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Sample records for rat hepatic stellate

  1. Anticytoproliferative effect of Vitamin C on rat hepatic stellate cell

    PubMed Central

    Su, Min; Chao, Guo; Liang, Minqing; Song, Jianhua; Wu, Ka

    2016-01-01

    This study was conducted to investigate the potential therapeutical benefit of Vitamin (VC), a potent antioxidant, on suppressing proliferation of immortalized rat liver stellate cell line (HSC-T6) in vitro, and to discuss the underlying mechanism. HSC-T6 was co-treated with different concentrations of VC (50, 100, 200 μmol/L) on designed time points. Then, cell viability was assessed by using MTT analysis, and the changes of cytomorphology was observed with apoptosis-specific TUNEL and immunohistochemical stains, as well as the intracellular target genes was determined by using RT-PCR, respectively. As the outcomes, VC-treated HSC-T6 showed significantly inhibited cell growth in a dose-dependent manner when compared to the vehicle control. Cytologically, VC increased TUNEL-labeled positive cells in cultured HSC-T6, which the cell count was greater than vehicle control. Meanwhile, VC-treated HSC-T6 showed elevated immunoreactive for TGF-β1-labeled cells. Moreover, VC contributed to down-regulated expressions of intracellular c-myc, cyclin D1, mTOR mRNAs in HSC-T6. Collectively, these preliminary findings have demonstrated that VC-mediated anti-proliferative effect on HSCs is involved in molecular mechanisms of promoting apoptosis and blocking endogenous collagenation. PMID:27398165

  2. Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

    SciTech Connect

    Zan, Yanlu; Zhang, Yuxia; Tien, Po

    2013-06-07

    Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

  3. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells

    PubMed Central

    Huang, Shiow-Chyn; Kuo, Ping-Chung; Hung, Hsin-Yi; Pan, Tai-Long; Chen, Fu-An; Wu, Tian-Shung

    2016-01-01

    Three new γ-ionylideneacetic acid derivatives, phellinulins A–C (1–3), were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis. PMID:27164091

  4. Fibrogenic response of hepatic stellate cells in ovariectomised rats exposed to ketogenic diet.

    PubMed

    Bobowiec, R; Wojcik, M; Jaworska-Adamu, J; Tusinska, E

    2013-02-01

    The discrepancy about the role of estrogens in hepatic fibrogenesis and lack of studies addressed of ketogenic diet (KD) on hepatic stellate cells (HSC), prompted us to investigate the activity of HSC in control, KD- and thioacetamide (TAA)-administrated rats with different plasma concentration of estradiol (E2). HSC were isolated by the collagenase perfusion methods and separated by the Percoll gradient centrifugation. After the 4(th) and 8(th) day of incubation, lysates of HSC and the media were collected for further analysis. The HSC derived from KD-rats released remarkably more transforming growth factor (TGF)-β1 than cells obtained from animals fed with a standard diet. The ovariectomy of KD-rats markedly intensified the secretion of this fibrogenic cytokine on the 8(th) day of incubation (201.33 ±1 7.15 pg/ml). In HSC of rats exposed to E2, the TGF-β1 concentration did not exceed 157 ± 34.39 pg/ml. In respect to the collagen type I, the HSC obtained from ovariectomised KD-rats released an augmented amount of this ECM protein after the 8(th) day of culture (1.83 ± 0.14 U/ml). In the same time, higher quantities of ASMA appeared in the KD rats (1.41 ± 0.3 pg/mg protein). Exposition of rats to E2 did not markedly decrease the amount of ASMA. In summary, KD was able to induce morphological and functional changes in HSC, especially derived from rats deprived of ovarian estrogens. However, the preservation of E2 in ovariectomised rats didn't substantially alter the activation of HSC.

  5. Effect of lipid on proliferation and activation of rat hepatic stellate cells (I)

    PubMed Central

    Lu, Lun-Gen; Zeng, Min-De; Li, Ji-Qiang; Hua, Jing; Fan, Jian-Gao; Fan, Zhu-Ping; Qiu, De-Kai

    1998-01-01

    AIM: To study the effect of lipid (triglyceride and very low-density lipoprotein, VLDL) on proliferation and activation of rat hepatic stellate cells (HSC). METHODS: HSC were isolated and cultured from liver of Wistar rats by in situperfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. HSC proliferation was examined with MTT colorimetric assay. RESULT: Triglyceride of 12.5 mg/L had a promoting effect on proliferation of HSC (P < 0.05), 25, 50, 100 and 200 mg/L had no effects (P > 0.05), but 400 mg/L had an inhibiting effect (P < 0.01). VLDL of 6.25 and 12.5 mg/L had no effect on proliferation of HSC (P > 0.05), but increased concentration of VLDL could promote the HSC proliferation (P < 0.05). CONCLUSION: Lipid had an effect on proliferation of HSC. Triglyceride and VLDL may promote HSC proliferation and may be associated with fatty liver and hepatic fibrogenesis. PMID:11819353

  6. Berberine Inhibition of Fibrogenesis in a Rat Model of Liver Fibrosis and in Hepatic Stellate Cells.

    PubMed

    Wang, Ning; Xu, Qihe; Tan, Hor Yue; Hong, Ming; Li, Sha; Yuen, Man-Fung; Feng, Yibin

    2016-01-01

    Aim. To examine the effect of berberine (BBR) on liver fibrosis and its possible mechanisms through direct effects on hepatic stellate cells (HSC). Methods. The antifibrotic effect of BBR was determined in a rat model of bile duct ligation- (BDL-) induced liver fibrosis. Multiple cellular and molecular approaches were introduced to examine the effects of BBR on HSC. Results. BBR potently inhibited hepatic fibrosis induced by BDL in rats. It exhibited cytotoxicity to activated HSC at doses nontoxic to hepatocytes. High doses of BBR induced apoptosis of activated HSC, which was mediated by loss of mitochondrial membrane potential and Bcl-2/Bax imbalance. Low doses of BBR suppressed activation of HSC as evidenced by the inhibition of α-smooth muscle actin (α-SMA) expression and cell motility. BBR did not affect Smad2/3 phosphorylation but significantly activated 5' AMP-activated protein kinase (AMPK) signalling, which was responsible for the transcriptional inhibition by BBR of profibrogenic factors α-SMA and collagen in HSC. Conclusion. BBR is a promising agent for treating liver fibrosis through multiple mechanisms, at least partially by directly targeting HSC and by inhibiting the AMPK pathway. Its value as an antifibrotic drug in patients with liver disease deserves further investigation. PMID:27239214

  7. Sedum mexicanum Britt. Induces Apoptosis of Primary Rat Activated Hepatic Stellate Cells

    PubMed Central

    Lee, Shou-Lun; Chin, Ting-Yu; Lai, Ching-Long; Wang, Wen-Han

    2015-01-01

    Background. Liver fibrosis is a significant liver disease in Asian countries. Sedum mexicanum Britt. (SM) has been claimed to have antihepatitis efficacy. In traditional folk medicine, a solution of boiling water-extracted SM (SME) is consumed to prevent and treat hepatitis. However, its efficacy has not yet been verified. The purpose of this study was to investigate the in vitro effect of SME on hepatoprotection. Methods. Hepatic stellate cells (HSCs) and hepatocytes (HCs) were isolated from the livers of the rats by enzymatic digestion and density gradient centrifugation. Results. Treating the HCs and aHSCs with SME caused a dose-dependent decrease in the viability of aHSCs but not that of HCs. In addition, treatment with SME resulted in apoptosis of aHSCs, as determined by DAPI analysis and flow cytometry. SME also increased the amount of cleaved caspase-3, cleaved caspase-9, and cleaved poly ADP-ribose polymerase (PARP) in aHSCs. Furthermore, SME treatment induced a dose-dependent reduction in Bcl-2 expression and increased the expression of Bax in aHSCs. Conclusions. SME did not cause cytotoxicity in HCs, but it induced apoptosis in aHSCs through the mitochondria-dependent caspase-3 pathway. Therefore, SME may possess therapeutic potential for liver fibrosis. PMID:26078767

  8. Establishment and characterization of an immortalized rat hepatic stellate cell line

    PubMed Central

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Fu-Shun

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis and portal hypertension. This study established a new rat HSC cell line LSC-1. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate rat HSC. Cells have been maintained in culture for multiple passages. LSC-1 cell biological characteristics were studied. LSC-1 cell have been maintained in culture over 100 passages. This new HSC cell line express telomerase reverse transcriptase (TRT) and p53, suggesting that it is immortalized spontaneously. LSC-1 cells have a doubling time of 46 hours and their growth is serum-dependent. Karyotypic analysis revealed that LSC-1 cells possess normal chromosome phenotype. Moreover, LSC-1 cells do not grow in soft agar or induce tumors in nude mice, suggesting that they are not transformed. LSC-1 cells express desmin, glial fibrillary acidic proteins (GFAP), collagen type I and III, α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1), platelet derived growth factor B (PDGF-B) and inducible nitric oxide synthase (iNOS). TGF-β1 stimulation increased collagen type I and III expression in LSC-1 cells. Additionally, LSC-1 cells proliferate in response to PDGF-BB, and contract in response to endothelin-1 (ET-1). In summary, LSC-1 cells exhibit activated HSC phenotype characteristics, and therefore are useful tool to study the pathogenesis of liver cirrhosis and portal hypertension. PMID:26722391

  9. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    PubMed

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

  10. Study on antifibrotic effects of curcumin in rat hepatic stellate cells.

    PubMed

    Lin, Yun-Lian; Lin, Chia-Yu; Chi, Chin-Wen; Huang, Yi-Tsau

    2009-07-01

    Suppression of activation or fibrogenesis and induction of apoptosis, in hepatic stellate cells (HSCs) have been proposed as therapeutic strategies against liver fibrosis. Curcumin, an active compound isolated from yellow curry pigment of turmeric (Curcuma longa Linn), has been demonstrated to be an effective anti-inflammatory and antioxidant compound. In this study, we investigated the in vitro antifibrogenic effects of curcumin on HSCs at the concentration range of (1-40 microM). A cell line of rat HSCs (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1). The inhibitory effects of curcumin (1.25 approximately 10 microM) on fibrosis-related markers including alpha-smooth muscle actin (alpha-SMA) and collagen were assessed. In addition, the induction effects of curcumin (20 approximately 40 microM) on apoptosis in HSC-T6 cells were also assessed by Hoechst and propidium iodide stains. Curcumin (1.25 approximately 10 microM) concentration-dependently suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells, without cytotoxicity. Whereas, higher concentrations of curcumin (20 approximately 40 microM) induced cell apoptosis and cytochrome c release in HSC-T6 cells. Our results suggest that curcumin exerted antifibrotic effects, possibly through two different mechanisms depending on its concentrations. At lower concentrations (1.25 approximately 10 microM), curcumin exerted antifibrogenic effects, whereas at higher concentrations (20 approximately 40 microM), curcumin exerted induction of apoptosis in HSCs. PMID:19152370

  11. Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells

    PubMed Central

    Moore, Cathy C; Lakner, Ashley M; Yengo, Christopher M; Schrum, Laura W

    2011-01-01

    AIM: To identify and characterize the function of nonmuscle myosin II (NMM II) isoforms in primary rat hepatic stellate cells (HSCs). METHODS: Primary HSCs were isolated from male Sprague-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM II isoform (II-A, II-B and II-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM II protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM IIisoform expression determined by immunohistochemistry. Using a selective myosin II inhibitor and siRNA-mediated knockdown of each isoform, NMM II functionality in primary rat HSCs was determined by contraction and migration assays. RESULTS: NMM II-A and II-B mRNA expression was increased in culture-activated HSCs (Day 14) with significant increases seen in all pair-wise comparisons (II-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; II-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (II-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; II-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No significant differences were observed in NMM II-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM II-A and II-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro studies showed increased expression of NMM II-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM II-A and II-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM II-A and II-B to migration and contraction, NMM II-A and II-B expression were downregulated with siRNA. NMM II

  12. Tetrandrine stimulates the apoptosis of hepatic stellate cells and ameliorates development of fibrosis in a thioacetamide rat model

    PubMed Central

    Yin, Ming-Fu; Lian, Li-Hua; Piao, Dong-Ming; Nan, Ji-Xing

    2007-01-01

    AIM: To investigate the therapeutic effect of tetrandrine on liver fibrosis induced by thioacetamide in rats in vivo and in vitro. METHODS: In vitro study: we investigated the effect of tetrandrine on the apoptosis of rat hepatic stellate cells transformed by simian virus 40 (T-HSC/Cl-6), which retains the features of activated cells. In vivo study: hepatic fibrosis was induced in rats by thioacetamide. Tetrandrine was given orally to rats at doses of 5, 10 or 20 mg/kg for 4 wk compared with intraperitoneal injection of interferon-г. RESULTS: In vitro study: 5, 10 or 25 μg/mL of tetrandrine-induced activation of caspase-3 in t-HSC/Cl-6 cells occurred dose-dependently. In vivo study: tetrandrine treatment as well as interferon-г significantly ameliorated the development of fibrosis as determined by lowered serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil) and the levels of liver hydroxyproline (Hyp), hyaluronic acid (HA), laminin (LN) and also improved histological findings. The effects of tetrandrine at the concentration of 20 mg/kg were better than the other concentration groups. CONCLUSION: Tetrandrine promotes the apoptosis of activated HSCs in vitro. Tetrandrine administration can prevent liver fibrosis and liver damage induced by thioacetamide in rats in vivo, indicating that it might exert a direct effect on rat HSCs. PMID:17451202

  13. Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

    PubMed Central

    Su, Li-Jen; Chang, Chia-Chuan; Yang, Chih-Hsueh; Hsieh, Shur-Jong; Wu, Yi-Chin; Lai, Jin-Mei; Tseng, Tzu-Ling; Huang, Chi-Ying F.; Hsu, Shih-Lan

    2013-01-01

    Background Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl4)-induced liver injury rats. Methods Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated. Results Oral administration of MGP significantly alleviated DMN- or CCl4-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. Conclusions The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis. PMID:23335984

  14. DNMT1-mediated PTEN hypermethylation confers hepatic stellate cell activation and liver fibrogenesis in rats

    SciTech Connect

    Bian, Er-Bao; Huang, Cheng; Ma, Tao-Tao; Tao, Hui; Zhang, Hui; Cheng, Chang; Lv, Xiong-Wen; Li, Jun

    2012-10-01

    Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation. Highlights: ► PTEN methylation status and loss of PTEN expression ► DNMT1 mediated PTEN hypermethylation. ► Hypermethylation of PTEN contributes to the activation of ERK and AKT pathways.

  15. Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.

    PubMed

    Reimann, T; Hempel, U; Krautwald, S; Axmann, A; Scheibe, R; Seidel, D; Wenzel, K W

    1997-02-10

    The transdifferentiation of hepatic stellate cells into myofibroblast-like cells and the proliferation of the transdifferentiated cells are controlled by TGF-beta1. Little is known about the intracellular signal transducers of TGF-beta1. In this paper we show that in cultured hepatic stellate cells TGF-beta1 induces activation of Ras, Raf-1, MEK and MAPK p42 and p44. The activation of MAPK depends on the activation of MEK. Our data exclude that the observed effects are mediated by a bFGF or PDGF autocrine loop. PMID:9038360

  16. Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells

    PubMed Central

    Vincent, K; Jones, E; Arthur, M; Smart, D; Trim, J; Wright, M; Mann, D

    2001-01-01

    BACKGROUND—Activation of hepatic stellate cells (HSCs) to a myofibroblastic phenotype is a key event in liver fibrosis. Identification of transcription factors with activities that are modulated during HSC activation will improve our understanding of the molecular events controlling HSC activation.
AIMS—To determine if changes in E-box DNA binding activity occur during in vitro and in vivo activation of rat and human HSCs and to investigate mechanisms underlying any observed changes.
METHODS—Nuclear extracts were prepared from rat HSCs isolated and cultured from normal and carbon tetrachloride injured rat livers and from HSCs isolated from human liver. EMSA analysis of E-box DNA binding activity was performed on nuclear extracts to determine changes during HSC activation. Western and northern blot analysis of MyoD and Id1 basic helix-loop-helix (bHLH) proteins was performed to confirm expression in HSC.
RESULTS—HSC activation was associated with inducible expression of two low mobility E-box binding complexes that were immunoreactive with an anti-MyoD antibody. MyoD mRNA expression was found at similar levels in freshly isolated and activated HSCs; in contrast, MyoD protein expression was elevated in activated HSCs. Activation of rat HSCs was accompanied by reduced expression of the inhibitory bHLH protein Id1.
CONCLUSIONS—In vitro and in vivo activation of rat and human HSCs is accompanied by induction of MyoD binding to E-box DNA sequences which appears to be mechanistically associated with elevated MyoD protein expression and reduced expression of the inhibitory Id1 protein. Clarification of the role of MyoD and Id1 proteins in HSC activation and liver fibrogenesis is now required.


Keywords: liver fibrosis; hepatic stellate cell; basic helix-loop-helix transcription factors; MyoD; Id1 PMID:11600477

  17. Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells.

    PubMed

    Huang, Yue-Hong; Chen, Yun-Xin; Zhang, Li-Juan; Chen, Zhi-Xin; Wang, Xiao-Zhong

    2014-09-01

    Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin‑10 (rIL‑10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid‑expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of α-smooth muscle actin (α-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL‑transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen.

  18. Caffeine protects against alcohol-induced liver fibrosis by dampening the cAMP/PKA/CREB pathway in rat hepatic stellate cells.

    PubMed

    Wang, Qi; Dai, Xuefei; Yang, Wanzhi; Wang, He; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun; Lv, Xiongwen

    2015-04-01

    Alcoholic liver fibrosis (ALF) is characterized by hyperplasia of extracellular matrix under long-term alcohol stimulation. Hepatic stellate cell (HSC) activation plays an important role in promoting hepatic fibrogenesis. Caffeine, as the main active component of coffee and tea, was widely consumed in daily life. It was always a thought that caffeine can reduce the probability of suffering from liver diseases. In this study, we attempt to validate the hypothesis that caffeine inhibits activation of HSCs which were isolated from rat ALF model. The rats were gavaged by ethanol to establish ALF model and then treated with different concentrations of caffeine or colchicine. Serum was collected to measure the contents of serum alanine aminotransferase (ALT), aspartate transaminase (AST), hyaluronic acid (HA), laminin (LN), N-terminal peptide of type III procollagen (PIIINP) and type IV collagen (CIV). Then liver tissues were obtained for hematoxylin-eosin staining and Sirius-red staining. Others were treated through liver perfusion to isolate primary rat HSCs. Interestingly, we found that caffeine significantly decreased ALT, AST, HA, LN, PIIINP and CIV levels and reversed liver fibrosis in rat ALF models. Results of immunohistochemistry, real-time PCR and western blot indicated that caffeine could reduce fibrosis and inhibit cAMP/PKA/CREB signal pathway in HSC. Caffeine has a preventive effect on ALF. The mechanism may be interpreted that caffeine inhibits the cAMP/PKA/CREB signal pathway through adenosine A2A receptors in HSC.

  19. Hepatic stellate cells contribute to progenitor cells and liver regeneration.

    PubMed

    Kordes, Claus; Sawitza, Iris; Götze, Silke; Herebian, Diran; Häussinger, Dieter

    2014-12-01

    Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP(+) HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell-based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin-handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid-synthesizing and -transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell-based liver regeneration.

  20. Hepatic stellate cells contribute to progenitor cells and liver regeneration

    PubMed Central

    Kordes, Claus; Sawitza, Iris; Götze, Silke; Herebian, Diran; Häussinger, Dieter

    2014-01-01

    Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP+ HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell–based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin–handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid–synthesizing and –transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell–based liver regeneration. PMID:25401473

  1. Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury.

    PubMed

    Tennakoon, Anusha Hemamali; Izawa, Takeshi; Wijesundera, Kavindra Kumara; Golbar, Hossain M; Tanaka, Miyuu; Ichikawa, Chisa; Kuwamura, Mitsuru; Yamate, Jyoji

    2013-11-01

    Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1-10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.

  2. Dicliptera Chinensis polysaccharides target TGF-β/Smad pathway and inhibit stellate cells activation in rats with dimethylnitrosamine-induced hepatic fibrosis.

    PubMed

    Zhang, X; Zhang, J; Jia, L; Xiao, S

    2016-01-27

    This study aims to study impact of Dicliptera chinensis polysaccharide (DCP) on hepatic fibrosis (HF) and activation of hepatic stellate cells (HSCs). Liver fibrosis model was induced by intraperitoneal injection of dimethyl nitrosamines (DMN) in rat. Rats in treatment group were administrated with different concentrations of DCP (0, 100, 300 mg/kg) by intraperitoneal injection. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to assess histo-pathological change. α-SMA, TGF-β1 and pSmad 2/3 were assayed by immuno-histochemistry. HSC-T6 cells were stimulated by recombined rat TGF-β1 (1 ng/mL) to simulate an activating model in vitro and then interfered with DCP (concentration of 0, 25, 50, 100, 200, 400 µg/ml). MTT assay was used to determine cell proliferation and western blotting was used to detect α-SMA and pSmad 2/3 expression. Results demonstrated that DCP alleviated DMN-induced liver fibrosis in rat and significantly down-regulated TGF-β1 expression, pSmad2/3 and α-SMA in liver tissue in a dose-dependent way. DCP inhibited proliferation and activation of TGF-β1-stimulated HSC-T6 in vitro and significantly down-regulated α-SMA and pSmad2/3 expression. In conclusion, this study revealed that DCP attenuates progression of liver fibrosis through suppressing TGF-β/Smad pathway. DCP is a potential botanical polysaccharide to management liver fibrosis.

  3. The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

    PubMed

    Calleja, Miguel Angel; Vieites, Jose María; Montero-Meléndez, Trinidad; Montero-Meterdez, Trinidad; Torres, María Isabel; Faus, María José; Gil, Angel; Suárez, Antonio

    2013-02-14

    Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

  4. Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells.

    PubMed

    De Bleser, P J; Xu, G; Rombouts, K; Rogiers, V; Geerts, A

    1999-11-26

    Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimer's disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl(4)-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-beta (TGF-beta) increased the production of H(2)O(2). Treatment with catalase decreased TGF-beta expression. Addition of H(2)O(2) resulted in increased TGF-beta production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H(2)O(2). A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1, 2,4-triazole increased TGF-beta production. A paradoxical decrease of TGF-beta production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-beta production. TGF-beta decreased the capacity of the SCs to remove H(2)O(2.) An increase in the capacity to remove H(2)O(2) was observed when TGF-beta was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-beta production and 2) that cellular GSH levels discriminate whether H(2)O(2) is the result of oxidative stress or acts as second messenger in the TGF-beta signal transduction pathway.

  5. Short Hairpin RNA Causes the Methylation of Transforming Growth Factor-β Receptor II Promoter and Silencing of the Target Gene in Rat Hepatic Stellate Cells

    PubMed Central

    Kim, Jin-Wook; Zhang, Yan-Hong; Zern, Mark A; Rossi, John J.; Wu, Jian

    2008-01-01

    Small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in plant and animal cells. RNA dependent DNA methylation (RdDM) accounts for TGS in plants, but it is unclear whether siRNA induces RdDM in mammalian cells. To determine whether stable expression of short hairpin siRNA (shRNA) induces DNA methylation in mammalian cells, we transduced rat hepatic stellate SBC10 cells with lentiviral vectors which encode an U6 promoter-driven shRNA expression cassette homologous to the transforming growth factor-β receptor (TGFβRII) promoter region. Sequencing analysis of bisulfite-modified genomic DNA showed the methylation of cytosine residues both in CpG dinucleotides and non-CpG sites around the target region of the TGFβRII promoter in SBC10 cells transduced with the promoter-targeting lentiviral vector. In these cells, real-time RT-PCR showed a decrease in TGFβRII mRNA levels which were reversed by treatment with 5-aza-2-deoxycytidine. Our results demonstrate that recombinant lentivirus-mediated shRNA delivery resulted in the methylation of the homologous promoter area in mammalian cells, and this approach may be used as a tool for transcriptional gene silencing by epigenetic modification of mammalian cell promoters. PMID:17533113

  6. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats

    PubMed Central

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  7. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats.

    PubMed

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  8. MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARγ expression: A rat fibrosis model study.

    PubMed

    Lu, Le; Wang, Jinlong; Lu, Hongwei; Zhang, Guoyu; Liu, Yang; Wang, Jiazhong; Zhang, Yafei; Shang, Hao; Ji, Hong; Chen, Xi; Duan, Yanxia; Li, Yiming

    2015-09-25

    Hepatic stellate cells (HSCs) are the primary sources of extracellular matrix (ECM) in normal and fibrotic liver. Peroxisome proliferator-activated receptor gamma (PPARγ) maintains HSCs in a quiescent state, and its downregulation induces HSC activation. MicroRNAs (miRNAs) can induce PPARγ mRNA degradation, but the mechanism by which miRNAs regulate PPARγ in rat HSCs is unclear. This study aimed to investigate some miRNAs which putatively bind to the 3'-untranslated region (3'-UTR) of PPARγ mRNA, and increase expression of ECM genes in rat HSCs. In carbon tetrachloride injection (CCl4) and common bile duct ligation (CBDL) liver fibrosis models, miRNAs miR-130a, miR-130b, miR-301a, miR-27b and miR-340 levels were found to be increased and PPARγ expression decreased. Overexpression of miR-130a and miR-130b enhanced cell proliferation by involving Runx3. MiR-130a and miR-130b decreased PPARγ expression by targeting the 3'-UTR of PPARγ mRNA in rat HSC-T6 cells. Transforming growth factor-β1 (TGF-β1) may mediate miR-130a and miR-130b overexpression, PPARγ downregulation, and ECM genes overexpression in cell culture. These findings suggest that miR-130a and miR-130b are involved in downregulation of PPARγ in liver fibrosis. PMID:26255201

  9. Anti-fibrotic effect of thymoquinone on hepatic stellate cells.

    PubMed

    Ghazwani, Mohammed; Zhang, Yifei; Gao, Xiang; Fan, Jie; Li, Jiang; Li, Song

    2014-02-15

    Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl4-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect. PMID:24182989

  10. Anti-fibrotic effect of thymoquinone on hepatic stellate cells.

    PubMed

    Ghazwani, Mohammed; Zhang, Yifei; Gao, Xiang; Fan, Jie; Li, Jiang; Li, Song

    2014-02-15

    Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl4-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect.

  11. Carvedilol Improves Inflammatory Response, Oxidative Stress and Fibrosis in the Alcohol-Induced Liver Injury in Rats by Regulating Kuppfer Cells and Hepatic Stellate Cells

    PubMed Central

    Leitão, Renata Ferreira de Carvalho; Brito, Gerly Anne de Castro; Miguel, Emilio de Castro; Guedes, Paulo Marcos Matta; de Araújo, Aurigena Antunes

    2016-01-01

    -α, procollagen type I (PCI), procollagen type III (PCIII), and NF-κB were decreased in the alcohol-CARV 5 mg/kg group relative to the alcohol-only group. Conclusions CARV can reduce the stress oxidative, inflammatory response and fibrosis in ethanol-induced liver injury in a rat model by downregulating signalling of Kuppfer cells and hepatic stellate cells (HSCs) through suppression of inflammatory cytokines. PMID:26891124

  12. Molecular magnetic resonance imaging of activated hepatic stellate cells with ultrasmall superparamagnetic iron oxide targeting integrin αvβ3 for staging liver fibrosis in rat model

    PubMed Central

    Zhang, Caiyuan; Liu, Huanhuan; Cui, Yanfen; Li, Xiaoming; Zhang, Zhongyang; Zhang, Yong; Wang, Dengbin

    2016-01-01

    Purpose To evaluate the expression level of integrin αvβ3 on activated hepatic stellate cells (HSCs) at different stages of liver fibrosis induced by carbon tetrachloride (CCl4) in rat model and the feasibility to stage liver fibrosis by using molecular magnetic resonance imaging (MRI) with arginine-glycine-aspartic acid (RGD) peptide modified ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) specifically targeting integrin αvβ3. Materials and methods All experiments received approval from our Institutional Animal Care and Use Committee. Thirty-six rats were randomly divided into three groups of 12 subjects each, and intraperitoneally injected with CCl4 for either 3, 6, or 9 weeks. Controls (n=10) received pure olive oil. The change in T2* relaxation rate (ΔR2*) pre- and postintravenous administration of RGD-USPIO or naked USPIO was measured by 3.0T clinical MRI and compared by one-way analysis of variance or the Student’s t-test. The relationship between expression level of integrin αvβ3 and liver fibrotic degree was evaluated by Spearman’s ranked correlation. Results Activated HSCs were confirmed to be the main cell types expressing integrin αvβ3 during liver fibrogenesis. The protein level of integrin αv and β3 subunit expressed on activated HSCs was upregulated and correlated well with the progression of liver fibrosis (r=0.954, P<0.001; r=0.931, P<0.001, respectively). After injection of RGD-USPIO, there is significant difference in ΔR2* among rats treated with 0, 3, 6, and 9 weeks of CCl4 (P<0.001). The accumulation of iron particles in fibrotic liver specimen is significantly greater for RGD-USPIO than naked USPIO after being injected with equal dose of iron. Conclusion Molecular MRI of integrin αvβ3 expressed on activated HSCs by using RGD-USPIO may distinguish different liver fibrotic stages in CCl4 rat model and shows promising to noninvasively monitor the progression of the liver fibrosis and therapeutic response to

  13. Angiotensin II increases mRNA levels of all TGF-beta isoforms in quiescent and activated rat hepatic stellate cells.

    PubMed

    Moreno-Alvarez, Paola; Sosa-Garrocho, Marcela; Briones-Orta, Marco A; González-Espinosa, Claudia; Medina-Tamayo, Jaciel; Molina-Jijón, Eduardo; Pedraza-Chaverri, José; Macías-Silva, Marina

    2010-10-01

    AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

  14. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  15. MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARγ expression: A rat fibrosis model study

    SciTech Connect

    Lu, Le; Wang, Jinlong; Lu, Hongwei; Zhang, Guoyu; Liu, Yang; Wang, Jiazhong; Zhang, Yafei; Shang, Hao; Ji, Hong; Chen, Xi; Duan, Yanxia; Li, Yiming

    2015-09-25

    Hepatic stellate cells (HSCs) are the primary sources of extracellular matrix (ECM) in normal and fibrotic liver. Peroxisome proliferator-activated receptor gamma (PPARγ) maintains HSCs in a quiescent state, and its downregulation induces HSC activation. MicroRNAs (miRNAs) can induce PPARγ mRNA degradation, but the mechanism by which miRNAs regulate PPARγ in rat HSCs is unclear. This study aimed to investigate some miRNAs which putatively bind to the 3′-untranslated region (3′-UTR) of PPARγ mRNA, and increase expression of ECM genes in rat HSCs. In carbon tetrachloride injection (CCl{sub 4}) and common bile duct ligation (CBDL) liver fibrosis models, miRNAs miR-130a, miR-130b, miR-301a, miR-27b and miR-340 levels were found to be increased and PPARγ expression decreased. Overexpression of miR-130a and miR-130b enhanced cell proliferation by involving Runx3. MiR-130a and miR-130b decreased PPARγ expression by targeting the 3′-UTR of PPARγ mRNA in rat HSC-T6 cells. Transforming growth factor-β1 (TGF-β1) may mediate miR-130a and miR-130b overexpression, PPARγ downregulation, and ECM genes overexpression in cell culture. These findings suggest that miR-130a and miR-130b are involved in downregulation of PPARγ in liver fibrosis. - Highlights: • MiR-130a and miR-130b are increased and PPARγ is decreased in liver fibrosis models. • MiR-130a and miR-130b decreased PPARγ by targeting the 3′-UTR of PPARγ mRNA. • MiR-130a and miR-130b enhanced HSC cell proliferation by involving Runx3. • TGF-β1 may mediate miR-130a and miR-130b overexpression.

  16. Pharmacological Intervention in Hepatic Stellate Cell Activation and Hepatic Fibrosis

    PubMed Central

    Schon, Hans-Theo; Bartneck, Matthias; Borkham-Kamphorst, Erawan; Nattermann, Jacob; Lammers, Twan; Tacke, Frank; Weiskirchen, Ralf

    2016-01-01

    The activation and transdifferentiation of hepatic stellate cells (HSCs) into contractile, matrix-producing myofibroblasts (MFBs) are central events in hepatic fibrogenesis. These processes are driven by autocrine- and paracrine-acting soluble factors (i.e., cytokines and chemokines). Proof-of-concept studies of the last decades have shown that both the deactivation and removal of hepatic MFBs as well as antagonizing profibrogenic factors are in principle suitable to attenuate ongoing hepatic fibrosis. Although several drugs show potent antifibrotic activities in experimental models of hepatic fibrosis, there is presently no effective pharmaceutical intervention specifically approved for the treatment of liver fibrosis. Pharmaceutical interventions are generally hampered by insufficient supply of drugs to the diseased liver tissue and/or by adverse effects as a result of affecting non-target cells. Therefore, targeted delivery systems that bind specifically to receptors solely expressed on activated HSCs or transdifferentiated MFBs and delivery systems that can improve drug distribution to the liver in general are urgently needed. In this review, we summarize current strategies for targeted delivery of drugs to the liver and in particular to pro-fibrogenic liver cells. The applicability and efficacy of sequestering molecules, selective protein carriers, lipid-based drug vehicles, viral vectors, transcriptional targeting approaches, therapeutic liver- and HSC-specific nanoparticles, and miRNA-based strategies are discussed. Some of these delivery systems that had already been successfully tested in experimental animal models of ongoing hepatic fibrogenesis are expected to translate into clinically useful therapeutics specifically targeting HSCs. PMID:26941644

  17. Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

    PubMed Central

    Wang, Yu-Gang; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wen-Ying; Wang, Na; Shi, Min

    2015-01-01

    AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation. METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclinD1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy. RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediated through p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase (acetyl K68) and nuclear factor-κB p65 (acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models. CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis. PMID:26217084

  18. Growth arrest and decrease of alpha-SMA and type I collagen expression by palmitic acid in the rat hepatic stellate cell line PAV-1.

    PubMed

    Abergel, Armand; Sapin, Vincent; Dif, Nicolas; Chassard, Christophe; Darcha, Claude; Marcand-Sauvant, Julie; Gaillard-Martinie, Brigitte; Rock, Edmond; Dechelotte, Pierre; Sauvant, Patrick

    2006-05-01

    Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by alpha-smooth muscle actin (alpha-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75 muM) alone or in combination with retinol (2 muM) significantly decreased cell proliferation and alpha-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.

  19. Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Wang Xueqing; Huang Guangcun; Mei Shuang; Qian Jin; Ji Juling; Zhang Jinsheng

    2009-03-06

    Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) and P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.

  20. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells.

    PubMed

    Feng, Yi; Ying, Hai-Yan; Qu, Ying; Cai, Xiao-Bo; Xu, Ming-Yi; Lu, Lun-Gen

    2016-09-01

    Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.

  1. Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression.

    PubMed

    Caliari, Steven R; Perepelyuk, Maryna; Soulas, Elizabeth M; Lee, Gi Yun; Wells, Rebecca G; Burdick, Jason A

    2016-06-13

    The extracellular matrix (ECM) presents an evolving set of mechanical cues to resident cells. We developed methacrylated hyaluronic acid (MeHA) hydrogels containing both stable and hydrolytically degradable crosslinks to provide cells with a gradually softening (but not fully degradable) milieu, mimicking physiological events such as fibrosis regression. To demonstrate the utility of this cell culture system, we studied the phenotype of rat hepatic stellate cells, the major liver precursors of fibrogenic myofibroblasts, within this softening environment. Stellate cells that were mechanically primed on tissue culture plastic attained a myofibroblast phenotype, which persisted when seeded onto stiff (∼20 kPa) hydrogels. However, mechanically primed stellate cells on stiff-to-soft (∼20 to ∼3 kPa) hydrogels showed reversion of the myofibroblast phenotype over 14 days, with reductions in cell area, expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA), and Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) nuclear localization when compared to stellate cells on stiff hydrogels. Cells on stiff-to-soft hydrogels did not fully revert, however. They displayed reduced expression of glial fibrillary acidic protein (GFAP), and underwent abnormally rapid re-activation to myofibroblasts in response to re-stiffening of the hydrogels through introduction of additional crosslinks. These features are typical of stellate cells with an intermediate phenotype, reported to occur in vivo with fibrosis regression and re-injury. Together, these data suggest that mechanics play an important role in fibrosis regression and that integrating dynamic mechanical cues into model systems helps capture cell behaviors observed in vivo.

  2. The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

    PubMed Central

    Loo, Christine K C; Pereira, Tamara N; Pozniak, Katarzyna N; Ramsing, Mette; Vogel, Ida; Ramm, Grant A

    2015-01-01

    The precise embryological origin and development of hepatic stellate cells is not established. Animal studies and observations on human fetuses suggest that they derive from posterior mesodermal cells that migrate via the septum transversum and developing diaphragm to form submesothelial cells beneath the liver capsule, which give rise to mesenchymal cells including hepatic stellate cells. However, it is unclear if these are similar to hepatic stellate cells in adults or if this is the only source of stellate cells. We have studied hepatic stellate cells by immunohistochemistry, in developing human liver from autopsies of fetuses with and without malformations and growth restriction, using cellular Retinol Binding Protein-1 (cRBP-1), Glial Fibrillary Acidic Protein (GFAP), and α-Smooth Muscle Actin (αSMA) antibodies, to identify factors that influence their development. We found that hepatic stellate cells expressing cRBP-1 are present from the end of the first trimester of gestation and reduce in density throughout gestation. They appear abnormally formed and variably reduced in number in fetuses with abnormal mesothelial Wilms Tumor 1 (WT1) function, diaphragmatic hernia and in ectopic liver nodules without mesothelium. Stellate cells showed similarities to intravascular cells and their presence in a fetus with diaphragm agenesis suggests they may be derived from circulating stem cells. Our observations suggest circulating stem cells as well as mesothelium can give rise to hepatic stellate cells, and that they require normal mesothelial function for their development. PMID:26265759

  3. (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat hepatic stellate cells.

    PubMed

    Roth, S; Michel, K; Gressner, A M

    1998-04-01

    Cultured parenchymal liver cells (PC) were recently recognized to contain (latent) transforming growth factor beta (TGF-beta) while the expression of TGF-beta mRNA remains controversial. This study was designed to analyze PC in different microenvironments (liver in situ, highly purified, isolated, and cultured PC) regarding the qualitative and quantitative content of mature and latent TGF-beta protein (immunostainings, enzyme-linked immunosorbent assay [ELISA], and enzyme-labeled fluorescence [ELF] technique). The results were compared with its gene expression (reverse-transcription polymerase chain reaction [RT-PCR]). In all microenvironments, PC contained latent TGF-beta, which was partially activated after cell isolation and culture. The amount of total TGF-beta (mature plus latent) of latency-associated peptide (LAP) and of latent TGF-beta binding protein (LTBP) were shown to decrease during culture. In contrast, TGF-beta2 and TGF-beta3 mRNA and LTBP-1 and -3 mRNA expression were first detectable after culture. Permeabilization of cell membranes in whole liver and of isolated PC with streptolysin O or carbon tetrachloride, respectively, released TGF-beta, a part of which was integrated in the large latent complex as estimated by analytical gel filtration chromatography. The TGF-beta released by damaged PC induces paracrine effects on hepatic stellate cell cultures. It stimulates hyaluronan synthesis and antagonizes the effect of mitogenic factor(s) of PC on [3H]thymidine incorporation. The results strongly suggest that the main part of hepatocellular TGF-beta is not generated by de novo synthesis but from uptake into the liver in vivo. The immunodetection of preexisting mature TGF-beta after isolation of the cells is probably caused by intracellular activation of latent TGF-beta. The injury-dependent discharge of TGF-beta from PC might be an important mechanism for initiation and perpetuation of various forms of chronic human liver diseases.

  4. MicroRNA‑200a suppresses epithelial‑to‑mesenchymal transition in rat hepatic stellate cells via GLI family zinc finger 2.

    PubMed

    Yu, Fujun; Zheng, Yihu; Hong, Weilong; Chen, Bicheng; Dong, Peihong; Zheng, Jianjian

    2015-12-01

    Hepatic stellate cells (HSCs) have an important role in liver fibrosis. Epithelial‑to‑mesenchymal transition (EMT), which is promoted by the Hedgehog (Hh) signaling pathway, is involved in the activation of HSCs. MicroRNAs (miRNAs/miRs) have been reported to be involved in the progression of liver fibrosis. A previous study indicated that the activation of HSCs was suppressed by miR‑200a via targeting transforming growth factor‑β2 and β‑catenin. However, whether miR‑200a is able to regulate the EMT in HSCs has remained elusive. The present study revealed that miR‑200a was decreased in vitro and in vivo during liver fibrosis. Furthermore, miR‑200a overexpression resulted in the inhibition of proliferation, α‑SMA expression and extracellular matrix production of activated HSCs. Of note, miR‑200a overexpression reduced myofibroblastic markers, including α‑SMA, type I collagen and desmin, and increased the epithelial cell marker E‑cadherin. These results were further confirmed by immunofluorescence staining. Further study showed that the expression of genes associated with Hh signaling, including Hhip, Shh and Gli1, were not affected by miR‑200a. However, Gli2, a downstream signaling protein of the Hh pathway, was inhibited by miR‑200a and confirmed as a target of miR‑200a using a dual luciferase reporter assay. In addition, the inhibition of the Hh pathway by miR‑200a resulted in an increase of BMP‑7 and Id2 as well as a reduction of Snai1 and S100A4. Collectively, the results of the present study demonstrated that miR‑200a suppressed the EMT process in HSCs, at least in part, via Gli2. PMID:26499180

  5. Aqueous Date Flesh or Pits Extract Attenuates Liver Fibrosis via Suppression of Hepatic Stellate Cell Activation and Reduction of Inflammatory Cytokines, Transforming Growth Factor-β1 and Angiogenic Markers in Carbon Tetrachloride-Intoxicated Rats

    PubMed Central

    Al-Rasheed, Nouf M.; Attia, Hala A.; Mohamad, Raeesa A.; Al-Rasheed, Nawal M.; Al-Amin, Maha A.; AL-Onazi, Asma

    2015-01-01

    Previous data indicated the protective effect of date fruit extract on oxidative damage in rat liver. However, the hepatoprotective effects via other mechanisms have not been investigated. This study was performed to evaluate the antifibrotic effect of date flesh extract (DFE) or date pits extract (DPE) via inactivation of hepatic stellate cells (HSCs), reducing the levels of inflammatory, fibrotic and angiogenic markers. Coffee was used as reference hepatoprotective agent. Liver fibrosis was induced by injection of CCl4 (0.4 mL/kg) three times weekly for 8 weeks. DFE, DPE (6 mL/kg), coffee (300 mg/kg), and combination of coffee + DFE and coffee + DPE were given to CCl4-intoxicated rats daily for 8 weeks. DFE, DPE, and their combination with coffee attenuated the elevated levels of inflammatory cytokines including tumor necrosis factor-α, interleukin-6, and interleukin-1β. The increased levels of transforming growth factor-β1 and collagen deposition in injured liver were alleviated by both extracts. CCl4-induced expression of α-smooth muscle actin was suppressed indicating HSCs inactivation. Increased angiogenesis was ameliorated as revealed by reduced levels and expression of vascular endothelial growth factor and CD31. We concluded that DFE or DPE could protect liver via different mechanisms. The combination of coffee with DFE or DPE may enhance its antifibrotic effects. PMID:25945106

  6. The improving effects on hepatic fibrosis of interferon-γ liposomes targeted to hepatic stellate cells

    NASA Astrophysics Data System (ADS)

    Li, Qinghua; Yan, Zhiqiang; Li, Feng; Lu, Weiyue; Wang, Jiyao; Guo, Chuanyong

    2012-07-01

    No satisfactory anti-fibrotic therapies have yet been applied clinically. One of the main reasons is the inability to specifically target the responsible cells to produce an available drug concentration and the side-effects. Exploiting the key role of the activated hepatic stellate cells (HSCs) in both hepatic fibrogenesis and over-expression of platelet-derived growth factor receptor-β (PDGFR-β), we constructed targeted sterically stable liposomes (SSLs) modified by a cyclic peptide (pPB) with affinity for the PDGFR-β to deliver interferon (IFN)-γ to HSCs. The pPB-SSL-IFN-γ showed satisfactory size distribution. In vitro pPB-SSL could be taken up by activated HSCs. The study of tissue distribution via living-body animal imaging showed that the pPB-SSL-IFN-γ mostly accumulated in the liver until 24 h. Furthermore, the pPB-SSL-IFN-γ showed more significant remission of hepatic fibrosis. In vivo the histological Ishak stage, the semiquantitative score for collagen in fibrotic liver and the serum levels of collagen type IV-C in fibrotic rats treated with pPB-SSL-IFN-γ were less than those treated with SSL-IFN-γ, IFN-γ and the control group. In vitro pPB-SSL-IFN-γ was also more effective in suppressing activated HSC proliferation and inducing apoptosis of activated HSCs. Thus the data suggest that pPB-SSL-IFN-γ might be a more effective anti-fibrotic agent and a new opportunity for clinical therapy of hepatic fibrosis.

  7. Indole-3-carbinol enhances the resolution of rat liver fibrosis and stimulates hepatic stellate cell apoptosis by blocking the inhibitor of κB kinase α/inhibitor of κB-α/nuclear factor-κB pathway.

    PubMed

    Ping, Jie; Gao, Ai-mei; Qin, Hai-quan; Wei, Xiao-ning; Bai, Jing; Liu, Lian; Li, Xiao-hai; Li, Rui-wen; Ao, Ying; Wang, Hui

    2011-11-01

    Hepatic stellate cells (HSC) play a pivotal role in liver fibrosis, and the clearance of activated HSC by apoptosis is associated with the resolution of liver fibrosis. The development of strategies that promote this process in a selective way is therefore important. We evaluated the effects of indole-3-carbinol (I3C), a nutritional component derived from vegetables from the Brassica family, on liver fibrosis and HSC apoptosis. The in vivo therapeutic effects of I3C were monitored in three rat models of liver fibrosis induced by porcine serum, bile duct ligation, or multiple hepatotoxic factors, and its proapoptotic effect and molecular mechanism were studied in vitro in HSC-T6, a rat HSC line. The results showed that I3C treatment significantly reduced the number of activated HSC in the livers of rats with liver fibrosis. In histopathology, I3C reduced hepatocyte degeneration and necrosis, accelerated collagen degradation, and promoted the reversal of liver fibrosis. I3C prescribed to HSC-T6 resulted in morphologic alterations typical of apoptosis and DNA cleavage to a nucleosomal ladder. Moreover, I3C significantly increased the HSC-T6 apoptosis rate and the expression ratio of Bax to Bcl-2. High-throughput protein array analysis indicated that the tumor necrosis factor-α/nuclear factor-κB (NF-κB) signal pathway participated in I3C-induced HSC-T6 apoptosis. Western blot and electrophoretic mobility-shift assay confirmed that I3C inhibited the phosphorylation of inhibitor of κB kinase α and inhibitor of κB-α and NF-κB DNA binding activity. In conclusion, I3C could promote the reverse process of liver fibrosis in vivo and induce apoptosis of activated HSC in vitro, which indicates the use of I3C as a potential therapeutic agent in liver fibrosis treatment.

  8. Hepatic uptake of (TH)retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

    SciTech Connect

    Blomhoff, R.; Norum, K.R.; Berg, T.

    1985-11-05

    We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.

  9. Oxidative Stress and Hepatic Stellate Cells: A PARADOXICAL RELATIONSHIP

    PubMed Central

    Gandhi, Chandrashekhar R.

    2016-01-01

    In physiology, reactive oxygen species (ROS) are produced by most cells for normal function and as a defense mechanism against foreign particles, microbes and viruses. Hepatic macrophages (Kupffer cells), sinusoidal endothelial cells, hepatocytes and hepatic stellate cells (HSCs) are all capable of generating ROS in physiology and pathology. ROS are also produced by infiltrating inflammatory cells during acute and chronic liver injury. Increased levels of ROS have been implicated in apoptotic/necrotic death of hepatocytes, and liver failure. In contrast to causing injury to hepatocytes, ROS and lipid peroxidation products induce transdifferentiation of the quiescent HSCs into an activated highly proliferative myofibroblast-like phenotype. ROS and lipid peroxidation products also stimulate the synthesis of extracellular matrix (ECM) by activated HSCs. Deposition of excessive amounts of ECM is the primary mechanism of fibrosis and cirrhosis of the liver, and interactions between ROS and HSCs appear to play a major role in this pathology. Although these findings suggest that HSCs are resistant to the injurious actions of ROS, there is compelling evidence demonstrating ROS-induced death of activated HSCs. Detailed mechanistic understanding of such paradoxical interactions between ROS and HSCs will be critical for developing therapies for chronic fibrotic liver disease.

  10. Curcumin-induced recovery from hepatic injury involves induction of apoptosis of activated hepatic stellate cells.

    PubMed

    Priya, S; Sudhakaran, P R

    2008-10-01

    Hepatic stellate cells (HSCs) undergo activation and transdifferentiation to myofibroblast like cells in liver injury, leading to liver fibrosis. During recovery from injury, activated HSCs may either revert back to quiescent state or undergo apoptosis or both. In the present study, we have examined whether recovery from hepatic injury involves apoptosis of activated HSCs and tested whether curcumin (the yellow pigment from Curcuma longa Linn.) promotes recovery from hepatic injury by inducing apoptosis of these cells. Hepatic injury was induced by CCl4 and apoptosis was studied in HSCs isolated from liver by MTT assay, DNA fragmentation, and DAPI and annexin staining. Hepatic recovery was assessed by measuring hepatic marker activities, such as serum GOT, GPT and protein. Hepatic recovery occurred within 4 weeks after inducing injury in untreated control, whereas curcumin treatment caused hepatic recovery within 2 weeks, as evidenced by the reduction of hepatic marker activities to near normal levels. HSCs isolated from liver of animals treated with curcumin showed maximum apoptotic marker activities in 2nd week, whereas in HSCs from untreated control recovering from injury, maximum apoptosis was observed in 4th week. Induction of apoptosis in vivo during hepatic recovery was also suggested by increase in caspase-3 activity. Treatment of isolated HSCs in culture with curcumin caused apoptosis during later stages confirming that curcumin induced apoptosis of activated HSCs and not in unactivated quiescent HSCs. These results suggested that hepatoprotective effect of curcumin causing recovery from injury involved apoptosis of activated HSCs. PMID:19069843

  11. Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

    PubMed

    Lu, Jing; Chen, Baian; Li, Shengli; Sun, Quan

    2014-06-01

    Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease.

  12. Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

    PubMed

    Lu, Jing; Chen, Baian; Li, Shengli; Sun, Quan

    2014-06-01

    Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease. PMID:24735816

  13. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

    SciTech Connect

    Parekkadan, Biju; Poll, Daan van; Megeed, Zaki; Kobayashi, Naoya; Tilles, Arno W.; Berthiaume, Francois; Yarmush, Martin L.

    2007-11-16

    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-{alpha} abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

  14. Mechanisms of liver fibrosis associated with experimental Fasciola hepatica infection: roles of Fas2 proteinase and hepatic stellate cell activation.

    PubMed

    Marcos, Luis A; Terashima, Angélica; Yi, Pedro; Andrade, Roy; Cubero, Francisco J; Albanis, Efsevia; Gotuzzo, Eduardo; Espinoza, Jose R; Friedman, Scott L

    2011-02-01

    We have evaluated the possible mechanisms of liver fibrosis caused by Fasciola hepatica in an animal model and in culture using immortalized human stellate cells. Liver biopsies of F. hepatica-infected rats were performed at wk 8 and 16. Serum-starved LX-2 cells, a human stellate cell line, were exposed to increasing concentrations of Fas2 antigen. The expression of key fibrosis-related genes was evaluated by qRT-PCR. There was a significant correlation between fibrogenic gene expression and both intensity and duration of infection. LX-2 cells exposed to Fas2 showed progressively increased expression of mRNAs for Collagen I, alpha-smooth muscle-actin, platelet-derived growth factor beta receptor, and tissue inhibitor of metalloproteinase II; inhibition of Fas2 cysteine proteinase activity by E-64 abrogated these increases, suggesting that the protease activity of Fas2 is involved in fibrogenic stimulation. In summary, F. hepatica infection is associated with up-regulation of mRNAs associated with hepatic fibrogenesis in vivo and in activated hepatic stellate cells.

  15. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells

    PubMed Central

    Siegmund, Sören V.; Schlosser, Monika; Schildberg, Frank A.; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A.; Strassburg, Christian P.; Schwabe, Robert F.

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  16. Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells.

    PubMed

    Lian, Naqi; Jiang, Yuanyuan; Zhang, Feng; Jin, Huanhuan; Lu, Chunfeng; Wu, Xiafei; Lu, Yin; Zheng, Shizhong

    2015-07-01

    Accumulating evidence indicates that Hedgehog (Hh) signaling becomes activated in chronic liver injury and plays a role in the pathogenesis of hepatic fibrosis. Hepatic stellate cells (HSCs) are Hh-responsive cells and activation of the Hh pathway promotes transdifferentiation of HSCs into myofibroblasts. Targeting Hh signaling may be a novel therapeutic strategy for treatment of liver fibrosis. We previously reported that curcumin has potent antifibrotic effects in vivo and in vitro, but the underlying mechanisms are not fully elucidated. This study shows that curcumin downregulated Patched and Smoothened, two key elements in Hh signaling, but restored Hhip expression in rat liver with carbon tetrachloride-induced fibrosis and in cultured HSCs. Curcumin also halted the nuclear translocation, DNA binding, and transcription activity of Gli1. Moreover, the Hh signaling inhibitor cyclopamine, like curcumin, arrested the cell cycle, induced mitochondrial apoptosis, reduced fibrotic gene expression, restored lipid accumulation, and inhibited invasion and migration in HSCs. However, curcumin's effects on cell fate and fibrogenic properties of HSCs were abolished by the Hh pathway agonist SAG. Furthermore, curcumin and cyclopamine decreased intracellular levels of adenosine triphosphate and lactate, and inhibited the expression and/or function of several key molecules controlling glycolysis. However, SAG abrogated the curcumin effects on these parameters of glycolysis. Animal data also showed that curcumin downregulated glycolysis-regulatory proteins in rat fibrotic liver. These aggregated data therefore indicate that curcumin modulated cell fate and metabolism by disrupting the Hh pathway in HSCs, providing novel molecular insights into curcumin reduction of HSC activation.

