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Sample records for rat hepatic stellate

  1. Inhibitory Effect of Tanshinone IIA on Rat Hepatic Stellate Cells

    PubMed Central

    Liu, Ya-Wei; Huang, Yi-Tsau

    2014-01-01

    Background Anti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs) is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C19H18O3, Tan IIA) is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation. Materials and Methods The cell line of rat hepatic stellate cells (HSC-T6) was stimulated with lipopolysaccharide (LPS) (100 ng/ml). Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM), then induced by LPS (100 ng/ml). NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38). Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells. Results All concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells. Conclusion Our results demonstrated that Tan IIA decreased LPS-induced HSC activation. PMID:25076488

  2. Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

    PubMed Central

    Shimizu, I; Mizobuchi, Y; Yasuda, M; Shiba, M; Ma, Y; Horie, T; Liu, F; Ito, S

    1999-01-01

    Background—Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. 
Aims—To examine the inhibitory effect of oestradiol on stellate cell activation. 
Methods—In vivo, hepatic fibrosis was induced in rats by dimethylnitrosamine or pig serum. In vitro, rat stellate cells were activated by contact with plastic dishes resulting in their transformation into myofibroblast-like cells. 
Results—In the dimethylnitrosamine and pig serum models, treatment with oestradiol at gestation related doses resulted in a dose dependent suppression of hepatic fibrosis with restored content of hepatic retinyl palmitate, reduced collagen content, lower areas of stellate cells which express α smooth muscle actin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation. 
Conclusion—The antifibrogenic role of oestradiol in the liver may contribute to the sex associated differences in the progression from hepatic fibrosis to cirrhosis. 

 Keywords: hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate PMID:9862839

  3. Aloe emodin suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.

    PubMed

    Woo, Sun Wook; Nan, Ji-Xing; Lee, Sung Hee; Park, Eun-Jeon; Zhao, Yu Zhe; Sohn, Dong Hwan

    2002-04-01

    We have studied the inhibitory effect of aloe emodin on hepatic stellate cells activation and proliferation, as these cells play a key role in the pathogenesis of hepatic fibrosis. Rat hepatic stellate cells were activated by contact with plastic dishes, resulting in their transformation into myofibroblast-like cells. Primary hepatic stellate cells were exposed to aloe emodin (1-10 microg/ml). Possible cytotoxic effects were measured on stellate cells and hepatocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of aloe emodin on production of type I collagen and smooth muscle cell alpha-actin were examined at the same concentration, by quantitative immunoprecipitation. Antiproliferative effects were examined by bromodeoxyuridine incorporation. Aloe emodin at 10 microg/ml restored the morphological changes characteristic of activated primary stellate cells, reduced DNA synthesis to 95% of control hepatic stellate cells at 10 microg/ml without affecting cell viability, and inhibited type I collagen production and smooth muscle alpha-actin expression by 86.77% and 99%, respectively, which suggest that aloe emodin is a potent inhibitor of stellate cell transformation.

  4. 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

  5. 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.

  6. 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

  7. All-trans and 9-cis retinoic acid alter rat hepatic stellate cell phenotype differentially

    PubMed Central

    Hellemans, K; Grinko, I; Rombouts, K; Schuppan, D; Geerts, A

    1999-01-01

    BACKGROUND—Hepatic stellate cells exert specific functions in the liver: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids.
AIMS—To examine the influence of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9RA) on extracellular matrix production and proliferation of activated hepatic stellate cells.
METHODS—Cells were isolated using collagenase/pronase, purified by centrifugation in nycodenz, and cultured for two weeks. At this time point the cells exhibited the activated phenotype. Cells were exposed to various concentrations of ATRA and 9RA. The expression of procollagens I, III, and IV, of fibronectin and of laminin were analysed by immunoprecipitation and northern hybridisation.
RESULTS—ATRA exerted a significant inhibitory effect on the synthesis of procollagens type I, III, and IV, fibronectin, and laminin, but did not influence stellate cell proliferation, whereas 9RA showed a clear but late effect on proliferation. 9RA increased procollagen I mRNA 1.9-fold, but did not affect the expression of other matrix proteins.
CONCLUSION—Results showed that ATRA and 9RA exert different, often contrary effects on activated stellate cells. These observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or in animals subjected to fibrogenic stimuli.


Keywords: hepatic stellate cells; retinoic acid; extracellular matrix proteins; proliferation PMID:10369717

  8. 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.

  9. Antifibrotic effects of triptolide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

    PubMed

    Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

    2011-07-01

    Triptolide (C₃₈H₄₂O₆N₂, TP, a diterpene triepoxide derived from Tripterygium wilfordii Hook F.), is a potent immunosuppresive and antiinflammatory agent. The present study investigated whether TP exerted antihepatofibrotic effects in vitro and in vivo. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or transforming growth factor (TGF)-β1. The inhibitory effects of TP on the nuclear factor-κB (NFκB) signaling cascade and fibrosis markers, including α-smooth muscle actin (α-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats. The rats were randomly assigned to one of three groups: control rats, DMN rats receiving vehicle only and DMN rats receiving TP (20 μg/kg). Treatment was given by gavage twice daily for 3 weeks starting 1 week after the start of DMN administration. TP (5-100 nM) concentration-dependently inhibited the NFκB transcriptional activity induced by TNF-α, lipopolysaccharide and phorbol 12-myristate 13-acetate in HSC-T6 cells. In addition, TP also suppressed TNF-α and TGF-β1-induced collagen deposition and α-SMA secretion in HSC-T6 cells. In vivo, TP treatment significantly reduced hepatic fibrosis scores, collagen contents, IL-6 and TNF-α levels, and the number of α-SMA and NFκB-positive cells in DMN rats. The results showed that TP exerted antifibrotic effects in both HSC-T6 cells and DMN rats.

  10. Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

    PubMed

    Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

    2006-05-01

    Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition

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

    PubMed

    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.

  12. Apoptosis of rat hepatic stellate cells induced by diallyl trisulfide and proteomics profiling in vitro.

    PubMed

    Zhang, Yajie; Zhou, Xiaoming; Xu, Lipeng; Wang, Lulu; Liu, Jinling; Ye, Jing; Qiu, Pengxin; Liu, Qinghua

    2016-11-18

    Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 μg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive - propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.

  13. The Transcriptomic Response of Rat Hepatic Stellate Cells to Endotoxin: Implications for Hepatic Inflammation and Immune Regulation

    PubMed Central

    Tandon, Ashish; Gandhi, Chandrashekhar R.

    2013-01-01

    With their location in the perisinusoidal space of Disse, hepatic stellate cells (HSCs) communicate with all of the liver cell types both by physical association (cell body as well as cytosolic processes penetrating into sinusoids through the endothelial fenestrations) and by producing several cytokines and chemokines. Bacterial lipopolysaccharide (LPS), circulating levels of which are elevated in liver diseases and transplantation, stimulates HSCs to produce increased amounts of cytokines and chemokines. Although recent research provides strong evidence for the role of HSCs in hepatic inflammation and immune regulation, the number of HSC-elaborated inflammatory and immune regulatory molecules may be much greater then known at the present time. Here we report time-dependent changes in the gene expression profile of inflammatory and immune-regulatory molecules in LPS-stimulated rat HSCs, and their validation by biochemical analyses. LPS strongly up-regulated LPS-response elements (TLR2 and TLR7) but did not affect TLR4 and down-regulated TLR9. LPS also up-regulated genes in the MAPK, NFκB, STAT, SOCS, IRAK and interferon signaling pathways, numerous CC and CXC chemokines and IL17F. Interestingly, LPS modulated genes related to TGFβ and HSC activation in a manner that would limit their activation and fibrogenic activity. The data indicate that LPS-stimulated HSCs become a major cell type in regulating hepatic inflammatory and immunological responses by altering expression of numerous relevant genes, and thus play a prominent role in hepatic pathophysiology including liver diseases and transplantation. PMID:24349206

  14. Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells.

    PubMed

    Wang, Zhi-Ming; Zhou, Le-Yuan; Liu, Bin-Bin; Jia, Qin-An; Dong, Yin-Ying; Xia, Yun-Hong; Ye, Sheng-Long

    2014-10-01

    The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (P<0.001), migration rate and the number of invasive HCC cells (P=0.001). Co-injection of HCC cells and activated HSCs into rats significantly increased the weight of the resulting HCC tumors (P<0.01). The paracrine activity of activated HSCs markedly altered the gene expression profile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

  15. 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

  16. Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity.

    PubMed

    El-Mansy, A A; Mazroa, S A; Hamed, W S; Yaseen, A H; El-Mohandes, E A

    2016-01-01

    The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.

  17. 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.

  18. Proliferative and oxidative response of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats exposed to ketogenic diet.

    PubMed

    Wójcik, M; Wessely-Szponder, J; Kosior-Korzecka, U

    2014-01-01

    Ketogenic diet (KD) is considered in the context of its anti-epileptic effects, but its influence on liver dysfunction has not been elucidated yet. The study was aimed to investigate the activity of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats fed with KD, in respect of NO and superoxide generation by these cells as well as their proliferative activity in vitro. We also sought to characterize the plasma FFA profiles in control and ketogenic rats. Hep and HSC were isolated by the collagenase perfusion method and separated by the Percoll gradient centrifugation. After the 4th, 8th and 12th day of incubation, the media were collected for further analysis. NO generation increased within the time of incubation both in Hep and HSC isolated from KD-rats. In HSC group NO production raised significantly from 2.65 ± 0.07 μM/10(6) cells on 4th day of incubation to 5.49 ± 1.2 μM/10(6) cells on 12th day of incubation. In respect to O2⁻· generation experimental Hep and HSC provide considerably higher quantities of this free radical until 12th day of incubation (2.5 ± 0.07 and 3.2 ± 0.3 nM/10(6) cells, respectively). Although KD exerts anti-proliferative effect on hepatocytes, in respect to HSC it intensifies their proliferative activity. Furthermore, as we estimated on the basis of NO and O2⁻. generation both Hep and HSC exposed to KD are the source of free radicals.

  19. Hsian-tsao (Mesona procumbens Heml.) prevents against rat liver fibrosis induced by CCl(4) via inhibition of hepatic stellate cells activation.

    PubMed

    Shyu, Ming-Huan; Kao, Tzu-Chien; Yen, Gow-Chin

    2008-12-01

    In this study, the protective effect of extract of Hsian-tsao (Mesona procumbens) (EHT) against liver fibrogenesis in carbon tetrachloride (CCl(4))-injured rats was evaluated. The inhibitory effect of oleanolic acid (OA) and ursolic acid (UA), which are the active compounds in EHT, on the activation of hepatic stellate cells (HSC) was also determined. The results showed that EHT at a dosage of 1.2g/kg of b.w. significantly reduced the liver injury induced by CCl(4) in rats. It also decreased the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the deposition of collagen in the liver. Oral administration of EHT reduced the levels of alpha-smooth muscle actin (alpha-SMA) and the activity of metalloproteinases (MMPs) in rats injured by treatment with CCl(4). In addition, we performed experiments with the rat hepatic stellate cell line HSC-T6 in which we induced the expression of MMP-2 and alpha-SMA with phorbol-12-myristate-13-acetate (PMA). Treating these cells with OA (20microM) or UA (10microM) caused a decrease in the levels of both proteins. Taken together, our data indicate that EHT can efficiently inhibit CCl(4)-induced liver fibrosis in rats. EHT may therefore be a useful functional food for preventing liver fibrosis.

  20. Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells

    PubMed Central

    Chen, Ying-Hua; Lan, Tian; Li, Jing; Qiu, Chun-Hui; Wu, Teng; Gou, Hong-Ju; Lu, Min-Qiang

    2012-01-01

    AIM: To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis. METHODS: Male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 d and were treated with Gardenia jasminoides by gavage. The effects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells (LX-2) in vitro. RESULTS: Treatment with Gardenia jasminoides decreased serum alanine aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 146.6 ± 15 U/L vs 77 ± 6.5 U/L, P = 0.0007) and aspartate aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 188 ± 35.2 U/L vs 128 ± 19 U/L, P = 0.005) as well as hydroxyproline (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue, P = 0.004) after BDL. Furthermore, Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1 (TGF-β1), collagen type I (Col I) and α-smooth muscle actin (α-SMA). Gardenia jasminoides significantly suppressed the upregulation of TGF-β1, Col I and α-SMA in LX-2 exposed to recombinant TGF-β1. Moreover, Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent. PMID:23326120

  1. Cadmium induces alpha(1)collagen (I) and metallothionein II gene and alters the antioxidant system in rat hepatic stellate cells.

    PubMed

    del Carmen, Escobar Ma; Souza, Verónica; Bucio, Leticia; Hernández, Elizabeth; Damián-Matsumura, Pablo; Zaga, Verónica; Gutiérrez-Ruiz, Ma Concepción

    2002-01-15

    The mechanism of cadmium-mediated hepatotoxicity has been the subject of numerous investigations, principally in hepatocytes. Although, some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process in parenchymal cells. However, there is no information about the effect of cadmium in other hepatic cell types, such as stellate cells (fat storing cells, Ito cells, perisinusoidal cells, parasinusoidal cells, lipocytes). Hepatic stellate cells (HSC) express a quiescent phenotype in a healthy liver and acquire an activated phenotype in liver injury. These cells play an important role in the fibrogenic process. The objective of this study was to investigate the effect of a 24 h treatment of low Cd concentrations in glutathione content, lipid peroxidation damage, cytosolic free Ca, antioxidant enzyme activities: glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase along with the capacity of this heavy metal to induce metallothionein II and alpha(1)collagen (I) in an hepatic stellate cell line (CFSC-2G). Cd-treated cells increased lipid peroxidation and the content of cytosolic free calcium, decreased glutathione content and superoxide dismutase, glutathione peroxidase and catalase activity. Cd was able to induce the expression of the metallothionein II and alpha(1)collagen (I) gene, that was not described in this cell type. Cadmium may act as a pro-fibrogenic agent in the liver probably by inducing oxidative damage by enhancing lipid peroxidation and altering the antioxidant system of the cells. Although, the exact role metallothionein induction plays in this process is unknown, it probably, provides a cytosolic pool of potential binding sites to sequester ionic Cd, thereby decreasing its toxicity.

  2. (+)-Catechin attenuates activation of hepatic stellate cells.

    PubMed

    Bragança de Moraes, Cristina Machado; Bitencourt, Shanna; de Mesquita, Fernanda Cristina; Mello, Denizar; de Oliveira, Leticia Paranhos; da Silva, Gabriela Viegas; Lorini, Vinicius; Caberlon, Eduardo; de Souza Basso, Bruno; Schmid, Julia; Ferreira, Gabriela Acevedo; de Oliveira, Jarbas Rodrigues

    2014-04-01

    (+)-Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti-inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)-catechin on hepatic stellate cells. (+)-Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator-activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro-inflammatory cytokines were not influenced by (+)-catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)-catechin can reduce hepatic stellate cell activation.

  3. 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.

  4. Cluster of differentiation 147 is a key molecule during hepatocellular carcinoma cell-hepatic stellate cell cross-talk in the rat liver.

    PubMed

    Ma, Tianyou; Wang, Zhilun; Yang, Zhantian; Chen, Jinghong

    2015-07-01

    The cross-talk between hepatocellular carcinoma (HCC) cells and activated hepatic stellate cells (HSCs) is considered to be important for modulating the biological behavior of tumor cells. However, the molecular links between inflammation and cancer in the activation of HSCs remain to be elucidated. The present study demonstrated that cluster of differentiation (CD)147 is a key molecule involved in the interaction between HCC cells and HSCs. The effects of conditioned medium from human HCC cells on the activation of the human HSC line, LX-2, were assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blotting, RT-qPCR and gelatin zymography were also used to investigate the effects of CD147 on the activation of LX-2. The expression levels of α-smooth muscle actin (α-SMA) and CD147 were assessed in a co-culture system of LX-2 and FHCC-98 cells by immunofluorescence staining and immunoblotting. In hepatic tissues from a rat model of fibrosis, immunohistochemistry and immunoblotting were performed to detect the expression levels of α-SMA and CD147. Tumor-conditioned medium and CD147 promoted cell proliferation, activated LX-2 cells, increased the expression levels of α-SMA, collagen I and tissue inhibitor of metalloproteinase-1 (TIMP-1), and increased the secretion of matrix metalloproteinase (MMP)-2. The HSCs, which were induced in the co-culture system of HCC cells and HSCs exhibited marked expression levels of CD147. In the hepatic tissue of rat models of fibrosis induced by CCl4, marked expression levels of CD147 were observed in the activated HSCs. Therefore, CD147 promoted the activation of HSCs and was a key molecule during HCC cell-HSC cross-talk in the rat liver.

  5. Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

    PubMed

    Marcos, Ricardo; Correia-Gomes, Carla

    2016-12-01

    Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

  6. 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.

  7. 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.

  8. A Novel Matrine Derivative WM130 Inhibits Activation of Hepatic Stellate Cells and Attenuates Dimethylnitrosamine-Induced Liver Fibrosis in Rats.

    PubMed

    Xu, Yang; Peng, Zhangxiao; Ji, Weidan; Li, Xiang; Lin, Xuejing; Qian, Liqiang; Li, Xiaoya; Chai, Xiaoyun; Wu, Qiuye; Gao, Quangen; Su, Changqing

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is a critical event in process of hepatic fibrogenesis and cirrhosis. Matrine, the active ingredient of Sophora, had been used for clinical treatment of acute/chronic liver disease. However, its potency was low. We prepared a high potency and low toxicity matrine derivate, WM130 (C30N4H40SO5F), which exhibited better pharmacological activities on antihepatic fibrosis. This study demonstrated that WM130 results in a decreased proliferative activity of HSC-T6 cells, with the half inhibitory concentration (IC50) of 68 μM. WM130 can inhibit the migration and induce apoptosis in HSC-T6 cells at both concentrations of 68 μM (IC50) and 34 μM (half IC50). The expression of α-SMA, Collagen I, Collagen III, and TGF-β1 could be downregulated, and the protein phosphorylation levels of EGFR, AKT, ERK, Smad, and Raf (p-EGFR, p-AKT, p-ERK, p-Smad, and p-Raf) were also decreased by WM130. On the DMN-induced rat liver fibrosis model, WM130 can effectively reduce the TGF-β1, AKT, α-SMA, and p-ERK levels, decrease the extracellular matrix (ECM) formation, and inhibit rat liver fibrosis progression. In conclusion, this study demonstrated that WM130 can significantly inhibit the activation of HSC-T6 cells and block the rat liver fibrosis progression by inducing apoptosis, suppressing the deposition of ECM, and inhibiting TGF-β/Smad and Ras/ERK pathways.

  9. Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in rat hepatic stellate cells.

    PubMed

    Koide, Shigeki; Kobayashi, Yoshimasa; Oki, Yutaka; Nakamura, Hirotoshi

    2004-09-01

    Prostaglandin (PG) E2 inhibits hepatic stellate cell (HSC) mitogenesis. PGE-specific receptors are divided into four subtypes that are coupled either to Ca2+ mobilization (EP1 and EP3) or to the stimulation of adenyl cyclase (EP2 and EP4). The aims of the current study were to identify PGE receptor subtypes in cultured rat HSC and to examine which PGE receptor subtype(s) mediates the inhibitory effect of PGE2 on platelet-derived growth factor (PDGF)-stimulated proliferation. Reverse transcription-polymerase chain reaction analysis was performed to detect PGE receptor subtype mRNA expression. Cell proliferation was determined by measuring [3H]thymidine incorporation, and intracellular cyclic AMP was measured by radioimmunoassay. Cultured rat HSC expressed mRNAs for all four subtypes of PGE receptor. PGE2- and EP2-selective agonist produced dose-dependent inhibitory effects on PDGF-stimulated proliferation. Neither EP1-, EP3-, nor EP4-selective agonists showed any inhibitory effect. An adenylate cyclase inhibitor strongly blunted the inhibition of DNA synthesis elicited by PGE2 and the EP2 agonist. The EP2 agonist generated higher and more prolonged increases in intracellular cyclic AMP than the EP4 agonist. Activation of the PGE EP2 receptor has an antiproliferative effect in HSC that may be mediated by cyclic AMP-related signal transduction pathways.

  10. 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

  11. JNK1 and JNK2 regulate α-SMA in hepatic stellate cells during CCl4 -induced fibrosis in the rat liver.

    PubMed

    Hong, Il-Hwa; Park, Sang-Joon; Goo, Moon-Jung; Lee, Hye-Rim; Park, Jin-Kyu; Ki, Mi-Ran; Kim, Sang-Hyeob; Lee, Eun-Mi; Kim, Ah-Young; Jeong, Kyu-Shik

    2013-10-01

    Following liver injuries, hepatic stellate cells (HSCs) express α-SMA. Mitogen activated protein kinase (MAPK) signaling pathways mediate α-SMA expression in distinct cell types. However, the regulation of α-SMA expression by MAPKs in HSCs has been rarely studied. We aimed to study the role of MAPKs in the activation of HSCs during liver fibrosis. Liver fibrosis of rats was induced by carbon tetrachloride. HSC-T6 cells, murine embryonic fibroblasts, JNK1(-/-) and JNK2(-/-) cells were used for in vitro studies. Immunohistochemistry and immunoblot analysis were used. We have found that the expression of JNK and α-SMA co-localized in HSCs during liver fibrosis, but ERK and p38 expressed in macrophages. The expression of α-SMA was up-regulated by JNK1 and JNK2 in non-stress condition. Under TGF-β stimulation, however, the level α-SMA expression was increased by only JNK1, but not significantly changed by JNK2. We suggest that JNKs are responsible for α-SMA regulation, and especially JNK1 has a major role in up-regulation of α-SMA expression in HSCs under stress condition induced by TGF-β during liver fibrosis.

  12. Induction of apoptosis by tanshinone I via cytochrome c release in activated hepatic stellate cells.

    PubMed

    Kim, Ji Young; Kim, Kyoung Mi; Nan, Ji-Xing; Zhao, Yu Zhe; Park, Pil-Hoon; Lee, Sang Jun; Sohn, Dong Hwan

    2003-04-01

    Hepatic stellate cells play central roles in hepatic fibrosis. The therapeutic goal in hepatic fibrosis is to halt or reverse fibrosis. Apoptosis is suggested to eliminate activated hepatic stellate cells in fibrosis. Salvia miltiorrhiza is a traditional medicine used to improve blood circulation and treat chronic hepatitis and hepatic fibrosis. We investigated the effect of tanshinone I, an ingredient of Salvia miltiorrhiza, on the apoptotic death of rat hepatic stellate cells transformed by simian virus 40 (T-HSC/Cl-6), which retains the features of activated stellate cells. Treatment of T-HSC/Cl-6 cells with tanshinone I resulted in the induction of typical DNA fragmentation and DNA ladder formation in a concentration- and time-dependent manner. The induction of apoptosis was confirmed by flow cytometric analysis. Treatment of T-HSC/Cl-6 cells with tanshinone I caused activation of caspase-3 and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Tanshinone I induced mitochondrial membrane dipolarization and the release of cytochrome c from mitochondria into the cytosol. In conclusion, our results demonstrate that tanshinone I induces apoptosis of T-HSC/Cl-6 cells and that tanshinone I-induced apoptosis involves caspase activation through cytochrome c release and loss of mitochondrial membrane potential.

  13. Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133.

    PubMed Central

    Houglum, K; Lee, K S; Chojkier, M

    1997-01-01

    Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in

  14. 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

  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. The role of curcumin in streptozotocin-induced hepatic damage and the trans-differentiation of hepatic stellate cells.

    PubMed

    Mustafa, Hesham N

    2016-04-01

    Diabetic patients frequently suffer from non-alcoholic steatohepatitis. The current study aimed to investigate the role of curcumin and the response of hepatic stellate cells in streptozotocin (STZ)-induced hepatic damage. Sixty male rats were divided into three groups. The normal control injected with a citrate buffer vehicle and the diabetic control group which was injected intraperitoneally (IP) with a single-dose of streptozotocin (50mg/kg body weight) and a diabetic group was treated with an oral dose of curcumin at 80 mg/kg body weight daily for 60 days. Curcumin effectively counteracts oxidative stress-mediated hepatic damage and improves biochemical parameters. Alpha-smooth muscle actin (α-SMA) was significantly reduced, and insulin antibodies showed strong positive immunoreactivity with curcumin administration. These results optimistically demonstrate the potential use of curcumin, which is attributed to its antiradical/antioxidant activities and its potential β-cell regenerative properties. Also, it has the capability to encourage the trans-differentiation of hepatic stellate cells into insulin-producing cells for a period of time. In addition, as it is an anti-fibrotic mediator that inhibits hepatic stellate cell activation and the transition to myofibroblast-like cells, this suggests the possibility of considering curcumin's novel therapeutic effects in reducing hepatic dysfunction in diabetic patients.

  18. Suppression of hepatic stellate cell activation through downregulation of gremlin1 expression by the miR-23b/27b cluster

    PubMed Central

    Wang, Hu; Ni, Yi-Ran; Wang, Jie; Wu, Jiang-Feng; Liu, Chang-Bai

    2016-01-01

    The imbalance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways is a critical step in promoting hepatic stellate cell activation during hepatic fibrogenesis. Gremlin1 may impair the balance. Something remains unclear about the regulatory mechanisms of gremlin1 action on hepatic stellate cell activation and hepatic fibrosis. In the current study, gremlin1 overexpression promotes activation of hepatic stellate cells. Knockdown of gremlin1 with siRNAs suppresses hepatic stellate cell activation and attenuates hepatic fibrosis in rat model. Our results also show that miR-23b/27b cluster members bind to 3′-untranslated region of gremlin1 resulting in reduction of transforming growth factor β, α-smooth muscle actin and collagenI α1/2 gene expression. Our findings suggest that gremlin1 promotes hepatic stellate cell activation and hepatic fibrogenesis through impairment of the balance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways. The miR-23b/27b cluster suppresses activation of hepatic stellate cells through binding gremlin1 to rectify the imbalance. PMID:27863390

  19. The antifibrogenic effect of (-)-epigallocatechin gallate results from the induction of de novo synthesis of glutathione in passaged rat hepatic stellate cells.

    PubMed

    Yumei, Fu; Zhou, Yajun; Zheng, Shizhong; Chen, Anping

    2006-07-01

    Hepatic stellate cells (HSC) are the major players during hepatic fibrogenesis. Overproduction of extracellular matrix (ECM) is a characteristic of activated HSC. Transforming growth factor-beta (TGF-beta) is the most potent fibrogenic cytokine while connective tissue growth factor (CTGF) mediates the production of TGF-beta-induced ECM in activated HSC. HSC activation and hepatic fibrogenesis are stimulated by oxidative stress. Glutathione (GSH) is the most important intracellular antioxidant. The aim of this study is to explore the mechanisms of (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, in the inhibition of ECM gene expression in activated HSC. It is hypothesized that EGCG inhibits ECM gene expression in activated HSC by interrupting TGF-beta signaling through attenuating oxidative stress. It is found that EGCG interrupts TGF-beta signaling in activated HSC by suppressing gene expression of type I and II TGF-beta receptors. EGCG inhibits CTGF gene expression, leading to the reduction in the abundance of ECM, including alphaI(I) procollagen. Exogenous CTGF dose dependently eliminates the antifibrogenic effect. EGCG attenuates oxidative stress in passaged HSC by scavenging reactive oxygen species and reducing lipid peroxidation. De novo synthesis of GSH is a prerequisite for EGCG to interrupt TGF-beta signaling and to reduce the abundance of alphaI(I) procollagen in activated HSC in vitro. Taken together, our results demonstrate that the interruption of TGF-beta signaling by EGCG results in the suppression of gene expression of CTGF and ECM in activated HSC in vitro. In addition, our results, for the first time, demonstrate that the antioxidant property of EGCG derived from de novo synthesis of intracellular GSH plays a critical role in its antifibrogenic effect. These results provide novel insights into the mechanisms of EGCG as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.

  20. Subcellular localization of (latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stellate cells.

    PubMed

    Roth-Eichhorn, S; Kühl, K; Gressner, A M

    1998-12-01

    Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF-beta1,-2,-3, beta1-LAP, and LTBP-1 after cultivation using double-immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-beta1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for beta1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-beta, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle--actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent

  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. Post‑translational modification of CREB‑1 decreases collagen I expression by inhibiting the TGF‑β1 signaling pathway in rat hepatic stellate cells.

    PubMed

    Deng, Xiaoling; Deng, Liang; Wang, Pei; Cheng, Chunwei; Xu, Keshu

    2016-12-01

    Liver fibrosis is a reversible wound‑healing response to liver damage. Following liver injury, activated hepatic stellate cells (HSCs) cause excessive deposition of collagen and other components of the extracellular matrix, which may eventually impair liver function. Transforming growth factor‑β1 (TGF‑β1) is critical for the development of liver fibrosis, and acts by activating HSCs, stimulating matrix deposition and inducing other profibrotic responses. Certain studies have demonstrated that the transcription factor, cyclic adenosine monophosphate (cAMP)‑response element binding protein‑1 (CREB‑1) is important in the inhibition of fibrosis; however, the underlying mechanism remains to be elucidated. The present study hypothesized that CREB‑1 inhibits fibrosis via blocking the TGF‑β1 signaling pathway in HSCs. Treatment with the cAMP‑elevating agent, forskolin stimulated CREB‑1 phosphorylation and transcription activation in HSCs. In addition, treatment with the histone deacetylase inhibitor, trichostatin A extended CREB‑1 phosphorylation. The present study used a pRSV‑CREB‑1 expression vector to upregulate CREB‑1 gene expression. The results indicated that activated CREB‑1 reduced TGF‑β1‑stimulated expression of collagen I, activated Smad2/3 and upregulated expression of Smad7. In addition, activated CREB‑1 attenuated the activation of extracellular signal‑regulated kinase 1/2 induced by TGF‑β1, expression of Ras homolog gene family, member A (RhoA) and Rho‑associated coiled‑coil containing protein kinase 1. Thus, post‑translational modification of CREB‑1 inhibited the profibrotic effects of TGF‑β1 in HSCs via Smad‑dependent and ‑independent signaling pathways.

  4. 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.

  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. 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.

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

    PubMed

    Gandhi, Chandrashekhar R

    2012-01-01

    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.

  8. Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx

    PubMed Central

    Gong, Jin; Tu, Wei; Han, Jian; He, Jiayi; Liu, Jingmei; Han, Ping; Wang, Yunwu; Li, Mengke; Liu, Mei; Liao, Jiazhi; Tian, Dean

    2016-01-01

    Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, but its involvement in hepatic fibrogenesis remains unclear. Special AT-rich binding protein 1 (SATB1) has been implicated in reprogramming chromatin organization and transcription profiles in many cancers and non-cancer-related conditions. We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus (HBV)-infected patients and HBV transgenic (HBV-Tg) mouse model. Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model. Moreover, we suggested HBV encoded x protein (HBx) induced SATB1 expression through activation of JNK and ERK pathways. Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells (HSCs) by secretion of connective tissue growth factor (CTGF), Interleukin-6 (IL-6) and platelet derived growth factor-A (PDGF-AA). Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells in HBV-related fibrosis. PMID:27883059

  9. Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro

    PubMed Central

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

    2004-01-01

    AIM: To investigate the effects of platelet-derived growth factor (PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells. METHODS: Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group. RESULTS: The expression levels of Fas between the 4 groups had no significant differences (P > 0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091 ± 0.007 vs 0.385 ± 0.051, P < 0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126 ± 0.008 vs 0.210 ± 0.024, P < 0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P > 0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513 ± 0.016 vs 0.400 ± 0.022, P < 0.01). No significant difference was found between group I and group P (P > 0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449 ± 0.028 vs 0.513 ± 0.016, P < 0.05). CONCLUSION: PDGF may promote proliferation of HSCs but is

  10. Hepatic stellate cell interferes with NK cell regulation of fibrogenesis via curcumin induced senescence of hepatic stellate cell.

    PubMed

    Jin, Huanhuan; Jia, Yan; Yao, Zhen; Huang, Jingjing; Hao, Meng; Yao, Shunyu; Lian, Naqi; Zhang, Feng; Zhang, Chenxi; Chen, Xingran; Bian, Mianli; Shao, Jiangjuan; Wu, Li; Chen, Anping; Zheng, Shizhong

    2017-05-01

    Hepatic fibrosis, a common scarring response to various forms of chronic liver injury, is a precursor to cirrhosis and liver cancer. During liver fibrosis, hepatic stellate cells (HSCs) initially activate and proliferate, which are responsible for the secretion of extracellular matrix components. However, these cells eventually senesce and are cleared by natural killer (NK) cells. Our previous researches have shown that the natural product curcumin could promote the senescence of activated HSC. In this study, we investigated how NK cells target senescent HSC and assessed the effect of this process on liver fibrosis. We found that senescent HSC induced by curcumin are susceptible to NK cells killing, due to the increased expression of NK cell activating ligand major histocompatibility complex class I chain-related genes A (MICA) and UL16-binding proteins 2 (ULBP2), but not Poliovirus Receptor (PVR). Further studies displayed that the interaction between NK cells and senescent LX2 cells stimulated granule exocytosis. Moreover, the inhibition of granule exocytosis weakened the cytotoxicity of NK cells and promoted the accumulation of senescent LX2 cells. Therefore, these aggregated data indicated that NK cells mediated clearance of senescent LX2 cells and granule exocytosis could play a protective role in the improvement of liver fibrosis.

  11. Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

    PubMed

    Narmada, Balakrishnan Chakrapani; Kang, Yuzhan; Venkatraman, Lakshmi; Peng, Qiwen; Sakban, Rashidah Binte; Nugraha, Bramasta; Jiang, Xuan; Bunte, Ralph M; So, Peter T C; Tucker-Kellogg, Lisa; Mao, Hai-Quan; Yu, Hanry

    2013-05-01

    Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

  12. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

    2005-04-01

    After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

  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. Hepatic Stellate Cells: Partners in Crime for Liver Metastases?

    PubMed Central

    Kang, Ningling; Gores, Gregory; Shah, Vijay

    2011-01-01

    Hepatic stellate cells were recently postulated as a component of the prometastatic liver microenvironment because they can transdifferentiate into highly proliferative and motile myofibroblasts that are implicated in the desmoplastic reaction and metastatic growth. This review focuses on bidirectional interactions between tumor cells and HSC in the liver microenvironment and discusses mechanisms whereby tumor derived factors activate HSC, and in turn, activated HSC promote metastatic growth. Bidirectional interactions between tumors and HSC may function as an “amplification loop” to further enhance metastatic growth in the liver. The activation of HSC is a complex process regulated by multiple factors such as TGF-β and PDGF signaling pathways, which may present as therapeutic targets in the prevention and treatment of liver metastases. Targeting HSC/myofibroblasts with TGF-β or PDGF antagonists in coordination with chemotherapy, radiotherapy or surgery may prove to be effective at reducing liver metastases and increasing the survival benefit of patients by targeting both tumor cells and the tumor microenvironment. PMID:21520207

  15. An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

    PubMed Central

    Qian, Hui; Deng, Xing; Huang, Zhao-Wei; Wei, Ji; Ding, Chen-Hong; Feng, Ren-Xin; Zeng, Xin; Chen, Yue-Xiang; Ding, Jin; Qiu, Lei; Hu, Zhen-Lin; Zhang, Xin; Wang, Hong-Yang; Zhang, Jun-Ping; Xie, Wei-Fen

    2015-01-01

    Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases. PMID:26169608

  16. 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

  17. Catalase ameliorates hepatic fibrosis by inhibition of hepatic stellate cells activation.

    PubMed

    Dong, Yuwei; Qu, Ying; Xu, Mingyi; Wang, Xingpeng; Lu, Lungen

    2014-01-01

    Catalase, an endogenous antioxidant enzyme, is thought to have rescue effects on hepatic fibrosis. In this study, the regulation of catalase in CCl₄-induced hepatic fibrogenesis was investigated. Our results indicated that catalase expression was decreased upon CCl₄ treatment in a time-dependent manner, while the expression of several profibrosis and proangiogenic factors, including transforming growth factor (TGF)-beta 1, vascular endothelial growth factor (VEGF), and angiopoietin 1 were significantly increased. To assess the role of catalase in hepatic fibrosis, catalase was overexpressed in HSC-T6 cells. This overexpression resulted in the inhibition of cell proliferation, migratory activity, and alpha-smooth muscle actin (alpha-SMA) expression, key features that characterize activation of hepatic stellate cells (HSC). Overexpression of catalase led to a decrease in the secretion of collagen type 1 and angiopoietin 1. These results indicate that loss of catalase activity is involved in the pathogenesis of hepatic fibrosis caused by the activation of HSCs.

