keap1-Nrf2 protein-protein interaction inhibitors and its application to p62 peptides.

PubMed

Lu, Meng-Chen; Yuan, Zhen-Wei; Jiang, Yong-Lin; Chen, Zhi-Yun; You, Qi-Dong; Jiang, Zheng-Yu

2016-04-01

Protein-protein interactions (PPIs) as drug targets have been gaining growing interest, though developing drug-like small molecule PPI inhibitors remains challenging. Peptide PPI inhibitors, which can provide informative data on the PPI interface, are good starting points to develop small molecule modulators. Computational methods combining molecular dynamics simulations and binding energy calculations could give both the structural and the energetic perspective of peptide PPI inhibitors. Herein, we set up a computational workflow to investigate Keap1-Nrf2 peptide PPI inhibitors and predict the activity of novel sequences. Furthermore, we applied this method to investigate p62 peptides as PPI inhibitors of Keap1-Nrf2 and explored the activity change induced by the phosphorylation of serine. Our results showed that because of the unfavorable solvation effects, the binding affinity of the phosphorylated p62 peptide is lower than the Nrf2 ETGE peptide. Our research results not only provide a useful method to investigate the Keap1-Nrf2 peptide inhibitors, but also give a good example to show how to incorporate computational methods into the study of peptide PPI inhibitors. Besides, applying this method to p62 peptides provides a detailed explanation for the expression of cytoprotective Nrf2 targets induced by p62 phosphorylation, which may benefit the further study of the crosstalk between the Keap1-Nrf2 pathway and p62-mediated selective autophagy.

Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Seiji; Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582; Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813

Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels,more » implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.« less

Hypochlorous acid-induced heme oxygenase-1 gene expression promotes human endothelial cell survival

PubMed Central

Wei, Yong; Liu, Xiao-ming; Peyton, Kelly J.; Wang, Hong; Johnson, Fruzsina K.; Johnson, Robert A.

2009-01-01

Hypochlorous acid (HOCl) is a unique oxidant generated by the enzyme myeloperoxidase that contributes to endothelial cell dysfunction and death in atherosclerosis. Since myeloperoxidase localizes with heme oxygenase-1 (HO-1) in and around endothelial cells of atherosclerotic lesions, the present study investigated whether there was an interaction between these two enzymes in vascular endothelium. Treatment of human endothelial cells with the myeloperoxidase product HOCl stimulated a concentration- and time-dependent increase in HO-1 protein that resulted in a significant rise in carbon monoxide (CO) production. The induction of HO-1 protein was preceded by a prominent increase in HO-1 mRNA and total and nuclear factor-erythroid 2-related factor 2 (Nrf2). In addition, HOCl induced a significant rise in HO-1 promoter activity that was blocked by mutating the antioxidant response element (ARE) in the promoter or by overexpressing a dominant-negative mutant of Nrf2. The HOCl-mediated induction of Nrf2 or HO-1 was blocked by the glutathione donor N-acetyl-l-cysteine but was unaffected by ascorbic or uric acid. Finally, treatment of endothelial cells with HOCl stimulated mitochondrial dysfunction, caspase-3 activation, and cell death that was potentiated by the HO inhibitor, tin protoporphyrin-IX, or by the knockdown of HO-1, and reversed by the exogenous administration of biliverdin, bilirubin, or CO. These results demonstrate that HOCl induces HO-1 gene transcription via the activation of the Nrf2/ARE pathway to counteract HOCl-mediated mitochondrial dysfunction and cell death. The ability of HOCl to activate HO-1 gene expression may represent a critical adaptive response to maintain endothelial cell viability at sites of vascular inflammation and atherosclerosis. PMID:19625608

IDH1 R132H mutation regulates glioma chemosensitivity through Nrf2 pathway.

PubMed

Li, Kaishu; Ouyang, Leping; He, Mingliang; Luo, Ming; Cai, Wangqing; Tu, Yalin; Pi, Rongbiao; Liu, Anmin

2017-04-25

Numerous studies have reported that glioma patients with isocitrate dehydrogenase 1(IDH1) R132H mutation are sensitive to temozolomide treatment. However, the mechanism of IDH1 mutations on the chemosensitivity of glioma remains unclear. In this study, we investigated the role and the potential mechanism of Nrf2 in IDH1 R132H-mediated drug resistance. Wild type IDH1 (R132H-WT) and mutant IDH1 (R132H) plasmids were constructed. Stable U87 cells and U251 cells overexpressing IDH1 were generated. Phenotypic differences between IDH1-WT and IDH1 R132H overexpressing cells were evaluated using MTT, cell colony formation assay, scratch test assay and flow cytometry. Expression of IDH1 and its associated targets, nuclear factor-erythroid 2-related factor 2 (Nrf2), NAD(P)H quinine oxidoreductase 1 (NQO1), multidrug resistant protein 1 (MRP1) and p53 were analyzed. The IDH1 R132H overexpressing cells were more sensitive to temozolomide than WT and the control, and Nrf2 was significantly decreased in IDH1 R132H overexpressing cells. We found that knocking down Nrf2 could decrease resistance to temozolomide. The nuclear translocation of Nrf2 in IDH1 R132H overexpressing cells was lower than the WT and the control groups after temozolomide treatment. When compared with WT cells, NQO1 expression was reduced in IDH1 R132H cells, especially after temozolomide treatment. P53 was involved in the resistance mechanism of temozolomide mediated by Nrf2 and NQO1. Nrf2 played an important role in IDH1 R132H-mediated drug resistance. The present study provides new insight for glioma chemotherapy with temozolomide.