  17. Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells

    PubMed Central

    YOSHIZATO, Katsutoshi; THUY, Le Thi Thanh; SHIOTA, Goshi; KAWADA, Norifumi

    2016-01-01

    Cytoglobin (CYGB), a new member of the globin family, was discovered in 2001 as a protein associated with stellate cell activation (stellate cell activation-associated protein [STAP]). Knowledge of CYGB, including its crystal, gene, and protein structures as well as its physiological and pathological importance, has increased progressively. We investigated the roles of oxygen (O2)-binding CYGB as STAP in hepatic stellate cells (HSCs) to understand the part played by this protein in their pathophysiological activities. Studies involving CYGB-gene-deleted mice have led us to suppose that CYGB functions as a regulator of O2 homeostasis; when O2 homeostasis is disrupted, HSCs are activated and play a key role(s) in hepatic fibrogenesis. In this review, we discuss the rationale for this hypothesis. PMID:26972599

  18. HEPATIC STELLATE CELL LIPID DROPLETS: A SPECIALIZED LIPID DROPLET FOR RETINOID STORAGE

    PubMed Central

    Blaner, William S.; O’Byrne, Sheila M.; Wongsiriroj, Nuttaporn; Kluwe, Johannes; D’Ambrosio, Diana; Jiang, Hongfeng; Schwabe, Robert F.; Hillman, Elizabeth M.C.; Piantedosi, Roseann; Libien, Jenny

    2009-01-01

    The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease. PMID:19071229

  19. Depletion of β-arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway.

    PubMed

    Sun, Wu-Yi; Song, Yang; Hu, Shan-Shan; Wang, Qing-Tong; Wu, Hua-Xun; Chen, Jing-Yu; Wei, Wei

    2013-05-01

    β-Arrestins are multifunctional adaptor proteins. Recently, some new roles of β-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of β-arrestin1, β-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of β-arrestin2 in the hepatic tissues, but not β-arrestin1. Further, as hepatic fibrosis worsens, β-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β-arrestin1 and β-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of β-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β-arrestin2 mRNA (siβ-arrestin2) into HSCs led to a 68% and 70% reduction of β-arrestin2 mRNA and protein expression, respectively. siβ-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siβ-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The

  20. Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver

    PubMed Central

    Friedman, Scott L.

    2010-01-01

    The hepatic stellate cell has surprised and engaged physiologists, pathologists, and hepatologists for over 130 years, yet clear evidence of its role in hepatic injury and fibrosis only emerged following the refinement of methods for its isolation and characterization. The paradigm in liver injury of activation of quiescent vitamin A-rich stellate cells into proliferative, contractile, and fibrogenic myofibroblasts has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression. But this simple paradigm has now yielded to a remarkably broad appreciation of the cell's functions not only in liver injury, but also in hepatic development, regeneration, xenobiotic responses, intermediary metabolism, and immunoregulation. Among the most exciting prospects is that stellate cells are essential for hepatic progenitor cell amplification and differentiation. Equally intriguing is the remarkable plasticity of stellate cells, not only in their variable intermediate filament phenotype, but also in their functions. Stellate cells can be viewed as the nexus in a complex sinusoidal milieu that requires tightly regulated autocrine and paracrine cross-talk, rapid responses to evolving extracellular matrix content, and exquisite responsiveness to the metabolic needs imposed by liver growth and repair. Moreover, roles vital to systemic homeostasis include their storage and mobilization of retinoids, their emerging capacity for antigen presentation and induction of tolerance, as well as their emerging relationship to bone marrow-derived cells. As interest in this cell type intensifies, more surprises and mysteries are sure to unfold that will ultimately benefit our understanding of liver physiology and the diagnosis and treatment of liver disease. PMID:18195085

  1. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

    PubMed

    Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-10-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.

  2. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6[S

    PubMed Central

    Eichmann, Thomas O.; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G.; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-01-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. PMID:26330055

  3. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

    PubMed

    Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-10-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. PMID:26330055

  4. Cytoglobin as a Marker of Hepatic Stellate Cell-derived Myofibroblasts

    PubMed Central

    Kawada, Norifumi

    2015-01-01

    Myofibroblasts play important roles in inflammation, fibrosis and tumorigenesis in chronically inflamed liver. Liver myofibroblasts originate from hepatic stellate cells, portal fibroblasts or mesothelial cells, and they are localized in and around fibrotic septum and portal tracts. Liver myofibroblasts are the source of extracellular matrix materials, including type I collagen and multiple fibrogenic growth factors, such as transforming growth factor-β and vascular endothelial growth factor. Although a detailed characterization of the function of individual myofibroblasts has not been conducted, owing to the lack of appropriate cell markers, recent lineage-tracing technology has revealed the limited contribution of myofibroblasts that are derived from portal fibroblasts to various types of liver fibrosis, as compared with the contribution of hepatic stellate cells. In addition, cytoglobin, which is the fourth globin in mammals and function as a local gas sensor, provides a new perspective on the involvement of stellate cells in fibrosis and carcinogenesis, possibly through its anti-oxidative properties and is a promising new marker that discriminates between myofibroblasts derived from stellate cells and those from portal fibroblasts. PMID:26617531

  5. Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression

    PubMed Central

    Soliman, Elwy M.; Rodrigues, Michele Angela; Gomes, Dawidson Assis; Sheung, Nina; Yu, Jin; Amaya, Maria Jimina; Nathanson, Michael H.; Dranoff, Jonathan A.

    2010-01-01

    Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca2+ signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca2+ signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca2+ chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca2+ signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca2+ signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca2+ signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca2+ signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca2+ are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis. PMID:19131107

  6. Canonical Wnt signaling maintains the quiescent stage of hepatic stellate cells

    SciTech Connect

    Kordes, Claus Sawitza, Iris; Haeussinger, Dieter

    2008-02-29

    It is well known that hepatic stellate cells (HSC) develop into cells, which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that {beta}-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119, an inhibitor of the glycogen synthase kinase 3{beta}, led to reduced {beta}-catenin phosphorylation, induced nuclear translocation of {beta}-catenin, elevated glutamine synthetase production, impeded synthesis of {alpha}-smooth muscle actin and Wnt5a, but promoted the expression of glial fibrillary acidic protein, Wnt10b, and paired-like homeodomain transcription factor 2c. In addition, canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that {beta}-catenin-dependent Wnt signaling maintains the quiescent state of HSC and, similar to stem and progenitor cells, influences their developmental fate.

  7. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line.

    PubMed

    Miyamae, Yusaku; Nishito, Yukina; Nakai, Naomi; Nagumo, Yoko; Usui, Takeo; Masuda, Seiji; Kambe, Taiho; Nagao, Masaya

    2016-08-12

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. PMID:27270032

  8. Inhibitory effects of capsaicin on hepatic stellate cells and liver fibrosis.

    PubMed

    Yu, Fu-Xiang; Teng, Yin-Yan; Zhu, Qian-Dong; Zhang, Qi-Yu; Tang, Yin-He

    2014-10-01

    Hepatic stellate cells (HSCs) play an important role in the process of liver fibrosis. In this study, we investigated the inhibitory effects of capsaicin on HSCs and liver fibrosis. Cultured HSCs were incubated with various concentrations of capsaicin. Cell proliferation was examined using a cell counting kit. Production of hydrogen peroxide was determined using a 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay. The mRNA and protein expression of target genes was analyzed by reverse transcription PCR and Western blot analysis, respectively. Cell apoptosis was evaluated by annexin V-FITC and propidium iodide (PI) costaining followed by flow cytometric analysis. A CCl4 rat liver fibrosis model was used to assess in vivo effects of capsaicin by histological examination and measurement of liver fibrosis markers, including hydroxyproline content, serum type III collagen, and hyaluronic acid (HA) levels. Our results show that capsaicin dose-dependently inhibited cell proliferation, suppressed cell activation, and decreased hydrogen peroxide production in cultured HSCs. Capsaicin reduced the mRNA levels of tissue inhibitors of metalloproteinase 1 (TIMP-1) and transforming growth factor-β1 (TGF-β1) in HSCs. Moreover, capsaicin-induced cell apoptosis was associated with increased expression of Bax, cytochrome c (cyt c), and caspase-3, but reduced levels of Bcl-2. The animal studies further revealed that capsaicin efficiently reduced the extent of liver fibrosis, inhibited HSC proliferation, and promoted cell apoptosis. Our findings suggest that capsaicin might inhibit fibrogenesis by inhibiting the activities of HSCs.

  9. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

    PubMed Central

    Bataller, Ramón; Schwabe, Robert F.; Choi, Youkyung H.; Yang, Liu; Paik, Yong Han; Lindquist, Jeffrey; Qian, Ting; Schoonhoven, Robert; Hagedorn, Curt H.; Lemasters, John J.; Brenner, David A.

    2003-01-01

    Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II–induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox–/– mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox–/– mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47phox–/– mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis. PMID:14597764

  10. Hepatic Stellate Cells and microRNAs in Pathogenesis of Liver Fibrosis

    PubMed Central

    Kitano, Mio; Bloomston, P. Mark

    2016-01-01

    microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by either blocking translation or inducing degradation of target mRNA. miRNAs play essential roles in diverse biological and pathological processes, including development of hepatic fibrosis. Hepatic stellate cells (HSCs) play a central role in development of hepatic fibrosis and there are intricate regulatory effects of miRNAs on their activation, proliferation, collagen production, migration, and apoptosis. There are multiple differentially expressed miRNAs in activated HSCs, and in this review we aim to summarize current data on miRNAs that participate in the development of hepatic fibrosis. Based on this review, miRNAs may serve as biomarkers for diagnosis of liver disease, as well as markers of disease progression. Most importantly, dysregulated miRNAs may potentially be targeted by novel therapies to treat and reverse progression of hepatic fibrosis. PMID:26999230

  11. EPAC activation inhibits acetaldehyde-induced activation and proliferation of hepatic stellate cell via Rap1.

    PubMed

    Yang, Yan; Yang, Feng; Wu, Xiaojuan; Lv, Xiongwen; Li, Jun

    2016-05-01

    Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 μmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde-induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me-cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde-induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde-induced HSCs via Rap1. PMID:26854595

  12. Autophagy Releases Lipid That Promotes Fibrogenesis by Activated Hepatic Stellate Cells in Mice and in Human Tissues

    PubMed Central

    HERNÁNDEZ–GEA, VIRGINIA; GHIASSI–NEJAD, ZAHRA; ROZENFELD, RAPHAEL; GORDON, RONALD; FIEL, MARIA ISABEL; YUE, ZHENYU; CZAJA, MARK J.; FRIEDMAN, SCOTT L.

    2012-01-01

    BACKGROUND & AIMS The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells. METHODS We analyzed hepatic stellate cells from C57BL/6 wild-type, Atg7F/F, and Atg7F/F-GFAP-Cre mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl4 or thioacetamide (TAA); liver tissues and stellate cells were analyzed. Autophagy was blocked in fibrogenic cells from liver and other tissues using small interfering RNAs against Atg5 or Atg7 and chemical antagonists. Human pulmonary fibroblasts were isolated from samples of lung tissue from patients with idiopathic pulmonary fibrosis or from healthy donors. RESULTS In mice, induction of liver injury with CCl4 or TAA increased levels of autophagy. We also observed features of autophagy in activated stellate cells within injured human liver tissue. Loss of autophagic function in cultured mouse stellate cells and in mice following injury reduced fibrogenesis and matrix accumulation; this effect was partially overcome by providing oleic acid as an energy substrate. Autophagy also regulated expression of fibrogenic genes in embryonic, lung, and renal fibroblasts. CONCLUSIONS Autophagy of activated stellate cells is required for hepatic fibrogenesis in mice. Selective reduction of autophagic activity in fibrogenic cells in liver and other tissues might be used to treat patients with fibrotic diseases. PMID:22240484

  13. Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion*

    PubMed Central

    Ramezani-Moghadam, Mehdi; Wang, Jianhua; Ho, Vikki; Iseli, Tristan J.; Alzahrani, Badr; Xu, Aimin; Van der Poorten, David; Qiao, Liang; George, Jacob; Hebbard, Lionel

    2015-01-01

    Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin. PMID:25575598

  14. Metadoxine prevents damage produced by ethanol and acetaldehyde in hepatocyte and hepatic stellate cells in culture.

    PubMed

    Gutiérrez-Ruiz, M C; Bucio, L; Correa, A; Souza, V; Hernández, E; Gómez-Quiroz, L E; Kershenobich, D

    2001-11-01

    Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to improve liver function tests in alcoholic patients. In the present work we have investigated the effect of metadoxine on some parameters of cellular damage in hepatocytes and hepatic stellate cells in culture treated with ethanol and acetaldehyde. HepG2 and CFSC-2G cells were treated with 50 mM ethanol or 175 microM acetaldehyde as initial concentration in the presence or absence of 10 microg ml(-1) of metadoxine. Twenty-four hours later reduced and oxidized glutathione content, lipid peroxidation damage, collagen secretion and IL-6, IL-8 and TNF- alpha secretion were determined. Our results suggest that metadoxine prevents glutathione depletion and the increase in lipid peroxidation damage caused by ethanol and acetaldehyde in HepG2 cells. In hepatic stellate cells, metadoxine prevents the increase in collagen and attenuated TNF- alpha secretion caused by acetaldehyde. Thus, metadoxine could be useful in preventing the damage produced in early stages of alcoholic liver disease as it prevents the redox imbalance of the hepatocytes and prevents TNF- alpha induction, one of the earliest events in hepatic damage. PMID:11712874

  15. Lactoferrin protects against chemical-induced rat liver fibrosis by inhibiting stellate cell activation.

    PubMed

    Tung, Yu-Tang; Tang, Ting-Yu; Chen, Hsiao-Ling; Yang, Shang-Hsun; Chong, Kowit-Yu; Cheng, Winston T K; Chen, Chuan-Mu

    2014-01-01

    Liver diseases, which can be caused by alcohol abuse, chemical intoxication, viral hepatitis infection, and autoimmune disorders, are a significant health issue because they can develop into liver fibrosis and cirrhosis. Lactoferrin (LF), a siderophilic protein with 2 iron-binding sites, has been demonstrated to possess a multitude of biological functions, including antiinflammation, anticancer, and antimicrobial effects, as well as immunomodulatory-enhancing functions. In the current study, we induced hepatotoxicity in rats with dimethylnitrosamine (DMN) to establish a situation that would enable us to evaluate the hepatoprotective effects of LF against hepatic injury. Our results showed that DMN-induced hepatic pathological damage significantly decreased the body weight and liver index, increased the mRNA and protein levels of collagen α-1(I) (ColIα-1) and α-smooth muscle actin, and increased the hydroxyproline content. However, treatment with LF significantly increased body weight and liver index, decreased the mRNA and protein levels of ColIα-1 and α-smooth muscle actin, and suppressed the hydroxyproline content when compared with the DMN-treated group. Liver histopathology also showed that low-dose LF (100mg/kg of body weight) or high-dose LF (300 mg/kg of body weight) could significantly reduce the incidences of liver lesions induced by DMN. These results suggest that the LF exhibits potent hepatoprotection against DMN-induced liver damage in rats and that the hepatoprotective effects of LF may be due to the inhibition of collagen production and to stellate cell activation. PMID:24731632

  16. CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway.

    PubMed

    Saiman, Yedidya; Agarwal, Ritu; Hickman, DaShawn A; Fausther, Michel; El-Shamy, Ahmed; Dranoff, Jonathan A; Friedman, Scott L; Bansal, Meena B

    2013-09-01

    Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension. PMID:23812037

  17. Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

    PubMed

    Birukawa, Naoko Kubo; Murase, Kazuyuki; Sato, Yasushi; Kosaka, Akemi; Yoneda, Akihiro; Nishita, Hiroki; Fujita, Ryosuke; Nishimura, Miyuki; Ninomiya, Takafumi; Kajiwara, Keiko; Miyazaki, Miyono; Nakashima, Yusuke; Ota, Sigenori; Murakami, Yuya; Tanaka, Yasunobu; Minomi, Kenjiro; Tamura, Yasuaki; Niitsu, Yoshiro

    2014-07-18

    Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

  18. Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF-κB and the PI3K/Akt Signaling Pathway.

    PubMed

    Zhang, De-Quan; Sun, Peng; Jin, Quan; Li, Xia; Zhang, Yu; Zhang, Yu-Jing; Wu, Yan-Ling; Nan, Ji-Xing; Lian, Li-Hua

    2016-01-01

    In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t-HSC/Cl-6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t-HSC/Cl-6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS-induced protein expression of α-SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)-κB in LPS-activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF-κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.

  19. TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways

    SciTech Connect

    Fang, Ling Zhan, Shuxiang; Huang, Cheng; Cheng, Xi; Lv, Xiongwen; Si, Hongfang; Li, Jun

    2013-11-01

    TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl{sub 4}-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 mRNA and protein in the fibrotic livers from CCl{sub 4}-treated rats. • Increasing expression of TRPM7 mRNA and protein during HSC activation. • Blockade of TRPM7 inhibited the PDGF-BB induced proliferation of HSC-T6 cells. • Blockade of TRPM7 decreased α-SMA and Col1α1 expressions in activated HSC-T6 cells. • TRPM7 up-regulation contributes to the activation of ERK and AKT pathways.

  20. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

    NASA Astrophysics Data System (ADS)

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-04-01

    Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

  1. Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

    PubMed Central

    2016-01-01

    Fibroblast growth factors (FGFs) are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs). Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis. PMID:27699175

  2. Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

    PubMed Central

    2016-01-01

    Fibroblast growth factors (FGFs) are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs). Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.

  3. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

    PubMed Central

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-01-01

    Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

  4. Thymoquinone attenuates liver fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate cells.

    PubMed

    Bai, Ting; Lian, Li-Hua; Wu, Yan-Ling; Wan, Ying; Nan, Ji-Xing

    2013-02-01

    Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. In the present study, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/Cl-6. T-HSC/Cl-6 cells were treated with TQ (3.125, 6.25 and 12.5μM) prior to LPS (1μg/ml). Our data demonstrated that TQ effectively decreased activated T-HSC/Cl-6 cell viability. TQ significantly attenuated the expression of CD14 and Toll-like receptor 4 (TLR4). TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase-protein kinase B (Akt) phosphorylation. The expression of α-SMA and collagen-I were significantly decreased by TQ. Furthermore, TQ decreased X linked inhibitor of apoptosis (XIAP) and cellular FLIP (c-FLIPL) expression, which are related with the regulation of apoptosis. Furthermore, TQ significantly increased the survival against LPS challenge in d-galactosamine (d-GlaN)-sensitized mice, and decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were in line with in vitro results. Our data demonstrated that TQ attenuates liver fibrosis partially via blocking TLR4 expression and PI3K phosphorylation on the activated HSCs. Therefore, TQ may be a potential candidate for the therapy of hepatic fibrosis.

  5. Suppression of hepatic stellate cell activation by microRNA-29b

    SciTech Connect

    Sekiya, Yumiko; Ogawa, Tomohiro; Yoshizato, Katsutoshi; Ikeda, Kazuo; Kawada, Norifumi

    2011-08-19

    Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.

  6. Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation.

    PubMed

    Sun, Xu; He, Yong; Ma, Tao-Tao; Huang, Cheng; Zhang, Lei; Li, Jun

    2014-03-01

    Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl4-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.

  7. Tetrandrine regulates hepatic stellate cell activation via TAK1 and NF-κB signaling.

    PubMed

    Li, Xia; Jin, Quan; Wu, Yan-Ling; Sun, Peng; Jiang, Shuang; Zhang, Yu; Zhang, De-Quan; Zhang, Yu-Jing; Lian, Li-Hua; Nan, Ji-Xing

    2016-07-01

    We investigated the anti-fibrotic mechanism of tetrandrine, a bisbenzylisoquinoline alkaloid from the Chinese herb, Stephania tetrandra, on the immortalized HSC-T6 rat hepatic stellate cell line. Tetrandrine (0.39-50μM) dose- and time-dependently inhibited HSC-T6 cell viability within 24h and exhibited almost no cytotoxicity at concentrations lower than 6.25μM in the presence of tumor necrosis factor-α (TNF-α). At a much high concentration (50μM), tetrandrine caused fatal cytotoxity in both HSCs and hepatocytes. TNF-α time-dependently increased α-smooth muscle actin (α-SMA) expression, while a lower concentration of tetrandrine (6.25μM) prior to TNF-α treatment reduced the expression of α-SMA and TNFR-1-associated death domain (TRADD). TNF-α treatment induced TGF-β-activated kinase-1 (TAK1) and c-Jun N-terminal kinase (JNK) phosphorylation, which were attenuated by tetrandrine. Furthermore, TNF-α treatment activated nuclear factor-κB (NF-κB) nuclear translocation and IκB-α degradation. Tetrandrine treatment prior to TNF-α reduced nuclear phosphorylated and total NF-κB p65, while the cytosolic IκB-α and NF-κB p65 levels significantly increased. In addition, treatment with only tetrandrine induced the cleavage of caspase-3 and PARP within a range of higher concentrations. Tetrandrine-induced apoptosis was confirmed by the TUNEL assay and flow-cytometric analysis. Treatment with only tetrandrine markedly reduced α-SMA expression, except for at lower concentrations of tetrandrine. A higher concentration of tetrandrine (25μM) induced a significant increase in JNK and extracellular signal-regulated kinase (ERK) phosphorylation, NF-κB nuclear translocation and IκB-α degradation. In conclusion, the anti-fibrogenic effects of tetrandrine on HSCs involved a dosage-dependent signaling pathway, based on the tetrandrine concentration, by regulating TAK1, JNK and NF-κB. The present data provides strong evidence for the anti-fibrotic dosage

  8. The use of nanoparticles to deliver nitric oxide to hepatic stellate cells for treating liver fibrosis and portal hypertension.

    PubMed

    Duong, Hien T T; Dong, Zhixia; Su, Lin; Boyer, Cyrille; George, Jacob; Davis, Thomas P; Wang, Jianhua

    2015-05-20

    Polymeric nanoparticles are designed to transport and deliver nitric oxide (NO) into hepatic stellate cells (HSCs) for the potential treatment of both liver fibrosis and portal hypertension. The nanoparticles, incorporating NO donor molecules (S-nitrosoglutathione compound), are designed for liver delivery, minimizing systemic delivery of NO. The nanoparticles are decorated with vitamin A to specifically target HSCs. We demonstrate, using in vitro and in vivo experiments, that the targeted nanoparticles are taken up specifically by rat primary HSCs and the human HSC cell line accumulating in the liver. When nanoparticles, coated with vitamin A, release NO in liver cells, we find inhibition of collagen I and α-smooth muscle actin (α-SMA), fibrogenic genes associated with activated HSCs expression in primary rat liver and human activated HSCs without any obvious cytotoxic effects. Finally, NO-releasing nanoparticles targeted with vitamin A not only attenuate endothelin-1 (ET-1) which elicites HSC contraction but also acutely alleviates haemodynamic disorders in bile duct-ligated-induced portal hypertension evidenced by decreasing portal pressure (≈20%) and unchanging mean arterial pressure. This study clearly shows, for the first time, the potential for HSC targeted nanoparticle delivery of NO as a treatment for liver diseases with proven efficacy for alleviating both liver fibrosis and portal hypertension.

  9. Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy.

    PubMed

    Zhang, Zhengping; Wang, Chunming; Zha, Yinhe; Hu, Wei; Gao, Zhongfei; Zang, Yuhui; Chen, Jiangning; Zhang, Junfeng; Dong, Lei

    2015-03-24

    Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

  10. Oleoylethanolamide, an endogenous PPAR-α ligand, attenuates liver fibrosis targeting hepatic stellate cells

    PubMed Central

    Chen, Junde; Li, Lei; Zheng, Zihan; Ren, Jie; Qiu, Yan

    2015-01-01

    Oleoylethanolamide (OEA), an endocannabinoid-like molecule, was revealed to modulate lipid metabolism through a peroxisome proliferator-activated receptor-α (PPAR-α) mediated mechanism. In present study, we further investigated the activities and mechanisms of OEA in ameliorating hepatic fibrosis in Sv/129 mice induced by a methionine choline-deficient (MCD) diet or thioacetamide (TAA) treatment. Liver fibrosis development was assessed by Hematoxylin-eosin and Sirius red staining. Treatment with OEA (5 mg/kg/day, intraperitoneal injection, i.p.) significantly attenuated the progress of liver fibrosis in both two experimental animal models by blocking the activation of hepatic stellate cells (HSCs). Gene expression analysis of hepatic tissues indicated that OEA inhibited the expression of α-smooth muscle action (α-SMA) and collagen matrix, fibrosis markers, and genes involved in inflammation and extracellular matrix remodeling. In vitro studies showed that OEA inhibited transforming growth factor β1-stimulated HSCs activation through suppressing Smad2/3 phosphorylation, α-SMA expression and myofibroblast transformation. These improvements could not be observed in PPAR-α knockout mice models with OEA administration, which suggested all the anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating hepatic fibrosis development through the inhibition of HSCs activation in liver and therefore may be a potential therapeutic agent for liver fibrosis. PMID:26729705

  11. Class II HDAC Inhibition Hampers Hepatic Stellate Cell Activation by Induction of MicroRNA-29

    PubMed Central

    Mannaerts, Inge; Eysackers, Nathalie; Onyema, Oscar O.; Van Beneden, Katrien; Valente, Sergio; Mai, Antonello; Odenthal, Margarete; van Grunsven, Leo A.

    2013-01-01

    Background The conversion of a quiescent vitamin A storing hepatic stellate cell (HSC) to a matrix producing, contractile myofibroblast-like activated HSC is a key event in the onset of liver disease following injury of any aetiology. Previous studies have shown that class I histone deacetylases (HDACs) are involved in the phenotypical changes occurring during stellate cell activation in liver and pancreas. Aims In the current study we investigate the role of class II HDACs during HSC activation. Methods We characterized the expression of the class II HDACs freshly isolated mouse HSCs. We inhibited HDAC activity by selective pharmacological inhibition with MC1568, and by repressing class II HDAC gene expression using specific siRNAs. Results Inhibition of HDAC activity leads to a strong reduction of HSC activation markers α-SMA, lysyl oxidase and collagens as well as an inhibition of cell proliferation. Knock down experiments showed that HDAC4 contributes to HSC activation by regulating lysyl oxidase expression. In addition, we observed a strong up regulation of miR-29, a well-known anti-fibrotic miR, upon treatment with MC1568. Our in vivo work suggests that a successful inhibition of class II HDACs could be promising for development of future anti-fibrotic compounds. Conclusions In conclusion, the use of MC1568 has enabled us to identify a role for class II HDACs regulating miR-29 during HSC activation. PMID:23383282

  12. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation

    PubMed Central

    Caliari, Steven R.; Perepelyuk, Maryna; Cosgrove, Brian D.; Tsai, Shannon J.; Lee, Gi Yun; Mauck, Robert L.; Wells, Rebecca G.; Burdick, Jason A.

    2016-01-01

    Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics. PMID:26906177

  13. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation

    NASA Astrophysics Data System (ADS)

    Caliari, Steven R.; Perepelyuk, Maryna; Cosgrove, Brian D.; Tsai, Shannon J.; Lee, Gi Yun; Mauck, Robert L.; Wells, Rebecca G.; Burdick, Jason A.

    2016-02-01

    Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

  14. Xanthohumol uptake and intracellular kinetics in hepatocytes, hepatic stellate cells, and intestinal cells.

    PubMed

    Wolff, Horst; Motyl, Magdalena; Hellerbrand, Claus; Heilmann, Jörg; Kraus, Birgit

    2011-12-28

    Xanthohumol (XN) is the major prenylated chalcone of hops and hence an ingredient of beer. Despite many advances in understanding of the pharmacology of XN, one largely unresolved issue is its low bioavailability in the human organism. Also, not much is known about its actual concentrations and pharmacokinetics in liver and intestinal cells. Therefore, the uptake, intracellular distribution, and kinetics of XN were studied in various cell types, namely, hepatocellular carcinoma cells (HuH-7), hepatic stellate cells (HSC), primary cultured hepatocytes, and colorectal adenocarcinoma cells (Caco-2). Fluorescent microscopy allowed for the first time visualization and tracing of the uptake and intracellular distribution of XN. A rapid accumulation of XN concentrations that were up to >60-fold higher than the concentration present in the ambient culture medium was observed. Fluorescence recovery after photobleaching experiments revealed that most XN molecules are bound to cellular proteins, which may alter properties of cellular factors. PMID:22088086

  15. Hedgehog-mediated paracrine interaction between hepatic stellate cells and marrow-derived mesenchymal stem cells

    SciTech Connect

    Lin Nan Tang Zhaofeng; Deng Meihai; Zhong Yuesi; Lin Jizong; Yang Xuhui; Xiang Peng; Xu Ruiyun

    2008-07-18

    During liver injury, bone marrow-derived mesenchymal stem cells (MSCs) can migrate and differentiate into hepatocytes. Hepatic stellate cell (SC) activation is a pivotal event in the development of liver fibrosis. Therefore, we hypothesized that SCs may play an important role in regulating MSC proliferation and differentiation through the paracrine signaling pathway. We demonstrate that MSCs and SCs both express hedgehog (Hh) pathway components, including its ligands, receptors, and target genes. Transwell co-cultures of SCs and MSCs showed that the SCs produced sonic hedgehog (Shh), which enhanced the proliferation and differentiation of MSCs. These findings demonstrate that SCs indirectly modulate the activity of MSCs in vitro via the Hh pathway, and provide a plausible explanation for the mechanisms of transplanted MSCs in the treatment of liver fibrosis.

  16. Role of ethanol in the regulation of hepatic stellate cell Function

    PubMed Central

    Wang, Jian-Hua; Batey, Robert G; George, Jacob

    2006-01-01

    Evidence has accumulated to suggest an important role of ethanol and/or its metabolites in the pathogenesis of alcohol-related liver disease. In this review, the fibrogenic effects of ethanol and its metabolites on hepatic stellate cells (HSCs) are discussed. In brief, ethanol interferes with retinoid metabolism and its signaling, induces the release of fibrogenic cytokines such as transforming growth factor β-1 (TGFβ-1) from HSCs, up-regulates the gene expression of collagen I and enhances type I collagen protein production by HSCs. Ethanol further perpetuates an activated HSC phenotype through extracellular matrix remodeling. The underlying pathophysiologic mechanisms by which ethanol exerts these pro-fibrogenic effects on HSCs are reviewed. PMID:17109512

  17. Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines

    PubMed Central

    2012-01-01

    Background Crosstalk between malignant hepatocytes and the surrounding peritumoral stroma is a key modulator of hepatocarcinogenesis and therapeutic resistance. To examine the chemotherapy resistance of these two cellular compartments in vitro, we evaluated a well-established hepatic tumor cell line, HepG2, and an adult hepatic stellate cell line, LX2. The aim was to compare the chemosensitization potential of arsenic trioxide (ATO) in combination with sorafenib or fluorouracil (5-FU), in both hepatic tumor cells and stromal cells. Methods Cytotoxicity of ATO, 5-FU, and sorafenib, alone and in combination against HepG2 cells and LX2 cells was measured by an automated high throughput cell-based proliferation assay. Changes in survival and apoptotic signaling pathways were analyzed by flow cytometry and western blot. Gene expression of the 5-FU metabolic enzyme, thymidylate synthase, was analyzed by real time PCR. Results Both HepG2 and LX2 cell lines were susceptible to single agent sorafenib and ATO at 24 hr (ATO IC50: 5.3 μM in LX2; 32.7 μM in HepG2; Sorafenib IC50: 11.8 μM in LX2; 9.9 μM in HepG2). In contrast, 5-FU cytotoxicity required higher concentrations and prolonged (48–72 hr) drug exposure. Concurrent ATO and 5-FU treatment of HepG2 cells was synergistic, leading to increased cytotoxicity due in part to modulation of thymidylate synthase levels by ATO. Concurrent ATO and sorafenib treatment showed a trend towards increased HepG2 cytotoxicity, possibly due to a significant decrease in MAPK activation in comparison to treatment with ATO alone. Conclusions ATO differentially sensitizes hepatic tumor cells and adult hepatic stellate cells to 5-FU and sorafenib. Given the importance of both of these cell types in hepatocarcinogenesis, these data have implications for the rational development of anti-cancer therapy combinations for the treatment of hepatocellular carcinoma (HCC). PMID:22963400

  18. Neferine inhibits cultured hepatic stellate cell activation and facilitates apoptosis: A possible molecular mechanism.

    PubMed

    Ding, Hui; Shi, Jinghong; Wang, Ying; Guo, Jia; Zhao, Juhui; Dong, Lei

    2011-01-10

    Neferine is a major alkaloid component of "Lian Zi Xin", embryos of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae. Previous studies have shown that neferine has an inhibitory effect on pulmonary fibrosis through its anti-inflammatory and anti-oxidative activities and inhibition of cytokines and NF-κB. However, it is unknown whether neferine also has an inhibitory effect on liver fibrosis through inhibition of TGF-β1 and collagen I and facilitation of apoptosis of hepatic stellate cells. This study examined the effects of neferine on cultured hepatic stellate (HSC-T6) cells and explored its possible action mechanisms by means of MTT assay, enzyme-linked immunosorbent assay, flow-cytometric annexin V-PI assay and Hoechst 33258 staining, as well as real-time PCR and western blotting. The results showed that neferine administration (2, 4, 6, 8 and 10μmol/l) significantly decreased the TGF-β1 and collagen I produced in HSC-T6 cells, and increased the HSC-T6 cell apoptosis in a dose-dependent manner. Neferine treatment for 48h at concentrations of 6 and 10μmol/l significantly increased Bax and caspase 3 mRNAs and proteins, and reduced Bcl2 and alpha-smooth muscle actin (α-SMA) mRNAs and proteins. Our data indicate that neferine efficiently inhibits cultured HSC-T6 cell activation and induces apoptosis by increasing Bax and caspase 3 expression via the mitochondrial pathway.

  19. Neferine inhibits cultured hepatic stellate cell activation and facilitates apoptosis: A possible molecular mechanism.

    PubMed

    Ding, Hui; Shi, Jinghong; Wang, Ying; Guo, Jia; Zhao, Juhui; Dong, Lei

    2011-01-10

    Neferine is a major alkaloid component of "Lian Zi Xin", embryos of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae. Previous studies have shown that neferine has an inhibitory effect on pulmonary fibrosis through its anti-inflammatory and anti-oxidative activities and inhibition of cytokines and NF-κB. However, it is unknown whether neferine also has an inhibitory effect on liver fibrosis through inhibition of TGF-β1 and collagen I and facilitation of apoptosis of hepatic stellate cells. This study examined the effects of neferine on cultured hepatic stellate (HSC-T6) cells and explored its possible action mechanisms by means of MTT assay, enzyme-linked immunosorbent assay, flow-cytometric annexin V-PI assay and Hoechst 33258 staining, as well as real-time PCR and western blotting. The results showed that neferine administration (2, 4, 6, 8 and 10μmol/l) significantly decreased the TGF-β1 and collagen I produced in HSC-T6 cells, and increased the HSC-T6 cell apoptosis in a dose-dependent manner. Neferine treatment for 48h at concentrations of 6 and 10μmol/l significantly increased Bax and caspase 3 mRNAs and proteins, and reduced Bcl2 and alpha-smooth muscle actin (α-SMA) mRNAs and proteins. Our data indicate that neferine efficiently inhibits cultured HSC-T6 cell activation and induces apoptosis by increasing Bax and caspase 3 expression via the mitochondrial pathway. PMID:20969858

  20. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway

    PubMed Central

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  1. Tetramethylpyrazine Inhibits Activation of Hepatic Stellate Cells through Hedgehog Signaling Pathways In Vitro

    PubMed Central

    Hu, Jue; Cao, Gang; Wu, Xin; Cai, Hao; Cai, Baochang

    2015-01-01

    Background and Aim. Tetramethylpyrazine (TMP), a major alkaloid isolated from Ligusticum chuanxiong, has been reported in hepatic fibrosis models. However, the action mechanism remains unclear. In the present study, effects of tetramethylpyrazine (TMP) against hepatic stellate cell (HSC) activation as well as the possible mechanisms were evaluated. Methods. Western blot assay was used to detect TMP effects on protein expression of Smo, Patched, Hhip, and Gli and to investigate the effects of TMP on Cyclin D1, Cyclin E1, CDK2, Bcl-2, Bax, and caspase expression with cyclopamine supplementation. Results. Our results showed that TMP significantly inhibits the expression of Cyclin D1, Cyclin E1, and Cyclin-dependent kinase CDK2 and changes the HSC cycle by inhibiting the proliferation of HSC. Moreover, TMP has also been shown to decrease the expression of Bcl-2 and increase the expression of Bax in HSC-T6 cells. Furthermore, TMP can inhibit the expression of connective tissue growth factor (CTGF), and the inhibitory effect was intensified after the application of joint treatment with TMP and cyclopamine. Conclusion. TMP may be an effective Hh signaling pathway inhibitor for hepatic fibrosis treatment. PMID:26380286

  2. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway.

    PubMed

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  3. Sorafenib induces autophagic cell death and apoptosis in hepatic stellate cell through the JNK and Akt signaling pathways.

    PubMed

    Hao, Huiyao; Zhang, Di; Shi, Junli; Wang, Yan; Chen, Lei; Guo, Yongze; Ma, Junji; Jiang, Xiaoyu; Jiang, Huiqing

    2016-03-01

    Increasing hepatic stellate cell (HSC) death is an attractive approach for limiting liver fibrosis. We investigated the molecular mechanisms underlying the effects of sorafenib on HSCs. LX2 cells were incubated with sorafenib and a variety of inhibitors of apoptosis, autophagy, and necrosis. Electron microscopy was used to observe autophagosomes. Inhibitors and siRNA were used to examine the role of the Akt/mTOR/p70S6K and JNK pathways. Ultrastructural analysis revealed that rat HSCs treated with 5 μmol/l sorafenib accumulated residual digested material and empty or autophagic vacuoles. Incubating LX2 cells with lysosomal protease inhibitors increased the accumulation of LC3-II, indicating that sorafenib enhances autophagic flux in HSCs. Autophagy may precede apoptosis. Lower concentrations of sorafenib and a shorter treatment time resulted in the dominance of autophagic cell death over apoptosis. Further analysis showed that Beclin 1 is inactivated by the caspases induced by sorafenib during apoptosis. Inhibition of autophagy in LX2 cells using 3-methyladenine treatment or siRNA-mediated knockdown of Atg5 resulted in a marked increase in apoptosis. Finally, sorafenib induced programmed cell death by attenuation and activation of Akt/mTOR/p70S6K and JNK signaling. Sorafenib-induced cell death is mediated by both autophagy and apoptosis. Elucidation of the signaling pathways activated by sorafenib could potentially lead to novel antifibrosis therapies for chronic liver diseases. PMID:26629768

  4. Hop bitter acids exhibit anti-fibrogenic effects on hepatic stellate cells in vitro.

    PubMed

    Saugspier, Michael; Dorn, Christoph; Thasler, Wolfgang E; Gehrig, Manfred; Heilmann, Jörg; Hellerbrand, Claus

    2012-04-01

    Female inflorescences of the hop plant Humulus lupulus L. contain a variety of secondary metabolites with bitter acids (BA) as quantitatively dominating secondary metabolites. The use of hops in beer brewing has a long history due to the antibacterial effects of the BA and their typical bitter taste. Furthermore, hop cones are used in traditional medicine and for pharmaceutical purposes. Recent studies indicate that BA may affect activity of the transcription factor NFκB. NFκB plays a key role in the activation process of hepatic stellate cells (HSC), which is the key event of hepatic fibrosis. The aim of this study was to investigate the effect of BA on HSC (activation) and their potential to inhibit molecular processes involved in the pathogenesis of hepatic fibrosis. HSC were isolated from murine and human liver tissue and incubated with a characterized fraction of bitter acids purified from a CO(2) hop extract. At a concentration of 25μg/ml BA started to induce LDH leakage. Already at lower concentrations BA lead to a dose dependent inhibition of HSC proliferation and inhibited IκB-α-phosphorylation, nuclear p65 translocation and binding activity in a dose dependent way (up to 10μg/ml). Accordingly, the same BA-doses inhibited the expression of pro-inflammatory and NFκB regulated genes as MCP-1 and RANTES, but did not affect expression of genes not related to NFκB signaling. In addition to the effect on activated HSC, BA inhibited the in vitro activation process of freshly isolated HSC as evidenced by delayed expression of collagen I and α-SMA mRNA and protein. Together, these findings indicate that BA inhibit NFκB activation, and herewith the activation and development of profibrogenic phenotype of HSC. Thus, bitter acids appear as potential functional nutrients for the prevention or treatment hepatic fibrosis in chronic liver disease.

  5. Activation of PPARγ/P53 signaling is required for curcumin to induce hepatic stellate cell senescence

    PubMed Central

    Jin, H; Lian, N; Zhang, F; Chen, L; Chen, Q; Lu, C; Bian, M; Shao, J; Wu, L; Zheng, S

    2016-01-01

    Activation of quiescent hepatic stellate cells (HSCs) is the major event in hepatic fibrogenesis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Although inhibition of cell proliferation and induction of apoptosis are potential strategies to block the activation of HSCs, a better understanding of the senescence of activated HSCs can provide a new therapeutic strategy for prevention and treatment of liver fibrosis. The antioxidant curcumin, a phytochemical from turmeric, has been shown to suppress HSC activation in vitro and in vivo. The current work was aimed to evaluate the effect of curcumin on senescence of activated HSCs and to elucidate the underlying mechanisms. In this study, curcumin promoted the expression of senescence marker Hmga1 in rat fibrotic liver. In addition, curcumin increased the number of senescence-associated β-galactosidase-positive HSCs in vitro. At the same time, curcumin induced HSC senescence by elevating the expression of senescence markers P16, P21 and Hmga1, concomitant with reduced abundance of HSC activation markers α-smooth muscle actin and α1(I)-procollagen in cultured HSCs. Moreover, curcumin affected the cell cycle and telomerase activity. We further demonstrated that P53 pharmacological inhibitor pifithrin-α (PFT-α) or transfection with P53 siRNA abrogated the curcumin-induced HSC senescence in vitro. Meanwhile, curcumin disruption of P53 leading to increased senescence of activated HSCs was further verified in vivo. Further studies indicated that curcumin promoted the expression of P53 through a PPARγ activation-dependent mechanism. Moreover, promoting PPARγ transactivating activity by a PPARγ agonist 15d-PGJ2 markedly enhanced curcumin induction of senescence of activated HSCs. However, the PPARγ antagonist PD68235 eliminated curcumin induction of HSC senescence. Taken together, our results provided a novel insight into the mechanisms underlying curcumin inhibition of HSC

  6. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  7. Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

    PubMed Central

    Sahin, Hacer; Wasmuth, Hermann E.; Kisseleva, Tatiana; Brenner, David A.; Trautwein, Christian; Weiskirchen, Ralf; Scholten, David

    2013-01-01

    Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology. PMID:23437087

  8. Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells.

    PubMed

    Wu, Lin; Mao, Chengqiong; Ming, Xin

    2016-01-01

    Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis and, more recently, from fatty liver diseases. Activation and proliferation of hepatic stellate cells (HSCs) represent a key aspect of fibrogenesis and are associated with progressive reduction of HSC apoptosis. Bcl-x, an antiapoptotic member of Bcl-2 gene family, plays a role in apoptosis regulation in mammalian cells. Through alternative splicing, the Bcl-x gene yields two major protein isoforms with opposing functions, antiapoptotic Bcl-xL and proapoptotic Bcl-xS. This study aimed to investigate the role of Bcl-x and its alternate splicing in HSC apoptosis. The results indicated that the expression of Bcl-xL was dramatically higher than Bcl-2 in activated human HSCs. The relative expression of Bcl-xL over Bcl-xS increased gradually when HSCs were activated in cell culture, which was consistent with the increase in apoptosis resistance of activated HSCs. Redirection of Bcl-x splicing by an antisense oligonucleotide from the antiapoptotic isoform to the proapoptotic isoform induced death of HSCs without other apoptosis stimuli. We conclude that Bcl-x plays a role in regulation of HSC apoptosis and modulation of Bcl-x alternative splicing may become a novel molecular therapy for liver fibrosis. PMID:27579319

  9. Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells

    PubMed Central

    Wu, Lin; Mao, Chengqiong

    2016-01-01

    Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis and, more recently, from fatty liver diseases. Activation and proliferation of hepatic stellate cells (HSCs) represent a key aspect of fibrogenesis and are associated with progressive reduction of HSC apoptosis. Bcl-x, an antiapoptotic member of Bcl-2 gene family, plays a role in apoptosis regulation in mammalian cells. Through alternative splicing, the Bcl-x gene yields two major protein isoforms with opposing functions, antiapoptotic Bcl-xL and proapoptotic Bcl-xS. This study aimed to investigate the role of Bcl-x and its alternate splicing in HSC apoptosis. The results indicated that the expression of Bcl-xL was dramatically higher than Bcl-2 in activated human HSCs. The relative expression of Bcl-xL over Bcl-xS increased gradually when HSCs were activated in cell culture, which was consistent with the increase in apoptosis resistance of activated HSCs. Redirection of Bcl-x splicing by an antisense oligonucleotide from the antiapoptotic isoform to the proapoptotic isoform induced death of HSCs without other apoptosis stimuli. We conclude that Bcl-x plays a role in regulation of HSC apoptosis and modulation of Bcl-x alternative splicing may become a novel molecular therapy for liver fibrosis. PMID:27579319

  10. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    PubMed

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis. PMID:23525364

  11. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    PubMed

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis.

  12. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver

    PubMed Central

    He, Wenhua; Shi, Feng; Zhou, Zhi-Wei; Li, Bimin; Zhang, Kunhe; Zhang, Xinhua; Ouyang, Canhui; Zhou, Shu-Feng; Zhu, Xuan

    2015-01-01

    NADPH oxidases (NOXs) are a predominant mediator of redox homeostasis in hepatic stellate cells (HSCs), and oxidative stress plays an important role in the pathogenesis of liver fibrosis. Ursolic acid (UA) is a pentacyclic triterpenoid with various pharmacological activities, but the molecular targets and underlying mechanisms for its antifibrotic effect in the liver remain elusive. This study aimed to computationally predict the molecular interactome and mechanistically investigate the antifibrotic effect of UA on oxidative stress, with a focus on NOX4 activity and cross-linked signaling pathways in human HSCs and rat liver. Drug–drug interaction via chemical–protein interactome tool, a server that can predict drug–drug interaction via chemical–protein interactome, was used to predict the molecular targets of UA, and Database for Annotation, Visualization, and Integrated Discovery was employed to analyze the signaling pathways of the predicted targets of UA. The bioinformatic data showed that there were 611 molecular proteins possibly interacting with UA and that there were over 49 functional clusters responding to UA. The subsequential benchmarking data showed that UA significantly reduced the accumulation of type I collagen in HSCs in rat liver, increased the expression level of MMP-1, but decreased the expression level of TIMP-1 in HSC-T6 cells. UA also remarkably reduced the gene expression level of type I collagen in HSC-T6 cells. Furthermore, UA remarkably attenuated oxidative stress via negative regulation of NOX4 activity and expression in HSC-T6 cells. The employment of specific chemical inhibitors, SB203580, LY294002, PD98059, and AG490, demonstrated the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in the regulatory effect of UA on NOX4 activity and expression. Collectively, the antifibrotic effect of UA is partially due to the oxidative stress attenuating effect through manipulating NOX4 activity and expression. The results

  13. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver.

    PubMed

    He, Wenhua; Shi, Feng; Zhou, Zhi-Wei; Li, Bimin; Zhang, Kunhe; Zhang, Xinhua; Ouyang, Canhui; Zhou, Shu-Feng; Zhu, Xuan

    2015-01-01

    NADPH oxidases (NOXs) are a predominant mediator of redox homeostasis in hepatic stellate cells (HSCs), and oxidative stress plays an important role in the pathogenesis of liver fibrosis. Ursolic acid (UA) is a pentacyclic triterpenoid with various pharmacological activities, but the molecular targets and underlying mechanisms for its antifibrotic effect in the liver remain elusive. This study aimed to computationally predict the molecular interactome and mechanistically investigate the antifibrotic effect of UA on oxidative stress, with a focus on NOX4 activity and cross-linked signaling pathways in human HSCs and rat liver. Drug-drug interaction via chemical-protein interactome tool, a server that can predict drug-drug interaction via chemical-protein interactome, was used to predict the molecular targets of UA, and Database for Annotation, Visualization, and Integrated Discovery was employed to analyze the signaling pathways of the predicted targets of UA. The bioinformatic data showed that there were 611 molecular proteins possibly interacting with UA and that there were over 49 functional clusters responding to UA. The subsequential benchmarking data showed that UA significantly reduced the accumulation of type I collagen in HSCs in rat liver, increased the expression level of MMP-1, but decreased the expression level of TIMP-1 in HSC-T6 cells. UA also remarkably reduced the gene expression level of type I collagen in HSC-T6 cells. Furthermore, UA remarkably attenuated oxidative stress via negative regulation of NOX4 activity and expression in HSC-T6 cells. The employment of specific chemical inhibitors, SB203580, LY294002, PD98059, and AG490, demonstrated the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in the regulatory effect of UA on NOX4 activity and expression. Collectively, the antifibrotic effect of UA is partially due to the oxidative stress attenuating effect through manipulating NOX4 activity and expression. The results suggest that

  14. Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

    PubMed Central

    O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

    2014-01-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

  15. Chloroquine improved carbon tetrachloride-induced liver fibrosis through its inhibition of the activation of hepatic stellate cells: role of autophagy.

    PubMed

    He, Wei; Wang, Bin; Yang, Jing; Zhuang, Yun; Wang, Liangzhi; Huang, Xiaodan; Chen, Jianping

    2014-01-01

    Autophagy is involved in the activation of hepatic stellate cells (HSCs), which is the key process of liver fibrosis. We reasoned that chloroquine, based on its ability to inhibit autophagy, might exert beneficial effects in liver fibrosis. Liver fibrosis in rats was induced by carbon tetrachloride (CCl4). Rats were divided into three groups, a normal control group (N group), model group (M group), and chloroquine group (CQ group). Liver fibrosis in the rats was evaluated by hematoxyline-eosin (H&E) and Masson staining. The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB) were determined using an automated biochemistry analyzer. Total hepatic hydroxyproline levels were determined with a kit. The expressions of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) were detected by immunofluorescence staining, and the expressions of LC3-II and p62 were determined by Western blotting. Compared with N group, M group showed impaired liver function, liver fibrosis, increased hydroxyproline content, up-regulated expressions of α-SMA and TGF-β1, which have been reported to be pro-fibrogenic genes in vivo, and increased autophagy flux as indicated by the accumulation of LC3-II and degradation of p62. These changes were attenuated by chloroquine treatment. Chloroquine exerts beneficial effects in CCl4-induced liver fibrosis. The mechanism of action includes inhibition of autophagy pathways and inhibition of activation of HSCs. PMID:25177034

  16. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    SciTech Connect

    Iyer, Soumya C; Kannan, Anbarasu; Gopal, Ashidha; Devaraj, Niranjali; Halagowder, Devaraj

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  17. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage

    PubMed Central

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-01-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (ENKO) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in ENKO mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  18. Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence.

    PubMed

    Yoneda, Akihiro; Sakai-Sawada, Kaori; Niitsu, Yoshiro; Tamura, Yasuaki

    2016-02-01

    Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition.

  19. Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence.

    PubMed

    Yoneda, Akihiro; Sakai-Sawada, Kaori; Niitsu, Yoshiro; Tamura, Yasuaki

    2016-02-01

    Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition. PMID:26812497

  20. Etoposide Induces Apoptosis in Activated Human Hepatic Stellate Cells via ER Stress

    PubMed Central

    Wang, Chen; Zhang, Feng; Cao, Yu; Zhang, Mingming; Wang, Aixiu; Xu, Mingcui; Su, Min; Zhang, Ming; Zhuge, Yuzheng

    2016-01-01

    The activation of hepatic stellate cells (HSCs) plays a vital role in the progression of liver fibrosis, and the induction of HSCs apoptosis may attenuate or reverse fibrogenesis. The therapeutic effects of etoposide(VP-16), a widely used anticancer agent, on HSCs apoptosis and liver fibrosis resolution are still unclear. Here, we report that VP-16 reduced the proliferation of LX-2 cells and led to significantly high levels of apoptosis, as indicated by Annexin V staining and the proteolytic cleavage of the executioner caspase-3 and PARP. Additionally, the unfolded protein response regulators CHOP, BIP, caspase-12, p-eIF2α and IRE1α, which are considered endoplasmic reticulum (ER) stress markers, were upregulated by VP-16. The strong inhibitory effect of VP-16 on LX-2 cells was mainly dependent on ER stress, which activated JNK signaling pathway. Remarkably, VP-16 treatment decreased the expression of α-SMA and type I collagen and simultaneously increased the ratio of matrix metalloproteinases (MMPs) to tissue inhibitor of matrix metalloproteinases (TIMPs). In contrast, VP-16 induced significantly more apoptosis in HSCs than in normal hepatocytes. Taken together, our findings demonstrate that VP-16 exerts a proapoptotic effect on LX-2 cells and has an antifibrogenic effect on collagen deposition, suggesting a new strategy for the treatment of liver fibrosis. PMID:27680712

  1. Inhibition of endothelin-1-mediated contraction of hepatic stellate cells by FXR ligand.

    PubMed

    Li, Jiang; Kuruba, Ramalinga; Wilson, Annette; Gao, Xiang; Zhang, Yifei; Li, Song

    2010-11-11

    Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands.

  2. Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts.

    PubMed

    Bosselut, Nelly; Housset, Chantal; Marcelo, Paulo; Rey, Colette; Burmester, Thorsten; Vinh, Jöelle; Vaubourdolle, Michel; Cadoret, Axelle; Baudin, Bruno

    2010-03-01

    In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF-HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2-D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast-related typical functions. Among them, cofilin-1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF-HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real-time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF-HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.

  3. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

    PubMed

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-02-13

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings.

  4. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

    PubMed

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-03-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  5. Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis

    PubMed Central

    McKee, Chad; Sigala, Barbara; Soeda, Junpei; Mouralidarane, Angelina; Morgan, Maelle; Mazzoccoli, Gianluigi; Rappa, Francesca; Cappello, Francesco; Cabibi, Daniela; Pazienza, Valerio; Selden, Claire; Roskams, Tania; Vinciguerra, Manlio; Oben, Jude A.