  18. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    PubMed

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B

    2017-03-15

    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  19. Prolyl oligopeptidase attenuates hepatic stellate cell activation through induction of Smad7 and PPAR-γ

    PubMed Central

    Zhou, Da; Wang, Jing; He, Ling-Nan; Li, Bing-Hang; Ding, Yong-Nian; Chen, Yuan-Wen; Fan, Jian-Gao

    2017-01-01

    Prolyl oligopeptidase (POP) is a serine endopeptidase widely distributed in vivo with high activity in the liver. However, its biological functions in the liver have remained largely elusive. A previous study by our group has shown that POP produced N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) and thereby exerted an anti-fibrogenic effect on hepatic stellate cells (HSCs) in vitro. It was therefore hypothesized that POP may affect the activation state of HSCs and has an important role in liver fibrosis. The HSC-T6 immortalized rat liver stellate cell line was treated with the POP inhibitor S17092 or transfected with recombinant lentivirus to overexpress POP. Cell proliferation and apoptosis were determined using a Cell Counting Kit-8 and flow cytometry, respectively. The activation status of HSCs was determined by examination of the expression of α-smooth muscle actin (α-SMA), collagen I, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGF)-β-Smad signaling and peroxisome proliferator activated receptor-γ (PPAR-γ). Inhibition by S17092 decreased, whereas lentiviral expression increased the activity of POP and cell proliferation, while neither of the treatments affected cell apoptosis. Of note, S17092 significantly increased, whereas POP overexpression decreased the expression of α-SMA and MCP-1 without affecting the expression of collagen I and TGF-β1. Furthermore, S17092 caused a reduction, whereas POP overexpression caused an upregulation of Smad7 protein and PPAR-γ, but not phosphorylated-Smad2/3 expression. In conclusion, POP attenuated the activation of HSCs through inhibition of TGF-β signaling and induction of PPAR-γ, which may have therapeutic potential in liver fibrosis. PMID:28352366

  20. Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

    PubMed Central

    Liu, Qiong; Chen, Jing; Wang, Xu; Yu, Liang; Hu, Li-hong; Shen, Xu

    2010-01-01

    Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation. Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis. PMID:20644552

  1. Bone marrow-derived pancreatic stellate cells in rats.

    PubMed

    Sparmann, Gisela; Kruse, Marie-Luise; Hofmeister-Mielke, Nicole; Koczan, Dirk; Jaster, Robert; Liebe, Stefan; Wolff, Daniel; Emmrich, Jörg

    2010-03-01

    Origin and fate of pancreatic stellate cells (PSCs) before, during and after pancreatic injury are a matter of debate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow (BM) origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein (eGFP)-expressing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride (DBTC) was used to induce acute pancreatic inflammation with subsequent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation.

  2. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.

    PubMed

    Olaso, E; Ikeda, K; Eng, F J; Xu, L; Wang, L H; Lin, H C; Friedman, S L

    2001-11-01

    Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

  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.

  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. Decoding the role of the nuclear receptor SHP in regulating hepatic stellate cells and liver fibrogenesis

    PubMed Central

    Cipriani, Sabrina; Carino, Adriana; Masullo, Dario; Zampella, Angela; Distrutti, Eleonora; Fiorucci, Stefano

    2017-01-01

    The small heterodimer partner (SHP) is an orphan nuclear receptor that lacks the DNA binding domain while conserves a putative ligand-binding site, thought that endogenous ligands for this receptor are unknown. Previous studies have determined that SHP activation protects against development of liver fibrosis a process driven by trans-differentiation and activation of hepatic stellate cells (HSCs), a miofibroblast like cell type, involved in extracellular matrix (ECM) deposition. To dissect signals involved in this activity we generated SHP-overexpressing human and rat HSCs. Forced expression of SHP in HSC-T6 altered the expression of 574 genes. By pathway and functional enrichment analyses we detected a cluster of 46 differentially expressed genes involved in HSCs trans-differentiation. Using a isoxazole scaffold we designed and synthesized a series of SHP agonists. The most potent member of this group, ISO-COOH (EC50: 9 μM), attenuated HSCs trans-differentiation and ECM deposition in vitro, while in mice rendered cirrhotic by carbon tetrachloride (CCl4) or α-naphthyl-isothiocyanate (ANIT), protected against development of liver fibrosis as measured by morphometric analysis and expression of α-SMA and α1-collagen mRNAs. In aggregate, present results identify SHP as a counter-regulatory signal for HSCs transactivation and describe a novel class of SHP agonists endowed with anti-fibrotic activity. PMID:28117422

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

    PubMed

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

    2009-03-01

    Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca(2+) signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca(2+) signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca(2+) chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca(2+) 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 Ca(2+) signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca(2+) signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca(2+) 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 Ca(2+) 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.

  7. The antioxidant (-)-epigallocatechin-3-gallate inhibits rat hepatic stellate cell proliferation in vitro by blocking the tyrosine phosphorylation and reducing the gene expression of platelet-derived growth factor-beta receptor.

    PubMed

    Chen, Anping; Zhang, Li

    2003-06-27

    During hepatic fibrogenesis, quiescent hepatic stellate cells (HSC) become active and trans-differentiate into myofibroblast-like cells. This process coincides with an increase in cell proliferation, loss of stored vitamin A droplets, and excessive production and deposition of extracellular matrix components. HSC activation is coupled with the sequential expression of cytokine receptors, including platelet-derived growth factor-beta receptor (PDGF-betaR). Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays critical roles in activation of HSC during hepatic fibrogenesis. We have recently demonstrated that the antioxidant (-)-epigallocatechin gallate (EGCG), a major component in green tea extracts, significantly inhibited the proliferation of passaged HSC. The aim of the present study is to elucidate the underlying mechanisms. Since PDGF is a potent mitogen for HSC and mediates the early proliferative response, it was hypothesized that EGCG might inhibit HSC proliferation by interfering with the PDGF signal transduction. In this report, we demonstrated that EGCG, in two steps, significantly and effectively inhibited the proliferation of primary and passaged HSC. The polyphenolic compound initiated its inhibitory action by rapidly blocking the phosphorylation of tyrosines in PDGF-betaR elicited by PDGF in serum. This action was short lived, persisting for a few hours. In addition, this antioxidant inhibited the gene expression of PDGF-betaR by blocking the activation of transcription factors activator protein-1 and NF-kappaB, which were required for the gene transcription. The latter action remained effective for no less than 48 hours. These results provided a novel insight into the mechanisms by which EGCG inhibits HSC growth. The inhibitory effect of the natural antioxidant, its long history of beverage consumption without adverse health effects, and higher potent antioxidant capability make it a good

  8. Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis.

    PubMed

    Bingül, İlknur; Başaran-Küçükgergin, Canan; Aydın, A Fatih; Çoban, Jale; Doğan-Ekici, Işın; Doğru-Abbasoğlu, Semra; Uysal, Müjdat

    2016-07-01

    The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis.

  9. Inhibitory Effects of microRNA 19b in Hepatic Stellate Cell-Mediated Fibrogenesis

    PubMed Central

    Lakner, Ashley M.; Steuerwald, Nury M.; Walling, Tracy L.; Ghosh, Sriparna; Li, Ting; McKillop, Iain H.; Russo, Mark W.; Bonkovsky, Herbert L.; Schrum, Laura W.

    2012-01-01

    Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers. CONCLUSIONS miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis. PMID:22278637

  10. 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

  11. Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis.

    PubMed

    Hintermann, Edith; Bayer, Monika; Ehser, Janine; Aurrand-Lions, Michel; Pfeilschifter, Josef M; Imhof, Beat A; Christen, Urs

    2016-07-03

    Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.

  12. Effect of Maotai liquor in inducing metallothioneins and on hepatic stellate cells

    PubMed Central

    Cheng, Ming-Liang; Wu, Jun; Wang, Hai-Qin; Xue, Lie-Ming; Tan, Ying-Zhi; Ping, Liu; Li, Cheng-Xiu; Huang, Neng-Hui; Yao, Yu-Mei; Ren, Lan-Zheng; Ye, Lan; Li, Ling; Jia, Mei-Lin

    2002-01-01

    AIM: To explore the possible mechanism why drinking Maotai liquor dose not cause hepatic fibrosis. METHODS: After being fed with Maotai for 56 days consecutively, the male SD rats were decollated for detecting the biological indexes, and the livers were harvested to examine the liver indexes and the level of hepatic metallothioneins (MT). Hepatic stellate cells (HSC) proliferation and collagen generation were also observed. RESULTS: Hepatic MT contents were 216.0 ng·g-1 ± 10.8 ng·g-1 in the rats of Maotai group and 10.0 ng·g-1 ± 2.8 ng·g-1 in the normal control group, which was increased obviously in Maotain group (P < 0.05). In the rats with grade CCL2 poisoning induced by Maotai, hepatic MT content was 304.8 ng·g-1 ± 12.1 ng·g-1 whereas in the controls with grade CCL4 poisoning, it was 126.4 ng·g-1 ± 4.8 ng·g-1 (P < 0.05). MDA was 102.0 nmol·g-1 ± 3.4 nmol·g-1 in Maotai group and 150.8 nmol·g-1 ± 6.7 nmol·g-1 in the control group (P < 0.05). When both of the groups were suffering from grade CCL4 poisoning, hepatic MT contents was negatively correlated with MDA (r = -0.8023, n = 20, P < 0.01). The 570 nmA values of each tube with HSC regeneration at concentrations of 0, 10, 50, 100, and 200 g·L-1 of Maotai were 0.818, 0.742, 0.736, 0.72, 0.682, and 0.604, respectively. From the concentration of 10 g·L-1, Maotai began to show obvious inhibitory effects against HSC, and the inhibition was concentration-dependent (P < 0.05, P < 0.01). Type I collagen contents in HSC were 61.4, 59.9, 50.1, 49.2, 48.7, 34.4 μg·g-1 at concentrations of 0, 10, 50, 100, and 200 g·L-1 of Maotai. At the concentration of 100-200 g·L-1, Maotai had obvious inhibitory effect against the secretion of type I collagen (P < 0.05). Gene expression analysis was conducted on cells with Maotai concentrations of 0, 50, 100 g·L-1 respectively and the ash values of β-actin gene expression were 0.88, 0.74, and 0.59, respectively, suggesting that at the concentration of 100 g·L-1

  13. Effect of Kruppel-like factor 4 on Notch pathway in hepatic stellate cells.

    PubMed

    Xue, Yin-Kai; Tan, Jun; Dou, Dong-Wei; Chen, Ding; Chen, Lu-Jia; Ren, Huan-Ping; Chen, Li-Bo; Xiong, Xin-Gao; Zheng, Hai

    2016-12-01

    The relationship between Kruppel-like factor 4 (KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms. Fifty SPF BALB/c mice were randomly divided into two groups. A liver fibrosis model was established in 25 mice as the experimental group, and the remaining 25 mice served as controls. On the day 0, 7, 14, and 35, liver tissues were removed for immunofluorescent detection. The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells, and KLF4 and Notch-associated factor expression was detected by qRT-PCR. Additionally, the hepatic stellate cell line LX-2 was used to establish control and experimental groups, and was cultured in vitro. LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately, whereas those in the control group were given isotonic culture medium. After 48 h, KLF4 expression was examined by Western blotting. After transient transfection of LX-2 cells to increase KLF4, the expression of Notch factor was examined. Immunofluorescence analysis showed that, with the aggravation of liver fibrosis, the absorbance (A) values of KLF4 were decreased (day 0: 980.73±153.19; day 7: 1087.99±230.23; day 14: 390.95±93.56; day 35: 245.99±87.34). The expression of Notch pathway- related factors (Notch-1, Notch-2, and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression. With the increase of KLF4 expression, Notch-2 (0.73±0.13) and Jagged-1 (0.43±0.12) expression decreased, whereas Notch-1 level was not detectable. When the Notch pathway was inhibited, KLF4 levels generally increased (18.12±1.31). Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells, which may provide a reference for the treatment of hepatic fibrosis.

  14. 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

  15. 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.

  16. MicroRNA-34a Promotes Hepatic Stellate Cell Activation via Targeting ACSL1

    PubMed Central

    Yan, Gangli; Li, Binbin; Xin, Xuan; Xu, Midie; Ji, Guoqing; Yu, Hongyu

    2015-01-01

    Background The incidence of liver fibrosis remains high due to the lack of effective therapies. Our previous work found that microRNA (miR)-34a expression was increased, while acy1-CoA synthetase long-chain family member1 (ACSL1) was decreased, in a dimethylnitrosamine (DNS)-induced hepatic fibrosis rat model. We hypothesized that miR-34a may play a role in the process of hepatic fibrosis by targeting ACSL1. Material/Methods From days 2 to 14, cultured primary hepatic stellate cells (HSCs) underwent cell morphology, immunocytochemical staining, and quantitative reverse transcription PCR (RT-qPCR) for alpha smooth muscle actin (α-SMA), desmin, rno-miR-34a, and ACSL1 expression. Wild-type and mutant luciferase reporter plasmids were constructed according to the predicted miR-34a binding site on the 3′-untranslated region (UTR) of the ACSL1 mRNA and then transfected into HEK293 cells. rno-miR-34a was silenced in HSCs to confirm that rno-miR-34a negatively regulates ACSL1 expression. mRNA and protein expression of α-SMA, type I collagen, and desmin were assayed in miR-34a-silenced HSCs. Results HSCs were deemed quiescent during the first 3 days and activated after 10 days. rno-miR-34a expression increased, and ACSL1 expression decreased, from day 2 to 7 to 14. rno-miR-34a was shown to specifically bind to the 3′-UTR of ACSL1. miR-34a-silenced HSCs showed higher ACSL1and lower α-SMA, type I collagen, and desmin expression than that of matching negative controls and non-transfected cells. Conclusions miR-34a appears to play an important role in the process of liver fibrosis by targeting ACSL1 and may show promise as a therapeutic molecular target for hepatic fibrosis. PMID:26437572

  17. 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

  18. Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1.

    PubMed

    Chen, H-Y; Chen, Z-X; Huang, R-F; Lin, N; Wang, X-Z

    2014-10-27

    We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.

  19. Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

    PubMed

    Zheng, Jianjian; Wu, Cunzao; Xu, Ziqiang; Xia, Peng; Dong, Peihong; Chen, Bicheng; Yu, Fujun

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-β1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

  20. Epigallocatechin-3-gallate, a polyphenol component of green tea, suppresses both collagen production and collagenase activity in hepatic stellate cells.

    PubMed

    Nakamuta, Makoto; Higashi, Nobuhiko; Kohjima, Motoyuki; Fukushima, Marie; Ohta, Satoshi; Kotoh, Kazuhiro; Kobayashi, Naoya; Enjoji, Munechika

    2005-10-01

    Catechins such as epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), and epigallocatechin (EGC) are polyphenol components of green tea. EGCG is the major component and has been reported to possess a wide range of biological properties including anti-fibrogenic activity. In hepatic fibrosis, activated hepatic stellate cells (HSCs) play a central role. In this study, we investigated the effect of catechins, including EGCG, on collagen production and collagenase activity in rat primary HSCs and activated human HSC-derived TWNT-4 cells. EGCG (50 microM) suppressed type I collagen production in rat HSCs more than ECG (50 microM) did; however, EGC (50 microM) did not show suppressive effects. EGCG also inhibited both collagen production and collagenase activity (active matrix metalloproteinase-1 [MMP-1]) in a dose-dependent manner, but did not affect the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production in TWNT-4 cells. Real-time PCR unexpectedly revealed that EGCG enhanced the transcription of type I collagen and TIMP-1, but did not affect the transcription of alpha-smooth muscle actin (alpha-SMA), and reduced the transcription MMP-1 in TWNT-4 cells. These findings demonstrated that EGCG inhibited collagen production regardless of enhanced collagen transcription and suppressed collagenase activity, and suggested that EGCG might have therapeutic potential for liver fibrosis.

  1. MircoRNA-145 promotes activation of hepatic stellate cells via targeting krüppel-like factor 4

    PubMed Central

    Men, Ruoting; Wen, Maoyao; Zhao, Mingyue; Dan, Xuelian; Yang, Zongze; Wu, Wenchao; Wang, Maggie Haitian; Liu, Xiaojing; Yang, Li

    2017-01-01

    Krüppel-like Factor 4 (KLF4), a target gene of miR-145, can negatively regulate lung fibrosis. However, the potential role of KLF4 and miR-145 in hepatic stellate cells (HSCs) activation or in hepatic fibrosis keeps unclear. This study aims to characterize miR-145 and KLF4 in activated HSCs and liver cirrhotic, and the underlying molecular basis. miR-145 was significantly up-regulated, while KLF4 was dramatically down-regulated during the activation of rat primary HSCs and TGF-βtreated HSCs. Furthermore, miR-145 mimics induced and inhibition of miR-145 reduced α-SMA and COL-I expression in primary HSCs. Additionally, the mRNA and protein levels of KLF4 in the liver of cirrhotic patients and rats were significantly down-regulated. α-SMA and COL-I were increased after inhibition of KLF4 by specific shRNA in primary HSCs. Forced KLF4 expression led to a reduction of α-SMA and COL-I expression in HSCs. miR-145 promotes HSC activation and liver fibrosis by targeting KLF4. PMID:28091538

  2. 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.

  3. Dihydroartemisinin counteracts fibrotic portal hypertension via farnesoid X receptor-dependent inhibition of hepatic stellate cell contraction.

    PubMed

    Xu, Wenxuan; Lu, Chunfeng; Zhang, Feng; Shao, Jiangjuan; Yao, Shunyu; Zheng, Shizhong

    2017-01-01

    Portal hypertension is a frequent pathological symptom occurring especially in hepatic fibrosis and cirrhosis. Current paradigms indicate that inhibition of hepatic stellate cell (HSC) activation and contraction is anticipated to be an attractive therapeutic strategy, because activated HSC dominantly facilitates an increase in intrahepatic vein pressure through secreting extracellular matrix and contracting. Our previous in vitro study indicated that dihydroartemisinin (DHA) inhibited contractility of cultured HSC by activating intracellular farnesoid X receptor (FXR). However, the effect of DHA on fibrosis-related portal hypertension still requires clarification. In this study, gain- and loss-of-function models of FXR in HSC were established to investigate the mechanisms underlying DHA protection against chronic CCl4 -caused hepatic fibrosis and portal hypertension. Immunofluorescence staining visually showed a decrease in FXR expression in CCl4 -administrated rat HSC but an increase in that in DHA-treated rat HSC. Serum diagnostics and morphological analyses consistently indicated that DHA exhibited hepatoprotective effects on CCl4 -induced liver injury. DHA also reduced CCl4 -caused inflammatory mediator expression and inflammatory cell infiltration. These improvements were further enhanced by INT-747 but weakened by Z-guggulsterone. Noteworthily, DHA, analogous to INT-747, significantly lowered portal vein pressure and suppressed fibrogenesis. Experiments on mice using FXR shRNA lentivirus consolidated the results above. Mechanistically, inhibition of HSC activation and contraction was found as a cellular basis for DHA to relieve portal hypertension. These findings demonstrated that DHA attenuated portal hypertension in fibrotic rodents possibly by targeting HSC contraction via a FXR activation-dependent mechanism. FXR could be a target molecule for reducing portal hypertension during hepatic fibrosis.

  4. Activated Hepatic Stellate Cells Are Dependent on Self-collagen, Cleaved by Membrane Type 1 Matrix Metalloproteinase for Their Growth

    PubMed Central

    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

    2014-01-01

    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. PMID:24867951

  5. 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.

  6. 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.

  7. 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.

  8. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

    PubMed Central

    Saile, B.; Knittel, T.; Matthes, N.; Schott, P.; Ramadori, G.

    1997-01-01

    During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable

  9. 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

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

    PubMed

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

    2016-04-11

    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.

  11. 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

  12. 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.

  13. Early activated hepatic stellate cell-derived molecules reverse acute hepatic injury

    PubMed Central

    Chang, Wen-Ju; Song, Lu-Jun; Yi, Tuo; Shen, Kun-Tang; Wang, Hong-Shan; Gao, Xiao-Dong; Li, Min; Xu, Jian-Min; Niu, Wei-Xin; Qin, Xin-Yu

    2015-01-01

    AIM: To test whether hepatic stellate cells (HSCs) at different activation stages play different roles in acetaminophen (APAP)-induced acute liver injury (ALI). METHODS: HSCs were isolated from mouse liver and cultured in vitro. Morphological changes of initiation HSCs [HSCs (5d)] and perpetuation HSCs [HSCs (p3)] were observed by immunofluorescence and transmission electron microscopy. The protective effects of HSC-derived molecules, cell lysates and HSC-conditioned medium (HSC-CM) were tested in vivo by survival and histopathological analyses. Liver injury was determined by measuring aminotransferase levels in the serum and by histologic examination of tissue sections under a light microscope. Additionally, to determine the molecular mediators of the observed protective effects of initiation HSCs, we examined HSC-CM using a high-density protein array. RESULTS: HSCs (5d) and HSCs (p3) had different morphological and phenotypic traits. HSCs (5d) presented a star-shaped appearance with expressing α-SMA at non-uniform levels between cells. However, HSCs (p3) evolved into myofibroblast-like cells without lipid droplets and expressed a uniform and higher level of α-SMA. HSC-CM (5d), but not HSC-CM (p3), provided a significant survival benefit and showed a dramatic reduction of hepatocellular necrosis and panlobular leukocyte infiltrates in mice exposed to APAP. However, this protective effect was abrogated at higher cell masses, indicating a therapeutic window of effectiveness. Furthermore, the protein array screen revealed that HSC-CM (5d) was composed of many chemokines and growth factors that correlated with inflammatory inhibition and therapeutic activity. When compared with HSC-CM (p3), higher levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-1γ, hepatocyte growth factor, interleukin-10, and matrix metalloproteinase-2, but lower levels of stem cell factor and Fas-Ligand were observed in HSC-CM (5d). CONCLUSION: These data indicated

  14. 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.

  15. Diallyl Trisulfide Suppresses Oxidative Stress-Induced Activation of Hepatic Stellate Cells through Production of Hydrogen Sulfide.

    PubMed

    Zhang, Feng; Jin, Huanhuan; Wu, Li; Shao, Jiangjuan; Zhu, Xiaojing; Chen, Anping; Zheng, Shizhong

    2017-01-01

    Accumulating data reveal that garlic has beneficial effects against chronic liver disease. We previously reported that diallyl trisulfide (DATS), the primary organosulfur compound in garlic, reduced fibrosis and attenuated oxidative stress in rat fibrotic liver. The present study was aimed at elucidating the underlying mechanisms. The primary rat hepatic stellate cells (HSCs) were cultured and stimulated with hydrogen peroxide (H2O2) for inducing HSC activation under oxidative stress. We examined the effects of DATS on the profibrogenic properties and oxidative stress in H2O2-treated HSCs. The results showed that DATS suppressed and reduced fibrotic marker expression in HSCs. DATS arrested cell cycle at G2/M checkpoint associated with downregulating cyclin B1 and cyclin-dependent kinase 1, induced caspase-dependent apoptosis, and reduced migration in HSCs. Moreover, intracellular levels of reactive oxygen species and lipid peroxide were decreased by DATS, but intracellular levels of glutathione were increased in HSCs. Furthermore, DATS significantly elevated hydrogen sulfide (H2S) levels within HSCs, but iodoacetamide (IAM) reduced H2S levels and significantly abrogated DATS production of H2S within HSCs. IAM also abolished all the inhibitory effects of DATS on the profibrogenic properties and oxidative stress in HSCs. Altogether, we demonstrated an H2S-associated mechanism underlying DATS inhibition of profibrogenic properties and alleviation of oxidative stress in HSCs. Modulation of H2S production may represent a therapeutic remedy for liver fibrosis.

  16. Diallyl Trisulfide Suppresses Oxidative Stress-Induced Activation of Hepatic Stellate Cells through Production of Hydrogen Sulfide

    PubMed Central

    Zhang, Feng; Jin, Huanhuan; Wu, Li; Shao, Jiangjuan; Zhu, Xiaojing; Chen, Anping

    2017-01-01

    Accumulating data reveal that garlic has beneficial effects against chronic liver disease. We previously reported that diallyl trisulfide (DATS), the primary organosulfur compound in garlic, reduced fibrosis and attenuated oxidative stress in rat fibrotic liver. The present study was aimed at elucidating the underlying mechanisms. The primary rat hepatic stellate cells (HSCs) were cultured and stimulated with hydrogen peroxide (H2O2) for inducing HSC activation under oxidative stress. We examined the effects of DATS on the profibrogenic properties and oxidative stress in H2O2-treated HSCs. The results showed that DATS suppressed and reduced fibrotic marker expression in HSCs. DATS arrested cell cycle at G2/M checkpoint associated with downregulating cyclin B1 and cyclin-dependent kinase 1, induced caspase-dependent apoptosis, and reduced migration in HSCs. Moreover, intracellular levels of reactive oxygen species and lipid peroxide were decreased by DATS, but intracellular levels of glutathione were increased in HSCs. Furthermore, DATS significantly elevated hydrogen sulfide (H2S) levels within HSCs, but iodoacetamide (IAM) reduced H2S levels and significantly abrogated DATS production of H2S within HSCs. IAM also abolished all the inhibitory effects of DATS on the profibrogenic properties and oxidative stress in HSCs. Altogether, we demonstrated an H2S-associated mechanism underlying DATS inhibition of profibrogenic properties and alleviation of oxidative stress in HSCs. Modulation of H2S production may represent a therapeutic remedy for liver fibrosis. PMID:28303169

  17. Adoptive transfer of hepatic stellate cells ameliorates liver ischemia reperfusion injury through enriching regulatory T cells.

    PubMed

    Feng, Min; Wang, Quanrongzi; Wang, Hao; Wang, Meng; Guan, Wenxian; Lu, Ling

    2014-04-01

    Our previous study indicated that adoptive transferred regulatory T cells (Tregs) attenuated liver ischemia reperfusion injury (IRI). Recent studies demonstrated that hepatic stellate cells (HSCs) were producers of induced Tregs (iTregs) via retinoic acid. This study aimed to investigate the role of adoptive transferred HSCs in liver IRI. Mice were treated with gradient doses of HSCs before surgery at 24h or 72h. The levels of serum aminotransferases and hepatic cytokines were evaluated after reperfusion. Meanwhile, hepatic Tregs and their subsets were analyzed by flow cytometry. We found that adoptive transferred HSCs attenuated liver IRI. Administration of HSCs expanded the number of hepatic iTregs and natural Tregs (nTregs) after reperfusion. In addition, we found that the increased Tregs were almost Helios-Tregs before surgery. These Helios-Tregs were considered as iTregs and protected liver from IRI partially. Furthermore, adoptive transferred HSCs stabilized nTregs and prevented nTregs from reducing after reperfusion. These nTregs also attenuated liver IRI partially. Depletion of Tregs abolished the protective effect of HSCs. Thus, we conclude that adoptive transferred HSCs ameliorate liver IRI in Tregs-dependent manner.

  18. Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

    PubMed Central

    Chen, Jennifer Y.; Newcomb, Benjamin; Zhou, Chan; Pondick, Joshua V.; Ghoshal, Sarani; York, Samuel R.; Motola, Daniel L.; Coant, Nicolas; Yi, Jae Kyo; Mao, Cungui; Tanabe, Kenneth K.; Bronova, Irina; Berdyshev, Evgeny V.; Fuchs, Bryan C.; Hannun, Yusuf; Chung, Raymond T.; Mullen, Alan C.

    2017-01-01

    Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide. PMID:28322247

  19. 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.

  20. 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.

  1. 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.

  2. Antrodia cinnamomea profoundly exalted the reversion of activated hepatic stellate cells by the alteration of cellular proteins.

    PubMed

    Chen, Yi-Ren; Chang, Kai-Ting; Tsai, May-Jywan; Lee, Chia-Hung; Huang, Kao-Jean; Cheng, Henrich; Ho, Yen-Peng; Chen, Jian-Chyi; Yang, Hsueh-Hui; Weng, Ching-Feng

    2014-07-01

    The direct modulation of Antrodia cinnamomea (AC) on the prominent role of liver fibrosis-hepatic stellate cells (HSCs) in situ remains unclear. Firstly, the administration of A. cinnamomea mycelial extract (ACME) could improve liver morphology and histological changes including collagen formation and GPT activity in the liver of thioacetamide (TAA)-injured rats. The morphology and fatty acid restore of TAA-induced HSCs (THSCs) returned to the non-chemical induced HSCs (NHSCs) type as measured by immunofluorescence and Oil Red O staining. PPARγ was upregulated associated with the lowering of α-SMA protein in NHSC-ACME. ACME inhibited the MMP-2 activity in NHSCs by gelatin Zymography. After LC-MS/MS, the cytoskeleton (tubulin, lamin A) and heat shock protein 8 in NHSC-ACME, and guanylate kinase, brain-specific kinase, SG-II and p55 proteins were downregulated in THSC-ACME. Whereas MHC class II, SMC6 protein, and phospholipase D were upregulated in NHSC-ACME. Furthermore, PKG-1 was downregulated in NHSC-ACME and upregulated in THSC-ACME. SG-II and p55 proteins were downregulated in NHSC-ACME and THSC-ACME by Western blotting. Taken together, the beneficial effect of A. cinnamomea on the induction of HSC cellular proteins is potentially applied as an alternative and complementary medicine for the prevention and amelioration of a liver injury.

  3. Human T cell microparticles circulate in blood of hepatitis patients and induce fibrolytic activation of hepatic stellate cells

    PubMed Central

    Kornek, Miroslaw; Popov, Yury; Libermann, Towia A.; Afdhal, Nezam H.; Schuppan, Detlef

    2010-01-01

    Microparticles (MP) are small cell membrane vesicles which are released from cells during apoptosis or activation. While circulating platelet MP have been studied in some detail, the existence and functional role of T cell MP remain elusive. We show that blood from patients with active hepatitis C (ALT>100 IU/ml) contains elevated numbers of T cell MP compared to patients with mild hepatitis C (ALT<40 IU/ml) and healthy controls. T cell MP fuse with cell membranes of hepatic stellate cells (HSC), the major effector cells for excess matrix deposition in liver fibrosis and cirrhosis. MP uptake is partly ICAM-1 dependent and leads to activation of NFkB and ERK1/2 and subsequent upregulation of fibrolytic genes in HSC, to downregulation of procollagen α1(I) mRNA, and blunting of profibrogenic activities of TGFβ1. Ex vivo the induced fibrolytic activity is evident in MP derived from activated CD4+ T cells, and highest with MP from activated and apoptotic CD8+ T cells. Mass spectrometry, FACS analysis and function blocking antibodies revealed CD147/Emmprin as candidate transmembrane molecule in HSC fibrolytic activation by CD8+ T cell MP. We conclude that 1) circulating T cell MP are a novel diagnostic marker for inflammatory liver diseases, and 2) in vivo induction of T cell MP may be a novel strategy to induce regression of liver fibrosis. PMID:20979056

  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. 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

  6. 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

  7. Upregulation of SIRT1-AMPK by thymoquinone in hepatic stellate cells ameliorates liver injury.

    PubMed

    Yang, Yong; Bai, Ting; Yao, You-Li; Zhang, De-Quan; Wu, Yan-Ling; Lian, Li-Hua; Nan, Ji-Xing

    2016-11-16

    Thymoquinone (TQ) is a biologically active compound isolated from the seeds of Nigella sativa L. (Ranuculaceae). This study investigated the hepato-protective effect of TQ on liver injury through AMP-activated protein kinase (AMPK) signaling in hepatic stellate cells (HSCs). In vitro, TGF-β time-dependently attenuated liver kinase B-1 (LKB1) and AMPK phosphorylation, which were blocked by pretreatment with TQ and AICAR (an activator of AMPK). TQ significantly inhibited collagen-Ι, α-SMA, TIMP-1 and enhanced MMP-13 expression, contributing to prevent TGF-β-induced human HSCs activation. Moreover, TQ induced peroxisome proliferator activated receptor-γ (PPAR-γ) expression, which was inhibited by genetic deletion of AMPK. In vivo, C57BL/6 mice were fed with ethanol diet for 10 days, then administering a single dose of ethanol (5g/kg body weight) via gavage. TQ (20 or 40mg/kg) were given by gavage every day. TQ attenuated the increases in serum aminotransferase and hepatic triglyceride in mice fed with ethanol, while significantly activated LKB1 and AMPK phosphorylation. In addition, TQ enhanced the sirtuin 1 (SIRT1) expression. In conclusion, we demonstrate that AMPK pathway is a key therapeutic target for controlling liver injury and TQ confers hepato-protection against TGF-β-induced the activation of HSCs and ethanol-induced liver injury.

  8. Hepatic Stellate Cells Preferentially Induce Foxp3+ Regulatory T Cells by Production of Retinoic Acid

    PubMed Central

    Dunham, Richard M.; Thapa, Manoj; Velazquez, Victoria M.; Elrod, Elizabeth J.; Denning, Timothy L.; Pulendran, Bali

    2013-01-01

    The liver has long been described as immunosuppressive, although the mechanisms underlying this phenomenon are incompletely understood. Hepatic stellate cells (HSCs), a population of liver nonparenchymal cells, are potent producers of the regulatory T cell (Treg)–polarizing molecules TGF-β1 and all-trans retinoic acid, particularly during states of inflammation. HSCs are activated during hepatitis C virus infection and may therefore play a role in the enrichment of Tregs during infection. We hypothesized that Ag presentation in the context of HSC activation will induce naive T cells to differentiate into Foxp3+ Tregs. To test this hypothesis, we investigated the molecular interactions between murine HSCs, dendritic cells, and naive CD4+ T cells. We found that HSCs alone do not present Ag to naive CD4+ T cells, but in the presence of dendritic cells and TGF-β1, preferentially induce functional Tregs. This Treg induction was associated with retinoid metabolism by HSCs and was dependent on all-trans retinoic acid. Thus, we conclude that HSCs preferentially generate Foxp3+ Tregs and, therefore, may play a role in the tolerogenic nature of the liver. PMID:23359509

  9. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets

    PubMed Central

    Zhang, Chong-Yang; Yuan, Wei-Gang; He, Pei; Lei, Jia-Hui; Wang, Chun-Xu

    2016-01-01

    Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells (HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis. PMID:28082803

  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.

  11. Succinate causes α-SMA production through GPR91 activation in hepatic stellate cells.

    PubMed

    Li, Ying Hui; Woo, Sung Hoon; Choi, Dae Hee; Cho, Eun-Hee

    2015-08-07

    Succinate acts as an extracellular signaling molecule as well as an intermediate in the citric acid cycle. It binds to and activates its specific G protein-coupled receptor 91 (GPR91). GPR91 is present in hepatic stellate cells (HSCs), but its role in hepatic fibrogenesis remains unclear. Cultured HSCs treated with succinate showed increased protein expression of GPR91 and α-smooth muscle actin (α-SMA), markers of fibrogenic response. Succinate also increased mRNA expression of α-SMA, transforming growth factor β (TGF-β), and collagen type I. Transfection of siRNA against GPR91 abrogated succinate-induced increases in α-SMA expression. Malonate, an inhibitor of succinate dehydrogenase (SDH), increased succinate levels in cultured HSCs and increased GPR91 and α-SMA expression. Feeding mice a methionine- and choline-deficient (MCD) diet is a widely used technique to create an animal model of nonalcoholic steatohepatitis (NASH). HSCs cultured in MCD media showed significantly decreased SDH activity and increased succinate concentration and GPR91 and α-SMA expression. Similarly, palmitate treatment significantly decreased SDH activity and increased GPR91 and α-SMA expression. Finally, C57BL6/J mice fed the MCD diet had elevated succinate levels in their plasma. The MCD diet also decreased SDH activity, increased succinate concentration, and increased GPR91 and α-SMA expression in isolated HSCs. Collectively, our results show that succinate plays an important role in HSC activation through GPR91 induction, and suggest that succinate and GPR91 may represent new therapeutic targets for modulating hepatic fibrosis.

  12. Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver.

    PubMed

    Van Rossen, Elke; Vander Borght, Sara; van Grunsven, Leo Adrianus; Reynaert, Hendrik; Bruggeman, Veerle; Blomhoff, Rune; Roskams, Tania; Geerts, Albert

    2009-03-01

    Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.

  13. Alteration and localization of glycan-binding proteins in human hepatic stellate cells during liver fibrosis.

    PubMed

    Zhong, Yaogang; Qin, Yannan; Dang, Liuyi; Jia, Liyuan; Zhang, Zhiwei; Wu, Haoxiang; Cui, Jihong; Bian, Huijie; Li, Zheng

    2015-10-01

    Glycan-binding proteins (GBPs) play an important role in cell adhesion, bacterial/viral infection, and cellular signaling pathways. However, little is known about the precision alteration of GBPs referred to pathological changes in hepatic stellate cells (HSCs) during liver fibrosis. Here, the carbohydrate microarrays were used to probe the alteration of GBPs in the activated HSCs and quiescent HSCs. As a result, 12 carbohydrates (e.g. Gal, GalNAc, and Man-9Glycan) showed increased signal, while seven carbohydrates (e.g. NeuAc, Lac, and GlcNAc-O-Ser) showed decreased signal in activated HSCs. Three carbohydrates (Gal, GalNAc, and NeuAc) were selected and subsequently used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HSCs and liver tissues by cy/histochemistry; the results showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HSCs may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti-fibrotic strategies.

  14. 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.