IDH1 R132H mutation regulates glioma chemosensitivity through Nrf2 pathway

PubMed Central

Luo, Ming; Cai, Wangqing; Tu, Yalin; Pi, Rongbiao; Liu, Anmin

2017-01-01

Purpose Numerous studies have reported that glioma patients with isocitrate dehydrogenase 1(IDH1) R132H mutation are sensitive to temozolomide treatment. However, the mechanism of IDH1 mutations on the chemosensitivity of glioma remains unclear. In this study, we investigated the role and the potential mechanism of Nrf2 in IDH1 R132H-mediated drug resistance. Methods Wild type IDH1 (R132H-WT) and mutant IDH1 (R132H) plasmids were constructed. Stable U87 cells and U251 cells overexpressing IDH1 were generated. Phenotypic differences between IDH1-WT and IDH1 R132H overexpressing cells were evaluated using MTT, cell colony formation assay, scratch test assay and flow cytometry. Expression of IDH1 and its associated targets, nuclear factor-erythroid 2-related factor 2 (Nrf2), NAD(P)H quinine oxidoreductase 1 (NQO1), multidrug resistant protein 1 (MRP1) and p53 were analyzed. Results The IDH1 R132H overexpressing cells were more sensitive to temozolomide than WT and the control, and Nrf2 was significantly decreased in IDH1 R132H overexpressing cells. We found that knocking down Nrf2 could decrease resistance to temozolomide. The nuclear translocation of Nrf2 in IDH1 R132H overexpressing cells was lower than the WT and the control groups after temozolomide treatment. When compared with WT cells, NQO1 expression was reduced in IDH1 R132H cells, especially after temozolomide treatment. P53 was involved in the resistance mechanism of temozolomide mediated by Nrf2 and NQO1. Conclusions Nrf2 played an important role in IDH1 R132H-mediated drug resistance. The present study provides new insight for glioma chemotherapy with temozolomide. PMID:28427200

Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

PubMed Central

Biswas, Madhurima; Kwong, Erick K.; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y.

2013-01-01

Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF-Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrfl attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. PMID:23623971

Activation of Nrf2 by H2O2: de novo synthesis versus nuclear translocation.

PubMed

Covas, Gonçalo; Marinho, H Susana; Cyrne, Luísa; Antunes, Fernando

2013-01-01

The most common mechanism described for the activation of the transcription factor Nrf2 is based on the inhibition of its degradation in the cytosol followed by its translocation to the nucleus. Recently, Nrf2 de novo synthesis was proposed as an additional mechanism for the rapid upregulation of Nrf2 by hydrogen peroxide (H2O2). Here, we describe a detailed protocol, including solutions, pilot experiments, and experimental setups, which allows exploring the role of H2O2, delivered either as a bolus or as a steady state, in endogenous Nrf2 translocation and synthesis. We also show experimental data, illustrating that H2O2 effects on Nrf2 activation in HeLa cells are strongly dependent both on the H2O2 concentration and on the method of H2O2 delivery. The de novo synthesis of Nrf2 is triggered within 5min of exposure to low concentrations of H2O2, preceding Nrf2 translocation to the nucleus which is slower. Evidence of de novo synthesis of Nrf2 is observed only for low H2O2 steady-state concentrations, a condition that is prevalent in vivo. This study illustrates the applicability of the steady-state delivery of H2O2 to uncover subtle regulatory effects elicited by H2O2 in narrow concentration and time ranges. Copyright © 2013 Elsevier Inc. All rights reserved.

ROLE OF NRF2 IN THE OXIDATIVE STRESS-DEPENDENT HYPERTENSION ASSOCIATED WITH THE DEPLETION OF DJ-1

PubMed Central

Cuevas, Santiago; Yang, Yu; Konkalmatt, Prasad; Asico, Laureano; Feranil, Jun; Jones, John; Villar, Van Anthony; Armando, Ines; Jose, Pedro A.

2015-01-01

Renal dopamine 2 receptor dysfunction is associated with oxidative stress and high blood pressure. We have reported that DJ-1, an oxidative stress response protein, is positively regulated by dopamine 2 receptor in the kidney. The transcription factor Nrf2 regulates the expression of several antioxidant genes. We tested the hypothesis that Nrf2 is involved in the renal DJ-1-mediated inhibition of reactive oxygen species production. We have reported that silencing dopamine 2 receptor in mouse renal proximal tubule cells decreases the expression of DJ-1. We now report that silencing DJ-1 or dopamine 2 receptor in mouse proximal tubule cells and mouse kidneys, decreases Nrf2 expression and activity and increases reactive oxygen species production; blood pressure is also increased in mice in which renal DJ-1 or dopamine 2 receptor is silenced. DJ-1−/− mice have decreased renal Nrf2 expression and activity, and increased nitro-tyrosine levels an dopamine 2 receptor d blood pressure. Silencing Nrf2 in mouse proximal tubule cells does not alter the expression of DJ-1 or dopamine 2 receptor, indicating that Nrf2 is downstream of dopamine 2 receptor and DJ-1. A Nrf2 inducer, bardoxolone, normalizes the systolic blood pressure and renal malondialdehyde levels in DJ-1−/− mice without affecting them in their wild-type littermates. Because Nrf2 ubiquitination is increased in DJ-1−/− mice, we conclude that the protective effect of DJ-1 on renal oxidative stress is mediated, in part, by preventing Nrf2 degradation. Moreover, renal dopamine 2 receptor and DJ-1 are necessary for normal Nrf2 activity to keep a normal redox balance and blood pressure. PMID:25895590

Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy.

PubMed

Gu, Junlian; Cheng, Yanli; Wu, Hao; Kong, Lili; Wang, Shudong; Xu, Zheng; Zhang, Zhiguo; Tan, Yi; Keller, Bradley B; Zhou, Honglan; Wang, Yuehui; Xu, Zhonggao; Cai, Lu

2017-02-01

We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM. © 2017 by the American Diabetes Association.

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

PubMed

Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

2012-12-01

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

Fucoxanthin enhances HO-1 and NQO1 expression in murine hepatic BNL CL.2 cells through activation of the Nrf2/ARE system partially by its pro-oxidant activity.