    2015-01-01

    Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD. PMID:25744849

  6. Phospholipase D1 decreases type I collagen levels in hepatic stellate cells via induction of autophagy.

    PubMed

    Seo, H-Y; Jang, B-K; Jung, Y-A; Lee, E-J; Kim, H-S; Jeon, J-H; Kim, J-G; Lee, I-K; Kim, M-K; Park, K-G

    2014-06-20

    Hepatic stellate cells (HSCs) are major players in liver fibrogenesis. Accumulating evidence shows that suppression of autophagy plays an important role in the development and progression of liver disease. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA) and choline, was recently shown to modulate autophagy. However, little is known about the effects of PLD1 on the production of type I collagen that characterizes liver fibrosis. Here, we examined whether PLD1 regulates type I collagen levels in HSCs through induction of autophagy. Adenovirus-mediated overexpression of PLD-1 (Ad-PLD1) reduced type I collagen levels in the activated human HSC lines, hTERT and LX2. Overexpression of PLD1 in HSCs led to induction of autophagy as demonstrated by increased LC3-II conversion and formation of LC3 puncta, and decreased p62 abundance. Moreover, inhibiting the induction of autophagy by treating cells with bafilomycin or a small interfering (si)RNA for ATG7 rescued Ad-PLD1-induced suppression of type I collagen accumulation in HSCs. The effects of PLD on type I collagen levels were not related to TGF-β/Smad signaling. Furthermore, treatment of cells with PA induced autophagy and inhibited type I collagen accumulation. The present study indicates that PLD1 plays a role in regulating type I collagen accumulation through induction of autophagy. PMID:24802400

  7. Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

    PubMed Central

    Xiong, Wu-Jun; Hu, Li-Juan; Jian, Yi-Cheng; Wang, Li-Jing; Jiang, Ming; Li, Wei; He, Yi

    2012-01-01

    AIM: To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions. METHODS: HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model was also analyzed with Western blotting. RESULTS: Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of

  8. Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

    PubMed

    Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping

    2013-02-01

    This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

  9. Nilotinib induces apoptosis and autophagic cell death of activated hepatic stellate cells via inhibition of histone deacetylases.

    PubMed

    Shaker, Mohamed E; Ghani, Ayaz; Shiha, Gamal E; Ibrahim, Tarek M; Mehal, Wajahat Z

    2013-08-01

    Increasing hepatic stellate cell (HSC) death is a very attractive approach for limiting liver fibrosis. Tyrosine kinase inhibitors have been shown to have anti-fibrotic properties, but the mechanisms are poorly understood. Here, we identified the mechanism of action of the second-generation tyrosine kinase inhibitor nilotinib in inducing HSC death. Human HSC line (LX-2) and rat HSCs were treated with nilotinib and its predecessor, imatinib, in the absence or presence of various blockers, known to interfere with death signaling pathways. Nilotinib, but not imatinib, induced progressive cell death of activated, but not quiescent, HSCs in a dose-dependent manner. Activated HSCs died through apoptosis, as denoted by increased DNA fragmentation and caspase activation, and through autophagy, as indicated by the accumulation of autophagic markers, light chain (LC)3A-II and LC3B-II. Although inhibition of caspases with Z-VAD-FMK suppressed nilotinib-induced HSCs' apoptosis, there was no increase in HSCs' survival, because autophagy was exacerbated. However, blocking the mitochondrial permeability transition pore (mPTP) opening with cyclosporin A completely abolished both apoptosis and autophagy due to nilotinib. Moreover, nilotinib treatment decreased the protein expression of histone deacetylases 1, 2 and 4. Interestingly, pretreament with C646, a selective p300/CBP histone acetyl transferase inhibitor, resulted in diverting nilotinib-induced apoptosis and autophagy towards necrosis. In conclusion, the identification of mPTP as a target of nilotinib in activated HSCs suggests coordination with histone deacetylases inhibition to induce apoptosis and autophagy. Thus, our study provides novel insights into the anti-fibrotic effects of nilotinib.

  10. Celecoxib derivative OSU-03012 inhibits the proliferation and activation of hepatic stellate cells by inducing cell senescence.

    PubMed

    Zhang, Jun; Wang, Miao; Zhang, Zuowei; Luo, Zhongguang; Liu, Fei; Liu, Jie

    2015-04-01

    Liver fibrosis may lead to portal hypertension, liver failure or hepatocellular carcinoma, and predominantly results from the proliferation and activation of hepatic stellate cells. OSU‑03012, a non‑cyclooxygenase‑inhibiting celecoxib derivative, has been previously demonstrated to promote apoptosis in certain cell types, however, its function in hepatic fibrosis remains unclear. In the current study, the inhibitory effect of OSU‑03012 on the proliferation of the LX2 human hepatic stellate cell line was evaluated by cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction was performed in order to examine the expression of α‑smooth muscle actin and type I collagen, which are representative of LX2 cell activation. The senescence of LX2 cells was measured by senescence‑associated β‑galactosidase staining, and the cell cycle and apoptosis levels were assessed by flow cytometry. The impact of senescence‑associated signaling on protein expression was assessed by western blot analysis. OSU‑03012 was observed to inhibit cell proliferation and prevent the secretion of profibrotic factors in LX2 cells in a dose‑dependent manner. Furthermore, the results demonstrated that OSU‑03012 inhibited the proliferation and activation of LX2 via the induction of cell senescence at the G1 phase, rather than via cell apoptosis. The induction of senescence may be via the upregulation of p16, p21 and p27. In conclusion, the current study provided insight into the pharmacological mechanisms of OSU‑03012 in preventing the proliferation and activation of hepatic stellate cells through cell senescence. The current study supports the theory that OSU‑03012 is a novel agent for potential use against liver fibrosis.

  11. Up-regulation of interleukin-22 mediates liver fibrosis via activating hepatic stellate cells in patients with hepatitis C.

    PubMed

    Wu, Li-Yuan; Liu, Shuhong; Liu, Yuan; Guo, Chaonan; Li, Hanwei; Li, Wenshu; Jin, Xueyuan; Zhang, Keming; Zhao, Ping; Wei, Lai; Zhao, Jingmin

    2015-05-01

    Interleukin-22 (IL-22) is known to play a critical role in liver immunity. However, the role of IL-22 in HCV-associated liver fibrosis is poorly understood. In this study, patients with HCV infection disclosed significant increases in peripheral numbers of IL-22-producing cells as well as in IL-22 plasma levels. In the liver, the increased intrahepatic IL-22(+) cells were positively correlated with fibrotic staging scores and clinical progression from CHC to cirrhosis. Moreover, the majority of IL-22(+) cells were located in fibrotic areas in the liver of patients with cirrhosis and co-localized with α-smooth muscle actin (α-SMA) positive hepatic stellate cells (HSCs). In vitro, administration of IL-22 was accompanied with inhibited LX-2 cell apoptosis, promoted LX-2 cell proliferation, increased expression of α-SMA, and up-regulated collagen production by LX-2 cells. Collectively, our data provide evidence that IL-22 may contribute to the fibrogenesis of HCV-associated liver fibrosis by activating HSCs.

  12. Dihydroartemisinin alleviates bile duct ligation-induced liver fibrosis and hepatic stellate cell activation by interfering with the PDGF-βR/ERK signaling pathway.

    PubMed

    Chen, Qin; Chen, Lianyun; Kong, Desong; Shao, Jiangjuan; Wu, Li; Zheng, Shizhong

    2016-05-01

    Liver fibrosis represents a frequent event following chronic insult to trigger wound healing responses in the liver. Activation of hepatic stellate cells (HSCs), which is a pivotal event during liver fibrogenesis, is accompanied by enhanced expressions of a series of marker proteins and pro-fibrogenic signaling molecules. Artemisinin, a powerful antimalarial medicine, is extracted from the Chinese herb Artemisia annua L., and can inhibit the proliferation of cancer cells. Dihydroartemisinin (DHA), the major active metabolite of artemisinin, is able to attenuate lung injury and fibrosis. However, the effect of DHA on liver fibrosis remains unclear. The aim of this study was to investigate the effect of DHA on bile duct ligation-induced injury and fibrosis in rats. DHA improved the liver histological architecture and attenuated collagen deposition in the fibrotic rat liver. Experiments in vitro showed that DHA inhibited the proliferation of HSCs and arrested the cell cycle at the S checkpoint by altering several cell-cycle regulatory proteins. Moreover, DHA reduced the protein expressions of a-SMA, α1 (I) collagen and fibronectin, being associated with interference of the platelet-derived growth factor β receptor (PDGF-βR)-mediated ERK pathway. These data collectively revealed that DHA relieved liver fibrosis possibly by targeting HSCs via the PDGF-βR/ERK pathway. DHA may be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis. PMID:27038258

  13. Distribution of vitamin A-storing lipid droplets in hepatic stellate cells in liver lobules--a comparative study.

    PubMed

    Higashi, Nobuyo; Senoo, Haruki

    2003-03-01

    To investigate the storage mechanisms of vitamin A, we examined the liver of adult polar bears and arctic foxes, which physiologically store a large amount of vitamin A, by high-performance liquid chromatography (HPLC), transmission electron microscopy (TEM) morphometry, gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, staining with hematoxylin-eosin (H&E), Masson's trichrome, and Ishii and Ishii's silver impregnation. HPLC revealed that the polar bears and arctic foxes contained 1.8-1.9 x 10(4) nmol total retinol (retinol plus retinyl esters) per gram liver. In the arctic foxes, the composition of the retinyl esters was found to be 51.1% palmitate, 26.6% oleate, 15.4% stearate, and 7% linoleate. The hepatic stellate cells of the arctic animals were demonstrated by TEM to contain the bulk of the vitamin A-lipid droplets in their cytoplasm. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The gradient was expressed as a symmetric crescendo-decrescendo profile starting at the periportal zone, peaking at the middle zone, and sloping down toward the central zone in the liver lobule. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. The gradient and the composition of the retinyl esters in storing vitamin A were not changed by differences in the vitamin A amount in the livers. These results indicate that the heterogeneity of vitamin A-storage capacity in hepatic stellate cells of arctic foxes and polar bears is genetically determined. PMID:12552640

  14. The bHLH Transcription Factor Hand2 Marks Hepatic Stellate Cells in Zebrafish: Analysis of Stellate Cell Entry into the Developing Liver

    PubMed Central

    Yin, Chunyue; Evason, Kimberley J.; Maher, Jacquelyn J.; Stainier, Didier Y.R.

    2012-01-01

    Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity and interactions between these two cell types are potentially critical for their development and function. Here we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene expression profile, and increased proliferation and matrix production in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development. Conclusion Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior. PMID:22488653

  15. Relevance of activated hepatic stellate cells in predicting the development of pediatric liver allograft fibrosis.

    PubMed

    Venturi, Carla; Reding, Raymond; Quinones, Jorge Abarca; Sokal, Etienne; Rahier, Jacques; Bueno, Javier; Sempoux, Christine

    2016-06-01

    Activated hepatic stellate cells (HSCs) are the main collagen-producing cells in liver fibrogenesis. With the purpose of analyzing their presence and relevance in predicting liver allograft fibrosis development, 162 liver biopsies of 54 pediatric liver transplantation (LT) recipients were assessed at 6 months, 3 years, and 7 years after LT. The proportion of activated HSCs, identified by α-smooth muscle actin (ASMA) immunostaining, and the amount of fibrosis, identified by picrosirius red (PSR%) staining, were determined by computer-based morphometric analysis. Fibrosis was also staged by using the semiquantitative liver allograft fibrosis score (LAFSc), specifically designed to score fibrosis in the pediatric LT population. Liver allograft fibrosis displayed progression over time by PSR% (P < 0.001) and by LAFSc (P < 0.001). The ASMA expression decreased in the long term, with inverse evolution with respect to fibrosis (P < 0.01). Patients with ASMA-positive HSCs area ≥ 8% at 6 months (n = 20) developed a higher fibrosis proportion compared to those with ASMA-positive HSCs area ≤ 8% (n = 34) at the same period of time and in the long term (P = 0.03 and P < 0.01, respectively), but not at 3 years (P = 0.8). ASMA expression ≥ 8% at 6 months was found to be an independent risk factor for 7-year fibrosis development by PSR% (r(2) = 0.5; P < 0.01) and by LAFSc (r(2) = 0.3; P = 0.03). Furthermore, ASMA expression ≥ 8% at 3 years showed an association with the development of fibrosis at 7 years (P = 0.02). In conclusion, there is a high proportion of activated HSCs in pediatric LT recipients. ASMA ≥ 8% at 6 months seems to be a risk factor for early and longterm fibrosis development. In addition, activated HSCs showed inverse evolution with respect to fibrosis in the long term. Liver Transplantation 22 822-829 2016 AASLD. PMID:26851053

  16. Gallic Acid Induces Necroptosis via TNF–α Signaling Pathway in Activated Hepatic Stellate Cells

    PubMed Central

    Chang, Ya Ju; Hsu, Shih Lan; Liu, Yi Ting; Lin, Yu Hsuan; Lin, Ming Hui; Huang, Shu Jung; Ho, Ja-an Annie; Wu, Li-Chen

    2015-01-01

    Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA), a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs), the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF–α–mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH). Elevated oxidative stress triggered the production of TNF–α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3), and the inactivation of caspase–8. Calmodulin and calpain–1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP). The TNF–α antagonist (SPD–304) and the RIP1 inhibitor (necrostatin–1, Nec–1) confirmed GA-induced TNFR1–mediated necroptosis. The inhibition of RIP1 by Nec–1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling–triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis. PMID:25816210

  17. PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells.

    PubMed

    Pirazzi, Carlo; Valenti, Luca; Motta, Benedetta Maria; Pingitore, Piero; Hedfalk, Kristina; Mancina, Rosellina Margherita; Burza, Maria Antonella; Indiveri, Cesare; Ferro, Yvelise; Montalcini, Tiziana; Maglio, Cristina; Dongiovanni, Paola; Fargion, Silvia; Rametta, Raffaela; Pujia, Arturo; Andersson, Linda; Ghosal, Saswati; Levin, Malin; Wiklund, Olov; Iacovino, Michelina; Borén, Jan; Romeo, Stefano

    2014-08-01

    Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease. PMID:24670599

  18. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    PubMed Central

    Gao, Run-Ping; Brigstock, David R

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase. PMID:19673024

  19. [Morphological features of the rat stellate ganglion during early postnatal development].

    PubMed

    Korzina, M B; Korobkin, A A; Vasil'eva, O A; Masliukov, P M

    2010-01-01

    The aim of this work was to study the anatomical characteristics of the stellate ganglion (SG) and the morphometric characteristics of its neurons in rats of different age groups (newborn, 10-, 20-, 30-, 60- and 180-day-old) using anatomical and histological methods. The results obtained indicated that in rats since birth there were three variants of branch origin from the medial margin of SG. No differences were observed in these variants between right and left SG. The sizes of both SG and its neurons increased during the first two months of postnatal development. The density of neurons in SG sections decreased from the moment of birth until the six months of age. The number of SG neurons did not change significantly in the postnatal ontogenesis. Thus, SG in rats is anatomically formed by the moment of birth, while the sizes and morphometric characteristics of SG neurons become finally stabilized by the second month of age. PMID:20572389

  20. Modulation of catecholamine-synthesizing enzymes in adrenal medulla and stellate ganglia by treadmill exercise of stressed rats.

    PubMed

    Gavrilovic, Ljubica; Spasojevic, Natasa; Dronjak, Sladjana

    2012-03-01

    The sympatho-adrenal system represents one of the main systems involved in the response to stressful events because its stress-induced activation results in an increased release of catecholamines. Exercise training acts as an important modulator of sympatho-adrenal system, adrenal medulla and stellate ganglia being two components of this system. This study aimed at investigating physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase in the adrenal medulla and stellate ganglia of chronically psychosocially stressed adult rats exposed daily to 20-min treadmill exercise for 12 weeks, using TaqMan RT-PCR assay. Chronic psychosocial stress decreased gene expression of the examined enzymes in the adrenal medulla and treadmill exercise did not lead to further modulation of the corresponding gene expression. On the other hand, chronic psychosocial stress produced a significant increase of TH (about 51%) and DBH (about 103%) gene expression in stellate ganglia, while treadmill exercise decreased gene expression of these enzymes to control levels in psychosocially stressed rats. Our data indicate that treadmill exercise leads to a decreased gene transcription of catecholamine biosynthetic enzymes in stellate ganglia and attenuation of cardiac noradrenaline production in stressful situations. Reduction of catecholamine synthesis in stellate ganglia may be linked to the beneficial effects of treadmill exercise on cardiovascular system in stressed animals.

  1. Liver Fibrosis and Protection Mechanisms Action of Medicinal Plants Targeting Apoptosis of Hepatocytes and Hepatic Stellate Cells

    PubMed Central

    Moreno-Cuevas, Jorge E.; González-Garza, Maria Teresa; Rodríguez-Montalvo, Carlos; Cruz-Vega, Delia Elva

    2014-01-01

    Following chronic liver injury, hepatocytes undergo apoptosis leading to activation of hepatic stellate cells (HSC). Consequently, activated HSC proliferate and produce excessive extracellular matrix, responsible for the scar formation. The pandemic trend of obesity, combined with the high incidence of alcohol intake and viral hepatitis infections, highlights the urgent need to find accessible antifibrotic therapies. Treatment strategies should take into account the versatility of its pathogenesis and act on all the cell lines involved to reduce liver fibrosis. Medicinal plants are achieving popularity as antifibrotic agents, supported by their safety, cost-effectiveness, and versatility. This review will describe the role of hepatocytes and HSC in the pathogenesis of liver fibrosis and detail the mechanisms of modulation of apoptosis of both cell lines by twelve known hepatoprotective plants in order to reduce liver fibrosis. PMID:25505905

  2. Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells.

    PubMed

    Wang, Xiaoyun; Hu, Fengyu; Hu, Xuchu; Chen, Wenjun; Huang, Yan; Yu, Xinbing

    2014-08-01

    Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 μg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies

  3. Curcumin, the main active constituent of turmeric (Curcuma longa L.), induces apoptosis in hepatic stellate cells by modulating the abundance of apoptosis-related growth factors.

    PubMed

    He, Ya-Jun; Kuchta, Kenny; Lv, Xia; Lin, Yu; Ye, Guo-Rong; Liu, Xu-You; Song, Hui-Dong; Wang, Le-Xin; Kobayashi, Yuta; Shu, Jian-Chang

    2015-11-01

    In order to elucidate the mechanism of action of curcumin against hepatic fibrosis, cultured rat hepatic stellate cells (HSC) (HSC-T6) were incubated with curcumin for 24 h, after which apoptosis was measured by flow-cytometry. The protein levels of the pro-apoptotic factors Fas and p53b as well as of the anti-apoptotic factor Bcl-2 were monitored by immunocytochemical ABC staining after incubation with curcumin for 24 h. In the case of 20 μM curcumin, not only was the respective apoptosis index increased, but also the abundance of the pro-apoptotic factors Fas and p53 were amplified, whereas that of the anti-apoptotic factor Bcl-2 decreased. All these effects were highly reproducible (P<0.05). Consequently, curcumin has an up-regulating effect on pro-apoptotic factors like Fas and p53 as well as a down-regulating effect of the anti-apoptotic factor Bcl-2, thus inducing apoptosis in HSC.

  4. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  5. Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells

    PubMed Central

    Kweon, Soo-Mi; Chi, Feng; Higashiyama, Reiichi; Lai, Keane; Tsukamoto, Hidekazu

    2016-01-01

    PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6–8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5’ Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho β-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC. PMID:27214381

  6. Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells.

    PubMed

    Kweon, Soo-Mi; Chi, Feng; Higashiyama, Reiichi; Lai, Keane; Tsukamoto, Hidekazu

    2016-01-01

    PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6-8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5' Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho β-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC. PMID:27214381

  7. Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice

    PubMed Central

    Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna; Chowdhury, Abhijit; Boyer, James L.; Santra, Amal

    2013-01-01

    Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 μg/ gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-β1, PDGF-Rβ, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro (α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis. PMID:21134390

  8. Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice

    SciTech Connect

    Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna; Chowdhury, Abhijit; Boyer, James L.; Santra, Amal

    2011-02-15

    Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 {mu}g/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including {alpha}-smooth muscle actin, transforming growth factor-{beta}1, PDGF-R{beta}, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro({alpha}) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.

  9. Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice.

    PubMed

    Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna; Chowdhury, Abhijit; Boyer, James L; Santra, Amal

    2011-02-15

    Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 μg/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-β1, PDGF-Rβ, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro(α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.

  10. Therapeutic targeting of the PDGF and TGF-beta-signaling pathways in hepatic stellate cells by PTK787/ZK22258.

    PubMed

    Liu, Yuqing; Wen, Xiao Ming; Lui, Eric Lik Hang; Friedman, Scott L; Cui, Wei; Ho, Nancy Pei Shan; Li, Lei; Ye, Tao; Fan, Sheung Tat; Zhang, Hui

    2009-10-01

    Stimulation of hepatic stellate cells (HSCs) by platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) is an essential pathway of proliferation and fibrogenesis, respectively, in liver fibrosis. We provide evidence that PTK787/ZK222584 (PTK/ZK), a potent tyrosine kinase inhibitor that blocks vascular endothelial growth factor receptor (VEGFR), significantly inhibits PDGF receptor expression, as well as PDGF-simulated HSC proliferation, migration and phosphorylation of ERK1/2, Akt and p70S6 kinase. Interestingly, PTK/ZK also antagonizes the TGF-beta1-induced expression of VEGF and VEGFR1. Furthermore, PTK/ZK downregulates TGF-beta receptor expression, which is associated with reduced Akt, ERK and p38MAPK phosphorylation. Furthermore, PDGF-induced TGF-beta1 expression is inhibited by PTK/ZK. These findings provide evidence that PTK/ZK targets multiple essential pathways of stellate cell activation that provoke proliferation and fibrogenesis. Our study underscores the potential use of PTK/ZK as an antifibrotic drug in chronic liver disease. PMID:19668241

  11. Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial-mesenchymal transition in liver injury.

    PubMed

    Li, Yuchang; Wang, Jiaohong; Asahina, Kinji

    2013-02-01

    In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-β signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial-mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts. PMID:23345421

  12. Interferon-γ Triggers Hepatic Stellate Cell-Mediated Immune Regulation through MEK/ERK Signaling Pathway

    PubMed Central

    Gu, Xiaodong; Wang, Yan; Xiang, Jianbin; Chen, Zongyou; Wang, Lianfu; Lu, Lina; Qian, Shiguang

    2013-01-01

    Hepatic stellate cells (HSCs) interact with immune cells to actively participate in regulating immune response in the liver which is mediated by the effector molecules, including B7-H1. We demonstrated here that expression of B7-H1 on HSCs was markedly enhanced by interferon-(IFN-) γ stimulation. IFN-γ stimulated HSCs inhibited T-cell proliferation via induction of T-cell apoptosis (22.1% ± 1.6%). This immunosuppressive effect was inhibited by preincubation with an anti-B7-H1 antibody, or inhibitor of the MEK/ERK pathway inhibited IFN-γ mediated expression of B7-H1. Thus, regulation of B7-H1 expression on HSCs by IFN-γ represents an important mechanism that regulates immune responses in the liver favoring tolerogenicity rather than immunogenicity. Involvement of MEK/ERK pathway provides a novel target for therapeutic approaches. PMID:23737812

  13. Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells.

    PubMed

    Mao, Qiang; Xie, Zhizhi; Wang, Xiaoyun; Chen, Wenjun; Ren, Mengyu; Shang, Mei; Lei, Huali; Tian, Yanli; Li, Shan; Liang, Pei; Chen, Tingjin; Liang, Chi; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2015-02-01

    Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

  14. Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model.

    PubMed

    Karthikeyan, Swathi; Potter, James J; Geschwind, Jean-Francois; Sur, Surojit; Hamilton, James P; Vogelstein, Bert; Kinzler, Kenneth W; Mezey, Esteban; Ganapathy-Kanniappan, Shanmugasundaram

    2016-01-15

    Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

  15. Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

    SciTech Connect

    Chen, Chao; Wu, Chao-Qun; Zhang, Zong-Qi; Yao, Ding-Kang; Zhu, Liang

    2011-07-15

    Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced {alpha}-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.

  16. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

    PubMed Central

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Rao, Kummara Madhusudana; Park, Soo Yong; Chung, Ildoo; Ha, Chang-Sik; Kim, Sang-Woo; Yun, Yang H.; Jung, Youngmi

    2016-01-01

    Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis. PMID:27001906

  17. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression.

    PubMed

    Hyun, Jeongeun; Wang, Sihyung; Kim, Jieun; Rao, Kummara Madhusudana; Park, Soo Yong; Chung, Ildoo; Ha, Chang-Sik; Kim, Sang-Woo; Yun, Yang H; Jung, Youngmi

    2016-01-01

    Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl4)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl4-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis. PMID:27001906

  18. TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

    SciTech Connect

    Fang, Ling; Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Ma, Taotao; Liu, Xuejiao; Zhu, Qian; Zhan, Shuxiang; Li, Jun

    2014-10-15

    Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

  19. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  20. Inhibitory effects of armepavine against hepatic fibrosis in rats

    PubMed Central

    Weng, Ting-Chun; Shen, Chien-Chang; Chiu, Yung-Tsung; Lin, Yun-Lian; Kuo, Cheng-Deng; Huang, Yi-Tsau

    2009-01-01

    Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C19H23O3N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 μM) concentration-dependently attenuated TNF-α- and LPS-stimulated α-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-α-induced collagen collagen deposition, NFκB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1α2, TGF-β1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-κB activation pathways. PMID:19723340

  1. Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes.

    PubMed

    Kasuya, Junichi; Sudo, Ryo; Masuda, Genta; Mitaka, Toshihiro; Ikeda, Mariko; Tanishita, Kazuo

    2015-03-01

    In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.

  2. Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO

    PubMed Central

    Sumpter, Tina L.; Dangi, Anil; Matta, Benjamin M.; Huang, Chao; Stolz, Donna B.; Vodovotz, Yoram; Thomson, Angus W.; Gandhi, Chandrashekhar R.

    2012-01-01

    Hepatic stellate cells (HSCs) are critical for hepatic wound repair and tissue remodeling. They also produce cytokines and chemokines that may contribute to the maintenance of hepatic immune homeostasis and the inherent tolerogenicity of the liver. The functional relationship between HSCs and the professional migratory APCs in the liver, i.e. dendritic cells (DCs), has not been evaluated. Here, we report that murine liver DCs co-localize with HSCs in vivo under normal, steady-state conditions, and cluster with HSCs in vitro. In vitro, HSCs secrete high levels of DC chemoattractants, such as MIP1α and MCP-1, as well as cytokines that modulate DC activation, including TNFα, IL-6 and IL-1β. Culture of HSCs with conventional liver myeloid (m) DCs resulted in increased IL-6 and IL-10 secretion compared to that of either cell population alone. Co-culture also resulted in enhanced expression of co-stimulatory (CD80, CD86) and co-inhibitory (B7-H1) molecules on mDCs. HSC-induced mDC maturation required cell-cell contact and could be blocked, in part, by neutralizing MIP1α or MCP-1. HSC-induced mDC maturation was dependent on activation of STAT3 in mDCs and in part on HSC-secreted IL-6. Despite up-regulation of co-stimulatory molecules, mDCs conditioned by HSCs demonstrated impaired ability to induce allogeneic T cell proliferation, which was independent of B7-H1, but dependent upon HSC-induced STAT3 activation and subsequent up-regulation of IDO. In conclusion, by promoting IDO expression, HSCs may act as potent regulators of liver mDCs and function to maintain hepatic homeostasis and tolerogenicity. PMID:22962681

  3. IGF-I induces senescence of hepatic stellate cells and limits fibrosis in a p53-dependent manner

    PubMed Central

    Nishizawa, Hitoshi; Iguchi, Genzo; Fukuoka, Hidenori; Takahashi, Michiko; Suda, Kentaro; Bando, Hironori; Matsumoto, Ryusaku; Yoshida, Kenichi; Odake, Yukiko; Ogawa, Wataru; Takahashi, Yutaka

    2016-01-01

    Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) and cirrhosis determines patient prognosis; however, effective treatment for fibrosis has not been established. Oxidative stress and inflammation activate hepatic stellate cells (HSCs) and promote fibrosis. In contrast, cellular senescence inhibits HSCs’ activity and limits fibrosis. The aim of this study was to explore the effect of IGF-I on NASH and cirrhotic models and to clarify the underlying mechanisms. We demonstrate that IGF-I significantly ameliorated steatosis, inflammation, and fibrosis in a NASH model, methionine-choline-deficient diet-fed db/db mice and ameliorated fibrosis in cirrhotic model, dimethylnitrosamine-treated mice. As the underlying mechanisms, IGF-I improved oxidative stress and mitochondrial function in the liver. In addition, IGF-I receptor was strongly expressed in HSCs and IGF-I induced cellular senescence in HSCs in vitro and in vivo. Furthermore, in mice lacking the key senescence regulator p53, IGF-I did not induce cellular senescence in HSCs or show any effects on fibrosis. Taken together, these results indicate that IGF-I induces senescence of HSCs, inactivates these cells and limits fibrosis in a p53-dependent manner and that IGF-I may be applied to treat NASH and cirrhosis. PMID:27721459

  4. The hop constituent xanthohumol exhibits hepatoprotective effects and inhibits the activation of hepatic stellate cells at different levels

    PubMed Central

    Weiskirchen, Ralf; Mahli, Abdo; Weiskirchen, Sabine; Hellerbrand, Claus

    2015-01-01

    Xanthohumol is the principal prenylated flavonoid of the female inflorescences of the hop plant. In recent years, various beneficial xanthohumol effects including anti-inflammatory, antioxidant, hypoglycemic activities, and anticancer effects have been revealed. This review summarizes present studies indicating that xanthohumol also inhibits several critical pathophysiological steps during the development and course of chronic liver disease, including the activation and pro-fibrogenic genotype of hepatic stellate cells. Also the various mechanism of action and molecular targets of the beneficial xanthohumol effects will be described. Furthermore, the potential use of xanthohumol or a xanthohumol-enriched hop extract as therapeutic agent to combat the progression of chronic liver disease will be discussed. It is notable that in addition to its hepatoprotective effects, xanthohumol also holds promise as a therapeutic agent for treating obesity, dysregulation of glucose metabolism and other components of the metabolic syndrome including hepatic steatosis. Thus, therapeutic xanthohumol application appears as a promising strategy, particularly in obese patients, to inhibit the development as well as the progression of non-alcoholic fatty liver disease. PMID:25999863

  5. Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells.

    PubMed Central

    Senoo, H; Smeland, S; Malaba, L; Bjerknes, T; Stang, E; Roos, N; Berg, T; Norum, K R; Blomhoff, R

    1993-01-01

    Rat liver stellate cells were cocultured with HepG2 human hepatoma cells, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was studied by cryoimmunoelectron microscopy. In stellate cells, human RBP was found on the cell surface and within endosomes. The transfer of human RBP from HepG2 cells to stellate cells was blocked by addition of RBP antibodies to the culture medium. Very little uptake of RBP was observed when fibroblasts were cocultured with HepG2 cells. In a series of experiments, RBP was bound to its putative cell surface receptor at 4 degrees C, and the stellate cells were washed and then incubated at 37 degrees C in order to allow them to internalize a pulse of RBP. About 50% of the RBP was internalized after 6 min of incubation. The RBP-positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membranes. After 2 hr, however, most RBP was localized in intracellular vesicles at a distance from the vesicular membranes, suggesting that RBP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with 125I-RBP at 4 degrees C and cell-associated radioactivity was determined. The calculated dissociation constant for the specific binding was 12.7 +/- 3.2 nM. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cells bound about 30 times more 125I-labeled RBP than did parenchymal cells (based on mass of cell protein). These data suggest that RBP mediates the paracrine transfer of retinol from hepatocytes to perisinusoidal stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis. Images Fig. 1 Fig. 2 PMID:8386378

  6. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells.

    PubMed

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José; Delpino, María Victoria

    2015-12-14

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

  7. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells

    PubMed Central

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José

    2015-01-01

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway. PMID:26667834

  8. Morin, a dietary flavonoid, exhibits anti-fibrotic effect and induces apoptosis of activated hepatic stellate cells by suppressing canonical NF-κB signaling.

    PubMed

    MadanKumar, Perumal; NaveenKumar, Perumal; Devaraj, Halagowder; NiranjaliDevaraj, Sivasithamparam

    2015-03-01

    In experimental liver fibrosis, activated hepatic stellate cells (HSCs) play a central role and thus, induction of apoptosis of activated HSCs is a promising therapeutic strategy for liver fibrosis. The present study was designed to elucidate the molecular mechanisms of the pro-apoptotic effects of morin, a dietary flavonoid, in vitro and in vivo. Culture-activated human HSCs (LX-2 cells) were treated with morin (50 μM) for 24 and 48 h, and the mechanism of cell death induced by morin was evaluated. Also, the anti-fibrotic and pro-apoptotic effect of morin in diethylnitrosamine (DEN)-induced fibrotic rats were determined. Morin induced apoptosis in cultured LX-2 cells by preventing the nuclear translocation of nuclear factor-κBp65 (NF-κBp65) by inhibiting NF-κB activation via inhibition of IκBα degradation and thereby suppressing anti-apoptotic proteins and activating caspases. In fibrotic rats, morin treatment resulted in inhibition of canonical NF-κB signaling and induction of apoptosis, mainly by downregulating Bcl-2, upregulating Bax and cyt c and by activation of caspase-9 and caspase-3. Translocation of phosphatidylserine to the outer membrane, altered nuclear morphology and DNA fragmentation confirmed the induction of apoptosis by morin. Overall, morin treatment ameliorated experimental liver fibrosis, most likely through induction of apoptosis by inhibiting canonical NF-κB signaling in activated HSCs. It is therefore postulated that morin is a potential therapeutic candidate for liver fibrosis.

  9. Curcumin inhibits srebp-2 expression in activated hepatic stellate cells in vitro by reducing the activity of specificity protein-1.

    PubMed

    Kang, Qiaohua; Chen, Anping

    2009-12-01

    Elevated levels of cholesterol/low-density lipoprotein (LDL) are a risk factor for the development of nonalcoholic steatohepatitis and its associated hepatic fibrosis. However, underlying mechanisms remain elusive. We previously reported that curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity, leading to the inhibition of the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis. We recently showed that curcumin suppressed gene expression of LDL receptor in activated HSCs in vitro by repressing gene expression of the transcription factor sterol regulatory element binding protein-2 (SREBP-2), leading to the reduction in the level of intracellular cholesterol in HSCs and to the attenuation of the stimulatory effects of LDL on HSCs activation. The current study aimed at exploring molecular mechanisms by which curcumin inhibits srebp-2 expression in HSCs. Promoter deletion assays, mutagenesis assays, and EMSAs localize a specificity protein-1 (SP-1) binding GC-box in the srebp-2 promoter, which is responsible for enhancing the promoter activity and responding to curcumin in HSCs. Curcumin suppresses gene expression of SP-1 and reduces its trans-activation activity, which are mediated by the activation of PPARgamma. The inhibitory effect of curcumin on SP-1 binding to the GC-box is confirmed by chromatin immuno-precipitation. In summary, our results demonstrate that curcumin inhibits srebp-2 expression in cultured HSCs by activating PPARgamma and reducing the SP-1 activity, leading to the repression of ldlr expression. These results provide novel insights into molecular mechanisms by which curcumin inhibits LDL-induced HSC activation.

  10. N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis.

    PubMed

    Kamada, Yoshihiro; Mori, Kanako; Matsumoto, Hitoshi; Kiso, Shinichi; Yoshida, Yuichi; Shinzaki, Shinichiro; Hiramatsu, Naoki; Ishii, Mayuko; Moriwaki, Kenta; Kawada, Norifumi; Takehara, Tetsuo; Miyoshi, Eiji

    2012-06-01

    N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions. PMID:22294551

  11. FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis

    PubMed Central

    Verbeke, Len; Mannaerts, Inge; Schierwagen, Robert; Govaere, Olivier; Klein, Sabine; Vander Elst, Ingrid; Windmolders, Petra; Farre, Ricard; Wenes, Mathias; Mazzone, Massimiliano; Nevens, Frederik; van Grunsven, Leo A.; Trebicka, Jonel; Laleman, Wim

    2016-01-01

    Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis. PMID:27634375

  12. FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis.

    PubMed

    Verbeke, Len; Mannaerts, Inge; Schierwagen, Robert; Govaere, Olivier; Klein, Sabine; Vander Elst, Ingrid; Windmolders, Petra; Farre, Ricard; Wenes, Mathias; Mazzone, Massimiliano; Nevens, Frederik; van Grunsven, Leo A; Trebicka, Jonel; Laleman, Wim

    2016-01-01

    Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis. PMID:27634375

  13. The herbal compound Songyou Yin (SYY) inhibits hepatocellular carcinoma growth and improves survival in models of chronic fibrosis via paracrine inhibition of activated hepatic stellate cells

    PubMed Central

    Xue, Tong-Chun; Zhang, Quan-Bao; Zhang, Ke-Zhi; Zhang, Qiang-Bo; You, Yang; Tian, Hui; Qin, Lun-Xiu; Tang, Zhao-You

    2015-01-01

    Chronic fibrosis is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathological progression of hepatic fibrosis has been linked to cellular processes that promote tumor growth and metastasis. Several recent studies have highlighted the cross-talk between tumor cells and activated hepatic stellate cells (aHSCs) in HCC. The herbal compound Songyou Yin (SYY) is known to attenuate hepatoma cell invasion and metastasis via down-regulation of cytokine secretion by aHSCs. However the underlying mechanism of SYY treatment in reversal of hepatic fibrosis and metastasis of liver cancers is not known. In the current study, a nude mouse model with liver fibrosis bearing orthotopic xenograft was established and we found that SYY could reduce associated fibrosis, inhibit tumor growth and improve survival. In the subcutaneous tumor model with fibrosis, we found that SYY could inhibit liver cancer. In vitro, hepatoma cells incubated with conditioned media (CM) from SYY treated aHSCs showed reduced proliferation, decrease in colony formation and invasive potential. SYY treated group showed altered gene expression, with 1205 genes up-regulated and 1323 genes down-regulated. Gene cluster analysis indicated that phosphatidylinositol-3-kinase (PI3K) was one of the key genes altered in the expression profiles. PI3K related markers were all significantly down-regulated. ELISA also indicated decreased secretion of cytokines which were regulated by PI3K/AKT signaling after SYY treatment in the hepatic stellate cell line, LX2. These data clearly demonstrate that SYY therapy inhibits HCC invasive and metastatic potential and improves survival in nude mice models with chronic fibrosis background via inhibition of cytokine secretion by activated hepatic stellate cells. PMID:26517671

  14. Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

    PubMed Central

    Tu, Wei; Ye, Jin; Wang, Zhi-Jun

    2016-01-01

    AIM To investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis. METHODS The expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed. RESULTS The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. CONCLUSION ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling. PMID:27784964

  15. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    PubMed

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-01

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade. PMID:26089141

  16. ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation.

    PubMed

    Zhang, Kun; Chang, Yanan; Shi, Zhemin; Han, Xiaohui; Han, Yawei; Yao, Qingbin; Hu, Zhimei; Cui, Hongmei; Zheng, Lina; Han, Tao; Hong, Wei

    2016-07-20

    Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis.

  17. Hydrogels with Differential and Patterned Mechanics to Study Stiffness-Mediated Myofibroblastic Differentiation of Hepatic Stellate Cells

    PubMed Central

    Guvendiren, Murat; Perepelyuk, Maryna; Wells, Rebecca G.; Burdick, Jason A.

    2014-01-01

    The differentiation of hepatic stellate cells (HSCs) into myofbroblasts is a key event in liver fibrosis. Due to the local stiffening of the extracellular matrix (ECM) during fibrosis, it is of great interest to develop mimics that can be used to investigate the cellular response to changes in mechanics. Here, we used a step-wise hydrogel crosslinking technique, where macromolecules are crosslinked using a sequence of addition then UV light-mediated radical crosslinking, to generate hydrogels with tunable stiffness. Freshly isolated HSCs remained rounded with lipid droplets and high levels of PPARγ expression on soft substrates (E~2 kPa); however, HSCs spread, lost their lipid droplets, and expressed high levels of α-smooth muscle actin (α-SMA) and type I collagen on stiff substrates (E~ 24 kPa). Similarly, fully differentiated cells reverted to a quiescent state when plated on soft substrates. Stiffness-induced differentiation of HSCs was enhanced in the presence of exogenous TGF-β1, a dominant signal in fibrosis. When the UV-induced secondary crosslinking was restricted with a photomask to spatially control mechanics, HSCs responded based on the local hydrogel stiffness, although they remained quiescent on stiff substrates if the stiff feature size was not sufficient to allow cell spreading. This hydrogel system permits the investigation of HSC response to materials with diverse levels and spatially heterogeneous mechanical properties. PMID:24361340

  18. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis

    PubMed Central

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis. PMID:26713258

  19. Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells.

    PubMed

    Zhong, Chunge; Jiang, Chunyu; Xia, Xichun; Mu, Teng; Wei, Lige; Lou, Yuntian; Zhang, Xiaoshu; Zhao, Yuqing; Bi, Xiuli

    2015-07-01

    Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNF-α. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade.

  20. Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells

    PubMed Central

    Taschler, Ulrike; Schreiber, Renate; Chitraju, Chandramohan; Grabner, Gernot F.; Romauch, Matthias; Wolinski, Heimo; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Lass, Achim; Zimmermann, Robert

    2015-01-01

    Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl ester (RE) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL deficiency and hormone-sensitive lipase (HSL) deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades RE even in the presence of TGs, implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs. PMID:25732851

  1. The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells.

    PubMed

    Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Götze, Silke; Kordes, Claus; Sawitza, Iris; Hoffmann, Michèle J; Franke, Manuel; Schulz, Wolfgang A; Scheller, Jürgen; Piekorz, Roland P; Häussinger, Dieter; Ahmadian, Mohammad R

    2016-04-15

    Hepatic stellate cells (HSCs) were recently identified as liver-resident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3Kα/δ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

  2. Long non-coding RNA APTR promotes the activation of hepatic stellate cells and the progression of liver fibrosis

    SciTech Connect

    Yu, Fujun; Zheng, Jianjian; Mao, Yuqing; Dong, Peihong; Li, Guojun; Lu, Zhongqiu; Guo, Chuanyong; Liu, Zhanju; Fan, Xiaoming

    2015-08-07

    In this study, we aimed at assessing a role of Alu-mediated p21 transcriptional regulator (APTR) in hepatofibrogenesis. APTR was upregulated in fibrotic liver samples and activated hepatic stellate cells (HSCs). Knockdown of APTR inhibited the activation of HSCs in vitro and mitigated the accumulation of collagen in vivo. Importantly, APTR silencing could abrogate TGF-β{sub 1}-induced upregulation of α-SMA in HSCs. In addition, inhibition of cell cycle and cell proliferation by APTR knockdown was attenuated by p21 siRNA1 in primary HSCs. Finally, serum APTR levels were increased in patients with liver cirrhosis, indicating a potential biomarker for liver cirrhosis. Collectively, evidence is proposed for a new biological role of APTR in hepatofibrogenesis. - Highlights: • APTR is upregulated in fibrotic liver tissues and activated HSCs. • APTR silencing inhibits HSC activation and the progression of liver fibrosis. • Antifibrotic effect of APTR silencing is achieved by increasing p21.

  3. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    PubMed

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  4. ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation

    PubMed Central

    Zhang, Kun; Chang, Yanan; Shi, Zhemin; Han, Xiaohui; Han, Yawei; Yao, Qingbin; Hu, Zhimei; Cui, Hongmei; Zheng, Lina; Han, Tao; Hong, Wei

    2016-01-01

    Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis. PMID:27435808

  5. TIMP-1 mediates TGF-β-dependent crosstalk between hepatic stellate and cancer cells via FAK signaling.

    PubMed

    Park, Sang-A; Kim, Min-Jin; Park, So-Yeon; Kim, Jung-Shin; Lim, Woosung; Nam, Jeong-Seok; Yhong Sheen, Yhun

    2015-01-01

    Transforming growth factor-β (TGF-β) signaling plays a key role in progression and metastasis of HCC. This study was undertaken to gain the proof of concept of a small-molecule inhibitor of TGF-β type I receptor kinase, EW-7197 as a potent anti-cancer therapy for HCC. We identified tissue inhibitors of metalloproteinases-1 (TIMP-1) as one of the secreted proteins of hepatic stellate cells (HSCs) and a key mediator of TGF-β-mediated crosstalk between HSCs and HCC cells. TGF-β signaling led to increased expression of TIMP-1, which activates focal adhesion kinase (FAK) signaling via its interaction with CD63. Inhibition of TGF-β signaling using EW-7197 significantly attenuated the progression and intrahepatic metastasis of HCC in an SK-HEP1-Luc orthotopic-xenograft mouse model. In addition, EW-7197 inhibited TGF-β-stimulated TIMP-1 secretion by HSCs as well as the TIMP-1-induced proliferation, motility, and survival of HCC cells. Further, EW-7197 interrupted TGF-β-mediated epithelial-to-mesenchymal transition and Akt signaling, leading to significant reductions in the motility and anchorage-independent growth of HCC cells. In conclusion, we found that TIMP-1 mediates TGF-β-regulated crosstalk between HSCs and HCC cells via FAK signaling. In addition, EW-7197 demonstrates potent in vivo anti-cancer therapeutic activity and may be a potential new anti-cancer drug of choice to treat patients with liver cancer. PMID:26549110

  6. HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis

    PubMed Central

    Son, Mi Kwon; Ryu, Ye-Lim; Jung, Kyung Hee; Lee, Hyunseung; Lee, Hee Seung; Yan, Hong Hua; Park, Heon Joo; Ryu, Ji-Kan; Suh, Jun–Kyu; Hong, Sungwoo; Hong, Soon-Sun

    2013-01-01

    Hepatic stellate cells (HSCs) are the primary source of matrix components in liver disease such as fibrosis. Phosphatidylinositol 3-kinase (PI3K) signaling in HSCs has been shown to induce fibrogenesis. In this study, we evaluated the anti-fibrotic activity of a novel imidazopyridine analogue (HS-173) in human HSCs as well as mouse liver fibrosis. HS-173 strongly suppressed the growth and proliferation of HSCs and induced the arrest at the G2/M phase and apoptosis in HSCs. Furthermore, it reduced the expression of extracellular matrix components such as collagen type I, which was confirmed by an in vivo study. We also observed that HS-173 blocked the PI3K/Akt signaling pathway in vitro and in vivo. Taken together, HS-173 suppressed fibrotic responses such as cell proliferation and collagen synthesis by blocking PI3K/Akt signaling. Therefore, we suggest that this compound may be an effective therapeutic agent for ameliorating liver fibrosis through the inhibition of PI3K signaling. PMID:24326778

  7. Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2

    PubMed Central

    Xu, Jianfeng; Li, Jie; Hong, Zaifa; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy. PMID:26758420

  8. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis.

    PubMed

    Bansal, Ruchi; van Baarlen, Joop; Storm, Gert; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as "master producers" and macrophages as "master regulators", are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling.

  9. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

    PubMed Central

    Bansal, Ruchi; van Baarlen, Joop; Storm, Gert; Prakash, Jai

    2015-01-01

    Hepatic stellate cells (HSCs) known as “master producers” and macrophages as “master regulators”, are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling. PMID:26658360

  10. Periostin down-regulation attenuates the pro-fibrogenic response of hepatic stellate cells induced by TGF-β1.

    PubMed

    Hong, Li; Shejiao, Dai; Fenrong, Chen; Gang, Zhao; Lei, Dong

    2015-10-01

    Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)-β1-induced HSC activation remains unclear. We used RT-PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α-smooth muscle actin (α-SMA), collagen I, TGF-β1, p-Smad2 and p-Smad3 were determined by western blot. Our study found that periostin was up-regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA-periostin suppressed TGF-β1-induced HSC proliferation. The HSC transfected with siRNA-periostin significantly inhibited TGF-β1-induced expression levels of α-SMA and collagen I. Furthermore, TGF-β1 stimulated the expression of periostin, and siRNA-periostin attenuated TGF-β1-induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF-β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.

  11. CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism

    PubMed Central

    Wilhelm, Annika; Aldridge, Victoria; Haldar, Debashis; Naylor, Amy J; Weston, Christopher J; Hedegaard, Ditte; Garg, Abhilok; Fear, Janine; Reynolds, Gary M; Croft, Adam P; Henderson, Neil C; Buckley, Christopher D; Newsome, Philip N

    2016-01-01

    Introduction CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. Objective To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. Design CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248−/− and wild-type controls with carbon tetrachloride (CCl4) treatment. Results Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248−/− mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248−/− mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248−/− HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248−/− HSC was confirmed by significantly reduced c-fos expression in CD248−/− HSC compared with wild-type HSC. Conclusions Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury. PMID:26078290

  12. The Role of Lipin-1 in the Regulation of Fibrogenesis and TGF-β Signaling in Hepatic Stellate Cells.

    PubMed

    Jang, Chang Ho; Kim, Kyu Min; Yang, Ji Hye; Cho, Sam Seok; Kim, Seung Jung; Shin, Sang Mi; Cho, Il Je; Ki, Sung Hwan

    2016-09-01

    The adipogenic transcriptional regulation was reported to inhibit transdifferentiation of hepatic stellate cells (HSCs), which constitute the main fibrogenic cell type in the liver. Lipin-1 exhibits a dual function: an enzyme that catalyzes the conversion of phosphatidate to diacylglycerol and a transcriptional regulator. However, the involvement of Lipin-1 in the regulation of transforming growth factor-β (TGF-β) signaling and fibrogenesis in HSCs is not fully understood. Here, we showed that Lipin-1 was downregulated in activated primary HSCs and TGF-β-treated LX-2 cells, immortalized human HSC cell lines. The downregulation of Lipin-1 by TGF-β was not dependent on altered mRNA stability but rather on protein stability. Treatment of LX-2 cells with the proteasome inhibitor led to the accumulation of Lipin-1. Moreover, we observed a significant increase in Lipin-1 polyubiquitination. Overexpression of Lipin-1 attenuated TGF-β-induced fibrogenic gene expression. In addition, Lipin-1 inhibited TGF-β-mediated activation of Sma and Mad-related family (SMAD), a major transcription factor that transduces intracellular signals from TGF-β. Resveratrol, a well-known natural polyphenolic antioxidant, is known to inhibit liver fibrosis, although its mechanism of action remains unknown. Our data showed that resveratrol significantly increased the levels of Lipin-1 protein and mRNA in HSCs. Further investigation revealed that resveratrol blocked the polyubiquitination of Lipin-1. Resveratrol inhibited TGF-β-induced fibrogenic gene expression. TGF-β-induced SMAD binding element-luciferase reporter activity was significantly diminished by resveratrol with a simultaneous decrease in SMAD3 phosphorylation. Consistently, knockdown of the Lipin-1 gene using siRNA abolished the inhibitory effect of resveratrol. We conclude that Lipin-1 can antagonize HSC activation through the inhibition of TGF-β/SMAD signaling and that resveratrol may affect Lipin-1 gene induction and

  13. AMP-activated protein kinase inhibits TGF-β-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300.

    PubMed

    Lim, Joong-Yeon; Oh, Min-A; Kim, Won Ho; Sohn, Hee-Young; Park, Sang Ick

    2012-03-01

    Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate-activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)-β-induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX-2) and rat (CFSC-2G) HSC lines with TGF-β to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), metformin, or adiponectin lowered TGF-β-induced expression of COL1A and myofibroblast marker alpha-smooth muscle actin (α-SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α-SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad-dependent transcription, it did not affect TGF-β-stimulated phosphorylation, nuclear localization, or DNA-binding activity of Smad2/3. AICAR rather attenuated TGF-β-induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK-induced antifibrotic function in HSCs.

  14. Intercellular communication within the rat anterior pituitary gland: X. Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland.

    PubMed

    Shirasawa, Nobuyuki; Mabuchi, Yoshio; Sakuma, Eisuke; Horiuchi, Osamu; Yashiro, Takashi; Kikuchi, Motoshi; Hashimoto, Yasuo; Tsuruo, Yoshihiro; Herbert, Damon C; Soji, Tsuyoshi

    2004-05-01

    Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells

  15. Interleukin-22 ameliorates liver fibrogenesis by attenuating hepatic stellate cell activation and downregulating the levels of inflammatory cytokines

    PubMed Central

    Lu, Dong-Hong; Guo, Xiao-Yun; Qin, Shan-Yu; Luo, Wei; Huang, Xiao-Li; Chen, Mei; Wang, Jia-Xu; Ma, Shi-Jia; Yang, Xian-Wen; Jiang, Hai-Xing

    2015-01-01

    AIM: To investigate the effect of interleukin (IL)-22 on hepatic fibrosis in mice and the possible mechanism involved. METHODS: Liver fibrosis was induced in male BALB/c mice by CCl4. Recombinant IL-22 (rmIL-22) was administered intraperitoneally in CCl4-treated mice. Fibrosis was assessed by histology and Masson staining. The activation of hepatic stellate cells (HSCs) was investigated by analysis of α-smooth muscle actin expression. The frequencies of T helper (Th) 22 cells, Th17 cells and Th1 cells, the expression of inflammatory cytokines [IL-22, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-1β] and transcription factors [aryl hydrocarbon receptor (AHR), RAR-related orphan receptor (RORγt), T-bet] mRNA in the liver were investigated. In addition, the plasma levels of IL-22, IL-17A, IFN-γ, TNF-α, IL-6 and IL-1β were evaluated. RESULTS: Significant elevations in circulating Th22 cells, Th17 cells, Th1 cells, IL-22, IL-17A, and IFN-γ were observed in the hepatic fibrosis group compared with the control group (P < 0.01). Treatment with rmIL-22 in mice with hepatic fibrosis ameliorated the severity of hepatic fibrosis, which was confirmed by lower hepatic fibrosis pathological scores (P < 0.01). RmIL-22 decreased the frequencies of Th22 cells (6.71% ± 0.97% vs 8.09% ± 0.74%, P < 0.01), Th17 cells (4.34% ± 0.37% vs 5.71% ± 0.24%, P < 0.01), Th1 cells (3.09% ± 0.49% vs 4.91% ± 0.73%, P < 0.01), and the levels of IL-22 (56.23 ± 3.08 vs 70.29 ± 3.01, P < 0.01), IL-17A (30.74 ± 2.77 vs 45.68 ± 2.71, P < 0.01), and IFN-γ (74.78 ± 2.61 vs 124.89 ± 2.82, P < 0.01). Down-regulation of IL-22, IL-17A, IFN-γ, TNF-α, IL-6, IL-1β, AHR RORγt, and T-bet gene expression in the liver was observed in the rmIL-22 group (P < 0.01). CONCLUSION: The frequencies of Th22, Th17 and Th1 cells are elevated in hepatic fibrosis. RmIL-22 can attenuate HSC activation and down-regulate the levels of inflammatory cytokines, thereby ameliorating

  16. Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-beta1 inhibits fibrogenic activities of hepatic stellate cells.