  15. 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

  16. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

    PubMed

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

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

  17. Hepatic Stellate Cells Regulate Immune Response via Induction of Myeloid Suppressor Cells

    PubMed Central

    Chou, Hong-Shiue; Hsieh, Ching-Chuan; Yang, Horng-Ren; Wang, Lianfu; Arakawa, Yusuke; Brown, Kathleen; Wu, Qingyu; Lin, Feng; Peters, Marion; Fung, John J.; Lu, Lina; Qian, Shiguang

    2011-01-01

    Although organ transplants have been applied for decades, outcomes of somatic cell transplants remain disappointing, presumably due to lack of appropriate supporting stromal cells. Thus, cotransplantation with liver stromal cells, hepatic stellate cells (HSC), achieves long-term survival of islet allografts in mice via induction of effector T cell apoptosis and generation of regulatory T (Treg) cells. In this study, we provide evidence both in vitro and in vivo that HSC can promote generation of myeloid-derived suppressor cells (MDSC). HSC-induced MDSC demonstrate potent immune inhibitory activity. Induction of MDSC is dependent on intact IFN-γ signaling pathway in HSC, and is mediated by soluble factors, suggesting that the specific tissue stromal cells, such as HSC, play a crucial role in regulating immune response via inflammation-induced generation of MDSC. Large amounts of MDSC can be propagated in vitro from bone marrow derived myeloid precursor cells under the influence of HSC. Cotransplantation with in vitro generated MDSC can effectively protects islet allografts from host immune attack. Local delivery of potent immune suppressor cells for cell transplants holds a great clinical application potential. PMID:21374665

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Metformin-mediated Bambi expression in hepatic stellate cells induces prosurvival Wnt/β-catenin signaling.

    PubMed

    Subramaniam, Nanthakumar; Sherman, Mara H; Rao, Renuka; Wilson, Caroline; Coulter, Sally; Atkins, Annette R; Evans, Ronald M; Liddle, Christopher; Downes, Michael

    2012-04-01

    AMP-activated protein kinase (AMPK) regulates lipid, cholesterol, and glucose metabolism in specialized metabolic tissues, such as muscle, liver, and adipose tissue. Agents that activate AMPK, such as metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), have beneficial effects on liver glucose and lipid metabolism. In addition, AMPK activation in proliferating hepatic stellate cells (HSC) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their effects, our aim was to examine the effects of AMPK activation in quiescent HSCs with these two agents on HSC function. We found that phospho-AMPK levels were markedly upregulated by both AICAR and metformin in quiescent HSCs. However, although AICAR treatment induced cell death, cells treated with metformin did not differ from untreated controls. AICAR-mediated HSC cell death was paralleled by loss of expression of the TGF-β decoy receptor Bambi, whereas metformin increased Bambi expression. Transfection of siRNA-Bambi into HSCs also induced cell death, mimicking the effects of AICAR, whereas overexpression of Bambi partially rescued AICAR-treated cells. As Bambi has previously been shown to promote cell survival through Wnt/β-catenin signaling, a reporter incorporating binding sites for a downstream target of this pathway was transfected into HSCs and was induced. We conclude that although AICAR and metformin both activate AMPK in quiescent HSCs, AICAR rapidly induced cell death, whereas metformin-treated cells remained viable. The finding that metformin increases Bambi expression and activates Wnt/β-catenin signaling provides a possible mechanistic explanation for this observation. These results suggest that AICAR and metformin may confer disease-specific therapeutic benefits.

  4. NS5ATP13 Promotes Liver Fibrogenesis via Activation of Hepatic Stellate Cells.

    PubMed

    Li, Yaru; Liu, Shunai; Han, Ming; Lu, Hongping; Wang, Qi; Zhang, Yu; Tursun, Kelbinur; Li, Zhongshu; Feng, Shenghu; Cheng, Jun

    2017-01-29

    Liver fibrosis is a reversible wound-healing response to any etiology of chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) is the key event in liver fibrogenesis. Generally, persistent activation and proliferation of HSCs results in liver fibrosis progression, while primary mechanisms of liver fibrosis resolution are apoptosis and reversion to a quiescent phenotype of activated HSCs. NS5ATP13 (HCV NS5A-transactivated protein 13) is involved in nucleologenesis and tumorigenesis, but its role in liver fibrosis and HSC activation remains unclear. This study found that NS5ATP13 was upregulated in both fibrotic liver tissues and activated human HSCs induced by TGF-β1. Moreover, NS5ATP13 enhanced extracellular matrix (ECM) production and HSC activation, with or without TGF-β1 treatment, likely involving the TGF-β1/Smad3 signaling pathway. Additionally, NS5ATP13 boosted HSC proliferation by inhibiting cell apoptosis. Furthermore, HCV NS5A promoted the profibrogenic effect of NS5ATP13 partly through TGF-β1 and NF-κB p65 (RelA) upregulation. Meanwhile, NS5ATP13 was required for the pro-fibrogenic effect of NF-κB. Moreover, NS5ATP13 and NF-κB phosphorylation as well as HSC activation were reduced by CX-4945, a CK2 specific inhibitor. These findings indicated that NS5ATP13 acts as a profibrogenic factor, providing a potential target for antifibrotic therapies. This article is protected by copyright. All rights reserved.

  5. 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

  6. Transcriptome-based repurposing of apigenin as a potential anti-fibrotic agent targeting hepatic stellate cells

    PubMed Central

    Hicks, Daniel F.; Goossens, Nicolas; Blas-García, Ana; Tsuchida, Takuma; Wooden, Benjamin; Wallace, Michael C.; Nieto, Natalia; Lade, Abigale; Redhead, Benjamin; Cederbaum, Arthur I; Dudley, Joel T.; Fuchs, Bryan C.; Lee, Youngmin A.; Hoshida, Yujin; Friedman, Scott L.

    2017-01-01

    We have used a computational approach to identify anti-fibrotic therapies by querying a transcriptome. A transcriptome signature of activated hepatic stellate cells (HSCs), the primary collagen-secreting cell in liver, and queried against a transcriptomic database that quantifies changes in gene expression in response to 1,309 FDA-approved drugs and bioactives (CMap). The flavonoid apigenin was among 9 top-ranked compounds predicted to have anti-fibrotic activity; indeed, apigenin dose-dependently reduced collagen I in the human HSC line, TWNT-4. To identify proteins mediating apigenin’s effect, we next overlapped a 122-gene signature unique to HSCs with a list of 160 genes encoding proteins that are known to interact with apigenin, which identified C1QTNF2, encoding for Complement C1q tumor necrosis factor-related protein 2, a secreted adipocytokine with metabolic effects in liver. To validate its disease relevance, C1QTNF2 expression is reduced during hepatic stellate cell activation in culture and in a mouse model of alcoholic liver injury in vivo, and its expression correlates with better clinical outcomes in patients with hepatitis C cirrhosis (n = 216), suggesting it may have a protective role in cirrhosis progression.These findings reinforce the value of computational approaches to drug discovery for hepatic fibrosis, and identify C1QTNF2 as a potential mediator of apigenin’s anti-fibrotic activity. PMID:28256512

  7. Hypoxia-Inducible Factor-1alpha and MAPK Co-Regulate Activation of Hepatic Stellate Cells upon Hypoxia Stimulation

    PubMed Central

    Guan, Fei; Xiao, Yan; Deng, Jing; Chen, Huoying; Chen, Xiaolin; Li, Jianrong; Huang, Hanju; Shi, Chunwei

    2013-01-01

    Background Hepatic stellate cell (HSC) plays a key role in pathogenesis of liver fibrosis. During liver injury, hypoxia in local micro-environment is inevitable. Hif-1α is the key transcriptional regulation factor that induces cell’s adaptive responses to hypoxia. Recently, it was reported that MAPK is involved in regulation of Hif-1α activity. Aims To explore whether Hif-1α regulates HSC activation upon hypoxia, and whether MAPK affects Hif-1α-regulated signaling cascades, thus providing new targets for preventing liver fibrosis. Methods Hif-1α expression in livers of Schistosomajaponicum infected BALB/c mice was detected with western blot and immunohistochemistry. A rat cell line of HSC, HSC-T6, was cultured in 1% oxygen. HSC activation, including F-actin reorganization, increase of vimentin and α-SMA, was detected with western blot or immunocytochemistry. Cells were transfected with specific siRNA to Hif-1α, expression of activation markers, transcription of fibrosis-promoting cytokines, secretion of collagen I were detected with western blot, Real Time PCR and ELISA. Lysate from HSC-T6 cells pretreated with PD98059, a specific MEK1 pharmacological inhibitor, was subjected to detect Hif-1α ubiquitination and nuclear translocation with western blot and immunoprecipitation. Results and Conclusions Hif-1α apparently increased in liver tissues of Schistosomajaponicum infected mice. 1% O2 induced F-actin reorganization, increase of Hif-1α, vimentin and α-SMA in HSC-T6 cells. Hif-1α Knockdown inhibited HSC-T6 activation, transcription of IL-6, TGF-β and CTGF and secretion of collagen I from HSC-T6 cells upon hypoxia. Inhibition of MAPK phosphorylation enhanced Hif-1α ubiquitination, and inhibited Hif-1α translocation into nucleus. Conclusively, Hif-1α and MAPK participate in HSC activation upon hypoxia. PMID:24040163

  8. 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.

  9. Metformin reduces hepatic resistance and portal pressure in cirrhotic rats.

    PubMed

    Tripathi, Dinesh M; Erice, Eva; Lafoz, Erica; García-Calderó, Héctor; Sarin, Shiv K; Bosch, Jaime; Gracia-Sancho, Jordi; García-Pagán, Juan Carlos

    2015-09-01

    Increased hepatic vascular resistance is the primary factor in the development of portal hypertension. Metformin ameliorates vascular cells function in several vascular beds. Our study was aimed at evaluating the effects, and the underlying mechanisms, of metformin on hepatic and systemic hemodynamics in cirrhotic rats and its possible interaction with the effects of propranolol (Prop), the current standard treatment for portal hypertension. CCl4-cirrhotic rats received by gavage metformin 300 mg/kg or its vehicle once a day for 1 wk, before mean arterial pressure (MAP), portal pressure (PP), portal blood flow (PBF), hepatic vascular resistance, and putative molecular/cellular mechanisms were measured. In a subgroup of cirrhotic rats, the hemodynamic response to acute Prop (5 mg/kg iv) was assessed. Effects of metformin ± Prop on PP and MAP were validated in common bile duct ligated-cirrhotic rats. Metformin-treated CCl4-cirrhotic rats had lower PP and hepatic vascular resistance than vehicle-treated rats, without significant changes in MAP or PBF. Metformin caused a significant reduction in liver fibrosis (Sirius red), hepatic stellate cell activation (α-smooth muscle actin, platelet-derived growth factor receptor β polypeptide, transforming growth factor-βR1, and Rho kinase), hepatic inflammation (CD68 and CD163), superoxide (dihydroethidium staining), and nitric oxide scavenging (protein nitrotyrosination). Prop, by decreasing PBF, further reduced PP. Similar findings were observed in common bile duct ligated-cirrhotic rats. Metformin administration reduces PP by decreasing the structural and functional components of the elevated hepatic resistance of cirrhosis. This effect is additive to that of Prop. The potential impact of this pharmacological combination, otherwise commonly used in patients with cirrhosis and diabetes, needs clinical evaluation.

  10. Herbal compound “Songyou Yin” attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells

    PubMed Central

    2013-01-01

    Background Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound “Songyou Yin” (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells. Methods Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA). Results Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-β1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY. Conclusions SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells. PMID:23622143

  11. 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

  12. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  13. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

    PubMed

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

    2015-12-25

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

  14. CCN1/CYR61 overexpression in hepatic stellate cells induces ER stress-related apoptosis.

    PubMed

    Borkham-Kamphorst, Erawan; Steffen, Bettina T; Van de Leur, Eddy; Haas, Ute; Tihaa, Lidia; Friedman, Scott L; Weiskirchen, Ralf

    2016-01-01

    CCN1/CYR61 is a matricellular protein of the CCN family, comprising six secreted proteins specifically associated with the extracellular matrix (ECM). CCN1 acts as an enhancer of the cutaneous wound healing process by preventing hypertrophic scar formation through induction of myofibroblast senescence. In liver fibrosis, the senescent cells are primarily derived from activated hepatic stellate cells (HSC) that initially proliferate in response to liver damage and are the major source of ECM. We investigate here the possible use of CCN1 as a senescence inducer to attenuate liver fibrogenesis by means of adenoviral gene transfer in primary HSC, myofibroblasts (MFB) and immortalized HSC lines (i.e. LX-2, CFSC-2G). Infection with Ad5-CMV-CCN1 induced large amounts of CCN1 protein in all these cells, resulting in an overload of the endoplasmic reticulum (ER) and in a compensatory unfolded protein response (UPR). The UPR resulted in upregulation of ER chaperones including BIP/Grp78, Grp94 and led to an activation of IRE1α as evidenced by spliced XBP1 mRNA with IRE1α-induced JNK phosphorylation. The UPR arm PERK and eIF2a was phosphorylated, combined with significant CHOP upregulation. Ad5-CMV-CCN1 induced HSC apoptosis that was evident by proteolytic cleavage of caspase-12, caspase-9 and the executor caspase-3 and positive TUNEL stain. Remarkably, Ad5-CMV-CCN1 effectively reduced collagen type I mRNA expression and protein. We conclude that the matricellular protein CCN1 gene transfer induces HSC apoptosis through ER stress and UPR.

  15. Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes.

    PubMed Central

    Théret, N.; Musso, O.; L'Helgoualc'h, A.; Clément, B.

    1997-01-01

    Activation of matrix metalloproteinase (MMP)-2, the 72-kd collagenase IV/gelatinase A, is involved in extracellular matrix remodeling. It has been suggested that a membrane-type MMP (MT-MMP-1) and the tissue inhibitor of metalloproteinase (TIMP)-2 are involved in MMP-2 processing, but the exact mechanism(s) of its activation remains unclear. We have investigated the role of cell-cell cooperation in the activation of pro-MMP-2 in the liver, using pure cultures and co-cultures of hepatocytes and hepatic stellate cells (HSCs). Northern blot analysis and in situ hybridization showed that, in both pure and co-cultures, HSCs, but not hepatocytes, expressed MMP-2, TIMP-2, and MT-MMP-1 mRNA. Zymography analyses revealed the latent form of MMP-2 in medium from 2-day-old pure HSC cultures with higher amounts in medium from hepatocyte/HSC co-cultures. When hepatocytes were added to 10-day-old HSC cultures, the activated form of MMP-2 was detected, concomitantly with the deposition of an abundant extracellular matrix. Incubation of plasma membrane-enriched fractions from hepatocytes with conditioned medium from pure HSC cultures generated the activated species of MMP-2 (62 and 59 kd). Activation of pro-MMP-2 by hepatocyte membranes was inhibited by EDTA, heat, and trypsin but not by serine proteinase inhibitors. These data show that the co-expression of TIMP-2, MMP-2, and MT-MMP-1 by HSCs does not lead to secretion of the activated form of MMP-2. Hepatocytes, which do not express MMP-2, TIMP-2, or MT-MMP-1, induce MMP-2 activation through a plasma membrane-dependent mechanism(s), thus suggesting that cell-cell interactions are involved in this process in vivo. Images Figure 1 Figure 2 Figure 3 PMID:9006321

  16. The biphasic nature of hypoxia-induced directional migration of activated human hepatic stellate cells.

    PubMed

    Novo, Erica; Povero, Davide; Busletta, Chiara; Paternostro, Claudia; di Bonzo, Lorenzo Valfrè; Cannito, Stefania; Compagnone, Alessandra; Bandino, Andrea; Marra, Fabio; Colombatto, Sebastiano; David, Ezio; Pinzani, Massimo; Parola, Maurizio

    2012-03-01

    Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

  17. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

    PubMed Central

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

    2015-01-01

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

  18. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

    PubMed

    Coll, Mar; El Taghdouini, Adil; Perea, Luis; Mannaerts, Inge; Vila-Casadesús, Maria; Blaya, Delia; Rodrigo-Torres, Daniel; Affò, Silvia; Morales-Ibanez, Oriol; Graupera, Isabel; Lozano, Juan José; Najimi, Mustapha; Sokal, Etienne; Lambrecht, Joeri; Ginès, Pere; van Grunsven, Leo A; Sancho-Bru, Pau

    2015-06-22

    Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

  19. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells

    PubMed Central

    Coll, Mar; Taghdouini, Adil El; Perea, Luis; Mannaerts, Inge; Vila-Casadesús, Maria; Blaya, Delia; Rodrigo-Torres, Daniel; Affò, Silvia; Morales-Ibanez, Oriol; Graupera, Isabel; Lozano, Juan José; Najimi, Mustapha; Sokal, Etienne; Lambrecht, Joeri; Ginès, Pere; van Grunsven, Leo A.; Sancho-Bru, Pau

    2015-01-01

    Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs. PMID:26096707

  20. Chrysin attenuates liver fibrosis and hepatic stellate cell activation through TGF-β/Smad signaling pathway.

    PubMed

    Balta, Cornel; Herman, Hildegard; Boldura, Oana Maria; Gasca, Ionela; Rosu, Marcel; Ardelean, Aurel; Hermenean, Anca

    2015-10-05

    We investigated the protective effect of chrysin on chronic liver fibrosis in mice and the potential mechanism underlying TGF-β1-mediated hepatic stellate cells (HSCs) activation on fibrogenesis. Experimental fibrosis was established by intraperitoneal injection of mice with 20% v/v, 2 ml/kg CCl4 twice a week, for 7 weeks. Mice were orally treated with 3 doses of chrysin (50, 100 and 200 mg/kg) or with vehicle as control. For the assessment of the spontaneous reversion of fibrosis, CCl4 treated animals were investigated after two weeks of recovery time. Silymarin was used as standard hepatoprotective flavonoid. Histopathological investigations showed that hepatic fibrosis grade was markedly reduced in the chrysin groups compared to the fibrotic one. Moreover, CCl4 activated HSCs induced an upregulation of smooth muscle actin (α-SMA), an increased number of TGF-β1 immunopositive cells and marked up-regulation of TGF-β1. α-SMA and TGF-β1 levels were significantly reduced in all chrysin treated groups in a dose-dependent manner, whereas the level of spontaneous reversal of fibrosis was lower compared to all flavonoid treated groups. Liver mRNA levels of Smad 2 in the 50, 100 and 200 mg/kg chrysin treated groups were significantly reduced by about 88.54%, 92.15% and 95.56% of the corresponding levels in the fibrosis mice group. The results were similar for mRNA levels of Smad 3. The protective response to silymarin was almost similar to that seen with the highest doses of chrysin. In this study, we have shown that chrysin has the efficacy to reverse CCl4-stimulated liver fibrosis by inhibition of HSCs activation and proliferation through TGF-β1/Smad pathway. These results suggest that chrysin may be useful in stopping or reversing the progression of liver fibrosis and might offer the possibility to develop a new therapeutic drug, useful in treatment of chronic liver diseases.

  1. Suberoylanilide hydroxamic acid suppresses hepatic stellate cells activation by HMGB1 dependent reduction of NF-κB1

    PubMed Central

    Wang, Wenwen; Yan, Min; Ji, Qiuhong; Lu, Jinbiao; Ji, Yuhua

    2015-01-01

    Hepatic stellate cells (HSCs) activation is essential to the pathogenesis of liver fibrosis. Exploring drugs targeting HSC activation is a promising anti-fibrotic strategy. In the present study, we found suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, prominently suppressed the activation phenotype of a human hepatic stellate cell line—LX2. The production of collagen type I and α-smooth muscle actin (α-SMA) as well as the proliferation and migration of LX2 cells were significantly reduced by SAHA treatment. To determine the molecular mechanisms underlying this suppression, genome wild gene regulation by SAHA was determined by Affymetrix 1.0 human cDNA array. Upon SAHA treatment, the abundance of 331 genes was up-regulated and 173 genes was down-regulated in LX2 cells. Bioinformatic analyses of these altered genes highlighted the high mobility group box 1 (HMGB1) pathway was one of the most relevant pathways that contributed to SAHA induced suppression of HSCs activation. Further studies demonstrated the increased acetylation of intracellular HMGB1 in SAHA treated HSCs, and this increasing is most likely to be responsible for SAHA induced down-regulation of nuclear factor kappa B1 (NF-κB1) and is one of the main underlying mechanisms for the therapeutic effect of SAHA for liver fibrosis. PMID:26557438

  2. Aucubin and its hydrolytic derivative attenuate activation of hepatic stellate cells via modulation of TGF-β stimulation.

    PubMed

    Lv, Pei-Yu; Feng, Han; Huang, Wei-Hua; Tian, Ying-Ying; Wang, Ya-Qin; Qin, Yu-Hua; Li, Xiao-Hui; Hu, Kai; Zhou, Hong-Hao; Ouyang, Dong-Sheng

    2017-03-01

    Eucommia ulmoides is an important traditional Chinese medicine and has been used as a tonic with a long history. Aucubin is an active component extracted from Eucommia ulmoides, which has liver-protection effects. However the mechanisms are still unclear. To investigate the inhibitory effects and the underlying mechanisms of aucubin on TGF-β1-induced activation of hepatic stellate cells and ECM deposition, Human hepatic stellate cells (LX-2 cells) were incubated with TGF-β1 to evaluate the anti-fibrotic effect of aucubin. Western blot was used to investigate the expression of α-SMA, Col I, Col III, MMP-2 and TIMP-1. ROS production was monitored using DCFH-DA probe, and NOX4 expression was detected by Real-time PCR. Results indicated that TGF-β1 stimulated the activation and ECM deposition of LX-2 cells. Compared with the control group, aucubin and aucubigenin both reduced the protein expression of α-SMA, Col I, Col III and MMP-2 in LX-2 cells. Aucubin and aucubigenin also suppressed the generation of ROS and down-regulated the NOX4 mRNA expression. Taken together, aucubin and aucubigenin both inhibit the activation and ECM deposition of LX-2 cells activated by TGF-β1. Aucubin and aucubigenin are potential therapeutic candidate drugs for liver fibrosis.

  3. Identification of cromolyn sodium as an anti-fibrotic agent targeting both hepatocytes and hepatic stellate cells.

    PubMed

    Choi, Joon-Seok; Kim, Jun Ki; Yang, Yoon Jung; Kim, Yeseul; Kim, Pilhan; Park, Sang Gyu; Cho, Eun-Young; Lee, Dae Ho; Choi, Jin Woo

    2015-12-01

    Liver fibrosis and cirrhosis, the late stage of fibrosis, are threatening diseases that lead to liver failure and patient death. Although aberrantly activated hepatic stellate cells (HSCs) are the main cause of disease initiation, the symptoms are primarily related to damaged hepatocytes. Thus, damaged hepatocytes, as well as HSCs, need to be simultaneously considered as therapeutic targets to develop more efficient treatments. Here, we suggest cromolyn sodium as an anti-fibrotic agent to commonly modulate hepatocytes and hepatic stellate cells. The differentially expressed genes from 6 normal and 40 cirrhotic liver tissues which were collected from GEO data were assessed by pharmacokinetic analysis using a connectivity map to identify agents that commonly revert abnormal hepatocytes and HSCs to normal conditions. Based on a series of analyses, a few candidates were selected. Candidates were tested in vitro to determine their anti-fibrotic efficacy on HSCs and hepatocytes. Cromolyn, which was originally developed as a mast cell stabilizer, showed the potential to ameliorate activated HSCs in vitro. The activation and collagen accumulation for HSC cell lines LX2 and HSC-T6 were reduced by 50% after cromolyn treatment at a low concentration without apoptosis. Furthermore, cromolyn treatment compromised the TGF-β-induced epithelial mesenchyme transition and replicative senescence rate of hepatocytes, which are generally associated with fibrogenesis. Taken together, cromolyn may be the basis for an effective cure for fibrosis and cirrhosis because it targets both HSCs and hepatocytes.

  4. Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

    PubMed

    Nagahara, Teruya; Shiraha, Hidenori; Sawahara, Hiroaki; Uchida, Daisuke; Takeuchi, Yasuto; Iwamuro, Masaya; Kataoka, Junro; Horiguchi, Shigeru; Kuwaki, Takeshi; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yamamoto, Kazuhide

    2015-09-01

    Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1

  5. 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

  6. 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.

  7. Resveratrol inhibits dimethylnitrosamine-induced hepatic fibrosis in rats.

    PubMed

    Lee, Eun-Sil; Shin, Mi-Ok; Yoon, Sik; Moon, Jeon-Ok

    2010-06-01

    Resveratrol, a phytoalexin found in grapes and red wines, has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of resveratrol on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Oral administration of resveratrol (20 mg/kg daily for 4 weeks) remarkably prevented the DMN-induced loss in body and liver weight, and inhibited the elevation of serum alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin levels. Resveratrol also increased serum albumin and hepatic glutathione levels and reduced the hepatic level of malondialdehyde due to its antioxidant effect. Furthermore, DMN-induced elevation of hydroxyproline content was reduced in the resveratrol treated rats, the result of which was consistent with the reduction in type I collagen mRNA expression and the histological analysis of liver tissue stained with Sirius red. The reduction in hepatic stellate cell activation, as assessed by alpha-smooth muscle actin staining, and the reduction in transforming growth factor-beta1 mRNA expression were associated with resveratrol treatment. In conclusion, resveratrol exhibited in vivo hepatoprotective and antifibrogenic effects against DMN-induced liver injury, suggesting that resveratrol may be useful in the prevention of the development of hepatic fibrosis.

  8. Association of interleukin-15-induced peripheral immune activation with hepatic stellate cell activation in persons coinfected with hepatitis C virus and HIV.

    PubMed

    Allison, Robert D; Katsounas, Antonios; Koziol, Deloris E; Kleiner, David E; Alter, Harvey J; Lempicki, Richard A; Wood, Brad; Yang, Jun; Fullmer, Brandie; Cortez, Karoll J; Polis, Michael A; Kottilil, Shyam

    2009-08-15

    Hepatic stellate cells (HSCs) mediate hepatitis C virus (HCV)-related liver fibrosis, and increased HSC activation in human immunodeficiency virus (HIV)/HCV coinfection may be associated with accelerated fibrosis. We examined the level of HSC activation in HIV/HCV-coinfected and HCV-monoinfected subjects and its relationship to the level of activation and gene expression of peripheral immune cells in coinfected subjects. HSC activation levels positively correlated with peripheral CD4+ and CD8+ T cell immune activation and were associated with enhanced interleukin-15 (IL-15) gene expression, suggesting a pathogenic role for IL-15-driven immunomediated hepatic fibrosis. Future strategies that reduce immune activation and HSC activation may delay progression of liver fibrosis.

  9. EZH2-mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis.

    PubMed

    Yang, Yang; Chen, Xiao-Xia; Li, Wan-Xia; Wu, Xiao-Qin; Huang, Cheng; Xie, Juan; Zhao, Yu-Xin; Meng, Xiao-Ming; Li, Jun

    2017-03-23

    EZH2, a histone H3 lysine-27-specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway-stimulated fibroblasts in vitro and in vivo by repressing Dkk-1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl4 -induced rat liver and primary HSCs as well as TGF-β1-treated HSC-T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF-β1-induced proliferation of HSC-T6 cells and the expression of α-SMA. In addition, knockdown of Dkk1 promoted TGF-β1-induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk-1 through trimethylation of H3K27me3 in TGF-β1-treated HSC-T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2-mediated repression of Dkk1 promotes the activation of Wnt/β-catenin pathway, which is an essential event for HSC activation.

  10. 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

  11. 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.

  12. Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

    PubMed Central

    Cao, Sheng; Yaqoob, Usman; Das, Amitava; Shergill, Uday; Jagavelu, Kumaravelu; Huebert, Robert C.; Routray, Chittaranjan; Abdelmoneim, Soha; Vasdev, Meher; Leof, Edward; Charlton, Michael; Watts, Ryan J.; Mukhopadhyay, Debabrata; Shah, Vijay H.

    2010-01-01

    PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor β (PDGFRβ) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-β–dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRβ-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions. PMID:20577048

  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. 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

  15. A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

    SciTech Connect

    Schnabl, Bernd Brandl, Katharina; Fink, Marina; Gross, Philipp; Taura, Kojiro; Gaebele, Erwin; Hellerbrand, Claus; Falk, Werner

    2008-10-17

    Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NF{kappa}B and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.

  16. 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

  17. 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.

  18. [Effects of desmopressin acetate and pituitrin on proliferation, contraction, and secretion of hepatic stellate cells].

    PubMed

    Li, X; Zhang, X T; Zhang, M Y; Wang, G C; Feng, H; Zhang, C Q

    Objective: To investigate the effects of desmopressin acetate and pituitrin on the proliferation, contraction, and secretion of hepatic stellate cells (HSCs). Methods: The human HSC cell line LX-2 was selected as the research model. And three groups were designed: blank control group, desmopressin acetate group (three subgroups: 1×10(-10)mol/L, 1×10(-9)mol/L, and 1×10(-8)mol/L desmopressin acetate), and pituitrin group (three subgroups: 0.1 U/L, 1.0 U/L, and 10.0 U/L pituitrin). Water-soluble tetrazolium salt (WST)-1 assay was used to evaluate cell proliferation; collagen gel contraction assay was used to assess cell contraction; enzyme-linked immunosorbent assay (ELISA) was used to identify cell secretion. The data was subjected to one-way analysis of variance. Results: (1) The results of WST-1 assay showed that the values of A450in three desmopressin acetate subgroups (1×10(-10)mol/L, 1×10(-9)mol/L, and 1×10(-8)mol/L) were 0.459±0.017, 0.467±0.024, and 0.436±0.015, respectively. And the values of A450 in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 0.495±0.011, 0.507±0.015, and 0.501±0.009, respectively. Compared with the control group, the desmopressin acetate at high concentration significantly inhibited the cell proliferation (P< 0.05), but the pituitrin at three different concentrations significantly promoted the cell proliferation (P< 0.05). (2) The collagen gel area ratios in three desmopressin acetate subgroups (1×10(-10)mol/L, 1×10(-9)mol/L, and 1×10(-8)mol/L) were 77.07±4.42, 75.85±3.70, and 72.74±3.92, respectively. And the collagen gel area ratios in three pituitrin subgroups (0.1 U/L, 1.0 U/L, and 10.0 U/L) were 57.83±3.96, 50.28±6.69, and 43.56±7.68, respectively. Compared with the control group, the pituitrin at three different concentrations significantly reduced the collagen gel area (P< 0.01). (3) The matrix metalloproteinase(MMP)-2 concentrations in three desmopressin acetate subgroups (1×10(-10)mol/L, 1

  19. 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.

  20. Epigallocatechin-3-gallate, a green-tea polyphenol, suppresses Rho signaling in TWNT-4 human hepatic stellate cells.

    PubMed

    Higashi, Nobuhiko; Kohjima, Motoyuki; Fukushima, Marie; Ohta, Satoshi; Kotoh, Kazuhiro; Enjoji, Munechika; Kobayashi, Naoya; Nakamuta, Makoto

    2005-06-01

    Epigallocatechin-3-gallate (EGCG), a major constituent of the polyphenoids in green tea, has been reported to possess a wide range of biologic activities, including antifibrogenesis. Activated hepatic stellate cells (HSCs) are central to hepatic fibrosis, and Rho (a small GTPase)-signaling pathways have been implicated in the activation and proliferation of HSCs. In this study, we investigated the effect of EGCG on Rho-signaling pathways in activated human HSC-derived TWNT-4 cells. EGCG inhibited stress-fiber formation, an indicator of Rho activation, and changed the distribution of alpha-smooth-muscle actin. These inhibitory effects of EGCG were restored by overexpression of constitutively active Rho. A pull-down assay revealed that activated Rho (GTP-bound state) was strongly inhibited by ECGC and accompanied by suppressed phosphorylation of focal adhesion kinase, which is a regulator of Rho-signaling pathways. 5-Bromo-2'-deoxy-uridine incorporation demonstrated that ECGC (100 micromol/L suppressed cell growth by 80%, and terminal deoxynucleotidyl transferase viotin-deoxyruidine triphosphate nick end-labeling revealed that EGCG (100 micromol/L) caused apoptosis in half of the total cells. EGCG also strongly inhibited lysophoaphatidic acid (an activator of Rho) and induced phosphorylation of mitogen-activated protein kinases (Erk1/2, c-jun kinase, and p38). These findings demonstrate that EGCG regulates the structure and growth of HSCs by way of Rho-signaling pathways and suggest that EGCG has therapeutic potential in the setting of liver fibrosis.

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

    PubMed

    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.

  2. 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

  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 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

  5. 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.

  6. Microarray data and pathway analyses for primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Borradaile, Nica M

    2017-02-01

    As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided.

  7. Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages.

    PubMed

    Olaso, Elvira; Arteta, Beatriz; Benedicto, Aitor; Crende, Olatz; Friedman, Scott L

    2011-12-01

    Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-β1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.

  8. 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

  9. 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

  10. Protective effects of L-carnosine on CCl4 -induced hepatic injury in rats.

    PubMed

    Alsheblak, Mehyar Mohammad; Elsherbiny, Nehal M; El-Karef, Amro; El-Shishtawy, Mamdouh M

    2016-03-01

    The present study was undertaken to investigate the possible protective effect of L-carnosine (CAR), an endogenous dipeptide of alanine and histidine, on carbon tetrachloride (CCl4)-induced hepatic injury. Liver injury was induced in male Sprague-Dawley rats by intraperitoneal (i.p.) injections of CCl4, twice weekly for six weeks. CAR was administered to rats daily, at dose of 250 mg/kg, i.p. At the end of six weeks, blood and liver tissue specimens were collected. Results show that CAR treatment attenuated the hepatic morphological changes, necroinflammation and fibrosis induced by CCl4, as indicated by hepatic histopathology scoring. In addition, CAR treatment significantly reduced the CCl4-induced elevation of liver-injury parameters in serum. CAR treatment also combatted oxidative stress; possibly by restoring hepatic nuclear factor erythroid 2-related factor 2 (Nrf-2) levels. Moreover, CAR treatment prevented the activation of hepatic stellate cells (HSCs), as indicated by reduced α-smooth muscle actin (α-SMA) expression in the liver, and decreased hepatic inflammation as demonstrated by a reduction in hepatic tumor necrosis factor-α (TNF-α) and restoration of interleukin-10 (IL-10) levels. In conclusion, CCl4-induced hepatic injury was alleviated by CAR treatment. The results suggest that these beneficial, protective effects are due, at least in part, to its anti-oxidant, anti-inflammatory and anti-fibrotic activities.

  11. 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.

  12. 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

    2015-07-08

    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.

  13. The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx.

    PubMed

    Failli, P; Ruocco, C; De Franco, R; Caligiuri, A; Gentilini, A; Giotti, A; Gentilini, P; Pinzani, M

    1995-11-01

    Platelet-derived growth factor (PDGF) is a key mitogen for hepatic stellate cells (HSC) and has been shown to be implicated in liver tissue repair and fibrogenesis. In this study the relationship between PDGF-induced intracellular Ca2+ concentration ([Ca2+]i) increase and mitogenesis in cultured human HSC was evaluated. In high-density cell cultures (80-90% subconfluence), PDGF induced a significant increase in [Ca2+]i, characterized by a short-lasting peak phase, which was followed by a long-lasting plateau phase. The plateau phase was abolished in the absence of extracellular Ca2+. However, in low-density cell cultures (30-40% subconfluence), the plateau phase was absent or markedly less pronounced. In parallel sets of experiments, PDGF was significantly less effective in inducing mitogenesis in low-density cell cultures than in high-density cell cultures and was totally ineffective in the absence of extracellular Ca2+. These results suggest that 1) spatial and time dynamics of PDGF-induced [Ca2+]i increase are dependent on cell density and 2) PDGF-induced mitogenesis requires extracellular Ca2+ influx.

  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. The XBP1 Arm of the Unfolded Protein Response Induces Fibrogenic Activity in Hepatic Stellate Cells Through Autophagy

    PubMed Central

    Kim, Rosa S.; Hasegawa, Daisuke; Goossens, Nicolas; Tsuchida, Takuma; Athwal, Varinder; Sun, Xiaochen; Robinson, Christopher L.; Bhattacharya, Dipankar; Chou, Hsin-I; Zhang, David Y.; Fuchs, Bryan C.; Lee, Youngmin; Hoshida, Yujin; Friedman, Scott L.

    2016-01-01

    Autophagy and the unfolded protein response (UPR) both promote activation of hepatic stellate cells (HSC), however the link between the two stimuli remains unclear. Here we have explored the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways and sought to establish the interdependence between autophagy and the UPR during HSC activation. XBP1 induction accompanied both culture-based HSC activation and ER stress induced by tunicamycin. Ectopic overexpression of XBP1 induced collagen 1-alpha expression in HSCs, which was inhibited by knockdown of ATG7, a critical autophagy mediator. Genome-wide transcriptomic profiling indicated an upregulation of collagen synthesis pathways, but not of the transforming growth factor (TGF)-b pathway, a canonical fibrogenic driver, suggesting that XBP1 activates a specific subset of fibrogenesis pathways independent of TGF-β1. XBP1 target gene signatures were significantly induced in rodent liver fibrosis models (n = 3–5) and in human samples of non-alcoholic fatty liver disease (NAFLD) (n = 72–135). Thus, XBP1-mediated UPR contributes to fibrogenic HSC activation and is functionally linked to cellular autophagy. PMID:27996033

  16. Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt

    PubMed Central

    Han, Chang Yeob; Koo, Ja Hyun; Kim, Sung Hoon; Gardenghi, Sara; Rivella, Stefano; Strnad, Pavel; Hwang, Se Jin; Kim, Sang Geon

    2016-01-01

    Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin. PMID:28004654

  17. Activated hepatic stellate cells play pivotal roles in hepatocellular carcinoma cell chemoresistance and migration in multicellular tumor spheroids

    PubMed Central

    Song, Yeonhwa; Kim, Se-hyuk; Kim, Kang Mo; Choi, Eun Kyung; Kim, Joon; Seo, Haeng Ran

    2016-01-01

    Most Hepatocellular carcinoma (HCC) are resistant to conventional chemotherapeutic agents and remain an unmet medical need. Recently, multiple studies on the crosstalk between HCC and their tumor microenvironment have been conducted to overcome chemoresistance in HCC. In this study, we formed multicellular tumor spheroids (MCTS) to elucidate the mechanisms of environment-mediated chemoresistance in HCC. We observed that hepatic stellate cells (HSCs) in MCTS significantly increased the compactness of spheroids and exhibited strong resistance to sorafenib and cisplatin relative to other types of stromal cells. Increased collagen 1A1 (COL1A1) expression was apparent in activated HSCs but not in fibroblasts or vascular endothelial cells in MCTS. Additionally, COL1A1 deficiency, which was increased by co-culture with HSCs, decreased the cell-cell interactions and thereby increased the therapeutic efficacy of anticancer therapies in MCTS. Furthermore, losartan, which can inhibit collagen I synthesis, attenuated the compactness of spheroids and increased the therapeutic efficacy of anticancer therapies in MCTS. Meanwhile, activated HSCs facilitated HCC migration by upregulating matrix metallopeptidase 9 (MMP9) in MCTS. Collectively, crosstalk between HCC cells and HSCs promoted HCC chemoresistance and migration by increasing the expression of COL1A1 and MMP9 in MCTS. Hence, targeting HSCs might represent a promising therapeutic strategy for liver cancer therapy. PMID:27853186

  18. 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.