PubMed

Liu, Cheng-Ling; Chiu, Yu-Ting; Hu, Miao-Lin

2011-10-26

To determine whether fucoxanthin, a major carotenoid in brown sea algae, may activate cellular antioxidant enzymes via up-regulation of the Nrf2/antioxidant-response element (ARE) pathway, we incubated mouse hepatic BNL CL.2 cells with fucoxanthin (0.5-20 μM) for 0-24 h. We found that fucoxanthin (≥5 μM) significantly increased cellular reactive oxygen species (ROS) at 6 h of incubation, whereas preincubation with α-d-tocopherol (30 μM) significantly attenuated the increase of ROS, indicating the pro-oxidant nature of fucoxanthin. Fucoxanthin significantly increased the phosphorylation of ERK and p38 and markedly increased nuclear Nrf2 protein accumulation after incubation for 12 h. Moreover, fucoxanthin significantly enhanced binding activities of nuclear Nrf2 with ARE and increased mRNA and protein expression of HO-1 and NQO1 after incubation for 12 h. siRNA inhibition of Nrf2 led to markedly decreased HO-1 and NQO1 protein expression. Thus, fucoxanthin may exert its antioxidant activity, at least partly, through its pro-oxidant actions.

Butein induction of HO-1 by p38 MAPK/Nrf2 pathway in adipocytes attenuates high-fat diet induced adipose hypertrophy in mice.

PubMed

Wang, Zheng; Ka, Sun-O; Lee, Youngyi; Park, Byung-Hyun; Bae, Eun Ju

2017-03-15

Adipose tissue inflammation and oxidative stress are key components in the development of obesity and insulin resistance. Heme oxygenase (HO)-1 in adipocytes protects against obesity and adipose dysfunction. In this study, we report the identification of butein, a flavonoid chalcone, as a novel inducer of HO-1 expression in adipocytes in vitro and in vivo. Butein upregulated HO-1 mRNA and protein expression in 3T3-L1 adipocytes, accompanied by Kelch-Like ECH-Associated Protein (Keap) 1 degradation and increase in the nuclear level of nuclear factor erythroid 2-related factor 2 (Nrf2). Butein modulation of Keap1 and Nrf2 as well as HO-1 upregulation was reversed by pretreatment with p38 MAPK inhibitor SB203580, indicating the involvement of p38 MAPK in butein activation of Nrf2 in adipocytes. In addition, HO-1 activation by butein led to the inhibitions of reactive oxygen species and adipocyte differentiation, as evidenced by the fact that butein repression of reactive oxygen species and adipogenesis was reversed by pretreatment with HO-1 inhibitor SnPP. Induction of HO-1 expression by butein was also demonstrated in the adipose tissue of C57BL/6 mice fed a high-fat diet administered along with butein for three weeks, and correlated with the inhibitions of adiposity and adipose tissue inflammation, which were reversed by co-administration of SnPP. Altogether, our results demonstrate that butein activates the p38 MAPK/Nrf2/HO-1 pathway to act as a potent inhibitor of adipose hypertrophy and inflammation in a diet-induced obesity model and thus has potential for suppressing obesity-linked metabolic syndrome. Copyright © 2017 Elsevier B.V. All rights reserved.

Ameliorating mitochondrial dysfunction restores carbon ion-induced cognitive deficits via co-activation of NRF2 and PINK1 signaling pathway.

PubMed

Liu, Yang; Yan, Jiawei; Sun, Cao; Li, Guo; Li, Sirui; Zhang, Luwei; Di, Cuixia; Gan, Lu; Wang, Yupei; Zhou, Rong; Si, Jing; Zhang, Hong

2018-07-01

Carbon ion therapy is a promising modality in radiotherapy to treat tumors, however, a potential risk of induction of late normal tissue damage should still be investigated and protected. The aim of the present study was to explore the long-term cognitive deficits provoked by a high-linear energy transfer (high-LET) carbon ions in mice by targeting to hippocampus which plays a crucial role in memory and learning. Our data showed that, one month after 4 Gy carbon ion exposure, carbon ion irradiation conspicuously resulted in the impaired cognitive performance, neurodegeneration and neuronal cell death, as well as the reduced mitochondrial integrity, the disrupted activities of tricarboxylic acid cycle flux and electron transport chain, and the depressed antioxidant defense system, consequently leading to a decline of ATP production and persistent oxidative damage in the hippocampus region. Mechanistically, we demonstrated the disruptions of mitochondrial homeostasis and redox balance typically characterized by the disordered mitochondrial dynamics, mitophagy and glutathione redox couple, which is closely associated with the inhibitions of PINK1 and NRF2 signaling pathway as the key regulators of molecular responses in the context of neurotoxicity and neurodegenerative disorders. Most importantly, we found that administration with melatonin as a mitochondria-targeted antioxidant promoted the PINK1 accumulation on the mitochondrial membrane, and augmented the NRF2 accumulation and translocation. Moreover, melatonin pronouncedly enhanced the molecular interplay between NRF2 and PINK1. Furthermore, in the mouse hippocampal neuronal cells, overexpression of NRF2/PINK1 strikingly protected the hippocampal neurons from carbon ion-elicited toxic insults. Thus, these data suggest that alleviation of the sustained mitochondrial dysfunction and oxidative stress through co-modulation of NRF2 and PINK1 may be in charge of restoration of the cognitive impairments in a mouse model of high-LET carbon ion irradiation. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Deficiency in the nuclear factor E2-related factor 2 renders pancreatic β-cells vulnerable to arsenic-induced cell damage

PubMed Central

Yang, Bei; Fu, Jingqi; Zheng, Hongzhi; Xue, Peng; Yarborough, Kathy; Woods, Courtney G; Hou, Yongyong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