    PubMed

    Arias, Monica; Lahme, Birgit; Van de Leur, Eddy; Gressner, Axel M; Weiskirchen, Ralf

    2002-06-01

    Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.

  17. Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling

    PubMed Central

    Bai, Liang; Tao, Yongqing; Wang, Suying; Zhi, Dexian

    2016-01-01

    Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-XL, and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis. PMID:27110557

  18. Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling.

    PubMed

    Wang, Liwen; Yue, Zhiwei; Guo, Mengzheng; Fang, Lianying; Bai, Liang; Li, Xinyu; Tao, Yongqing; Wang, Suying; Liu, Qiang; Zhi, Dexian; Zhao, Hui

    2016-01-01

    Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-X L , and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis. PMID:27110557

  19. Bone marrow cell transplantation is associated with fibrogenic cells apoptosis during hepatic regeneration in cholestatic rats.

    PubMed

    Nunes de Carvalho, Simone; da Cunha Lira, Dalvaci; Costa Cortez, Erika Afonso; de Andrade, Daniela Caldas; Thole, Alessandra Alves; Stumbo, Ana Carolina; de Carvalho, Lais

    2013-04-01

    Liver fibrosis is accompanied by hepatocyte death and proliferation of α-SMA(+) fibrogenic cells (activated hepatic stellate cells and myofibroblasts), which synthesize extracellular matrix components that contribute to disorganization of the hepatic parenchyma and loss of liver function. Therefore, apoptosis of these fibrogenic cells is important to hepatic regeneration. This study aimed to analyze the effect of cell therapy using bone marrow mononuclear cell (BMMNC) transplantation on α-SMA expression and on apoptosis of hepatic cells during liver fibrosis induced by bile duct ligation (BDL). Livers were collected from normal rats, fibrotic rats after 14 and 21 days of BDL, and rats that received BMMNC at 14 days of BDL and were analyzed after 7 days. Apoptosis in fibrogenic cells was analyzed by immunoperoxidase, confocal microscopy, and Western blotting, and liver regeneration was assessed by proliferating cell nuclear antigen staining. Results showed that caspase-3 and proliferating cell nuclear antigen expression were significantly increased in the BMMNC-treated group. Additionally, confocal microscopy analysis showed cells coexpressing α-SMA and caspase-3 in these animals, suggesting fibrogenic cell death. These results suggest a novel role for BMMNC in liver regeneration during fibrotic disease by stimulating fibrogenic cells apoptosis and hepatocyte proliferation, probably through secretion of specific cytokines that modulate the hepatic microenvironment toward an antifibrogenic balance.

  20. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    PubMed

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  1. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    PubMed Central

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-01-01

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis. PMID:27420058

  2. Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.

    PubMed

    Ramani, Komal; Donoyan, Shant; Tomasi, Maria Lauda; Park, Sunhee

    2015-05-01

    Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.

  3. Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells.

    PubMed

    Park, Soo-Jung; Sohn, Hee-Young; Yoon, Jeongsook; Park, Sang Ick

    2009-10-01

    Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP(L/S) expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP(L/S) expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP(L/S)-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP(L/S), consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis. PMID:19470406

  4. MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

    SciTech Connect

    Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

    2014-04-18

    Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

  5. HIV and HCV Co-Culture Promotes Profibrogenic Gene Expression through an Epimorphin-Mediated ERK Signaling Pathway in Hepatic Stellate Cells.

    PubMed

    Shi, Lei; Qin, Enqiang; Zhou, Junnian; Zhao, Juanjuan; Nie, Weimin; Jiang, Tianjun; Chen, Weiwei; Wu, Dan; Huang, Lei; Liu, Liying; Lv, Liping; Zhao, Min; Zhang, Zheng; Wang, Fusheng

    2016-01-01

    Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been a major cause of mortality in the highly active anti-retroviral therapy (HAART) era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs), remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2) in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1). The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1) in an extracellular signal-regulated kinase (ERK)-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection. PMID:27362846

  6. HIV and HCV Co-Culture Promotes Profibrogenic Gene Expression through an Epimorphin-Mediated ERK Signaling Pathway in Hepatic Stellate Cells

    PubMed Central

    Shi, Lei; Qin, Enqiang; Zhou, Junnian; Zhao, Juanjuan; Nie, Weimin; Jiang, Tianjun; Chen, Weiwei; Wu, Dan; Huang, Lei; Liu, Liying; Lv, Liping; Zhao, Min; Zhang, Zheng; Wang, Fusheng

    2016-01-01

    Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been a major cause of mortality in the highly active anti-retroviral therapy (HAART) era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs), remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2) in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1). The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1) in an extracellular signal-regulated kinase (ERK)-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection. PMID:27362846

  7. Anti-fibrotic effects of the Masson pine pollen aqueous extract on hepatic fibrosis rat model

    PubMed Central

    Cong, Tao; Jin, Xue-Yuan; Zhao, Lin; Ma, Long; Li, Rui-Sheng; Zhao, Ping; Guo, Chang-Jiang

    2015-01-01

    Aim: To observe the antifibrotic effects of Masson Pine Pollen aqueous extract. Methods: Adult Sprague-Dawley rats were randomly divided into control (CG), hepatic fibrosis model (MG), MPPAE low dose (LG), MPPAE high dose (HG), and MPP original powder (MPPOP; OG) groups. Each group was treated with specific protocols and sacrificed 8 weeks later. Multiple indicators such as serum transaminase, HE staining of the liver tissue, and relevant indexes to fibrosis were determined. Results: Severe hyperplasia of fibrous connective tissues was observed in livers of the MG group rats, while aspartate transaminase and alanine transaminase levels and collagen content obviously increased, superoxide dismutase and glutathione peroxidase activities and MMPs expression decreased, malondialdehyde (MDA) and 8-hydroxy-2’-deoxyguanosine concentrations increased, while mRNA expressions of hepatic stellate cell (HSC)-related cytokines such as transforming growth factor-β1 and platelet-derived growth factor, transcription factors such as nuclear factor-κB p65, and signaling protein α-smooth muscle actin were all increased significantly. Conclusions: MPPAE effectively inhibited the fibrotic process in this CCl4-induced hepatic fibrosis rat model. It may be associated with synergic functions of antioxidant activity, inhibitory activity on HSC proliferation, collagen synthesis, and MMPs expression induction. PMID:26191155

  8. Curcumin eliminates oxidized LDL roles in activating hepatic stellate cells by suppressing gene expression of lectin-like oxidized LDL receptor-1.

    PubMed

    Kang, Qiaohua; Chen, Anping

    2009-11-01

    Type II diabetes mellitus (T2DM) is often accompanied by non-alcoholic steatohepatitis (NASH) and associated with hypercholesterolemia, that is, increased levels of plasma low-density lipoprotein (LDL) and oxidized LDL (ox-LDL). Approximately one-third of NASH develops hepatic fibrosis. The role of hypercholesterolemia in T2DM and NASH-associated hepatic fibrogenesis remains obscure. We previously reported that the phytochemical curcumin inhibited the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis, and protected the liver from fibrogenesis in vitro and in vivo. The aims of this study are to evaluate the role of ox-LDL in activation of HSCs, to assess curcumin effects on eliminating the role of ox-LDL, and to further explore the underlying mechanisms. In this report, we observe that ox-LDL alters the expression of genes closely relevant to HSC activation, which is eliminated by curcumin. Curcumin suppresses gene expression of lectin-like oxidized LDL receptor-1 (LOX-1), leading to the blockade of the transport of extracellular ox-LDL into cells. This suppressive effect of curcumin results from the interruption of Wnt signaling and the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma). In conclusion, these results support our initial hypothesis and demonstrate that ox-LDL stimulates HSC activation, which is eliminated by curcumin by suppressing lox-1 expression by interrupting Wnt signaling and stimulating PPARgamma activity. These results provide novel insights into the role of ox-LDL in T2DM and NASH-associated hepatic fibrogenesis and mechanisms by which curcumin suppresses ox-LDL-induced HSC activation, as well as the implication of curcumin in the treatment of T2DM and NASH-associated hepatic fibrosis. PMID:19736547

  9. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell.

    PubMed

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S; Bachem, M G; Sun, Zi-Lin

    2016-01-01

    We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated "already primed by diabetic environment" ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2'-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis.

  10. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell

    PubMed Central

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S.; Bachem, M. G.; Sun, Zi-Lin

    2016-01-01

    We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated “already primed by diabetic environment” ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2′-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis. PMID:26697502

  11. Synaptic relationships between a multipolar stellate cell and a pyramidal neuron in the rat visual cortex. A combined Golgi-electron microscope study.

    PubMed

    Peters, A; Proskauer, C C

    1980-04-01

    Two synapsing and impregnated neurons in the rat visual cortex have been examined by a combined Golgi-electron microscope technique in which the Golgi precipitate is replaced by gold particles. One of the neurons is a stellate cell with smooth dendrites and a well impregnated axon, while the other is a layer III pyramidal neuron. Light microscopy showed some boutons from the axonal plexus of the stellate cell closely apposed to the soma and dendrites of the pyramid and it was predicted that synapses were present at these sites. An electron microscopic examination of serial thin sections, in which the profiles of the impregnated neurons are marked by their content of gold particles, showed most of these predicted synapses to exist. Indeed, axon terminals of the stellate cell formed five symmetric synapses with the cell body of the pyramid, one with the apical dendritic shaft and three with basal dendrites. Reasons are given for believing these synapses to be inhibitory. In addition, it was found that one of the axon terminals of the stellate cell synapsed with one of that cell's own dendrites. The significance of this finding is discussed.

  12. Matrix metalloproteinase-1 induction by diethyldithiocarbamate is regulated via Akt and ERK/miR222/ETS-1 pathways in hepatic stellate cells

    PubMed Central

    Liu, Tianhui; Wang, Ping; Cong, Min; Zhang, Dong; Liu, Lin; Li, Hongyi; Zhai, Qingling; Li, Zhuo; Jia, Jidong; You, Hong

    2016-01-01

    Matrix metalloproteinase-1 (MMP-1) plays an important role in fibrolysis by degrading excessively deposited collagen I and III. We previously demonstrated that diethyldithiocarbamate (DDC) up-regulates MMP-1 in hepatic stellate cells via the ERK1/2 and Akt signalling pathways. In the current study, we attempted to further explore the molecular mechanisms involved in the regulation of MMP-1. We treated a co-cultured system that included hepatocytes (C3A) and hepatic stellate cells (LX-2) with DDC. The data revealed that the transcriptional factor ETS-1, which is an important regulator of MMP-1, was up-regulated in LX-2 cells following DDC treatment. Furthermore, the up-regulation of MMP-1 by DDC has been abrogated through employing si-ETS-1 to block expression of ETS-1. We found that DDC significantly inhibited the expression of miR-222 in LX-2 cells. We transfected miR-222 mimic into LX-2 cells and then co-cultured the cells with C3A. The up-regulation of ETS-1 and MMP-1 in LX-2 cells treated with DDC were inhibited after miR-222 mimic transfection. These data indicate that DDC up-regulated MMP-1 in LX-2 cells through the miR-222/ETS-1 pathway. Finally, we treated the co-cultured system with an Akt inhibitor (T3830) and an ERK1/2 inhibitor (U0126). Both T3830 and U0126 blocked the suppression of miR-222 by DDC in LX-2. Collectively, these data indicate that DDC up-regulated MMP-1 in LX-2 cells through the Akt and ERK/miR-222/ETS-1 pathways. Our study provides experimental data that will aid the control of the process of fibrolysis in liver fibrosis prevention and treatment. PMID:27412967

  13. Matrix metalloproteinase-1 induction by diethyldithiocarbamate is regulated via Akt and ERK/miR222/ETS-1 pathways in hepatic stellate cells.

    PubMed

    Liu, Tianhui; Wang, Ping; Cong, Min; Zhang, Dong; Liu, Lin; Li, Hongyi; Zhai, Qingling; Li, Zhuo; Jia, Jidong; You, Hong

    2016-08-01

    Matrix metalloproteinase-1 (MMP-1) plays an important role in fibrolysis by degrading excessively deposited collagen I and III. We previously demonstrated that diethyldithiocarbamate (DDC) up-regulates MMP-1 in hepatic stellate cells via the ERK1/2 and Akt signalling pathways. In the current study, we attempted to further explore the molecular mechanisms involved in the regulation of MMP-1. We treated a co-cultured system that included hepatocytes (C3A) and hepatic stellate cells (LX-2) with DDC. The data revealed that the transcriptional factor ETS-1, which is an important regulator of MMP-1, was up-regulated in LX-2 cells following DDC treatment. Furthermore, the up-regulation of MMP-1 by DDC has been abrogated through employing si-ETS-1 to block expression of ETS-1. We found that DDC significantly inhibited the expression of miR-222 in LX-2 cells. We transfected miR-222 mimic into LX-2 cells and then co-cultured the cells with C3A. The up-regulation of ETS-1 and MMP-1 in LX-2 cells treated with DDC were inhibited after miR-222 mimic transfection. These data indicate that DDC up-regulated MMP-1 in LX-2 cells through the miR-222/ETS-1 pathway. Finally, we treated the co-cultured system with an Akt inhibitor (T3830) and an ERK1/2 inhibitor (U0126). Both T3830 and U0126 blocked the suppression of miR-222 by DDC in LX-2. Collectively, these data indicate that DDC up-regulated MMP-1 in LX-2 cells through the Akt and ERK/miR-222/ETS-1 pathways. Our study provides experimental data that will aid the control of the process of fibrolysis in liver fibrosis prevention and treatment.

  14. A rat model for hepatitis E virus

    PubMed Central

    Mishra, Niraj; Verbeken, Erik; Ramaekers, Kaat; Dallmeier, Kai

    2016-01-01

    ABSTRACT Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo. Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans). As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains). Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies. PMID:27483350

  15. Plumbagin Ameliorates CCl4-Induced Hepatic Fibrosis in Rats via the Epidermal Growth Factor Receptor Signaling Pathway

    PubMed Central

    Chen, Si; Chen, Yi; Chen, Bi; Cai, Yi-jing; Zou, Zhuo-lin; Wang, Jin-guo; Lin, Zhuo; Wang, Xiao-dong; Fu, Li-yun; Hu, Yao-ren; Chen, Yong-ping; Chen, Da-zhi

    2015-01-01

    Epidermal growth factor (EGF) and its signaling molecules, EGFreceptor (EGFR) and signal transducer and activator of transcription factor 3 (STAT3), have been considered to play a role in liver fibrosis and cirrhosis. Plumbagin (PL) is an extracted component from the plant and has been used to treat different kinds of cancer. However, its role in regulation of EGFR and STAT3 during liver fibrosis has not been investigated. In this study, the effects of PL on the regulation of EGFR and STAT3 were investigated in carbon tetrachloride (CCl4) induced liver fibrosis and hepatic stellate cells (HSC-T6). PL significantly attenuated liver injury and fibrosis in CCl4 treated rats. At concentrations of 2 to 6 μM, PL did not induce significant cytotoxicity of HSC-T6 cells. Moreover, PL reduced phosphorylation of EGFR and STAT3 in both fibrotic liver and heparin-binding EGF-like growth factor (HB-EGF) treated HSC-T6 cells. Furthermore, PL reduced the expression of α-SMA, EGFR, and STAT3 in both fibrotic liver and HB-EGF treated HSC-T6 cells. In conclusion, plumbagin could ameliorate the development of hepatic fibrosis through its downregulation of EGFR and STAT3 in the liver, especially in hepatic stellate cells. PMID:26550019

  16. Ontogeny of the rat hepatic adrenoceptors

    SciTech Connect

    McMillian, M.K.

    1985-01-01

    Hepatic alpha-1, alpha-2, and beta-2 adrenoceptors were characterized during development of the rat through Scatchard analysis of (/sup 3/H)-prazosin, (/sup 3/H)-rauwolscine and (/sup 125/I)-pindolol binding to washed particle membrane preparations. Major changes in adrenoceptor number occur shortly before birth and at weaning. The fetal rat liver is characterized by a large number of alpha-2 adrenoceptors which falls 10-20 fold at birth. The number of hepatic beta adrenoceptors decreases 30-50% during the third week after birth increases slightly at weaning, then decreases gradually in the adult. Hepatic alpha-1 adrenoceptor number increases 3-5 fold at weaning to become the predominant adrenoceptor in the adult rat liver. The basis for the fall in alpha-2 number at birth remains unclear. The fall in beta receptor number at the end of the second week post-natally appears dependent on increased insulin and corticosterone secretion as well as increased NE release form nerve terminals. The basis for the increase in beta number at weaning and the sex-dependent loss of beta function but not receptor number in the adult rat remains unknown. The dramatic increases in alpha-1 number and function at weaning are dependent on increased adrenocortical secretion, adrenalectomy prevents the normal. This effect of adrenocorticoids might be mediated through glycogen, as glycogen depletion during fasting decreases alpha-1 receptor number and function at weaning are dependent on increased adrenocortical secretion, adrenalectomy prevents the normal. This effect of adrenocorticoids might be mediated through glycogen, as glycogen depletion during fasting decreases alpha-1 receptor number and function. These findings suggest that hepatic adrenoceptor number adapts from the low carbohydrate diet of the suckling rat to the high carbohydrate diet of the adult at weaning.

  17. Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

    SciTech Connect

    Fowell, Andrew J.; Collins, Jane E.; Duncombe, Dale R.; Pickering, Judith A.; Rosenberg, William M.C.; Benyon, R. Christopher

    2011-04-08

    Research highlights: {yields} Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis. {yields} We used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. {yields} Specific silencing of TIMP-1, but not TIMP-2, significantly reduces HSC proliferation and is associated with reduced Akt phosphorylation. {yields} TIMP-1 is localised in part to the HSC nucleus. {yields} TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. -- Abstract: Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover

  18. Septin4_i1 Regulates Apoptosis in Hepatic Stellate Cells through Peroxisome Proliferator-Activated Receptor-γ/Akt/B-Cell Lymphoma 2 Pathway

    PubMed Central

    Zhu, Dandan; Wang, Jianxin; Sun, Xiaolei; Chen, Jinling; Pan, Jing; Xu, Tianhua; Qin, Yongwei; He, Xingxin; Huang, Caiqun

    2015-01-01

    Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-β/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression. PMID:25527525

  19. Septin4_i1 regulates apoptosis in hepatic stellate cells through peroxisome proliferator-activated receptor-γ/Akt/B-cell lymphoma 2 pathway.

    PubMed

    Zhu, Dandan; Wang, Jianxin; Sun, Xiaolei; Chen, Jinling; Duan, Yinong; Pan, Jing; Xu, Tianhua; Qin, Yongwei; He, Xingxin; Huang, Caiqun

    2015-03-01

    Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-β/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression. PMID:25527525

  20. Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis

    PubMed Central

    Sun, Lina; Fan, Zhiwen; Chen, Junliang; Tian, Wenfang; Li, Min; Xu, Huihui; Wu, Xiaoyan; Shao, Jing; Bian, Yaoyao; Fang, Mingming; Xu, Yong

    2016-01-01

    Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis. PMID:27323886

  1. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells

    PubMed Central

    Reiner, Andrew H.; Coll, Mar; Verhulst, Stefaan; Mannaerts, Inge; Øie, Cristina I.; Smedsrød, Bård; Najimi, Mustapha; Sokal, Etienne; Luttun, Aernout; Sancho-Bru, Pau; Collas, Philippe; van Grunsven, Leo A.

    2015-01-01

    Background & Aims Liver fibrogenesis – scarring of the liver that can lead to cirrhosis and liver cancer – is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. Material and methods Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. Results We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. Conclusions Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation. PMID:26353929

  2. Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

    PubMed Central

    Fickert, Peter; Fuchsbichler, Andrea; Moustafa, Tarek; Wagner, Martin; Zollner, Gernot; Halilbasic, Emina; Stöger, Ulrike; Arrese, Marco; Pizarro, Margarita; Solís, Nancy; Carrasco, Gonzalo; Caligiuri, Alessandra; Sombetzki, Martina; Reisinger, Emil; Tsybrovskyy, Oleksiy; Zatloukal, Kurt; Denk, Helmut; Jaeschke, Hartmut; Pinzani, Massimo; Trauner, Michael

    2009-01-01

    The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR−/−) by CCl4 intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl4-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands. PMID:19910507

  3. Multivesicular stellate cells in primary biliary cirrhosis.

    PubMed

    Cameron, R G; Neuman, M G; Shear, N H; Blendis, L M

    1997-09-01

    Stellate cells have only recently received attention in patients with primary biliary cirrhosis (PBC). We used electron microscopy and morphometry to perform a qualitative and quantitative examination of lipid-storing activity of stellate cells in liver biopsies of 26 patients with noncirrhotic and cirrhotic PBC. Parallel with this study, a comparative analysis of the morphology of stellate cells in 51 patients with livers of normal histology was performed. There was a marked increase in the total number of lipid vesicles in stellate cells in all PBC patients when compared to livers with normal histology. Multiple multivesicular stellate cells were seen in the livers of 21 out of 26 patients with PBC. There were 11 to 28 lipid vesicles per multivesicular stellate cell from 1 micromol/L to 5 micromol/L in diameter per lipid vesicle. Hepatocytes showed little or no steatosis in 24 out of 26 (92%) PBC patients. Multivesicular stellate cells were not seen in female patients with normal liver histology. These results suggest that there is an alteration in hepatic lipid-storage that involves stellate cells in PBC, which could be an early manifestation of this disease. Its significance remains to be determined.

  4. Multivesicular stellate cells in primary biliary cirrhosis.

    PubMed

    Cameron, R G; Neuman, M G; Shear, N; Blendis, L M

    1997-10-01

    Stellate cells have only recently received attention in patients with primary biliary cirrhosis (PBC). We have used electron microscopy and morphometry to perform a qualitative and quantitative examination of lipid-storing activity of stellate cells in liver biopsies of 26 patients with noncirrhotic and cirrhotic PBC. In parallel with this study, a comparative analysis of the morphology of stellate cells in 51 patients with livers of normal histology was performed. There was a marked increased in the total number of lipid vesicles in stellate cells in all PBC patients when compared with livers with normal histology. Multiple multivesicular stellate cells were seen in the livers of 21 of 26 patients with PBC. There were 11 to 28 lipid vesicles per multivesicular stellate cell in sizes of 1 microm to 5 microm in diameter per lipid vesicle. Hepatocytes showed little or no steatosis in 24 of 26 (92%) PBC patients. Multivesicular stellate cells were not seen in female patients with normal liver histology. These results suggest that there is an alteration in hepatic lipid storage that involves stellate cells in PBC that could be an early manifestation of this disease. Its significance remains to be elucidated.

  5. Low Cardiac Output Leads Hepatic Fibrosis in Right Heart Failure Model Rats

    PubMed Central

    Fujimoto, Yoshitaka; Urashima, Takashi; Shimura, Daisuke; Ito, Reiji; Kawachi, Sadataka; Kajimura, Ichige; Akaike, Toru; Kusakari, Yoichiro; Fujiwara, Masako; Ogawa, Kiyoshi; Goda, Nobuhito; Ida, Hiroyuki; Minamisawa, Susumu

    2016-01-01

    Background Hepatic fibrosis progresses with right heart failure, and becomes cardiac cirrhosis in a severe case. Although its causal factor still remains unclear. Here we evaluated the progression of hepatic fibrosis using a pulmonary artery banding (PAB)-induced right heart failure model and investigated whether cardiac output (CO) is responsible for the progression of hepatic fibrosis. Methods and Results Five-week-old Sprague-Dawley rats divided into the PAB and sham-operated control groups. After 4 weeks from operation, we measured CO by echocardiography, and hepatic fibrosis ratio by pathological examination using a color analyzer. In the PAB group, CO was significantly lower by 48% than that in the control group (78.2±27.6 and 150.1±31.2 ml/min, P<0.01). Hepatic fibrosis ratio and serum hyaluronic acid, an index of hepatic fibrosis, were significantly increased in the PAB group than those in the control group (7.8±1.7 and 1.0±0.2%, P<0.01, 76.2±27.5 and 32.7±7.5 ng/ml, P<0.01). Notably, the degree of hepatic fibrosis significantly correlated a decrease in CO. Immunohistological analysis revealed that hepatic stellate cells were markedly activated in hypoxic areas, and HIF-1α positive hepatic cells were increased in the PAB group. Furthermore, by real-time PCR analyses, transcripts of profibrotic and fibrotic factors (TGF-β1, CTGF, procollargen I, procollargen III, MMP 2, MMP 9, TIMP 1, TIMP 2) were significantly increased in the PAB group. In addition, western blot analyses revealed that the protein level of HIF-1α was significantly increased in the PAB group than that in the control group (2.31±0.84 and 1.0±0.18 arbitrary units, P<0.05). Conclusions Our study demonstrated that low CO and tissue hypoxia were responsible for hepatic fibrosis in right failure heart model rats. PMID:26863419

  6. Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

    PubMed

    Resheq, Yazid J; Li, Ka-Kit; Ward, Stephen T; Wilhelm, Annika; Garg, Abhilok; Curbishley, Stuart M; Blahova, Miroslava; Zimmermann, Henning W; Jitschin, Regina; Mougiakakos, Dimitrios; Mackensen, Andreas; Weston, Chris J; Adams, David H

    2015-03-15

    Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

  7. S-Adenosylmethionine suppresses the expression of Smad3/4 in activated human hepatic stellate cells via Rac1 promoter methylation

    PubMed Central

    BIAN, KANGQI; ZHANG, FENG; WANG, TINGTING; ZOU, XIAOPING; DUAN, XUHONG; CHEN, GUANGXIA; ZHUGE, YUZHENG

    2016-01-01

    The aim of the present study was to investigate whether S-adenosylmethionine (SAM) was able to suppress activated human hepatic stellate cells (HSCs). Human LX-2 HSCs were cultured with SAM or NSC23766, and were transfected with plasmids encoding ras-related C3 botulinum toxin substrate 1 (Rac1) protein or an empty expression vector. Cell proliferation was detected by Cell Counting Kit-8. Cell migration and invasion were determined using the Transwell assay. The expression levels of Rac1 and Smad3/4 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) or western blotting. The methylation status of Rac1 promoters was measured by methylation-specific PCR. The results demonstrated that SAM and NSC23766 suppressed the expression of Smad3/4 in LX-2 cells. The overexpression of Rac1 enhanced the proliferation, migration and invasion of LX-2 cells. In addition, compared with the control groups, a marked increase was observed in the protein expression levels of Smad3/4 in the LX-2 cells transfected with Rac1 plasmids. The methylation-specific PCR findings showed that SAM increased the methylation of Rac1 promoters. The results of the present study suggested that Rac1 enhanced the expression of Smad3/4 in activated HSCs; however, this increase may be suppressed by SAM-induced methylation of Rac1 promoters. PMID:26986629

  8. Fuzheng Huayu Recipe Ameliorates Liver Fibrosis by Restoring Balance between Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Hepatic Stellate Cells.

    PubMed

    Pan, Qin; Wang, Yu-Qin; Li, Guang-Ming; Duan, Xiao-Yan; Fan, Jian-Gao

    2015-01-01

    Activation of hepatic stellate cells (HSCs) depending on epithelial-to-mesenchymal transition (EMT) reflects the key event of liver fibrosis. Contrastively, mesenchymal-to-epithelial transition (MET) of HSCs facilitates the fibrosis resolution. Here we investigated the effect of Fuzheng Huayu (FZHY) recipe, a Chinese herbal decoction made of Radix Salviae Miltiorrhizae, Semen Persicae, Cordyceps sinensis, Pollen Pini, and Gynostemma pentaphyllum, on liver fibrosis concerning the balance of EMT and MET in HSCs. In contrast to the increased TGF-β 1/BMP-7 ratio in activated HSCs, FZHY administration induced significant upregulation of BMP-7 and downregulation of TGF-β 1 at both transcription and translation levels. Restoration of TGF-β 1/BMP-7 ratio inhibited the expression of p38 MAPK and phosphorylated p38 MAPK, resulting in the reversal of epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) as characterized by the abolishment of EMT markers (α-SMA and desmin) and reoccurrence of MET marker (E-cadherin). In vivo treatment of FZHY recipe also demonstrated the statistical reduction of activated HSCs with EMT phenotype, which attenuated the carbon tetrachloride- (CCl4-) induced liver fibrosis in a dose-dependent manner. These findings may highlight a novel antifibrotic role of FZHY recipe on the basis of rebalancing EMT and MET in HSCs. PMID:26881209

  9. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway.

    PubMed

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  10. Egg antigen p40 of Schistosoma japonicum promotes senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Xu, Tianhua; Zhu, Dandan; Wang, Jianxin; Huang, Caiqun; Lyu, Lei; Hu, Bin; Sun, Wei; Duan, Yinong

    2016-01-01

    Liver fibrosis is a serious disease that is characterized by the excess deposition of extracellular matrix (ECM) components. Activated hepatic stellate cells (HSCs) are a major source of ECM and serve as a key regulator in liver fibrogenesis. Inactivation of HSCs is essential for liver fibrotic regression. The present study explores the underlying mechanisms of Schistosoma japonicum egg antigen p40 (Sjp40) promoting senescence in HSCs and antifibrosis. For the first time we report that Sjp40 inhibits the activation and proliferation of an immortalized human HSC line (LX-2 cells) and promotes cellular senescence and cell cycle arrest. Sjp40 through action on the STAT3/p53/p21 pathway triggered cellular senescence, while knockdown of p53 or STAT3 partly restored cell senescence. In addition, Sjp40-induced cellular senescence caused LX-2 cells to be more sensitive to a human NK cell line (YT cells). Together these findings provide novel insights into the mechanism of antifibrosis and may have implications for the development of antifibrosis therapies. PMID:27468691

  11. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  12. Functional Study of miR-27a in Human Hepatic Stellate Cells by Proteomic Analysis: Comprehensive View and a Role in Myogenic Tans-Differentiation

    PubMed Central

    Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

    2014-01-01

    We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA. PMID:25265485

  13. MicroRNA-122 Inhibits the Production of Inflammatory Cytokines by Targeting the PKR Activator PACT in Human Hepatic Stellate Cells.

    PubMed

    Nakamura, Masato; Kanda, Tatsuo; Sasaki, Reina; Haga, Yuki; Jiang, Xia; Wu, Shuang; Nakamoto, Shingo; Yokosuka, Osamu

    2015-01-01

    MicroRNA-122 (miR-122) is one of the most abundant miRs in the liver. Previous studies have demonstrated that miR-122 plays a role in inflammation in the liver and functions in hepatic stellate cells (HSCs), which reside in the space of Disse. Here, we showed that the transient inhibition of PKR-activating protein (PACT) expression, by miR-122 or siRNA targeting of PACT, suppressed the production of proinflammatory cytokines, such as interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, in human HSC LX-2. Sequence and functional analyses confirmed that miR-122 directly targeted the 3'-untranslated region of PACT. Immunofluorescence analysis revealed that miR-122 blocked NF-κB-nuclear translocation in LX-2 cells. We also showed that conditioned medium from miR-122-transfected LX-2 cells suppressed human monocyte-derived THP-1 cell migration. Taken together, our study indicates that miR-122 may downregulate cytokine production in HSCs and macrophage chemotaxis and that the targeting of miR-122 may have therapeutic potential for preventing the progression of liver diseases. PMID:26636761

  14. Circulating miRNAs as Predictor Markers for Activation of Hepatic Stellate Cells and Progression of HCV-Induced Liver Fibrosis

    PubMed Central

    El-Ahwany, Eman; Nagy, Faten; Zoheiry, Mona; Shemis, Mohamed; Nosseir, Mona; Taleb, Hoda Abu; El Ghannam, Maged; Atta, Rafaat; Zada, Suher

    2016-01-01

    Introduction Liver fibrosis is the excessive accumulation of extracellular matrix that occurs by activation of hepatic stellate cells (HSCs), which has been identified as the major driver of liver fibrosis. Several studies confirmed that miRNAs have regulatory effects on the activation of HSCs by affecting the signaling pathways. The aim of this study was to develop non-invasive diagnostic markers by measuring different circulating miRNAs in serum as predictor markers for early diagnosis of liver fibrosis and its progression. Methods In this case-control study, we enrolled 66 subjects with chronic hepatitis C (CHC) with early stage of fibrosis and 65 subjects with CHC with late-stage fibrosis. Also, 40 subjects were included as normal controls. The six main miRNAs, i.e., miR-138, miR-140, miR-143, miR-325, miR-328, and miR-349, were measured using the reverse transcription-polymerase chain reaction. Results In the cases of CHC both with early and late stage of fibrosis, the circulating levels of the six main miRNAs were significantly higher than the levels in the control group. ROC analysis indicated that the sensitivity and specificity of miR-138 were 89.3% and 71.43%, respectively, in the early stage of fibrosis. In the late stage, the sensitivity and specificity of miR-138 were 89.3 and 93.02%, respectively, whereas, for miR-143, they were 75.0 and 88.4%, respectively. Conclusions Circulating miR-138 could serve as a non-invasive biomarker for the detection of early fibrosis. Also, miR-138 and miR-143 could be specific biomarkers for indicating the late stage of liver fibrosis. PMID:26955452

  15. The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

    PubMed

    Wojtalla, Alexandra; Herweck, Frank; Granzow, Michaela; Klein, Sabine; Trebicka, Jonel; Huss, Sebastian; Lerner, Raissa; Lutz, Beat; Schildberg, Frank Alexander; Knolle, Percy Alexander; Sauerbruch, Tilman; Singer, Manfred Vincenz; Zimmer, Andreas; Siegmund, Sören Volker

    2012-04-15

    The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

  16. Calcium channel blockers ameliorate iron overload-associated hepatic fibrosis by altering iron transport and stellate cell apoptosis.

    PubMed

    Zhang, Ying; Zhao, Xin; Chang, Yanzhong; Zhang, Yuanyuan; Chu, Xi; Zhang, Xuan; Liu, Zhenyi; Guo, Hui; Wang, Na; Gao, Yonggang; Zhang, Jianping; Chu, Li

    2016-06-15

    Liver fibrosis is the principal cause of morbidity and mortality in patients with iron overload. Calcium channel blockers (CCBs) can antagonize divalent cation entry into renal and myocardial cells and inhibit fibrogenic gene expression. We investigated the potential of CCBs to resolve iron overload-associated hepatic fibrosis. Kunming mice were assigned to nine groups (n=8 per group): control, iron overload, deferoxamine, high and low dose verapamil, high and low dose nimodipine, and high and low dose diltiazem. Iron deposition and hepatic fibrosis were measured in mouse livers. Expression levels of molecules associated with transmembrane iron transport were determined by molecular biology approaches. In vitro HSC-T6 cells were randomized into nine groups (the same groups as the mice). Changes in proliferation, apoptosis, and metalloproteinase expression in cells were detected to assess the anti-fibrotic effects of CCBs during iron overload conditions. We found that CCBs reduced hepatic iron content, intracellular iron deposition, the number of hepatic fibrotic areas, collagen expression levels, and hydroxyproline content. CCBs rescued abnormal expression of α1C protein in L-type voltage-dependent calcium channel (LVDCC) and down-regulated divalent metal transporter-1 (DMT-1) expression in mouse livers. In iron-overloaded HSC-T6 cells, CCBs reduced iron deposition, inhibited proliferation, induced apoptosis, and elevated expression of matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1). CCBs are potential therapeutic agents that can be used to address hepatic fibrosis during iron overload. They resolve hepatic fibrosis probably correlated with regulating transmembrane iron transport and inhibiting HSC growth. PMID:27095094

  17. Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats.

    PubMed

    Liu, Cheng; Tao, Qing; Sun, Mingyu; Wu, Jim Z; Yang, Wengang; Jian, Ping; Peng, Jinghua; Hu, Yiyang; Liu, Chenghai; Liu, Ping

    2010-12-01

    Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver

  18. Gene profile of chemokines on hepatic stellate cells of schistosome-infected mice and antifibrotic roles of CXCL9/10 on liver non-parenchymal cells.

    PubMed

    Liang, Yue-jin; Luo, Jie; Lu, Qiao; Zhou, Ying; Wu, Hai-wei; Zheng, Dan; Ren, Yong-ya; Sun, Ke-yi; Wang, Yong; Zhang, Zhao-song

    2012-01-01

    Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune

  19. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways

    SciTech Connect

    Zhang, Feng; Ni, Chunyan; Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li; Lu, Yin; Zheng, Shizhong

    2012-11-15

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H{sub 2}O{sub 2}), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H{sub 2}O{sub 2} at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H{sub 2}O{sub 2}-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H{sub 2}O{sub 2} stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H{sub 2}O{sub 2}-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation.

  20. Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

    PubMed

    Ye, Zhaoyang; Cheng, Kun; Guntaka, Ramareddy V; Mahato, Ram I

    2006-01-01

    Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

  1. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    SciTech Connect

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  2. Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

    PubMed Central

    Ge, Jingjing; Chang, Na; Zhao, Zhongxin; Tian, Lei; Duan, Xianghui; Yang, Lin; Li, Liying

    2016-01-01

    RNA-binding protein HuR mediates transforming growth factor (TGF)-β1-induced profibrogenic actions. Up-regulation of Sphingosine kinase 1 (SphK1) is involved in TGF-β1-induced activation of hepatic stellate cells (HSCs) in liver fibrogenesis. However, the molecular mechanism of TGF-β1 regulates SphK1 remains unclear. This study was designed to investigate the role of HuR in TGF-β1-induced SphK1 expression and identify a new molecular mechanism in liver fibrogenensis. In vivo, HuR expression was increased, translocated to cytoplasm, and bound to SphK1 mRNA in carbon tetrachloride- and bile duct ligation-induced mouse fibrotic liver. HuR mRNA expression had a positive correlation with mRNA expressions of SphK1 and fibrotic markers, α-smooth muscle actin (α-SMA) and Collagen α1(I), respectively. In vitro, up-regulation of SphK1 and activation of HSCs stimulated by TGF-β1 depended on HuR cytoplasmic accumulation. The effects of TGF-β1 were diminished when HuR was silenced or HuR cytoplasmic translocation was blocked. Meanwhile, overexpression of HuR mimicked the effects of TGF-β1. Furthermore, TGF-β1 prolonged half-life of SphK1 mRNA by promoting its binding to HuR. Pharmacological or siRNA-induced SphK1 inhibition abrogated HuR-mediated HSC activation. In conclusion, our data suggested that HuR bound to SphK1 mRNA and played a crucial role in TGF-β1-induced HSC activation. PMID:26912347

  3. Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/MyD88/NF-κB signaling pathway

    PubMed Central

    Liu, Min; Xu, Youwei; Han, Xu; Yin, Lianhong; Xu, Lina; Qi, Yan; Zhao, Yanyan; Liu, Kexin; Peng, Jinyong

    2015-01-01

    The present work aimed to investigate the activities and underlying mechanisms of dioscin against alcoholic liver fibrosis (ALF). In vivo liver fibrosis in mice was induced by an alcoholic liquid diet, and in vitro studies were performed on activated HSC-T6 and LX2 cells treated with lipopolysaccharide. Our results showed that dioscin significantly attenuated hepatic stellate cells (HSCs) activation, improved collagen accumulation, and attenuated inflammation through down-regulating the levels of myeloid differentiation factor 88 (MyD88), nuclear factor κB (NF-κB), interleukin (IL)-1, IL-6 and tumour necrosis factor-α by decreasing Toll-like receptor (TLR)4 expression both in vivo and in vitro. TLR4 overexpression was also decreased by dioscin, leading to the markedly down-regulated levels of MyD88, NF-κB, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) in cultured HSCs. Suppression of cellular MyD88 by ST2825 or abrogation of NF-κB by pyrrolidine dithiocarbamate eliminated the inhibitory effects of dioscin on the levels of TGF-β1, α-SMA and COL1A1. In a word, dioscin exhibited potent effects against ALF via altering TLR4/MyD88/NF-κB signaling pathway, which provided novel insights into the mechanisms of this compound as an antifibrogenic candidate for the treatment of ALF in the future. PMID:26655640

  4. Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways.

    PubMed

    Huang, Yan; Li, Xiaohui; Wang, Yarui; Wang, Huan; Huang, Cheng; Li, Jun

    2014-09-01

    Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1α1 and α-smooth muscle actin (α-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis

  5. Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

    PubMed Central

    Lan, Tian; Kisseleva, Tatiana; Brenner, David A.

    2015-01-01

    Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC. PMID:26222337

  6. Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1.

    PubMed

    Ge, Jingjing; Chang, Na; Zhao, Zhongxin; Tian, Lei; Duan, Xianghui; Yang, Lin; Li, Liying

    2016-02-25

    RNA-binding protein HuR mediates transforming growth factor (TGF)-β1-induced profibrogenic actions. Up-regulation of Sphingosine kinase 1 (SphK1) is involved in TGF-β1-induced activation of hepatic stellate cells (HSCs) in liver fibrogenesis. However, the molecular mechanism of TGF-β1 regulates SphK1 remains unclear. This study was designed to investigate the role of HuR in TGF-β1-induced SphK1 expression and identify a new molecular mechanism in liver fibrogenensis. In vivo, HuR expression was increased, translocated to cytoplasm, and bound to SphK1 mRNA in carbon tetrachloride- and bile duct ligation-induced mouse fibrotic liver. HuR mRNA expression had a positive correlation with mRNA expressions of SphK1 and fibrotic markers, α-smooth muscle actin (α-SMA) and Collagen α1(I), respectively. In vitro, up-regulation of SphK1 and activation of HSCs stimulated by TGF-β1 depended on HuR cytoplasmic accumulation. The effects of TGF-β1 were diminished when HuR was silenced or HuR cytoplasmic translocation was blocked. Meanwhile, overexpression of HuR mimicked the effects of TGF-β1. Furthermore, TGF-β1 prolonged half-life of SphK1 mRNA by promoting its binding to HuR. Pharmacological or siRNA-induced SphK1 inhibition abrogated HuR-mediated HSC activation. In conclusion, our data suggested that HuR bound to SphK1 mRNA and played a crucial role in TGF-β1-induced HSC activation.

  7. Hepatic stellate cells inhibit T cells through active TGFβ1 from a cell-surface-bound latent TGFβ1/GARP complex

    PubMed Central

    Li, Yan; Kim, Byung-Gyu; Qian, Shiguang; Letterio, John J.; Fung, John J.; Lu, Lina; Lin, Feng

    2016-01-01

    Hepatic stellate cells (HSCs) inhibit T cells, a process that could help the liver to maintain its immunoprivileged status. HSCs secrete latent TGFβ1, but the detailed mechanisms by which latent TGFβ1 is activated and whether it plays any role in HSC-mediated T-cell suppression remain unclear. Glycoprotein A repetitions predominant (GARP) is a surface marker of activated regulatory T cells (Tregs). GARP binds latent TGFβ1 for its activation, which is critical for Tregs to suppress effector T cells; however, it is still unclear whether GARP is present on HSCs and whether it has any impact on HSC function. In this study, we found that TGFβ1+/− HSCs, which produce reduced levels of TGFβ1, showed decreased potency in inhibiting T cells. We also found that pharmaceutical or genetic inhibition of the TGFβ1 signaling pathway reduced the T-cell-inhibiting activity of HSCs. In addition, using isolated primary HSCs, we demonstrated that GARP was constitutively expressed on HSCs. Blocking GARP function or knocking down GARP expression significantly impaired the potency of HSCs to suppress the proliferation of and IFNγ production from activated T cells, suggesting that GARP is important for HSCs to inhibit T cells. These results demonstrate the unexpected presence of GARP on HSCs and its significance in regard to the ability of HSCs to activate latent TGFβ1 and thereby inhibit T cells. Our study reveals a new mechanism for HSC-mediated immune regulation and potentially for other conditions, such as liver fibrosis, that involve HSC-secreted TGFβ1. PMID:26246140

  8. Effect of pentoxifylline on arachidonic acid metabolism, neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell.

    PubMed

    Cardoso, Carla C A; Paviani, Ernani R; Cruz, Lavínia A; Guma, Fátima C R; Borojevic, Radovan; Guaragna, Regina M

    2003-12-01

    In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A2 (PLA2), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1-2.5 microM), it increased the quantity of stored lipids. Retinol and PTF (5 microM and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA2 activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE2, but not of PGF2. However, when in presence of retinol, we observed a slightly higher increase in PGE2 and PGF2a production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC. PMID:14674680

  9. Role of TGF-β signaling in differentiation of mesothelial cells to vitamin A-poor hepatic stellate cells in liver fibrosis.

    PubMed

    Li, Yuchang; Lua, Ingrid; French, Samuel W; Asahina, Kinji

    2016-02-15

    Mesothelial cells (MCs) form a single layer of the mesothelium and cover the liver surface. A previous study demonstrated that, upon liver injury, MCs migrate inward from the liver surface and give rise to hepatic stellate cells (HSCs) in biliary fibrosis induced by bile duct ligation (BDL) or myofibroblasts in CCl4-induced fibrosis. The present study analyzed the role of transforming growth factor-β (TGF-β) signaling in mesothelial-mesenchymal transition (MMT) and the fate of MCs during liver fibrosis and its regression. Deletion of TGF-β type II receptor (Tgfbr2) gene in cultured MCs suppressed TGF-β-mediated myofibroblastic conversion. Conditional deletion of Tgfbr2 gene in MCs reduced the differentiation of MCs to HSCs and myofibroblasts in the BDL and CCl4 models, respectively, indicating that the direct TGF-β signaling in MCs is responsible to MMT. After BDL and CCl4 treatment, MC-derived HSCs and myofibroblasts were distributed near the liver surface and the thickness of collagen was increased in Glisson's capsule beneath the liver surface. Fluorescence-activated cell sorting analysis revealed that MC-derived HSCs and myofibroblasts store little vitamin A lipids and have fibrogenic phenotype in the fibrotic livers. MCs contributed to 1.4 and 2.0% of activated HSCs in the BDL and CCl4 models, respectively. During regression of CCl4-induced fibrosis, 20% of MC-derived myofibroblasts survived in the liver and deactivated to vitamin A-poor HSCs. Our data indicate that MCs participate in capsular fibrosis by supplying vitamin A-poor HSCs during a process of liver fibrosis and regression.

  10. Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats

    SciTech Connect

    Tsai, M.-K.; Lin, Y.-L.; Huang, Y.-T.

    2010-01-15

    Enhanced oxidative stress is associated with hepatic fibrosis. Salvianolic acids A (Sal A) and B (Sal B) have been reported to be strong polyphenolic antioxidants and free radical scavengers. The present study is to investigate if Sal A and B could attenuate oxidative stress and liver fibrosis in rats. A cell line of rat hepatic stellate cells (HSCs) was stimulated with platelet-derived growth factor (PDGF, 10 ng/ml). The inhibitory effects of Sal A and B on intracellular hydrogen peroxide levels were measured with dichlorofluorescein diacetate (DCF-DA) dye assay. alpha-Smooth muscle actin (alpha-SMA), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits were measured by Western blotting. Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg) twice per week for 6 weeks. Sal A (10 mg/kg), Sal B (50 mg/kg) or S-adenosylmethionine (SAMe, 10 mg/kg), was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. In vitro, PDGF increased the accumulation of hydrogen peroxide in HSCs, which was attenuated by Sal A (10 muM) and Sal B (200 muM). Sal A and B attenuated the PDGF-stimulated expressions of alpha-SMA and NADPH oxidase subunits gp91{sup phox} and p47{sup phox} in membrane fractions. In vivo studies showed that the hepatic levels of collagen, malondialdehyde, TNF-alpha, IL-6, and IL-1beta, fibrosis scores and protein expressions of alpha-SMA, heme-oxygenase-1, iNOS, and gp91{sup phox}, and serum levels of ALT, AST, IL-6, and IL-1beta were increased in TAA-intoxicated rats, all of which were attenuated by 4-week treatment of Sal A or Sal B. Our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through inhibition of NADPH oxidase. Sal A and B treatments were also effective against hepatic fibrosis in TAA-intoxicated rats.

  11. Tetramethylpyrazine reduces inflammation in liver fibrosis and inhibits inflammatory cytokine expression in hepatic stellate cells by modulating NLRP3 inflammasome pathway.

    PubMed

    Wu, Xiafei; Zhang, Feng; Xiong, Xin; Lu, Chunfeng; Lian, Naqi; Lu, Yin; Zheng, Shizhong

    2015-04-01

    Hepatic fibrosis is concomitant with liver inflammation, which has been highlighted as significant treatment of chronic liver disease. We previously demonstrated that tetramethylpyrazine (TMP), the effective component of Ligusticum chuanxiong Hort, can inhibit the activation of HSCs and consequential anti-hepatic fibrosis. In this study, our work demonstrated that TMP improved liver histological architecture, decreased hepatic enzyme levels and attenuated collagen deposition in the rat fibrotic liver. In addition, TMP significantly protected the liver from CCl4-caused injury and fibrogenesis by suppressing inflammation with reducing levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), NLRP3, nuclear factor-kappa B (NF-κB) and interleukin-1β (IL-1β). Experiments in vitro showed that TMP inhibited inflammatory cytokine expression in HSCs associated with disrupting platelet-derived growth factor-b receptor (PDGF-βR)/NLRP3/caspase1 pathway. These data collectively indicate that TMP can attenuate liver inflammation in liver fibrosis and possibly by targeting HSCs via PDGF-βR/NLRP3/caspase1 pathway. It provides novel mechanistic insights into TMP as a potential therapeutic remedy for hepatic fibrosis. PMID:25847612

  12. Effects of thyrotoxicosis and selective hepatic autonomic denervation on hepatic glucose metabolism in rats.

    PubMed

    Klieverik, Lars P; Sauerwein, Hans P; Ackermans, Mariëtte T; Boelen, Anita; Kalsbeek, Andries; Fliers, Eric

    2008-03-01

    Thyrotoxicosis is known to induce a broad range of changes in carbohydrate metabolism. Recent studies have identified the sympathetic and parasympathetic nervous system as major regulators of hepatic glucose metabolism. The present study aimed to investigate the pathogenesis of altered endogenous glucose production (EGP) in rats with mild thyrotoxicosis. Rats were treated with methimazole in drinking water and l-thyroxine (T(4)) from osmotic minipumps to either reinstate euthyroidism or induce thyrotoxicosis. Euthyroid and thyrotoxic rats underwent either a sham operation, a selective hepatic sympathetic denervation (Sx), or a parasympathetic denervation (Px). After 10 days of T(4) administration, all animals were submitted to a hyperinsulinemic euglycemic clamp combined with stable isotope dilution to measure EGP. Plasma triiodothyronine (T(3)) showed a fourfold increase in thyrotoxic compared with euthyroid animals. EGP was increased by 45% in thyrotoxic compared with euthyroid rats and correlated significantly with plasma T(3). In thyrotoxic rats, hepatic PEPCK mRNA expression was increased 3.5-fold. Relative suppression of EGP during hyperinsulinemia was 34% less in thyrotoxic than in euthyroid rats, indicating hepatic insulin resistance. During thyrotoxicosis, Sx attenuated the increase in EGP, whereas Px resulted in increased plasma insulin with unaltered EGP compared with intact animals, compatible with a further decrease in hepatic insulin sensitivity. We conclude that chronic, mild thyrotoxicosis in rats increases EGP, whereas it decreases hepatic insulin sensitivity. Sympathetic hepatic innervation contributes only to a limited extent to increased EGP during thyrotoxicosis, whereas parasympathetic hepatic innervation may function to restrain EGP in this condition. PMID:18182466

  13. Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells

    PubMed Central

    Robert, Sacha; Gicquel, Thomas; Bodin, Aude; Lagente, Vincent; Boichot, Elisabeth

    2016-01-01

    Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable

  14. Suppressive effect of microRNA-29b on hepatic stellate cell activation and its crosstalk with TGF-β1/Smad3.

    PubMed

    Liang, Chunli; Bu, Shurui; Fan, Xiaoming

    2016-07-01

    The microRNA (miR)-29 family is closely associated with fibrotic processes by virtue of its low expression in many tissues during organ fibrosis. The present study investigated whether miR-29b overexpression suppressed hepatic stellate cell (HSC) activation and its interactions with transforming growth factor (TGF)-β1/mothers against decapentaplegic homolog 3 (Smad3), a classical signal transduction pathway contributing to the activation of HSCs. The results showed that transfection of LX-2 (human HSC) cells with miR-29b mimic or pSUPER-Smad3 silencing (si)RNA resulted in significantly increased expression of miR-29b and decreased expression of Smad3. miR-29b overexpression inhibited proliferation of LX-2 cells 24 h after transfection. Both miR-29b overexpression and Smad3 silencing antagonized the effects of TGF-β1 on the expression of α-smooth muscle actin (α-SMA) and collagen type I (col-1). Furthermore, infection with miR-29b mimics suppressed Smad3 and TGF-β1 expression, suggesting that miR-29b inhibited LX-2 activation mediated by both Smad3 and TGF-β1. Nevertheless, primary miR-29a/b1, miR-29b2/c and mature miR-29b were downregulated by TGF-β1 and stimulated by Smad3 silencing, suggesting that TGF-β1/Smad3 signalling pathway regulate not just mature miR-29b but also its transcription. In summary, our results show overwhelming evidence corroborating the suppressive effect of miR-29b on TGF-β1-induced LX-2 cell activation. The results also revealed the existence of crosstalk between miR-29b and TGF-β1/Smad3 during LX-2 activation, suggesting a feedback loop between miR-29b and TGF-β1/Smad3 signalling that promotes liver fibrosis. Copyright © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.

  15. Epigenetic regulation of connective tissue growth factor by microRNA-214 delivery in exosomes from mouse or human hepatic stellate cells

    PubMed Central

    Chen, Li; Charrier, Alyssa; Zhou, Yu; Chen, Ruju; Yu, Bo; Agarwal, Kitty; Tsukamoto, Hidekazu; Lee, L. James; Paulaitis, Michael E; Brigstock, David R

    2013-01-01

    Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3′-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214 which otherwise inhibited CCN2 expression by directly binding to the CCN2 3′-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50–150nm in diameter, negatively charged (−26mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pre-treatment of the cells with the exosome inhibitor, GW4869. Co-culture of miR-214-transfected donor HSC with CCN2 3′-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild type CCN2 3′-UTR reporter but not of a mutant CCN2 3′-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3′-UTR. MiR-214 in LX-2 cells was shuttled via exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3′-UTR activity or expression of CCN2 downstream targets, including αSMA or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. Conclusion Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of epigenetic regulation by exosomal miRs. PMID:24122827

  16. miR-181b Promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients

    SciTech Connect

    Wang, Baocan; Li, Wenxi; Guo, Kun; Xiao, Yongtao; Wang, Yuqin; Fan, Jiangao

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer miR-181a and miR-181b, especially, miR-181b could be induced by transforming growth factor-beta 1 (TGF-{beta}1) in hepatic stellate cells. Black-Right-Pointing-Pointer miR-181b could promote HSC-T6 cell proliferation by directly targeting the negative cell regulator-p27 in HSC-T6 cell. Black-Right-Pointing-Pointer miR-181b was identified as potential serum diagnostic marker for liver cirrhosis patients. -- Abstract: MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because of the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-{beta}1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-{beta}1 pathway by a currently unknown mechanism.