  19. Angiogenin Secretion From Hepatoma Cells Activates Hepatic Stellate Cells To Amplify A Self-Sustained Cycle Promoting Liver Cancer

    PubMed Central

    Bárcena, Cristina; Stefanovic, Milica; Tutusaus, Anna; Martinez-Nieto, Guillermo A.; Martinez, Laura; García-Ruiz, Carmen; de Mingo, Alvaro; Caballeria, Juan; Fernandez-Checa, José C.; Marí, Montserrat; Morales, Albert

    2015-01-01

    Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management. PMID:25604905

  20. Long non-coding RNA PVT1 activates hepatic stellate cells through competitively binding microRNA-152

    PubMed Central

    Zheng, Jianjian; Yu, Fujun; Dong, Peihong; Wu, Limei; Zhang, Yuan; Hu, Yanwei; Zheng, Lei

    2016-01-01

    Epithelial-mesenchymal transition (EMT) process is considered as a key event in the activation of hepatic stellate cells (HSCs). Hedgehog (Hh) pathway is known to be required for EMT process. Long non-coding RNAs (lncRNAs) have been reported to be involved in a wide range of biological processes. Plasmacytoma variant translocation 1 (PVT1), a novel lncRNA, is often up-regulated in various human cancers. However, the role of PVT1 in liver fibrosis remains undefined. In this study, PVT1 was increased in fibrotic liver tissues and activated HSCs. Depletion of PVT1 attenuated collagen deposits in vivo. In vitro, PVT1 down-regulation inhibited HSC activation including the reduction of HSC proliferation, α-SMA and type I collagen. Further studies showed that PVT1 knockdown suppressed HSC activation was through inhibiting EMT process and Hh pathway. Patched1 (PTCH1), a negative regulator factor of Hh pathway, was enhanced by PVT1 knockdown. PTCH1 demethylation caused by miR-152 was responsible for the effects of PVT1 knockdown on PTCH1 expression. Notably, miR-152 inhibitor reversed the effects of PVT1 knockdown on HSC activation. Luciferase reporter assays and pull-down assays showed a direct interaction between miR-152 and PVT1. Collectively, we demonstrate that PVT1 epigenetically down-regulates PTCH1 expression via competitively binding miR-152, contributing to EMT process in liver fibrosis. PMID:27588491

  1. Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53.

    PubMed

    Liu, Wen-Ting; Jing, Ying-Ying; Yu, Guo-feng; Chen, Hong; Han, Zhi-peng; Yu, Dan-Dan; Fan, Qing-Min; Ye, Fei; Li, Rong; Gao, Lu; Zhao, Qiu-Dong; Wu, Meng-Chao; Wei, Li-Xin

    2016-01-01

    The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.

  2. 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

  3. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation.

    PubMed

    Harvey, Wendy A; Jurgensen, Kimberly; Pu, Xinzhu; Lamb, Cheri L; Cornell, Kenneth A; Clark, Reilly J; Klocke, Carolyn; Mitchell, Kristen A

    2016-02-17

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

  4. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation

    PubMed Central

    Harvey, Wendy A.; Jurgensen, Kimberly; Pu, Xinzhu; Lamb, Cheri L.; Cornell, Kenneth A.; Clark, Reilly J.; Klocke, Carolyn; Mitchell, Kristen A.

    2016-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10 nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multifaceted mechanism. PMID:26860701

  5. Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells.

    PubMed

    Martucci, Renata B; Ziulkoski, Ana L; Fortuna, Vitor A; Guaragna, Regina M; Guma, Fátima C R; Trugo, Luiz C; Borojevic, Radovan

    2004-05-15

    Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

  6. Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation

    PubMed Central

    Bárcena, Cristina; Stefanovic, Milica; Tutusaus, Anna; Joannas, Leonel; Menéndez, Anghara; García-Ruiz, Carmen; Sancho-Bru, Pau; Marí, Montserrat; Caballeria, Joan; Rothlin, Carla V.; Fernández-Checa, José C.; de Frutos, Pablo García; Morales, Albert

    2015-01-01

    Background & Aims Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase, and its ligand Gas6 are involved in cell differentiation, immune response and carcinogenesis. Methods HSCs were obtained from wild type and Axl−/− mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated wild type and Axl−/− mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. Results In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl KO mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. Conclusions: The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management. PMID:25908269

  7. Thrombospondin 1 acts as a strong promoter of transforming growth factor β effects via two distinct mechanisms in hepatic stellate cells

    PubMed Central

    Breitkopf, K; Sawitza, I; Westhoff, J H; Wickert, L; Dooley, S; Gressner, A M

    2005-01-01

    Background and aims: Thrombospondin 1 (TSP-1) is an important activator of latent transforming growth factor β (TGF-β) but little is known of the expression patterns and functions of TSP-1 in liver cells. We therefore analysed if and how TSP-1 acts on TGF-β during fibrogenesis. Methods and results: Using reverse transcription-polymerase chain reaction, we demonstrated that hepatocytes from normal liver expressed no TSP-1 mRNA whereas Kupffer cells and sinusoidal endothelial cells did. TSP-1 mRNA and protein were detected in quiescent and activated cultured hepatic stellate cells (HSC) and TSP-1 expression was highly inducible by platelet derived growth factor BB (PDGF-BB) and, to a lesser extent, by tumour necrosis factor α in activated HSC. Furthermore, addition of PDGF-BB directly led to enhanced TGF-β mRNA expression and a TSP-1 dependent increase in TGF-β/Smad signalling. Using either a peptide specifically blocking the interaction of TSP-1 with latent TGF-β or antibodies against TSP-1 not only abrogated activation of latent TGF-β but also reduced the effects of the active dimer itself. Conclusions: Our data suggest that TSP-1 expression is important for TGF-β effects and that it is regulated by the profibrogenic mediator PDGF-BB in HSC. Furthermore, the presence of TSP-1 seems to be a prerequisite for effective signal transduction by active TGF-β not only in rat HSC but also in other cell types such as human dermal fibroblasts. PMID:15831915

  8. 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

  9. Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

    PubMed

    Salloum, Shadi; Holmes, Jacinta A; Jindal, Rohit; Bale, Shyam S; Brisac, Cynthia; Alatrakchi, Nadia; Lidofsky, Anna; Kruger, Annie J; Fusco, Dahlene N; Luther, Jay; Schaefer, Esperance A; Lin, Wenyu; Yarmush, Martin L; Chung, Raymond T

    2016-12-01

    Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells.

  10. 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

  11. Anti-fibrotic effects of a methylenedioxybenzene compound, CW209292 on dimethylnitrosamine-induced hepatic fibrosis in rats.

    PubMed

    Oh, Se-Woong; Kim, Dae-Hoon; Ha, Jong-Ryul; Kim, Dae-Yong

    2009-08-01

    A series of methylenedioxybenzene compounds were synthesized and found to have hepatoprotective effects in chemical-induced hepatotoxicity models. The purpose of the present study was to investigate the anti-fibrotic effects of a synthetic methylenedioxybenzene compound, CW209292, using the dimethylnitrosamine (DMN)-induced chronic liver injury model in rats. Liver injuries were induced in Sprague Dawley rats by injection of DMN (intraperitoneally, 10 microl/kg) 3 times per week for 4 weeks. The rats were treated with CW209292 (per os, 25 or 75 mg/kg/d) for 4 weeks. Treatment of rats with DMN for 4 weeks resulted in significant decreases in serum albumin levels, whereas concomitant treatment with CW209292 prevented these decreases. CW209292 treatment also shortened prothrombin time prolonged by DMN, providing evidence that the agent was active in preserving liver function against DMN insult. DMN treatment caused marked increases in plasma bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), and hyaluronic acid levels; CW209292 treatment reversed these increases. CW209292 also significantly reduced hepatic hydroxyproline content as well as hepatic fibrosis and inflammation in histological examination. Additionally, immunochemically detectable hepatic collagen type IV and alpha-smooth muscle actin levels were decreased by CW209292 treatment. Proliferation of hepatic stellate cells isolated from DMN-treated rats was inhibited by CW209292. Furthermore, tumor growth factor (TGF)-beta1 mRNA expression was increased in DMN-treated rats, whereas CW209292 treatment prevented these increases. These results suggest that CW209292 exhibits anti-fibrotic effects in Sprague Dawley rats with DMN-induced hepatic fibrosis by blocking the mRNA expression of TGF-beta1 and subsequent inhibition of the proliferation of hepatic stellate cells.

  12. 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

  13. Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment

    PubMed Central

    Das, Amitava; Shergill, Uday; Thakur, Lokendra; Sinha, Sutapa; Urrutia, Raul; Mukhopadhyay, Debabrata

    2010-01-01

    Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 μg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 μM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These

  14. 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.

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

    PubMed

    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

    2016-07-13

    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.

  16. 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

  17. ROLE OF METHIONINE ADENOSYLTRANSFERASE α2 AND β PHOSPHORYLATION AND STABILIZATION IN HUMAN HEPATIC STELLATE CELL TRANS-DIFFERENTIATION

    PubMed Central

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

    2016-01-01

    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. PMID:25294683

  18. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

    PubMed

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A

    2015-01-01

    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

  19. Autophagy induced by purple pitanga (Eugenia uniflora L.) extract triggered a cooperative effect on inducing the hepatic stellate cell death.

    PubMed

    Denardin, Cristiane C; Martins, Leo A M; Parisi, Mariana M; Vieira, Moema Queiroz; Terra, Silvia R; Barbé-Tuana, Florencia M; Borojevic, Radovan; Vizzotto, Márcia; Emanuelli, Tatiana; Guma, Fátima Costa Rodrigues

    2017-04-01

    Activated hepatic stellate cells (HSC) are the major source of collagen I in liver fibrosis. Eugenia uniflora L. is a tree species that is widely distributed in South America. E. uniflora L. fruit-popularly known as pitanga-has been shown to exert beneficial properties. Autophagy contributes to the maintenance of cellular homeostasis and survival under stress situation, but it has also been suggested to be an alternative cell death pathway. Mitochondria play a pivotal role on signaling cell death. Mitophagy of damaged mitochondria is an important cell defense mechanism against organelle-mediated cell death signaling. We previously found that purple pitanga extract induced mitochondrial dysfunction, cell cycle arrest, and death by apoptosis and necrosis in GRX cells, a well-established activated HSC line. We evaluated the effects of 72-h treatment with crescent concentrations of purple pitanga extract (5 to 100 μg/mL) on triggering autophagy in GRX cells, as this is an important mechanism to cells under cytotoxic conditions. We found that all treated cells presented an increase in the mRNA expression of autophagy-related protein 7 (ATG7). Concomitantly, flow cytometry and ultrastructural analysis of treated cells revealed an increase of autophagosomes/autolysosomes that consequentially led to an increased mitophagy. As purple pitanga extract was previously found to be broadly cytotoxic to GRX cells, we postulated that autophagy contributes to this scenario, where cell death seems to be an inevitable fate. Altogether, the effectiveness on inducing activated HSC death can make purple pitanga extract a good candidate on treating liver fibrosis.

  20. Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

    PubMed

    Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

    2015-11-12

    Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.

  1. Differential Regulation of TGF-β/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi

    2012-01-01

    Current evidence suggests that regulation of extracellular matrix (ECM) accumulation by fibrogenic transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) signals involves different mechanisms in acute and chronic liver injuries, even though hepatic stellate cells (HSC) are the principal effecter in both cases. As a result of chronic liver damage, HSC undergo progressive activation to become myofibroblasts (MFB)-like cells. Our current review will discuss the differential regulation of TGF-β signaling between HSC and MFB in vitro and in vivo. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad-homology (MH) 1 and MH2 domains. TGF-β type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create COOH-terminally (C), linker (L), or dually (L/C) phosphorylated (p) isoforms. After acute liver injury, TGF-β and PDGF synergistically promote collagen synthesis in the activated HSC via pSmad2L/C and pSmad3L/C pathways. To avoid unlimited ECM deposition, Smad7 induced by TGF-β negatively regulates the fibrogenic TGF-β signaling. In contrast, TGF-β and PDGF can transmit the fibrogenic pSmad2L/C and mitogenic pSmad3L signals in MFB throughout chronic liver injury, because Smad7 cannot be induced by the pSmad3L pathway. This lack of Smad7 induction might lead to constitutive fibrogenesis in MFB, which eventually develop into accelerated liver fibrosis.

  2. Alteration of N-glycoproteins/N-glycosites in human hepatic stellate cells activated with transforming growth factor-β1.

    PubMed

    Qin, Y; Wang, Q; Zhong, Y; Zhao, F; Wu, F; Wang, Y; Ma, T; Liu, C; Bian, H; Li, Z

    2016-03-20

    Proteins N-glycosylation is significantly increased in the activated human hepatic stellate cells (HSCs) stimulated by transforming growth factor-β1 (TGF-β1) compared to the quiescent HSCs according to our previous study. However, little is known about the alteration of N-glycoprotein profiles in the activated HSCs. Profiles of N-glycopeptides / N-glycoproteins / N-glycosites in LX-2 cells, with and without activation by TGF-β1, were identified and compared using hydrazide chemistry enrichment coupled with liquid chromatography - mass spectrometry analysis. Western blot and immunohistochemistry were further used for validation. A total of 103 non-redundant N-glycopeptides, with 107 glycosylation sites from 86 N-glycoproteins, were identified in activated and quiescent LX-2 cells respectively. Among these, 23 proteins were known N-glycoproteins, and 58 were newly identified N-glycoproteins. In addition, 43 proteins (e.g., pigment epithelium-derived factor and clathrin heavy chain 1) were solely identified or up-regulated in the activated LX-2 cells, which participated in focal adhesion and glycosaminoglycan degradation pathways and were involved in interaction clusters of cytoskeletal proteins (e.g., myosin light chains and keratins). The increased expression of glucosamine (N-acetyl)-6-sulfatase and phospholipase C beta 2 and the decreased expression of zinc finger and BTB domain-containing protein 1 were validated in the activated compared to the quiescent LX-2 cells. In conclusion, increased expression of N-glycoproteins and N-glycosites play important roles in cellular contractility, signal transduction, and responses to stimuli in the activated HSCs, which might provide useful information for discovering novel molecular mechanism of HSC activation and therapeutic targets in liver fibrosis.

  3. Cyanidin-3-O-β-glucoside Purified from Black Rice Protects Mice against Hepatic Fibrosis Induced by Carbon Tetrachloride via Inhibiting Hepatic Stellate Cell Activation.

    PubMed

    Jiang, Xinwei; Guo, Honghui; Shen, Tianran; Tang, Xilan; Yang, Yan; Ling, Wenhua

    2015-07-15

    This study investigated whether cyanidin-3-O-β-glucoside (Cy-3-G), a predominant anthocyanin, could exert a protective role on liver injury and its further mechanisms of the anti-fibrosis actions in mice. The results demonstrated that the treatment of Cy-3-G (800 mg/kg diet) for 8 weeks significantly attenuated hepatotoxicity and fibrosis in carbon tetrachloride (CCl4) administered mice. Cy-3-G strongly down-regulated the expression of α-smooth muscle actin (α-SMA), desmin, and matrix metalloproteinase (MMPs), which showed its suppression effect on the activation of hepatic stellate cells (HSCs). In addition, Cy-3-G favorably regulated oxidative stress and apoptosis in liver. Furthermore, Cy-3-G ameliorated the infiltration of inflammatory cells such as neutrophils and leukocytes and meanwhile suppressed the production of pro-inflammatory cytokines and growth factors. In conclusion, daily intake of Cy-3-G could prevent liver fibrosis progression in mice induced by CCl4 through inhibiting HSC activation, which provides a basis for clinical practice of liver fibrosis prevention.

  4. Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/smad pathway

    PubMed Central

    HUANG, WEIJUAN; LI, LIN; TIAN, XIAOPENG; YAN, JINJIN; YANG, XINZHENG; WANG, XINLONG; LIAO, GUOZHEN; QIU, GENQUAN

    2015-01-01

    Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti-fibrotic effect by mediating the transforming growth factor β (TGF-β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF-β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H-proline incorporation assay and expression of α-smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF-β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI-1) and urokinase-type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5–80 μg/ml) significantly inhibited fetal bovine serum-induced cell proliferation in a dose-dependent manner. Cell invasion and activation of HSCs induced by TGF-β1 were disrupted by treatment with APE in a dose-dependent manner. TGF-β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF-β1-induced reduction in Smad-7 expression in a dose-dependent manner. The results further indicated that APE treatment not only

  5. The expression of HIV-1 Vpu in monocytes causes increased secretion of TGF-β that activates profibrogenic genes in hepatic stellate cells.

    PubMed

    Patel, Paresh; Khan, Nabab; Rani, Manjusha; Gupta, Deepti; Jameel, Shahid

    2014-01-01

    There is faster progression to fibrosis in persons with liver injury who are also infected with HIV. Other reports have suggested that HIV can directly infect and activate stellate cells, and the viral Tat and gp160 proteins also induce profibrogenic factors from peripheral blood mononuclear cells (PBMCs). We tested the role of HIV-1 Vpu accessory protein in promoting profibrogenic activation of hepatic stellate cells. Human stellate LX2 cells were cocultured with human monocytic U937 cells stably expressing the Vpu protein or latently infected U1 cells knocked down for Vpu expression, LX2 cells were also cultured with the supernatants from these cells. The expression of profibrogenic markers was evaluated in LX2 cells usingquantitative reverse transcription polymerase chain reaction (qRT-PCR),western blotting, immunofluorescence,flow cytometry and ELISA were used to confirm and quantitate protein expression. Monocytic cells expressing Vpu increased the expression of profibrogenic markers in LX2 cells. The culture supernatants of these cells contained increased levels of transforming growth factor beta (TGF-β), which correlated with increased activity of the AP-1 transcription factor. Antibodies against TGF-β or a TGF-β receptor inhibitor (SB431452) reversed Vpu-mediated profibrogenic activation of LX2 cells, suggesting that TGF-β mediated these effects. The cytokine macrophage migration inhibitory factor (MIF) attenuated Vpu-mediated TGF-β secretion and profibrogenic effects on LX2 cells. Besides its other roles in pathogenesis, Vpu is likely to contribute to hepatic fibrosis through this hitherto unknown mechanism.

  6. Activated hepatic stellate cells impair NK cell anti-fibrosis capacity through a TGF-β-dependent emperipolesis in HBV cirrhotic patients.

    PubMed

    Shi, Jijing; Zhao, Juanjuan; Zhang, Xin; Cheng, Yongqian; Hu, Jinhua; Li, Yuanyuan; Zhao, Xin; Shang, Qinghua; Sun, Yanling; Tu, Bo; Shi, Lei; Gao, Bin; Wang, Fu-Sheng; Zhang, Zheng

    2017-03-14

    Natural killer (NK) cells can induce liver fibrosis remission by killing hepatic stellate cells (HSCs) and producing interferon (IFN)-γ in a mouse model; however, their anti-fibrotic immune-characteristics and regulatory mechanisms by HSCs remain to be determined, especially in livers from HBV-infected liver cirrhosis (LC) patients. We analyzed frequency, phenotype and anti-fibrotic function of hepatic and peripheral NK subsets in 43 HBV-LC patients. We found that hepatic NK subsets from LC patients displayed a decreased frequency, activation status and anti-fibrotic activity compared with those from chronic hepatitis B patients, which were mainly mediated by increased intrahepatic tumour-growth factor (TGF)-β because blockade of TGF-β significantly reversed NK anti-fibrotic function in vitro. In vivo, hepatic NK cells were enriched in proximity to the α-smooth muscle actin (α-SMA+) area within mild fibrosis regions; while in severe fibrotic areas, they were either directly attached to or separated from the α-SMA+ region. NK cells from LC patients could enter HSCs to form emperipolesis (a cell-in-cell structure) and become apoptotic; anti-TGF-β treatment ameliorated this emperipolesis. This finding suggested a novel mechanism by which activated HSCs impair NK cells' anti-fibrosis capacity through a TGF-β-dependent emperipolesis in LC patients, providing an anti-fibrotic rational by enhancing NK cell activity.

  7. Activated hepatic stellate cells impair NK cell anti-fibrosis capacity through a TGF-β-dependent emperipolesis in HBV cirrhotic patients

    PubMed Central

    Shi, Jijing; Zhao, Juanjuan; Zhang, Xin; Cheng, Yongqian; Hu, Jinhua; Li, Yuanyuan; Zhao, Xin; Shang, Qinghua; Sun, Yanling; Tu, Bo; Shi, Lei; Gao, Bin; Wang, Fu-Sheng; Zhang, Zheng

    2017-01-01

    Natural killer (NK) cells can induce liver fibrosis remission by killing hepatic stellate cells (HSCs) and producing interferon (IFN)-γ in a mouse model; however, their anti-fibrotic immune-characteristics and regulatory mechanisms by HSCs remain to be determined, especially in livers from HBV-infected liver cirrhosis (LC) patients. We analyzed frequency, phenotype and anti-fibrotic function of hepatic and peripheral NK subsets in 43 HBV-LC patients. We found that hepatic NK subsets from LC patients displayed a decreased frequency, activation status and anti-fibrotic activity compared with those from chronic hepatitis B patients, which were mainly mediated by increased intrahepatic tumour-growth factor (TGF)-β because blockade of TGF-β significantly reversed NK anti-fibrotic function in vitro. In vivo, hepatic NK cells were enriched in proximity to the α-smooth muscle actin (α-SMA+) area within mild fibrosis regions; while in severe fibrotic areas, they were either directly attached to or separated from the α-SMA+ region. NK cells from LC patients could enter HSCs to form emperipolesis (a cell-in-cell structure) and become apoptotic; anti-TGF-β treatment ameliorated this emperipolesis. This finding suggested a novel mechanism by which activated HSCs impair NK cells’ anti-fibrosis capacity through a TGF-β-dependent emperipolesis in LC patients, providing an anti-fibrotic rational by enhancing NK cell activity. PMID:28291251

  8. 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.

  9. Salvianolic Acid B Attenuates Rat Hepatic Fibrosis via Downregulating Angiotensin II Signaling

    PubMed Central

    Li, Shu; Wang, Lina; Yan, Xiuchuan; Wang, Qinglan; Tao, Yanyan; Li, Junxia; Peng, Yuan; Liu, Ping; Liu, Chenghai

    2012-01-01

    The renin-angiotensin system (RAS) plays an important role in hepatic fibrosis. Salvianolic acid B (Sal B), one of the water-soluble components from Radix Salviae miltiorrhizae, has been used to treat hepatic fibrosis, but it is still not clear whether the effect of Sal B is related to angiotensin II (Ang II) signaling pathway. In the present study, we studied Sal B effect on rat liver fibrosis and Ang-II related signaling mediators in dimethylnitrosamine-(DMN-) induced rat fibrotic model in vivo and Ang-II stimulated hepatic stellate cells (HSCs) in vitro, with perindopril or losartan as control drug, respectively. The results showed that Sal B and perindopril inhibited rat hepatic fibrosis and reduced expression of Ang II receptor type 1 (AT1R) and ERK activation in fibrotic liver. Sal B and losartan also inhibited Ang II-stimulated HSC activation including cell proliferation and expression of type I collagen I (Col-I) and α-smooth muscle actin (α-SMA) production in vitro, reduced the gene expression of transforming growth factor beta (TGF-β), and downregulated AT1R expression and ERK and c-Jun phosphorylation. In conclusion, our results indicate that Sal B may exert an antihepatic fibrosis effect via downregulating Ang II signaling in HSC activation. PMID:23243430

  10. [Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells].

    PubMed

    Hao, L S; Liu, Y L; Zhang, G L; Chen, J; Song, X J; Wang, Y L; Wang, J; Jin, L M

    2017-01-20

    Objective: To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro. Methods: The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca(2+) concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups. Results: Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen

  11. 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

  12. Antihepatofibrotic Effects of Aqueous Extract of Prunella vulgaris on Carbon Tetrachloride-Induced Hepatic Fibrosis in Rats.

    PubMed

    Hu, Yi-Xiang; Yu, Chen-Huan; Wu, Fang; Yu, Wen-Ying; Zhong, Yu-Sen; Ying, Hua-Zhong; Yu, Bing

    2016-01-01

    Prunella vulgaris has been widely used in the folk medicine of Northeastern Asian countries for the treatment of acute liver injury and infectious hepatitis. In the present study, the protective effect of aqueous extract from P. vulgaris was investigated on carbon tetrachloride-induced hepatic fibrosis in vivo. Our data showed that the administration of aqueous extract from P. vulgaris at doses of 50, 100, and 200 mg/kg significantly reduced the elevated serum levels of alanine aminotransferase, aspartate aminotransferase, type III precollagen, and hyaluronic acid in rats with hepatic fibrosis. In addition, aqueous extract from P. vulgaris also reduced the incidence of liver lesions and the formation of fibrous septa, and remarkably decreased the serum levels of inflammatory cytokines, platelet derived growth factor, interleukin-4, interleukin-8, and tumor necrosis factor alpha. Furthermore, aqueous extract from P. vulgaris significantly inhibited the activation of hepatic stellate cells by regulating the expression of α smooth muscle actin, transforming growth factor β 1, and smad2 and also decreased the deposition of extracellular matrix proteins via regulating the expressions of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-2,-13. Real-time polymerase chain reaction further revealed that post-treatment with aqueous extract from P. vulgaris decreased the elevated levels of miR-34a and miR-199a-5p in hepatic fibrosis rats. These results demonstrated that aqueous extract from P. vulgaris alleviates carbon tetrachloride-induced hepatic fibrosis by inhibiting the activation of hepatic stellate cells, promoting collagenolysis and regulating fibrosis-related microRNAs.

  13. Ferric nitrilotriacetate (Fe-NTA)-induced reactive oxidative species protects human hepatic stellate cells from apoptosis by regulating Bcl-2 family proteins and mitochondrial membrane potential

    PubMed Central

    Liu, Mei; Li, Shu-Jie; Xin, Yong-Ning; Ji, Shu-Sheng; Xie, Rui-Jin; Xuan, Shi-Ying

    2015-01-01

    Reactive oxidative species (ROS)-induced apoptosis of human hepatic stellate (HSC) is one of the treatments for liver fibrosis. However, how ROS (reactive oxygen species) affect HSC apoptosis and liver fibrosis is still unknown. In our study, ROS in human HSC cell line LX-2 was induced by ferric nitrilotriacetate (Fe-NTA) and assessed by superoxide dismutase (SOD) activity and methane dicarboxylic aldehyde (MDA) level. We found that in LX2 cells Fe-NTA induced notable ROS, which played a protective role in HSCs cells apoptosis by inhibiting Caspase-3 activation. Fe-NTA-induced ROS increased mRNA and protein level of anti-apoptosis Bcl-2 and decreased mRNA protein level of pro-apoptosis gene Bax, As a result, maintaining mitochondrial membrane potential of HSCs. Fe-NTA-induced ROS play a protective role in human HSCs by regulating Bcl-2 family proteins and mitochondrial membrane potential. PMID:26770403

  14. 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.

  15. Soluble Egg Antigens of Schistosoma japonicum Induce Senescence of Activated Hepatic Stellate Cells by Activation of the FoxO3a/SKP2/P27 Pathway

    PubMed Central

    Chen, Jinling; Zhu, Dandan; Wang, Jianxin; Sun, Xiaolei; Chen, Liuting; Wu, Liting

    2016-01-01

    Background Liver fibrosis was viewed as a reversible process. The activation of hepatic stellate cells (HSCs) is a key event in the process of liver fibrosis. The induction of senescence of HSCs would accelerate the clearance of the activated HSCs. Previously, we demonstrated that soluble egg antigens (SEA) of Schistosoma japonicum promoted the senescence of HSCs via STAT3/P53/P21 pathway. In this paper, our study was aimed to explore whether there are other signaling pathways in the process of SEA-induced HSCs aging and the underlying effect of SKP2/P27 signal on senescent HSCs. Methodology/Principal findings Human hepatic stellate cell line, LX-2 cells, were cultured and stimulated with SEA. Western blot and cellular immunofluorescence analysis were performed to determine the expression of senescence-associated protein, such as P27, SKP2 and FoxO3a. Besides, RNA interfering was applied to knockdown the expression of related protein. The senescence of HSCs was determined by senescence-associated β-gal staining. We found that SEA increased the expression of P27 protein, whereas it inhibited the expression of SKP2 and FoxO3a. Knockdown of P27 as well as overexpression of SKP2 both suppressed the SEA-induced senescence of HSCs. In addition, the nuclear translocation of FoxO3a from the nucleus to the cytoplasm was induced by SEA stimulation. Conclusions/Significance The present study demonstrates that SEA promotes HSCs senescence through the FoxO3a/SKP2/P27 pathway. PMID:28036393

  16. 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

  17. Nod-like receptor protein 3 inflammasome activation by Escherichia coli RNA induces transforming growth factor beta 1 secretion in hepatic stellate cells

    PubMed Central

    Wang, Hui; Liu, Shu; Wang, Ying; Chang, Bing; Wang, Bingyuan

    2016-01-01

    Nod-like receptor protein 3 (NLRP3) inflammasome has been implicated in alcoholic liver disease. Chronic alcohol consumption enhances gut permeability and causes microbial translocation. The present study explored the activation of the NLRP3 inflammasome by Escherichia coli RNA in hepatic stellate cells (HSCs), and the potential role of NLRP3 inflammasome in hepatic fibrosis. E. coli RNA transfection induced HSC-T6 cells to secrete and express mature interleukin-1 beta (IL-1β), which was abolished by NLRP3 siRNA pretreatment. In addition, E. coli RNA transfection enhanced caspase-1 expression, whereas reduced caspase-1 precursor (pro-caspase-1) expression. E. coli RNA-stimulated transforming growth factor beta 1 (TGF-β1) overproduction in HSC-T6 cells, which was blocked by recombinant IL-1 receptor antagonist (rIL-1Ra) or nuclear factor κB inhibitor BAY 11-7082. Furthermore, E. coli RNA-induced overexpression of pro-fibrogenic factors was suppressed by rIL-1Ra or TGF-β receptor inhibitor A83-01. These results demonstrate that E. coli RNA can stimulate NLRP3 inflammasome activation, which leads to excessive production of pro-fibrogenic factors, suggesting that NLRP3 inflammasome activation in HSCs may play a role in hepatic fibrosis. PMID:26773180

  18. Glycyrrhizinate reduces portal hypertension in isolated perfused rat livers with chronic hepatitis

    PubMed Central

    Zhao, Xin; Deng, Bo; Xu, Xue-Yan; Yang, Shi-Jun; Zhang, Tao; Song, Yi-Jun; Liu, Xiao-Ting; Wang, Yue-Qi; Cai, Da-Yong

    2013-01-01

    AIM: To investigate the effects of diammonium glycyrrhizinate (Gly) on portal hypertension (PHT) in isolated portal perfused rat liver (IPPRL) with carbon tetrachloride (CCl4)-induced chronic hepatitis. METHODS: PHT model was replicated with CCl4 in rats for 84 d. Model was identified by measuring the ascetic amounts, hepatic function, portal pressure in vivo, splenic index, and pathological alterations. Inducible nitric oxide synthase (iNOS) in liver was assessed by immunohistochemistry. IPPRLs were performed at d0, d28, d56, and d84. After phenylephrine-induced constriction, Gly was geometrically used to reduce PHT. Gly action was expressed as median effective concentration (EC50) and area under the curve (AUC). Underlying mechanism was exploited by linear correlation between AUC values of Gly and existed iNOS in portal triads. RESULTS: PHT model was confirmed with ascites, splenomegaly, serum biomarkers of hepatic injury, and elevated portal pressure. Pathological findings had shown normal hepatic structure at d0, degenerations at d28, fibrosis at d56, cirrhosis at d84 in PHT rats. Pseudo lobule ratios decreased and collagen ratios increased progressively along with PHT development. Gly does dose-dependently reduce PHT in IPPRLs with CCl4-induced chronic hepatitis. Gly potencies were increased gradually along with PHT development, characterized with its EC50 at 2.80 × 10-10, 3.03 × 10-11, 3.77 × 10-11 and 4.65×10-11 mol/L at d0, d28, d56 and d84, respectively. Existed iNOS was located at hepatocyte at d0, stellate cells at d28, stellate cells and macrophages at d56, and macrophages in portal triads at d84. Macrophages infiltrated more into portal triads and expressed more iNOS along with PHT development. AUC values of Gly were positively correlated with existed iNOS levels in portal triads. CONCLUSION: Gly reduces indirectly PHT in IPPRL with CCl4-induced chronic hepatitis. The underlying mechanisms may relate to rescue NO bioavailability from macrophage

  19. 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

  20. Effect of sinapic acid against dimethylnitrosamine-induced hepatic fibrosis in rats.

    PubMed

    Shin, Dong-Su; Kim, Kung Wook; Chung, Hae Young; Yoon, Sik; Moon, Jeon-Ok

    2013-05-01

    Sinapic acid is a member of the phenylpropanoid family and is abundant in cereals, nuts, oil seeds, and berries. It exhibits a wide range of pharmacological properties. In this study, we investigated the hepatoprotective and antifibrotic effects of sinapic acid on dimethylnitrosamine (DMN)-induced chronic liver injury in rats. Sinapic acid remarkably prevented DMN-induced loss of body weight. This was accompanied by a significant increase in levels of serum alanine transaminase, aspartate transaminase, and liver malondialdehyde content. Furthermore, sinapic acid reduced hepatic hydroxyproline content, which correlated with a reduction in the expression of type I collagen mRNA and histological analysis of collagen in liver tissue. Additionally, the expression of hepatic fibrosis-related factors such as α-smooth muscle actin and transforming growth factor-β1 (TGF-β1), were reduced in rats treated with sinapic acid. Sinapic acid exhibited strong scavenging activity. In conclusion, we find that sinapic acid exhibits hepatoprotective and antifibrotic effects against DMN-induced liver injury, most likely due to its antioxidant activities of scavenging radicals, its capacity to suppress TGF-β1 and its ability to attenuate activation of hepatic stellate cells. This suggests that sinapic acid is a potentially useful agent for the protection against liver fibrosis and cirrhosis.

  1. 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

  2. Quercetin attenuates the activation of hepatic stellate cells and liver fibrosis in mice through modulation of HMGB1-TLR2/4-NF-κB signaling pathways.

    PubMed

    Li, Xi; Jin, Qianwen; Yao, Qunyan; Xu, Beili; Li, Zheng; Tu, Chuantao

    2016-11-02

    This study aimed to investigate the effects of quercetin on liver fibrogenesis in mice and to elucidate the underlying molecular mechanisms. Mice were administered with carbon tetrachloride (CCl4) for eight weeks to induce liver fibrosis and concomitantly orally treated with quercetin (50mgkg(-1)day(-1)). Here, we demonstrated that quercetin dramatically ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl4. Quercetin also inhibited the activation of hepatic stellate cells (HSC) in vivo and in vitro, as evaluated by α-smooth muscle actin (α-SMA) expression, which is a specific marker of HSC activation. Moreover, reduced fibrosis was associated with decreased high-mobility group box 1 (HMGB1), toll like receptor (TLR) 2 and TLR4 genes, and protein expression. Quercetin also inhibited the cytoplasmic translocation of HMGB1 in hepatocytes of fibrotic livers. Further investigation demonstrated that quercetin treatment significantly attenuated CCl4-induced nuclear translocation of the nuclear factor-κB (NF-κB) p65 and inhibited degradation of IκBα (an inhibitor of NF-κB) expression in the liver compared with vehicle-treated fibrotic mice. Considered together, our data indicate that quercetin has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be involved in modulating the HMGB1-TLR2/4-NF-κB signaling pathways.

  3. 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

  4. Deletion of the collagen-specific molecular chaperone Hsp47 causes endoplasmic reticulum stress-mediated apoptosis of hepatic stellate cells.