2012-01-01

Chronic human exposure to inorganic arsenic (iAs), a potent environmental oxidative stressor, is associated with increased prevalence of Type 2 diabetes, where impairment of pancreatic β-cell function is a key pathogenic factor. Nuclear factor E2-related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. However, persistent activation of Nrf2 in response to chronic oxidative stress, including inorganic arsenite (iAs3+) exposure, blunts glucose-triggered reactive oxygen species (ROS) signaling and impairs glucose-stimulated insulin secretion (GSIS). In the current study, we found that MIN6 pancreatic β-cells with stable knockdown of Nrf2 (Nrf2-KD) by lentiviral shRNA and pancreatic islets isolated from Nrf2-knockout (Nrf2−/−) mice exhibited reduced expression of several antioxidant and detoxification enzymes in response to acute iAs3+ exposure. As a result, Nrf2-KD MIN6 cells and Nrf2−/− islets were more susceptible to iAs3+ and monomethylarsonous acid (MMA3+)-induced cell damage, as measured by decreased cell viability, augmented apoptosis and morphological change. Pretreatment of MIN6 cells with Nrf2 activator tert-butylhydroquinone protected the cells from iAs3+-induced cell damage in an Nrf2-dependent fashion. In contrast, antioxidant N-acetyl cysteine protected Nrf2-KD MIN6 cells against acute cytotoxicity of iAs3+. The present study demonstrates that Nrf2-mediated antioxidant response is critical in the pancreatic β-cell defense mechanism against acute cytotoxicity by arsenic. The findings here, combined with our previous results on the inhibitory effect of antioxidants on ROS signaling and GSIS, suggest that Nrf2 plays paradoxical roles in pancreatic β-cell dysfunction induced by environmental arsenic exposure. PMID:23000044

Combination of rapamycin and garlic-derived S-allylmercaptocysteine induces colon cancer cell apoptosis and suppresses tumor growth in xenograft nude mice through autophagy/p62/Nrf2 pathway.

PubMed

Li, Siying; Yang, Guang; Zhu, Xiaosong; Cheng, Lin; Sun, Yueyue; Zhao, Zhongxi

2017-09-01

The natural plant-derived product S-allylmercapto-cysteine (SAMC) has been studied in cancer therapy as a single and combination chemotherapeutic agent. The present study was employed to verify the combination use of SAMC and rapamycin that is the mTOR inhibitor with anticancer ability but has limited efficacy due to drug resistance, and to explore the underlying mechanisms. We combined rapamycin and SAMC for colorectal cancer treatment in the HCT‑116 cancer cells and a xenograft murine model. The in vivo study was established by xenografting HCT‑116 cells in BALB/c nude mice. It was found that the combination therapy had enhanced tumor-suppressing ability with the upregulation of the Bax/Bcl-2 ratio as a consequence of activated apoptosis, inhibition of autophagic activity and prevention of Akt phosphorylation. The rapamycin and SAMC combination activated antioxidant transcription expressions of Nrf2 and downstream gene NQO1. Concomitantly, autophagosome cargo p62 was downregulated, indicating that the p62 played a negative-regulatory role between Nrf2 and autophagy. Our results show that the combination of SAMC and rapamycin enhanced the anticancer ability, which could be used for the treatment of colorectal cancer. The underling mechanism of autophagy/p62/Nrf2 pathway discovered may provide a new direction for drug development, especially for traditional Chinese medicines.

Aspirin suppresses neuronal apoptosis, reduces tissue inflammation, and restrains astrocyte activation by activating the Nrf2/HO-1 signaling pathway.

PubMed

Wei, Wang; Shurui, Chen; Zipeng, Zhou; Hongliang, Dai; Hongyu, Wang; Yuanlong, Li; Kang, Zhou; Zhaoliang, Shen; Yue, Guo; Chang, Liu; Mei, Xifan

2018-05-02

The nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element signaling pathway plays a substantial role in preventing oxidative stress-related diseases. Aspirin has been shown to exert several pharmacological effects by inducing the expression of the heme oxygenase-1 (HO-1) protein. However, the effects of aspirin on spinal cord injury (SCI) have rarely been studied. Therefore, we sought to investigate the neuroprotective effects of aspirin after SCI. We employed a spinal cord contusion model in Sprague-Dawley rats, and aspirin was administered intraperitoneally for 7 days. Nissl staining showed that the aspirin treatment significantly reduced the loss of motor neurons after SCI compared with vehicle-treated animals. The expression of Nrf2, quinine oxidoreductase 1, and HO-1 proteins was increased in aspirin-treated animals after SCI compared with the vehicle group. In addition, aspirin simultaneously decreased the expression of inflammation-related proteins, such as tumor necrosis factor-α and interleukin-6 after SCI. Moreover, the ratio of apoptotic neurons in the anterior horn and the levels of the apoptosis-related proteins caspase-3, cleaved caspase-3, and Bax were significantly decreased in the aspirin group compared with the vehicle group. Immunofluorescence staining was used to detect the colocalization of NeuN and HO-1, and the results showed that aspirin significantly increased expression of the HO-1 protein in neurons. In addition, western blots and immunofluorescence staining showed aspirin restrained astrocyte activation. In conclusion, aspirin induces neuroprotective effects by inhibiting astrocyte activation and apoptosis after SCI through the activation of the Nrf2/HO-1 signaling pathway.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

PubMed

Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

2017-01-01

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jin-Sun; Kim, Hee-Sun, E-mail: hskimp@ewha.ac.kr

2014-05-16

Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigatedmore » the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.« less

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress.