  17. Hepatic iron deprivation prevents spontaneous development of fulminant hepatitis and liver cancer in Long-Evans Cinnamon rats.

    PubMed

    Kato, J; Kobune, M; Kohgo, Y; Sugawara, N; Hisai, H; Nakamura, T; Sakamaki, S; Sawada, N; Niitsu, Y

    1996-08-15

    Several clinical studies have suggested that excess hepatic iron accumulation is a progressive factor in some liver diseases including chronic viral hepatitis and hemochromatosis. However, it is not known whether iron-induced hepatotoxicity may be directly involved in hepatitis, cirrhosis, and liver cancer. The Long-Evans Cinnamon (LEC) rat, which accumulates excess copper in the liver as in patients with Wilson's disease, is of a mutant strain displaying spontaneous hemolysis, hepatitis, and liver cancer. We found previously that LEC rats harbored an additional abnormality: accumulation of as much iron as copper in the liver. In the present study, we compared the occurrence of hepatitis and liver cancer in LEC rats fed an iron-deficient diet (ID) with those in rats fed a regular diet (RD). The RD group showed rapid increments of hepatic iron concentrations as the result of hemolysis, characteristics of fulminant hepatitis showing apoptosis, and a 53% mortality rate. However, no rats in the ID group died of fulminant hepatitis. Hepatic iron, especially "free" iron concentration and the extent of hepatic fibrosis in the ID group were far less than those of the RD group. At week 65, all rats in the RD group developed liver cancer, whereas none did in the ID group. These results suggest that the accumulation of iron, possibly by virtue of synergistic radical formation with copper, plays an essential role in the development of fulminant hepatitis, hepatic fibrosis, and subsequent hepatocarcinogenesis in LEC rats.

  18. Telmisartan prevents hepatic fibrosis and enzyme-altered lesions in liver cirrhosis rat induced by a choline-deficient L-amino acid-defined diet

    SciTech Connect

    Jin Haiyan; Yamamoto, Naoki; Uchida, Koichi; Terai, Shuji; Sakaida, Isao

    2007-12-28

    Rennin-angiotensin system is involved in liver fibrogenesis through activating hepatic stellate cells (HSCs). Telmisartan (Tel) is an angiotensin II type 1 receptor antagonist, could function as a selective peroxisome proliferator-activated receptor {gamma} activator. Here we studied the effect of Tel on liver fibrosis, pre-neoplastic lesions in vivo and primary HSCs in vitro. In vivo study, we used the choline-deficient L-amino acid-defined (CDAA)-diet induced rat NASH model. The rats were fed the CDAA diet for 8 weeks to induce liver fibrosis and pre-neoplastic lesions, and then co-administrated with Tel for another 10 weeks. Tel prevented liver fibrogenesis and pre-neoplastic lesions by down-regulating TGF{beta}1 and TIMP-1, 2 and increasing MMP-13 expression. Tel inhibited HSCs activation and proliferation. These results suggested that Tel could be a promising drug for NASH related liver fibrosis.

  19. The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

    PubMed

    Khanizadeh, S; Ravanshad, M; Hosseini, S Y; Davoodian, P; Zadeh, A N; Sabahi, F; Sarvari, J; Khanlari, Z; Hasani-Azad, M

    2016-01-01

    The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-β) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-β cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122. PMID:27640434

  20. Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4-/- mouse model

    PubMed Central

    Reiter, Florian P; Wimmer, Ralf; Wottke, Lena; Artmann, Renate; Nagel, Jutta M; Carranza, Manuel O; Mayr, Doris; Rust, Christian; Fickert, Peter; Trauner, Michael; Gerbes, Alexander L; Hohenester, Simon; Denk, Gerald U

    2016-01-01

    AIM: To study the interleukin-1 (IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4-/- (Abcb4-/-) mouse model. METHODS: Female and male Abcb4-/- mice from 6 to 13 mo of age were analysed for the degree of cholestasis (liver serum tests), extent of liver fibrosis (hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction (qPCR)]. For in vivo experiments, murine hepatic stellate cells (HSCs) were isolated via pronase-collagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/mL IL-1β with or without 2.5 μg/mL Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the BrdU assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis (FDH) assay. In vivo 8-wk-old Abcb4-/- mice with an already fully established hepatic phenotype were treated with Anakinra (1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, qPCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4-/- animals as defined by a lower hydroxyproline content (274 ± 64 μg/g vs 436 ± 80 μg/g liver, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test) and lower mRNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1 (TIMP) (1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 mRNA expression (1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1β mRNA expression levels (1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P < 0.001; Mann

  1. Hepatitis E Virus Genotype 3 in Wild Rats, United States

    PubMed Central

    Volk, Kylie; Van Den Bussche, Ronald A.

    2012-01-01

    The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related. PMID:22840202

  2. Hepatitis E virus genotype 3 in wild rats, United States.

    PubMed

    Lack, Justin B; Volk, Kylie; Van Den Bussche, Ronald A

    2012-08-01

    The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related. PMID:22840202

  3. Schistosoma japonicum egg antigen up-regulates fibrogenesis and inhibits proliferation in primary hepatic stellate cells in a concentration-dependent manner

    PubMed Central

    Liu, Ping; Wang, Mi; Lu, Xiao-Dan; Zhang, Shu-Juan; Tang, Wang-Xian

    2013-01-01

    AIM: To investigate the effects of different concentrations of Schistosoma japonicum (S. japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells (HSCs). METHODS: A mouse model of schistosomiasis-associated liver fibrosis (SSLF) was established by infecting mice with schistosomal cercaria via the abdomen. HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation, and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin (α-SMA) and desmin. The growth inhibitory effect and 50% inhibitory concentration (IC50) of S. japonicum egg antigen for primary HSCs (24 h) were determined using a cell counting kit-8 (CCK-8) assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMOL/LP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in HSCs in response to different concentrations of S. japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction. The levels of phospho-P38 (P-P38), phospho-Jun N-terminal kinase (P-JNK) and phospho-Akt (P-AKT) in HSCs were detected by Western blotting. RESULTS: An SSLF mouse model was established, and primary HSCs were successfully isolated and cultured. S. japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner. The IC50 of the S. japonicum egg antigen was 244.53 ± 35.26 μg/mL. S. japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs (P < 0.05), whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner (P < 0.05). A high concentration of S. japonicum egg antigen enhanced P-P38, P-JNK and P-AKT activation (P < 0.05). The changes in α-SMA and MMOL/LP-9 expression induced by S. japonicum egg antigen were closely correlated with P-P38 and P-JNK activation (P < 0.05). The attenuation of MMOL/LP-9

  4. Experimental models of hepatic fibrosis in the rat.

    PubMed

    Wasser, S; Tan, C E

    1999-01-01

    The rat is a frequently used experimental model in studies involving human disease. We review several methods of inducing hepatic fibrosis and cirrhosis in rats. These include induction by hepatotoxins and hepatocarcinogens such as carbon tetrachloride, dimethylnitrosamine, thioacetamide and furan; the hepatoxin-cum-nutrient, alcohol; a high fat-low choline-low protein diet; immunologic agents such as heterologous serum or bacterial cell wall products; and obstructive jaundice and biliary cirrhosis by common bile duct ligation. PMID:10374036

  5. Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

    PubMed Central

    Li, Jian-ping; Gao, Yan; Chu, Shi-feng; Zhang, Zhao; Xia, Cong-yuan; Mou, Zheng; Song, Xiu-yun; He, Wen-bin; Guo, Xiao-feng; Chen, Nai-hong

    2014-01-01

    Aim: To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl4-induced hepatic fibrosis in rats and to explore the mechanisms of the effects. Methods: Rats were given 6% alcohol in water and injected with CCl4 (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson's trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies. Results: In the alcohol- and CCl4-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl4-induced cell proliferation, reversed CCl4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl4-treated HSCs in vitro. Conclusion: Rg1 exerts protective effects in a rat model of alcohol- and CCl4-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes. PMID:24976156

  6. Complete Genome Sequence of a Rat Hepatitis E Virus Strain Isolated in the United States

    PubMed Central

    Debing, Yannick; Emerson, Suzanne U.; Purcell, Robert H.; Dallmeier, Kai

    2014-01-01

    Hepatitis E virus is a common cause of acute hepatitis in humans. Related viruses have been isolated from multiple animal species, including rats, but their impact on human health is unclear. We present the first full-length genome sequence of a rat hepatitis E virus strain isolated in the United States (LA-B350). PMID:25377700

  7. Senescence of activated stellate cells limits liver fibrosis

    PubMed Central

    Krizhanovsky, Valery; Yon, Monica; Dickins, Ross A.; Hearn, Stephen; Simon, Janelle; Miething, Cornelius; Yee, Herman; Zender, Lars; Lowe, Scott W.

    2011-01-01

    Summary Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to non-cancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage. PMID:18724938

  8. Effects of acute ethanol exposure on hepatic metallothionein, zinc and glutathione in male and female rats

    SciTech Connect

    Harris, J.; Harrell, B. )

    1991-03-15

    The purpose of this study was to determine the simultaneous responses of hepatic metallothionein (MT), zinc (Zn) and glutathione (GSH) in male and female rats to an acute ethanol dose. In male rats, hepatic MT has been shown to be induced by an acute ethanol dose. The Sprague-Dawley rats were fed the Lieber-DeCarli control diet for a five day period and then divided into 6 groups: baseline females and males, ethanol-treated females and males, pair-fed females and males. At t=0, baseline rats were killed, ethanol-treated rats were given ethanol by intragastric tube, and pair-fed rats were given ethanol by intragastric tube, and pair-fed rats were given an isocaloric sucrose solution by intragastric tube. At t=24 h, the ethanol-treated and pair-fed rats were killed. Livers were assayed for MT. Zn and GSH. Concentrations of blood ethanol (BEC) were significantly greater for male than female rats. A two way ANOVA with the independent variables being time and sex was performed to analyze differences for hepatic MT, Zn and GSH. For rats dosed with ethanol and killed 24 h later compared with rats at baseline, hepatic MT was significantly greater and hepatic Zn and GSH were not significantly different. Hepatic MT, Zn and GSH were not significantly different by sex. A significant correlation existed between hepatic Zn/g and MT/g. In conclusion, 24 h after an acute dose of ethanol, female as well as male rats responded with the induction of hepatic MT; and enough cysteine was available for the induction of hepatic MT and the maintenance of hepatic GSH levels. The measurement of Zn bound to hepatic MT rather than total hepatic Zn would be desirable to discern if changes in Zn distribution occur.

  9. Avocado oils and hepatic lipid metabolism in growing rats.

    PubMed

    Werman, M J; Neeman, I; Mokady, S

    1991-02-01

    The effect of various avocado oils on liver metabolism was studied in growing female rats. The rats were fed diets containing 10% (w/w) avocado oil for 4 wk. In comparison with rats fed refined avocado oil obtained from cored fruit by centrifugal separation, rats fed unrefined avocado oil obtained by organic solvent extraction from intact fruit, or its unsaponifiable components, showed a significant increase in total liver lipogenesis as well as in phospholipid and triglceride synthesis. Rats fed avocado-seed oil exhibited enhanced [1-14C]acetate incorporation into total liver lipids but showed the same distribution of label in the three main lipid classes as that of rats fed refined avocado oil. In addition, a significant reduction of triglycerides and protein content of plasma very-low-density lipoprotein and high-density lipoprotein fractions was observed in rats fed avocado-seed oil as compared with rats fed refined oil. Electron micrographs suggested that the alterations in hepatic lipogenesis are related to the marked proliferation of the smooth endoplasmic reticulum, which is known to be associated with induction of enzymes involved with lipid biosynthesis. The differences between the animals fed seed oil and those fed the unrefined oils, in the distribution of label within the main lipid classes, indicate that more than one factor is involved in the alterations caused by these oils.

  10. MicroRNA-146a-5p Negatively Regulates Pro-Inflammatory Cytokine Secretion and Cell Activation in Lipopolysaccharide Stimulated Human Hepatic Stellate Cells through Inhibition of Toll-Like Receptor 4 Signaling Pathways

    PubMed Central

    Chen, Yuhan; Zeng, Zhaochong; Shen, Xiaoyun; Wu, Zhifeng; Dong, Yinying; Cheng, Jason Chia-Hsien

    2016-01-01

    Lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway is demonstrated to be involved in the hepatic fibrosis. MicroRNA (miR)-146a-5p is a key regulator of the innate immune response. The functional significance of miR-146a-5p during the LPS/TLR4 mediated hepatic fibrosis process remains unclear. In this study, we found that TLR4 and α-smooth muscle actin (α-SMA) were up-regulated and miR-146a-5p was down-regulated in human hepatic stellate cell (HSC) line LX2 after LPS stimulation. Overexpression of miR-146a-5p inhibited LPS induced pro-inflammatory cytokines secretion through down-regulating the expression levels of TLR-4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor associated factor-6 (TRAF6) and phosphorylation of nuclear factor-kappa B (NF-κB). Knockdown of IRAK1 and TRAF6 also suppressed pro-inflammatory cytokine production by inhibiting NF-κB phosphorylation. In addition, miR-146a-5p mimic blocked LPS induced TRAF6 dependent c-Jun N-terminal kinase (JNK) and Smad2 activation as well as α-SMA production. Taken together, these results suggest that miR-146a-5p suppresses pro-inflammatory cytokine secretion and cell activation of HSC through inhibition of TLR4/NF-κB and TLR4/TRAF6/JNK pathway. PMID:27399683

  11. Susceptibility of laboratory rats against genotypes 1, 3, 4, and rat hepatitis E viruses.

    PubMed

    Li, Tian-Cheng; Yoshizaki, Sayaka; Ami, Yasushi; Suzaki, Yuriko; Yasuda, Shumpei P; Yoshimatsu, Kumiko; Arikawa, Jiro; Takeda, Naokazu; Wakita, Takaji

    2013-04-12

    To determine whether or not rats are susceptible to hepatitis E virus (HEV) infection, each of group containing three laboratory rats (Wistar) were experimentally inoculated with genotypes 1, 3, 4 and rat HEV by intravenous injection. Serum and stool samples were collected and used to detect HEV RNA and anti-HEV antibodies by RT-PCR and ELISA, respectively. The virus infection was monitored up to 3 months after inoculation. None of the serum or stool samples collected from the rats inoculated with G1, G3, or G4 HEV indicated positive sign for virus replication. Although no alteration was observed in ALT level, rat HEV RNA was detected in stools from both of the rats inoculated with rat HEV, and both rats were positive for anti-rat HEV IgG and IgM from 3 weeks after inoculation. These results demonstrated that rats are susceptible to rat HEV but not to G1, G3, and G4 HEV. We also confirm that the nude rats were useful for obtaining a large amount of rat HEV and that the rat HEV was transmitted by the fecal-oral route.

  12. MeCP2 silencing of LncRNA H19 controls hepatic stellate cell proliferation by targeting IGF1R.

    PubMed

    Yang, Jing-Jing; Liu, Li-Ping; Tao, Hui; Hu, Wei; Shi, Peng; Deng, Zi-Yu; Li, Jun

    2016-06-01

    Methyl-CpG-binding protein 2 (MeCP2) plays a key role in liver fibrosis. However, the potential mechanism of MeCP2 in liver fibrosis remains unclear. Early reports suggest that LncRNA H19 is important epigenetic regulator with critical roles in cell proliferation, but its role in hepatic fibrosis remains elusive. Sprague-Dawley rats liver fibrosis was generated by 12-weeks treatment with CCl4 intraperitoneal injection. HSC-T6 cells were used in vitro study. The expression levels of MeCP2, H19, IGF1R, α-SMA, and Col1A1 were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSC-T6 cells were transfected with MeCP2-siRNA, pEGF-C1-MeCP2, pEX-3-H19, and H19-siRNA. Finally, cell proliferation ability was assessed by the MTT assay. Here, we found that H19 was significantly down-regulated in HSCs and fibrosis tissues, and an opposite pattern is observed for MeCP2 and IGF1R. Silencing of MeCP2 blocked HSCs proliferation. Knockdown of MeCP2 elevated H19 expression in activated HSCs, and over-expression of MeCP2 inhibited H19 expression in activated HSCs. Moreover, we investigated the effect of H19 on IGF1R expression. Overexpression of H19 in HSCs repressed the expression of IGF1R, and an opposite pattern is observed for H19 silenced. In addition, we reported that overexpression of H19 inhibited the TGF-β1-induced proliferation of HSCs. Furthermore, MeCP2 negative regulation of H19 by targeting the protein IGF1R. Taken together, these results demonstrated that MeCP2 silencing of H19 can alter the IGF1R overexpression, thus contributing to HSCs proliferation. These data could suggest the development of combination therapies that target the MeCP2. PMID:27350269

  13. Clonorchis sinensis lysophospholipase inhibits TGF-β1-induced expression of pro-fibrogenic genes through attenuating the activations of Smad3, JNK2, and ERK1/2 in hepatic stellate cell line LX-2.

    PubMed

    Zhou, Lina; Shang, Mei; Shi, Mengchen; Zhao, Lu; Lin, Zhipeng; Chen, Tingjin; Wu, Yinjuan; Tang, Zeli; Sun, Hengchang; Yu, Jinyun; Huang, Yan; Yu, Xinbing

    2016-02-01

    Liver fibrosis is a wound healing response associated with chronic liver injury. Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis. Since helminths have the ability to live for decades in the host by establishing an adaptive relationship in the interplay with its hosts, we hypothesize that whether Clonochis sinensis LysophospholipaseA (CsLysoPLA), a component of excretory/secretory proteins, can attenuate the fibrogenic response by inhibiting activation of LX-2 cells, thereby balancing the pro-fibrotic and anti-fibrotic response during the Clonochis sinensis (C. sinensis) infection. In the present study, LX-2 cells were stimulated with CsLysoPLA in the presence of TGF-β1, and the expressions of collagen type I (COL1A1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 (MMP2) were decreased. In addition, CsLysoPLA significantly inhibited the proliferation and migration of LX-2 cells stimulated by TGF-β1. Pretreatment of LX-2 cells with CsLysoPLA attenuated the phosphorylation of Smad3 as well as JNK2 and ERK1/2 in response to the stimulation of TGF-β1. For the first time, our results showed an anti-fibrogenic effect of CsLysoPLA by attenuating the response of LX-2 cells to TGF-β1 through inhibiting the activations of Smad3, ERK1/2, and JNK2. PMID:26486942

  14. Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis. A model of Wilson's disease.

    PubMed

    Li, Y; Togashi, Y; Sato, S; Emoto, T; Kang, J H; Takeichi, N; Kobayashi, H; Kojima, Y; Une, Y; Uchino, J

    1991-05-01

    Long-Evans Cinnamon (LEC) rats, an inbred strain of a mutant rat isolated from Long-Evans rats, develop hereditary hepatitis. To elucidate the role of copper metabolism in the development of the hepatitis in LEC rats, we examined the copper concentration in the tissues and serum levels of copper and ceruloplasmin. Copper concentration in the liver of LEC rats was over 40 times that of normal Long-Evans Agouti (LEA) rats, while the serum ceruloplasmin and copper concentrations in LEC rats decreased significantly. The hepatocytes of LEC rats show steatosis in cytoplasm and pleomorphism of mitochondria, resembling the histologic features of the liver in Wilson's disease. These findings suggest that the hereditary hepatitis in LEC rats is closely associated with copper toxicity, and may be dealing with a rat form of Wilson's disease. Thus the LEC rats will provide a unique and useful animal model for clarifying the mechanism and for developing treatment strategies for Wilson's disease and other abnormal copper metabolism in humans.

  15. Renal and hepatic transporter expression in type 2 diabetic rats.

    PubMed

    Nowicki, Michael T; Aleksunes, Lauren M; Sawant, Sharmilee P; Dnyanmote, Ankur V; Mehendale, Harihara M; Manautou, José E

    2008-01-01

    Membrane transporters are critical for the uptake as well as elimination of chemicals and by-products of metabolism from the liver and kidneys. Since these proteins are important determinants of chemical disposition, changes in their expression in different disease states can modulate drug pharmacokinetics. The present study investigated alterations in the renal and hepatic expression of organic anion and cation transporters (Oats/Octs), multidrug resistance-associated proteins (Mrps), breast cancer resistance protein (Bcrp), P-glycoprotein (Pgp), and hepatic Na(+)-taurocholate cotransporting polypeptide (Ntcp) in type 2 diabetic rats. For this purpose, type 2 diabetes was induced by feeding male Sprague-Dawley rats a high fat diet followed by a single dose of streptozotocin (45 mg/kg, i.p., in 0.01 M citrate buffer pH 4.3) on day 14. Controls received normal diet and vehicle. Kidney and liver samples were collected on day 24 for generation of crude plasma membrane fractions and Western blot analysis of Oat, Oct, Mrp, Bcrp, Pgp, and Ntcp proteins. With regards to renal uptake transporters, type 2 diabetes increased levels of Oat2 (2.3-fold) and decreased levels of Oct2 to 50% of control kidneys. Conversely, efflux transporters Mrp2, Mrp4, and Bcrp were increased 5.4-fold, 2-fold, and 1.6-fold, respectively in type 2 diabetic kidneys with no change in levels of Mrp1, Mrp5, or Pgp. Studies of hepatic transporters in type 2 diabetic rats reveal that the protein level of Mrp5 was reduced to 4% of control livers with no change in levels of Bcrp, Mrp1, Mrp2, Mrp4, Ntcp, or Pgp. The changes reported in this study may have implications in type 2 diabetic patients.

  16. Calcium signalling in pancreatic stellate cells: Mechanisms and potential roles.

    PubMed

    Gryshchenko, Oleksiy; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

    2016-03-01

    Hepatic and pancreatic stellate cells may or may not be regarded as stem cells, but they are capable of remarkable transformations. There is less information about stellate cells in the pancreas than in the liver, where they were discovered much earlier and therefore have been studied longer and more intensively than in the pancreas. Most of the work on pancreatic stellate cells has been carried out in studies on cell cultures, but in this review we focus attention on Ca(2+) signalling in stellate cells in their real pancreatic environment. We review current knowledge on patho-physiologically relevant Ca(2+) signalling events and their underlying mechanisms. We focus on the effects of bradykinin in the initial stages of acute pancreatitis, an often fatal disease in which the pancreas digests itself and its surroundings. Ca(2+) signals, elicited in the stellate cells by the action of bradykinin, may have a negative effect on the outcome of the acute disease process and promote the development of chronic pancreatitis. The bradykinin-elicited Ca(2+) signals can be inhibited by blockade of type 2 receptors and also by blockade of Ca(2+)-release activated Ca(2+) channels. The potential benefits of such pharmacological inhibition for the treatment of pancreatitis are reviewed. PMID:26960936

  17. miR-1273g-3p modulates activation and apoptosis of hepatic stellate cells by directly targeting PTEN in HCV-related liver fibrosis.

    PubMed

    Niu, Xuemin; Fu, Na; Du, Jinghua; Wang, Rongqi; Wang, Yang; Zhao, Suxian; Du, Huijuan; Wang, Baoyu; Zhang, Yuguo; Sun, Dianxing; Nan, Yuemin

    2016-08-01

    MicroRNA (miRNA) play a pivotal role in the development of liver fibrosis. However, the functions of miRNA in hepatitis C virus (HCV)-related liver fibrosis remain unclear. In this study, we systematically analyzed the microarray data of the serum miRNA in patients with HCV-induced hepatic fibrosis. Among 41 dysregulated miRNA, miR-1273g-3p was the most significantly upregulated miRNA and correlated with the stage of liver fibrosis. Overexpression of miR-1273g-3p could inhibit translation of PTEN, increase the expression of α-SMA, Col1A1, and reduce apoptosis in HSCs. Hence, we conclude that miR-1273g-3p might affect the activation and apoptosis of HSCs by directly targeting PTEN in HCV-related liver fibrosis. PMID:27423040

  18. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    PubMed

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation.

  19. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    PubMed

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. PMID:25498792

  20. The induction of human myeloid derived suppressor cells through hepatic stellate cells is dose-dependently inhibited by the tyrosine kinase inhibitors nilotinib, dasatinib and sorafenib, but not sunitinib.

    PubMed

    Heine, Annkristin; Schilling, Judith; Grünwald, Barbara; Krüger, Achim; Gevensleben, Heidrun; Held, Stefanie Andrea Erika; Garbi, Natalio; Kurts, Christian; Brossart, Peter; Knolle, Percy; Diehl, Linda; Höchst, Bastian

    2016-03-01

    Increased numbers of immunosuppressive myeloid derived suppressor cells (MDSCs) correlate with a poor prognosis in cancer patients. Tyrosine kinase inhibitors (TKIs) are used as standard therapy for the treatment of several neoplastic diseases. However, TKIs not only exert effects on the malignant cell clone itself but also affect immune cells. Here, we investigate the effect of TKIs on the induction of MDSCs that differentiate from mature human monocytes using a new in vitro model of MDSC induction through activated hepatic stellate cells (HSCs). We show that frequencies of monocytic CD14(+)HLA-DR(-/low) MDSCs derived from mature monocytes were significantly and dose-dependently reduced in the presence of dasatinib, nilotinib and sorafenib, whereas sunitinib had no effect. These regulatory effects were only observed when TKIs were present during the early induction phase of MDSCs through activated HSCs, whereas already differentiated MDSCs were not further influenced by TKIs. Neither the MAPK nor the NFκB pathway was modulated in MDSCs when any of the TKIs was applied. When functional analyses were performed, we found that myeloid cells treated with sorafenib, nilotinib or dasatinib, but not sunitinib, displayed decreased suppressive capacity with regard to CD8+ T cell proliferation. Our results indicate that sorafenib, nilotinib and dasatinib, but not sunitinib, decrease the HSC-mediated differentiation of monocytes into functional MDSCs. Therefore, treatment of cancer patients with these TKIs may in addition to having a direct effect on cancer cells also prevent the differentiation of monocytes into MDSCs and thereby differentially modulate the success of immunotherapeutic or other anti-cancer approaches. PMID:26786874

  1. Mechanisms of action of acetaldehyde in the up-regulation of the human α2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

    PubMed

    Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M Raj

    2014-05-01

    Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

  2. Pancreatic Stellate Cell Models for Transcriptional Studies of Desmoplasia-Associated Genes

    PubMed Central

    Mathison, Angela; Liebl, Ann; Bharucha, Jinai; Mukhopadhyay, Debabrata; Lomberk, Gwen; Shah, Vijay; Urrutia, Raul

    2010-01-01

    Background Pancreatic stellate cells are emerging as key players in pathophysiopathological processes underlying the development of pancreatic disease, including pancreatitis and cancer. The cells are scarce in the pancreas making their isolation time and resource use consuming. Methods Therefore, with the ultimate goal of facilitating mechanistic studies, here we report the isolation, characterization, and immortalization of stellate cell lines from rat and mouse origin. Results These cell lines display morphological and molecular markers as well as non-tumorigenic characteristics similar to the frequently used hepatic counterparts. In addition, we have tested their robustness as a model for transcriptional regulatory studies. We find that these cells respond well to TGFβ signaling by triggering a distinct cascade of gene expression, some genes overlap with the TGFβ response of LX2 cells. These cells express several key chromatin proteins and epigenetic regulators involved in the regulation of gene expression, including co-repressors such as Sin3A (short-term repression), HP1 (long-term repression), as well as CBP/p300 (activation). Furthermore, these cells are well suited for Gal4-based transcriptional activation and repression assays. Conclusions The cell model reported here may therefore help fuel investigations in the field of signaling, transcription, and perhaps other studies on similarly exciting cellular processes. PMID:20847583

  3. 1,25-(OH){sub 2}-vitamin D{sub 3} prevents activation of hepatic stellate cells in vitro and ameliorates inflammatory liver damage but not fibrosis in the Abcb4{sup −/−} model

    SciTech Connect

    Reiter, Florian P.; Hohenester, Simon; Nagel, Jutta M.; Wimmer, Ralf; Artmann, Renate; Wottke, Lena; Makeschin, Marie-Christine; Mayr, Doris; Rust, Christian; Trauner, Michael; Denk, Gerald U.

    2015-04-03

    Background/Purpose of the study: Vitamin D{sub 3}-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D{sub 3}-administration has thus been proposed as a therapeutic approach. Vitamin D{sub 3} has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH){sub 2}-vitamin D{sub 3} inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. Methods: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen{sup ®}-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4{sup −/−} (Abcb4{sup −/−})-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. Results: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-β-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4{sup −/−}-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. Conclusion: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4{sup −/−}-mice, administration of calcitriol ameliorates inflammatory liver-damage but has

  4. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    PubMed Central

    Zhang, Wei; Chen, Xiao-Ping; Zhang, Wan-Guang; Zhang, Feng; Xiang, Shuai; Dong, Han-Hua; Zhang, Lei

    2009-01-01

    AIM: To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions. PMID:19195056

  5. Fish oil decreases hepatic lipogenic genes in rats fasted and refed on a high fructose diet.

    PubMed

    de Castro, Gabriela S; Cardoso, João Felipe R; Calder, Philip C; Jordão, Alceu A; Vannucchi, Helio

    2015-03-01

    Fasting and then refeeding on a high-carbohydrate diet increases serum and hepatic triacylglycerol (TAG) concentrations compared to standard diets. Fructose is a lipogenic monosaccharide which stimulates de novo fatty acid synthesis. Omega-3 (n-3) fatty acids stimulate hepatic β-oxidation, partitioning fatty acids away from TAG synthesis. This study investigated whether dietary n-3 fatty acids from fish oil (FO) improve the hepatic lipid metabolic response seen in rats fasted and then refed on a high-fructose diet. During the post-prandial (fed) period, rats fed a FO rich diet showed an increase in hepatic peroxisome proliferator-activated receptor α (PPAR-α) gene expression and decreased expression of carbohydrate responsive element binding protein (ChREBP), fatty acid synthase (FAS) and microsomal triglyceride transfer protein (MTTP). Feeding a FO rich diet for 7 days prior to 48 h of fasting resulted in lower hepatic TAG, lower PPAR-α expression and maintenance of hepatic n-3 fatty acid content. Refeeding on a high fructose diet promoted an increase in hepatic and serum TAG and in hepatic PPAR-α, ChREBP and MTTP expression. FO did not prevent the increase in serum and hepatic TAG after fructose refeeding, but did decrease hepatic expression of lipogenic genes and increased the n-3 fatty acid content of the liver. n-3 Fatty acids can modify some components of the hepatic lipid metabolic response to later feeding with a high fructose diet.

  6. Catheterization of the Hepatic Artery Via the Left Common Carotid Artery in Rats

    SciTech Connect

    Li Xiao; Wang Yixiang, J.; Zhou Xiangping Guan Yongsong; Tang Chengwei

    2006-12-15

    The commonly used approach for rat hepatic artery catheterization is via the gastroduodenal artery, which is ligated after the procedure. A new method of rat hepatic artery catheterization via the left common carotid artery (LCCA) is described. The LCCA is repaired after catheterization. The catheterization procedures included the following: (1) opening the rat's abdominal cavity and exposing the portion of abdominal aorta at the level of the celiac trunk; (2) separating and exposing the LCCA; inserting a microguidewire and microcatheter set into the LCCA via an incision; after placement into the descending aorta, the microguidewire and microcatheter are maneuvered into the hepatic artery under direct vision; (3) after transcatheter therapy, the catheter is withdrawn and the incision at the LCCA is repaired. This technique was employed on 60 male Sprague-Dawley rats with diethylnitrosamine-induced liver cancer, using a 3F microguidewire and microcatheter set. Selective hepatic artery catheterization was successfully performed in 57 rats. One rat died during the operation and five rats died within 7 days after the procedure. It is envisaged that as experience increases, the catheterization success rate will increase and the death rate will decrease. A new approach for selective hepatic artery catheterization via the LCCA in rats is introduced, which makes repeat catheterization of this artery possible and allows large embolization particles to be delivered by using a 3F catheter.

  7. BML-11, a lipoxin receptor agonist, protected carbon tetrachloride-induced hepatic fibrosis in rats.

    PubMed

    Zhou, Xiao-Yan; Yu, Zhong-Jian; Yan, Dan; Wang, Hong-Mei; Huang, Yong-Hong; Sha, Juan; Xu, Fang-Yun; Cai, Zhen-Yu; Min, Wei-Ping

    2013-10-01

    Inflammation plays an important role in the occurrence and development of fibrosis. Lipoxins (LXs) and BML-111 (lipoxin A4 agonist) have been approved for potent anti-inflammatory properties. Previously, we and others had showed LXs and BML-111 could protect acute hepatic injury, inhibit the growth and invasion of hepatic tumor. However, there are few reports dealing with their effects on hepatic fibrosis. To explore whether LXs and the analog could interrupt the process of hepatic fibrosis, the effects of BML-111 on tetrachloride-induced hepatic fibrosis were observed and the possible mechanism were discussed. Sprague-Dawley rats were induced liver fibrosis by carbon tetrachloride (CCl4) for 10 weeks with or without BML-111, and the histopathology and collagen content were employed to quantify hepatic necro-inflammation and fibrosis. Moreover, the expression levels of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and platelet-derived growth factor (PDGF) were examined via Western blot or ELISA. Rats treated with BML-111 improved hepatic necro-inflammation and inhibited hepatic fibrosis in association with reduction of α-SMA expression and decreased collagen deposition. Furthermore, BML-111 could downregulate the expressions of TGF-β1 and PDGF significantly. BML-111 played a critical protective role in CCl4-induced hepatic fibrosis through inhibiting the levels of TGF-β1 and PDGF in rats.

  8. Construction and characterization of an infectious cDNA clone of rat hepatitis E virus.

    PubMed

    Li, Tian-Cheng; Yang, Tingting; Yoshizaki, Sayaka; Ami, Yasushi; Suzaki, Yuriko; Ishii, Koji; Haga, Kei; Nakamura, Tomofumi; Ochiai, Susumu; Takaji, Wakita; Johne, Reimar

    2015-06-01

    Rat hepatitis E virus (HEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of rat HEV strain R63/DEU/2009 was cloned downstream of the T7 RNA polymerase promoter and capped genomic RNA generated by in vitro transcription was injected into nude rats. Rat HEV RNA could be detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. Rat HEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to rat HEV RNA detection in serum and faeces of nude rats, and to seroconversion in Wistar rats. In addition, rat HEV was isolated in PLC/PRF/5 cells from the rat HEV RNA-positive faecal suspension of nude rats and then passaged. The cell culture supernatant was infectious for nude rats. Genome analysis identified nine point mutations of the cell-culture-passaged virus in comparison with the originally cloned rat HEV genome. The results indicated that infectious rat HEV could be generated from the cDNA clone. As rats are widely used and well-characterized laboratory animals, studies on genetically engineered rat HEV may provide novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses.

  9. Construction and characterization of an infectious cDNA clone of rat hepatitis E virus.

    PubMed

    Li, Tian-Cheng; Yang, Tingting; Yoshizaki, Sayaka; Ami, Yasushi; Suzaki, Yuriko; Ishii, Koji; Haga, Kei; Nakamura, Tomofumi; Ochiai, Susumu; Takaji, Wakita; Johne, Reimar

    2015-06-01

    Rat hepatitis E virus (HEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of rat HEV strain R63/DEU/2009 was cloned downstream of the T7 RNA polymerase promoter and capped genomic RNA generated by in vitro transcription was injected into nude rats. Rat HEV RNA could be detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. Rat HEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to rat HEV RNA detection in serum and faeces of nude rats, and to seroconversion in Wistar rats. In addition, rat HEV was isolated in PLC/PRF/5 cells from the rat HEV RNA-positive faecal suspension of nude rats and then passaged. The cell culture supernatant was infectious for nude rats. Genome analysis identified nine point mutations of the cell-culture-passaged virus in comparison with the originally cloned rat HEV genome. The results indicated that infectious rat HEV could be generated from the cDNA clone. As rats are widely used and well-characterized laboratory animals, studies on genetically engineered rat HEV may provide novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses. PMID:25634930

  10. Hepatitis

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Hepatitis KidsHealth > For Kids > Hepatitis Print A A A ... an important digestive liquid called bile . What Is Hepatitis? Hepatitis is an inflammation (say: in-fluh-MAY- ...

  11. Mechanisms of Hepatic Fibrogenesis

    PubMed Central

    Friedman, Scott L.

    2010-01-01

    Substantial improvements in the treatment of chronic liver disease have accelerated interest in uncovering the mechanisms underlying hepatic fibrosis and its resolution. Activation of resident hepatic stellate cells into proliferative, contractile, and fibrogenic cells in liver injury remains a dominant theme driving the field. However, several new areas of rapid progress in the past 5–10 years also have taken root, including: (1) identification of different fibrogenic populations apart from resident stellate cells, for example, portal fibroblasts, fibrocytes, and bone-marrow– derived cells, as well as cells derived from epithelial mesenchymal transition; (2) emergence of stellate cells as finely regulated determinants of hepatic inflammation and immunity; (3) elucidation of multiple pathways controlling gene expression during stellate cell activation including transcriptional, post-transcriptional, and epigenetic mechanisms; (4) recognition of disease-specific pathways of fibrogenesis; (5) re-emergence of hepatic macrophages as determinants of matrix degradation in fibrosis resolution and the importance of matrix cross-linking and scar maturation in determining reversibility; and (6) hints that hepatic stellate cells may contribute to hepatic stem cell behavior, cancer, and regeneration. Clinical and translational implications of these advances have become clear, and have begun to impact significantly on the management and outlook of patients with chronic liver disease. PMID:18471545

  12. High prevalence of rat hepatitis E virus in wild rats in China.

    PubMed

    Li, Wei; Guan, Dawei; Su, Juan; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng; Ke, Chang Wen

    2013-08-30

    Serum samples from a total of 713 wild rats captured in Zhanjiang city in China from December 2011 to September 2012 were investigated for the prevalence of rat hepatitis E virus (HEV) by exploring rat HEV-specific antibodies and RNA. By an ELISA based on recombinant rat HEV-like particles (HEV-LPs), 23.3% (166/713) of the rats were positive for anti-HEV IgG, and 8.3% (59/713) were positive for anti-HEV IgM. The IgG-positive rates in Rattus norvegicus, Bandicota indica, Rattus flavipectus, Rattus rattoides losea, and Rattus rattus hainanus, were 27.8% (64/230), 23.0% (40/174), 19.9% (34/171), 21.5% (26/121), and 11.8% (2/17), while the IgM-positive rates were 8.3% (19/230), 6.9% (12/174), 8.2% (14/171), 10.7% (13/121), and 5.9% (1/17), respectively. The IgG-positive rate of the rats captured in rural areas, 24.1% (84/348), was higher than that in the central area of Zhanjiang city, 15.1% (32/212). The highest IgG-positive rates, as high as 45.3% (39/86), were detected in wild rats trapped in the garbage dump. Twelve of the 59 IgM-positive serum samples were positive for HEV RNA, which was detected in all of the wild rat species except R. rattus hainanus. A phylogenetic analysis of the partial genome of rat HEV ORF1 indicated that all of the 12 HEV strains belong to rat HEV, and no other genotype HEV were detected. The rat HEV from Zhangjiang city could be classified into three separated clusters, suggesting that the infection due to rat HEV with a variety of genome entities occurs extensively among wild rats in China.

  13. Angiotensin II enhances epithelial-to-mesenchymal transition through the interaction between activated hepatic stellate cells and the stromal cell-derived factor-1/CXCR4 axis in intrahepatic cholangiocarcinoma.

    PubMed

    Okamoto, Koichi; Tajima, Hidehiro; Nakanuma, Shinichi; Sakai, Seisho; Makino, Isamu; Kinoshita, Jun; Hayashi, Hironori; Nakamura, Keishi; Oyama, Katsunobu; Nakagawara, Hisatoshi; Fujita, Hideto; Takamura, Hiroyuki; Ninomiya, Itasu; Kitagawa, Hirohisa; Fushida, Sachio; Fujimura, Takashi; Harada, Shinichi; Wakayama, Tomohiko; Iseki, Shoichi; Ohta, Tetsuo

    2012-08-01

    We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facilitate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1α was released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and meta-stasis of tumor cells expressing AT-1 and CXCR4

  14. Petroselinum crispum extract attenuates hepatic steatosis in rats fed with fructose enriched diet.

    PubMed

    Nair, V Yuneesha; Balakrishanan, N; Antony Santiago, J Victor

    2015-01-01

    Non alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and ongoing research efforts are focused on understanding the underlying pathophysiology of hepatic steatosis with the anticipation that these efforts will identify novel therapeutic targets. This study investigated the Petroselinum crispum extract in hepatic steatosis in rats fed with fructose enriched diet. Rats were divided into the 4 groups: Group 1 rats received standard pellet diet with corn starch for the entire experimental period of 8 weeks. Group 2 rats received standard pellet diet and 2 gm/kg body weight crude Parsley leaf ethanol extract for the entire experimental period of 8 weeks. Group 3 rats received modified fructose diet. Group 4 rats received modified fructose diet and 2gm/kg crude Parsley leaf ethanol extract. Hepatic function and structure was evaluated in these rats. Modified fructose diet produced dyslipidemia, hepatic steatosis and infiltration of inflammatory cells in the liver and higher plasma hepatic markers. Petroselinum crispum extract reversed metabolic changes such as abnormal crispum extract attenuated chronic changes in modified fructose diet induced NAFLD (Tab. 2, Fig. 3, Ref. 43). PMID:26435020

  15. Petroselinum crispum extract attenuates hepatic steatosis in rats fed with fructose enriched diet.

    PubMed

    Nair, V Yuneesha; Balakrishanan, N; Antony Santiago, J Victor

    2015-01-01

    Non alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and ongoing research efforts are focused on understanding the underlying pathophysiology of hepatic steatosis with the anticipation that these efforts will identify novel therapeutic targets. This study investigated the Petroselinum crispum extract in hepatic steatosis in rats fed with fructose enriched diet. Rats were divided into the 4 groups: Group 1 rats received standard pellet diet with corn starch for the entire experimental period of 8 weeks. Group 2 rats received standard pellet diet and 2 gm/kg body weight crude Parsley leaf ethanol extract for the entire experimental period of 8 weeks. Group 3 rats received modified fructose diet. Group 4 rats received modified fructose diet and 2gm/kg crude Parsley leaf ethanol extract. Hepatic function and structure was evaluated in these rats. Modified fructose diet produced dyslipidemia, hepatic steatosis and infiltration of inflammatory cells in the liver and higher plasma hepatic markers. Petroselinum crispum extract reversed metabolic changes such as abnormal crispum extract attenuated chronic changes in modified fructose diet induced NAFLD (Tab. 2, Fig. 3, Ref. 43).

  16. Chlorogenic acid from honeysuckle improves hepatic lipid dysregulation and modulates hepatic fatty acid composition in rats with chronic endotoxin infusion

    PubMed Central

    Zhou, Yan; Ruan, Zheng; Wen, Yanmei; Yang, Yuhui; Mi, Shumei; Zhou, Lili; Wu, Xin; Ding, Sheng; Deng, Zeyuan; Wu, Guoyao; Yin, Yulong

    2016-01-01

    Chlorogenic acid as a natural hydroxycinnamic acid has protective effect for liver. Endotoxin induced metabolic disorder, such as lipid dysregulation and hyperlipidemia. In this study, we investigated the effect of chlorogenic acid in rats with chronic endotoxin infusion. The Sprague-Dawley rats with lipid metabolic disorder (LD group) were intraperitoneally injected endotoxin. And the rats of chlorogenic acid-LD group were daily received chlorogenic acid by intragastric administration. In chlorogenic acid-LD group, the area of visceral adipocyte was decreased and liver injury was ameliorated, as compared to LD group. In chlorogenic acid-LD group, serum triglycerides, free fatty acids, hepatic triglycerides and cholesterol were decreased, the proportion of C20:1, C24:1 and C18:3n-6, Δ9-18 and Δ6-desaturase activity index in the liver were decreased, and the proportion of C18:3n-3 acid was increased, compared to the LD group. Moreover, levels of phosphorylated AMP-activated protein kinase, carnitine palmitoyltransferase-I, and fatty acid β-oxidation were increased in chlorogenic acid-LD group compared to LD rats, whereas levels of fatty acid synthase and acetyl-CoA carboxylase were decreased. These findings demonstrate that chlorogenic acid effectively improves hepatic lipid dysregulation in rats by regulating fatty acid metabolism enzymes, stimulating AMP-activated protein kinase activation, and modulating levels of hepatic fatty acids. PMID:27013782

  17. Experimental hepatic fibrosis due to Capillaria hepatica infection (differential features presented by rats and mice).

    PubMed

    Andrade, Stelamares Boyda de; Andrade, Zilton A

    2004-06-01

    Rats and mice are among the most susceptible hosts for the helminth Capillaria hepatica. More information on the similarities and differences between the hepatic pathology presented by these two parasite hosts are needed, since they may represent good models for the study of hepatic fibrosis. Early changes are similar for both hosts and are represented by necro-inflammatory lesions around dead parasites and their eggs and diffuse and intense reactive hepatitis. Although worms remain alive longer in mice than in rats, hepatic changes are more rapidly and deeply modulated in the former, even leading to almost complete disappearance of fibrosis. As for the rats, the modulation of the focal lesions is followed by the formation of septal fibrosis, a process where fine and long fibrous septa appear connecting portal spaces and central veins in such a way as to form a final morphologic picture of cirrhosis. Hepatic functional changes usually present good correlations with the morphologic findings at the different phases of the infection evolution. Therefore C. hepatica infection in rats and mice represent two different models of hepatic fibrosis and these differences, if properly known and understood, can be explored to answer different questions regarding several aspects of hepatic fibrosis.

  18. Evidence for hepatic involvement in control of ad libitum food intake in rats.

    PubMed

    Friedman, M I; Sawchenko, P E

    1984-07-01

    Sectioning the hepatic branch of the anterior abdominal vagal trunk attenuated but did not abolish the normal nocturnal bias in the day-night distribution of food intake in female rats. Neither total daily food intake nor body weight was affected by hepatic vagotomy. This effect appeared to be specific to the hepatic branch of the nerve because sectioning the remaining (gastric and celiac) abdominal vagal branches did not influence daily feeding rhythms and appeared to be specific to feeding behavior, because the day-night rhythms of drinking behavior and wheel-running activity were not affected by hepatic vagotomy. In male rats, hepatic vagotomy also produced an increase in daytime food consumption but without commensurate reduction in nighttime eating. As a result, male rats with hepatic vagotomy displayed a modest chronic hyperphagia and body weight gain, which was associated primarily with increased linear growth. The effect of nerve section on daytime food intake was expressed quite rapidly. Daytime food intake increased within 8 h after hepatic vagotomy, which was produced at light onset by pulling on a previously implanted suture. Collectively, these results demonstrate that hepatic vagotomy changes daily feeding rhythms and suggest that the liver and perhaps its vagal innervation are involved in the control of ad libitum eating behavior.

  19. Efficacy of HGF carried by ultrasound microbubble-cationic nano-liposomes complex for treating hepatic fibrosis in a bile duct ligation rat model, and its relationship with the diffusion-weighted MRI parameters.

    PubMed

    Zhang, Shou-hong; Wen, Kun-ming; Wu, Wei; Li, Wen-yan; Zhao, Jian-nong

    2013-12-01

    Hepatic fibrosis is a major consequence of liver aggression. Finding novel ways for counteracting this damaging process, and for evaluating fibrosis with a non-invasive imaging approach, represent important therapeutic and diagnostic challenges. Hepatocyte growth factor (HGF) is an anti-fibrosis cell growth factor that induces apoptosis in activated hepatic stellate cells, reduces excessive collagen deposition, and stimulates hepatocyte regeneration. Thus, using HGF in gene therapy against liver fibrosis is an attractive approach. The aims of the present study were: (i) to explore the efficacy of treating liver fibrosis using HGF expression vector carried by a novel ultrasound microbubble delivery system; (ii) to explore the diagnostic interest of diffusion-weighted MRI (DWI-MRI) in evaluating liver fibrosis. We established a rat model of hepatic fibrosis. The rats were administered HGF linked to novel ultrasound micro-bubbles. Progression of hepatic fibrosis was evaluated by histopathology, hydroxyproline content, and DWI-MRI to determine the apparent diffusion coefficient (ADC). Our targeted gene therapy produced a significant anti-fibrosis effect, as shown by liver histology and significant reduction of hydroxyproline content. Moreover, using DWI-MRI, the b value (diffusion gradient factor) was equal to 300s/mm(2), and the ADC values significantly decreased as the severity of hepatic fibrosis increased. Using this methodology, F0-F2 could be distinguished from F3 and F4 (P<0.01). This is the first in vivo report of using an ultrasound microbubble-cationic nano-liposome complex for gene delivery. The data indicate that, this approach is efficient to counteract the fibrosis process. DWI-MRI appears a promising imaging technique for evaluating liver fibrosis.

  20. Age differences affecting induction of hepatic drug metabolizing enzymes by methaqualone and phenobarbital in the rat.

    PubMed

    Mathur, P P; Boren, J A; Smyth, R D; Reavey-Cantwell, N H

    1975-05-01

    Methaqualone pretreatment for 3 or 6 days caused an induction of hepatic enzymes in the young male rat as measured by a reduction in hexobarbital-hypnosis. However, methaqualone pretreatment had no effect on the hexobarbital-hypnotic response in older male rats. Phenobarbital was a more potent enzyme inducer than methaqualone, and caused induction of liver enzymes in both age groups.

  1. Dimethyl dimethoxy biphenyl dicarboxylate attenuates hepatic and metabolic alterations in high fructose-fed rats.

    PubMed

    Morsy, Mohamed A; Ibrahim, Mohamed A; Abd-Elghany, Manal I

    2016-01-01

    High fructose consumption is currently linked to metabolic disorders including insulin resistance and dyslipidemia as well as hepatic steatosis. Dimethyl dimethoxy biphenyl dicarboxylate (DDB) is a hepatoprotectant with antioxidant and anti-inflammatory properties. The aim of this study therefore is to evaluate the effect of DDB on high fructose-induced metabolic disturbances and hepatic steatosis in a rat model. Male Wistar rats were allocated into three groups: control, fructose-fed (10% in drinking water and 10% in diet), and fructose-fed DDB (300 mg/kg, orally)-treated groups. Rats were fed a high-fructose diet for 6 weeks, while DDB was administered for an additional 2 weeks. High-fructose consumption elevated serum glucose and insulin levels and impaired oral glucose tolerance test, revealing insulin resistance. It also increased serum triglycerides and alanine aminotransferase as well as visceral fat content and decreased serum high-density lipoprotein. Additionally, histopathological examination revealed that high fructose intake induced hepatic steatosis. These alterations were associated with increased serum uric acid as well as hepatic content of malondialdehyde and nitric oxide (NO) in addition to overexpression of inducible NO synthase (iNOS). DDB administration significantly ameliorated the high fructose-induced hepatic and metabolic alterations. In conclusion, DDB ameliorates high fructose-induced metabolic disorders and hepatic steatosis in rats. Such protection is, at least in part, due to the inhibition of lipid peroxidation, decrease in iNOS overexpression, and reduction of elevated uric acid.

  2. Betaine alleviates hepatic lipid accumulation via enhancing hepatic lipid export and fatty acid oxidation in rats fed with a high-fat diet.

    PubMed

    Xu, Li; Huang, Danping; Hu, Qiaolin; Wu, Jing; Wang, Yizhen; Feng, Jie

    2015-06-28

    To assess the effects of betaine on hepatic lipid accumulation and investigate the underlying mechanism, thirty-two male Sprague-Dawley rats weighing 100 (sd 2·50) g were divided into four groups, and started on one of four treatments: basal diet, basal diet with betaine administration, high-fat diet and high-fat diet with betaine administration. The results showed that no significant difference of body weight was found among experimental groups. Compared with high-fat diet-fed rats, a betaine supplementation decreased (P< 0·05) hepatic TAG accumulation induced by high-fat diet, which was also supported by hepatic histology results. Additionally, hepatic betaine-homocysteine methyltransferase concentration [corrected] as well as its mRNA abundance and lecithin level were found increased (P< 0·05) by betaine supplementation in both basal diet-fed rats and high-fat diet-fed rats. Betaine administration in high-fat diet-fed rats exhibited a higher (P< 0·05) concentration [corrected] of hepatic carnitine palmitoyltransferase 1 (CPT1) compared with high-fat diet-fed rats. High-fat diet inhibited (P< 0·05) the gene expression of hepatic PPARα and CPT1. However, betaine administration in high-fat diet-fed rats elevated (P< 0·05) the gene expression of PPARα and CPT1. Moreover, concentration, gene and protein expressions of hepatic fibroblast growth factor 21 (FGF21) were increased (P< 0·05) in response to betaine administration in high-fat diet group; meanwhile the gene expression of hepatic AMP-activated protein kinase was increased (P< 0·05) as well. The results suggest that betaine administration enhanced hepatic lipid export and fatty acid oxidation in high-fat diet-fed rats, thus effectively alleviating fat accumulation in the liver.

  3. Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats.

    PubMed

    Park, So Young; Kim, Chung Hyeon; Lee, Ji Yoon; Jeon, Jang Su; Kim, Min Ju; Chae, Song Hee; Kim, Hyoung Chin; Oh, Soo Jin; Kim, Sang Kyum

    2016-10-01

    In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1'- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

  4. Exposure to a northern contaminant mixture (NCM) alters hepatic energy and lipid metabolism exacerbating hepatic steatosis in obese JCR rats.

    PubMed

    Mailloux, Ryan J; Florian, Maria; Chen, Qixuan; Yan, Jin; Petrov, Ivan; Coughlan, Melanie C; Laziyan, Mahemuti; Caldwell, Don; Lalande, Michelle; Patry, Dominique; Gagnon, Claude; Sarafin, Kurtis; Truong, Jocelyn; Chan, Hing Man; Ratnayake, Nimal; Li, Nanqin; Willmore, William G; Jin, Xiaolei

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to 10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co

  5. Exposure to a Northern Contaminant Mixture (NCM) Alters Hepatic Energy and Lipid Metabolism Exacerbating Hepatic Steatosis in Obese JCR Rats

    PubMed Central

    Mailloux, Ryan J.; Florian, Maria; Chen, Qixuan; Yan, Jin; Petrov, Ivan; Coughlan, Melanie C.; Laziyan, Mahemuti; Caldwell, Don; Lalande, Michelle; Patry, Dominique; Gagnon, Claude; Sarafin, Kurtis; Truong, Jocelyn; Chan, Hing Man; Ratnayake, Nimal; Li, Nanqin; Willmore, William G.; Jin, Xiaolei

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co

  6. Exposure to a northern contaminant mixture (NCM) alters hepatic energy and lipid metabolism exacerbating hepatic steatosis in obese JCR rats.