    PubMed

    Kawasaki, Kunito; Ushioda, Ryo; Ito, Shinya; Ikeda, Kazuo; Masago, Yusaku; Nagata, Kazuhiro

    2015-02-06

    Chronic liver injury, often caused by alcoholism and viral hepatitis, causes liver fibrosis via the induction of collagen production. In liver fibrosis, hepatic stellate cells (HSCs) are activated and transform into myofibroblasts, which actively produce and secrete collagen into the extracellular matrix. Hsp47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for the maturation and secretion of collagen. Here, we used the Cre-LoxP system to disrupt the Hsp47 gene in isolated HSCs from Hsp47 floxed mice. Immature type I procollagen accumulated and partially aggregated in Hsp47-KO HSCs. This accumulation was augmented when autophagy was inhibited, which induced expression of the endoplasmic reticulum (ER) stress-inducible proteins BiP (immunoglobulin heavy chain-binding protein) and Grp94 (94-kDa glucose-regulated protein). The inhibition of autophagy in Hsp47-KO HSCs also induced CHOP (CCAAT/enhancer-binding protein homologous protein), which is an ER stress-induced transcription factor responsible for apoptosis. These data suggest that apoptosis is induced through ER stress by procollagen accumulation in Hsp47-KO HSCs when autophagy is inhibited. Thus, Hsp47 could be a promising therapeutic target in liver fibrosis.

  5. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

    PubMed

    Yin, Li; Sun, Mingzeng; Ilic, Zoran; Leffert, Hyam L; Sell, Stewart

    2002-02-01

    Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction.

  6. 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

  7. Starring stellate cells in liver immunology.

    PubMed

    Winau, Florian; Quack, Christian; Darmoise, Alexandre; Kaufmann, Stefan H E

    2008-02-01

    Stellate cells are star-shaped cells located in the liver and mediate a multitude of primarily non-immunological functions. They play a pivotal role in the metabolism of vitamin A and store 80% of total body retinol. Upon activation, stellate cells differentiate to myofibroblasts for production of extracellular matrix, leading to liver fibrosis. Moreover, activated stellate cells regulate liver blood flow through vasoconstriction implicated in portal hypertension. Earlier work demonstrated stellate cell derived secretion of chemokines and cytokines such as transforming growth factor beta (TGF-beta), suggesting an association with immunological processes. Indeed, recent evidence indicated that hepatic stellate cells perform potent APC function for stimulation of NKT cells as well as CD8 and CD4 T cells. Additionally, stellate cell mediated antigen presentation induced protective immunity against bacterial infection. Current experiments reveal that the presenting ability of stellate cells is the key to antigen-dependent T cell instruction by vitamin A derived retinoic acid. Finally, future studies will show whether in the firmament of immunology stellate cells will represent fixed or falling stars.

  8. 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.

  9. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    PubMed

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  10. The distribution of stellate cell descending axons in the rat cerebellum: a Golgi and a combined Golgi-electron microscopical study.

    PubMed Central

    Paula-Barbosa, M M; Tavares, M A; Ruela, C; Barroca, H

    1983-01-01

    Axonal descending branches of stellate cells in the molecular layer of the cerebellar cortex of the rat were studied by means of Golgi and combined Golgi-ultrastructural methods. Special attention was paid to those branches from more superficially located cell bodies. With the Golgi method, it was observed that the number of axons from stellate cells forming pericellular baskets and 'pinceaux' increases as their cell bodies come to lie deeper in the layer. With the combined Golgi-ultrastructural method, it was verified that the synaptic contacts established by these axons are identical to those of axons from basket cells, either contacting Purkinje cell bodies or lying around the axon initial segments, where they establish septate-like junctions. This overlapping of axonal territories between stellate and basket cells is in accordance with the hypothesis that these interneurons, although situated at different levels of the cerebellar molecular layer, may be genetically identical cells. Their diversity of form would depend on the cellular microenvironment present at the time of differentiation. Images Figs. 1-4 Figs. 5-7 Fig. 8 Fig. 9 Fig. 10 PMID:6668252

  11. Dynamic expression of extracellular signal-regulated kinase in rat liver tissue during hepatic fibrogenesis

    PubMed Central

    Zhang, Xiao-Lan; Liu, Jin-Ming; Yang, Chang-Chun; Zheng, Yi-Lin; Liu, Li; Wang, Zhan-Kui; Jiang, Hui-Qing

    2006-01-01

    AIM: To investigate whether extracellular signal-regulated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and Masson’s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMA expression (r = 0.958,P < 0.05). CONCLUSION: The expression of ERK1 protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a

  12. 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.

  13. 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

  14. 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

  15. Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway

    PubMed Central

    Yang, Ning; Shi, Juan-Juan; Wu, Feng-Ping; Li, Mei; Zhang, Xin; Li, Ya-Ping; Zhai, Song; Jia, Xiao-Li; Dang, Shuang-Suo

    2017-01-01

    AIM To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on α-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway. PMID:28275300

  16. Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

    PubMed Central

    Bahrami, Arya J.; Gunaje, Jagadambika J.; Hayes, Brian J.; Riehle, Kimberly J.; Kenerson, Heidi L.; Yeung, Raymond S.; Stempien-Otero, April S.; Campbell, Jean S.; Mahoney, William M.

    2014-01-01

    Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury. PMID:25290689

  17. Disruption of intermolecular disulfide bonds in PDGF-BB dimers by N-acetyl-L-cysteine does not prevent PDGF signaling in cultured hepatic stellate cells

    SciTech Connect

    Borkham-Kamphorst, Erawan; Meurer, Steffen K.; Gressner, Axel M.; Weiskirchen, Ralf . E-mail: rweiskirchen@ukaachen.de

    2005-12-30

    Oxidative stress is important in the pathogenesis of liver fibrosis through its induction of hepatic stellate cell (HSC) proliferation and enhancement of collagen synthesis. Reactive oxygen species have been found to be essential second messengers in the signaling of both major fibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-{beta} (TGF-{beta}), in cultured HSC and liver fibrosis. The non-toxic aminothiol N-acetyl-L-cysteine (NAC) inhibits cellular activation and attenuates experimental fibrosis in liver. Prior reports show that NAC is capable of reducing the effects of TGF-{beta} in biological systems, in cultured endothelial cells, and HSC through its direct reducing activity upon TGF-{beta} molecules. We here analyzed the effects of NAC on PDGF integrity, receptor binding, and downstream signaling in culture-activated HSC. We found that NAC dose-dependently induces disintegration of PDGF in vitro. However, even high doses (>20 mM) were not sufficient to prevent the phosphorylation of the PDGF receptor type {beta}, extracellular signal-regulated kinase, or protein kinase B (PKB/Akt). Therefore, we conclude that the PDGF monomer is still active. The described antifibrotic effects are therefore mainly attributable to the structural impairment of TGF-{beta} signaling components reported previously.

  18. No evidence of rat hepatitis E virus excretion into urine of rats.

    PubMed

    Li, Tian-Cheng; Yoshizaki, Sayaka; Ami, Yasushi; Suzaki, Yuriko; Johne, Reimar; Wakita, Takaji

    2016-08-31

    To investigate whether rat hepatitis E virus (rat HEV) is excreted into the urine of rats, we infected three Wistar and six nude rats with rat HEV and examined the rat HEV RNA in serum, fecal and urine samples. We detected rat HEV RNA in the serum and fecal samples of all rats but not in the urine. Our results suggest that in rats, rat HEV is not transmitted via urine.

  19. PPAR{gamma} agonists prevent TGF{beta}1/Smad3-signaling in human hepatic stellate cells

    SciTech Connect

    Zhao Caiyan; Chen, Wei; Yang Liu; Chen Lihong; Stimpson, Stephen A.; Diehl, Anna Mae . E-mail: annamae.diehl@duke.edu

    2006-11-17

    PPAR{gamma} agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPAR{gamma} agonists inhibit transforming growth factor (TGF){beta}1-activation of TGF{beta} receptor (TGF{beta}R)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1{alpha}I. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPAR{gamma} expression and transcriptional activity. These adipocytic HSC were then treated with TGF{beta}1 {+-} a TGF{beta}R-1 kinase inhibitor (SB431542) or a PPAR{gamma} agonist (GW7845). TGF{beta}1 caused dose- and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGF{beta}R-1 kinase inhibitor, the PPAR{gamma} agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPAR{gamma} agonists reflect their ability to inhibit TGF{beta}1-TGF{beta}R1 signaling that initiates ECM gene expression in quiescent HSC.

  20. 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.

  1. 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.

  2. 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

  3. The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

    PubMed Central

    Renga, Barbara; Francisci, Daniela; Schiaroli, Elisabetta; Carino, Adriana; Cipriani, Sabrina; D'Amore, Claudio; Sidoni, Angelo; Sordo, Rachele Del; Ferri, Ivana; Lucattelli, Monica; Lunghi, Benedetta; Baldelli, Franco; Fiorucci, Stefano

    2014-01-01

    Background The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life's cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders. Aim In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes. Methods LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors. Results Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2. Conclusions The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs. PMID:24736615

  4. 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

  5. Hepatic stellate cells increase the immunosuppressive function of natural Foxp3+ regulatory T cells via IDO-induced AhR activation.

    PubMed

    Kumar, Sudhir; Wang, Jiang; Thomson, Angus W; Gandhi, Chandrashekhar R

    2017-02-01

    Immunosuppressive, naturally occurring CD4(+)CD25(+)forkhead box p3(+) (Foxp3(+)) regulatory T cells (nTregs) offer potential for the treatment of immune-mediated inflammatory disorders. However, potential instability of ex vivo-expanded nTregs following their adoptive transfer may be a significant limitation. LPS-stimulated hepatic stellate cells (HSCs) induce expansion and enhance the suppressive function and stability of allogeneic nTregs We aimed to delineate mechanisms underlying HSC-induced expansion and increased potency of nTregs HSCs and nTregs were isolated from mouse livers and spleens, respectively. Following coculture with LPS-pretreated allogeneic HSCs (LPS/HSCs), proliferation of nTregs was measured by CFSE dilution, and Foxp3 expression and acetylation were determined by immunoprecipitation (IP) and Western blotting analysis. Expression of various genes associated with immunologic tolerance was determined by quantitative RT-PCR (qRT-PCR). LPS stimulation increased the expression and activity of the immunoregulatory enzyme IDO1 in HSCs, and LPS/HSCs stimulated aryl hydrocarbon receptor (AhR) signaling in cocultured nTregs Reciprocally, Tregs increased IDO1 expression in HSCs. IDO1(-/-) LPS/HSCs were inferior to WT LPS/HSCs in stimulating nTreg expansion. Pharmacologic inhibition of IDO1 in HSCs by 1-methyltryptophan (1MT) inhibited LPS/HSC-induced AhR signaling in nTregs, which was responsible for their expansion, Foxp3 expression, and stabilization of Foxp3 by increasing acetylation of lysine residues. Finally, HSCs cryopreserved, following 2-3 passages, were as potent as primary-cultured HSCs in expanding nTregs In conclusion, LPS/HSCs expand allogeneic nTregs through an IDO-dependent, AhR-mediated mechanism and increase their stability through lysine-acetylation of Foxp3. nTregs expanded by cryopreserved HSCs may have potential for clinical use.

  6. Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2) via Distinct Pathways

    PubMed Central

    Lv, Zhigang; Xu, Lieming

    2012-01-01

    Salvianolic acid B (SA-B) is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs) to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4) via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling. PMID:21860657

  7. Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells

    PubMed Central

    Shi, Hong-Bo; Lou, Jin-Li; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Duan, Zhong-Ping

    2017-01-01

    AIM To prepare a Gpm6a/ReelinGFPCreERT2 construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC). METHODS Gpm6a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit. RESULTS A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with BamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers. CONCLUSION The construct of Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function. PMID:28127196

  8. 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

  9. Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

    PubMed

    Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

    2009-02-01

    Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

  10. Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.

    PubMed

    Azzariti, Amalia; Mancarella, Serena; Porcelli, Letizia; Quatrale, Anna Elisa; Caligiuri, Alessandra; Lupo, Luigi; Dituri, Francesco; Giannelli, Gianluigi

    2016-12-01

    In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization.

  11. Standardized Salvia miltiorrhiza extract suppresses hepatic stellate cell activation and attenuates steatohepatitis induced by a methionine-choline deficient diet in mice.

    PubMed

    Lee, Hak Sung; Son, Woo-Chan; Ryu, Jae-Eun; Koo, Bon Am; Kim, Yeong Shik

    2014-06-17

    The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME) on gene and protein expression of non-alcoholic steatohepatitis (NASH)-related factors in activated human hepatic stellate cells (HSC), and in mice with steatohepatitis induced by a methionine-choline deficient (MCD) diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight) was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor β-1 (TGF-β1) or TGF-β1 plus SME (0.1-10 μg/mL). To investigate the effect of SME on reactive oxygen species (ROS)-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2) or H2O2 plus SME (0.1-100 μg/mL). MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-α), TGF-β1, interleukin-1β (IL-1β), C-reactive protein (CRP), α-smooth muscle actin (α-SMA), type I collagen, matrix metalloproteinase-2 (MMP-2) and MMP-9. TGF-β1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD) activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.

  12. Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Promotes Hepatic Stellate Cells Migration via Canonical NF-κB/MMP9 Pathway

    PubMed Central

    Wang, Aixiu; Wang, Chen; Cao, Yu; Zhang, Ming; Zhang, Mingming; Su, Min; Zou, Xiaoping; Xu, Guifang; Zhuge, Yuzheng

    2016-01-01

    In the liver, the signal and function of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) have mainly been assessed in association with liver regeneration. However, the effects of TWEAK on liver fibrosis have not been fully elucidated. To investigate the effects of TWEAK on human hepatic stellate cells (HSCs) and to explore the relevant potential mechanisms, human HSCs line—LX-2 were cultured with TWEAK. Cell migration was detected by transwell assay; cell viability was evaluated by Cell Counting Kit-8; the expression of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13 gene was identified by quantitative real-time polymerase chain reaction and western blotting; the activity of matrix metalloproteinases (MMPs) was tested by enzyme-linked immuno sorbent assay; small interfering RNA transfection was applied for depletion of MMP9 and p65. The result of transwell assay revealed that TWEAK promoted LX-2 migration. Subsequently, our data testified that the expression and activity of MMP9 was induced by TWEAK in LX-2 cells, which enhanced the migration. Furthermore, our findings showed that TWEAK upregulated the phosphorylation of IκBα and p65 protein to increase MMP9 expression in LX-2 cells. Meanwhile, the alpha-smooth muscle actin, vimentin and desmin expression were upregulated following TWEAK treatment. The results in the present study revealed that TWEAK promotes HSCs migration via canonical NF-κB/MMP9 pathway, which possibly provides a molecular basis targeting TWEAK for the therapy of liver fibrosis. PMID:27907201

  13. 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

  14. Human Immunodeficiency Virus (HIV)-1 Infects Human Hepatic Stellate Cells and Promotes Collagen I and Monocyte Chemoattractant Protein-1 Expression: Implications for the Pathogenesis of HIV/Hepatitis C Virus–Induced Liver Fibrosis

    PubMed Central

    Tuyama, Ana C.; Hong, Feng; Saiman, Yedidya; Wang, Chuansheng; Ozkok, Derya; Mosoian, Arevik; Chen, Ping; Chen, Benjamin K.; Klotman, Mary E.; Bansal, Meena B.

    2010-01-01

    Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) develop more rapid fibrosis than those infected with HCV only. In HIV/HCV-coinfected patients, fibrosis progression correlates with HIV RNA levels, suggesting a direct role of HIV in liver fibrogenesis. Chemokine (C-C motif) receptor 5 (CCR5) and cysteine-X-cysteine receptor 4 (CXCR4), the two major coreceptors required for HIV entry into cells, are expressed on activated hepatic stellate cells (HSCs), the principle fibrogenic cell type in the liver. We therefore examined whether HIV can infect HSCs, explored the potential mechanisms of viral entry, and assessed the impact of infection as reflected by the ability of HSCs to transfer virus to T lymphocytes and elicit a proinflammatory and profibrogenic response. We report that the laboratory-adapted viruses HIV-IIIB (CXCR4-tropic or X4) and HIV-BaL (CCR5-tropic or R5) and primary HIV isolates can infect both a human stellate cell line, LX-2, and primary human HSCs. HIV entry and gene expression in HSCs was confirmed using HIV–green fluorescent protein (GFP) expression viral constructs in the presence or absence of the reverse-transcriptase inhibitor azidothymidine. CD4 expression on a subset of primary HSCs was demonstrated using fluorescence-activated cell sorting and immunofluorescence staining. Blocking experiments in the presence of anti-CD4, anti-CXCR4, and anti-CCR5 revealed that HIV entry into HSCs is predominantly CD4/chemokine coreceptor-independent. HIV infection promoted HSC collagen I expression and secretion of the proinflammatory cytokine monocyte chemoattractant protein-1. Furthermore, infected LX-2 cells were capable of transferring GFP-expressing virus to T lymphocytes in a coculture system. Conclusion Taken together, our results suggest a potential role of HIV in liver fibrosis/inflammation mediated through effects on HSCs. The role of early highly active antiretroviral therapy initiation in patients with HIV

  15. 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.

  16. Novel Hepatitis E Virus Genotype in Norway Rats, Germany

    PubMed Central

    Johne, Reimar; Heckel, Gerald; Plenge-Bönig, Anita; Kindler, Eveline; Maresch, Christina; Reetz, Jochen; Schielke, Anika

    2010-01-01

    Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E–like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus. PMID:20735931

  17. Magnolol Attenuates Concanavalin A-induced Hepatic Fibrosis, Inhibits CD4(+) T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3/Smad4 Signalling.

    PubMed

    Zhang, Hongjun; Ju, Baoling; Zhang, Xiaoli; Zhu, Yanfei; Nie, Ying; Xu, Yuanhong; Lei, Qiuxia

    2016-12-29

    Magnolol is a pharmacological biphenolic compound extracted from Chinese herb Magnolia officinalis, which displays anti-inflammatory and antioxidant effects. This study was aimed at exploring the potential effect of magnolol on immune-related liver fibrosis. Herein, BALB/c mice were injected with concanavalin A (ConA, 8 mg/kg/week) up to 6 weeks to establish hepatic fibrosis, and magnolol (10, 20, 30 mg/kg/day) was given to these mice orally throughout the whole experiment. We found that magnolol preserved liver function and attenuated liver fibrotic injury in vivo. In response to ConA stimulation, the CD4(+) T cells preferred to polarizing towards CD4(+) T helper 17 (Th17) cells in liver. Magnolol was observed to inhibit Th17 cell differentiation in ConA-treated liver in addition to suppressing interleukin (IL)-17A generation. Hepatic stellate cells were activated in fibrotic liver as demonstrated by increased alpha smooth muscle actin (α-SMA) and desmin. More transforming growth factor (TGF)-β1 and activin A were secreted into the serum. Magnolol suppressed this abnormal HSC activation. Furthermore, the phosphorylation of Smad3 in its linker area (Thr179, Ser 204/208/213) was inhibited by magnolol. In vitro, the recombinant IL-17A plus TGF-β1 or activin A induced activation of human LX2 HSCs and promoted their collagen production. Smad3/Smad4 signalling pathway was activated in LX2 cells exposed to the fibrotic stimuli, as illustrated by the up-regulated phospho-Smad3 and the enhanced interaction between Smad3 and Smad4. These alterations were suppressed by magnolol. Collectively, our study reveals a novel antifibrotic effect of magnolol on Th17 cell-mediated fibrosis.

  18. 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.

  19. 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

  20. Inhibition of the SphK1/S1P signaling pathway by melatonin in mice with liver fibrosis and human hepatic stellate cells.

    PubMed

    González-Fernández, Bárbara; Sánchez, Diana I; Crespo, Irene; San-Miguel, Beatriz; Álvarez, Marcelino; Tuñón, María J; González-Gallego, Javier

    2016-11-01

    The sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) system is involved in different pathological processes, including fibrogenesis. Melatonin abrogates activation of hepatic stellate cells (HSCs) and attenuates different profibrogenic pathways in animal models of fibrosis, but it is unknown if protection associates with its inhibitory effect on the SphK1/S1P axis. Mice in treatment groups received carbon tetrachloride (CCl4 ) 5 μL g(-1) body wt i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg kg(-1)  day(-1) i.p, beginning 2 weeks after the start of CCl4 administration. At both 4 and 6 weeks following CCl4 treatment, liver mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased significantly. S1P production, and expression of S1P receptor (S1PR)1, S1PR3 and acid sphingomyelinase (ASMase) were significantly elevated. However, there was a decreased expression of S1PR2 and S1P lyase (S1PL). Melatonin attenuated liver fibrosis, as shown by a significant inhibition of the expression of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β and collagen (Col) Ι. Furthermore, melatonin inhibited S1P production, lowered expression of SphK1, S1PR1, SP1R3, and ASMase, and increased expression of S1PL. Melatonin induced a reversal of activated human HSCs cell line LX2, as evidenced by a reduction in α-SMA, TGF-β, and Col I expression. Melatonin-treated cells also exhibited an inhibition of the SphK1/S1P axis. Antifibrogenic effect of SphK1 inhibition was confirmed by treatment of LX2 cells with PF543. Abrogation of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in liver fibrogenesis. © 2016 BioFactors, 2016.

  1. 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.

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

    PubMed

    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

  3. Fibrogenic actions of acetaldehyde are β-catenin dependent but Wingless independent: a critical role of nucleoredoxin and reactive oxygen species in human hepatic stellate cells.

    PubMed

    Arellanes-Robledo, Jaime; Reyes-Gordillo, Karina; Shah, Ruchi; Domínguez-Rosales, José Alfredo; Hernández-Nazara, Zamira Helena; Ramirez, Francesco; Rojkind, Marcos; Lakshman, M Raj

    2013-12-01

    We investigated whether the fibrogenic actions of acetaldehyde, the immediate oxidation product of ethanol, are mediated via Wingless (WNT) and/or β-catenin pathways in human hepatic stellate cells (HSC). First, we show that both β-catenin small inhibitory RNA and a dominant negative-MYC expression vector markedly down-regulated the expressions of fibrogenic genes in freshly isolated HSC. We further show that acetaldehyde up-regulated platelet-derived growth factor receptor beta mRNA and protein expressions ranging from 4.0- to 7.2-fold (P<0.001). Acetaldehyde induced MYC and collagen type-1 alpha-2 mRNA and protein expressions were WNT independent because DKK1, an antagonist of the canonical WNT/β-catenin pathway, completely failed to block these inductions. Acetaldehyde increased phospho-glycogen synthase kinase-3 beta (GSK3B) protein by 31% (P<0.01), whereas phospho-β-catenin protein decreased by 50% (P ≤ 0.01). Significantly, in contrast to 43% (P<0.01) inhibition of β-catenin nuclear translocation in nucleoredoxin (NXN)-overexpressed HSC, acetaldehyde profoundly stimulated β-catenin nuclear translocation by 51%, (P<0.01). Acetaldehyde also increased the cellular reactive oxygen species level 2-fold (P<0.001) with a concomitant 2-fold (P<0.001) increase in 4-hydroxynonenal adducts. Conversely, there was a 44% decrease (P<0.001) in glutathione levels with a concomitant 76% (P<0.001) decrease in the level of NXN/ disheveled (DVL) complex. Based on these findings, we conclude that actions of acetaldehyde are mediated by a mechanism that inactivates NXN by releasing DVL, leading to the inactivation of GSK3B, and thereby blocks β-catenin phosphorylation and degradation. Thus, the stabilized β-catenin translocates to the nucleus where it up-regulates the fibrogenic pathway genes. This novel mechanism of action of acetaldehyde has the potential for therapeutic interventions in liver fibrosis induced by alcohol.

  4. 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.

  5. Foetal programming by methyl donor deficiency produces steato-hepatitis in rats exposed to high fat diet.

    PubMed

    Bison, Anaïs; Marchal-Bressenot, Aude; Li, Zhen; Elamouri, Ilef; Feigerlova, Eva; Peng, Lu; Houlgatte, Remi; Beck, Bernard; Pourié, Gregory; Alberto, Jean-Marc; Umoret, Remy; Conroy, Guillaume; Bronowicki, Jean-Pierre; Guéant, Jean-Louis; Guéant-Rodriguez, Rosa-Maria

    2016-11-17

    Non-alcoholic steatohepatitis (NASH) is a manifestation of metabolic syndrome, which emerges as a major public health problem. Deficiency in methyl donors (folate and vitamin B12) during gestation and lactation is frequent in humans and produces foetal programming effects of metabolic syndrome, with small birth weight and liver steatosis at day 21 (d21), in rat pups. We investigated the effects of fetal programming on liver of rats born from deficient mothers (iMDD) and subsequently subjected to normal diet after d21 and high fat diet (HF) after d50. We observed increased abdominal fat, ASAT/ALAT ratio and angiotensin blood level, but no histological liver abnormality in d50 iMDD rats. In contrast, d185 iMDD/HF animals had hallmarks of steato-hepatitis, with increased markers of inflammation and fibrosis (caspase1, cleaved IL-1β, α1(I) and α2(I) collagens and α-SMA), insulin resistance (HOMA-IR and Glut 2) and expression of genes involved in stellate cell stimulation and remodelling and key genes triggering NASH pathomechanisms (transforming growth factor beta super family, angiotensin and angiotensin receptor type 1). Our data showed a foetal programming effect of MDD on liver inflammation and fibrosis, which suggests investigating whether MDD during pregnancy is a risk factor of NASH in populations subsequently exposed to HF diet.

  6. Foetal programming by methyl donor deficiency produces steato-hepatitis in rats exposed to high fat diet

    PubMed Central

    Bison, Anaïs; Marchal-Bressenot, Aude; Li, Zhen; Elamouri, Ilef; Feigerlova, Eva; Peng, Lu; Houlgatte, Remi; Beck, Bernard; Pourié, Gregory; Alberto, Jean-Marc; Umoret, Remy; Conroy, Guillaume; Bronowicki, Jean-Pierre; Guéant, Jean-Louis; Guéant-Rodriguez, Rosa-Maria

    2016-01-01

    Non-alcoholic steatohepatitis (NASH) is a manifestation of metabolic syndrome, which emerges as a major public health problem. Deficiency in methyl donors (folate and vitamin B12) during gestation and lactation is frequent in humans and produces foetal programming effects of metabolic syndrome, with small birth weight and liver steatosis at day 21 (d21), in rat pups. We investigated the effects of fetal programming on liver of rats born from deficient mothers (iMDD) and subsequently subjected to normal diet after d21 and high fat diet (HF) after d50. We observed increased abdominal fat, ASAT/ALAT ratio and angiotensin blood level, but no histological liver abnormality in d50 iMDD rats. In contrast, d185 iMDD/HF animals had hallmarks of steato-hepatitis, with increased markers of inflammation and fibrosis (caspase1, cleaved IL-1β, α1(I) and α2(I) collagens and α-SMA), insulin resistance (HOMA-IR and Glut 2) and expression of genes involved in stellate cell stimulation and remodelling and key genes triggering NASH pathomechanisms (transforming growth factor beta super family, angiotensin and angiotensin receptor type 1). Our data showed a foetal programming effect of MDD on liver inflammation and fibrosis, which suggests investigating whether MDD during pregnancy is a risk factor of NASH in populations subsequently exposed to HF diet. PMID:27853271

  7. Vitamin D ameliorates hepatic ischemic/reperfusion injury in rats.

    PubMed

    Seif, Ansam Aly; Abdelwahed, Doaa Mohamed

    2014-09-01

    Vitamin D, most commonly associated with the growth and remodeling of bone, has been shown to ameliorate ischemia/reperfusion injury (IRI) in some tissues, yet its underlying mechanism remains elusive. This study was designed to examine the protective effect of vitamin D, if any, against hepatic IRI in rats and the underlying mechanism involved. Adult female Wistar rats were randomly divided into control, sham-operated (sham), ischemia/reperfusion (I/R), and ischemic-reperfused vitamin D-treated (vit D) groups. Rats in the I/R and vit D groups were subjected to partial (70%) hepatic ischemia for 45 min, followed by 1 h of reperfusion. Vitamin D was given to rats orally in a dose of 500 IU/kg daily for 2 weeks before being subjected to I/R. Markers of liver damage, oxidative stress, inflammation and apoptosis were evaluated. Hepatic morphology was also examined. Vit D-treated rats had significantly lower serum levels of alanine aminotransferase, aspartate aminotransferase, and γ glutamyl transferase compared to rats in the I/R group. Also, vit D-treated rats showed a significant decrease in malondialdehyde, interleukin-1 beta, interleukin-6, tumor necrosis factor-α, nuclear factor κB, B cell leukemia/lymphoma 2-associated X protein, cytochrome c, and caspase-3 levels, with higher levels of glutathione peroxidase and B cell lymphoma 2 protein levels in liver tissues compared to I/R rats. Histological examination showed less damaged liver tissues with amelioration of apoptotic signs in the vit D group compared to the I/R group. In conclusion, vitamin D supplementation ameliorates hepatic IRI mostly by alleviating the inflammatory-apoptotic response mediated by the oxidative reperfusion injury insult.

  8. 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

  9. Melatonin reduces hepatic mitochondrial dysfunction in diabetic obese rats.

    PubMed

    Agil, Ahmad; El-Hammadi, Mazen; Jiménez-Aranda, Aroa; Tassi, Mohamed; Abdo, Walied; Fernández-Vázquez, Gumersindo; Reiter, Russel J

    2015-08-01

    Hepatic mitochondrial dysfunction is thought to play a role in the development of liver steatosis and insulin resistance, which are both common characteristics of obesity and type 2 diabetes mellitus (T2DM). It was hypothesized that the antioxidant properties of melatonin could potentially improve the impaired functions of hepatic mitochondria in diabetic obese animals. Male Zucker diabetic fatty (ZDF) rats and lean littermates (ZL) were given either melatonin (10 mg/kg BW/day) orally for 6 wk (M-ZDF and M-ZL) or vehicle as control groups (C-ZDF and C-ZL). Hepatic function was evaluated by measurement of serum alanine transaminase and aspartate transaminase levels, liver histopathology and electron microscopy, and hepatic mitochondrial functions. Several impaired functions of hepatic mitochondria were observed in C-ZDF in comparison with C-ZL rats. Melatonin treatment to ZDF rats decreases serum levels of ALT (P < 0.001), alleviates liver steatosis and vacuolation, and also mitigates diabetic-induced mitochondrial abnormalities, glycogen, and lipid accumulation. Melatonin improves mitochondrial dysfunction in M-ZDF rats by increasing activities of mitochondrial citrate synthase (P < 0.001) and complex IV of electron transfer chain (P < 0.05) and enhances state 3 respiration (P < 0.001), respiratory control index (RCR) (P < 0.01), and phosphorylation coefficient (ADP/O ratio) (P < 0.05). Also melatonin augments ATP production (P < 0.05) and diminishes uncoupling protein 2 levels (P < 0.001). These results demonstrate that chronic oral melatonin reduces liver steatosis and mitochondria dysfunction in ZDF rats. Therefore, it may be beneficial in the treatment of diabesity.

  10. Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells.

    PubMed

    Horiguchi, Kotaro; Fujiwara, Ken; Kouki, Tom; Kikuchi, Motoshi; Yashiro, Takashi

    2008-12-01

    Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anterior pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.

  11. MicroRNA-19b reduces hepatic stellate cell proliferation by targeting GRB2 in hepatic fibrosis models in vivo and in vitro as part of the inhibitory effect of estradiol.

    PubMed

    Ge, Shanfei; Xie, Jianping; Liu, Fei; He, Jinni; He, Jinwen

    2015-11-01

    Estradiol (E2) is a major determinant of gender-based differences in the development of hepatic fibrosis. MicroRNAs (miRNAs) are endogenous 19-25 nucleotide, noncoding, single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs and play an important role in liver fibrosis. The mechanisms underlying the regulation of miRNAs by E2 remain largely unknown. In this study, miR-19b levels were higher and were associated with lower GRB2 mRNA and protein levels in female rats more than in male rats. We also showed that miR-19b levels were down-regulated, were associated with the up-regulation of GRB2 mRNA and protein levels in PS (porcine serum-induced hepatic fibrosis) versus NS (normal control) groups and were up-regulated when associated with the down-regulation of GRB2 mRNA and protein levels in PS + E2 versus PS and in aHSC + E2 (estradiol treated aHSC) versus aHSC groups. MiR-19b expression inhibited cell proliferation in aHSCs, and also down-regulated GRB2 protein expression. The overexpression of miR-19b inhibited cell growth and suppressed COL1A1 protein levels by decreasing the levels of GRB2. However, the forced expression of GRB2 partly rescued the effect of miR-19b in the cells, attenuated cell proliferation, and suppressed the GRB2 protein level by up-regulating the levels of GRB2. Taken together, these findings will shed light on the role of miR-19b in regulating aHSC proliferation via the miR-19b/GRB2 axis. This newly identified miR-19b/GRB2 interaction provided novel insights into the suppressive effect of E2 on HSC proliferation and might facilitate the development of therapies targeting hepatic fibrosis.

  12. 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.

  13. Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells.

    PubMed

    Li, Ying Hui; Choi, Dae Hee; Lee, Eun Hye; Seo, Su Ryeon; Lee, Seungkoo; Cho, Eun-Hee

    2016-05-06

    Sirtuin 3 (SIRT3) is an NAD(+)-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment.

  14. Cathepsin H-Mediated Degradation of HDAC4 for Matrix Metalloproteinase Expression in Hepatic Stellate Cells: Implications of Epigenetic Suppression of Matrix Metalloproteinases in Fibrosis through Stabilization of Class IIa Histone Deacetylases.

    PubMed

    Yang, Zemin; Liu, Yu; Qin, Lan; Wu, Pengfei; Xia, Zanxian; Luo, Mei; Zeng, Yilan; Tsukamoto, Hidekazu; Ju, Zongyun; Su, Danmei; Kang, Han; Xiao, Zhixiong; Zheng, Sujun; Duan, Zhongping; Hu, Richard; Wang, Qiang; Pandol, Stephen J; Han, Yuan-Ping

    2017-02-01

    In three-dimensional extracellular matrix, mesenchymal cells including hepatic stellate cells (HSCs) gain the ability to express matrix metalloproteinases (MMPs) on injury signals. In contrast, in myofibroblastic HSCs in fibrotic liver, many MMP genes are silenced into an epigenetically nonpermissive state. The mechanism by which the three-dimensional extracellular matrix confers the MMP genes into an epigenetically permissive state has not been well characterized. In continuation of previous work, we show here that the up-regulation of MMP genes is mediated through degradation of class IIa histone deacetylases (HDACs) by certain cysteine cathepsins (Cts). In three-dimensional extracellular matrix culture, CtsH, among other cysteine cathepsins, was up-regulated and localized as puncta in the nuclear and cytoplasmic compartments in a complex with HDAC4 for its degradation. Conversely, along with HSC trans-differentiation, CtsH and CtsL were progressively down-regulated, whereas HDAC4 was concurrently stabilized. The inhibition of cysteine cathepsins by specific proteinase inhibitors or chloroquine, which raises cellular pH, restored HDAC4. Recombinant CtsH could break down HDAC4 in the transfected cells and in vitro at acidic pH. In human cirrhotic liver, activated HSCs express high levels of class IIa HDACs but little CtsH. We propose that cysteine cathepsin-mediated degradation of class IIa HDACs plays a key role in the modulation of MMP expression/suppression and HSC functions in tissue injury and fibrosis.

  15. 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.

  16. Chinese medicinal herbs in treating model rats with hepatic fibrosis.

    PubMed

    Zhou, Yun-Xiao; Chen, Jiu; Li, Jian-Ping; Wang, Yan-Li; Jin, Xiao-Dong

    2009-12-30

    The objective of this study was to examine the effects of Chinese medicine formula-Yu Zhang Dan (YZD, composed of Herba Lysimachiae, Rhizoma Polygoni Cuspidati, Radix Curcumae) on the model rats with hepatic fibrosis. Forty male Sprague-Dawley (SD) rats were used in the present study, and they were separated randomly into 4 groups: a normal control group (Group A, n=5), a model control (Group B, n=15), a high dose of YZD (Group C, n=10), and a low dose of YZD (Group D, n=10). Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl(4)). The variation of the serum alanine transaminase (ALT), aspartate aminotransferase (AST), hyaluronate acid (HA), laminin (LN), type • • procollagen peptide (P• •NP), L-Glutathione (GSH) was respectively measured with radioimmunoassay (RIA) and detection of transforming growth factor-beta 1 (TGF-β1) and smooth muscle alpha actin (α-SMA) was conducted with immunohistochemistry. The ALT, AST HA, LN and PIII NP levels in the serum of the model control group were significantly higher than those of the normal control group (P<0.05), and both of the high dose of YZD and low dose of YZD significantly decreased the ALT, AST HA, LN and PIII NP levels of the model rats (P<0.05). The TGF-β1 and α-SMA levels of the model control group were significantly higher than those of the normal control group (P<0.05), and both of the high dose of YZD and low dose of YZD significantly decreased the TGF-β1 levels of the model rats (P<0.05) , and only the high dose of YZD significantly decreased the α-SMA levels of the model rats (P<0.05). The expression of TGF-β1 and α-SMA in the liver tissues of the rats were in the cytoplasm of the cells. It may be through decreasing the ALT, AST, HA, LN and PIII NP levels in the serum of the model rats and decreasing the expression of TGF-β1 and α-SMA in the liver tissues of the model rats that YZD significantly relieved the hepatic fibrosis.