PubMed

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-06-27

Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. The cytoprotective effect of chamomile was examined on H(2)O(2)-induced cellular stress in RAW 264.7 murine macrophages. RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H(2)O(2). Treatment with 50μM H(2)O(2) for 6h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20μg/mL for 16h followed by H(2)O(2) treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. Copyright © 2012 Elsevier Inc. All rights reserved.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

PubMed Central

Bhaskaran, Natarajan; Shukla, Sanjeev; Kanwal, Rajnee; Srivastava, Janmejai K; Gupta, Sanjay

2012-01-01

Aims Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. Main Methods The cytoprotective effect of chamomile was examined on H2O2-induced cellular stress in RAW 264.7 murine macrophages. Key Findings RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H2O2. Treatment with 50 μM H2O2 for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20 μg/mL for 16 h followed by H2O2 treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. Significance These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties. PMID:22683429

Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Do, Minh Truong; Kim, Hyung Gyun; Khanal, Tilak

2013-09-01

Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancermore » A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells. The inactivation of AMPK by siRNA, DN-AMPK or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner. These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anticancer therapy in response to metformin treatment. - Highlights: • Metformin inhibits HO-1 expression in cancer cells. • Metformin attenuates Raf-ERK-Nrf2 signaling. • Suppression of HO-1 by metformin is independent of AMPK. • HO-1 inhibition contributes to anti-proliferative effects of metformin.« less

Genetic polymorphism of the Nrf2 promoter region is associated with vitiligo risk in Han Chinese populations.

PubMed

Song, Pu; Li, Kai; Liu, Ling; Wang, Xiaowen; Jian, Zhe; Zhang, Weigang; Wang, Gang; Li, Chunying; Gao, Tianwen

2016-10-01

The nuclear factor erythroid-derived two-like 2-antioxidant response element (Nrf2-ARE) pathway and its downstream antioxidant enzyme heme oxygenase-1 (HMOX1 or HO-1) play essential roles in H2 O2 -induced oxidative damage in human melanocytes. However, the link between Nrf2 promoter polymorphisms and susceptibility to oxidative stress-related diseases such as vitiligo is unknown. This study evaluated the association of the Nrf2 and HO-1 genes polymorphisms with vitiligo susceptibility. In this case-control study of 1136 Han Chinese vitiligo patients and 1200 controls, Nrf2 (rs35652124 and rs6721961) and HO-1 (rs2071746) genes were genotyped by PCR-restriction fragment length polymorphism analysis. Overall, a significantly decreased risk of vitiligo was found to be associated with Nrf2 rs35652124 CC and combined (CT+CC) genotypes [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.50-0.83 and OR, 0.84, 95% CI 0.71-0.99, respectively], as well as among subgroups: female, onset age ≤20 and never smoker. We subsequently found that Nrf2 rs35652124 C allele had higher transcriptional activity in the luciferase reporter assay compared with Nrf2 rs35652124 T allele. Furthermore, we investigated serum HO-1 activity was associated with the rs35652124 CT+CC genotype and lower in patients than in controls (P = 0.024). Logistic regression analysis showed a dose-response relationship between lower vitiligo risk and increased HO-1 activity in rs35652124 CT+CC genotype carriers (Ptrend < 0.05). These findings indicate that the C allele of rs35652124 located in the promoter region of Nrf2 gene is associated with protective effect on vitiligo in a Han Chinese population. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Hederagenin Induces Apoptosis in Cisplatin-Resistant Head and Neck Cancer Cells by Inhibiting the Nrf2-ARE Antioxidant Pathway.

PubMed

Kim, Eun Hye; Baek, Seungho; Shin, Daiha; Lee, Jaewang; Roh, Jong-Lyel

2017-01-01

Acquired resistance to cisplatin is the most common reason for the failure of cisplatin chemotherapy. Hederagenin, triterpenoids extracted from ivy leaves, exhibits antitumor activity in various types of cancer. However, the therapeutic potential of hederagenin in head and neck cancer (HNC) has remained unclear. Therefore, we examined the effects of hederagenin in cisplatin-resistant HNC cells and characterized its molecular mechanisms of action in this context. We evaluated the effects of hederagenin treatment on cell viability, apoptosis, reactive oxygen species (ROS) production, glutathione levels, mitochondrial membrane potential (Δ Ψ m), and protein and mRNA expression in HNC cells. The antitumor effect of hederagenin in mouse tumor xenograft models was also analyzed. Hederagenin selectively induced cell death in both cisplatin-sensitive and cisplatin-resistant HNC cells by promoting changes in Δ Ψ m and inducing apoptosis. Hederagenin inhibited the Nrf2-antioxidant response element (ARE) pathway and activated p53 in HNC cells, thereby enhancing ROS production and promoting glutathione depletion. These effects were reversed by the antioxidant trolox. Hederagenin activated intrinsic apoptotic pathways via cleaved PARP, cleaved caspase-3, and Bax. The selective inhibitory effects of hederagenin were confirmed in cisplatin-resistant HNC xenograft models. These data suggest that hederagenin induces cell death in resistant HNC cells via the Nrf2-ARE antioxidant pathway.

Differential expression of the Nrf2-linked genes in pediatric septic shock.

PubMed

Grunwell, Jocelyn R; Weiss, Scott L; Cvijanovich, Natalie Z; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Shanley, Thomas P; Bigham, Michael T; Fitzgerald, Julie; Howard, Kelli; Frank, Erin; Harmon, Kelli; Wong, Hector R

2015-09-17

Experimental data from animal models of sepsis support a role for a transcription factor, nuclear erythroid-related factor 2 p45-related factor 2 (Nrf2), as a master regulator of antioxidant and detoxifying genes and intermediary metabolism during stress. Prior analysis of a pediatric septic shock transcriptomic database showed that the Nrf2 response is a top 5 upregulated signaling pathway in early pediatric septic shock. We conducted a focused analysis of 267 Nrf2-linked genes using a multicenter, genome-wide expression database of 180 children with septic shock 10 years of age or younger and 53 healthy controls. The analysis involved RNA isolated from whole blood within 24 h of pediatric intensive care unit admission for septic shock and a false discovery rate of 5 %. We compared differentially expressed genes from (1) patients with septic shock and healthy controls and (2) across validated gene expression-based subclasses of pediatric septic shock (endotypes A and B) using several bioinformatic methods. We found upregulation of 123 Nrf2-linked genes in children with septic shock. The top gene network represented by these genes contained primarily enzymes with oxidoreductase activity involved in cellular lipid metabolism that were highly connected to the peroxisome proliferator activated receptor and the retinoic acid receptor families. Endotype A, which had higher organ failure burden and mortality, exhibited a greater downregulation of Nrf2-linked genes than endotype B, with 92 genes differentially regulated between endotypes. Our findings indicate that Nrf2-linked genes may contribute to alterations in oxidative signaling and intermediary metabolism in pediatric septic shock.