    PubMed

    Mailloux, Ryan J; Florian, Maria; Chen, Qixuan; Yan, Jin; Petrov, Ivan; Coughlan, Melanie C; Laziyan, Mahemuti; Caldwell, Don; Lalande, Michelle; Patry, Dominique; Gagnon, Claude; Sarafin, Kurtis; Truong, Jocelyn; Chan, Hing Man; Ratnayake, Nimal; Li, Nanqin; Willmore, William G; Jin, Xiaolei

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to 10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co

  7. Zinc supplementation decreases hepatic copper accumulation in LEC rat: a model of Wilson's disease.

    PubMed

    Gonzalez, Blanca P Esparza; Niño Fong, Rodolfo; Gibson, Candace J; Fuentealba, I Carmen; Cherian, M George

    2005-01-01

    The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.

  8. The response of hepatic transcriptome to dietary cholesterol in Prague hereditary hypercholesterolemic (PHHC) rat.

    PubMed

    Vlachová, M; Heczková, M; Jirsa, M; Poledne, R; Kovář, J

    2014-01-01

    To understand the pathogenesis of hypercholesterolemia in Prague hereditary hypercholesterolemic (PHHC) rat, we analyzed the response of hepatic transcriptome to dietary cholesterol in PHHC and control Wistar rats. Male PHHC and Wistar rats were fed chow (C), 5 % fat (palm kernel oil) (CF) or 1 % cholesterol + 5 % fat (CHOL) diet for three weeks. Hepatic transcriptome was analyzed using Affymetrix GeneChip arrays. No differences were found in the effect of both control diets (C and CF) on lipid metabolism and gene expression of 6500 genes. Therefore, these data were pooled for further analysis. Dietary cholesterol induced accumulation of cholesterol and triacylglycerols in the liver in both strains and hypercholesterolemia in PHHC rats. However, there were no differences in response of hepatic transcriptome to CHOL diet. On the other hand, several genes were found to be differently expressed between both strains independently of the diet. Two of those genes, Apof and Aldh1a7, were studied in more detail, and their role in pathogenesis of hypercholesterolemia in PHHC rats could not been corroborated. In conclusion, the hypercholesterolemia in PHHC rats is due to physiological response of hepatic transcriptome to dietary cholesterol in different genetic background.

  9. Female spontaneously diabetic Torii fatty rats develop nonalcoholic steatohepatitis-like hepatic lesions

    PubMed Central

    Ishii, Yukihito; Motohashi, Yu; Muramatsu, Makoto; Katsuda, Yoshiaki; Miyajima, Katsuhiro; Sasase, Tomohiko; Yamada, Takahisa; Matsui, Tohru; Kume, Shinichi; Ohta, Takeshi

    2015-01-01

    AIM: To investigate the histological features of the liver in spontaneously diabetic Torii (SDT) fatty rats compared with age-matched Sprague-Dawley (SD) rats. METHODS: Female SDT Leprfa (SDT fatty) rats and age-matched SD rats were fed ad libitum. Body weight and biochemical parameters, such as serum glucose, triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels as well as fatty acid and TG accumulation in the liver were evaluated at 8 wk of age in the non-fasting state and at 8-wk intervals from 8 to 40 wk of age. Histopathological examinations of the liver were performed using hematoxylin and eosin and Sirius Red staining as well as double staining for ED-1 and toluidine blue. The expression of genes involved in TG synthesis, inflammation, and fibrosis was examined in the liver. RESULTS: SDT fatty rats showed significantly increased body weight compared with SD rats. Serum glucose, TG, and TC levels were significantly higher in SDT fatty rats compared with SD rats. The serum AST and ALT levels in SDT fatty rats were significantly elevated at 8 wk of age compared with the levels in SD rats. Hepatic TG content was marked in SDT fatty rats from 8 to 32 wk of age. Histopathologically, severe hepatosteatosis accompanied by inflammation was observed at 8 wk of age, and fibrosis started to occur at 32 wk of age. Furthermore, Sirius Red and ED-1 staining were increased in the liver at 32 wk of age. Hepatic gene expression related to TG synthesis, inflammation and fibrosis tended to increase in SDT fatty rats compared with SD rats, and the gene expression related to TG secretion was decreased in SDT fatty rats compared with SD rats. CONCLUSION: Female SDT fatty rats have the potential to become an important animal model of nonalcoholic steatohepatitis with type 2 diabetes and obesity. PMID:26290633

  10. Lung Matrix Metalloproteinase Activation following Partial Hepatic Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Ferrigno, Andrea; Rizzo, Vittoria; Tarantola, Eleonora

    2014-01-01

    Purpose. Warm hepatic ischemia-reperfusion (I/R) injury can lead to multiorgan dysfunction. The aim of the present study was to investigate whether acute liver I/R does affect the function and/or structure of remote organs such as lung, kidney, and heart via modulation of extracellular matrix remodelling. Methods. Male Sprague-Dawley rats were subjected to 30 min partial hepatic ischemia by clamping the hepatic artery and the portal vein. After a 60 min reperfusion, liver, lung, kidney, and heart biopsies and blood samples were collected. Serum hepatic enzymes, creatinine, urea, Troponin I and TNF-alpha, and tissue matrix metalloproteinases (MMP-2, MMP-9), myeloperoxidase (MPO), malondialdehyde (MDA), and morphology were monitored. Results. Serum levels of hepatic enzymes and TNF-alpha were concomitantly increased during hepatic I/R. An increase in hepatic MMP-2 and MMP-9 activities was substantiated by tissue morphology alterations. Notably, acute hepatic I/R affect the lung inasmuch as MMP-9 activity and MPO levels were increased. No difference in MMPs and MPO was observed in kidney and heart. Conclusions. Although the underlying mechanism needs further investigation, this is the first study in which the MMP activation in a distant organ is reported; this event is probably TNF-alpha-mediated and the lung appears as the first remote organ to be involved in hepatic I/R injury. PMID:24592193

  11. Hepatic metabolism of ibuprofen in rats under acute hypobaric hypoxia.

    PubMed

    Gola, Shefali; Keshri, Gaurav Km; Gupta, Asheesh

    2013-09-01

    Hypobaric hypoxia induced at high altitude causes a subnormal oxygen concentration in cells which affects the drug metabolic and pharmacokinetic (PK) capacity of the body. The metabolism and PK of drugs like ibuprofen may be impaired under hypoxia and may require a different than usual therapeutic dose regimen to ensure safe therapy. The present investigation was undertaken to evaluate the effect of acute hypobaric hypoxia (AHH) on hepatic metabolism and PK of ibuprofen in rats. Animals were exposed to simulated altitude of 7620 m (∼25,000 ft) for AHH exposure (6 and 24 h) in a decompression chamber and were administrated with single dose of ibuprofen (80 mg/kg body weight, p.o.). The results showed that GST activity was significantly reduced at 6 h (15%) and 24 h (23%) (p<0.05) in hypoxic group as compared to normoxic. A significant increase by 20-24% (p<0.05) in AST level was observed after AHH exposure. LDH activity also exhibited significant increase (p<0.05) after 24h of AHH. A significant down-regulated CYP2C9 level and mild histopathological changes were observed after 24h of AHH. Furthermore, PK variables viz. elimination half-life (T½) and mean residence time (MRT) of ibuprofen exhibited significant increase by 42% and 51% (p<0.05) respectively after 24h of AHH. Thus, results suggest that AHH exposure of 24h significantly affects phase II conjugation pathway, CYP2C9 level, AST level, liver histology and PK parameters. This asserts that AHH can impair disposition of ibuprofen however, it requires further investigation under chronic hypobaric hypoxic conditions.

  12. An epoxysuccinic acid derivative(loxistatin)-induced hepatic injury in rats and hamsters

    SciTech Connect

    Fukushima, K.; Arai, M.; Kohno, Y.; Suwa, T.; Satoh, T. )

    1990-08-01

    Loxistatin is a possible therapeutic agent of muscular dystrophy. A single oral administration of loxistatin to male rats caused focal necrosis of the liver with inflammatory cell infiltration. The severity of the lesions was dose-dependent up to 200 mg/kg and also manifest by an increase in serum alanine aminotransferase and aspartate aminotransferase activities. Hepatic glutathione (GSH) levels decreased with a maximum 20% depletion within 5 hr after the oral administration of loxistatin. Pretreatment with diethyl maleate did not potentiate the loxistatin-induced hepatic injury. On the other hand, the hepatoprotective effect of cysteamine was observed when cysteamine was administered 24 hr before loxistatin dosing, but the effect was not observed when the antidote was administered concomitantly with loxistatin. Pretreatment of rats with phenobarbital or trans-stilbene oxide provided partial protection against the hepatotoxic effect of loxistatin. Pretreatment with SKF-525A resulted in increased hepatic injury, while pretreatment with piperonyl butoxide, cimetidine, or 3-methylcholanthrene had no effect on hepatic damage by loxistatin. Five hours after (14C)loxistatin administration to rats, the covalent binding of the radioactivity to proteins was greatest in the liver, followed by the kidney, then muscle and blood to a lesser extent. (14C)Loxistatin acid, the pharmacologically active form of loxistatin, irreversibly bound to rat liver microsomal proteins; more binding occurred when the NADPH-generating system was omitted and when the microsomes were boiled first. GSH did not alter the extent of irreversible binding, whereas N-ethylmaleimide decreased the binding of (14C)loxistatin acid to rat liver microsomal proteins by 75%. Unlike the rat, administration of loxistatin to hamsters caused neither hepatic injury nor hepatic GSH depletion.

  13. [Induction of rat hepatic CYP2E1 expression by arecoline in vivo].

    PubMed

    Huang, Xiang-tao; Xiao, Run-mei; Wang, Ming-feng; Wang, Jun-jun; Chen, Yong

    2016-01-01

    The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg x kg(-1) x d(-1)) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepaticdoes not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- ranslational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human. PMID:27405178

  14. IL-13 promotes the proliferation of rat pancreatic stellate cells through the suppression of NF-{kappa}B/TGF-{beta}{sub 1} pathway

    SciTech Connect

    Shinozaki, Satoshi; Mashima, Hirosato; Ohnishi, Hirohide; Sugano, Kentaro

    2010-02-26

    In chronic pancreatitis, pancreatic stellate cells (PSCs) play a central role in tissue fibrogenesis. Transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}) and the Th2 lymphokines such as interleukin (IL)-13 are major profibrogenic cytokines in many organs. Activated PSCs produce various inflammatory cytokines including TGF-{beta}{sub 1}. In this study, we investigated whether IL-13 affects pancreatic fibrogenesis by modulating the functions of PSCs. IL-13 promoted PSCs proliferation without activation through the suppression of autocrine TGF-{beta}{sub 1}. IL-13 enhanced Stat6 phosphorylation in PSCs but Stat6 was not involved in the suppression of TGF-{beta}{sub 1}. IL-13 inhibited the transcriptional activity of NF-{kappa}B, and the expression of mutant I-{kappa}B reproduced the suppression of autocrine TGF-{beta}{sub 1} and promoted PSCs proliferation. Taken together, we demonstrated that IL-13 promotes PSCs proliferation through the suppression of the transcriptional activity of NF-{kappa}B, resulting in the decrease of autocrine TGF-{beta}{sub 1}. This finding provides an unequivocal evidence of IL-13 participation in pancreatic fibrosis, illustrating a new strategy for chronic pancreatitis.

  15. Variable severity and Ia antigen expression in streptococcal-cell-wall-induced hepatic granulomas in rats.

    PubMed

    Allen, J B; Wilder, R L

    1987-03-01

    We have previously reported that a single intraperitoneal injection of an aqueous suspension of group A streptococcal cell wall (SCW) fragments induces extensive hepatic granulomas in LEW/N female rats, but not in F344/N female rats. To further understand the mechanisms underlying these differences, we compared granuloma development and class II major histocompatibility complex antigen (Ia) expression in histocompatible LEW/N, F344/N, and CAR/N female rats in response to SCW fragments of four different average molecular sizes. In LEW/N female rats, the smallest fragments (less than 5 megadaltons) induced the most severe hepatic inflammatory disease, with development of widespread granulomas composed of macrophages, lymphocytes, and a peripheral rim of fibroblasts. The largest fragments (greater than 500 megadaltons) induced equivocal disease. Fragments of intermediate size induced granulomas of intermediate severity. The extent of granuloma development, the intensity of Ia antigen expression, and the amount of SCW antigen deposited in the liver qualitatively paralleled each other. In contrast, injection of the most granulomagenic SCW fragments into F344/N and CAR/N rats did not induce granulomas. Although these rat strains are histocompatible with the LEW/N (i.e., RTL.1) strain, hepatic Ia antigen expression in these strains was not increased significantly above basal levels. The amount of SCW antigen in the livers of the resistant rat strains appeared similar to the amount in the susceptible LEW/N strain. These data indicate that granuloma development is dependent on the size of the SCW fragment and host genetic background and that Ia expression directly parallels the severity of the hepatic disease. In addition, the data suggest that non-major histocompatibility complex genetic loci play a major role in regulating the development of the hepatic disease.

  16. Attenuated Effects of Deep-Sea Water on Hepatic Apoptosis in STZ-Induced Diabetic Rats.

    PubMed

    Hsu, Tsai-Ching; Chiu, Chun-Ching; Lin, Hsou-Lin; Kao, Tseng-Wei; Chen, Li-Jeng; Wu, Li-Yi; Huang, Chih-Yang; Tzang, Bor-Show

    2015-06-30

    Diabetes mellitus (DM) is a metabolic disorder and increasing evidences have indicated a connection between DM and hepatic abnormality. Deep-sea water (DSW) has been applied in many fields, especially in medicine; herein, we investigated the influence of DSW on hepatic apoptosis in streptozocin (STZ)-induced diabetes rats. Our experimental results firstly demonstrated the beneficial effects of 1×DSW, 2×DSW and 3×DSW in alleviating hepatic apoptosis in STZ-induced diabetic rats. We demonstrated that 1×DSW, 2×DSW and 3×DSW significantly suppressed the caspase-3 activity and TUNEL-positive cells in livers of STZ-induced diabetic rats. Significant reductions of both Fas-dependent and mitochondrial-dependent apoptotic molecules were also detected in livers of STZ-induced diabetic rats receiving DSW. Additionally, apoptotic signaling molecules such as phosphorylated IκB-α and NF-κB were significantly reduced in livers of DSW-treated STZ-induced diabetic rats. These findings indicate hepatic protective effects of DSW on DM and suggest DSW as a possible ingredient for health food. PMID:26014125

  17. Attenuated Effects of Deep-Sea Water on Hepatic Apoptosis in STZ-Induced Diabetic Rats.

    PubMed

    Hsu, Tsai-Ching; Chiu, Chun-Ching; Lin, Hsou-Lin; Kao, Tseng-Wei; Chen, Li-Jeng; Wu, Li-Yi; Huang, Chih-Yang; Tzang, Bor-Show

    2015-06-30

    Diabetes mellitus (DM) is a metabolic disorder and increasing evidences have indicated a connection between DM and hepatic abnormality. Deep-sea water (DSW) has been applied in many fields, especially in medicine; herein, we investigated the influence of DSW on hepatic apoptosis in streptozocin (STZ)-induced diabetes rats. Our experimental results firstly demonstrated the beneficial effects of 1×DSW, 2×DSW and 3×DSW in alleviating hepatic apoptosis in STZ-induced diabetic rats. We demonstrated that 1×DSW, 2×DSW and 3×DSW significantly suppressed the caspase-3 activity and TUNEL-positive cells in livers of STZ-induced diabetic rats. Significant reductions of both Fas-dependent and mitochondrial-dependent apoptotic molecules were also detected in livers of STZ-induced diabetic rats receiving DSW. Additionally, apoptotic signaling molecules such as phosphorylated IκB-α and NF-κB were significantly reduced in livers of DSW-treated STZ-induced diabetic rats. These findings indicate hepatic protective effects of DSW on DM and suggest DSW as a possible ingredient for health food.

  18. Endogenous carbon monoxide downregulates hepatic cystathionine-γ-lyase in rats with liver cirrhosis

    PubMed Central

    GUO, SHI-BIN; DUAN, ZHI-JUN; WANG, QIU-MING; ZHOU, QIN; LI, QING; SUN, XIAO-YU

    2015-01-01

    The aim of the present study was to investigate the effect of endogenous carbon monoxide (CO) on the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) pathway in cirrhotic rat livers. The rats were allocated at random into four groups: Sham, cirrhosis, cobalt protoporphyrin (CoPP) and zinc protoporphyrin IX (ZnPP). The expression of hepatic CSE mRNA was evaluated using a quantitative polymerase chain reaction, while CSE protein expression was determined using immunohistochemical analysis. Hematoxylin and eosin staining was performed for the histological evaluation of liver fibrosis. The levels of H2S, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and carboxyhemoglobin (COHb) in the arterial blood were determined, in addition to the portal vein pressure. The mRNA and protein expression levels of hepatic CSE and the serum levels of H2S were significantly decreased in the cirrhosis group compared with those in the sham group (P<0.05). Compared with the cirrhosis group, rats in the ZnPP group had significantly lower levels of serum ALT, AST and TBIL, arterial COHb and hepatic fibrosis, while hepatic CSE expression and the production of H2S were significantly increased (P<0.05). The CoPP group exhibited decreased hepatic CSE expression and H2S production, but aggravated hepatic function and fibrosis (P<0.05). In conclusion, the H2S/CSE pathway is involved in the formation of liver cirrhosis and serves a crucial function in protecting liver cells against the progression of liver fibrosis. Endogenous CO downregulates hepatic CSE mRNA and protein expression and the production of H2S in rats with liver cirrhosis. PMID:26668593

  19. The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

    PubMed Central

    Erion, Derek M.; Lapworth, Amanda; Amor, Paul A.; Bai, Guoyun; Vera, Nicholas B.; Clark, Ronald W.; Yan, Qingyun; Zhu, Yimin; Ross, Trenton T.; Purkal, Julie; Gorgoglione, Matthew; Zhang, Guodong; Bonato, Vinicius; Baker, Levenia; Barucci, Nicole; D’Aquila, Theresa; Robertson, Alan; Aiello, Robert J.; Yan, Jiangli; Trimmer, Jeff; Rolph, Timothy P.; Pfefferkorn, Jeffrey A.

    2014-01-01

    Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase

  20. The hepatoselective glucokinase activator PF-04991532 ameliorates hyperglycemia without causing hepatic steatosis in diabetic rats.

    PubMed

    Erion, Derek M; Lapworth, Amanda; Amor, Paul A; Bai, Guoyun; Vera, Nicholas B; Clark, Ronald W; Yan, Qingyun; Zhu, Yimin; Ross, Trenton T; Purkal, Julie; Gorgoglione, Matthew; Zhang, Guodong; Bonato, Vinicius; Baker, Levenia; Barucci, Nicole; D'Aquila, Theresa; Robertson, Alan; Aiello, Robert J; Yan, Jiangli; Trimmer, Jeff; Rolph, Timothy P; Pfefferkorn, Jeffrey A

    2014-01-01

    Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase

  1. The simultaneous occurrence of human norovirus and hepatitis E virus in a Norway rat (Rattus norvegicus).

    PubMed

    Wolf, Sandro; Reetz, Jochen; Johne, Reimar; Heiberg, Ann-Charlotte; Petri, Samuel; Kanig, Hanna; Ulrich, Rainer G

    2013-07-01

    Wild rats can be reservoirs and vectors for several human pathogens. An initial RT-PCR screening of the intestinal contents of Norway rats trapped in the sewer system of Copenhagen, Denmark, for caliciviruses revealed the presence of a human norovirus in one of 11 rodents. Subsequent phylogenetic analysis of the ~4.0-kb 3'-terminus of the norovirus genome resulted in the identification of a recombinant GI.b/GI.6 strain. The simultaneous detection of hepatitis E virus-like particles in the feces of this rat by transmission electron microscopy was confirmed by RT-PCR and sequence determination, resulting in the identification of a novel rat hepatitis E virus. PMID:23443935

  2. Effect of chronic renal insufficiency on hepatic and renal udp-glucuronyltransferases in rats.

    PubMed

    Yu, Chuanhui; Ritter, Joseph K; Krieg, Richard J; Rege, Bhaskar; Karnes, Thomas H; Sarkar, Mohamadi A

    2006-04-01

    Significant evidence exists regarding altered CYP450 enzymes in chronic renal insufficiency (CRI), although none exists for the phase II enzymes. The objective of this study was to investigate the effect of CRI on hepatic and renal UDP-glucuronyltransferase (UGT) enzymes. Three groups of rats were included: CRI induced by the 5/6th nephrectomy model, control, and control pair-fed (CPF) rats. UGT activities were determined in liver and kidney microsomes by the 3- and 17-glucuronidation of beta-estradiol (E2-3G and E2-17G), glucuronidation of 4-methylumbelliferone (4-MUG), and 3-glucuronidation of morphine (M3G). UGT isoforms responsible for these catalytic activities were screened using recombinant rat UGT1A1, UGT1A2, UGT1A3, UGT1A7, UGT2B2, UGT2B3, and UGT2B8. UGT protein levels were examined by Western blot analysis using polyclonal antibodies. There was no significant difference between CRI and CPF rats in hepatic and/or renal E2-3G (UGT1A1), E2-17G (UGT2B3), 4-MUG (UGT1A6), and M3G (UGT2B1) formation. Formation of E2-17G and 4-MUG in the liver and E2-3G and 4-MUG in the kidney was significantly reduced (p < 0.05) in CPF and CRI rats compared with control rats. The down-regulated glucuronidation activities were accompanied by corresponding reductions in protein content of specific UGT isoforms. These results suggest that CRI does not seem to influence the protein levels or catalytic activity of most of the major hepatic or renal UGT enzymes. The observed down-regulation of hepatic and renal UGTs in CRI and CPF rats could be caused by restricted food intake in these groups of rats.

  3. Protective effects of fish oil, allopurinol, and verapamil on hepatic ischemia-reperfusion injury in rats

    PubMed Central

    Messiha, Basim Anwar Shehata; Abo-Youssef, Amira M.

    2015-01-01

    Background: The major aim of this work was to study the protective effects of fish oil (FO), allopurinol, and verapamil on hepatic ischemia-reperfusion (IR)-induced injury in experimental rats. Materials and Methods: Sixty male Wistar albino rats were randomly assigned to six groups of 10 rats each. Group 1 served as a negative control. Group 2 served as hepatic IR control injury. Groups 3, 4, 5, and 6 received N-acetylcysteine (standard), FO, allopurinol, and verapamil, respectively, for 3 consecutive days prior to ischemia. All animals were fasted for 12 h, anesthetized and underwent midline laparotomy. The portal triads were clamped by mini-artery clamp for 30 min followed by reperfusion for 30 min. Blood samples were withdrawn for estimation of serum alanine transaminase (ALT) and aspartate transaminase (AST) activities as well as hepatic thiobarbituric acid reactive substances, reduced glutathione, myeloperoxidase, and total nitrate/nitrite levels, in addition to histopathological examination. Results: Fish oil, allopurinol, and verapamil reduced hepatic IR injury as evidenced by significant reduction in serum ALT and AST enzyme activities. FO and verapamil markedly reduced oxidative stress as compared to control IR injury. Levels of inflammatory biomarkers in liver were also reduced after treatment with FO, allopurinol, or verapamil. In accordance, a marked improvement of histopathological findings was observed with all of the three treatments. Conclusion: The findings of this study prove the benefits of FO, allopurinol, and verapamil on hepatic IR-induced liver injury and are promising for further clinical trials. PMID:26283828

  4. The effects of tramadol on hepatic ischemia/reperfusion injury in rats

    PubMed Central

    Mahmoud, Mona F.; Gamal, Samar; Shaheen, Mohamed A.; El-Fayoumi, Hassan M.

    2016-01-01

    Objectives: Tramadol is a centrally acting synthetic analgesic. It has a cardioprotective effect against myocardial ischemia-reperfusion (I/R) injury in isolated rat heart. We hypothesized that tramadol may exert a similar protective effect on hepatic I/R injury. Hence, the current investigation was designed to study the possible protective effects of tramadol on experimentally-induced hepatic I/R injury in rats. Materials and Methods: Tramadol was administered 30 min before ischemia following which the rats were subjected to 45 min of ischemia followed by 1 h of reperfusion. Results: Tramadol attenuated hepatic injury induced by I/R as evidenced by the reduction of transaminases, structural changes, and apoptotic cell death. It decreased the level of inflammatory markers such as tumor necrosis factor-alpha (TNF-α), TNF-α/interleukin-10 (IL-10) ratio, and nuclear factor-κB gene expression. It also increased the anti-inflammatory cytokine, IL-10 levels in hepatic tissues. Furthermore, it reduced oxidative stress parameters except manganese superoxide dismutase activity. Conclusion: The results suggest that tramadol has hepatoprotective effects against hepatic I/R injury via anti-inflammatory, antiapoptotic, and antioxidant effects. PMID:27298497

  5. Influence of Moxifloxacin on Hepatic Redox Status and Plasma Biomarkers of Hepatotoxicity and Nephrotoxicity in Rat.

    PubMed

    Ore, Ayokanmi; Olayinka, Ebenezer Tunde

    2015-01-01

    Moxifloxacin is a broad spectrum fluoroquinolone antibacterial agent. We examined the hepatic redox status and plasma biomarkers of nephrotoxicity and hepatotoxicity in rat following administration of moxifloxacin (MXF). Twenty-four Wistar rats, 180-200 g, were randomized into four groups (I-IV). Animals in group I (control) received 1 mL of distilled water, while animals in groups II, III, and IV received 1 mL each of MXF equivalent to 4 mg/kg b.w., 8 mg/kg b.w., and 16 mg/kg b.w., respectively. After seven days, plasma urea, bilirubin, and creatinine were significantly (P < 0.05) elevated in the MXF-treated animals. Activities of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase were significantly increased in the plasma of MXF-treated animals compared to control. Also plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides increased significantly in the MXF-treated groups relative to control. Moreover, MXF triggered a significant decrease in hepatic catalase, superoxide dismutase, and glutathione-S transferase activities. Likewise, MXF caused a decrease in the hepatic levels of glutathione and vitamin C. A significant increase in hepatic MDA content was also observed in the MXF-treated animals relative to control. Overall, our data suggest that the half-therapeutic, therapeutic, and twice the therapeutic dose of MXF induced nephrotoxicity, hepatotoxicity, and altered hepatic redox balance in rats. PMID:26550491

  6. Influence of Moxifloxacin on Hepatic Redox Status and Plasma Biomarkers of Hepatotoxicity and Nephrotoxicity in Rat

    PubMed Central

    Ore, Ayokanmi; Olayinka, Ebenezer Tunde

    2015-01-01

    Moxifloxacin is a broad spectrum fluoroquinolone antibacterial agent. We examined the hepatic redox status and plasma biomarkers of nephrotoxicity and hepatotoxicity in rat following administration of moxifloxacin (MXF). Twenty-four Wistar rats, 180–200 g, were randomized into four groups (I–IV). Animals in group I (control) received 1 mL of distilled water, while animals in groups II, III, and IV received 1 mL each of MXF equivalent to 4 mg/kg b.w., 8 mg/kg b.w., and 16 mg/kg b.w., respectively. After seven days, plasma urea, bilirubin, and creatinine were significantly (P < 0.05) elevated in the MXF-treated animals. Activities of alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase were significantly increased in the plasma of MXF-treated animals compared to control. Also plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides increased significantly in the MXF-treated groups relative to control. Moreover, MXF triggered a significant decrease in hepatic catalase, superoxide dismutase, and glutathione-S transferase activities. Likewise, MXF caused a decrease in the hepatic levels of glutathione and vitamin C. A significant increase in hepatic MDA content was also observed in the MXF-treated animals relative to control. Overall, our data suggest that the half-therapeutic, therapeutic, and twice the therapeutic dose of MXF induced nephrotoxicity, hepatotoxicity, and altered hepatic redox balance in rats. PMID:26550491

  7. Influence of zinc on the ontogeny of hepatic metallothionein in the fetal rat

    SciTech Connect

    Charles-Shannon, V.L.; Sasser, L.B.; Burbank, D.K.; Kelman, B.J.

    1981-10-01

    The ontogeny of hepatic metallothioneins (Mt) in fetal tissue as related to dietary and hepatic Zn was investigated. Sixty 6-month-old female rats were divided into two groups and given either double-distilled water or water containing 700 )g of Zn per milliliter. Dams from each group were killed on 16, 19, or 21 days of gestation, and maternal and fetal livers were removed. Mt content of the tissue was estimated by Piotrowski's Hg-saturation method. Results established the presence of an endogenous hepatic Mt in the fetal rat as early as 16 days of gestation. We further demonstrated a marked progressive increase in fetal Mt from Day 16 through gestation accompanied by a decrease in maternal hepatic Mt. It is suggested that Zn increased fetal Mt by inducing fetal synthesis, redistributing fetal Mt, or increasing Mt transport to the fetus, because both fetal and maternal hepatic Mt were increased. Fetal hepatic Mt concentration was several times greater than maternal Mt at corresponding stages of gestation. Mt may serve to either ensure adequate storage of Zn or Cu for fetal development or protect the fetus against metal toxicity, but the significance of these high endogenous levels of fetal Mt are not clear at this time.

  8. Dietary copper triggers onset of fulminant hepatitis in the Long-Evans cinnamon rat model

    PubMed Central

    Siaj, Ramsi; Sauer, Vanessa; Stöppeler, Sandra; Spiegel, Hans-Ullrich; Köhler, Gabriele; Zibert, Andree; Schmidt, Hartmut HJ

    2012-01-01

    AIM: To investigate the impact of dietary copper given at different time points on the onset of fulminant hepatitis. METHODS: The Long-Evans cinnamon (LEC) rat model of Wilson’s disease (WD) was used to study the impact of high dietary copper (hCu) on the induction of fulminant hepatitis at early or late time points of life. High Cu diet was started in rat pups or in adults (month 5) for three months. Animals that received reduced dietary copper (rCu) throughout their lifetime served as a control. Hepatitis-associated serum markers (alanine aminotransferase, aspartate transaminase, bilirubin) were analyzed in animal groups receiving hCu or rCu. Liver copper content and liver histology were revealed at sacrifice. A set of 5 marker genes previously found to be affected in injured liver and which are related to angiogenesis (Vegfa), fat metabolism (Srebf1), extracellular matrix (Timp1), oxidative stress (Hmox1), and the cell cycle (Cdkn1a) were analyzed by real-time polymerase chain reaction. RESULTS: Regardless of the time point when hCu was started, LEC rats (35/36) developed fulminant hepatitis and died. Animals receiving rCu (36/36) remained healthy, did not develop hepatitis, and survived long term without symptoms of overt disease, although liver copper accumulated in adult animals (477 ± 75 μg/g). With regard to start of hCu, onset of fulminant hepatitis was significantly (P < 0.001) earlier in adults (35 ± 9 d) that showed pre-accumulation of liver copper as compared to the pup group (77 ± 15 d). Hepatitis-associated serum markers, liver copper and liver histology, as well as gene expression, were affected in LEC rats receiving hCu. However, except for early and rapid onset of hepatitis, biochemical and molecular markers were similar at the early and late time points of disease. CONCLUSION: Rapid onset of fulminant hepatitis in asymptomatic LEC rats with elevated liver copper suggests that there is a critical threshold of liver copper which is important

  9. Effect of polybrominated biphenyls on hepatic excretory function in rats and mice.

    PubMed

    Cagen, S Z; Gibson, J E

    1978-04-01

    The purpose of this investigation was to determine the influence of polybrominated biphenyls (PBBs) on hepatic excretory function in developing and adult rats and mice. Prenatal or postnatal dietary exposure to PBBs (50 ppm in diet of pregnant or lactating mother or in diet of rat weanlings) resulted in elevated liver weight in developing rats. In 15-day-old rats that had been treated with PBBs increased liver weight correlated to enhanced ouabain transport from plasma into bile. Liver weight was also elevated in 21, 35, and 49-day-old rats exposed to PBBs, but this effect was not associated with stimulation of ouabain transport in these animals. However, adult rats fed 100 ppm PBBs for two weeks had significantly lower plasma concentrations of sulfobromophthalein (BSP) and increased biliary excretion of BSP, when compared to controls. PBBs-fed adult rats also excreted a greater percentage conjugated BSP (BSP-GSH) into bile. Two week dietary treatment of 100, 150, and 200 ppm PBBs resulted in enhanced initial disappearance of indocyanine green (ICG) from plasma of adult mice. However, dietary doses of 100 and 200 ppm PBBs to adult mice was not associated with enhanced capacity for ouabain excretion. In contrast, treatment with PBBs through the mother's diet (50 ppm) resulted in an almost twofold increase in cumulative ouabain excretion in 15-day-old mice. The results suggest that PBBs stimulate hepatic drug elimination in rats and mice, but the magnitude of the effect is dependent on age and transported compound.

  10. Hepatitis

    MedlinePlus

    ... has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool to ... risk for severe disease. Others A variety of viruses can affect the liver Signs and Symptoms Hepatitis ...

  11. Effects of hepatic enzyme inducers on thyroxine (T4) catabolism in primary rat hepatocytes

    EPA Science Inventory

    Nuclear receptor agonists such as phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 3-methylcholantrene (3-MC) decrease circulating thyroxine (T4) concentrations in rats. It is suspected that this decrease occurs through the induction of hepatic metabolizing en...

  12. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  13. Sasa borealis Stem Extract Attenuates Hepatic Steatosis in High-Fat Diet-induced Obese Rats

    PubMed Central

    Song, Yuno; Lee, Soo-Jung; Jang, Sun-Hee; Ha, Ji Hee; Song, Young Min; Ko, Yeoung-Gyu; Kim, Hong-Duck; Min, Wongi; Kang, Suk Nam; Cho, Jae-Hyeon

    2014-01-01

    The aim of the current study is to examine the improving effect of Sasa borealis stem (SBS) extract extracts on high-fat diet (HFD)-induced hepatic steatosis in rats. To determine the hepatoprotective effect of SBS, we fed rats a normal regular diet (ND), HFD, and HFD supplemented with 150 mg/kg body weight (BW) SBS extracts for five weeks. We found that the body weight and liver weight of rats in the HFD + SBS group were significantly lower than those in the HFD group. Significantly lower serum total cholesterol (TC) and triglyceride (TG) concentrations were observed in the SBS-supplemented group compared with the HFD group. We also found that the HFD supplemented with SBS group showed dramatically reduced hepatic lipid accumulation compared to the HFD alone group, and administration of SBS resulted in dramatic suppression of TG, TC in the HFD-induced fatty liver. In liver gene expression within the SBS treated group, PPARα was significantly increased and SREBP-1c was significantly suppressed. SBS induced a significant decrease in the hepatic mRNA levels of PPARγ, FAS, ACC1, and DGAT2. In conclusion, SBS improved cholesterol metabolism, decreased lipogenesis, and increased lipid oxidation in HFD-induced hepatic steatosis in rats, implying a potential application in treatment of non-alcoholic fatty liver disease. PMID:24905748

  14. Sasa borealis stem extract attenuates hepatic steatosis in high-fat diet-induced obese rats.

    PubMed

    Song, Yuno; Lee, Soo-Jung; Jang, Sun-Hee; Ha, Ji Hee; Song, Young Min; Ko, Yeoung-Gyu; Kim, Hong-Duck; Min, Wongi; Kang, Suk Nam; Cho, Jae-Hyeon

    2014-06-01

    The aim of the current study is to examine the improving effect of Sasa borealis stem (SBS) extract extracts on high-fat diet (HFD)-induced hepatic steatosis in rats. To determine the hepatoprotective effect of SBS, we fed rats a normal regular diet (ND), HFD, and HFD supplemented with 150 mg/kg body weight (BW) SBS extracts for five weeks. We found that the body weight and liver weight of rats in the HFD + SBS group were significantly lower than those in the HFD group. Significantly lower serum total cholesterol (TC) and triglyceride (TG) concentrations were observed in the SBS-supplemented group compared with the HFD group. We also found that the HFD supplemented with SBS group showed dramatically reduced hepatic lipid accumulation compared to the HFD alone group, and administration of SBS resulted in dramatic suppression of TG, TC in the HFD-induced fatty liver. In liver gene expression within the SBS treated group, PPARα was significantly increased and SREBP-1c was significantly suppressed. SBS induced a significant decrease in the hepatic mRNA levels of PPARγ, FAS, ACC1, and DGAT2. In conclusion, SBS improved cholesterol metabolism, decreased lipogenesis, and increased lipid oxidation in HFD-induced hepatic steatosis in rats, implying a potential application in treatment of non-alcoholic fatty liver disease. PMID:24905748

  15. Capillaria hepatica-induced hepatic fibrosis in rats: paradoxical effect of repeated infections.

    PubMed

    Oliveira, Ludmila; de Souza, Márcia Maria; Andrade, Zilton A

    2004-01-01

    Multiple exposures to parasitic agents are considered an important factor in the genesis of the most severe forms of the diseases they cause. Capillaria hepatica-induced septal fibrosis of the liver in rats usually runs without signs of portal hypertension or hepatic failure. After determining the hepatic profile of 15 animals during the course of a single infection, we submitted 20 rats to multiple Capillaria hepatica infections to determine whether repeated exposures would augment fibrosis production, transforming septal hepatic fibrosis into a true cirrhosis. Ten single-infection rats served as controls. A total of 5 exposures, with 45-day intervals, were made. Histological changes were followed by means of surgical liver biopsies, collected prior to infection and to each re-infection. Functional changes were minimal and transient. Although a slight recrudescence of fibrosis was observed after the first two re-infections and when the single-infected control group was re-infected at the end of the experiment, subsequent re-infections failed to increase the amount of fibrosis. On the contrary, there occurred quantitative and qualitative evidence of collagen degradation and suppression of parasite development. These paradoxical results are in keeping with the hypothesis that a complex immunological modulation participates in the mechanism of hepatic fibrosis induced by Capillaria hepatica infection in rats.

  16. HEPATIC GENE EXPRESSION PROFILES OF RATS EXPOSED TO PERFLUOROOCTANE SULFONATE (PFOS) IN UTERO

    EPA Science Inventory

    Hepatic Gene Expression Profiles of Rats Exposed to Perfluorooctanesulfonate (PFOS) in utero.
    J.A. Bjork1, J.M. Berthiaume1, C. Lau2, J. L. Butenhoff3, and K.B. Wallace1

    1Department of Biochemistry & Molecular Biology, University of Minnesota School of Medicine, Dulut...

  17. Artesunate ameliorates hepatic fibrosis induced by bovine serum albumin in rats through regulating matrix metalloproteinases.

    PubMed

    Xu, Yajie; Liu, Wendong; Fang, Buwu; Gao, Sinan; Yan, Jing

    2014-12-01

    The effect of Artesunate on anti-hepatic fibrosis was discovered by our team for the first time. In order to investigate the effect of Artesunate on hepatic fibrosis induced by Bovine serum albumin (BSA) in rats and understand the initiatory mechanism of its effect, several experiments were conducted in this assay. HE staining and Masson׳s Trichrome staining were employed in observation of morphological changes. The content of hydroxyproline in the hepatic tissue was determined by using an acid hydrolyzation method. In addition, the expression of Matrix metalloproteinase-13 (MMP-13) and type I collagen were tested by western blotting respectively. The expression of Matrix metalloproteinase-2(MMP-2), Matrix metalloproteinase-9 (MMP-9) were determined by Gelatin Zymography Assay. Also, we use immunohistochemical studies to measure the expression of α-SMA. The final results indicated that Artesunate could dramatically attenuate the extent of hepatic fibrosis showed by histopathological sections of hepatic tissues, significantly decrease the content of hydroxyproline and efficiently inhibit the protein expression of MMP-2, MMP-9, α-SMA and type I collagen. Artesunate could as well promote the expression of MMP-13 at the same time. In conclusion, the results not only suggested that Artesunate could ameliorate hepatic fibrosis, but also suggested the anti-fibrogenic mechanisms of Artesunate might be associated with inhibiting the activation of HSCs, decreasing the expression of MMP-2, MMP-9 and increasing the expression of MMP-13.These results would bring new insights for the treatment for hepatic fibrosis.

  18. The role of hepatic mitochondria in the regulation of glucose metabolism in BHE rats

    SciTech Connect

    Kim, M.J.C.

    1988-01-01

    The interacting effects of dietary fat source and thyroxine treatment on the hepatic mitochondrial function and glucose metabolism were studied. In the first study, three different sources of dietary fatty acids and thyroxine treatment were used to investigate the hepatic mitochondrial thermotropic behavior in two strains of rat. The NIDDM BHE and Sprague-Dawley rats were used. Feeding coconut oil increased serum T{sub 4} levels and T{sub 4} treatment increased serum T{sub 3} levels in the BHE rats. In the mitochondria from BHE rats fed coconut oil and treated with T{sub 4}, the transition temperature disappeared due to a decoupling of succinate supported respiration. This was not observed in the Sprague-Dawley rats. In the second study, two different sources of dietary fat and T{sub 4} treatment were used to investigate hepatic mitochondrial function. Coconut oil feeding increased Ca{sup ++}Mg{sup ++}ATPase and Mg{sup ++}ATPase. T{sub 4} treatment had potentiated this effect. T{sub 4} increased the malate-aspartate shuttle and {alpha}-glycerophosphate shuttle activities. In the third study, the glucose turnover rate from D-({sup 14}C-U)/(6-{sup 3}H)-glucose and gluconeogeneis from L-({sup 14}C-U)-alanine was examined. Dietary fat or T{sub 4} did not affect the glucose mass. T{sub 4} increased the irreversible fractional glucose turnover rate.

  19. Dexamethasone pretreatment attenuates lung and kidney injury in cholestatic rats induced by hepatic ischemia/reperfusion.

    PubMed

    Zhou, Liangyi; Yao, Xiangqing; Chen, Yanling

    2012-02-01

    Hepatic ischemia followed by reperfusion (IR) results in mild to severe organ injury, in which tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) seem to be involved. Thus, we aim to assess the influence of hepatic ischemia/reperfusion injury on remote organs in addition to cholestasis and consider the possible efficacy of steroid pretreatment in reducing the injury. A common bile duct ligation model was done on 24 male Sprague-Dawley rats. After 7 days, the rats were divided randomly into control group, IR group, and dexamethasone (DEX) group. The IR group showed significant increases in serum alanine aminotransferase, aspartate aminotransferase, and creatinine levels compared with the control and DEX groups. By ELISA techniques, higher levels of TNF-α and IL-1β in lung and kidney tissues were measured in the IR group than in the control and DEX groups, these were verified by immunohistochemistry. The lung histology of the IR group rats showed neutrophil infiltration, interstitial edema, and alveolar wall thickening. Kidney histology of the IR group rats showed vacuolization of the proximal tubular epithelial cells and tubular dilatation with granular eosinophilic casts. Better morphological aspects were observed in the DEX-pretreated animals. Minimal lesions were observed in the control. The results suggest that hepatic ischemia/reperfusion injury in cholestatic rats induced lung and kidney injuries. Pretreatment with dexamethasone reduced the IR-induced injury in addition to cholestasis.

  20. Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines.

    PubMed

    Jirintai, Suljid; Tanggis; Mulyanto; Suparyatmo, Joseph Benedictus; Takahashi, Masaharu; Kobayashi, Tominari; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2014-06-24

    Although rat hepatitis E virus (HEV) has been identified in wild rats, no cell culture systems for this virus have been established. A recent report suggesting the presence of antibodies against rat HEV in human sera encouraged us to cultivate rat HEV in human cells. When liver homogenates obtained from wild rats (Rattus rattus) in Indonesia were inoculated onto human hepatocarcinoma cells, the rat HEV replicated efficiently in PLC/PRF/5, HuH-7 and HepG2 cells, irrespective of its genetic group (G1-G3). The rat HEV particles released from cultured cells harbored lipid-associated membranes on their surface that were depleted by treatment with detergent and protease, with the buoyant density in sucrose shifting from 1.15-1.16 g/ml to 1.27-1.28 g/ml. A Northern blotting analysis revealed genomic RNA of 7.0 kb and subgenomic RNA of 2.0 kb in the infected cells. The subgenomic RNA of G1-G3 each possessed the extreme 5'-end sequence of GUAGC (nt 4933-4937), downstream of the highly conserved sequence of GAAUAACA (nt 4916-4923). The establishment of culture systems for rat HEV would allow for extended studies of the mechanisms of viral replication and functional roles of HEV proteins. Further investigation is required to clarify the zoonotic potential of rat HEV.

  1. Effect of Experimentally Induced Hepatic and Renal Failure on the Pharmacokinetics of Topiramate in Rats

    PubMed Central

    Matar, Kamal M.; Tayem, Yasin I.

    2014-01-01

    We aimed to investigate the effect of induced hepatic and renal failure on the pharmacokinetics of topiramate (TPM) in rats. Twenty-four Sprague-Dawley rats were used in this study. Renal or hepatic failure was induced by a single i.p. dose of 7.5 mg/kg cisplatin (n = 8) or 0.5 mL/kg carbon tetrachloride (CCl4) (n = 8), respectively. Three days after cisplatin dose or 24 h after CCl4 dose, the rats were administered a single oral dose of 20 mg/kg TPM. The plasma samples were quantified by LC-MS/MS method. Compared to control, plasma concentration-time profile in CCl4-treated and, to a lesser extent, in cisplatin-treated rats decreased more slowly particularly in the elimination phase. TPM oral clearance (CL/F) in CCl4-treated group was significantly lower than that in control (P < 0.001), whereas AUC0−∞, T1/2, and Vd/F were significantly higher in CCl4-treated rats compared to the control (P < 0.01). The CL/F was not significantly different between cisplatin-treated rats and control (P > 0.05). However, in cisplatin-treated rats, the T1/2 and Vd/F were significantly higher than that in the control group (P < 0.01). Both conditions failed to cause a significant effect on Cmax or Tmax. The present findings suggest that induced hepatic or renal failure could modify the pharmacokinetic profile of TPM in the rat. PMID:25009818

  2. Vitamin E supplementation does not prevent ethanol-reduced hepatic retinoic acid levels in rats.

    PubMed

    Chung, Jayong; Veeramachaneni, Sudipta; Liu, Chun; Mernitz, Heather; Russell, Robert M; Wang, Xiang-Dong

    2009-09-01

    Chronic, excessive ethanol intake can increase retinoic acid (RA) catabolism by inducing cytochrome P450 2E1 (CYP2E1). Vitamin E (VE) is an antioxidant implicated in CYP2E1 inhibition. In the current study, we hypothesized that VE supplementation inhibits CYP2E1 and decreases RA catabolism, thereby preventing ethanol-induced hepatocyte hyperproliferation. For 1 month, 4 groups of Sprague-Dawley rats were fed a Lieber-DeCarli liquid ethanol (36% of the total energy) diet as follows: either ethanol alone (Alc group) or ethanol in combination with 0.1 mg/kg body weight of all-trans-RA (Alc + RA group), 2 mg/kg body weight of VE (Alc + VE group), or both together (Alc + RA + VE group). Control rats were pair-fed a liquid diet with an isocaloric amount of maltodextrin instead of ethanol. The ethanol-fed groups had 3-fold higher hepatic CYP2E1 levels, 50% lower hepatic RA levels, and significantly increased hepatocyte proliferation when compared with the controls. The ethanol-fed rats given VE had more than 4-fold higher hepatic VE concentrations than the ethanol-fed rats without VE, but this did not prevent ethanol induction of CYP2E1, lower hepatic retinoid levels, or hepatocellular hyperproliferation. Furthermore, VE supplementation could not prevent RA catabolism in liver microsomal fractions of the ethanol-fed rats in vitro. These results show that VE supplementation can neither inhibit ethanol-induced changes in RA catabolism nor prevent ethanol-induced hepatocyte hyperproliferation in the rat liver.

  3. Alteration of hepatic structure and oxidative stress induced by intravenous nanoceria

    SciTech Connect

    Tseng, Michael T.; Lu, Xiaoqin; Duan, Xiaoxian; Hardas, Sarita S.; Sultana, Rukhsana; Wu, Peng; Unrine, Jason M.; Graham, Uschi; Butterfield, D. Allan; Grulke, Eric A.; Yokel, Robert A.

    2012-04-15

    Beyond the traditional use of ceria as an abrasive, the scope of nanoceria applications now extends into fuel cell manufacturing, diesel fuel additives, and for therapeutic intervention as a putative antioxidant. However, the biological effects of nanoceria exposure have yet to be fully defined, which gave us the impetus to examine its systemic biodistribution and biological responses. An extensively characterized nanoceria (5 nm) dispersion was vascularly infused into rats, which were terminated 1 h, 20 h or 30 days later. Light and electron microscopic tissue characterization was conducted and hepatic oxidative stress parameters determined. We observed acute ceria nanoparticle sequestration by Kupffer cells with subsequent bioretention in parenchymal cells as well. The internalized ceria nanoparticles appeared as spherical agglomerates of varying dimension without specific organelle penetration. In hepatocytes, the agglomerated nanoceria frequently localized to the plasma membrane facing bile canaliculi. Hepatic stellate cells also sequestered nanoceria. Within the sinusoids, sustained nanoceria bioretention was associated with granuloma formations comprised of Kupffer cells and intermingling CD3{sup +} T cells. A statistically significant elevation of serum aspartate aminotransferase (AST) level was seen at 1 and 20 h, but subsided by 30 days after ceria administration. Further, elevated apoptosis was observed on day 30. These findings, together with increased hepatic protein carbonyl levels on day 30, indicate ceria-induced hepatic injury and oxidative stress, respectively. Such observations suggest a single vascular infusion of nanoceria can lead to persistent hepatic retention of particles with possible implications for occupational and therapeutic exposures. -- Highlights: ► Time course study on nanoceria induced hepatic alterations in rats. ► Serum AST elevation indicated acute hepatotoxicity. ► Ceria is retained for up to 30 days in Kupffer cells

  4. Butyrate protects rat liver against total hepatic ischemia reperfusion injury with bowel congestion.

    PubMed

    Liu, Bin; Qian, Jianmin; Wang, Qingbao; Wang, Fangrui; Ma, Zhenyu; Qiao, Yingli

    2014-01-01

    Hepatic ischemia/reperfusion (I/R) injury is an unavoidable consequence of major liver surgery, especially in liver transplantation with bowel congestion, during which endotoxemia is often evident. The inflammatory response aggravated by endotoxin after I/R contributes to liver dysfunction and failure. The purpose of the present study was to investigate the protective effect of butyrate, a naturally occurring four-carbon fatty acid in the body and a dietary component of foods such as cheese and butter, on hepatic injury complicated by enterogenous endotoxin, as well as to examine the underlying mechanisms involved. SD rats were subjected to a total hepatic ischemia for 30 min after pretreatment with either vehicle or butyrate, followed by 6 h and 24 h of reperfusion. Butyrate preconditioning markedly improved hepatic function and histology, as indicated by reduced transaminase levels and ameliorated tissue pathological changes. The inflammatory factors levels, macrophages activation, TLR4 expression, and neutrophil infiltration in live were attenuated by butyrate. Butyrate also maintained the intestinal barrier structures, reversed the aberrant expression of ZO-1, and decreased the endotoxin translocation. We conclude that butyrate inhibition of endotoxin translocation, macrophages activation, inflammatory factors production, and neutrophil infiltration is involved in the alleviation of total hepatic I/R liver injury in rats. This suggests that butyrate should potentially be utilized in liver transplantation.

  5. Acute and chronic effect of ethanol on hepatic albumin synthesis in rat liver in vitro

    SciTech Connect

    Ohtake, H.; Kato, S.; Murawaki, Y.; Kishimoto, Y.; Wakushima, T.; Hirayama, C.

    1986-08-01

    To study the effects of ethanol and its metabolite on albumin metabolism, we examined the hepatic albumin synthesis and secretion in male Wistar rats in vitro, following acute and chronic ethanol administration. After acute ethanol administration, proalbumin synthesis in rat liver in vitro, declined to 47% of the control level at 4 hrs, the lowest level, and increased thereafter to slightly higher than the control level at 16 hrs. On the other hand, chronic ethanol administration for 4 weeks, increased proalbumin synthesis to 1.5 times that of the control level. In the acute ethanol group, a significant negative correlation was observed between proalbumin radioactivity and the concentration of hepatic ethanol and acetaldehyde. The variation between proalbumin radioactivity and hepatic ethanol concentration was wider than the variation between proalbumin and hepatic acetaldehyde. In the chronic ethanol group, ethanol was not detected in the liver. No significant differences from the proalbumin/albumin ratio were seen at any time point after acute or chronic ethanol administration. These findings suggest that the effects of ethanol on hepatic albumin synthesis differ with the method of ethanol administration, and acetaldehyde and/or ethanol is involved in the reduction in albumin synthesis, however, proalbumin-albumin conversion is not disturbed.