  17. Evaluation of two experimental models of hepatic encephalopathy in rats.

    PubMed

    García-Moreno, L M; Conejo, N M; González-Pardo, H; Aller, M A; Nava, M P; Arias, J; Arias, J L

    2005-01-01

    The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

  18. D-penicillamine-induced granulomatous hepatitis in brown Norway rats.

    PubMed

    Metushi, Imir G; Zhu, Xu; Uetrecht, Jack

    2014-08-01

    The mechanism of idiosyncratic drug reactions (IDRs) remains poorly understood. D-penicillamine treatment is associated with a wide range of autoimmune reactions including liver injury. An animal model which utilizes brown Norway (BN) rats has been used to investigate the mechanism of D-penicillamine-induced IDRs because it mimics the autoimmune reactions that occur in humans. The purpose of this study was to investigate the type of liver injury that results from D-penicillamine treatment in BN rats. We had previously noted that D-penicillamine caused histological changes in the liver, but there was no increase in alanine transaminase (ALT), and we assumed that there was no significant injury. However, we subsequently discovered that D-penicillamine inhibits the ALT assay. In the present study, we found that treatment of BN rats with a low doses of D-penicillamine (10 or 15 mg/day) resulted in a mild increases in glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities; however, this was not associated with histological changes. A higher dose of D-penicillamine (20 mg/day) resulted in 63% of the rats developing a skin rash, and these rats had elevated serum GLDH and SDH levels with histopathological changes characteristic of granulomatous hepatitis. This included large clusters of leukocytes in the form of granulomas that contained neutrophils, macrophages, and CD8 T cells. These changes did not occur in the rats that did not get sick. This model may be a good model to investigate the characteristics of drug-induced granulomatous hepatitis.

  19. The stellate cell system (vitamin A-storing cell system).

    PubMed

    Senoo, Haruki; Mezaki, Yoshihiro; Fujiwara, Mutsunori

    2017-03-15

    Past, present, and future research into hepatic stellate cells (HSCs, also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells) are summarized and discussed in this review. Kupffer discovered black-stained cells in the liver using the gold chloride method and named them stellate cells (Sternzellen in German) in 1876. Wake rediscovered the cells in 1971 using the same gold chloride method and various modern histological techniques including electron microscopy. Between their discovery and rediscovery, HSCs disappeared from the research history. Their identification, the establishment of cell isolation and culture methods, and the development of cellular and molecular biological techniques promoted HSC research after their rediscovery. In mammals, HSCs exist in the space between liver parenchymal cells (PCs) or hepatocytes and liver sinusoidal endothelial cells (LSECs) of the hepatic lobule, and store 50-80% of all vitamin A in the body as retinyl ester in lipid droplets in the cytoplasm. SCs also exist in extrahepatic organs such as pancreas, lung, and kidney. Hepatic (HSCs) and extrahepatic stellate cells (EHSCs) form the stellate cell (SC) system or SC family; the main storage site of vitamin A in the body is HSCs in the liver. In pathological conditions such as liver fibrosis, HSCs lose vitamin A, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. The morphology of these cells also changes from the star-shaped HSCs to that of fibroblasts or myofibroblasts.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Isolation and characterization of hepatic mast cells from cholestatic rats

    PubMed Central

    Hargrove, Laura; Graf-Eaton, Allyson; Kennedy, Lindsey; Demieville, Jennifer; Owens, Jennifer; Hodges, Kyle; Ladd, Brittany; Francis, Heather

    2016-01-01

    Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 hrs and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse coated Dynal® beads. MCs were stained with toluidine blue, and in isolated MCs, the expression of FCεRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF-receptors and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1 and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCεRI, chymase, tryptase, RMCPI and RMCPII, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats + NaCl and these parameters decreased in cells treated with MC supernatants from BDL + cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary

  5. Hepatic injury after whole-liver irradiation in the rat

    SciTech Connect

    Geraci, J.P.; Jackson, K.L.; Mariano, M.S.; Leitch, J.M.

    1985-03-01

    Radiation-induced hepatic injury in rats, which is characterized by marked ascites accompanied by liver necrosis, fibrosis, and vein lesions, is described in this study. These adverse sequelae are produced within 30 days after irradiation if there is surgical removal of two-thirds of the liver immediately after whole-liver irradiation. The LD/sub 50/30/ day and median survival time after liver irradiation and two-thirds partial hepatectomy is 24 Gy and 17 days, respectively. Death is preceded by reduction in liver function as measured by (/sup 131/I)-labeled rose bengal clearance. Prior to death, liver sepsis and endotoxemia were detected in most irradiated, partially hepatectomized animals. Pretreatment of the animals with endotoxin and/or antibiotic decontamination of the GI tract resulted in increased survival time, but no irradiated, partially hepatectomized animal survived beyond 63 days. This suggests that sepsis and endotoxemia resulting from the bacteria in the intestine are the immediate cause of death after 30-Gy liver irradiation and partial hepatectomy. It is concluded that the hepatectomized rat model is an economical and scientifically manageable experimental system to study a form of radiation hepatitis that occurs in compromised human livers.

  6. 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

  7. Does hepatic vagus nerve modulate the progression of biliary fibrosis in rats?

    PubMed

    Hajiasgharzadeh, Khalil; Tavangar, Seyed Mohammad; Javan, Mohammad; Dehpour, Ahmad R; Mani, Ali R

    2014-10-01

    Recent studies have shown that vagus nerve activation inhibits cytokine production in a variety of non-neural cells though activation of α7 nicotinic acetylcholine receptor (α7nAChR). Since chronic inflammation plays a pivotal role in liver fibrosis, this study was designed to investigate the role of hepatic vagus nerve in the progression of hepatic fibrosis in rats. Cirrhosis was induced by chronic ligation of the bile duct. Hepatic hydroxyproline level, portal pressure, serum transaminase level, hepatic TIMP-1 (tissue inhibitor of metalloproteinase-1) and MCP-1 (monocyte chemoattractant peptide-1) expression were measured in order to assess the progression of liver cirrhosis. α7nAChR expression was assessed using RT-PCR as well as immunostaining. RT-PCR analysis of the liver showed that α7nAChR mRNA is expressed in rat liver. Immunostaining study demonstrated that hepatic α7nAChR is mainly expressed in the hepatocytes of cirrhotic liver with minimum α7nAChR expression in biliary epithelium or myofibroblasts. Bile duct ligation was associated with portal hypertension, increased hepatic hydroxyproline level as well as TIMP-1 and MCP-1 expression in the liver. However neither selective hepatic vagotomy nor methyllycaconitine (an α7nAChR antagonist) could significantly affect development of portal hypertension or hepatic fibrosis in rats. Selective hepatic vagotomy could only attenuate serum aspartate aminotransferase level in bile duct ligated rats but did not have a significant effect on hepatic inflammation as assessed by MCP-1 mRNA expression. Our study provides evidence against a crucial role for the hepatic vagus nerve as an intrinsic protective mechanism in modulation of hepatic fibrosis in a rat model of biliary cirrhosis.

  8. 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

  9. Hepatic oxidative stress, genotoxicity and vascular dysfunction in lean or obese Zucker rats.

    PubMed

    Løhr, Mille; Folkmann, Janne K; Sheykhzade, Majid; Jensen, Lars J; Kermanizadeh, Ali; Loft, Steffen; Møller, Peter

    2015-01-01

    Metabolic syndrome is associated with increased risk of cardiovascular disease, which could be related to oxidative stress. Here, we investigated the associations between hepatic oxidative stress and vascular function in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24 and 37 weeks of age. Obese Zucker rats had more hepatic fat accumulation than their lean counterparts. Nevertheless, the obese rats had unaltered age-related level of hepatic oxidatively damaged DNA in terms of formamidopyrimidine DNA glycosylase (FPG) or human oxoguanine DNA glycosylase (hOGG1) sensitive sites as measured by the comet assay. There were decreasing levels of oxidatively damaged DNA with age in the liver of lean rats, which occurred concurrently with increased expression of Ogg1. The 37 week old lean rats also had higher expression level of Hmox1 and elevated levels of DNA strand breaks in the liver. Still, both strain of rats had increased protein level of HMOX-1 in the liver at 37 weeks. The external and lumen diameters of mesenteric arteries increased with age in obese Zucker rats with no change in media cross-sectional area, indicating outward re-modelling without hypertrophy of the vascular wall. There was increased maximal response to acetylcholine-mediated endothelium-dependent vasodilatation in both strains of rats. Collectively, the results indicate that obese Zucker rats only displayed a modest mesenteric vascular dysfunction, with no increase in hepatic oxidative stress-generated DNA damage despite substantial hepatic steatosis.

  10. Fish Oil Decreases Hepatic Lipogenic Genes in Rats Fasted and Refed on a High Fructose Diet

    PubMed Central

    de Castro, Gabriela S.; Cardoso, João Felipe R.; Calder, Philip C.; Jordão, Alceu A.; Vannucchi, Helio

    2015-01-01

    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. PMID:25751821

  11. 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

    2015-03-05

    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.

  12. Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

    PubMed Central

    Zhang, Li-Mei; Liu, Dian-Wu; Liu, Jian-Bo; Zhang, Xiao-Lin; Wang, Xiao-Bo; Tang, Long-Mei; Wang, Li-Qin

    2005-01-01

    AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intraven-ously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals. RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the

  13. Hepatic intralobular mapping of fructose metabolism in the rat liver.

    PubMed Central

    Burns, S P; Murphy, H C; Iles, R A; Bailey, R A; Cohen, R D

    2000-01-01

    Detailed mapping of glucose and lactate metabolism along the radius of the hepatic lobule was performed in situ in rat livers perfused with 1.5 mM lactate before and during the addition of 5 mM fructose. The majority of fructose uptake occurred in the periportal region; 45% of fructose taken up in the periportal half of the lobular volume being converted into glucose. Periportal lactate uptake was markedly decreased by addition of fructose. Basal perivenous lactate output, which was derived from glucose synthesized periportally, was increased in the presence of fructose. During fructose infusion there was a small decrease in cell pH periportally, but acidification of up to 0.5 pH units perivenously. The evidence suggests that in situ the apparent direct conversion of fructose into lactate represents, to a substantial extent, the result of periportal conversion of fructose into glucose and the subsequent uptake and glycolysis to lactate in the perivenous zone of some of that glucose. (31)P NMR spectroscopy showed that the cellular concentration of phosphomonoesters changes very little periportally during fructose infusion, but there was an approximate twofold increase perivenously, presumably due to the accumulation of fructose 1-phosphate. It may be inferred that fructokinase activity is expressed throughout the hepatic lobule. PMID:10880353

  14. In vivo and in vitro 31P magnetic resonance spectroscopic studies of the hepatic response of healthy rats and rats with acute hepatic damage to fructose loading.

    PubMed

    Lu, W; Locke, S J; Brauer, M

    1994-05-01

    The hepatic response to a fructose challenge for control rats, and rats subjected to an acute sublethal dose of carbon tetrachloride (CCl4) or bromobenzene (BB), was compared using dynamic in vivo 31P MRS. Fructose loading conditions were used in which control rats showed only a modest increase in hepatic phosphomonoester (PME), and a small decrease in ATP, Pi, and intracellular pH after fructose administration. Both CCl4 and BB-treated rats showed a much greater fructose-induced accumulation of PME than did controls. Trolox C, a free radical scavenger, prevented most of this PME increase. BB-treated rats, given sufficient time to recover from the hepatotoxic insult, responded to the fructose load similarly to controls. Liver aldolase activities of control, toxicant-treated rats, and toxicant plus Trolox C-treated rats correlated inversely with PME accumulation after fructose loading (correlation coefficient: -0.834, P < 0.05). Perchloric acid extracts of rat livers studied by in vitro 31P MRS confirmed that the PME accumulation after fructose loading is mainly due to an increase in fructose 1-phosphate. These studies are consistent with the aldolase-catalyzed cleavage of fructose 1-phosphate being rate-limiting in hepatic fructose metabolism, and that the CCl4 and BB treatment modify and inactivate the aldolase enzyme.

  15. Effect of O-ethylrutoside on serum and hepatic lipids in acute ethanal-treated rats.

    PubMed

    Wójcicki, J

    1977-01-01

    The serum total lipids, triglycerides, total cholesterol and free fatty acid concentrations, as well as hepatic triglyceride and cholesterol levels, were increased in acutely ethanol-treated rats. Treatment of ethanol-given animals with o-ethylrutoside resulted in a significant reduction in all examined fractions of serum lipids and in the hepatic total cholesterol level.

  16. Hepatitis

    MedlinePlus

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Hepatitis KidsHealth > For Teens > Hepatitis Print A A A ... to a liver condition called hepatitis . What Is Hepatitis? The liver is one of the body's powerhouses. ...

  17. Hepatitis

    MedlinePlus

    ... de los dientes Video: Getting an X-ray 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- ...

  18. 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.

  19. 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

  20. Hepatic Clearance Prediction of Nine Human Immunodeficiency Virus Protease Inhibitors in Rat.

    PubMed

    De Bruyn, Tom; Augustijns, Patrick F; Annaert, Pieter P

    2016-02-01

    This study aimed to determine the rate-limiting step in the overall hepatic clearance of the marketed human immunodeficiency virus (HIV) protease inhibitors (PI) in rats by predicting the experimentally determined hepatic in vivo clearance of these drugs based on in vitro clearance values for uptake and/or metabolism. In vitro uptake and metabolic clearance values were determined in suspended rat hepatocytes and rat liver microsomes, respectively. In vivo hepatic clearance was determined after intravenous bolus administration in rats. Excellent in vitro-in vivo correlation (IVIVC; R(2) = 0.80) was observed when metabolic intrinsic Cl values were used, which were determined in vitro at a single concentration corresponding to the blood concentration observed in rats in vivo at the mean residence time. On the contrary, poor IVIVC was observed when in vitro metabolic Cl values based on full Michaelis-Menten profiles were used. In addition, the use of uptake Cl values or a combination of both uptake and metabolic clearance data led to poor predictions of in vivo clearance. Although our findings indicate a key role for metabolism in the hepatic clearance of several HIV PI in rats, subsequent simulations revealed that inhibition of hepatic uptake can lead to altered hepatic clearance for several of these drugs.

  1. Chlorogenic acid from honeysuckle improves hepatic lipid dysregulation and modulates hepatic fatty acid composition in rats with chronic endotoxin infusion.

    PubMed

    Zhou, Yan; Ruan, Zheng; Wen, Yanmei; Yang, Yuhui; Mi, Shumei; Zhou, Lili; Wu, Xin; Ding, Sheng; Deng, Zeyuan; Wu, Guoyao; Yin, Yulong

    2016-03-01

    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.

  2. 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).

  3. 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.

  4. 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.

  5. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

    PubMed

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-11-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

  6. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

    PubMed Central

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-01-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases. PMID:27882225

  7. Lung and liver injury following hepatic ischemia/reperfusion in the rat is increased by exogenous lipopolysaccharide which also increases hepatic TNF production in vivo and in vitro.

    PubMed

    Colletti, L M; Green, M

    2001-10-01

    Hepatic ischemia/reperfusion (I/R) results in tumor necrosis factor (TNF) release. Kupffer cells (KC) are one source of this TNF. This study investigates the effects of hepatic I/R combined with lipopolysaccharide (LPS) on the lung and liver injury that follow hepatic I/R and on hepatic release of TNF, epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory protein-2 (MIP-2). The effects of these experimental conditions on TNF production by primary rat KC in vitro were also investigated. Rats were subjected to hepatic I/R alone, hepatic I/R + LPS, sham laparotomy alone, or sham laparotomy + LPS and pulmonary MPO, pulmonary microvascular permeability, hepatic neutrophil influx, hepatic injury, and hepatic TNF, ENA-78, and MIP-2 production were measured. These experiments demonstrated that hepatic I/R in conjunction with LPS results in a more severe lung and liver injury and increased hepatic TNF, ENA-78, and MIP-2 release. The effects of these experimental conditions on rat KC TNF production demonstrated that hepatic I/R + LPS results in a more significant release of TNF as compared to LPS alone or I/R alone. Hepatic I/R plus LPS results in a more severe lung and liver injury and is likely secondary to a more significant and prolonged release of TNF by KC. This may provide a mechanism for development of multiple organ system failure in some patients undergoing hepatic resection, hepatic transplantation, complex vascular operations, or in the setting of hypovolemic shock. Portal endotoxemia related to mesenteric venous congestion or other systemic insults may have a significant impact on post-operative complications and recovery in the setting of a local or global hepatic I/R injury.

  8. Effects of raloxifene on portal hypertension and hepatic encephalopathy in cirrhotic rats.

    PubMed

    Chang, Ching-Chih; Lee, Wen-Shin; Chuang, Chiao-Lin; Hsin, I-Fang; Hsu, Shao-Jung; Chang, Ting; Huang, Hui-Chun; Lee, Fa-Yauh; Lee, Shou-Dong

    2017-05-05

    Raloxifene, a selective estrogen receptor modulator, has been used extensively for osteoporosis. In addition to the effect of osteoporosis treatment, emerging evidences show that raloxifene affects the vascular function in different tissues. Cirrhosis is characterized with portal hypertension and complicated with hepatic encephalopathy. Portal hypertension affects portal-systemic shunt which leads to hepatic encephalopathy that the vascular modulation might influence severity of hepatic encephalopathy. Herein, we evaluated the impact of raloxifene on bile duct ligation (BDL)-induced cirrhotic rats. The female Sprague-Dawley rats received BDL plus ovariectomy or sham-operation. Four weeks later, rats were divided into 2 subgroups respectively to receive of raloxifene (10mg/kg/day) or saline (vehicle) for 14 days. On the 43th day, motor activities and hemodynamic parameters were measured. Hepatic and vascular mRNA and protein expressions were determined. The histopathological change of liver was examined. We found that the liver biochemistry, ammonia level and motor activity were similar between cirrhotic rats with or without raloxifene administration. The hemodynamic parameters were not significantly different except that raloxifene reduced portal venous inflow. Raloxifene exacerbated hepatic fibrosis and up-regulated hepatic endothelin-1 and cyclooxygenase 2 protein expressions. In addition, raloxifene modulated the mRNA expressions of endothelial nitric oxide synthase, cyclooxygenase and endothelin-1 in the superior mesenteric artery and collateral vessel. In conclusion, raloxifene aggravates hepatic fibrosis and decreases portal venous inflow in cirrhotic rats without adversely affecting portal hypertension and hepatic encephalopathy. The modulation of hepatic and vascular endothelin-1, endothelial nitric oxide synthase and cyclooxygenase expressions may play a role in the mechanism.

  9. Stereoselective propranolol metabolism in two drug induced rat hepatic microsomes.

    PubMed

    Li, Xin; Zeng, Su

    2000-02-01

    AIM:To study the influence of inducers BNF and PB on the stereoselective metabolism of propranolol in rat hepatic microsomes.METHODS:Phase I metabolism of propranolol was studied by using the microsomes induced by BNF and PB and the non induced microsome as the control.The enzymatic kinetic parameters of propranolol enantiomers were calculated by regression analysis of Lineweaver-Burk plots. Propranolol concentrations were assayed by HPLC.RESULTS:A RP-HPLC method was developed to determine propranolol concentration in rat hepatic microsomes. The linearity equations for R(+)propranolol and S(-) propranolol were A = 705.7C+311.2C (R = 0.9987) and a = 697.2C+311.4C (R = 0.9970) respectively. Recoveries of each enantiomer were 98.9%, 99.5%, 101.0% at 60&mgr;mol/L, 120&mgr;mol/L, 240&mgr;mol/L respectively. At the concentration level of 120&mgr;mol/L, propranolol enantiomers were metabolized at different rates in different microsomes. The concentration ratio R(+)/S(-) of control and PB induced microsomes increased with time, whereas that of microsome induced by BNF decreased. The assayed enzyme parameters were: 1. Km. Control group: R(+)30 plus minus 8, S(-)18 plus minus 5; BNF group: R(+)34 plus minus 3, S(-)39 plus minus 7; PB group: R(+)38 plus minus 17, S(-)36 plus minus 10. 2. Vmax. Control group: R(+)1.5 plus minus 0.2, S(-)2.9 plus minus 0.3; BNF group: R(+)3.8 plus minus 0.3, S(-)3.3 plus minus 0.5; PB group: R(+)0.07 plus minus 0.03, S(-)1.94 plus minus 0.07. 3. Clint. Control group: R(+)60 plus minus 3, S(-)170 plus minus 30; BNF group: R(+)111.0 plus minus 1, S(-) 84 plus minus 5; PB group: R(+)2.0 plus minus 2, S(-)56.0 plus minus 1. The enzyme parameters compared with unpaired t tests showed that no stereoselectivity was observed in enzymatic affinity of three microsomes to enantiomers and their catalytic abilities were quite different and had stereoselectivities.Compared with the control, microsome induced by BNF enhanced enzyme activity to propranolol R

  10. Characterization of liver stellate cell retinyl ester storage.

    PubMed Central

    Trøen, G; Nilsson, A; Norum, K R; Blomhoff, R

    1994-01-01

    The stellate cells of the liver are the main storage site of retinyl esters in the body. During cultivation in vitro of stellate cells isolated from rat and rabbit livers were observed that the cells rapidly loose their retinyl ester content. Freshly isolated stellate cells contain about 144 nmol of total retinol/mg of protein, while cells cultivated for 14 days contained below 0.1 nmol/mg of protein. When 3-day-old cultures were incubated for 6 h with 2 microM retinol, the cellular content increased from 5.6 to approx. 9.4 nmol of total retinyl esters/mg of protein. In contrast, little retinyl ester accumulated in 10-20-day-old cultures incubated with 2 microM retinol. At 50 microM retinol, however, the retinyl ester level did increase both with 3-day-old cultures and 10-20-day-old cultures. In parallel experiments with cultured fibroblasts esterification characteristics similar to those seen in older cultures of stellate cells were observed. When 10-day-old cultures of stellate cells were incubated with retinol alone, or in combination with palmitic acid, linoleic acid or oleic acid, the total storage of retinyl esters increased by 20-150%. In most cases, the fatty acid supplemented in the medium was found to be the dominant fatty acid esterified with retinol. Cultures of stellate cells were then exposed to a physiological concentration (1.3 microM) of radioactive retinol free in solution or bound to retinol-binding protein. With 3-day-old cultures, as well as older cultures, the cellular content of unesterified retinol was 10-20 times higher when free retinol was added compared with addition of retinol bound to retinol-binding protein. However, 2-3-fold as much radioactive retinyl esters were recovered in cells incubated with retinol-retinol-binding protein compared with retinol free in solution. These results show that retinol delivered to stellate cells from retinol-binding protein is preferentially esterified, and that the complex is handled differently to

  11. Hepatitis

    MedlinePlus

    ... clotting problems or chronic liver disease. previous continue Hepatitis B and Hepatitis C Although hep A is a ... does — through direct contact with infected body fluids. Hepatitis B and C are even more easily passed in ...

  12. Hepatitis

    MedlinePlus

    ... A if they've been vaccinated against it. Hepatitis B Hepatitis B is a more serious infection. It may lead ... of which cause severe illness and even death. Hepatitis B virus (HBV) is transmitted from person to person ...

  13. Hepatitis

    MedlinePlus

    ... Issues Listen Español Text Size Email Print Share Hepatitis Page Content Article Body Hepatitis means “inflammation of ... it has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool ...

  14. Eggshell membrane ameliorates hepatic fibrogenesis in human C3A cells and rats through changes in PPARγ-Endothelin 1 signaling

    PubMed Central

    Jia, Huijuan; Aw, Wanping; Saito, Kenji; Hanate, Manaka; Hasebe, Yukio; Kato, Hisanori

    2014-01-01

    Our previous nutrigenomic findings indicate that eggshell membrane (ESM) may prevent liver fibrosis. Here we investigated the effects and mechanisms underlying ESM intervention against liver injury by using DNA microarray analysis and comparative proteomics. In vitro hydrolyzed ESM attenuated the TGFβ1-induced procollagen production of human hepatocyte C3A cells and inhibited the expression of Endothelin 1 (EDN1) and its two receptors, and extracellular matrix components. In vivo male Wistar rats were allocated into a normal control group, a CCl4 group (hypodermic injection of 50% CCl4 2×/wk) and an ESM group (20 g ESM/kg diet with CCl4 injection) for 7 wks. Dietary ESM ameliorated the elevated activity of ALT/AST, oxidative stress and collagen accumulation in liver, accompanied by the down-regulated expression of Edn1 signaling and notable profibrogenic genes and growth factors as well as peroxisome proliferator-activated receptor gamma (PPARγ). Concomitantly, the decreased expressions of Galectin-1 and Desmin protein in the ESM group indicated the deactivation of hepatic stellate cells (HSCs). Through a multifaceted integrated omics approach, we have demonstrated that ESM can exert an antifibrotic effect by suppressing oxidative stress and promoting collagen degradation by inhibiting HSCs' transformation, potentially via a novel modulation of the PPARγ-Endothelin 1 interaction signaling pathway. PMID:25503635

  15. NDRG2 ameliorates hepatic fibrosis by inhibiting the TGF-β1/Smad pathway and altering the MMP2/TIMP2 ratio in rats.

    PubMed

    Yang, Jiandong; Zheng, Jin; Wu, Lin; Shi, Ming; Zhang, Hongtao; Wang, Xing; Xia, Ning; Wang, Desheng; Liu, Xinping; Yao, Libo; Li, Yan; Dou, Kefeng

    2011-01-01

    Liver fibrosis is a worldwide clinical issue. It has been well established that activated hepatic stellate cells (HSCs) are responsible for excessive extracellular matrix (ECM) deposition in chronically damaged livers. The identification of key elements that control HSCs activation will help to further our understanding of liver fibrosis and improve the outcome of clinical treatment. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of liver fibrosis as NDRG2 mRNA and protein levels were reduced during HSCs activation. In addition, enhanced NDRG2 expression reduced Smad3 transcription and phosphorylation, which inhibited HSCs activation by blocking the TGF-β1 signal. Moreover, NDRG2 contributed to an increase in the ratio of matrix metalloproteinase 2 (MMP2) to tissue inhibitor of matrix metalloproteinase 2 (TIMP2), which may facilitate the degradation of the ECM. In dimethylnitrosamine (DMN)-induced fibrotic rat livers, adenovirus-mediated NDRG2 overexpression resulted in decreased ECM deposition and improved liver function compared with controls. In conclusion, the present findings indicate that the modulation of NDRG2 is a promising strategy for the treatment of liver fibrosis.

  16. Tamarix gallica ameliorates thioacetamide-induced hepatic oxidative stress and hyperproliferative response in Wistar rats.

    PubMed

    Sehrawat, Anuradha; Sultana, Sarwat

    2006-04-01

    Tamarix gallica, a hepatic stimulant and tonic, was examined for its ability to inhibit thioacetamide (TAA)-induced hepatic oxidative stress, toxicity and early tumor promotion response in male Wistar rats. TAA (6.6 mmol/kg body wt. i.p) enhanced lipid peroxidation, hydrogen peroxide content, glutathione S-transferase and xanthine oxidase with reduction in the activities of hepatic antioxidant enzymes viz., glutathione peroxidase, superoxide dismutase and caused depletion in the level of hepatic glutathione content. A marked increase in liver damage markers was also observed. TAA treatment also enhanced tumor promotion markers, ornithine decarboxylase (ODC) activity and [3H] thymidine incorporation into hepatic DNA. Pretreatment of rats orally with Tamarix gallica extract (25 and 50 mg/kg body weight) prevented TAA-promoted oxidative stress and toxicity. Prophylaxis with Tamarix gallica significantly reduced the susceptibility of the hepatic microsomal membrane for iron-ascorbate induced lipid peroxidation, H2O2 content, glutathione S-transferase and xanthine oxidase activities. There was also reversal of the elevated levels of liver marker parameters and tumor promotion markers. Our data suggests that Tamarix gallica is a potent chemopreventive agent and may suppress TAA-mediated hepatic oxidative stress, toxicity, and tumor promotion response in rats.

  17. Effect of structurally diverse peroxisome proliferators on rat hepatic sulfotransferase.

    PubMed

    Witzmann, F; Coughtrie, M; Fultz, C; Lipscomb, J

    1996-01-05

    Exposure to perfluorocarboxylic acids, pthalate esters, and some hypolipidemic agents results in the proliferation of peroxisomes in the rodent liver. The structural diversity of these compounds suggests mechanistic diversity in their toxicity as well. To establish reliable biomarkers of peroxisome proliferation (PP) in compounds with distinct chemical toxicities, this study investigated the effect of in vivo exposure to perfluoro-n-octanoic acid, perfluoro-n-decanoic acid, di(2-ethylhexyl)phthalate (DEHP) and clofibrate on two-dimensional electrophoretic protein patterns of rat hepatic sulfotransferases, ST1A1, ST1C1 and ST2A1. After exposure to peroxisome proliferative doses, both ST1A1 and ST1C1 abundance in whole liver homogenates was significantly reduced, but only as a result of perfluorocarboxylic and exposure. The well-established PPs, DEHP and clofibrate had no effect on sulfotransferase expression whatsoever. The observed down-regulation of these STs is significant with respect to their normal detoxication activities and its potential correlation to carcinogenesis warrants further study. The present investigation supports previous studies that demonstrate the unique features of perfluorocarboxylic acid toxicity, relative to classic peroxisome proliferators and endorses the continued use of 2D protein-mapping of Sts and other proteins as biomarkers of chemical toxicity.

  18. [Effect of metformin on the formation of hepatic fibrosis in type 2 diabetic rats].

    PubMed

    Qiang, Gui-Fen; Zhang, Li; Xuan, Qi; Yang, Xiu-Ying; Shi, Li-Li; Zhang, Heng-Ai; Chen, Bai-Nian; Du, Guan-Hua

    2010-06-01

    The aim of this study is to investigate the effects of the metformin on the formation of hepatic fibrosis in type 2 diabetic rats and discuss its mechanism of liver-protecting activity. After SD rats were fed with high-fat and high-sucrose diet for four weeks, low-dose streptozotocin (STZ) was injected intraperitoneally to make the animal mode of type 2 diabetes. Then, all diabetic rats was fed with the high-fat diet and metformin (ig, 100 mg x kg(-1)) was given orally to metformin group for four months. After the last administration, fasting blood glucose was determined. The livers were removed to calculate the hepatic coefficient and to make HE and Picro acid-Sirius red staining, immunohistochemistry (alpha-SMA and TGFbeta1) and TUNEL staining in order to evaluate the effect of metformin on the hepatic fibrosis. The animal model of type 2 diabetes with hepatic fibrosis was successfully made. Metformin can significantly alleviate the lesions of hepatic steatosis and fibrosis, markedly reduce the expressions of alpha-SMA and TGFbeta1 in liver tissue of type 2 diabetic rats. However, TUNEL staining result suggested that metformin could not reduce apoptosis of hepatocytes. The results suggest that metformin can inhibit the formation of hepatic fibrosis in type 2 diabetes.

  19. Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachloride-induced hepatic fibrosis.

    PubMed

    Zhen, Mao-Chuan; Wang, Qian; Huang, Xiao-Hui; Cao, Liang-Qi; Chen, Xi-Ling; Sun, Kai; Liu, Yun-Jian; Li, Wen; Zhang, Long-Juan

    2007-12-01

    The aim of the study was to examine the effects of epigallocatechin-3-gallate (EGCG) on hepatic fibrogenesis and on cultured hepatic stellate cells (HSCs). The rat model of carbon tetrachloride (CCl(4))-induced hepatic fibrosis was used to assess the effect of daily intraperitoneal injections of EGCG on the indexes of fibrosis. Histological and hepatic hydroxyproline examination revealed that EGCG significantly arrested progression of hepatic fibrosis. EGCG caused significant amelioration of liver injury (reduced activities of serum alanine aminotransferase and aspartate aminotransferase). The development of CCl(4)-induced hepatic fibrosis altered the redox state with a decreased hepatic glutathione and increased the formation of lipid peroxidative products, which were partially normalized by treatment with EGCG, respectively. Moreover, EGCG markedly attenuated HSC activation as well as matrix metalloproteinase (MMP)-2 activity. In cultured stellate cell, the expression of MMP-2 mRNA and protein were substantially reduced by EGCG treatment. Concanavalin A-induced activation of secreted MMP-2 was inhibited by EGCG through the influence of membrane type 1-MMP activity. These results demonstrate that administration of EGCG may be useful in the treatment and prevention of hepatic fibrosis.

  20. High Sucrose Intake Ameliorates the Accumulation of Hepatic Triacylglycerol Promoted by Restraint Stress in Young Rats.

    PubMed

    Corona-Pérez, Adriana; Díaz-Muñoz, Mauricio; Rodríguez, Ida Soto; Cuevas, Estela; Martínez-Gómez, Margarita; Castelán, Francisco; Rodríguez-Antolín, Jorge; Nicolás-Toledo, Leticia

    2015-11-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. Stress promotes the onset of the NAFLD with a concomitant increment in the activity of the hepatic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1). However, the interaction between the stress and a carbohydrate-enriched diet for the development of NAFLD in young animals is unknown. In the present study, we evaluated the impact of chronic stress on the hepatic triacylglycerol level of young rats fed or not with a high sucrose-diet. For doing this, 21-day old male Wistar rats were allocated into 4 groups: control (C), chronic restraint stress (St), high-sucrose diet (S30), and chronic restraint stress plus a 30 % sucrose diet (St + S30). Chronic restraint stress consisted of 1-hour daily session, 5 days per week and for 4 weeks. Rats were fed with a standard chow and tap water (C group) or 30 % sucrose diluted in water (S30 group). The St + S30 groups consumed less solid food but had an elevated visceral fat accumulation in comparison with the St group. The St group showed a high level of serum corticosterone and a high activity of the hepatic 11β-HSD-1 concomitantly to the augmentation of hepatic steatosis signs, a high hepatic triacylglycerol content, and hepatic oxidative stress. Conversely, the high-sucrose intake in stressed rats (St + S30 group) reduced the hepatic 11β-HSD-1 activity, the level of serum corticosterone, and the hepatic triacylglycerol content. Present findings show that a high-sucrose diet ameliorates the triacylglycerol accumulation in liver promoted by the restraint stress in young male rats.

  1. 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.

  2. 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

  3. 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

  4. Green tea catechins, alleviate hepatic lipidemic-oxidative injury in Wistar rats fed an atherogenic diet.

    PubMed

    Ramesh, E; Jayakumar, T; Elanchezhian, R; Sakthivel, M; Geraldine, P; Thomas, P A

    2009-06-15

    In the present study, the efficacy of green tea catechins (GTC from the plant Camellia sinensis), with epigallocatechin gallate (EGCG), as the major component, was studied in relation to hepatic oxidative abnormalities in atherosclerotic rats. When male albino Wistar rats were fed an atherogenic diet for 30 days and then treated with saline for 7 or 15 days, there was a significant decline in hepatic mean activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase), and non-enzymatic antioxidants (reduced glutathione, vitamins C and E) while there was a significant elevation in the mean level of hepatic malondialdehyde (MDA), in comparison to the values noted in control rats fed a normal diet. In addition, a concomitant increase in the activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) was noted, when compared to the values in control rats. Following intraperitoneal administration of GTC (100mg/kg) for 7 or 15 days to rats fed the atherogenic diet, significantly higher mean activities of enzymatic and non-enzymatic antioxidants and lower mean levels of MDA in hepatic tissue and lower mean activities of AST, ALT, ALP and LDH in serum were observed, compared to the values in the rats fed the atherogenic diet and treated with saline. Histopathological studies were performed to provide direct evidence of the atherogenic diet-induced hepatic changes and of the hepatoprotective effect of GTC. These results suggest that EGCG as a major component of green tea catechins may protect against the hepatic abnormalities occurring in Wistar rats fed an atherogenic diet.

  5. Reduction in hepatic non-esterified fatty acid concentration after long-term treatment with atorvastatin lowers hepatic triglyceride synthesis and its secretion in sucrose-fed rats.

    PubMed

    Funatsu, Toshiyuki; Goto, Masahide; Kakuta, Hirotoshi; Suzuki, Masanori; Ida, Motoko; Nishijima, Satomi; Tanaka, Hideyuki; Yasuda, Shuhei; Miyata, Keiji

    2002-02-28

    The mechanism by which atorvastatin lowers plasma triglyceride (TG) levels is mainly through a decrease in hepatic TG secretion. However, it is not clear why atorvastatin, which does not inhibit TG synthesis in vitro, decreases hepatic TG secretion without a prospective increase in hepatic TG concentration. For the investigation of the mechanisms that underlie the hypotriglyceridemic effects of atorvastatin, we characterized the effect of either a single or an 11 day administration of atorvastatin in sucrose-induced hypertriglyceridemic rats. Atorvastatin (30 mg/kg p.o.) strongly decreased the rate of both very-low-density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B secretion. The inhibitor also decreased hepatic TG concentration. Hepatic TG synthesis activity was also decreased by atorvastatin, and its activity was correlated with both hepatic and plasma TG concentration. There was also a strong correlation between the hepatic TG synthesis and hepatic non-esterified fatty acid (NEFA) concentration (r(2)=0.815). These effects required chronic administration of the inhibitor and were not observed by acute treatment. Repeated administration of atorvastatin also strongly reduced hepatic acyl-coenzyme A synthase mRNA levels. These results suggest that the reduced hepatic NEFA most likely lowers hepatic TG synthesis and TG secretion in sucrose-fed hypertriglyceridemic rats.