Edaravone attenuates brain damage in rats after acute CO poisoning through inhibiting apoptosis and oxidative stress.

PubMed

Li, Qin; Bi, Ming Jun; Bi, Wei Kang; Kang, Hai; Yan, Le Jing; Guo, Yun-Liang

2016-03-01

Acute carbon monoxide (CO) poisoning is the most common cause of death from poisoning all over the world and may result in neuropathologic and neurophysiologic changes. Acute brain damage and delayed encephalopathy are the most serious complication, yet their pathogenesis is poorly understood. The present study aimed to evaluate the neuroprotective effects of Edaravone against apoptosis and oxidative stress after acute CO poisoning. The rat model of CO poisoning was established in a hyperbaric oxygen chamber by exposed to CO. Ultrastructure changes were observed by transmission electron microscopy (TEM). TUNEL stain was used to assess apoptosis. Immunohistochemistry and immunofluorescence double stain were used to evaluate the expression levels of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) protein and their relationship. By dynamically monitored the carboxyhemoglobin (HbCO) level in blood, we successfully established rat model of severe CO poisoning. Ultrastructure changes, including chromatin condensation, cytoplasm dissolution, vacuoles formation, nucleus membrane and cell organelles decomposition, could be observed after CO poisoning. Edaravone could improve the ultrastructure damage. CO poisoning could induce apoptosis. Apoptotic cells were widely distributed in cortex, striatum and hippocampus. Edaravone treatment attenuated neuronal apoptosis as compared with the poisoning group (P < 0.01). Basal expressions of HO-1 and Nrf-2 proteins were found in normal brain tissue. CO poisoning could activate HO-1/Nrf-2 pathway, start oxidative stress response. After the administration of Edaravone, the expression of HO-1 and Nrf-2 significantly increased (P < 0.01). These findings suggest that Edaravone may inhibit apoptosis, activate the Keapl-Nrf/ARE pathway, and thus improve the ultrastructure damage and neurophysiologic changes following acute CO poisoning. © 2014 Wiley Periodicals, Inc.

The effect of ex vivo CDDO-Me activation on nuclear factor erythroid 2-related factor 2 pathway in white blood cells from patients with septic shock.

PubMed

Noel, Sanjeev; Zheng, Laura; Navas-Acien, Ana; Fuchs, Ralph J

2014-11-01

Nuclear factor erythroid 2-related factor 2 (NRF2) has been shown to protect against experimental sepsis in mice and lipopolysaccharide (LPS)-induced inflammation in ex vivo white blood cells from healthy subjects by upregulating cellular antioxidant genes. The objective of this study was to test the hypothesis that ex vivo methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me) activates NRF2-regulated antioxidant genes in white blood cells from patients with septic shock and protects against LPS-induced inflammation and reactive oxidative species production. Peripheral blood was collected from 18 patients with septic shock who were being treated in medical and surgical intensive care units. Real-time polymerase chain reaction was used to quantify the expression of NRF2 target genes (NQO1, HO-1, GCLM, and FTL) and IL-6 in peripheral blood mononuclear cells (PBMCs), monocytes, and neutrophils after CDDO-Me treatment alone or after subsequent LPS exposure. Superoxide anion (O2) was measured to assess the effect of CDDO-Me pretreatment on subsequent LPS exposure. Treatment with CDDO-Me increased the gene expression of NQO1 (P = 0.04) and decreased the expression of HO-1 (P = 0.03) in PBMCs from patients with septic shock. Purified monocytes exhibited significant increases in the expression of NQO1 (P = 0.01) and GCLM (P = 0.003) after CDDO-Me treatment. Levels of other NRF2 target genes (HO-1 and FTL) remained similar to those of vehicle-treated cells. Peripheral blood mononuclear cells showed a trend toward increased IL-6 gene expression after CDDO-Me treatment, whereas purified monocytes showed a trend toward decreased IL-6. There was no discernible trend in the IL-6 expression subsequent to LPS treatment in either vehicle-treated or CDDO-Me-treated PBMCs and monocytes. Treatment with CDDO-Me significantly increased O2 production in PBMCs (P = 0.04). Although CDDO-Me pretreatment significantly attenuated O2 production to subsequent LPS exposure (P = 0.03), the change was comparable to that observed in vehicle-treated PBMCs. Pretreatment with CDDO-Me followed by LPS exposure had no significant effect on O2 levels in purified monocytes. These data suggest that the NRF2 pathway is differentially responsive to CDDO-Me activation in peripheral blood cells from patients with septic shock and results in increased O2 production. The data may also suggest a suppressed NRF2 pathway in white blood cells from critically ill patients.