  6. Transient effects of empty liposomes on hepatic macrophage populations in rats

    PubMed Central

    Pervin, Munmun; Golbar, Hossain M.; Bondoc, Alexandra; Izawa, Takeshi; Kuwamura, Mitsuru; Yamate, Jyoji

    2016-01-01

    Liposomes have been used as a vehicle for encapsulating chemicals or toxins in toxicological studies. We investigated the transient effects of empty liposomes on hepatic macrophages by applying a single intravenous injection at a dose of 10 ml/kg body weight in 6-week-old male F344 rats. One day after injection, the numbers of hepatic macrophages reacting to CD163, CD68, Iba-1, MHC class II, Gal-3 and CD204 were significantly increased in liposome-treated rats. CD163+ Kupffer cells and CD68+ macrophages with increased phagocytic activity in hepatic lobules were most sensitive. The histological architecture of the liver was not changed following liposome injection; however, hepatocytes showed increased proliferating activity, demonstrable with proliferation marker immunostaining and by an increase in gene profiles related to the cell cycle. In the liposome-treated rats, interestingly, AST and ALT values were significantly decreased, and MCP-1, IL-1β and TGF-β1 mRNAs were significantly increased. Collectively, the present study found that hepatic macrophages activated by liposomes can influence liver homeostasis. This information would be useful for background studies on liposomes. PMID:27182120

  7. Hepatic steatosis prevents heme oxygenase-1 induction by isoflurane in the rat liver

    PubMed Central

    Stoll, Patrick; Schwer, Christian I; Goebel, Ulrich; Buerkle, Hartmut; Hoetzel, Alexander; Schmidt, Rene

    2011-01-01

    AIM: To characterize the inductive effects of isoflurane (ISO) on hepatic heme oxygenase-1 (HO-1) in an animal model of hepatic steatosis. METHODS: Lean (LEAN) and obese (FAT) Zucker rats were randomized into 4 groups: 1: LEAN + pentobarbital sodium (PEN); 2: LEAN + ISO; 3: FAT + PEN; 4: FAT + ISO. The animals were mechanically ventilated for 6 h. In vitro analyses of liver tissue included determination of HO-1 mRNA and protein expression as well as measurement of HO enzyme activity and immunohistochemical analyses. RESULTS: Compared to PEN treatment, ISO administration profoundly induced hepatic HO-1 mRNA and protein expression and significantly increased HO enzyme activity in lean Zucker rats. In contrast, no difference in HO-1 gene expression was observed after ISO or PEN anesthesia in obese Zucker rats. CONCLUSION: The present study demonstrates that ISO is an inducer of hepatic HO-1 gene expression in non-steatotic organs but failed to upregulate HO-1 in steatotic livers. PMID:22072849

  8. [Effects of calcitriol and alfacalcidol on an osteoporosis model in rats with hepatic failure].

    PubMed

    Yamanishi, A; Ishibashi, Y; Kuriyama, K; Tachiiri, T; Kusajima, H; Kojima, E; Momo, K

    1999-01-01

    To predict the potential utility of calcitriol in human osteoporosis with hepatic dysfunction, we examined the effects of calcitriol and alfacalcidol in ovariectomized (OVX) aged-rats with CCl4-induced hepatic failure. In OVX+CCl4 rats, GOT, GTP, alkaline phosphatase and total bilirubin increased and hepatic enzyme activity (cytochrome b5 and P450) decreased. Repeated oral doses of calcitriol (0.1 and 0.2 microgram/kg) for 51 days inhibited a decrease in serum calcium concentration. This effect was more potent than that of alfacalcidol at the same dose. Both drugs tended to inhibit a decrease in femoral calcium contents. Calcitriol (0.2 microgram/kg) prevented a decrease in femoral bone density (dry and ash weight per volume), unlike alfacalcidol. Soft X-ray imaging analysis revealed that both drugs tended to inhibit the decrease in femoral bone density. There were no differences in the femoral bone strength between OVX+CCl4 and sham-operated rats. The serum calcitriol concentrations increased after the last doses of calcitriol, while they did not increase after the last dose of alfacalcidol. All these effects of calcitriol were related to the serum calcitriol levels. These results suggest that calcitriol, unlike alfacalcidol, may have a clinical therapeutic effect in osteoporosis with hepatic dysfunction.

  9. Regulation of biliary cholesterol secretion in the rat. Role of hepatic cholesterol esterification.

    PubMed Central

    Nervi, F; Bronfman, M; Allalón, W; Depiereux, E; Del Pozo, R

    1984-01-01

    Although the significance of the enterohepatic circulation of bile salts in the solubilization and biliary excretion of cholesterol is well established, little is known about the intrahepatic determinants of biliary cholesterol output. Studies were undertaken to elucidate some of these determinants in the rat. Feeding 1% diosgenin for 1 wk increased biliary cholesterol output and saturation by 400%. Bile flow, biliary bile salt, phospholipid and protein outputs remained in the normal range. When ethynyl estradiol (EE) was injected into these animals, biliary cholesterol output decreased to almost normal levels under circumstances of minor changes in the rates of biliary bile salt and phospholipid outputs. Similarly, when chylomicron cholesterol was intravenously injected into diosgenin-fed animals, biliary cholesterol output significantly decreased as a function of the dose of chylomicron cholesterol administered. Relative rates of hepatic cholesterol synthesis and esterification were measured in isolated hepatocytes. Although hepatic cholesterogenesis increased 300% in diosgenin-fed animals, the contribution of newly synthesized cholesterol to total biliary cholesterol output was only 19 +/- 9%, compared with 12 +/- 6% in control and 15 +/- 5% in diosgenin-fed and EE-injected rats. The rate of oleate incorporation into hepatocytic cholesterol esters was 30% inhibited in diosgenin-fed rats. When EE was injected into these animals, the rate of cholesterol esterification increased to almost 300%. To investigate further the interrelationship between hepatic cholesterol esterification and biliary cholesterol output, we studied 21 diosgenin-fed rats. Six of them received in addition EE and 10 received chylomicron cholesterol. The relationships between biliary cholesterol output as a function of both microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity and hepatic cholesterol ester concentration were significantly correlated in a reciprocal manner. From these

  10. Role of partial hepatectomy on Capillaria hepatica-induced hepatic fibrosis in rats.

    PubMed

    Santos, Carolina Cincurá Silva; Onofre-Nunes, Záira; Andrade, Zilton Araujo

    2007-01-01

    It is known that hepatic fibrosis may regress following partial hepatectomy, since the hepatic parenchyma regenerates very rapidly, but not the excess of fibrous tissue. The present study evaluated this hypothesis by observing the behavior of systematized septal fibrosis induced by either 30 or 90-day-old Capillaria hepatica infection, in rats subjected to partial hepatectomy. The results revealed that the morphology of the fibrosis was unaffected, but its relative quantity within the microscope field appeared significantly decreased, as a consequence of the increased liver tissue mass following regeneration.

  11. Investigation of hepatic fibrosis in rats with x-ray diffraction enhanced imaging

    SciTech Connect

    Li Hui; Zhang Lu; Wang Xueyan; Luo Shuqian; Wang Tailing; Wang Baoen; Zhao Xinyan

    2009-03-23

    X-ray diffraction enhanced imaging (DEI) is a phase contrast technique that generates excellent contrast of biological soft tissues compared to conventional absorption radiography. We explore the application of DEI in the diagnosis of hepatic fibrosis. The produced refraction contrast images of fibrous rat liver samples show clearly abnormal liver architectures. Moreover, by comparing to histological pictures, different stages of fibrosis are discriminated, and the corresponding morphological features are analyzed. Besides, quantitative analyses of texture features are presented. The results reported herein show that DEI can be a potential noninvasive technique to diagnose and stage hepatic fibrosis.

  12. Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450

    SciTech Connect

    Wyman, J.F.; Gollan, J.L.; Settle, W.; Farrell, G.C.; Correia, M.A.

    1986-01-01

    After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglogin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that heamoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of (tritium) haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic free haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.

  13. Impact of high altitude on the hepatic fatty acid oxidation and synthesis in rats

    SciTech Connect

    Ni, Qian; Shao, Yuan; Wang, Ying Zhen; Jing, Yu Hong; Zhang, You Cheng

    2014-04-04

    Highlights: • Acute exposure to high altitude (HA) increased hepatic fatty acid (FA) β-oxidation. • Acute exposure of rats to HA increased hepatic FA synthesis. • PPARα and AMPK can regulate the FA metabolism. • FA may be a key energy fuel and a compensation for CHO during acute exposure to HA. • The acute changes of FA metabolism may be a mechanism of acclimatization. - Abstract: High altitude (HA) affects energy metabolism. The impact of acute and chronic HA acclimatization on the major metabolic pathways is still controversial. In this study, we aimed to unveil the impact of HA on the key enzymes involved in the fatty acid (FA) metabolism in liver. Rats were exposed to an altitude of 4300 m for 30 days and the expressions of two key proteins involved in FA β-oxidation (carnitine palmitoyl transferase I, CPT-I; and peroxisome proliferator-activated receptor alpha, PPARα), two proteins involved in FA synthesis (acetyl CoA carboxylase-1, ACC-1; and AMP-activated protein kinase, AMPK), as well as the total ketone body in the liver and the plasma FFAs were examined. Rats without HA exposure were used as controls. We observed that the acute exposure of rats to HA (3 days) led to a significant increase in the expressions of CPT-I and PPARα and in the total hepatic ketone body. Longer exposure (15 days) caused a marked decrease in the expression of CPT-I and PPARα. By 30 days after HA exposure, the expression levels of CPT-I and PPARα returned to the control level. The hepatic ACC-1 level showed a significant increase in rats exposed to HA for 1 and 3 days. In contrast, the hepatic level of AMPK showed a significant reduction throughout the experimental period. Plasma FFA concentrations did not show any significant changes following HA exposure. Thus, increased hepatic FA oxidation and synthesis in the early phase of HA exposure may be among the important mechanisms for the rats to respond to the hypoxic stress in order to acclimatize themselves to the

  14. Polydatin supplementation ameliorates diet-induced development of insulin resistance and hepatic steatosis in rats.

    PubMed

    Zhang, Qi; Tan, Yingying; Zhang, Nan; Yao, Fanrong

    2015-01-01

    The pathophysiology of non-alcoholic fatty liver disease remains to be elucidated, and the currently available treatments are not entirely effective. Polydatin, a stilbenoid compound derived from the rhizome of Polygonum cuspidatum, has previously been demonstrated to possess hepatoprotective effects. The present study aimed to determine the effects of polydatin supplementation on hepatic fat accumulation and injury in rats fed a high-fat diet. In addition, the mechanisms underlying the protective effects of polydatin were examined. Male Sprague Dawley rats were randomly divided into four groups and received one of four treatment regimes for 12 weeks: Control diet, control diet supplemented with polydatin, high-fat diet, or high-fat diet supplemented with polydatin. Polydatin was supplemented in the drinking water at a concentration of 0.3% (wt/vol). The results of the present study showed that long-term high-fat feeding resulted in fatty liver in rats, which was manifested by excessive hepatic neutral fat accumulation and elevated plasma alanine aminotransferase and aspartate aminotransferase levels. Polydatin supplementation alleviated the hepatic pathological changes, and attenuated the insulin resistance, as shown by an improved homeostasis model assessment of basal insulin resistance values and a glucose tolerance test. Polydatin supplementation also corrected abnormal leptin and adiponectin levels. Specifically, polydatin supplementation enhanced insulin sensitivity in the liver, as shown by improved insulin receptor substrate 2 expression levels and Akt phosphorylation in the rat liver, following high-fat diet feeding. The results of the present study suggest that polydatin protects rats against high-fat feeding-induced insulin resistance and hepatic steatosis. Polydatin may be an effective hepatoprotective agent and a potential candidate for the prevention of fatty liver disease and insulin resistance. PMID:25333896

  15. Transfer of retinol from parenchymal to stellate cells in liver is mediated by retinol-binding protein

    SciTech Connect

    Blomhoff, R.; Berg, T.; Norum, K.R. )

    1988-05-01

    Newly absorbed chylomicron remnant retinyl ester is endocytosed by parenchymal liver cells, and retinol is subsequently transferred to perisinusoidal stellate cells in liver. In the present study, the authors have used several approaches to elucidate the mechanism for the paracrine transfer of retinol between liver parenchymal and stellate cells. In one series of experiments, chylomicrons labeled with ({sup 3}H)retinyl palmitate or with retinyl ({sup 3}H)palimtate were injected intravenously into rats. It was shown that the retinol as well as the palmitate moiety were initially taken up in parenchymal liver cells. However, only the retinol moiety was detected in stellate cells, indicating that the retinyl ester is hydrolyzed before retinol is transferred to stellate cells. It is well known that parenchymal liver cells secrete retinol bound to retinol-binding protein (RBP), and they have recently found that stellate cells do have RBP receptors. Here they report that antibodies against RBP completely block the transfer of retinol from parenchymal to stellate cells. These findings indicate that following uptake of chylomicron remnant retinyl ester in parenchymal cells, the retinyl ester is hydrolyzed, and retinol secreted from parenchymal cells on RBP is taken up by stellate cells by means of RBP receptors.

  16. Transfer of retinol from parenchymal to stellate cells in liver is mediated by retinol-binding protein.

    PubMed Central

    Blomhoff, R; Berg, T; Norum, K R

    1988-01-01

    Newly absorbed chylomicron remnant retinyl ester is endocytosed by parenchymal liver cells, and retinol is subsequently transferred to perisinusoidal stellate cells in liver. In the present study we have used several approaches to elucidate the mechanism for the paracrine transfer of retinol between liver parenchymal and stellate cells. In one series of experiments, chylomicrons labeled with [3H]retinyl palmitate or with retinyl [3H]palmitate were injected intravenously into rats. It was shown that the retinol as well as the palmitate moiety were initially taken up in parenchymal liver cells. However, only the retinol moiety was detected in stellate cells, indicating that the retinyl ester is hydrolyzed before retinol is transferred to stellate cells. It is well known that parenchymal liver cells secrete retinol bound to retinol-binding protein (RBP), and we have recently found that stellate cells do have RBP receptors. Here we report that antibodies against RBP completely block the transfer of retinol from parenchymal to stellate cells. These findings indicate that following uptake of chylomicron remnant retinyl ester in parenchymal cells, the retinyl ester is hydrolyzed, and retinol secreted from parenchymal cells on RBP is taken up by stellate cells by means of RBP receptors. PMID:3368453

  17. Frequent detection and characterization of hepatitis E virus variants in wild rats (Rattus rattus) in Indonesia.

    PubMed

    Mulyanto; Depamede, Sulaiman Ngongu; Sriasih, Made; Takahashi, Masaharu; Nagashima, Shigeo; Jirintai, Suljid; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2013-01-01

    One hundred sixteen rats (Rattus rattus) captured in Indonesia from 2011 to 2012 were investigated for the prevalence of hepatitis E virus (HEV)-specific antibodies and HEV RNA. Using an ELISA based on HEV genotype 4 with an ad hoc cutoff value of 0.500, 18.1 % of the rats tested positive for anti-HEV IgG. By nested RT-PCR, 14.7 % of the rats had rat HEV RNA, and none were positive for HEV genotype 1-4. A high HEV prevalence among rats was associated with lower sanitary conditions in areas with a high population density. Sixteen of the 17 HEV isolates obtained from infected rats showed >93.0 % nucleotide sequence identity within the 840-nucleotide ORF1-ORF2 sequence and were most closely related to a Vietnamese strain (85.9-87.9 % identity), while the remaining isolate differed from known rat HEV strains by 18.8-23.3 % and may belong to a novel lineage of rat HEV. These results suggest a wide distribution of rat HEV with divergent genomes. PMID:22983110

  18. Suppression of hepatic prostaglandin F2 alpha in rats by dietary alpha-tocopherol acetate is independent of total hepatic alpha-tocopherol.

    PubMed

    Duitsman, P K; Chen, H W; Cook, L R; Hendrich, S

    1992-09-01

    Groups of eight weanling female F344/N rats were fed semipurified diets that supplied 0, 50, 500, 5000, or 15,000 mg alpha-tocopherol acetate/kg diet, with and without 0.05% phenobarbital (PB) for 9 weeks. Both plasma and hepatic alpha-tocopherol levels, measured by HPLC, strongly correlated with alpha-tocopherol intake (r greater than 0.73, p less than 0.0001). Phenobarbital both depleted hepatic alpha-tocopherol and increased plasma alpha-tocopherol significantly. Although treatment with PB for 9 weeks significantly increased GST activity, PB did not affect hepatic prostaglandin (PG)F2 alpha status, as determined by radioimmunoassay. PGF2 alpha was significantly greater (by 52%) in rats fed no alpha-tocopherol than in rats fed 15,000 mg alpha-tocopherol acetate/kg diet. Hepatic PGF2 alpha status was correlated inversely but weakly with dietary alpha-tocopherol (r = -0.24, p less than 0.05). Hepatic PGF2 alpha status was not correlated with hepatic or plasma alpha-tocopherol status. This finding suggests either that there is a small depletion-resistant subcellular alpha-tocopherol pool which regulates PGF2 alpha production or that alpha-tocopherol alters PGF2 alpha production in vivo by an indirect mechanism.

  19. Stellate Cell Networks in the Teleost Pituitary

    PubMed Central

    Golan, Matan; Hollander-Cohen, Lian; Levavi-Sivan, Berta

    2016-01-01

    The folliculostellate cells of the mammalian pituitary are non-endocrine cells that are implicated in long-distance communication and paracrine signaling, but to date, these cells have yet to be characterized in teleosts. We found that the stellate cells of the teleost pituitary share many common attributes with mammalian folliculostellate cells. By labeling of stellate cells in live preparations of tilapia pituitaries we investigated their distribution, association with other endocrine cells and their anatomical and functional coupling. In the pars intermedia, stellate cells were arranged around neuronal bundles and their processes extended into the pars distalis. Within the pars distalis, stellate cells formed close associations with FSH cells and, to a lesser degree, with GH and LH cells, suggesting differential paracrine regulation of the two gonadotrope populations. The production of follistatin by stellate cells further corroborates the notion of a paracrine role on FSH release. We also found stellate cells to form gap junctions that enabled dye transfer to neighboring stellate cells, implicating that these cells form a large-scale network that connects distant parts of the pituitary. Our findings represent the first wide-scale study of stellate cells in teleosts and provide valuable information regarding their functional roles in pituitary function. PMID:27086978

  20. Stellate Cell Networks in the Teleost Pituitary.

    PubMed

    Golan, Matan; Hollander-Cohen, Lian; Levavi-Sivan, Berta

    2016-01-01

    The folliculostellate cells of the mammalian pituitary are non-endocrine cells that are implicated in long-distance communication and paracrine signaling, but to date, these cells have yet to be characterized in teleosts. We found that the stellate cells of the teleost pituitary share many common attributes with mammalian folliculostellate cells. By labeling of stellate cells in live preparations of tilapia pituitaries we investigated their distribution, association with other endocrine cells and their anatomical and functional coupling. In the pars intermedia, stellate cells were arranged around neuronal bundles and their processes extended into the pars distalis. Within the pars distalis, stellate cells formed close associations with FSH cells and, to a lesser degree, with GH and LH cells, suggesting differential paracrine regulation of the two gonadotrope populations. The production of follistatin by stellate cells further corroborates the notion of a paracrine role on FSH release. We also found stellate cells to form gap junctions that enabled dye transfer to neighboring stellate cells, implicating that these cells form a large-scale network that connects distant parts of the pituitary. Our findings represent the first wide-scale study of stellate cells in teleosts and provide valuable information regarding their functional roles in pituitary function. PMID:27086978

  1. The effects of acute ethanol exposure on inhibitors of hepatic regenerative activity in the rat.

    PubMed

    Zhang, M N; Gong, Y; Minuk, G Y

    2000-04-01

    The purpose of this study was to identify the mechanism(s) whereby acute ethanol exposure inhibits hepatic regenerative activity in the rat. Adult, male, Sprague-Dawley rats (200-250 g) were randomized to receive either ethanol (1 g/kg i.p. q 4 h) or an equal volume of saline (controls) for 24 h beginning 1 h prior to a 70% partial hepatectomy (PHx). At 0, 3, 6, 12 and 24 h post-PHx, rats were sacrificed (N = 4-6/group), and the expression of the following genes associated with inhibition of hepatocyte proliferation were documented; p53, p21, transforming growth factor-beta1 (TGF-beta1) and gamma aminobutyric acid transport protein (GABA-TP). Inhibition of hepatic regenerative activity was confirmed by 3H-thymidine incorporation into hepatic DNA at 24 h post-PHx. The results of the study revealed that in ethanol-treated rats, DNA synthesis was inhibited by 37% when compared to saline-treated controls (p < 0.01). Regarding suppressor gene expression, both p21 and TGF-beta1 mRNA expression in ethanol-treated rats were similar to those obtained in saline-treated controls. Although p53 mRNA expression differed in the two groups, in the ethanol-treated group, p53 mRNA expression was decreased rather than increased (relative to controls) at 24 h post-PHx, a finding not in keeping with inhibition of DNA synthesis. GABA-TP mRNA was strongly expressed prior to PHx in both ethanol- and saline-treated rats. Following PHx, GABA-TP mRNA expression decreased in both groups but remained low in the saline-treated group while returning to pre-PHx values in ethanol-treated rats. In summary, the results of this study indicate that the inhibitory effects of ethanol on hepatic regeneration are not associated with significant or the appropriate changes in mRNA expression of the p53, p21 or TGF-beta1 suppressor genes. On the other hand, transcriptional changes in GABA-TP gene expression post-PHx are in keeping with an inhibitory effect of GABA on hepatic regeneration.

  2. Gallic acid ameliorates hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose diet.

    PubMed

    Huang, Da-Wei; Chang, Wen-Chang; Wu, James Swi-Bea; Shih, Rui-Wen; Shen, Szu-Chuan

    2016-02-01

    Herein, we investigated the hypoglycemic effect of plant gallic acid (GA) on glucose uptake in an insulin-resistant cell culture model and on hepatic carbohydrate metabolism in rats with a high-fructose diet (HFD)-induced diabetes. Our hypothesis is that GA ameliorates hyperglycemia via alleviating hepatic insulin resistance by suppressing hepatic inflammation and improves abnormal hepatic carbohydrate metabolism by suppressing hepatic gluconeogenesis and enhancing the hepatic glycogenesis and glycolysis pathways in HFD-induced diabetic rats. Gallic acid increased glucose uptake activity by 19.2% at a concentration of 6.25 μg/mL in insulin-resistant FL83B mouse hepatocytes. In HFD-induced diabetic rats, GA significantly alleviated hyperglycemia, reduced the values of the area under the curve for glucose in an oral glucose tolerance test, and reduced the scores of the homeostasis model assessment of insulin resistance index. The levels of serum C-peptide and fructosamine and cardiovascular risk index scores were also significantly decreased in HFD rats treated with GA. Moreover, GA up-regulated the expression of hepatic insulin signal transduction-related proteins, including insulin receptor, insulin receptor substrate 1, phosphatidylinositol-3 kinase, Akt/protein kinase B, and glucose transporter 2, in HFD rats. Gallic acid also down-regulated the expression of hepatic gluconeogenesis-related proteins, such as fructose-1,6-bisphosphatase, and up-regulated expression of hepatic glycogen synthase and glycolysis-related proteins, including hexokinase, phosphofructokinase, and aldolase, in HFD rats. Our findings indicate that GA has potential as a health food ingredient to prevent diabetes mellitus.

  3. Chronic alcohol intake upregulates hepatic expression of carotenoid cleavage enzymes and PPAR in rats.

    PubMed

    Luvizotto, Renata A M; Nascimento, André F; Veeramachaneni, Sudipta; Liu, Chun; Wang, Xiang-Dong

    2010-10-01

    Excessive and chronic alcohol intake leads to a lower hepatic vitamin A status by interfering with vitamin A metabolism. Dietary provitamin A carotenoids can be converted into vitamin A mainly by carotenoid 15,15'-monooxygenase 1 (CMO1) and, to a lesser degree, carotenoid 9'10'-monooxygenase 2 (CMO2). CMO1 has been shown to be regulated by several transcription factors, such as the PPAR, retinoid X receptor, and thyroid receptor (TR). The regulation of CMO2 has yet to be identified. The impact of chronic alcohol intake on hepatic expressions of CMO1 and CMO2 and their related transcription factors are unknown. In this study, Fischer 344 rats were pair-fed either a liquid ethanol Lieber-DeCarli diet (n = 10) or a control diet (n = 10) for 11 wk. Hepatic retinoid concentration and expressions of CMO1, CMO2, PPARγ, PPARα, and TRβ as well as plasma thyroid hormones levels were analyzed. We observed that administering alcohol decreased hepatic retinoid levels but increased mRNA concentrations of CMO1, CMO2, PPARγ, PPARα, and TRβ and upregulated protein levels of CMO2, PPARγ, and PPARα. There was a positive correlation of PPARγ with CMO1 (r = 0.89; P < 0.0001) and both PPARγ and PPARα with CMO2 (r = 0.72, P < 0.001 and r = 0.62, P < 0.01, respectively). Plasma thyroid hormone concentrations did not differ between the control rats and alcohol-fed rats. This study suggests that chronic alcohol intake significantly upregulates hepatic expression of CMO1 and, to a much lesser extent, CMO2. This process may be due to alcohol-induced PPARγ expression and lower vitamin A status in the liver.

  4. Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

    PubMed

    Jackson, D P; Percy, D H; Morris, V L

    1984-09-01

    When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

  5. Echinococcus multilocularis: Single hepatic lesion experimentally established without metastasis in rats.

    PubMed

    Yamashita, Masamichi; Imagawa, Tomohiro; Nakaya, Kazuhiro; Sako, Yasuhito; Okamoto, Yoshiharu; Tsuka, Takeshi; Osaki, Tomohiro; Okamoto, Munehiro; Ito, Akira

    2013-10-01

    We herein describe the establishment of single hepatic lesions of Echinococcus multilocularis in rats. A 3mm incision was made on the liver with a surgical knife, and one small round vesicle of E. multilocularis (between 1 × 1 mm and <2 × 2 mm in diameter) was transplanted into the incision and covered with absorbable hemostat gauze. The presence and growth of the transplanted vesicle was monitored for 12 weeks using magnetic resonance imaging (MRI). Hepatic lesions, the metacestode of this parasite were confirmed in 12 of 17 infected rats (70.6%) by MRI and macroscopic examinations. The average size of the metacestodes with brood capsules at 12 weeks after the experimental transplantation of a single vesicle was 6.1 ± 2.5 mm × 4.4 ± 1.5mm. The smallest size of the metacestodes detected by MRI was approximately 3 × 3 mm. This new approach of establishing single hepatic metacestodes of E. multilocularis in experimental animals is expected to be useful for analyzing the immune-pathological mechanisms of hepatic AE. PMID:23916766

  6. Application of hepatic tolerance tests to the functional reserve assessment in rat models of fatty liver.

    PubMed

    Furuhama, K; Yabe, K

    1998-05-01

    The present study was designed to define whether maximal removal rate of indocyanine green (ICG Rmax), plasma cyclic 3',5'-adenosine monophosphate (cAMP) response to exogenous glucagon (peak to basal ratio of cAMP level: P/B cAMP) and plasma half-life of galactose (t1/2 galactose) can measure the hepatic functional reserve of fatty liver prepared in rats fed choline-deficient (9 weeks), 2% cholesterol (2 weeks) or 0.25% DL-ethionine (2 weeks) diet. Although changes in cholesterol and phospholipid values in serum during feeding periods differed among the models, histopathologic examinations in the liver of almost all animals revealed intermediate to severe fatty liver with or without fibrosis at each termination. ICG Rmax and P/B cAMP were significantly decreased in rats fed choline-deficient or DL-ethionine diet, implying reductions in hepatic functional mass and disturbances in hepatic cAMP production. Meanwhile, t1/2 galactose showed no change in any of the models, suggesting that glucose metabolisms in the models used may be preserved. These findings demonstrate that ICG Rmax and P/B cAMP can apply to measurement of hepatic surviving reserve of fatty liver with fibrosis.

  7. Genistein Attenuates Nonalcoholic Steatohepatitis and Increases Hepatic PPARγ in a Rat Model

    PubMed Central

    Susutlertpanya, Warinda; Werawatganon, Duangporn; Siriviriyakul, Prasong; Klaikeaw, Naruemon

    2015-01-01

    Nonalcoholic steatohepatitis (NASH) has become a global chronic liver disease, but no effective medicine has been proven to cure it. This study investigated the protective effects of genistein, a phytoestrogen, on NASH and examined whether it has any effect on hepatic PPARγ. Male Sprague-Dawley rats were divided into four groups: control group fed ad libitum with standard rat diet, NASH group fed ad libitum with high-fat diet to induce NASH and NASH + Gen8 group and NASH + Gen16 group fed with high-fat diet plus intragastric administration of 8 or 16 mg/kg genistein once daily. After 6 weeks, liver samples were collected to determine MDA, TNF-α, PPARγ, and histopathology. The findings were that levels of hepatic MDA and TNF-α increased in NASH group, but 16 mg/kg genistein reduced these levels significantly. Downregulation of hepatic PPARγ was observed in NASH group, but genistein significantly upregulated the expression of PPARγ in both NASH + Gen groups. The histological appearance of liver in NASH group presented pathological features of steatohepatitis which were diminished in both NASH + Gen groups. The results suggest that genistein attenuates the liver histopathology of NASH with upregulation of hepatic PPARγ, reduction of oxidative stress, and inhibition of inflammatory cytokine. PMID:26246839

  8. Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat

    SciTech Connect

    Sorrell, M.F.; Nauss, J.M.; Donohue, T.M. Jr.; Tuma, D.J.

    1983-03-01

    The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate (/sup 14/C)glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring (/sup 14/C)leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, (/sup 14/C)fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration.

  9. A return to ad libitum feeding following caloric restriction promotes hepatic steatosis in hyperphagic OLETF rats.

    PubMed

    Linden, Melissa A; Fletcher, Justin A; Meers, Grace M; Thyfault, John P; Laughlin, M Harold; Rector, R Scott

    2016-09-01

    Hyperphagic Otsuka Long-Evans Tokushima fatty (OLETF) rats develop obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD), but lifestyle modifications, such as caloric restriction (CR), can prevent these conditions. We sought to determine if prior CR had protective effects on metabolic health and NAFLD development following a 4-wk return to ad libitum (AL) feeding. Four-week-old male OLETF rats (n = 8-10/group) were fed AL for 16 wk (O-AL), CR for 16 wk (O-CR; ∼70% kcal of O-AL), or CR for 12 wk followed by 4 wk of AL feeding (O-AL4wk). CR-induced benefit in prevention of NAFLD, including reduced hepatic steatosis, inflammation, and markers of Kupffer cell activation/number, was largely lost in AL4wk rats. These findings occurred in conjunction with a partial loss of CR-induced beneficial effects on obesity and serum triglycerides in O-AL4wk rats, but in the absence of changes in serum glucose or insulin. CR-induced increases in hepatic mitochondrial respiration remained significantly elevated (P < 0.01) in O-AL4wk compared with O-AL rats, while mitochondrial [1-(14)C]palmitate oxidation, citrate synthase activity, and β-hydroxyacyl-CoA dehydrogenase activity did not differ among OLETF groups. NAFLD development in O-AL4wk rats was accompanied by increases in the protein content of the de novo lipogenesis markers fatty acid synthase and stearoyl-CoA desaturase-1 and decreases in phosphorylated acetyl-CoA carboxylase (pACC)/ACC compared with O-CR rats (P < 0.05 for each). The beneficial effects of chronic CR on NAFLD development were largely lost with 4 wk of AL feeding in the hyperphagic OLETF rat, highlighting the importance of maintaining energy balance in the prevention of NAFLD. PMID:27445343

  10. Trans-11 vaccenic acid reduces hepatic lipogenesis and chylomicron secretion in JCR:LA-cp rats.

    PubMed

    Wang, Ye; Jacome-Sosa, M Miriam; Ruth, Megan R; Goruk, Sue D; Reaney, Martin J; Glimm, David R; Wright, David C; Vine, Donna F; Field, Catherine J; Proctor, Spencer D

    2009-11-01

    Trans-11 vaccenic acid (VA) is the predominant trans isomer in ruminant fat and a major precursor to the endogenous synthesis of cis9,trans11-conjugated linoleic acid in humans and animals. We have previously shown that 3-wk VA supplementation has a triglyceride (TG)-lowering effect in a rat model of dyslipidemia, obesity, and metabolic syndrome (JCR:LA-cp rats). The objective of this study was to assess the chronic effect (16 wk) of VA on lipid homeostasis in both the liver and intestine in obese JCR:LA-cp rats. Plasma TG (P < 0.001), total cholesterol (P < 0.001), LDL cholesterol (P < 0.01), and nonesterified fatty acid concentrations, as well as the serum haptoglobin concentration, were all lower in obese rats fed the VA diet compared with obese controls (P < 0.05). In addition, there was a decrease in the postprandial plasma apolipoprotein (apo)B48 area under the curve (P < 0.05) for VA-treated obese rats compared with obese controls. The hepatic TG concentration and the relative abundance of fatty acid synthase and acetyl-CoA carboxylase proteins were all lower (P < 0.05) in the VA-treated group compared with obese controls. Following acute gastrointestinal infusion of a VA-triolein emulsion in obese rats that had been fed the control diet for 3 wk, the TG concentration was reduced by 40% (P < 0.05) and the number of chylomicron (CM) particles (apoB48) in nascent mesenteric lymph was reduced by 30% (P < 0.01) relative to rats infused with a triolein emulsion alone. In conclusion, chronic VA supplementation significantly improved dyslipidemia in both the food-deprived and postprandial state in JCR:LA-cp rats. The appreciable hypolipidemic benefits of VA may be attributed to a reduction in both intestinal CM and hepatic de novo lipogenesis pathways.

  11. Evaluation of the assumptions of an ontogeny model of rat hepatic cytochrome P450 activity.

    PubMed

    Alcorn, Jane; Elbarbry, Fawzy A; Allouh, Mohammed Z; McNamara, Patrick J

    2007-12-01

    We previously reported an ontogeny model of hepatic cytochrome P450 (P450) activity that predicts in vivo P450 elimination from in vitro intrinsic clearance. The purpose of this study was to conduct investigations into key assumptions of the P450 ontogeny model using the developing rat model system. We used two developmentally dissimilar enzymes, CYP2E1 and CYP1A2, and male rats (n = 4) at age groups representing critical developmental stages. Total body and liver weights and hepatic microsomal protein contents were measured. Following high-performance liquid chromatography analysis, apparent K(M) and V(max) estimates were calculated using nonlinear regression analysis for CYP2E1- and CYP1A2-mediated chlorzoxazone 6-hydroxylation and methoxyresorufin O-dealkylation, and V(max) estimates for p-nitrophenol and phenacetin hydroxylations, respectively. Hepatic scaling factors and V(max) values provided estimates for infant scaling factors (ISF). The data show microsomal protein contents increased with postnatal age and reached adult values after postnatal day (PD) 7. Apparent K(M) values were similar at all developmental stages except at < or =PD7. Developmental increases in probe substrate V(max) values did not correlate with the biphasic increase in immunoquantifiable P450. The activity of two different probe substrates for each P450 covaried as a function of age. A plot of observed ISF values as a function of age reflected the developmental pattern of rat hepatic P450. In summation, these observations diverge from several of the model's assumptions. Further investigations are required to explain these inconsistencies and to investigate whether the developing rat may provide a predictive paradigm for pediatric risk assessment for P450-mediated elimination processes.

  12. Protective effect of Indigofera aspalathoides against CCl4-induced hepatic damage in rats.

    PubMed

    Rajkapoor, B; Jayakar, B; Kavimani, S; Murugesh, N

    2006-01-01

    The alcoholic extract of stem of Indigofera aspalathoides was evaluated for its antihepatotoxic activity against CCl(4)-induced hepatic damage in rats. The activity was evaluated by using biochemical parameters, such as serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin and gama glutamate transpeptidase (GGTP). The histopathological changes of liver sample were compared with respective control. The extract showed remarkable hepatoprotective effect.

  13. Increased hepatic nicotine elimination after phenobarbital induction in the conscious rat

    SciTech Connect

    Foth, H.; Walther, U.I.; Kahl, G.F. )

    1990-09-15

    Elimination parameters of (14C)nicotine in conscious rats receiving nicotine (0.3 mg/kg) either intravenously or orally were studied. The oral availability of unchanged nicotine, derived by comparison of the respective areas under the concentration vs time curves (AUC), was 89%, indicating low hepatic extraction ratios of about 10%. Pretreatment of rats with phenobarbital (PB) markedly increased hepatic first-pass extraction of nicotine. The oral availability of unchanged nicotine in plasma dropped to 1.4% of the corresponding values obtained from PB-treated rats receiving nicotine iv. After PB pretreatment, the clearance of iv nicotine was increased approximately twofold over controls, much less than the observed more than ninefold increase of hepatic first-pass extraction. It is assumed that extrahepatic metabolism contributed significantly to the rapid removal of nicotine from the plasma. The elimination of cotinine, originating from nicotine administered either po or iv, was significantly increased by PB pretreatment, as determined by the ratio of corresponding AUCs. The pattern of nicotine metabolites in urine also indicated an increase in the rate of cotinine metabolic turnover. The amount of norcotinine in the organic extract of urine paralleled PB microsomal enzyme induction. The ratio between urinary concentrations of the normetabolite and cotinine correlated strongly with the PB-induced state of rat liver. This may be a suitable indicator of PB-inducible hepatic cytochrome P450 isoenzyme(s). Since smoking habits in man are feedback-regulated by nicotine plasma concentrations, a similar increase of nicotine elimination by microsomal enzyme induction in man may be of relevance for tobacco consumption.

  14. Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training.

    PubMed

    Du, Fang; Ding, Ye; Zou, Jun; Li, Zhili; Tian, Jijing; She, Ruiping; Wang, Desheng; Wang, Huijuan; Lv, Dongqiang; Chang, Lingling

    2015-01-01

    This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage. PMID:26000905

  15. Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training

    PubMed Central

    Zou, Jun; Li, Zhili; Tian, Jijing; She, Ruiping; Wang, Desheng; Wang, Huijuan; Lv, Dongqiang; Chang, Lingling

    2015-01-01

    This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage. PMID:26000905

  16. In vitro evaluation of hepatic and extra-hepatic metabolism of coumarins using rat subcellular fractions: correlation of in vitro clearance with in vivo data.

    PubMed

    Behera, Dayanidhi; Damre, Anagha; Varghese, Alice; Addepalli, Veeranjaneyulu

    2008-01-01

    7-Ethoxycoumarin (7-EC) and 7-hydroxycoumarin (7-HC) were chosen as model compounds to study hepatic and extra-hepatic metabolism in rat tissue subcellular (microsomal and S9) fractions and to scale the observed in vitro clearance to in vivo plasma clearance. 7-EC and 7-HC showed significant metabolic degradation in liver subcellular fractions as compared to subcellular fractions obtained from intestine, kidney, lung and brain. The total in vitro metabolic clearance for 7-EC and 7-HC was determined by adding the individual in vitro organ clearance values obtained in hepatic and extra-hepatic microsomes or S9 fractions. The predicted in vivo clearance for 7-HC was 63.6 and 81.6 ml/min/kg by in vitro scaling from microsomes and S9 fractions, respectively. For 7-EC, the values were 78.5 and 76.8 ml/min/kg, respectively. The predicted clearance was found to be reasonably accurate with slight over- and underprediction. Interestingly, the relative contribution of hepatic and extra-hepatic metabolism to the total clearance of 7-EC and 7-HC was remarkably high, ranging from 62-77% and 22-38%, respectively, of the total metabolic clearance. It is concluded that the model of multi-organ subcellular fractions is a useful in vitro tool for the prediction of in vivo metabolic clearance, as it can provide information about the relative contribution of extra-hepatic and hepatic metabolism to total metabolic clearance. PMID:19326776

  17. In vitro evaluation of hepatic and extra-hepatic metabolism of coumarins using rat subcellular fractions: correlation of in vitro clearance with in vivo data.

    PubMed

    Behera, Dayanidhi; Damre, Anagha; Varghese, Alice; Addepalli, Veeranjaneyulu

    2008-01-01

    7-Ethoxycoumarin (7-EC) and 7-hydroxycoumarin (7-HC) were chosen as model compounds to study hepatic and extra-hepatic metabolism in rat tissue subcellular (microsomal and S9) fractions and to scale the observed in vitro clearance to in vivo plasma clearance. 7-EC and 7-HC showed significant metabolic degradation in liver subcellular fractions as compared to subcellular fractions obtained from intestine, kidney, lung and brain. The total in vitro metabolic clearance for 7-EC and 7-HC was determined by adding the individual in vitro organ clearance values obtained in hepatic and extra-hepatic microsomes or S9 fractions. The predicted in vivo clearance for 7-HC was 63.6 and 81.6 ml/min/kg by in vitro scaling from microsomes and S9 fractions, respectively. For 7-EC, the values were 78.5 and 76.8 ml/min/kg, respectively. The predicted clearance was found to be reasonably accurate with slight over- and underprediction. Interestingly, the relative contribution of hepatic and extra-hepatic metabolism to the total clearance of 7-EC and 7-HC was remarkably high, ranging from 62-77% and 22-38%, respectively, of the total metabolic clearance. It is concluded that the model of multi-organ subcellular fractions is a useful in vitro tool for the prediction of in vivo metabolic clearance, as it can provide information about the relative contribution of extra-hepatic and hepatic metabolism to total metabolic clearance.

  18. Multiplicity and regulation of hepatic microsomal carboxylesterases in rats.

    PubMed

    Hosokawa, M; Maki, T; Satoh, T

    1987-06-01

    Three isozymes of carboxylesterase were purified from rat liver microsomes by using Sephadex G-150 gel filtration, DE-52 ion exchange, and chromatofocusing column chromatographies. These isozymes each showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were immunologically different from each other as determined by immunochemical blotting analysis and immunochemical inhibition of catalytic activity. The three isozymes were named RH1 (molecular weight 174,000, trimer, pl 6.0), RL1 (molecular weight 61,000, monomer, pl 6.5), and RL2 (molecular weight 61,000, monomer, pl 5.5). RL1 has the highest specific activities toward p-nitrophenylacetate and malathion. Acetanilide is a rather specific substrate for RL2, whereas RH1 has the highest specific activity for butanilicaine. RL1 has the highest specific activity for the hydrolysis of long-chain acyl-CoA. To investigate the hormonal regulation of carboxylesterase activities, we have quantitated RL1, RL2, and RH1 in liver microsomes from male and female rats using a radial immunodiffusion assay. The amount of RL1 in male rats was decreased by castration but recovered to almost the level in sham-operated rat liver microsomes after treatment of the castrated rats with testosterone. Conversely, in ovariectomized female rats, the amount of RL1 was increased as compared to that in sham-operated rats, and treatment of the ovariectomized rats with estradiol tended to decrease the quantity of RL1. In all cases of sex hormone treatment, the amount of RH1 remains unclear at present. However, the amount of RL2 may be, at least in part, regulated by estrogens. On the other hand, phenobarbital treatment of male and female rats caused a significant increase in the amounts of RH1 and RL2, whereas RL1 was not affected. It was concluded that the three isozymes differ considerably from each other in response to hormone treatment, inducibility, substrate specificity, and immunological properties

  19. Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats.

    PubMed

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-10-01

    Hepatocytes generated from human embryonic stem cells (hESCs) are considered to be an excellent candidate for restoring the liver function deficiencies. We have earlier standardized a three-step differentiation protocol to generate functional hepatocyte-like cells (HLCs) from hESCs, which expressed the major hepatic markers. We have also found that the HLCs remain stable and functional even after extended period of in vitro culture and cryopreservation. In the present study, we have aimed to investigate the therapeutic potential of cryopreserved-thawed hESC-derived hepatic progenitor cells following transplantation in carbon tetrachloride-induced fibrotic rat livers. Significant therapeutic effects, including improved hepatic histology and normal serum biochemistry of hepatic enzymes along with increased survival rate, were observed in the cell transplanted rats. This result is an encouraging indication to develop methods for clinical application of hESC-derived hepatic lineage cells.

  20. Different doses of partial liver irradiation promotes hepatic regeneration in rat.

    PubMed

    Liu, Ying; Shi, Changzheng; Cui, Meng; Yang, Zhenhua; Gan, Danhui; Wang, Yiming

    2015-01-01

    The aim of this study is to investigate whether partial liver irradiation promotes hepatic regeneration in rat. Left-half liver of rat was irradiated to 10 Gy, and the Right-half to 0, 5, 10 and 15 Gy, respectively. Then, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels were evaluated on 0 day, 15-day, 30-day, 45-day and 60-day after liver irradiation. Next, the serum HGF, NF-κB and TGF-β1 levels were also analyzed on 60-day after liver irradiation. Lastly, the cyclinD1 protein expression was appraised by western blots on 60-day after liver irradiation. ALT, AST and ALP levels were reduced compared with that of controls. The serum HGF, NF-κB and TGF-β1 levels, and the cyclinD1 protein expression in liver irradiation group were increased compared with that of controls group. However, hepatic regeneration of higher dose-irradiated cirrhotic liver was triggered a more enhanced regeneration, compared with that of higher doses group. In summary, these results suggest that different doses of partial liver irradiation promotes hepatic regeneration in rat.

  1. Effects of methapyrilene on rat hepatic xenobiotic metabolizing enzymes and liver morphology.

    PubMed

    Graichen, M E; Neptun, D A; Dent, J G; Popp, J A; Leonard, T B

    1985-02-01

    Short-term treatment of rats with hepatocarcinogens elicits a consistent pattern of phenotypic changes in hepatic drug metabolizing enzymes, the most striking of which is a marked increase in microsomal epoxide hydrolase (EH) activity. The antihistaminic drug methapyrilene induces a high incidence of hepatocellular carcinoma in F-344 rats. The studies reported here were designed to assess the effects of methapyrilene on hepatic EH activity, cytochrome P-450-dependent mixed-function oxidase activities, liver morphology, and liver-derived serum enzymes. Male F-344 rats were treated with three daily oral doses of methapyrilene-HCl, up to 300 mg/kg/day, and were sacrificed 48 hr after the last dose. Hepatic microsomal EH and cytosolic DT-diaphorase activities were increased in a dose-related fashion, to 420 and 230% of control, respectively. Cytochrome P-450 content and benzphetamine-N-demethylase and ethoxycoumarin-O-deethylase activities were concomitantly decreased to 35-50% of control. Serum gamma-glutamyl transpeptidase and alanine aminotransferase activities were elevated 22- to 27-fold, and serum bile acids to 36-fold by treatment with methapyrilene. Periportal lesions, characterized by inflammation, nuclear and nucleolar enlargement, bile duct hyperplasia, and hepatocellular necrosis, were observed following methapyrilene administration. The severity of the periportal lesion correlated with elevations in the serum chemistry parameters. The increases noted in microsomal EH activity supports the suggestion that this enzyme may be a useful biochemical marker for exposure to hepatocarcinogens. PMID:2859228

  2. Pregnancy and maternal iron deficiency stimulate hepatic CRBPII expression in rats.

    PubMed

    Cottin, Sarah C; Gambling, Lorraine; Hayes, Helen E; Stevens, Valerie J; McArdle, Harry J

    2016-06-01

    Iron deficiency impairs vitamin A (VA) metabolism in the rat but the mechanisms involved are unknown and the effect during development has not been investigated. We investigated the effect of pregnancy and maternal iron deficiency on VA metabolism in the mother and fetus. 54 rats were fed either a control or iron deficient diet for 2weeks prior to mating and throughout pregnancy. Another 15 female rats followed the same diet and were used as non-pregnant controls. Maternal liver, placenta and fetal liver were collected at d21 for total VA, retinol and retinyl ester (RE) measurement and VA metabolic gene expression analysis. Iron deficiency increased maternal hepatic RE (P<.05) and total VA (P<.0001), fetal liver RE (P<.05), and decreased placenta total VA (P<.05). Pregnancy increased Cellular Retinol Binding Protein (CRBP)-II gene expression by 7 fold (P=.001), decreased VA levels (P=.0004) and VA metabolic gene expression (P<.0001) in the liver. Iron deficiency increased hepatic CRBPII expression by a further 2 fold (P=.044) and RBP4 by~20% (P=.005), increased RBPR2 and decreased CRBPII, LRAT, and TTR in fetal liver, while it had no effect on VA metabolic gene expression in the placenta. Hepatic CRBPII expression is increased by pregnancy and further increased by iron deficiency, which may play an important role in VA metabolism and homeostasis. Maternal iron deficiency also alters VA metabolism in the fetus, which is likely to have consequences for development. PMID:27142737

  3. Pregnancy and maternal iron deficiency stimulate hepatic CRBPII expression in rats.

    PubMed

    Cottin, Sarah C; Gambling, Lorraine; Hayes, Helen E; Stevens, Valerie J; McArdle, Harry J

    2016-06-01

    Iron deficiency impairs vitamin A (VA) metabolism in the rat but the mechanisms involved are unknown and the effect during development has not been investigated. We investigated the effect of pregnancy and maternal iron deficiency on VA metabolism in the mother and fetus. 54 rats were fed either a control or iron deficient diet for 2weeks prior to mating and throughout pregnancy. Another 15 female rats followed the same diet and were used as non-pregnant controls. Maternal liver, placenta and fetal liver were collected at d21 for total VA, retinol and retinyl ester (RE) measurement and VA metabolic gene expression analysis. Iron deficiency increased maternal hepatic RE (P<.05) and total VA (P<.0001), fetal liver RE (P<.05), and decreased placenta total VA (P<.05). Pregnancy increased Cellular Retinol Binding Protein (CRBP)-II gene expression by 7 fold (P=.001), decreased VA levels (P=.0004) and VA metabolic gene expression (P<.0001) in the liver. Iron deficiency increased hepatic CRBPII expression by a further 2 fold (P=.044) and RBP4 by~20% (P=.005), increased RBPR2 and decreased CRBPII, LRAT, and TTR in fetal liver, while it had no effect on VA metabolic gene expression in the placenta. Hepatic CRBPII expression is increased by pregnancy and further increased by iron deficiency, which may play an important role in VA metabolism and homeostasis. Maternal iron deficiency also alters VA metabolism in the fetus, which is likely to have consequences for development.

  4. The calcium-activated potassium channel KCa3.1 is an important modulator of hepatic injury.

    PubMed

    Sevelsted Møller, Linda; Fialla, Annette Dam; Schierwagen, Robert; Biagini, Matteo; Liedtke, Christian; Laleman, Wim; Klein, Sabine; Reul, Winfried; Koch Hansen, Lars; Rabjerg, Maj; Singh, Vikrant; Surra, Joaquin; Osada, Jesus; Reinehr, Roland; de Muckadell, Ove B Schaffalitzky; Köhler, Ralf; Trebicka, Jonel

    2016-01-01

    The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury. PMID:27354175

  5. The calcium-activated potassium channel KCa3.1 is an important modulator of hepatic injury

    PubMed Central

    Sevelsted Møller, Linda; Fialla, Annette Dam; Schierwagen, Robert; Biagini, Matteo; Liedtke, Christian; Laleman, Wim; Klein, Sabine; Reul, Winfried; Koch Hansen, Lars; Rabjerg, Maj; Singh, Vikrant; Surra, Joaquin; Osada, Jesus; Reinehr, Roland; de Muckadell, Ove B. Schaffalitzky; Köhler, Ralf; Trebicka, Jonel

    2016-01-01

    The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury. PMID:27354175

  6. Cilostazol improves hepatic blood perfusion, microcirculation, and liver regeneration after major hepatectomy in rats.

    PubMed

    von Heesen, Maximilian; Dold, Stefan; Müller, Simon; Scheuer, Claudia; Kollmar, Otto; Schilling, Martin K; Menger, Michael D; Moussavian, Mohammed R

    2015-06-01

    Major hepatectomy or small-for-size liver transplantation may result in postoperative liver failure. So far, no treatment is available to improve liver regeneration. Herein, we studied whether cilostazol, a selective phosphodiesterase III inhibitor, is capable of improving liver regeneration after major hepatectomy. Sprague-Dawley rats (n = 74) were treated with cilostazol (5 mg/kg daily) or a glucose solution and underwent either 70% liver resection or a sham operation. Before and after surgery, hepatic arterial and portal venous blood flow and hepatic microvascular perfusion were analyzed. Liver morphology, function, and regeneration were studied with histology, immunohistochemistry, western blotting, and bile excretion analysis. Cilostazol significantly increased hepatic blood flow and microcirculation before and after hepatectomy in comparison with sham-operated controls. This was associated with an elevation of hepatic vascular endothelial growth factor expression, an increase of hepatocellular proliferation, and an acceleration of liver regeneration. Furthermore, cilostazol protected the tissue of the remnant liver as indicated by an attenuation of hepatocellular disintegration. In conclusion, cilostazol increases hepatic blood perfusion, microcirculation, and liver regeneration after a major hepatectomy. Thus, cilostazol may represent a novel strategy to reduce the rate of liver failure after both extended hepatectomy and small-for-size liver transplantation.

  7. Vescalagin from Pink Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Alleviates Hepatic Insulin Resistance and Ameliorates Glycemic Metabolism Abnormality in Rats Fed a High-Fructose Diet.

    PubMed

    Huang, Da-Wei; Chang, Wen-Chang; Wu, James Swi-Bea; Shih, Rui-Wen; Shen, Szu-Chuan

    2016-02-10

    This study investigates the ameliorative effect of vescalagin (VES) isolated from Pink wax apple fruit on hepatic insulin resistance and abnormal carbohydrate metabolism in high-fructose diet (HFD)-induced hyperglycemic rats. The results show that in HFD rats, VES significantly reduced the values of the area under the curve for glucose in an oral glucose tolerance test and the homeostasis model assessment of insulin resistance index. VES significantly enhanced the activity of hepatic antioxidant enzymes while reducing thiobarbituric acid-reactive substances in HFD rats. Western blot assay revealed that VES reduced hepatic protein expression involved in inflammation pathways while up-regulating expression of hepatic insulin signaling-related proteins. Moreover, VES up-regulated the expression of hepatic glycogen synthase and hepatic glycolysis-related proteins while down-regulating hepatic gluconeogenesis-related proteins in HFD rats. This study suggests some therapeutic potential of VES in preventing the progression of diabetes mellitus. PMID:26800576

  8. Vescalagin from Pink Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Alleviates Hepatic Insulin Resistance and Ameliorates Glycemic Metabolism Abnormality in Rats Fed a High-Fructose Diet.

    PubMed

    Huang, Da-Wei; Chang, Wen-Chang; Wu, James Swi-Bea; Shih, Rui-Wen; Shen, Szu-Chuan

    2016-02-10

    This study investigates the ameliorative effect of vescalagin (VES) isolated from Pink wax apple fruit on hepatic insulin resistance and abnormal carbohydrate metabolism in high-fructose diet (HFD)-induced hyperglycemic rats. The results show that in HFD rats, VES significantly reduced the values of the area under the curve for glucose in an oral glucose tolerance test and the homeostasis model assessment of insulin resistance index. VES significantly enhanced the activity of hepatic antioxidant enzymes while reducing thiobarbituric acid-reactive substances in HFD rats. Western blot assay revealed that VES reduced hepatic protein expression involved in inflammation pathways while up-regulating expression of hepatic insulin signaling-related proteins. Moreover, VES up-regulated the expression of hepatic glycogen synthase and hepatic glycolysis-related proteins while down-regulating hepatic gluconeogenesis-related proteins in HFD rats. This study suggests some therapeutic potential of VES in preventing the progression of diabetes mellitus.

  9. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    PubMed Central

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R.G.; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K.