  6. Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis

    PubMed Central

    Apte, M; Haber, P; Darby, S; Rodgers, S; McCaughan, G; Korsten, M; Pirola, R; Wilson, J

    1999-01-01

    BACKGROUND—The pathogenesis of pancreatic fibrosis is unknown. In the liver, stellate cells play a major role in fibrogenesis by synthesising increased amounts of collagen and other extracellular matrix (ECM) proteins when activated by profibrogenic mediators such as cytokines and oxidant stress. 
AIMS—To determine whether cultured rat pancreatic stellate cells produce collagen and other ECM proteins, and exhibit signs of activation when exposed to the cytokines platelet derived growth factor (PDGF) or transforming growth factor β (TGF-β). 
METHODS—Cultured pancreatic stellate cells were immunostained for the ECM proteins procollagen III, collagen I, laminin, and fibronectin using specific polyclonal antibodies. For cytokine studies, triplicate wells of cells were incubated with increasing concentrations of PDGF or TGF-β. 
RESULTS—Cultured pancreatic stellate cells stained strongly positive for all ECM proteins tested. Incubation of cells with 1, 5, and 10 ng/ml PDGF led to a significant dose related increase in cell counts as well as in the incorporation of 3H-thymidine into DNA. Stellate cells exposed to 0.25, 0.5, and 1 ng/ml TGF-β showed a dose dependent increase in α smooth muscle actin expression and increased collagen synthesis. In addition, TGF-β increased the expression of PDGF receptors on stellate cells. 
CONCLUSIONS—Pancreatic stellate cells produce collagen and other extracellular matrix proteins, and respond to the cytokines PDGF and TGF-β by increased proliferation and increased collagen synthesis. These results suggest an important role for stellate cells in pancreatic fibrogenesis. 

 Keywords: pancreatic fibrosis; stellate cell activation; cytokines PMID:10075961

  7. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ethan...

  8. Combined effect of sesamin and soybean phospholipid on hepatic fatty acid metabolism in rats.

    PubMed

    Ide, Takashi

    2014-05-01

    We studied the combined effect of sesamin (1:1 mixture of sesamin and episesamine) and soybean phospholipid on lipid metabolism in rats. Male rats were fed diets supplemented with 0 or 2 g/kg sesamin, and containing 0 or 50 g/kg soybean phospholipid, for 19 days. Sesamin and soybean phospholipid decreased serum triacylglycerol concentrations and the combination of these compounds further decreased the parameter in an additive fashion. Soybean phospholipid but not sesamin reduced the hepatic concentration of triacylglycerol. The combination failed to cause a strong decrease in hepatic triacylglycerol concentration, presumably due to the up-regulation of Cd36 by sesamin. Combination of sesamin and soybean phospholipid decreased the activity and mRNA levels of hepatic lipogenic enzymes in an additive fashion. Sesamin strongly increased the parameters of hepatic fatty acid oxidation enzymes. Soybean phospholipid increased hepatic activity of 3-hydroxyacyl-CoA dehydrogenase although it failed to affect the activity of other enzymes involved in fatty acid oxidation. Sesamin strongly increased hepatic concentration of carnitine. Sesamin and soybean phospholipid combination further increased this parameter, accompanying a parallel increase in mRNA expression of carnitine transporter. These changes can account for the strong decrease in serum triacylglycerol in rats fed a diet containing both sesamin and soybean phospholipid.

  9. Preventive effects of chronic exogenous growth hormone levels on diet-induced hepatic steatosis in rats

    PubMed Central

    2010-01-01

    Background Non-alcoholic fatty liver disease (NAFLD), which is characterized by hepatic steatosis, can be reversed by early treatment. Several case reports have indicated that the administration of recombinant growth hormone (GH) could improve fatty liver in GH-deficient patients. Here, we investigated whether chronic exogenous GH levels could improve hepatic steatosis induced by a high-fat diet in rats, and explored the underlying mechanisms. Results High-fat diet-fed rats developed abdominal obesity, fatty liver and insulin resistance. Chronic exogenous GH improved fatty liver, by reversing dyslipidaemia, fat accumulation and insulin resistance. Exogenous GH also reduced serum tumour necrosis factor-alpha (TNF-alpha) levels, and ameliorated hepatic lipid peroxidation and oxidative stress. Hepatic fat deposition was also reduced by exogenous GH levels, as was the expression of adipocyte-derived adipokines (adiponectin, leptin and resistin), which might improve lipid metabolism and hepatic steatosis. Exogenous GH seems to improve fatty liver by reducing fat weight, improving insulin sensitivity and correcting oxidative stress, which may be achieved through phosphorylation or dephosphorylation of a group of signal transducers and activators of hepatic signal transduction pathways. Conclusions Chronic exogenous GH has positive effects on fatty liver and may be a potential clinical application in the prevention or reversal of fatty liver. However, chronic secretion of exogenous GH, even at a low level, may increase serum glucose and insulin levels in rats fed a standard diet, and thus increase the risk of insulin resistance. PMID:20653983

  10. 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.

  11. Effects of acute hepatic and renal failure on pharmacokinetics of flunixin meglumine in rats.

    PubMed

    Hwang, Youn-Hwan; Yun, Hyo-In

    2011-01-01

    The aim of this study was to investigate the effects of hepatic and renal failure on the pharmacokinetics of flunixin in carbon tetrachloride (CCl(4))- and glycerol-treated rats. After intravenous administration of flunixin (2 mg/kg), the plasma concentration of flunixin was measured by high-performance liquid chromatography. Both acute hepatic and renal failure resulted in significantly increased area under the curve (AUC), prolonged elimination half-life (t(1/2β)), and reduced total body clearance (Cl(tot)) compared with respective controls (P<0.05). In conclusion, hepatic failure as well as renal failure modified the pharmacokinetics of flunixin.

  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. Hepatic regeneration after sublethal partial liver irradiation in cirrhotic rats.

    PubMed

    Gu, Ke; Lai, Song-Tao; Ma, Ning-Yi; Zhao, Jian-Dong; Ren, Zhi-Gang; Wang, Jian; Liu, Jin; Jiang, Guo-Liang

    2011-01-01

    Our previous animal study had demonstrated that partial liver irradiation (IR) could stimulate regeneration in the protected liver, which supported the measurements adopted in radiotherapy planning for hepatocellular carcinoma. The purpose of this present study is to investigate whether cirrhotic liver repopulation could be triggered by partial liver IR. The cirrhosis was induced by thioacetamide (TAA) in rats. After cirrhosis establishment, TAA was withdrawn. In Experiment 1, only right-half liver was irradiated with single doses of 5 Gy, 10 Gy and 15 Gy, respectively. In Experiment 2, right-half liver was irradiated to 15 Gy, and the left-half to 2.5 Gy, 5 Gy and 7.5 Gy, respectively. The regeneration endpoints, including liver index (LI); mitotic index (MI); liver proliferation index (LPI); PCNA-labeling index (PCNA-LI); serum HGF, VEGF, TGF-α and IL-6, were evaluated on 0 day, 30-day, 60-day, 90-day, 120-day and 150-day after IR. Serum and in situ TGF-β1 were also measured. In both experimental groups, the IR injuries were sublethal, inducing no more than 9% animal deaths. Upon TAA withdrawal, hepatic regeneration decelerated in the controls. In Experiment 1 except for LI, all other regeneration parameters were significantly higher than those in controls for both right-half and left-half livers. In Experiment 2 all regeneration parameters were also higher compared with those in controls for both half livers. Serum HGF and VEGF were increased compared with that of controls. Both unirradiated and low dose-irradiated cirrhotic liver were able to regenerate triggered by sublethal partial liver IR and higher doses and IR to both halves liver triggered a more enhanced regeneration.

  14. Hepatic ornithine decarboxylase induction by potato glycoalkaloids in rats.

    PubMed

    Caldwell, K A; Grosjean, O K; Henika, P R; Friedman, M

    1991-08-01

    The induction of hepatic ornithine decarboxylase (ODC) activity in rat livers by the potato glycoalkaloids alpha-solanine, alpha-chaconine, and their aglycone solanidine, has been studied. Ip administration of alpha-solanine at 7.5, 15 and 30 mg/kg body weight produced markedly elevated enzyme activity at 4 hr after treatment, with a linear dose response. The increase was four-fold at the lowest dose administered to 12-fold at the highest. ODC activity was measured at 1, 2, 3, 4, 5, 6, 8, and 24hr after alpha-solanine was given. A statistically significant increase in enzyme activity was evident at 3 hr after treatment; maximal activity occurred at 5 hr and was approximately 12 times greater than the dimethylsulphoxide (DMSO) control level. Elevated activities persisted for several hours, decreasing to about one-third of the maximal level at 8 hr. The relative effects of alpha-solanine, alpha-chaconine and solanidine on ODC activities were studied at 4 hr using an equimolar dose of 17 mM/kg body weight. ODC activity induced by alpha-chaconine was higher than that induced by alpha-solanine; the latter activity was two-thirds that of the former. The aglycone solanidine did not induce any increase in activity compared with the DMSO control. ODC activity with dexamethasone, a glucocorticoid, at 4 mg/kg body weight, followed a pattern similar to that of alpha-solanine. However, maximal activity occurred slightly earlier at 4 hr after treatment. The results show that the extent of induced ODC activity depends on the structure of the potato alkaloid.

  15. Comparative hepatic and renal toxicity of cadmium in male and female rats.

    PubMed

    Gubrelay, Udita; Mehta, Ashish; Singh, Maninder; Flora, S J S

    2004-01-01

    Rats (male and female) were exposed to 0.5 mg/kg and 1 mg/kg cadmium as cadmium chloride for 3 days and subsequently sacrificed for cadmium concentration and other biochemical variables indicative of hepatic and renal damage. The absorption of cadmium was supported by biochemical changes, which were significantly higher in females than in males. This could be due to higher rate of intestinal absorption of cadmium in females than males. Male and female rats both showed relatively higher cadmium concentration in kidneys than in liver. Female rats also showed the similar trend in tissue metal levels as compared to male rats. However, hepatic and renal histopathological observations showed that female rats suffered from severe hepatic injury like hydropic degeneration of hepatocytes, granulation, bile duct proliferation etc. In comparison to female rats, male rats did not show much remarkable changes. Renal damage was more prominent in female than male in the form of renal tubular damage; most of the tubular nuclei were pyknotic, congestion of the boundary of cortex and medulla etc. The results suggested that females were comparatively more vulnerable to the toxic effects of cadmium than males.

  16. 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

  17. Knockdown of Neuropeptide Y in the Dorsomedial Hypothalamus Promotes Hepatic Insulin Sensitivity in Male Rats.

    PubMed

    Li, Lin; de La Serre, C Barbier; Zhang, Ni; Yang, Liang; Li, Hong; Bi, Sheng

    2016-12-01

    Recent evidence has shown that alterations in dorsomedial hypothalamic (DMH) neuropeptide Y (NPY) signaling influence glucose homeostasis, but the mechanism through which DMH NPY acts to affect glucose homeostasis remains unclear. Here we report that DMH NPY descending signals to the dorsal motor nucleus of the vagus (DMV) modulate hepatic insulin sensitivity to control hepatic glucose production in rats. Using the hyperinsulinemic-euglycemic clamp, we revealed that knockdown of NPY in the DMH by adeno-associated virus-mediated NPY-specific RNAi promoted insulin's action on suppression of hepatic glucose production. This knockdown silenced DMH NPY descending signals to the DMV, leading to an elevation of hepatic vagal innervation. Hepatic vagotomy abolished the inhibitory effect of DMH NPY knockdown on hepatic glucose production, but this glycemic effect was not affected by vagal deafferentation. Together, these results demonstrate a distinct role for DMH NPY in the regulation of glucose homeostasis through the hepatic vagal efferents and insulin action on hepatic glucose production.

  18. 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.

  19. Atorvastatin reverses age-related reduction in rat hepatic PPARalpha and HNF-4.

    PubMed

    Sanguino, Elena; Roglans, Nuria; Alegret, Marta; Sánchez, Rosa M; Vázquez-Carrera, Manuel; Laguna, Juan C

    2005-08-01

    Old rats are resistant to fibrate-induced hypolipidemia owing to a reduction in hepatic peroxisome proliferator-activated receptor alpha (PPARalpha). We tested whether the age-related decrease in PPARalpha is prevented by atorvastatin (ATV), a hypolipidemic statin. We determined the activity and expression of Liver X receptor alpha (LXRalpha) and PPARalpha in the liver of 18-month-old rats treated with 10 mg kg(-1) of ATV for 21 days. We measured fatty acid oxidation (FAO), the expression of PPARalpha-target genes, liver triglyceride (TG) and cholesteryl ester (CE) contents and plasma concentrations of TG, cholesterol, glucose, nonesterified fatty acids (NEFA), insulin and leptin. While old female rats were practically unresponsive, ATV-treated old males showed lower liver TG (-41%) and CE (-48%), and plasma TG (-35%), glucose (-18%) and NEFA (-39%). Age-related alterations in LXRalpha expression and binding activity were reverted in ATV-treated old males. These changes were related to an increase in hepatic FAO (1.2-fold), and PPARalpha mRNA (2.2-fold), PPARalpha protein (1.6-fold), and PPARalpha-binding activity. Hepatic nuclear factor-4 (HNF-4) and chicken ovalbumin upstream-transcription factor-II participate in the transcriptional regulation of the PPARalpha gene, while peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) behaves as a PPAR coactivator. Ageing reduced the hepatic content of HNF-4 (74%) and PGC-1 (77%) exclusively in male rats. ATV administration to old males enhanced the hepatic expression and binding activity (two-fold) of HNF-4. ATV-induced changes in hepatic HNF-4 and PPARalpha may be responsible for the improvement of the lipid metabolic phenotype produced by ATV administration to senescent male rats.

  20. Olanzapine modulation of hepatic oxidative stress and inflammation in socially isolated rats.

    PubMed

    Todorović, Nevena; Tomanović, Nada; Gass, Peter; Filipović, Dragana

    2016-01-01

    Olanzapine, an atypical antipsychotic, is efficient in stress associated psychiatric diseases, but its effect on the liver, a primary organ for drug activation and detoxification, still remains unclear. The effect of olanzapine administration (7.5mg/kg/day), on rat hepatic glutathione (GSH)-dependent defense and proinflammatory cytokines following 6weeks of chronic social isolation (CSIS), which causes depressive- and anxiety-like behavior in adult male Wistar rats, was investigated. The subcellular distribution of nuclear factor-κB (NF-κB), cytosolic inducible nitric oxide synthase (iNOS) protein levels and hepatic histological alterations were also determined. Decreased GSH content and glutathione reductase activity associated with increased catalase and glutathione S-transferase activity following CSIS indicated hepatic oxidative stress. Moreover, CSIS caused NF-κB nuclear translocation and the concomitant increase in iNOS together with increase in interleukin-1beta and tumor necrosis factor alpha protein levels, but no effect on interleukin-6. Olanzapine treatment suppressed NF-κB activation and iNOS expression and caused modulation of GSH-dependent defense systems but failed to reverse CSIS-induced increase in hepatic proinflammatory cytokines. Portal inflammation, focal hepatocyte necrosis and an increased number of Kupffer cells in CSIS rats (vehicle- or olanzapine-treated) were found. Olanzapine-treated socially reared rats showed portal inflammation and focal hepatocyte necrosis. Data suggest that CSIS compromised GSH-dependent defense, triggered a proinflammatory response and histological alterations in rat liver. Olanzapine treatment partially reversed the alterations in hepatic GSH-dependent defense, but showed no anti-inflammatory effect suggesting that it may provide protective effect against hepatic CSIS-induced oxidative stress, but not against inflammation.

  1. Atorvastatin reverses age-related reduction in rat hepatic PPARα and HNF-4

    PubMed Central

    Sanguino, Elena; Roglans, Nuria; Alegret, Marta; Sánchez, Rosa M; Vázquez-Carrera, Manuel; Laguna, Juan C

    2005-01-01

    Old rats are resistant to fibrate-induced hypolipidemia owing to a reduction in hepatic peroxisome proliferator-activated receptor α (PPARα). We tested whether the age-related decrease in PPARα is prevented by atorvastatin (ATV), a hypolipidemic statin. We determined the activity and expression of Liver X receptor α (LXRα) and PPARα in the liver of 18-month-old rats treated with 10 mg kg−1 of ATV for 21 days. We measured fatty acid oxidation (FAO), the expression of PPARα-target genes, liver triglyceride (TG) and cholesteryl ester (CE) contents and plasma concentrations of TG, cholesterol, glucose, nonesterified fatty acids (NEFA), insulin and leptin. While old female rats were practically unresponsive, ATV-treated old males showed lower liver TG (−41%) and CE (−48%), and plasma TG (−35%), glucose (−18%) and NEFA (−39%). Age-related alterations in LXRα expression and binding activity were reverted in ATV-treated old males. These changes were related to an increase in hepatic FAO (1.2-fold), and PPARα mRNA (2.2-fold), PPARα protein (1.6-fold), and PPARα-binding activity. Hepatic nuclear factor-4 (HNF-4) and chicken ovalbumin upstream-transcription factor-II participate in the transcriptional regulation of the PPARα gene, while peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) behaves as a PPAR coactivator. Ageing reduced the hepatic content of HNF-4 (74%) and PGC-1 (77%) exclusively in male rats. ATV administration to old males enhanced the hepatic expression and binding activity (two-fold) of HNF-4. ATV-induced changes in hepatic HNF-4 and PPARα may be responsible for the improvement of the lipid metabolic phenotype produced by ATV administration to senescent male rats. PMID:15912134

  2. Methanethiol metabolism and its role in the pathogenesis of hepatic encephalopathy in rats and dogs.

    PubMed

    Blom, H J; Chamuleau, R A; Rothuizen, J; Deutz, N E; Tangerman, A

    1990-04-01

    The metabolism of methanethiol was studied in rats. Administration of a noncomatogenic dose of methanethiol through inspired air or injection into the upper colon resulted in an elevation of the concentrations of methanethiol mixed disulfides in serum (protein--S--S--CH3 and X--S--S--CH3, X yet unknown) and in urine (X--S--S--CH3). The concentrations of methanethiol mixed disulfides proved to be a relative measure of exposure to methanethiol. The levels of volatile sulfur compounds methanethiol, dimethylsulfide and dimethyldisulfide in the air expired by rats exposed to a noncomatogenic dose of methanethiol through the colon were also elevated. Rats with acute hepatic encephalopathy caused by liver ischemia also showed elevation of methanethiol mixed disulfide levels on challenge of methanethiol through the colon or inspired air, but to a significantly smaller extent than did the corresponding sham-operated rats. This suggests that the liver is at least partly responsible for formation of methanethiol mixed disulfides. No additional toxic effects were observed in the rats with ischemic livers on methanethiol exposition when compared with normal rats, suggesting that the liver does not play an essential role in methanethiol detoxification. Metabolism of methanethiol by blood to sulfate, for example, might be more important. In rats with acute hepatic encephalopathy caused by liver ischemia and in dogs suffering from hepatic encephalopathy resulting from chronic liver disease, large and significant increases in ammonia levels were measured. However, the mean levels of methanethiol mixed disulfides in rats and dogs with hepatic encephalopathy were not different from the mean normal levels in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. 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

  4. 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.

  5. Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

    PubMed

    Johne, Reimar; Dremsek, Paul; Kindler, Eveline; Schielke, Anika; Plenge-Bönig, Anita; Gregersen, Henrike; Wessels, Ute; Schmidt, Katja; Rietschel, Wolfram; Groschup, Martin H; Guenther, Sebastian; Heckel, Gerald; Ulrich, Rainer G

    2012-07-01

    Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

  6. Effects of perinatal exposure to nonylphenol on delivery outcomes of pregnant rats and inflammatory hepatic injury in newborn rats.

    PubMed

    Yu, J; Luo, Y; Yang, X F; Yang, M X; Yang, J; Yang, X S; Zhou, J; Gao, F; He, L T; Xu, J

    2016-12-08

    The current study aimed to investigate the effects of perinatal exposure to nonylphenol (NP) on delivery outcome of pregnant rats and subsequent inflammatory hepatic injury in newborn rats. The pregnant rats were divided into 2 groups: control group (corn oil) and NP exposure group. Thirty-four pregnant rats were administered NP or corn oil by gavage from the sixth day of pregnancy to 21 days postpartum, with blood samples collected at 12 and 21 days of pregnancy and 60 days after delivery. The NP concentration was measured by HPLC, with chemiluminescence used for detection of estrogen and progesterone levels. Maternal delivery parameters were also observed. Liver and blood of the newborn rats were collected and subjected to automatic biochemical detection of liver function and blood lipid analyzer (immunoturbidimetry), and ultrastructural observation of the hepatic microstructure, with the TNF-α and IL-1β hepatic tissue levels evaluated by immunohistochemistry. Compared with the control group, the pregnant and postpartum serum NP and estradiol levels of the mother rats in the NP group were significantly increased, together with lowered progesterone level, increased number of threatened abortion and dystocia, and fewer newborn rats and lower litter weight. Serum and hepatic NP levels of the newborn rats measured 60 days after birth were significantly higher than those of the control group, as well as lower testosterone levels and increased estradiol levels. When observed under electron microscope, the hepatocyte nuclei of the control group were large and round, with evenly distributed chromatin. The chromatin of hepatocytes in the NP group presented deep staining of the nuclei, significant lipid decrease in the cytoplasm, and the majority of cells bonded with lysate. The results of immunohistochemistry showed that there was almost no TNF-α or IL-1β expression in the hepatocytes of the control group, while the number of TNF-α-, PCNA-, and IL-1β-positive cells

  7. Effects of perinatal exposure to nonylphenol on delivery outcomes of pregnant rats and inflammatory hepatic injury in newborn rats

    PubMed Central

    Yu, J.; Luo, Y.; Yang, X.F.; Yang, M.X.; Yang, J.; Yang, X.S.; Zhou, J.; Gao, F.; He, L.T.; Xu, J.

    2016-01-01

    The current study aimed to investigate the effects of perinatal exposure to nonylphenol (NP) on delivery outcome of pregnant rats and subsequent inflammatory hepatic injury in newborn rats. The pregnant rats were divided into 2 groups: control group (corn oil) and NP exposure group. Thirty-four pregnant rats were administered NP or corn oil by gavage from the sixth day of pregnancy to 21 days postpartum, with blood samples collected at 12 and 21 days of pregnancy and 60 days after delivery. The NP concentration was measured by HPLC, with chemiluminescence used for detection of estrogen and progesterone levels. Maternal delivery parameters were also observed. Liver and blood of the newborn rats were collected and subjected to automatic biochemical detection of liver function and blood lipid analyzer (immunoturbidimetry), and ultrastructural observation of the hepatic microstructure, with the TNF-α and IL-1β hepatic tissue levels evaluated by immunohistochemistry. Compared with the control group, the pregnant and postpartum serum NP and estradiol levels of the mother rats in the NP group were significantly increased, together with lowered progesterone level, increased number of threatened abortion and dystocia, and fewer newborn rats and lower litter weight. Serum and hepatic NP levels of the newborn rats measured 60 days after birth were significantly higher than those of the control group, as well as lower testosterone levels and increased estradiol levels. When observed under electron microscope, the hepatocyte nuclei of the control group were large and round, with evenly distributed chromatin. The chromatin of hepatocytes in the NP group presented deep staining of the nuclei, significant lipid decrease in the cytoplasm, and the majority of cells bonded with lysate. The results of immunohistochemistry showed that there was almost no TNF-α or IL-1β expression in the hepatocytes of the control group, while the number of TNF-α-, PCNA-, and IL-1β-positive cells

  8. Repeated transplantation of hepatocytes prevents fulminant hepatitis in a rat model of Wilson's disease.

    PubMed

    Sauer, Vanessa; Siaj, Ramsi; Stöppeler, Sandra; Bahde, Ralf; Spiegel, Hans-Ullrich; Köhler, Gabriele; Zibert, Andree; Schmidt, Hartmut H J

    2012-02-01

    The outcome of consecutive hepatocyte transplants was explored in a rat model of Wilson's disease before the onset of fulminant hepatitis without preconditioning regimens. Rats received a high-copper diet in order to induce a rapid induction of liver failure. Sham-operated rats (15/15) developed jaundice and fulminant hepatitis, and they died within 4 weeks of first transplantation. Despite the continuation of a high dietary copper challenge, long-term survival was observed for a notable proportion of the transplanted animals (7/18). All survivors displayed normalized levels of hepatitis-associated serum markers and ceruloplasmin oxidase activity by posttransplant days 50 and 98, respectively. The liver copper concentrations, the liver histology, and the expression of marker genes were significantly restored within 4 months of transplantation in comparison with the control group. The high expression of a copper transporter gene (ATPase Cu++ transporting beta polypeptide) in the livers of the survivors indicated a high rate of repopulation by donor hepatocytes. Our data suggest that repeated cell transplantation can overcome the limitations of a single therapy session in rats with severe hepatic disease by functionally restoring the host liver without preconditioning.

  9. 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...

  10. 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...

  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. Effect of schisandrin B and sesamin mixture on CCl(4)-induced hepatic oxidative stress in rats.

    PubMed

    Chang, Chia-Yu; Chen, Ya-Ling; Yang, Suh-Ching; Huang, Guan-Cheng; Tsi, Daniel; Huang, Chi-Chang; Chen, Jiun-Rong; Li, Joe-Sharg

    2009-02-01

    To study the effects of schisandrin B and sesamin mixture on carbon tetrachloride (CCl(4))-induced hepatic oxidative stress in male Sprague-Dawley rats. The rats were randomly assigned to five groups: control group (olive oil injection), CCl(4) group (CCl(4) injection), silymarin group (CCl(4) injection combined with supplementation of silymarin, 7.5 mg/kg/day), low dose group (CCl(4) injection combined with supplementation of schisandrin B and sesamin mixture at a low dose, 43 mg/kg/day) and high dose group (CCl(4) injection combined with the supplementation of schisandrin B and sesamin mixture at a high dose, 215 mg/kg/day). The hepatic superoxide dismutase and glutathione peroxidase activities of rats in the low dose and high dose groups were increased significantly compared with those in the CCl(4) group. The hepatic reduced glutathione concentration in the silymarin, low dose and high dose groups were increased significantly (48%, 45% and 53%, respectively) when compared with those of the CCl(4) group. In addition, the concentration of glutathione in the erythrocytes of the low dose group was significantly higher than the CCl(4) group by 25%. These results suggest that the schisandrin B-sesamin mixture exerted a hepatoprotective effect by improving the antioxidative capacity in rats under CCl(4)-induced hepatic oxidative stress.

  13. Dietary medium-chain triglycerides attenuate hepatic lipid deposition in growing rats with protein malnutrition.

    PubMed

    Kuwahata, Masashi; Kubota, Hiroyo; Amano, Saki; Yokoyama, Meiko; Shimamura, Yasuhiro; Ito, Shunsuke; Ogawa, Aki; Kobayashi, Yukiko; Miyamoto, Ken-ichi; Kido, Yasuhiro

    2011-01-01

    The objective of this study was to investigate the effects of dietary medium-chain triglycerides (MCT) on hepatic lipid accumulation in growing rats with protein malnutrition. Weaning rats were fed either a low-protein diet (3%, LP) or control protein diet (20%, CP), in combination with or without MCT. The four groups were as follows: CP-MCT, CP+MCT, LP-MCT, and LP+MCT. Rats in the CP-MCT, CP+MCT and LP+MCT groups were pair-fed their respective diets based on the amount of diet consumed by the LP-MCT group. Rats were fed each experimental diet for 30 d. Four weeks later, the respiratory quotient was higher in the LP-MCT group than those in the other groups during the fasting period. Hepatic triglyceride content increased in the LP groups compared with the CP groups. Hepatic triglyceride content in the LP+MCT group, however, was significantly decreased compared with that in the LP-MCT group. Levels of carnitine palmitoyltransferase (CPT) 1a mRNA and CPT2 mRNA were significantly decreased in the livers of the LP-MCT group, as compared with corresponding mRNA levels of the other groups. These results suggest that ingestion of a low-protein diet caused fatty liver in growing rats. However, when rats were fed the low-protein diet with MCT, hepatic triglyceride deposition was attenuated, and mRNA levels encoding CPT1a and CPT2 were preserved at the levels of rats fed control protein diets.

  14. 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.

  15. 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

  16. 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.

  17. Hepatic drug metabolizing profile of Flinders Sensitive Line rat model of depression.

    PubMed

    Kotsovolou, Olga; Ingelman-Sundberg, Magnus; Lang, Matti A; Marselos, Marios; Overstreet, David H; Papadopoulou-Daifoti, Zoi; Johanson, Inger; Fotopoulos, Andrew; Konstandi, Maria

    2010-08-16

    The Flinders Sensitive Line (FSL) rat model of depression exhibits some behavioral, neurochemical, and pharmacological features that have been reported in depressed patients and has been very effective in screening antidepressants. Major factor that determines the effectiveness and toxicity of a drug is the drug metabolizing capacity of the liver. Therefore, in order to discriminate possible differentiation in the hepatic drug metabolism between FSL rats and Sprague-Dawley (SD) controls, their hepatic metabolic profile was investigated in this study. The data showed decreased glutathione (GSH) content and glutathione S-transferase (GST) activity and lower expression of certain major CYP enzymes, including the CYP2B1, CYP2C11 and CYP2D1 in FSL rats compared to SD controls. In contrast, p-nitrophenol hydroxylase (PNP), 7-ethoxyresorufin-O-dealkylase (EROD) and 16alpha-testosterone hydroxylase activities were higher in FSL rats. Interestingly, the wide spread environmental pollutant benzo(alpha)pyrene (B(alpha)P) induced CYP1A1, CYP1A2, CYP2B1/2 and ALDH3c at a lesser extend in FSL than in SD rats, whereas the antidepressant mirtazapine (MIRT) up-regulated CYP1A1/2, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2, mainly, in FSL rats. The drug also further increased ALDH3c whereas suppressed GSH content in B(alpha)P-exposed FSL rats. In conclusion, several key enzymes of the hepatic biotransformation machinery are differentially expressed in FSL than in SD rats, a condition that may influence the outcome of drug therapy. The MIRT-induced up-regulation of several drug-metabolizing enzymes indicates the critical role of antidepressant treatment that should be always taken into account in the designing of treatment and interpretation of insufficient pharmacotherapy or drug toxicity.

  18. Butyrate Protects Rat Liver against Total Hepatic Ischemia Reperfusion Injury with Bowel Congestion

    PubMed Central

    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. PMID:25171217

  19. Aqueous garlic extract attenuates hepatitis and oxidative stress induced by galactosamine/lipoploysaccharide in rats.

    PubMed

    El-Beshbishy, Hesham A

    2008-10-01

    Injection of D-galactosamine and lipopolysaccharide (DGaIN/LPS) is useful as an experimental model of acute hepatic damage. Juvenile rats were used for investigation. The hepatoprotective activity of aqueous garlic (Allium sativum) extract (AGE) at a dose of 300 mg/kg body weight for 14 days, intraperitoneal (i.p.) prior to the induction of DGalN/LPS, was investigated against DGalN/LPS-induced hepatitis in rats. DGalN/LPS (300 mg/kg body weight/30 microg/kg body weight, i.p.), induced hepatic damage that was manifested by a significant increase in the activities of marker enzymes [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and gamma glutamyl transferase (gamma GT)], bilirubin, lipid peroxides (LPO), tumor necrosis factor (TNF-alpha) and myeloperoxidase (MPO) activity level in serum. Also, the lipid profile in serum and liver homogenate including total cholesterol, triglycerides, free fatty acids and phospholipids were significantly deteriorated. The antioxidant enzyme activities (superoxide dismutase, SOD; reduced glutathione, GSH; catalase, CAT and glutathione peroxidase, GPX) in liver homogenate were significantly decreased in the DGalN/LPS. Pretreatment of rats with AGE reversed these altered parameters near to normal control values. Results of this study revealed that AGE could afford a significant protection in the alleviation of DGalN/LPS-induced hepatic damage.

  20. [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.

  1. 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

  2. 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

  3. Factors involved in changes in hepatic lipogenesis during development of the rat

    PubMed Central

    Lockwood, Elizabeth A.; Bailey, E.; Taylor, C. B.

    1970-01-01

    1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of `malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4–5 days and acetyl-CoA carboxylase 2–3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for `malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in `malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and `malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development. PMID:4248618

  4. 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

  5. Role of CTGF gene promoter methylation in the development of hepatic fibrosis

    PubMed Central

    Shi, Cuicui; Li, Guangming; Tong, Yanyan; Deng, Yilin; Fan, Jiangao

    2016-01-01

    Connective tissue growth factor (CTGF) plays a critical role in the hepatic stellate cells (HSCs)-mediated development of hepatic fibrosis. Nevertheless, the effects of CTGF gene promoter methylation in the pathogenesis of hepatic fibrosis remain largely unknown. In the current study, we isolated and overexpressed CTGF in primary HSCs. We analyzed the CTGF gene promoter methylation inHSCs that undergo a phenotypic change into myofibroblast-like cellsthat express α-smooth muscle actin (α-SMA) in vitro and in vivo in a CCl4-induced rat hepatic fibrosis model. We found that CTGF promoted the phenotypic changes of HSCs into myofibroblasts in vitro, while inhibition of CTGF promoter methylation augmented the process, suggesting that CTGF gene promoter methylation may negatively regulate hepatic fibrosis. In vivo, CCl4 induced hepatic fibrosis in rats, and the severity of hepatic fibrosis inversely correlated with the levels of CTGF gene promoter methylation in HSCs. Together, our data demonstrate that CTGF gene promoter methylation may prevent the development of hepatic fibrosis, and low level of CTGF gene promoter methylation in HSCs may be a predisposing factor for developing liver fibrotic disease. PMID:27069546

  6. Basis of aggravated hepatic lipid metabolism by chronic stress in high-fat diet-fed rat.

    PubMed

    Han, Ying; Lin, Min; Wang, Xiaobin; Guo, Keke; Wang, Shanshan; Sun, Mengfei; Wang, Jiao; Han, Xiaoyu; Fu, Ting; Hu, Yang; Fu, Jihua

    2015-03-01

    Our previous study has demonstrated that long-term stress, known as chronic stress (CS), can aggravate nonalcoholic fatty liver disease in high-fat diet (HFD)-fed rat. In this study, we tried to figure out which lipid metabolic pathways were impacted by CS in the HFD-fed rat. Male Sprague-Dawley rats (6 weeks of age, n = 8 per group) were fed with either standard diet or HFD with or without CS exposure for 8 weeks. Hepatic lipidosis, biochemical, hormonal, and lipid profile markers in serum and liver, and enzymes involved in de novo lipogenesis (DNL) of fatty acids (FAs) and cholesterol, β-oxidation, FAs uptake, triglycerides synthesis, and very low-density lipoprotein (VLDL) assembly in the liver were detected. CS exposure reduced hepatic lipidosis but further elevated hepatic VLDL content with aggravated dyslipidemia in the HFD-fed rats. There was a synergism between CS and HFD on VLDL production and dyslipidemia. PCR and western blot assays showed that CS exposure significantly promoted hepatic VLDL assembly in rats, especially in the HFD-fed rats, while it had little impact on DNL, β-oxidation, FAs uptake, and triglycerides synthesis in the HFD-fed rats. This phenomenon was in accordance with elevated serum glucocorticoid level. The critical influence of CS exposure on hepatic lipid metabolism in the HFD-fed rats is VLDL assembly which might be regulated by glucocorticoid.

  7. Suppression of rat hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fat.

    PubMed

    Blake, W L; Clarke, S D

    1990-12-01

    The objective of this research was to determine whether dietary polyunsaturated fatty acids suppress hepatic fatty acid synthase (FAS) mRNA levels by altering FAS gene transcription. Male Sprague-Dawley rats were meal-fed for 10 d a high glucose diet supplemented with 20% digestible energy as menhaden oil or tripalmitin. The transcription rate for FAS was determined by nuclear run-on analysis in hepatic nuclei isolated from rats 2 h postmeal. The values for transcription rates of FAS and S14 (a putative lipogenic protein) in rats fed menhaden oil were only 6 and 21%, respectively, of the rates in rats fed the tripalmitin diet (p less than 0.02). Gene transcription for beta-actin and phosphoenolpyruvate carboxykinase did not differ between treatments. The reduction in hepatic FAS mRNA levels caused by dietary polyunsaturated fats appears to be caused primarily by an inhibition of FAS transcription. The control of transcription by polyunsaturated fats appears not to be mediated by cAMP because the transcription rate for phosphoenolpyruvate carboxykinase (whose gene is very sensitive to cAMP stimulation) was unaffected by the source of dietary fat.

  8. Regulation of hepatic branched-chain α-ketoacid dehydrogenase complex in rats fed a high-fat diet.

    PubMed

    Kadota, Yoshihiro; Toyoda, Takanari; Kitaura, Yasuyuki; Adams, Sean H; Shimomura, Yoshiharu

    2013-12-01

    Branched-chain α-ketoacid (BCKA) dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of BCKA catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal models. In this study, we examined the regulation of hepatic BCKDC in rats with diet-induced obesity (DIO). Rats were fed a control or a 60% of energy high-fat diet (HFD) for twelve weeks. Concentrations of blood components and the activities and protein amounts of hepatic BCKDC and its specific kinase (BDK) were measured. The concentrations of plasma glucose, insulin, and corticosterone were significantly elevated in DIO rats compared to those fed the control diet, suggestive of insulin resistance. Blood BCAA concentrations were not increased. The activity of hepatic BCKDC that was present in the active form in the liver was higher in DIO rats compared to controls, although the total activity and the enzyme amount were not different between two diet groups. The activity of hepatic BDK and the abundance of BDK bound to the BCKDC were decreased in DIO rats. The total amount of hepatic BDK was also significantly decreased in DIO rats. In rats made obese through HFD feeding, in contrast to prior studies in rat models of type 2 diabetes, hepatic BDK was down-regulated and thereby hepatic BCKDC was activated, suggesting that DIO promotes liver BCKA catabolism. In this model there was no evidence that increased blood BCAAs drive DIO-associated insulin resistance, since concentrations of BCAAs were not altered by DIO.