Simple ortho- and para-hydroquinones as compounds neuroprotective against oxidative stress in a manner associated with specific transcriptional activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Satoh, Takumi; Saitoh, Sachie; Hosaka, Manami

2009-02-06

Electrophilic compounds protect neurons through the activation of the Keap1/Nrf2 pathway and the induction of phase-2 enzymes [T. Satoh, S.A. Lipton, Redox regulation of neuronal survival by electrophilic compounds, Trends Neurosci. 30 (2007) 38-45; T. Satoh, S. Okamoto, J. Cui, Y. Watanabe, K. Furuta, M. Suzuki, K. Tohyama, S.A. Lipton, Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc. Natl. Acad. Sci. USA 103 (2006) 768-773]. Hydroquinone-type electrophilic compounds such as tert-butyl hydroquinone (TBHQ) and carnosic acid (CA) have attracted special attention, because the oxidative conversion of 'hydroquinone' to 'quinone' is essential for the transcriptional activationmore » of the above-mentioned enzymes [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protect neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of specific cysteine, J. Neurochem. 104 (2008) 1161-1131; A.D. Kraft, D.A. Johnson, J.A. Johnson, Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult, J. Neurosci. 24 (2004) 1101-1112]. In the present study, we examined the relationship between electrophilicity and the protective effects afforded by electrophilic compounds. Electrophilicity was assessed in terms of the ability of a compound to bind to a cysteine on bovine serum albumin, by which we found that neuroprotective hydroquinones [TBHQ (para-) and CA (ortho-)] had distinctive patterns of cysteine binding compared with other electrophilic compounds. Further, we found that isomers of simple ortho- and para-hydroquinones such as 2-methylhydroquinone (para-) and 4-methyl-catechol (ortho-) [not in abstract] had similar properties of cysteine binding as TBHQ and CA, which compounds were associated with the transcriptional activation and an increase in the level of reduced glutathione. These results suggest that para- and ortho-dihydroquinones may be neuroprotective compounds active against oxidative stress.« less

A new flavonoid glycoside (APG) isolated from Clematis tangutica attenuates myocardial ischemia/reperfusion injury via activating PKCε signaling.

PubMed

Zhu, Yanrong; Di, Shouyin; Hu, Wei; Feng, Yingda; Zhou, Qing; Gong, Bing; Tang, Xinlong; Liu, Juntian; Zhang, Wei; Xi, Miaomiao; Jiang, Lin; Guo, Chao; Cao, Jingyi; Fan, Chongxi; Ma, Zhiqiang; Yang, Yang; Wen, Aidong

2017-03-01

Clematis tangutica has been shown to be beneficial for the heart; however, the mechanism of this effectremains unknown. Apigenin-7-O-β-D-(-6″-p-coumaroyl)-glucopyranoside (APG) is a new flavonoid glycoside isolated from Clematis tangutica. This study investigates the effects of APG on myocardial ischemia/reperfusion (IR) injury (IRI). An IRI model of primary myocardial cells and mice was used in this study. Compared with the IR group, APG preconditioning is protective against IRI in primary myocardial cells and in mice hearts in a dose-dependent manner. The cardioprotective mechanisms of APG may involve a significant PKCε translocation into the mitochondria and an activation of the Nrf2/HO-1 pathway, which respectively suppressesmitochondrial oxidative stress and inhibits apoptosis. In addition, PKCε-targeted siRNA and a PKCε specialized inhibitor (ε-V1-2) were used to inhibit PKCε expression and activity. The inhibition of PKCε reversed the cardioprotective effect of APG, with an inhibition of Nrf2/HO-1 activation and increased mitochondrial oxidative stress and cardiomyocyte apoptosis. In conclusion, PKCε activation plays an important role in the cardioprotective effects of APG. PKCε activation induced by APG preconditioning reduces mitochondrial oxidative stress and promotes Nrf2/HO-1-mediated anti-apoptosis signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis.

PubMed

Zhou, Richard; Lin, Jianjun; Wu, Defeng

2014-01-01

The mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins. The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro. The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1. Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice. The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study. © 2013.

Sulforaphane Induces Nrf2 and Protects Against CYP2E1-dependent Binge Alcohol –induced Liver Steatosis

PubMed Central

Zhou, Richard; Lin, Jianjun; Wu, Defeng

2013-01-01

Background The mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins. Method The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro. Results The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase -1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-Nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1. Conclusions Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol–induced fatty liver in mice. General significance The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study. PMID:24060752

In Vitro Cytotoxicity and Adaptive Stress Responses to Selected Haloacetic Acid and Halobenzoquinone Water Disinfection Byproducts.

PubMed

Procházka, Erik; Escher, Beate I; Plewa, Michael J; Leusch, Frederic D L

2015-10-19

The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts (DBPs). Some of these are mutagenic, genotoxic, teratogenic, and cytotoxic, as well as potentially carcinogenic both in vivo and in vitro. We investigated the in vitro biological activity of five DBPs: three monohaloacetic acids (monoHAAs) [chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA)] and two novel halobenzoquinones (HBQs) [2,6-dichloro-p-benzoquinone (DCBQ) and 2,6-dibromo-p-benzoquinone]. We focused particularly on cytotoxicity and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoxic to the Caco-2 cell line after a 4 h exposure, and all DBPs induced both of the adaptive stress response pathways, Nrf2/ARE and p53, in the micromolar range, as measured by two β-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA ∼ BAA > DCBQ ∼ DBBQ > CAA. Induction of oxidative stress was previously proposed to be the molecular initiating event (MIE) for both classes of DBPs. However, comparing the levels of activation of the two pathways uncovered that the Nrf2/ARE pathway was the more sensitive endpoint for HAAs, whereas the p53 pathway was more sensitive in the case of HBQs. Therefore, the DNA damage-responsive p53 pathway may be an important piece of information to fill in a gap in the adverse outcome pathway framework for the assessment of HBQs. Finally, we cautiously compared the potential risk of the two novel HBQs using a benchmarking approach to that of the well-studied CAA, which suggested that their relative risk may be lower than that of BAA and IAA.

Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biswas, Madhurima; Kwong, Erick K.; Park, Eujean

2013-08-01

Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 frommore » phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3.« less

Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPARγ, and HO-1

PubMed Central

Wu, Qing Qing; Wang, Yanxia; Senitko, Martin; Meyer, Colin; Wigley, W. Christian; Ferguson, Deborah A.; Grossman, Eric; Chen, Jianlin; Zhou, Xin J.; Hartono, John; Winterberg, Pamela; Chen, Bo; Agarwal, Anapam

2011-01-01

Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPARγ and Nrf2. Thus we report that BARD regulates PPARγ, not by acting as a ligand but by increasing the amount of PPARγ mRNA and protein. This should increase ligand-independent effects of PPARγ. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPARγ, we found increased Nrf2 and PPARγ on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity. PMID:21289052

Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2, PPARγ, and HO-1.