    2012-01-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2'-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats. PMID:23543859

  10. Effect of Octreotide on Hepatic Steatosis in Diet-Induced Obesity in Rats

    PubMed Central

    Li, Mao; Ye, Ting; Wang, Xiao-Xia; Li, Xian; Qiang, Ou; Yu, Tao; Tang, Cheng-Wei; Liu, Rui

    2016-01-01

    Background Non-alcoholic fatty liver disease (NAFLD) caused by liver lipid dysregulation is linked to obesity. Somatostatin (SST) and its analogs have been used to treat pediatric hypothalamic obesity. However, the application of such drugs for the treatment of NAFLD has not been evaluated. Objective This study aimed to investigate the expression levels of important regulators of hepatic lipid metabolism and the possible effect of the SST analog octreotide on these regulators. Methods SD rats were assigned to a control group and a high-fat diet group. Obese rats from the high-fat diet group were further divided into the obese and octreotide-treated groups. The body weight, plasma SST, fasting plasma glucose (FPG), insulin, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) levels were measured. Hepatic steatosis was evaluated based on the liver TG content, HE staining and oil red O staining. The SREBP-1c, ACC1, FAS, MTP, apoB and ADRP expression levels in the liver were also determined by RT-PCR, qRT-PCR, western blot or ELISA. Results The obese rats induced by high-fat diet expressed more SREBP-1c, FAS and ADRP but less MTP protein in the liver than those of control rats, whereas octreotide intervention reversed these changes and increased the level of apoB protein. Compared to the control group, obese rats showed increased liver ACC1, SREBP-1c and apoB mRNA levels, whereas octreotide-treated rats showed decreased mRNA levels of apoB and SREBP-1c. This was accompanied by increased body weight, liver TG contents, FPG, TG, TC, LDL-C, FFA, insulin and derived homeostatic model assessment (HOMA) values. Octreotide intervention significantly decreased these parameters. Compared to the control group, the obese group showed a decreasing trend on plasma SST levels, which were significantly increased by the octreotide intervention. Conclusion Octreotide can

  11. Hepatic uptake of gamma-butyrobetaine, a precursor of carnitine biosynthesis, in rats.

    PubMed

    Fujita, Masaharu; Nakanishi, Takeo; Shibue, Yuta; Kobayashi, Daisuke; Moseley, Richard H; Shirasaka, Yoshiyuki; Tamai, Ikumi

    2009-10-01

    Gamma-butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the beta-oxidation of fatty acids, and is converted to carnitine by gamma-butyrobetaine dioxygenase (BBD) predominantly in liver. We investigated the molecular mechanism of hepatic uptake of GBB in rat hepatocytes. Cellular localization of rat Octn2 (rOctn2:Slc22A5) was studied by Western blot analysis. Uptake of deuterated GBB (d(3)-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d(3)-GBB was quantified by use of liquid chromatography-tandem mass spectrometry. Western blot analysis demonstrated an expression of OCTN2 protein in hepatic basolateral membrane but not in bile canalicular membrane fraction. Furthermore, we found that d(3)-GBB was taken up by rOctn2 in an Na(+)-dependent manner with K(m) value of 13 microM. The apparent K(m) value for d(3)-GBB transport in freshly isolated rat hepatocytes was 9 microM. d(3)-GBB uptake by the rat hepatocytes was inhibited by gamma-aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 microM. Furthermore, d(3)-GBB uptake by rat hepatocytes was decreased by 45% with rat Gat2 (Slc6A13, a major liver GABA transporter) silenced by the microRNA method. Accordingly, the present study clearly demonstrates that GBB is taken up by hepatocytes for carnitine biosynthesis not only via Octn2 but also via the GABA transporter, possibly Gat2.

  12. Abnormal hepatic copper accumulation of spheroid composed of liver cells from LEC rats in vitro.

    PubMed

    Ueno, K; Yoshizawa, M; Satoh, T; Yoneda, S; Ohmichi, M; Yamazaki, M; Mori, Y; Suzuki, K T

    1995-11-01

    The LEC rat is a mutant strain displaying hereditary hepatitis, and shows abnormal accumulation of copper (Cu) similar to that occurring in Wilson's disease. We prepared a multicellular spheroid composed of LEC rat liver cells to investigate the mechanism for abnormal accumulation of Cu. These multicellular spheroids were prepared by detaching the monolayer on the collagen-conjugated thermo-responsive polymer coated culture dish at a temperature below the critical solution temperature and culturing on the non-adhesive substratum. Long-term cultured spheroids of LEC rat liver cells as well as SD rat liver cells were attempted. Non-parenchymal cells obtained by collagenase perfusion from the LEC liver were fewer than those from the SD liver. Cells from the LEC rat, over 11 weeks of age, did not form a cell sheet; however, a mixture of parenchymal cells from LEC rats over aged 11 weeks and non-parenchymal cells from SD rats of any age yielded intact spheroids. We examined the toxicity, the accumulation and distribution of Cu in spheroids. The accumulation of Cu in LEC spheroids was higher than that in SD spheroids. Results suggest that spheroids consisting of LEC liver cells are useful as an alternative model to in vivo tests to investigate the mechanism for abnormal accumulation of Cu in liver.

  13. Exercise and spirulina control non-alcoholic hepatic steatosis and lipid profile in diabetic Wistar rats

    PubMed Central

    2011-01-01

    Background Diabetes mellitus is associated with metabolic dysfunctions, including alterations in circulating lipid levels and fat tissue accumulation, which causes, among other pathologies, non-alcoholic fatty liver disease (NAFLD). Aim of the study The objective of this study was to analyse the effects of physical exercise and spirulina intake on the control of NAFLD in diabetic Wistar rats. Methods Diabetes was induced in the animals through intravenous administration of alloxan. The rats were divided into four groups: Diabetic Control (DC) - diabetic rats fed with a control diet and no physical exercise; Diabetic Spirulina (DS) - diabetic rats fed with a diet that included spirulina; Diabetic Spirulina and Exercise (DSE) - diabetic rats fed with a diet that included Spirulina and that exercised; and Diabetic Exercise (DE) - diabetic rats fed with a control diet and that exercised. Results The groups DS, DSE, and DE presented lower plasma concentrations of LDL cholesterol than DC, as well as lower levels of total liver lipids in groups DS, DSE, and DE in comparison to DC. Conclusion Thus, spirulina appears to be effective in reducing total circulating levels of LDL-cholesterol and hepatic lipids, alone or in conjunction with physical exercise in diabetic rats. PMID:21569626

  14. Quercetin Increases Hepatic Homocysteine Remethylation and Transsulfuration in Rats Fed a Methionine-Enriched Diet.

    PubMed

    Meng, Bin; Gao, Weina; Wei, Jingyu; Pu, Lingling; Tang, Zhenchuang; Guo, Changjiang

    2015-01-01

    This study was aimed at investigating the effects of quercetin on mRNA expression and activity of critical enzymes in homocysteine metabolism in rats fed a methionine-enriched diet. Rats were fed for 6 weeks the following diets, that is, control, 0.5% quercetin, 1.0% methionine, and 1.0% methionine plus 0.5% quercetin diets. Serum homocysteine was significantly increased after methionine treatment and decreased after the addition of quercetin. The mRNA expression of methionine synthase was significantly increased after methionine or methionine plus quercetin supplementation, while its enzymatic activity was significantly increased after methionine plus quercetin supplementation. The mRNA expression and enzymatic activity of cystathionine β-synthase and cystathionine γ-lyase were upregulated after quercetin, methionine, or quercetin plus methionine treatment and a more significant increase was observed for hepatic cystathionine β-synthase in the methionine plus quercetin treated rats, suggesting an interaction between methionine and quercetin. Meanwhile, hepatic ratio of S-adenosylmethionine to S-adenosylhomocysteine was significantly decreased in response to methionine supplementation and normalized after the addition of quercetin. It is concluded that quercetin reduces serum homocysteine by increasing remethylation and transsulfuration of homocysteine in rats exposed to a methionine-enriched diet.

  15. Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

    PubMed Central

    Rawat, A K

    1976-01-01

    Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'. PMID:1016246

  16. Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis.

    PubMed

    Heijne, Wilbert H M; Lamers, Robert-Jan A N; van Bladeren, Peter J; Groten, John P; van Nesselrooij, Joop H J; van Ommen, Ben

    2005-01-01

    This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org. PMID:16036859

  17. Effects of nanogold on the alleviation of carbon tetrachloride-induced hepatic injury in rats.

    PubMed

    Chen, Yi-Peng; Dai, Zong-Her; Liu, Pan-Chen; Chuu, Jiunn-Jye; Lee, Kuo-Yang; Lee, Shun-Lai; Chen, Yun-Ju

    2012-10-31

    Gold particles have been used in complementary medicine for decades, and many beneficial effects have been reported. Our present study sought to evaluate the therapeutic effects of nanogold in carbon tetrachloride (CCl₄)-injured liver of rats. Male SD rats were subjected to liver injury induction by CCl₄, then the rats were fed with zero to high dose (0, 1, 5 or 10 ppm) of nanogold water every day for 4 weeks. Biochemical analyses on liver functions were then performed to evaluate the therapeutic effects of nanogold. Our results revealed that gold nanoparticles lowered serum aspartate aminotransaminase (AST) and alanine aminotransferase and exerted serum total protein-recovering effects, which might be partially associated with the elevation of anti-inflammatory cytokine IL-10 level. In addition, serum triglyceride level fell after continuous ingestion of nanogold. Finally, the experimental animals recovered body weight after 4 weeks of nanogold ingestion. This is the first report indicating inflammation alleviating effects of nanogold on hepatic injury.

  18. The characterization and metabolism of rat hepatic nascent HLD subfractions

    SciTech Connect

    Winkler, K.E.

    1988-01-01

    Nascent HDL was isolated from recirculating rat liver perfusates and separated by heparin-sepharose chromatography into a non-retained fraction (A) and a fraction (B) that eluted with 0.5 M NaCl. Fractions A and B contained 70% and 30% of the nascent HDL protein, respectively. Livers perfused by the single-pass technique produced fractions A and B in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions A and B had more triglyceride (greater than 50% of total lipid). In a preliminary study designed to investigate whether nascent HDL-apo E was secreted by Kupffer cells or hepatocytes, label was targeted to Kupffer cells by perfusing rat livers with {sup 3}H-acetylated LDL or {sup 3}H-amino acids incorporated into large multilamellar vesicles. For metabolic studies, nascent HDL and nascent VLDL were isolated from rat livers that had been perfused with {sup 3}H-glycerol to label the triglyceride.

  19. Maternal obesity characterized by gestational diabetes increases the susceptibility of rat offspring to hepatic steatosis via a disrupted liver metabolome.

    PubMed

    Pereira, Troy J; Fonseca, Mario A; Campbell, Kristyn E; Moyce, Brittany L; Cole, Laura K; Hatch, Grant M; Doucette, Christine A; Klein, Julianne; Aliani, Michel; Dolinsky, Vernon W

    2015-07-15

    Maternal obesity is associated with a high risk for gestational diabetes mellitus (GDM), which is a common complication of pregnancy. The influence of maternal obesity and GDM on the metabolic health of the offspring is poorly understood. We hypothesize that GDM associated with maternal obesity will cause obesity, insulin resistance and hepatic steatosis in the offspring. Female Sprague-Dawley rats were fed a high-fat (45%) and sucrose (HFS) diet to cause maternal obesity and GDM. Lean control pregnant rats received low-fat (LF; 10%) diets. To investigate the interaction between the prenatal environment and postnatal diets, rat offspring were assigned to LF or HFS diets for 12 weeks, and insulin sensitivity and hepatic steatosis were evaluated. Pregnant GDM dams exhibited excessive gestational weight gain, hyperinsulinaemia and hyperglycaemia. Offspring of GDM dams gained more weight than the offspring of lean dams due to excess adiposity. The offspring of GDM dams also developed hepatic steatosis and insulin resistance. The postnatal consumption of a LF diet did not protect offspring of GDM dams against these metabolic disorders. Analysis of the hepatic metabolome revealed increased diacylglycerol and reduced phosphatidylethanolamine in the offspring of GDM dams compared to offspring of lean dams. Consistent with altered lipid metabolism, the expression of CTP:phosphoethanolamine cytidylyltransferase, and peroxisomal proliferator activated receptor-α mRNA was reduced in the livers of GDM offspring. GDM exposure programs gene expression and hepatic metabolite levels and drives the development of hepatic steatosis and insulin resistance in young adult rat offspring.

  20. Cryptotanshinone Ameliorates Hepatic Normothermic Ischemia and Reperfusion Injury in Rats by Anti-mitochondrial Apoptosis.

    PubMed

    Sun, Ping-Ping; Yuan, Fang; Xu, Jing; Sai, Ke; Chen, Jie; Guan, Su

    2014-01-01

    Cryptotanshinone (CT), isolated from the dried roots of Salvia militorrhiza, has been reported to have protective effects on myocardial and cerebral ischemia/reperfusion (I/R) injury both in vitro and in vivo. However, its effects and underlying mechanism on hepatic I/R injury remain unclear. To investigate its effects on hepatic I/R injury, thirty male Sprague-Dawley rats were randomized into 3 groups: a sham group, a vehicle-treated hepatic I/R group and a CT-treated (50 mg/kg) group. The hepatic I/R and CT-treated groups were subjected to 60 min of normothermic ischemia of the left lateral lobe of the liver, followed by 4 h of reperfusion. The animals were then sacrificed to collect the serum and the left liver lobe for assay. Hepatic function was protected, as evidenced by significantly reduced alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) levels in the CT-treated group as compared with I/R group. The terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) demonstrated significantly decreased apoptosis in the CT-administration animals. Western blotting demonstrated upregulation of the proapoptotic protein Bcl-2, as well as decreased levels of the activated form of caspase-3 and the cleaved form of its substrate, poly(ADP-ribose) polymerase (PARP) in the CT-treated group compared with those of the I/R group. In addition, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) was inhibited by CT. Our data suggest that CT attenuates hepatic I/R injury by inhibiting the intrinsic pathway of apoptosis, mediated partly through the inhibition of JNK and p38 MAPK phosporylation. PMID:25366482

  1. Hepatic AQP9 expression in male rats is reduced in response to PPARα agonist treatment.

    PubMed

    Lebeck, Janne; Cheema, Muhammad Umar; Skowronski, Mariusz T; Nielsen, Søren; Praetorius, Jeppe

    2015-02-01

    The peroxisome proliferator receptor α (PPARα) is a key regulator of the hepatic response to fasting with effects on both lipid and carbohydrate metabolism. A role in hepatic glycerol metabolism has also been found; however, the results are somewhat contradictive. Aquaporin 9 (AQP9) is a pore-forming transmembrane protein that facilitates hepatic uptake of glycerol. Its expression is inversely regulated by insulin in male rodents, with increased expression during fasting. Previous results indicate that PPARα plays a crucial role in the induction of AQP9 mRNA during fasting. In the present study, we use PPARα agonists to explore the effect of PPARα activation on hepatic AQP9 expression and on the abundance of enzymes involved in glycerol metabolism using both in vivo and in vitro systems. In male rats with free access to food, treatment with the PPARα agonist WY 14643 (3 mg·kg(-1)·day(-1)) caused a 50% reduction in hepatic AQP9 abundance with the effect being restricted to AQP9 expressed in periportal hepatocytes. The pharmacological activation of PPARα had no effect on the abundance of GlyK, whereas it caused an increased expression of hepatic GPD1, GPAT1, and L-FABP protein. In WIF-B9 and HepG2 hepatocytes, both WY 14643 and another PPARα agonist GW 7647 reduced the abundance of AQP9 protein. In conclusion, pharmacological PPARα activation results in a marked reduction in the abundance of AQP9 in periportal hepatocytes. Together with the effect on the enzymatic apparatus for glycerol metabolism, our results suggest that PPARα activation in the fed state directs glycerol into glycerolipid synthesis rather than into de novo synthesis of glucose. PMID:25477377

  2. Levocetirizine ameliorates high fructose diet-induced insulin resistance, vascular dysfunction and hepatic steatosis in rats.

    PubMed

    Shawky, Noha M; Shehatou, George S G; Abdel Rahim, Mona; Suddek, Ghada M; Gameil, Nariman M

    2014-10-01

    This study investigates the possible protective effects of levocetirizine against fructose-induced insulin resistance, hepatic steatosis and vascular dysfunction, in comparison to pioglitazone, a standard insulin sensitizer. Male Sprague Dawley rats (150-200 g) were divided into 4 groups. Three groups were fed on high fructose diets (HFD) containing 60% w/w fructose, while the fourth control group was fed on standard laboratory food for 8 weeks. AUCOGTT, AUCITT, fasting glucose, HOMA-IR, hepatic glutathione (GSH) and malondialdehyde (MDA) levels, serum total cholesterol, LDL-C, C-reactive protein (CRP) level and lactate dehydrogenase (LDH) activity and liver steatosis scores were significantly higher in HFD group compared to control group. Moreover, body weight gain, food intake, feeding efficiency, HOMA-β, Emax and pEC50 of acetylcholine-induced relaxations of aortic rings and hepatic superoxide dismutase (SOD) activity were significantly lower in HFD group than in control group. Treatment with levocetirizine caused significant decreases in AUCOGTT, AUCITT, HOMA-IR, hepatic GSH and MDA levels and serum CRP level and LDH activity and significant increases in hepatic SOD activity and HOMA-β when compared with the HFD group. Although levocetirizine failed to alter TC and LDL-C levels, it produced a significant increase in HDL-C level relative to control group. Levocetirizine was also able to improve acetylcholine-induced relaxations of aortic rings, indicating a protective effect against insulin resistance-induced endothelial damage comparable to that offered by pioglitazone. Moreover, levocetirizine substantially attenuated insulin resistance-associated liver macrovesicular steatosis. These findings demonstrate that levocetirizine ameliorates insulin resistance, improves glucose tolerance and attenuates insulin resistance-linked hepatic steatosis and vascular damage. PMID:25064340

  3. Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.

    PubMed

    Rodriguez, R J; Acosta, D

    1997-06-01

    Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.

  4. Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

    PubMed

    Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

    2012-11-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats. PMID:23014486

  5. Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

    PubMed

    Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

    2012-11-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

  6. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis.

    PubMed Central

    Herbst, H.; Wege, T.; Milani, S.; Pellegrini, G.; Orzechowski, H. D.; Bechstein, W. O.; Neuhaus, P.; Gressner, A. M.; Schuppan, D.

    1997-01-01

    The remodeling of extracellular matrix during chronic liver disease may partially be attributed to altered activity of matrix metalloproteinases and their tissue inhibitors (TIMPs). Expression of TIMP-1 and -2 was studied by in situ hybridization combined with immunohistochemistry in rat (acute and chronic carbon tetrachloride intoxication and secondary biliary fibrosis) and human livers and on isolated rat hepatic stellate cells. TIMP-1 and -2 transcripts appeared in rat livers within 1 to 3 hours after intoxication, pointing to a role in the protection against accidental activation of matrix metalloproteinases, and were present at high levels in all fibrotic rat and human livers predominantly in stellate cells. TIMP-2 RNA distribution largely matched with previously reported patterns of matrix metalloproteinase-2 (72-kd gelatinase) expression, suggesting generation of a TIMP-2/matrix metalloproteinase-2 complex (large inhibitor of metalloproteinases). Isolated stellate cells expressed TIMP-1 and -2 RNA. Addition of transforming growth factor-beta 1 enhanced TIMP-1 and matrix metalloproteinase-2 RNA levels in vitro, whereas TIMP-2-specific signals were reduced, likely to result in a stoichiometric excess of matrix-metalloproteinase-2 over TIMP-2. In the context of previous demonstrations of transforming growth factor-beta 1 and matrix metalloproteinase-2 in vivo, these patterns suggest an intrahepatic environment permitting only limited matrix degradation, ultimately resulting in redistribution of extracellular matrix with relative accumulation of collagen type 1. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:9137090

  7. The hepatic protective mechanism of Ginkgo biloba extract in rats with obstructive jaundice.

    PubMed

    Weng, Ming-Zhe; Zhou, Xian-Ping; Jia, Jian-Guang; Ding, Jing; Fang, Cui-Fu; Qin, Yi-Yu; Tao, Shao-Fu; Rao, Long-Hua; Li, Ji-Yu; Quan, Zhi-Wei

    2011-11-01

    The objective of our study was to examine the hepatic protective mechanism of Ginkgo biloba extract (GBE) in rats with obstructive jaundice (OJ). Twenty rats underwent bile duct ligation and received daily intraperitoneal injections of either control saline or Ginkgo biloba extract for 14 days. Ten sham-operated rats had their bile duct exposed but not ligated or sectioned. Serum alanine transaminase (ALT) was analyzed for liver function tests and liver damage was further assessed by histologic examination. The levels of endothelin 1 (ET-1) and nitric oxide (NO) in blood and liver homogenate were measured. The serum alanine transaminase was elevated in the bile duct ligation rats (BDL rats); GBE could significantly lower serum transaminase level and ameliorate liver histological damage. ET-1 and NO levels in both plasma and liver tissue were also elevated in common bile duct (CBD)-ligated rats, but this increase was significantly decreased by GBE treatment. Moreover, the degree of liver damage severity positively correlates with high levels of ET-1 and NO. GBE mediated the liver protective effect at least in part by suppressing overproduction of ET-1 and NO and restoring a proper balance between ET-1 and NO to some extent.

  8. Protective effect of Piper betle leaf extract against cadmium-induced oxidative stress and hepatic dysfunction in rats

    PubMed Central

    Milton Prabu, S.; Muthumani, M.; Shagirtha, K.

    2012-01-01

    The present study was undertaken to examine the attenuative effect of Piper betle leaf extract (PBE) against cadmium (Cd) induced oxidative hepatic dysfunction in the liver of rats. Pre-oral supplementation of PBE (200 mg/kg BW) treated rats showed the protective efficacy against Cd induced hepatic oxidative stress. Oral administration of Cd (5 mg/kg BW) for four weeks to rats significantly (P > 0.05) elevated the level of serum hepatic markers such as serum aspartate transaminase (AST), serum alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), bilirubin (TBRNs), oxidative stress markers viz., thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), protein carbonyls (PC) and conjugated dienes (CD) and significantly (P > 0.05) reduced the enzymatic antioxidants viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) and non-enzymatic antioxidants Viz., reduced glutathione (GSH), total sulfhydryls (TSH), vitamin C and vitamin E in the liver. Pre-oral supplementation of PBE (200 mg/kg BW) in Cd intoxicated rats, the altered biochemical indices and pathological changes were recovered significantly (P > 0.05) which showed ameliorative effect of PBE against Cd induced hepatic oxidative stress. From the above findings, we suggested that the pre-administration of P. betle leaf extract exhibited remarkable protective effects against cadmium-induced oxidative hepatic injury in rats. PMID:23961183

  9. Protective effect of naringenin on hepatic and renal dysfunction and oxidative stress in arsenic intoxicated rats.

    PubMed

    Mershiba, Sam Daniel; Dassprakash, M Velayutham; Saraswathy, Sundara Dhakshinamurthy

    2013-05-01

    Arsenic has a long history as a potent human poison, chronic exposure over a period of time may result in the manifestation of toxicity in practically all systems of the body. In the present investigation the efficacy of naringenin (NRG), a naturally occurring citrus flavanone against arsenic-induced hepatotoxic and nephrotoxic manifestations have been studied in rats. Arsenic trioxide was administered orally at the dose of 2 mg/kg/day with or without combination of NRG (20 or 50 mg/kg/day) for 28 days. At the end of the experimental period the hepatic and renal dysfunction was evaluated by histological examination, serum biomarkers and markers of oxidative stress; lipid peroxidation (LPO), reduced glutathione (GSH) and antioxidant enzymes. Arsenic intoxication increased serum bilirubin, urea, uric acid and creatinine levels, additionally enhanced the activities of hepatic marker enzymes aspartate transaminase, alanine transaminase and alkaline phosphatase. Also, the hepatic and renal tissues showed a marked elevation in LPO levels with a decrease in GSH content and the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase on arsenic treatment. Simultaneous treatment with NRG restored the activities of serum biomarkers and antioxidant enzymes in the tissues in a dose-dependent manner. Furthermore, the histopathological studies confirmed the protective effect of NRG co-treatment by reducing the pathological changes due to arsenic intoxication in both liver and kidney. Thus, our present study demonstrates that NRG has a potential to protect arsenic-induced oxidative hepatic and renal dysfunction.

  10. Comparative azo reductase activity of red azo dyes through caecal and hepatic microsomal fraction in rats.

    PubMed

    Singh, S; Das, M; Khanna, S K

    1997-09-01

    In order to study the rate of formation of toxic aromatic amines, anaerobic reduction of four red azo dyes viz. amaranth, carmoisine, fast Red E and ponceau 4R was investigated by incubating caecal content and hepatic microsomal fraction of rats with 37.5 microM concentration of dyes in sodium phosphate buffer pH 7.4 using NADPH generating system, glucose oxidase system and nitrogen as the gaseous phase. Caecal suspension exhibited higher azo reductase activity than that of hepatic microsomal fraction using any of the 4 azo dyes. Caecal microbes showed maximal azo reductase activity when ponceau 4R was used as a substrate followed by fast Red E and carmoisine, while with amaranth the activity was minimum. Similarly ponceau 4 R exhibited maximum hepatic microsomal azo reductase activity followed by fast Red E and carmoisine whereas, amaranth had minimum activity. Caecal flora possessed almost 17 fold higher degradative capability of ponceau 4 R and fast Red E colourants than the hepatic microsomal fraction. The higher reductive ability through caecal flora for ponceau 4R and fast Red E signifies the formation of more aromatic amines which may be re-absorbed through the intestine to be either eliminated through urine as conjugates or retained in the target tissues to elicit toxic effects.

  11. alpha-Tocopherol acetate supplementation enhances rat hepatic cytochrome PROD activity in the presence of phenobarbital induction.

    PubMed

    Lii, C K; Sung, W C; Ko, Y J; Chen, H W

    1998-01-01

    Hepatic cytochrome P-450 enzymes play important roles in bioactivation of chemical carcinogens, biotransformation of many endogenous compounds, and detoxification of numerous xenobiotics. These enzyme activities have been shown to be regulated by various dietary factors. In our previous study, hepatic cytochrome pentoxyresorufin O-dealkylase (PROD) activity was decreased in rats fed an alpha-tocopherol acetate-deficient diet compared with rats fed alpha-tocopherol acetate-adequate or -supplemented diets. The objective of the present study was to investigate whether the modulatory effect of dietary alpha-tocopherol acetate on hepatic cytochrome PROD activity is influenced by the presence of phenobarbital. Weanling male Sprague-Dawley rats were fed the AIN-76 diet for four days, fasted for two days, then fed semipurified diets that were alpha-tocopherol acetate deficient, adequate, or supplemented with 5 and 15 g/kg alpha-tocopherol acetate for four days. Liver and plasma alpha-tocopherol concentrations were dose dependently regulated by dietary alpha-tocopherol acetate level. Inhibition of lipid peroxidation by dietary alpha-tocopherol acetate was dose dependent. Hepatic total cytochrome P-450 content was significantly greater in rats fed diets supplemented with 5 and 15 g/kg alpha-tocopherol acetate than in rats fed an alpha-tocopherol-adequate diet (p < 0.05). Hepatic cytochrome PROD activity was significantly greater in rats fed diets supplemented with 5 and 15 g/kg alpha-tocopherol acetate than in rats fed alpha-tocopherol acetate-deficient and -adequate diets (p < 0.05). These results suggest that, in the presence of phenobarbital, dietary alpha-tocopherol acetate efficiently affects tissue alpha-tocopherol levels and inhibits lipid peroxidation and that diets supplemented with 5 or 15 g/kg alpha-tocopherol acetate enhance hepatic cytochrome PROD activity compared with alpha-tocopherol acetate-deficient or -adequate diets.

  12. Glucagon-like peptide-1 binding to rat hepatic membranes.

    PubMed

    Villanueva-Peñacarrillo, M L; Delgado, E; Trapote, M A; Alcántara, A; Clemente, F; Luque, M A; Perea, A; Valverde, I

    1995-07-01

    We have found [125I]glucagon-like peptide (GLP)-1(7-36)amide specific binding activity in rat liver and isolated hepatocyte plasma membranes, with an M(r) of approximately 63,000, estimated by cross-linking and SDS-PAGE. The specific binding was time- and membrane protein concentration-dependent, and equally displaced by unlabelled GLP-1(7-36)amide and by GLP-1(1-36)amide, achieving its ID50 at 3 x 10(-9) M of the peptides. GLP-1(7-36)amide did not modify the basal or the glucagon (10(-8) M)-stimulated adenylate cyclase in the hepatocyte plasma membranes. These data, together with our previous findings of a potent glycogenic effect of GLP-1(7-36)amide in isolated rat hepatocytes, led us to postulate that the insulin-like effects of this peptide on glucose liver metabolism could be mediated by a type of receptor probably different from that described for GLP-1 in pancreatic B-cells or, alternatively, by the same receptor which, in this tissue as well as in muscle, uses a different transduction system.

  13. Arsenic-induced hepatic mitochondrial toxicity in rats and its amelioration by dietary phosphate.

    PubMed

    Majumdar, Sangita; Karmakar, Subhra; Maiti, Anasuya; Choudhury, Monalisa; Ghosh, Aniruddha; Das, Asankur Sekhar; Mitra, Chandan

    2011-01-01

    The present study was aimed to test the hypothesis that inorganic phosphate may reduce arsenic toxicity by decreasing its intestinal transference. Co-administration of inorganic phosphate (6.56 M) and arsenic (6.07 mM) in the intestinal loops of rats, in situ, caused significant reduction of arsenic transference. Short-term arsenic exposure (3mg/kg body weight/day for 30 days) caused liver damage evidenced by activities of liver enzymes and necroinflammatory changes. These effects of arsenic were coupled with enhanced mitochondrial swelling, inhibition of cytochrome c oxidase, Ca(2+)-ATPase, a decrease in mitochondrial calcium content, changes in indices of hepatic mitochondrial oxidative stress and iNOS expression. Arsenic also increased hepatic caspase 3 activity and DNA fragmentation. All these apoptosis-related molecular changes caused by arsenic could be alleviated by supplementation with inorganic phosphate, which likely suggests a protective role of phosphate against arsenic-induced hepatotoxic changes.

  14. Induction of hepatic drug metabolizing enzymes by coal fly ash in rats

    SciTech Connect

    Srivastava, P.K.; Singh, Y.; Tyagi, S.R.; Misra, U.K.

    1987-12-01

    The effect of intratracheal administration of fly ash, its benzene-extracted residue and the benzene extract has been studied on the activities of hepatic mixed-function oxidases in the rat. Fly ash and its fractions significantly increased the levels of cytochrome P-450, cytochrome b/sub 5/, cytochrome b/sub 5/ reductase, NADPH-cytochrome c reductase, aminopyrine N-demethylase, aniline hydroxylase, and glutathione S-transferase in a dose-dependent manner. Phenobarbital or 3-methylcholanthrene treatment along with the administration of fly ash or its fractions showed an additive effect on the activities of the mixed-function oxidases. The observed effects were due to chemical component, i.e., organic and inorganic fractions of fly ash, and not due to its particulate nature. This was shown by the administration of glass beads, which did not cause any alteration in the activities of hepatic mixed-function oxidases.

  15. Hepatic capillariasis in rats: a new model for testing antifibrotic drugs.

    PubMed

    de Souza, M M; Silva, L M; Barbosa, A A; de Oliveira, I R; Paraná, R; Andrade, Z A

    2000-11-01

    Rats infected with the helminth Capillaria hepatica regularly develop septal hepatic fibrosis that may progress to cirrhosis in a relatively short time. Because of such characteristics, this experimental model was selected for testing drugs exhibiting antifibrosis potential, such as pentoxifylline, gadolinium chloride and vitamin A. Hepatic fibrosis was qualitatively and quantitatively evaluated in liver samples obtained by partial hepatectomy and at autopsy. The material was submitted to histological, biochemical and morphometric methods. A statistically significant reduction of fibrosis was obtained with pentoxifylline when administered intraperitoneally rather than intravenously. Gadolinium chloride showed moderate activity when administered prophylactically (before fibrosis had started), but showed a poor effect when fibrosis was well advanced. No modification of fibrosis was seen after vitamin A administration. Hydroxyproline content was correlated with morphometric measurements. The model appears to be adequate, since few animals die of the infection, fibrosis develops regularly in all animals, and the effects of different antifibrotic drugs and administration protocols can be easily detected.

  16. Arsenic-induced hepatic mitochondrial toxicity in rats and its amelioration by dietary phosphate.

    PubMed

    Majumdar, Sangita; Karmakar, Subhra; Maiti, Anasuya; Choudhury, Monalisa; Ghosh, Aniruddha; Das, Asankur Sekhar; Mitra, Chandan

    2011-01-01

    The present study was aimed to test the hypothesis that inorganic phosphate may reduce arsenic toxicity by decreasing its intestinal transference. Co-administration of inorganic phosphate (6.56 M) and arsenic (6.07 mM) in the intestinal loops of rats, in situ, caused significant reduction of arsenic transference. Short-term arsenic exposure (3mg/kg body weight/day for 30 days) caused liver damage evidenced by activities of liver enzymes and necroinflammatory changes. These effects of arsenic were coupled with enhanced mitochondrial swelling, inhibition of cytochrome c oxidase, Ca(2+)-ATPase, a decrease in mitochondrial calcium content, changes in indices of hepatic mitochondrial oxidative stress and iNOS expression. Arsenic also increased hepatic caspase 3 activity and DNA fragmentation. All these apoptosis-related molecular changes caused by arsenic could be alleviated by supplementation with inorganic phosphate, which likely suggests a protective role of phosphate against arsenic-induced hepatotoxic changes. PMID:21787675

  17. Hepatoprotective effect of biherbal ethanolic extract against paracetamol-induced hepatic damage in albino rats

    PubMed Central

    Anantha, Krishna Chaitanya D.; Siva, Reddy Challa; Manohar, Reddy A.

    2012-01-01

    Aim: The combined hepatoprotective effect of Bi-herbal ethanolic extract (BHEE) was evaluated against paracetamol induced hepatic damage in albino rats. Materials and Methods: Liver function tests and biochemical parameters were estimated using standard kits. Livers were quickly removed and fixed in 10% formalin and subjected to histopathological studies. Results: Ethanolic extract from the leaves of Aerva lanata and leaves of Achyranthes aspera at a dose level of 200 mg/kg, 400mg/kg body weight was administered orally once for 3 days. Substantially elevated serum marker enzymes such as SGOT, SGPT, ALP, due to paracetamol treatment were restored towards normal. Biochemical parameters like total protein, total bilirubin, total cholesterol, triglycerides, and urea were also restored towards normal levels. In addition, BHEE significantly decreased the liver weight of paracetamol intoxicated rats. Silymarin at a dose level of 25 mg/kg used as a standard reference also exhibited significant hepatoprotective activity against paracetamol induced hepatotoxicity. Conclusion: The results of this study strongly indicate that BHEE has got a potent hepatoprotective action against paracetamol induced hepatic damage in rats. PMID:23326091

  18. Failure of carnitine in improving hepatic nitrogen content in alcoholic and non‐alcoholic malnourished rats

    PubMed Central

    Rodrigues, Luciana P; Portari, Guilherme Vannucchi; Padovan, Gilberto João; Jordão, Alceu Afonso; Suen, Vivian M M; Sergio Marchini, Julio

    2010-01-01

    AIMS: To investigate the effect of carnitine supplementation on alcoholic malnourished rats' hepatic nitrogen content. METHODS: Malnourished rats, on 50% protein‐calorie restriction with free access to water (malnutrition group) and malnourished rats under the same conditions with free access to a 20% alcohol/water solution (alcohol group) were studied. After the undernourishment period (4 weeks with or without alcohol), both groups were randomly divided into two subgroups, one of them nutritionally recovered for 28 days with free access to a normal diet and water (recovery groups) and the other re‐fed with free access to diet and water plus carnitine (0.1 g/g body weight/day by gavage) (carnitine groups). No alcohol intake was allowed during the recovery period. RESULTS: The results showed: i) no difference between the alcohol/no alcohol groups, with or without carnitine, regarding body weight gain, diet consumption, urinary nitrogen excretion, plasma free fatty acids, lysine, methionine, and glycine. ii) Liver nitrogen content was highest in the carnitine recovery non‐alcoholic group (from 1.7 to 3.3 g/100 g, P<0.05) and lowest in alcoholic animals (about 1.5 g/100g). iii) Hepatic fat content (∼10 g/100 g, P>.05) was highest in the alcoholic animals. CONCLUSION: Carnitine supplementation did not induce better nutritional recovery. PMID:21049216

  19. Effect of body temperature on brain edema and encephalopathy in the rat after hepatic devascularization.

    PubMed

    Traber, P; DalCanto, M; Ganger, D; Blei, A T

    1989-03-01

    Brain edema is a fatal complication of fulminant hepatic failure and its pathogenesis remains unclear. To determine its presence in a model of ischemic hepatic failure, rats were subjected to a portacaval anastomosis followed by hepatic artery ligation. Brain water was measured using the sensitive gravimetric method. Preliminary studies revealed marked hypothermia in devascularized animals kept at room temperature (26.9 degrees +/- 2.8 degrees C). An additional group of devascularized rats was kept in an incubator. As expected for hypothermia, such animals had a lower arterial pressure and heart rate; the duration of encephalopathy was markedly prolonged. Water content of the cortical gray matter was only increased in normothermic devascularized rats: 80.14% +/- 0.31%, normal; 80.06% +/- 0.22%, portacaval shunt only; 80.42% +/- 0.26%, devascularized at room temperature; 81.29% +/- 0.38%, devascularized at controlled temperature (p less than 0.001). Such differences could not be detected using the dry-weight technique in whole cerebral hemispheres. Astrocyte changes in the cortical gray matter were noted in both edematous and nonedematous devascularized groups, coupled with the presence of vesicles containing horseradish peroxidase in the endothelial capillary cell. This suggests that in this model, brain edema may be due to both a cytotoxic mechanism and changes in the permeability of the blood-brain barrier. Future studies with this widely used model will require strict control of temperature to allow interpretation of experimental results. A therapeutic role for hypothermia in the management of brain edema deserves further attention. PMID:2914649

  20. Pathogenesis of hepatic septal fibrosis associated with Capillaria hepatica infection of rats.

    PubMed

    Santos, A B; Tolentino, M; Andrade, Z A

    2001-01-01

    Septal fibrosis is a common form of hepatic fibrosis, but its etiology and pathogenesis are poorly understood. Rats infected with the helminth Capillaria hepatica constitute a good experimental model of such fibrosis. To investigate the pathogenetic contribution of the several parasitic factors involved, the following procedures were performed in rats: a) regarding the role of eggs, these were isolated and injected either into the peritoneal cavity or directly into the liver parenchyma; b) for worms alone, 15-day-old infection was treated with mebendazole, killing the parasites before oviposition started; c) for both eggs and worms, rats at the 30th day of infection were treated with either mebendazole or ivermectin. Eggs only originated focal fibrosis from cicatricial granulomas, but no septal fibrosis. Worms alone induced a mild degree of perifocal septal fibrosis. Systematized septal fibrosis of the liver, similar to that observed in the infected controls, occurred only in the rats treated with mebendazole or ivermectin, with dead worms and immature eggs in their livers. Thus, future search for fibrogenic factors associated with C. hepatica infection in rats should consider lesions with both eggs and worms.

  1. Gum Arabic extracts protect against hepatic oxidative stress in alloxan induced diabetes in rats.

    PubMed

    Ahmed, Abdelkareem A; Fedail, Jaafar S; Musa, Hassan H; Kamboh, Asghar Ali; Sifaldin, Amal Z; Musa, Taha H

    2015-12-01

    Gum Arabic (GA) from Acacia seyal and Acacia senegal is a branched-chain polysaccharide which has strong antioxidant properties, and has been used to reduce the experimental toxicity. Yet, the effects of GA on oxidative stress in type I diabetic rats have not been reported. The aim of the study was to investigate the effects of GA on oxidative stress in Alloxan induced diabetes in rats. The rats were divided into 3 groups (n=20 of each): control group, diabetic group injected with allaoxan, and diabetic group given 15% GA in drinking water for 8 weeks. Oxidative damage to liver tissue was evaluated by measurement of key hepatic enzymes, lipid peroxidation, antioxidant enzymes and expression of oxidative stress genes. Activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were significantly (P<0.05) increased in GA group compared to diabetic and control groups. Treatment of GA decreased liver malondialdehyde (MDA), and increased glutathione (GSH). In addition, GA was significantly (P<0.05) reduced the activities of key liver enzymes, including alanine transaminase (ALT) and aspartate transaminase (AST). SOD, GPx and heat shock protein 70 (HSP70) mRNA were significantly increased in GA group compared to control and diabetic groups. Liver of all diabetic rats showed marked degeneration whereas slight degeneration was observed in GA treated rats compared to control. The results suggest that GA may protect liver by modulating the expression of oxidative stress genes, and thus can improve antioxidant status. PMID:26321624

  2. Antioxidant effects of proanthocyanidin from grape seed on hepatic tissue injury in diabetic rats

    PubMed Central

    Mansouri, Esrafil; Khorsandi, Layasadat; Abedi, Hassan Ali

    2014-01-01

    Objective(s): Diabetes plays an important role in the induction of the liver injury. Grape seed proanthocyanidin (GSP) have a wide range of medicinal properties against oxidative stress. In this study we evaluated antioxidant effects of GSP on liver in streptozotocin-induced diabetic rats. Materials and Methods: Thirty male Sprague–Dawley rats were divided into three groups: control, untreated diabetic and diabetic rats treated with GSP. Diabetes was induced in rats by intraperitoneal injection of streptozotocin (50 mg/kg). GSP were administered via oral gavage (200 mg/kg) for 4 weeks. Results: GSP produced significant hepatoprotective effects by decreasing activities of serum aminotransferases and alkaline phosphatase, and decreasing liver malondialdehyde and bilirubin (P<0.05) levels. It increased liver superoxide dismutase, catalase and glutathione peroxidase activities and albumin level (P<0.05). Administration of GSP significantly ameliorated structural changes induced in liver of diabetic rats. Conclusion: GSP have protective effects against hepatic tissue injury due to antioxidant properties. PMID:25140209

  3. A comparison of the inductive effects of phenobarbital, methaqualone, and methyprylon on hepatic mixed function oxidase enzymes in the rat.

    PubMed

    Reinke, L A; O'Connor, M F; Piepho, R W; Stohs, S J

    1975-05-01

    The effects of equal doses of three sedative-hypnotics, phenobarbital, methaqualone, and methyprylon, on the hepatic mixed function oxidase enzymes of the rat were investigated and compared. After 5 days of pretreatment, phenobarbital and methyprylon significantly increased aminopyrine demethylation, aniline hydroxylation, and cytochrome P-450 content in hepatic microsomes. Methaqualone pretreatment only increased hepatic aminopyrine demethylase activity and wet liver weights. After 29 days of pretreatment, phenobarbital significantly increases aminopyrine demethylase, aniline hydroxylase activity, liver weight and cytochrome P-450 content. Methaqualone only produced a significant increase in wet liver weight.

  4. Matrine induces the hepatic differentiation of WB-F344 rat hepatic progenitor cells and inhibits Jagged 1/HES1 signaling.

    PubMed

    Yang, Zhiyun; Wang, Li; Wang, Xianbo

    2016-10-01

    Matrine is a Chinese medicine, which is widely utilized for the attenuation of liver injuries and promotion of liver regeneration. It was previously observed that the in vivo administration of matrine promoted oval cell‑mediated liver regeneration in a rat model, suggesting that this compound may affect the differentiation of hepatic progenitor cells. The present study aimed to determine the mechanisms underlying this observation and to investigate the effect of matrine on the differentiation of the WB‑F344 rat hepatic progenitor cell line. Matrine was administered to rats, and rat serum was collected. WB‑F344 cells were cultured in the presence or absence of the rat serum for 24‑72 h, and the effects on cell viability and proliferation were assessed using acridine orange/propidium iodide staining and a 3‑(4,5‑dimethylthiazol‑2‑yl) ‑2,5‑diphenyltetrazolium bromide assay. The expression of albumin (ALB, a hepatocyte marker) and the notch signaling pathway ligand, Jagged 1, were assessed using immunohistochemistry and western blotting, and the mRNA transcription of ALB, Jagged 1 and hairy and enhancer of split‑1 (HES1, another notch signaling ligand) were measured using reverse transcription‑polymerase chain reaction analysis. The results showed that proliferation of the WB‑F344 cells was inhibited by matrine serum in a concentration‑ and time‑dependent manner. Matrine serum downregulated Jagged 1 and HES1, and upregulated ALB, indicating the induction of WB‑F344 cell differentiation. The effects of matrine serum were reversed by supplementing the culture medium with 0.1 mol/l parathyroid hormone, a Notch signaling pathway activator. In conclusion, matrine induced hepatic differentiation of the hepatic progenitor cells, likely by inhibiting the Jagged 1/HES1 signaling pathway.

  5. Age-dependent decrease in the hepatic uptake and biliary excretion of ouabain in rats.

    PubMed

    Ohta, M; Kanai, S; Sato, Y; Kitani, K

    1988-03-01

    The biliary excretion of i.v. injected ouabain was examined in male and female Wistar-derived rats in relation to age. The hepatic uptake velocity for ouabain was also determined in isolated hepatocyte preparations obtained from male rats of various ages. Biliary recovery values of ouabain (percent of the dose) were fairly comparable for young male and female rats (3-4 month old). Recovery progressively decreased with age, the first 10-min recoveries at 24 months being about one-third those of respective young values in both sexes. A significant linear relation was demonstrated between the first 10-min recovery (Y, percent of the dose) and rat age (X, month), yielding the relations of Y = 17.75-0.43X for males and Y = 18.99-0.43X for females respectively. Similarly, the initial uptake velocity (Y, nmol/mg/min) for ouabain decreased in a linear fashion with age (X, month), yielding a significant negative correlation (Y = 0.704-0.0021X, r = -0.839, P less than 0.005, N = 21) at an ouabain concentration of 8 microM. Kinetic studies using non-linear regression analysis revealed a significantly lower Vmax value (0.533 +/- 0.041 nmol/mg/min) in old (24-29 months) rats compared to the young (4-4.5 months) value (1.193 +/- 0.105 nmol per mg/min, P less than 0.05), while the affinity constant (Km, microM) did not differ significantly between young and old animals (203.12 +/- 25.42 microM in young rats vs 283.68 +/- 28.90 microM in old rats, mean +/- SE, 0.05 less than P less than 0.1). The results of the present study suggest that the age-dependent decrease in the biliary recovery of i.v. injected ouabain in rats can be largely explained by the decrease with age in the hepatic uptake of ouabain. Furthermore, the results provide further support for our previous thesis that the decrease in the lateral mobility of hepatocyte plasma membrane proteins, as revealed by the fluorescence recovery after photobleaching technique, may play a significant role in the age

  6. Stereoselective hepatic disposition of a diastereomeric pair of alphavbeta3 antagonists in rat.

    PubMed

    Prueksaritanont, T; Xu, X; Deluna, P; Yamazaki, M; Lin, J H

    2003-11-01

    1. The study investigated mechanisms underlying the stereoselective hepatic disposition observed in rats of a zwitterionic diastereomeric pair ((3S)-3-[(3R or 3S)-2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]pyrrolidin-1-yl]-3-quinolin-3-ylpropanoic acid) with different lipophilicities. 2. In a recirculating isolated rat liver system, the more hydrophilic diastereomer II possessed biliary clearance, CLb, and bile-to-liver concentration ratio higher (about 10-30-fold) than the lipophilic zwitterion I, whereas both I and II exhibited comparably high concentration ratios between liver and perfusate. Although MK-571, a known multidrug resistance protein (MRP) inhibitor, significantly inhibited the CLb of both compounds, it did not inhibit their canalicular transport, as evident by unchanged concentration ratios between bile and liver of either I or II. 3. Following an intravenous infusion of I or II to Sprague-Dawley rats, the biliary clearance calculated either based on plasma (CL(b,p)) or liver concentration (CL(b,l)), of II was much higher than that of I (about 5-50-fold). In rats lacking multidrug resistance protein 2 (Mrp2) (Eisai hyperbilirubinemic rat, EHBR), the biliary excretion rate and CL(b,p) of II were also higher than the corresponding values for I. However, both CL(b,p) or CL(b,l) of either I or II were not reduced in EHBR, as compared with control SD rats. 4. In the in vitro rat canalicular membrane vesicle study, I and II exhibited no differences in their inhibitory effect on the Mrp2 mediated ATP-dependent [3H]DNP-SG initial uptake (no inhibition at 10 microM and only about 40% inhibition at 100 microM). 5. Collectively, these results suggested that (1) the difference in the hepatic disposition between the two isomers was due primarily to the difference in their transport mechanism across the canalicular membrane and (2) Mrp2 did not play a major role in the observed differences in the biliary excretion of the diastereomers I and II in

  7. Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

    PubMed

    Senoo, Haruki; Mezaki, Yoshihiro; Morii, Mayako; Hebiguchi, Taku; Miura, Mitsutaka; Imai, Katsuyuki

    2013-11-01

    Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A. PMID:23765517

  8. Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

    PubMed

    Senoo, Haruki; Mezaki, Yoshihiro; Morii, Mayako; Hebiguchi, Taku; Miura, Mitsutaka; Imai, Katsuyuki

    2013-11-01

    Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A.

  9. Suppressive Effect of High Hydrogen Generating High Amylose Cornstarch on Subacute Hepatic Ischemia-reperfusion Injury in Rats

    PubMed Central

    TANABE, Hiroki; SASAKI, Yumi; YAMAMOTO, Tatsuro; KIRIYAMA, Shuhachi; NISHIMURA, Naomichi

    2012-01-01

    We examined whether feeding high hydrogen generating resistant starch could suppress subacute hepatic ischemia-reperfusion injury. Rats were fed a control diet with or without 20% high amylose cornstarch (HAS) supplementation for 14 days. On day 12, rats were subject to ischemia-reperfusion treatment. Portal hydrogen concentration was higher in the HAS group compared with the control group. Increased plasma alanine and aspartate aminotransferase activities due to ischemia-reperfusion treatment tended to decrease, and a significant reduction was observed by HAS feeding when compared with the control group. In conclusion, HAS, which enhances hydrogen generation in the hindgut, alleviated subacute hepatic ischemia-reperfusion injury. PMID:24936356

  10. Hepatic morphological changes and oxidative stress in chronic streptozotocin-diabetic rats.

    PubMed

    Evelson, Pablo; Susemihl, Carlota; Villarreal, Irene; Llesuy, Susana; Rodríguez, Ricardo; Peredo, Horacio; Lemberg, Abraham; Perazzo, Juan; Filinger, Ester

    2005-01-01

    Oxidative stress (OS) is a biological entity quoted as responsible for several pathologies including diabetes. Diabetes mellitus (DM) has been also associated to human cirrhosis. The present work was designed to study the occurrence of OS as well as morphologic alterations in rat livers following induction of DM. Two groups of rats were used: Control and Diabetic. DM was induced in the second group by streptozotocin (STZ) in a single dose of 60 mg/kg, injected i.p. The occurrence of OS was determined in liver homogenates by measuring the hydroperoxide-initiated chemiluminescence and the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Liver sinusoids were morphometrically analyzed. In conclusion, livers from the diabetic group did not show evidence of the occurrence of OS, as it would be expected, but dilation of hepatic sinusoids was documented and it was significantly different from control group. PMID:16010244

  11. Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver.

    PubMed

    Minguetti-Câmara, V C; Constantin, J; Suzuki-Kemmelmeier, F; Ishii-Iwamoto, E L; Bracht, A

    1998-02-01

    The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 micromol min[-1] g liver[-1]; 20-200 microM) in each of the four experimental protocols. The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when

  12. Protective Effects of Green Tea Extract against Hepatic Tissue Injury in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Abolfathi, Ali Akbar; Mohajeri, Daryoush; Rezaie, Ali; Nazeri, Mehrdad

    2012-01-01

    Although diabetic hepatopathy is potentially less common, it may be appropriate for addition to the list of target organ conditions related to diabetes. This study was designed to evaluate the hepatoprotective properties of green tea extract (GTE) in STZ-induced diabetes in rats. Wistar rats were made diabetic through single injection of STZ (75 mg/kg i.p.). The rats were randomly divided into four groups of 10 animals each: Group 1, healthy control; Group 2, nondiabetics treated with GTE administered orally (1.5%, w/v); Group 3, diabetics; Group 4, diabetics treated with GTE (1.5%, w/v) for 8 weeks. Serum biomarkers were assessed to determine hepatic injury. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were measured to assess free radical activity in the liver tissue. Hepatic antioxidant activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) were also determined. The biochemical findings were matched with histopathological verifications. Liver MDA content and serum levels of ALT, AST, ALP, and bilirubin in Group 3 significantly increased compared to Group 1 (P < 0.05) and significantly decreased in Group 4 compared to Group 3 (P < 0.05). Serum albumin level and GSH, SOD, CAT, and GSH-Px contents of the liver in Group 3 were significantly decreased compared to Group 1 (P < 0.05) and were significantly increased in Group 4 compared to Group 3 (P < 0.05). Histopathologically, the changes were in the same direction with biochemical findings. This study proved the hepatoprotective activity of GTE in experimentally induced diabetic rats. PMID:22956978

  13. Protective effect of thymoquinone against lead-induced hepatic toxicity in rats.

    PubMed

    Mabrouk, Aymen; Bel Hadj Salah, Imen; Chaieb, Wafa; Ben Cheikh, Hassen

    2016-06-01

    Lead (Pb) intoxication is a worldwide health problem which frequently affects the liver. This study was carried out to investigate the potential protective effect of thymoquinone (TQ), the major active ingredient of volatile oil of Nigella sativa seeds, against Pb-induced liver damage. Adult male rats were randomized into four groups: Control group received no treatment, Pb group was exposed to 2000 ppm Pb acetate in drinking water, Pb-TQ group was cotreated with Pb plus TQ (5 mg/kg/day, per orally), and TQ group receiving only TQ. All treatments were applied for 5 weeks. Results indicated that Pb exposure increased hepatic Pb content, damaged hepatic histological structure (necrotic foci, hepatic strands disorganization, hypertrophied hepatocytes, cytoplasmic vacuolization, cytoplasmic loss, chromatin condensation, mononuclear cell infiltration, congestion, centrilobular swelling), and changed liver function investigated by plasma biochemical parameters (AST, ALT, ALP, γ-GT, LDH). Pb treatment also decreased total antioxidant status level and increased lipid peroxidation in the liver. Supplementation with TQ remarkably improved the Pb-induced adverse effects without significantly reducing the metal accumulation in the liver. In conclusion, our results indicate, for the first time, a protective effect of TQ against Pb-induced hepatotoxicity and suggest that this component might be clinically useful in Pb intoxication. PMID:26971798