PubMed

Wu, Qing Qing; Wang, Yanxia; Senitko, Martin; Meyer, Colin; Wigley, W Christian; Ferguson, Deborah A; Grossman, Eric; Chen, Jianlin; Zhou, Xin J; Hartono, John; Winterberg, Pamela; Chen, Bo; Agarwal, Anapam; Lu, Christopher Y

2011-05-01

Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-γ (PPARγ), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPARγ and Nrf2. Thus we report that BARD regulates PPARγ, not by acting as a ligand but by increasing the amount of PPARγ mRNA and protein. This should increase ligand-independent effects of PPARγ. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPARγ, we found increased Nrf2 and PPARγ on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity.

Effect of cellular ubiquitin levels on the regulation of oxidative stress response and proteasome function via Nrf1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Donghee; Ryu, Kwon-Yul

The polyubiquitin genes Ubb and Ubc are upregulated under oxidative stress induced by arsenite [As(III)]. However, the role of ubiquitin (Ub) under As(III) exposure is not known in detail. In a previous study, we showed that the reduced viability observed in Ubc{sup −/−} mouse embryonic fibroblasts under As(III) exposure was not due to dysregulation of the Nrf2–Keap1 pathway, which prompted us to investigate another NFE2 family protein, nuclear factor erythroid 2-related factor 1 (Nrf1). In this study, we found that Ub deficiency due to Ubc knockdown in N2a cells reduced cell viability and proteasome activity under As(III) exposure. Furthermore, mRNAmore » levels of the proteasome subunit Psma1 were also reduced. In addition, Ub deficiency led to the nuclear accumulation of the p65 isoform of Nrf1 under As(III) exposure. Interestingly, the overexpression of p65-Nrf1 recapitulated the phenotypes of Ub-deficient N2a cells under As(III) exposure. On the other hand, Nrf1 knockdown suppressed the death of Ub-deficient N2a cells upon exposure to As(III). Therefore, the levels of p65-Nrf1 may play an important role in the maintenance of cell viability under oxidative stress induced by As(III). - Highlights: • N2a cells exhibit reduced viability upon exposure to As(III) via Ubc knockdown. • As(III)-induced proteasomal regulation is impaired in Ub-deficient N2a cells. • Ub deficiency leads to the nuclear accumulation of p65-Nrf1 under As(III) exposure. • p65 expression recapitulates As(III)-induced phenotypes of Ub-deficient N2a cells. • Nrf1 knockdown suppressed As(III)-induced death of Ub-deficient N2a cells.« less

Docosahexaenoic Acid (DHA) Provides Neuroprotection in Traumatic Brain Injury Models via Activating Nrf2-ARE Signaling.

PubMed

Zhu, Wei; Ding, Yuexia; Kong, Wei; Li, Tuo; Chen, Hongguang

2018-04-16

In this study, we explored the neuroprotective effects of docosahexaenoic acid (DHA) in traumatic brain injury (TBI) models. In this study, we first confirmed that DHA was neuroprotective against TBI via the NSS test and Morris water maze experiment. Western blot was conducted to identify the expression of Bax, caspase-3, and Bcl-2. And the cell apoptosis of the TBI models was validated by TUNEL staining. Relationships between nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway-related genes and DHA were explored by RT-PCR and Western blot. Rats of the DHA group performed remarkably better than those of the TBI group in both NSS test and water maze experiment. DHA conspicuously promoted the expression of Bcl-2 and diminished that of cleaved caspase-3 and Bax, indicating the anti-apoptotic role of DHA. Superoxide dismutase (SOD) activity and cortical malondialdehyde content, glutathione peroxidase (GPx) activity were renovated in rats receiving DHA treatment, implying that the neuroprotective influence of DHA was derived from lightening the oxidative stress caused by TBI. Moreover, immunofluorescence and Western blot experiments revealed that DHA facilitated the translocation of Nrf2 to the nucleus. DHA administration also notably increased the expression of the downstream factors NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1(HO-1). DHA exerted neuroprotective influence on the TBI models, potentially through activating the Nrf2- ARE pathway.

Sulforaphane attenuation of experimental diabetic nephropathy involves GSK-3 beta/Fyn/Nrf2 signaling pathway.

PubMed

Shang, Guoguo; Tang, Xinjun; Gao, Pan; Guo, Fanli; Liu, Hongpeng; Zhao, Zhonghua; Chen, Qi; Jiang, Tao; Zhang, Nong; Li, Hui

2015-06-01

Sulforaphane (SFN), the bioactive component of cruciferous vegetables, is a potent indirect antioxidant. Oxidative stress and activation of glycogen synthase kinase 3beta (GSK3β) are two major contributors to the pathogenesis of diabetic nephropathy (DN). Here, we investigated whether and how SFN affected GSK3β in experimental models of DN in vivo and in vitro. SFN treatment obviously prevented the increase in urine albumin excretion, matrix expansion, transforming growth factor-β1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Simultaneously, the level of 8-oxo-deoxyguanosine, an indicator of oxidative damage, was markedly lowered in SFN-treated diabetic rats, together with a significant reduction in activity of the GSK-3β/Fyn axis and an evident activation of Nrf2 signaling. Similarly, antifibrotic effects of SFN, parallel to enhanced inhibitory Ser9-phosphorylation of GSK3β and Fyn/Nrf2 nuclear export/import, were observed in the cultured rat mesangial cells (RMC) exposed to high glucose. The salutary effects of SFN on high-glucose-stimulated RMC were abolished by overexpression of GSK3β while being rescued by lithium chloride, a well-known GSK3β inhibitor. Taken together, our findings suggested that SFN ameliorated experimental diabetic nephropathy, at least in part, via GSK3β/Fyn/Nrf2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.