  9. The protective effects of Phyllanthus emblica Linn. extract on ethanol induced rat hepatic injury.

    PubMed

    Pramyothin, Pornpen; Samosorn, Patcharavadee; Poungshompoo, Somlak; Chaichantipyuth, Chaiyo

    2006-10-11

    This study was undertaken to investigate the protective effects of Phyllanthus emblica Linn. (PE) extract on ethanol induced rat hepatic injury. PE (0.5 and 1 mg/ml) increased cell viability of rat primary cultured hepatocytes being treated with ethanol (96 microl/m) by increasing % MTT and decreasing the release of transaminase. Hepatotoxic markers studied in rats included serum transaminases (AST and ALT), serum triglyceride (STG), hepatic triglyceride (HTG), TNF-alpha and IL-1beta together with histopathological examination. Pretreatment of rats with PE at oral dose of 25, 50 and 75 mg/kg or SL (silymarin, a reference hepatoprotective agent) at 5 mg/kg, 4 h before ethanol, lowered the ethanol induced levels of AST, ALT and IL-1beta. The 75 mg/kg PE dose gave the best result similar to SL. Treatment of rats with PE (75 mg/kg/day) or SL (5 mg/kg/day) for 7 days after 21 days with ethanol (4 g/kg/day, p.o.) enhanced liver cell recovery by bringing the levels of AST, ALT, IL-1beta back to normal. Histopathological studies confirmed the beneficial roles of PE and SL against ethanol induced liver injury in rats.

  10. 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

  11. Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy.

    PubMed

    Méndez, Marta; Méndez-López, Magdalena; López, Laudino; Aller, María A; Arias, Jaime; Arias, Jorge L

    2011-05-01

    Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory.

  12. Loss and recovery of liver regeneration in rats with fulminant hepatic failure.

    PubMed

    Eguchi, S; Lilja, H; Hewitt, W R; Middleton, Y; Demetriou, A A; Rozga, J

    1997-10-01

    We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobes necrosis. After this procedure, lack of regenerative response in the residual viable liver tissue (omental lobes) was associated with elevated plasma hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1) levels and delayed expression of HGF and c-met mRNA in the remnant liver. Here, we investigated whether syngeneic isolated hepatocytes transplanted in the spleen will prolong survival and facilitate liver regeneration in FHF rats. Inbred male Lewis rats were used. Group I rats (n = 46) received intrasplenic injection of 2 x 10(7) hepatocytes and 2 days later FHF was induced. Group II FHF rats (n = 46) received intrasplenic injection of saline. Rats undergoing partial hepatectomy of 68% (PH; n = 30) and a sham operation (SO; n = 30) served as controls. In 20 FHF rats (10 rats/group), survival time was determined. The remaining 72 FHF rats (36 rats/group) were used for physiologic studies (liver function and regeneration and plasma growth factor levels). In Group I rats survival was longer than that of Group II controls (73 +/- 22 hr vs. 33 +/- 9 hr; P < 0. 01). During the first 36 hr, Group I rats had lower blood ammonia, lactate, total bilirubin, PT, and PTT values, lower activity of liver enzymes, and higher monoethylglycinexylidide (MEGX) production than Group II rats. In Group I rats, livers increased in weight at a rate similar to that seen in PH controls and showed distinct mitotic and DNA synthetic activity (incorporation of bromodeoxyuridine and proliferation cell nuclear antigen expression). Plasma HGF and TGF-beta1 levels in these rats decreased and followed the pattern seen in PH rats; additionally, c-met expression in the remnant liver was accelerated. Hepatocyte transplantation prolonged survival in FHF rats and facilitated liver regeneration. Even though the remnant liver increased

  13. Preconditioning somatothermal stimulation on Qimen (LR14) reduces hepatic ischemia/reperfusion injury in rats

    PubMed Central

    2014-01-01

    Background In human beings or animals, ischemia/reperfusion (I/R) injury of the liver may occur in many clinical conditions, such as circulating shock, liver transplantation and surgery and several other pathological conditions. I/R injury has a complex pathophysiology resulting from a number of contributing factors. Therefore, it is difficult to achieve effective treatment or protection by individually targeting the mediators. This study aimed at studying the effects of local somatothermal stimulation preconditioning on the right Qimen (LR14) on hepatic I/R injury in rats. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups. The rats were preconditioned with thermal tolerance study, which included one dose of local somatothermal stimulation (LSTS) on right Qimen (LR14) at an interval of 12 h, followed by hepatic ischemia for 60 min and then reperfusion for 60 min. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have been used to assess the liver functions, and liver tissues were taken for the measurements such as malondialdehyde (MDA), glutathione (GSH), catalase (CAT), superoxidase dismutase (SOD), and myeloperoxidase (MPO). Results The results show that the plasma ALT and AST activities were higher in the I/R group than in the control group. In addition, the plasma ALT and AST activities decreased in the groups that received LSTS. The hepatic SOD levels reduced significantly by I/R injury. Moreover, the hepatic MPO activity significantly increased by I/R injury while it decreased in the groups given LSTS. Conclusions Our findings show that LSTS provides a protective effects on the liver from the I/R injury. Therefore, LSTS might offer an easy and inexpensive intervention for patients who have suffered from I/R of the liver especially in the process of hepatotomy and hepatic transplantation. PMID:24417801

  14. The hepatoprotective effects of Hypericum perforatum L. on hepatic ischemia/reperfusion injury in rats.

    PubMed

    Bayramoglu, Gokhan; Bayramoglu, Aysegul; Engur, Selin; Senturk, Hakan; Ozturk, Nilgun; Colak, Suat

    2014-05-01

    Little is known about the effective role of Hypericum perforatum on hepatic ischemia-reperfusion (I/R) injury in rats. Hence, albino rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. Hypericum perforatum extract (HPE) at the dose of 50 mg/kg body weight (HPE50) was intraperitonally injected as a single dose, 15 min prior to ischemia. Rats were sacrificed at the end of reperfusion period and then, biochemical investigations were made in serum and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) levels. At the same time alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were assayed in serum samples and compared statistically. While the ALT, AST, LDH activities and MDA levels were significantly increased, CAT and GPx activities significantly decreased in only I/R-induced control rats compared to normal control rats (p < 0.05). Treatment with HPE50 significantly decreased the ALT, AST, LDH activities and MDA levels, and markedly increased activities of CAT and GPx in tissue homogenates compared to I/R-induced rats without treatment-control group (p < 0.05). In oxidative stress generated by hepatic ischemia-reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidant defence systems in the body.

  15. Enhancement by Wy-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine-initiated hepatic tumorigenesis in the rat.

    PubMed Central

    Reddy, J. K.; Rao, M. S.

    1978-01-01

    Diethylnitrosamine (DEN), at a concentration of 100 parts/10(6) in drinking water for 14 days, caused the development, by 48 weeks, of very few liver tumours in 5 of 18 (27%) male F=344 rats fed control diet. When the DEN treatment was followed one week later by continuous feeding of the hypolipidemic hepatic peroxisome proliferator, Wy-14,643, at 0.1% dietary level, all of 28 rats (100%) developed, between 38 and 48 weeks, a significantly higher number of liver tumours. Furthermore, laparotomy at 22 weeks revealed that several rats fed Wy-14,643 after DEN initiation had developed visible liver nodules, suggesting that Wy-14,643 also accelerates the appearance of these tumours. Administration of another peroxisome proliferator, clofibrate, at 0.5% level in the diet after DEN initiation, also caused a substantial enhancement of liver tumorigenesis. The enhancement of liver-tumour development by clofibrate, however, was less than that by Wy-14,643. The marked enhancing effect of Wy-14,643 may be due to its profound hepatomegalic and peroxisome proliferative properties. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:728341

  16. Nuclear ploidy of neonatal rat livers: effects of two hepatic carcinogens (mirex and dimethylnitrosamine)

    SciTech Connect

    Carlson, J.; Abraham, R.

    1985-01-01

    The effect of two hepatic carcinogens, dimethylnitrosamine (DMN) (genotoxic) and mirex (epigenetic), on polyploidization in 12-d-old neonatal rats was investigated by Coulter counteranalysis and (/sup 3/H) thymidine uptake in isolated hepatic nuclear classes. DMN disturbed the normal ploidy development in the neonatal liver and the proportion of nuclei in the ploidy classes by inducing the premature formation of a significant population of tetraploids with a concommitant reduction in diploids. A great proportion of the replicative activity was present in tetraploid nuclei as measured by the incorporation of (/sup 3/H) thymidine. The labeling index and number of mitoses were also increased. In contrast to DMN, mirex had no influence on polyploidization. The neonatal rats used in these studies thus offer an opportunity to investigate in vivo the mode of action of genotoxic versus epigenetic compounds with reference to their effect on DNA.

  17. 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.

  18. Effects of creatine supplementation on biomarkers of hepatic and renal function in young trained rats.

    PubMed

    Souza, William Marciel; Heck, Thiago Gomes; Wronski, Evanio Castor; Ulbrich, Anderson Zampier; Boff, Everton

    2013-11-01

    Creatine supplementation has been widely used by athletes and young physical exercise practioneers in order of increasing muscle mass and enhancing athletic performance, but their use/overuse may represent a health risk on hepatic and renal impaired function. In this study, we evaluated the effects of 40 days of oral creatine supplementation on hepatic and renal function biomarkers in a young animal model. Wistar rats (5 weeks old) were divided in five groups (n = 7): control (CONTR), oral creatine supplementation (CREAT), moderate exercise training (EXERC), moderate exercise training plus oral creatine supplementation (EXERC + CREAT) and pathological group (positive control for liver and kidney injury) by the administration of rifampicin (RIFAMPICIN). Exercise groups were submitted to 60 min/day of swimming exercise session with a 4% of body weight workload for six weeks. The EXERC + CREAT showed the higher body weight at the end of the training protocol. The CREAT and EXERC + CREAT group showed an increase in hepatic (Aspartate transaminase and gamma-glutamyl transpeptidase) and renal (urea and creatinine) biomarkers levels (p < 0.05). Our study showed that the oral creatine supplementation promoted hepatic and renal function challenge in young rats submitted to moderate exercise training.

  19. Effect of dietary selenium concentration and duration of selenium feeding on hepatic glutathione concentrations in rats

    SciTech Connect

    LeBoeuf, R.A.; Zentner, K.L.; Hoekstra, W.G.

    1985-11-01

    Studies were conducted in rats to determine the effect of dietary selenium (Se) concentration on hepatic glutathione concentrations and enzyme activities associated with the maintenance of the cellular glutathione status. Male rats were fed 0.1, 3.0, or 6.0 ppm Se as Na/sub 2/SeO/sub 3/ for 2, 4, or 6 weeks at which time they were killed and analyses were performed. Both 3.0 and 6.0 ppm Se caused a significant dose-dependent increase in hepatic-reduced glutathione (GSH) by 4 weeks of feeding compared to 0.1 ppm Se. The increase in GSH was preceded by significant, dose-dependent increases in oxidized glutathione (GSSG) as well as the GSSG to GSH ratio. Increases in GSSG and the GSSG to GSH ratio as well as in glutathione reductase and glucose-6-phosphate dehydrogenase activities were observed by 2 weeks of high Se feeding. The current findings substantiate previous results demonstrating effects of high Se on hepatic glutathione concentrations and further suggest that increased cellular GSSG concentrations or the GSSG to GSH ratio caused by 3.0 and 6.0 ppm dietary Se signals for adaptive changes in hepatic glutathione metabolism.

  20. Regulation of development of hepatic glucokinase in the neonatal rat by the diet.

    PubMed

    Walker, D G; Eaton, S W

    1967-11-01

    1. Feeding a high-glucose diet to weanling rats showed that high hepatic glucokinase activities could be induced at 18 days of age, i.e. 2 days after development of the enzyme begins. 2. The normal development of glucokinase activity can be retarded by weaning rats on to carbohydrate-free, high-fat and high-protein diets. 3. Precocious development of the enzyme before 16 days of age cannot be induced by oral glucose administration. 4. It is concluded that the ability to synthesize glucokinase develops very rapidly and that the nature of the diet determines the normal developmental pattern.

  1. Simultaneous in vivo assessment of cardiac and hepatic metabolism in the diabetic rat using hyperpolarized MRS

    PubMed Central

    Ball, Daniel R.; Ball, Vicky; Dodd, Michael S.; Miller, Jack J.; Heather, Lisa C.; Tyler, Damian J.

    2016-01-01

    Understanding and assessing diabetic metabolism is vital for monitoring disease progression and improving treatment of patients. In vivo assessments, using MRI and MRS, provide non‐invasive and accurate measurements, and the development of hyperpolarized 13C spectroscopy in particular has been demonstrated to provide valuable metabolic data in real time. Until now, studies have focussed on individual organs. However, diabetes is a systemic disease affecting multiple tissues in the body. Therefore, we have developed a technique to simultaneously measure metabolism in both the heart and liver during a single acquisition. A hyperpolarized 13C MRS protocol was developed to allow acquisition of metabolic data from the heart and liver during a single scan. This protocol was subsequently used to assess metabolism in the heart and liver of seven control male Wistar rats and seven diabetic rats (diabetes was induced by three weeks of high‐fat feeding and a 30 mg/kg injection of streptozotocin). Using our new acquisition, we observed decreased cardiac and hepatic pyruvate dehydrogenase flux in our diabetic rat model. These diabetic rats also had increased blood glucose levels, decreased insulin, and increased hepatic triglycerides. Decreased production of hepatic [1‐13C]alanine was observed in the diabetic group, but this change was not present in the hearts of the same diabetic animals. We have demonstrated the ability to measure cardiac and hepatic metabolism simultaneously, with sufficient sensitivity to detect metabolic alterations in both organs. Further, we have non‐invasively observed the different reactions of the heart and liver to the metabolic challenge of diabetes. PMID:27779334

  2. 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.

  3. Effects of anti-ulcer agents on ethanol-induced gastric mucosal lesions in D-galactosamine-induced hepatitis rats.

    PubMed

    Taniguchi, Hiroyuki; Yomota, Eiji; Nogi, Koji; Onoda, Yuichi

    2002-01-01

    Patients with hepatic injury have an increased incidence of gastric ulcers and erosions. In this study, the effect of D-galactosamine(GalN)-induced hepatitis on ethanol-induced gastric mucosal lesions and the protective effect of anti-ulcer agents in rats were examined. Subcutaneous injection of GalN (1 g/kg) remarkably increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities suggesting induction of hepatic injury. Gastric mucosal lesions induced by ethanol were significantly aggravated in GalN-induced hepatitis rats. Orally administered ecabet (CAS 86408-72-2; 20-200 mg/kg) dose dependently inhibited ethanol-induced gastric mucosal lesions in GalN-induced hepatitis rats. Sucralfate (CAS 54182-58-0) tended to inhibit the gastric mucosal lesions at a dose of 200 mg/kg but teprenone (CAS 6809-52-5), cimetidine (CAS 51481-61-9) and rebamipide (CAS 90098-04-7) had little effect. All anti-ulcer agents had no effect on the serum ALT and AST activities increased by GalN pretreatment. These results indicate that the gastric mucosa of GalN-induced hepatitis rats is more susceptible to injury induced by luminal irritants such as ethanol. Ecabet potently inhibited gastric mucosal lesions suggesting its clinical utility for the gastric mucosal damage in patients with hepatic injury.

  4. 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.

  5. 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.

  6. 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

    2016-06-29

    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.

  7. 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

  8. Experimental Study on Transarterial Administration of GRGDSP Combined with Transarterial Chemoembolization in Rats with Hepatic Carcinoma

    SciTech Connect

    Qian Jun Yin Jun; Liang Huimin; Wang Yong; Feng Gansheng

    2008-03-15

    Purpose. To evaluate the effects of transarterial administration of an integrin antagonist, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), combined with transarterial chemoembolization (TACE) to treat hepatic carcinoma in rats. Methods. Walker-256 tumor was implanted beneath the liver capsule in 26 Wistar rats. Animal subjects were assigned to groups based on which treatment was injected into the hepatic artery: group A, GRGDSP + TACE; group B, TACE alone; and group C, normal saline. Magnetic resonance imaging (MRI), tumor pathology, and immunohistochemistry were performed to assess each treatment. Results. The ratios of the post-treatment to pretreatment tumor volumes (V2/V1) in groups A, B, and C were 4.42 {+-} 0.48, 6.98 {+-} 1.09, and 13.00 {+-} 1.68, respectively. The metastatic potential of the tumors was assessed by tumor cell nest counts, which were 5.00 {+-} 1.25, 6.63 {+-} 1.60, and 7.22 {+-} 1.92 in groups A, B, and C, respectively. Microvessel density (MVD) was quantified by measuring von Willebrand factor density values, which were 0.18 {+-} 0.02, 0.22 {+-} 0.02, and 0.23 {+-} 0.02 in groups A, B, and C, respectively. Conclusions. Transarterial infusion of GRGDSP combined with TACE noticeably inhibited the growth of hepatic carcinoma and intrahepatic metastases in rats.

  9. Bamboo salt attenuates CCl4-induced hepatic damage in Sprague-Dawley rats.

    PubMed

    Zhao, Xin; Song, Jia-Le; Kil, Jeung-Ha; Park, Kun-Young

    2013-08-01

    Bamboo salt, a Korean folk medicine, is prepared with solar salt (sea salt) and baked several times at high temperatures in a bamboo case. In this study, we compared the preventive effects of bamboo salt and purified and solar salts on hepatic damage induced by carbon tetrachloride in Sprague-Dawley rats. Compared with purified and solar salts, bamboo salts prevented hepatic damage in rats, as evidenced by significantly reduced serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase (P < 0.05). Bamboo salt (baked 9×) triggered the greatest reduction in these enzyme levels. In addition, it also reduced the levels of the proinflammatory cytokines interleukin (IL)-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. Histopathological sections of liver tissue demonstrated the protective effect of bamboo salt, whereas sections from animals treated with the other salt groups showed a greater degree of necrosis. We also performed reverse transcription-polymerase chain reaction and western blot analyses of the inflammation-related genes iNOS, COX-2, TNF-α, and IL-1β in rat liver tissues. Bamboo salt induced a significant decrease (~80%) in mRNA and protein expression levels of COX-2, iNOS, TNF-α, and IL-1β, compared with the other salts. Thus, we found that baked bamboo salt preparations could prevent CCl4-induced hepatic damage in vivo.

  10. Agmatine protects rat liver from nicotine-induced hepatic damage via antioxidative, antiapoptotic, and antifibrotic pathways.

    PubMed

    El-Sherbeeny, Nagla A; Nader, Manar A; Attia, Ghalia M; Ateyya, Hayam

    2016-12-01

    Tobacco smoking with its various forms is a global problem with proved hazardous effects to human health. The present work was planned to study the defending role of agmatine (AGM) on hepatic oxidative stress and damage induced by nicotine in rats. Thirty-two rats divided into four groups were employed: control group, nicotine-only group, AGM group, and AGM-nicotine group. Measurements of serum hepatic biochemical markers, lipid profile, and vascular cell adhesion molecule-1 were done. In addition, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) activity, and nitrate/nitrite (NOx) levels were estimated in the liver homogenates. Immunohistochemistry for Bax and transforming growth factor beta (TGF-β1) and histopathology of the liver were also included. Data of the study demonstrated that nicotine administration exhibited marked liver deterioration, an increase in liver enzymes, changes in lipid profile, and an elevation in MDA with a decline in levels of SOD, GSH, and NOx (nitrate/nitrite). Also, levels of proapoptotic Bax and profibrotic TGF-β1 showed marked elevation in the liver. AGM treatment to rats in nicotine-only group ameliorated all the previous changes. These findings indicate that AGM could successfully overcome the nicotine-evoked hepatic oxidative stress and tissue injury, apoptosis, and fibrosis.

  11. Protective effect of morin on dimethylnitrosamine-induced hepatic fibrosis in rats.

    PubMed

    Lee, Hee-Seung; Jung, Kyung Hee; Park, In-Suh; Kwon, Sung Won; Lee, Don-Haeng; Hong, Soon-Sun

    2009-04-01

    Morin, a plant-derived flavonoid, has been reported to exhibit a wide range of pharmacological properties. In this study, we investigated the protective effect of morin on hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. Oral administration of morin remarkably prevented weight loss in the body and liver from DMN and inhibited the elevation of serum alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin levels. For the evaluation of hepatic fibrosis-related factors, we investigated expressions of collagen type I, transforming growth factor beta(1) (TGF-beta(1)), and alpha-smooth muscle actin (alpha-SMA) in mRNA and protein levels. We observed that morin significantly reduced the expression of collagen type I, TGF-beta(1), and alpha-SMA on hepatic fibrosis induced by DMN. Taken together, this study demonstrated that morin showed hepatoprotective and antifibrogenic effects against DMN-induced hepatic injury. This suggests that morin may be useful in preventing the development of hepatic fibrosis and cirrhosis.

  12. Cerebral Epiphyseal Proteins and Melatonin Modulate the Hepatic and Renal Antioxidant Defense of Rats

    PubMed Central

    Bharti, Vijay K.; Srivastava, R. S.; Subramaian, P.; Warren Spence, D.; Pandi-Perumal, S. R.; Brown, Gregory M.

    2011-01-01

    The cerebral epiphysis (pineal gland) secrets melatonin and number of other proteins and peptides. It was thus hypothesized that antioxidant properties of epiphyseal proteins and melatonin could potentially benefit from exogenous therapies. In view of the therapeutic potential of these proteins, the present experiment was conducted to investigate the effect of buffalo epiphyseal proteins (BEP, at 100 μg/kg BW, i.p.) and melatonin (MEL, at 10 mg/kg BW, i.p) on changes in hepatic and renal antioxidant enzymes of adult female Wistar rats. Buffalo epiphyseal proteins significantly (P < .05) increased hepatic lipid peroxidation (LPO), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), reduced glutathione (GSH), and renal LPO, catalase (CAT), GR, GSH, GPx levels as compared to control animals. Similarly, MEL treatment significantly (P < .05) up-regulated hepatic SOD and GPx activity, whereas CAT, GR, GPx, and GSH levels in renal tissues were increased while SOD and LPO remained unaffected. Buffalo epiphyseal protein treatment produced greater effects on hepatic GPx and renal CAT and GSH levels than did MEL. These findings support the conclusion that buffalo epiphyseal proteins and melatonin activate a number of antioxidant mechanisms in hepatic and renal tissues. PMID:21660111

  13. Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry.

    PubMed

    Seymour, E Mitchell; Singer, Andrew A M; Kirakosyan, Ara; Urcuyo-Llanes, Daniel E; Kaufman, Peter B; Bolling, Steven F

    2008-06-01

    Elevated plasma lipids, glucose, insulin, and fatty liver are among components of metabolic syndrome, a phenotypic pattern that typically precedes the development of Type 2 diabetes. Animal studies show that intake of anthocyanins reduces hyperlipidemia, obesity, and atherosclerosis and that anthocyanin-rich extracts may exert these effects in association with altered activity of tissue peroxisome proliferator-activated receptors (PPARs). However, studies are lacking to test this correlation using physiologically relevant, whole food sources of anthocyanins. Tart cherries are a rich source of anthocyanins, and whole cherry fruit intake may also affect hyperlipidemia and/or affect tissue PPARs. This hypothesis was tested in the Dahl Salt-Sensitive rat having insulin resistance and hyperlipidemia. For 90 days, Dahl rats were pair-fed AIN-76a-based diets supplemented with either 1% (wt:wt) freeze-dried whole tart cherry or with 0.85% additional carbohydrate to match macronutrient and calorie provision. After 90 days, the cherry-enriched diet was associated with reduced fasting blood glucose, hyperlipidemia, hyperinsulinemia, and reduced fatty liver. The cherry diet was also associated with significantly enhanced hepatic PPAR-alpha mRNA, enhanced hepatic PPAR-alpha target acyl-coenzyme A oxidase mRNA and activity, and increased plasma antioxidant capacity. In conclusion, physiologically relevant tart cherry consumption reduced several phenotypic risk factors that are associated with risk for metabolic syndrome and Type 2 diabetes. Tart cherries may represent a whole food research model of the health effects of anthocyanin-rich foods and may possess nutraceutical value against risk factors for metabolic syndrome and its clinical sequelae.

  14. 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

  15. Surgical technique of orthotopic liver transplantation in rats: the Kamada technique and a new splint technique for hepatic artery reconstruction.

    PubMed

    Ishii, Eiichi; Shimizu, Akira; Takahashi, Mikiko; Terasaki, Mika; Kunugi, Shinobu; Nagasaka, Shinya; Terasaki, Yasuhiro; Ohashi, Ryuji; Masuda, Yukinari; Fukuda, Yuh

    2013-01-01

    Orthotopic liver transplantation (OLT) in rats is technically feasible and useful for the assessment of clinical liver transplantation and analysis of inflammatory liver diseases. OLT in rats was pioneered by Lee et al. in 1973 using hand-suture techniques of all vessels. This model has not been widely used due to the long operative time and technical demand. The cuff method was introduced by Kamada in 1979, and today, the Kamada technique is the one most commonly used worldwide. However, this technique does not include hepatic artery reconstruction, although this procedure is routinely performed in clinical transplantation. Nevertheless, several techniques for hepatic artery reconstruction in rat OLT have been reported recently, and our group also developed a simple splint technique from recipient right renal artery to donor celiac axis bearing the hepatic artery. In the present article, we describe the Kamada technique, as a standard surgical method for rat OLT. In addition, we also describe our splint technique for hepatic artery reconstruction. Then, we compare the features of Kamada technique and our splint technique for hepatic artery reconstruction and all other surgical techniques currently in use for rat OLT. The widespread use of the rat OLT model should help to provide full assessment of transplant immunology and the mechanism and treatment of inflammatory liver diseases.

  16. Effect of chlorpromazine on hepatic transport of indocyanine green in rats.

    PubMed

    Tsao, S C; Sawada, Y; Iga, T; Hanano, M

    1983-03-15

    The effect of chlorpromazine hydrochloride (CPZ) on the hepatic transport of indocyanine green (ICG) was studied in the rat, in an attempt to elucidate the mechanisms of hepatotoxicity of CPZ in vivo, by comparing the pharmacokinetic parameters of ICG after bolus and chronic administration of CPZ. Delays were shown in both plasma disappearance and biliary excretion of ICG in the CPZ-treated rats (10 and 15 mg/kg intraportal bolus administration). Significant decreases were observed in the pharmacokinetic parameters, V2 and total body clearance (CLtot) in CPZ 10 mg/kg treated rats and k34, V2 and CLtot in CPZ 15 mg/kg treated rats, while a significant increase was observed in k21 in both CPZ-treated groups; V1 was not altered. The apparent liver-to-plasma concentration ratio (Kp,app) of ICG at 50 min after i.v. administration was decreased significantly in CPZ 15 mg/kg treated rats when compared to control rats, suggesting an alteration in the distribution of ICG to the liver by CPZ. Bile flow rates decreased immediately after bolus intraportal administration of CPZ in both CPZ-treated groups, and they then returned progressively to the basal levels. The output of bile acids was also inhibited by CPZ in a time-dependent and reversible manner and the bile acid independent fraction of bile flow was decreased significantly in both CPZ-treated groups. Chronic treatment with CPZ (10 or 20 mg/kg, i.p., per day for 3 weeks) did not alter either the pharmacokinetic parameters or the bile secretion profile of ICG, although there were significant decreases in body and liver weights in CPZ-treated groups. This may have been due to the rapid metabolism and excretion of CPZ in the rat when compared to humans. It is proposed that the acute toxic effect of CPZ on hepatic transport of ICG in the rat may be due mainly to the time-dependent and reversible cholestasis induced by CPZ, and that chronic treatment with CPZ may not alter the hepatic transport of ICG in the rat.

  17. 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.

  18. 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

  19. Diminished acute phase response and increased hepatic inflammation of aged rats in response to intraperitoneal injection of lipopolysaccharide.

    PubMed

    Gomez, Christian R; Acuña-Castillo, Claudio; Pérez, Claudio; Leiva-Salcedo, Elías; Riquelme, Denise M; Ordenes, Gamaliel; Oshima, Kiyoko; Aravena, Mauricio; Pérez, Viviana I; Nishimura, Sumiyo; Sabaj, Valeria; Walter, Robin; Sierra, Felipe

    2008-12-01

    Aging is associated with a deterioration of the acute phase response to inflammatory challenges. However, the nature of these defects remains poorly defined. We analyzed the hepatic inflammatory response after intraperitoneal administration of lipopolysaccharide (LPS) given to Fisher 344 rats aged 6, 15, and 22-23 months. Induction of the acute phase proteins (APPs), haptoglobin, alpha-1-acid glycoprotein, and T-kininogen was reduced and/or retarded with aging. Initial induction of interleukin-6 in aged rats was normal, but the later response was increased relative to younger counterparts. An exacerbated hepatic injury was observed in aged rats receiving LPS, as evidenced by the presence of multiple microabscesses in portal tracts, confluent necrosis, higher neutrophil accumulation, and elevated serum levels of alanine aminotransferase, relative to younger animals. Our results suggest that aged rats displayed a reduced expression of APPs and increased hepatic injury in response to the inflammatory insult.

  20. Study on pretreatment of FPS-1 in rats with hepatic ischemia-reperfusion injury.

    PubMed

    Lin, Shiqing; Liu, Kexuan; Wu, Weikang; Chen, Chao; Wang, Zhi; Zhang, Xuanhong

    2009-01-01

    This study was designed to determine whether FPS-1, the water-soluble polysaccharide isolated from fuzi, protected against hepatic damage in hepatic ischemia-reperfusion injury in rats, and its mechanism. SD rats were subjected to 60 min of hepatic ischemia, followed by 120 min reperfusion. FPS-1 (160 mg/kg/day) was administered orally for 5 days before ischemia-reperfusion injury in treatment group. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin (ALB) were assayed to evaluate liver functions. Liver samples were taken for histological examination and determination of malondialdehyde (MDA), superoxide dismutase (SOD), that catalase (CAT) in liver. Na(+)-K(+)-ATPase and Ca(2+)-ATPase in mitochondria were measured with colorimetry method. Morphological changes were also investigated by using both light microscopy and electron microscopy (EM). In addition, apoptosis and oncosis were detected by Annexin V-FITC/PI immunofluorescent flow cytometry analysis. Serum AST and ALT levels were elevated in groups exposed to ischemia-reperfusion (p < 0.05). Ischemia-reperfusion caused a marked increase in MDA level, and significant decreases in hepatic SOD and CAT (p < 0.05). Na(+)-K(+)-ATPase and Ca(2+)-ATPase were reduced in ischemia-reperfusion groups compared to the sham group (p < 0.05). Oncosis and apoptosis were also observed in ischemia-reperfusion groups. Pretreatment with FPS-1 reversed all these biochemical parameters as well as histological alterations, evidently by increased SOD, CAT, reduced MDA and histological scores compared to the model group (p < 0.05). FPS-1 could attenuate the necrotic states by the detection of immunofluorescent flow cytometry analysis. Pretreatment with FPS-1 reduced hepatic ischemia-reperfusion injury through its potent antioxidative effects and attenuation of necrotic states.

  1. Hepatic energy metabolism correlated with pathology score in rats chronically fed ethanol

    SciTech Connect

    Takahasi, H.; Geoffrion, Y.; Butler, K.W.; French, S.W. )

    1989-02-09

    We repeatedly measured in vivo levels of hepatic phosphorylated metabolites in alcohol-fed rats using non-invasive 31P MRS to evaluate their relation to the severity of the pathologic changes of alcoholic liver disease (ALD). Five pairs of Wister rats were pair-fed with a liquid diet plus ethanol or an isocaloric amount of dextrose via an implanted intragastric tube for up to 6 months (mo). For MRS, the rats were sedated, air was provided by a face mask, and the diet was infused to maintain high blood alcohol levels. {sup 31}P MRS spectra were obtained using a CYCLOPS pulse-acquire sequence, 200 scans, and a 2s recycle delay. The inorganic phosphate (Pi)/ATP peak area ratio was found to be consistently higher in the alcohol-fed rats (E) compared to the pair-fed controls (C). The sugar phosphate/ATP ratio tended to be higher in E when compared to C. Cytosolic pH measured by the chemical shift of the Pi peak showed no significant differences between E and C. The monthly liver biopsies from E showed more severe steatosis, necrosis, and fibrosis with time of feeding and the Pi/ATP ratio was positively correlated with the scored histological changes. These results suggest that a low hepatic energetic state is associated with the progression of ALD pathology.

  2. Direct pathway for hepatic glycogenesis predominates in meal-fed rats

    SciTech Connect

    Huang, M.T.; Veech, R.L.

    1987-05-01

    The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. The rate of glycogen synthesis in livers of rats meal-fed for seven days was found to be about 1 umol/g/min. Plasma glucose concentration in the portal vein was generally below 8 mM before meal-feeding and could reach up to 12 mM at the end of the meal-feeding. Studies on the hepatic-portal (H-P) difference of plasma glucose showed that liver released glucose in the fasted state and could either extract or release glucose after feeding, depending on plasma glucose concentration in the portal vein. The cross-over concentration for the transition was found to be 8 mM. The relative importance of the direct vs indirect pathway for the replenishment of hepatic glycogen was determined by injecting (3-/sup 3/H,U-/sup 14/C)-glucose into the portal vein at the end of meal-feeding. Six minutes after the injection, the ratio of /sup 3/H//sup 14/C in glycogen-glucose was found to be 83-92% of the ratio in liver free glucose. The H-P differences of glucose, lactate, pyruvate, and alanine during feeding were determined. It was found that the H-P difference of (glc) was about 9 times greater than the combined total of ..delta.. (lac), ..delta.. (pyr), and ..delta.. (ala) as early as 10 minutes after the onset of feeding. It is concluded that the direct pathway for the replenishment of hepatic glycogen is predominant and can account for more than 80% of the total glycogen synthesized in vivo in the postprandial state, in contrast to the result of < 30% reported previously by Newgard et al in acute traumatized rats.

  3. Hepatic and extrahepatic uptake of HDL-derived plasma cholesterol in exercised and sedentary rats

    SciTech Connect

    Padmanathan, S.; Green, M.H.; Kris-Etherton, P.M.

    1986-03-01

    The present investigation was designed to study high density lipoprotein (HDL)-derived plasma cholesterol (C) turnover in hepatic and extrahepatic tissues of sedentary (S) and exercised (E) rats. 4-week-old Long Evans rats were exercised for 1 hr, 6 days weekly, for a period of 38 weeks, on a motor-driven treadmill at 0.8 mph at a 12% grade. Animals were injected with HDL that was labelled in vitro with /sup 3/H-cholesteryl ester. Serial blood samples and tissues were collected. HDL-C concentration was lower in E vs S rats (23.0 +/- 1.2 and 26.6 +/- 1.9 mg/dl, p < 0.01). While total plasma C was not different, liver C was higher in S vs E rats (8.2 +/- 0.8 and 7.2 +/- 0.5 mg/g). Adrenal C was higher in E vs S rats (29.5 +/- 2.3 and 20.7 +/- 2.3 mg/g, p < 0.01). Multicompartmental analysis of plasma and tissue tracer response led to development of an 8-component model (5 physiological; 3 nonphysiological) that depicted HDL-derived plasma C turnover. Plasma fraction of tracer declined more rapidly in E vs S rats. E rats cleared nonphysiological tracer more rapidly than S rats, but delayed release of tracer into the plasma longer. Fractional rate of tracer uptake into adrenals, liver, testes, and carcass was greater in E rats. There was a greater fractional turnover rate of tracer in adrenals and liver in S vs E rats. Hence HDL-derived plasma C turnover is altered with vigorous exercise.

  4. Partial Portal Vein Arterialization Attenuates Acute Bile Duct Injury Induced by Hepatic Dearterialization in a Rat Model

    PubMed Central

    Wei, Jishu; Wu, Junli; Gao, Wentao; Li, Qiang; Jiang, Kuirong

    2016-01-01

    Hepatic infarcts or abscesses occur after hepatic artery interruption. We explored the mechanisms of hepatic deprivation-induced acute liver injury and determine whether partial portal vein arterialization attenuated this injury in rats. Male Sprague-Dawley rats underwent either complete hepatic arterial deprivation or partial portal vein arterialization, or both. Hepatic ischemia was evaluated using biochemical analysis, light microscopy, and transmission electron microscopy. Hepatic ATP levels, the expression of hypoxia- and inflammation-associated genes and proteins, and the expression of bile transporter genes were assessed. Complete dearterialization of the liver induced acute liver injury, as evidenced by the histological changes, significantly increased serum biochemical markers, decreased ATP content, increased expression of hypoxia- and inflammation-associated genes and proteins, and decreased expression of bile transporter genes. These detrimental changes were extenuated but not fully reversed by partial portal vein arterialization, which also attenuated ductular reaction and fibrosis in completely dearterialized rat livers. Collectively, complete hepatic deprivation causes severe liver injury, including bile infarcts and biloma formation. Partial portal vein arterialization seems to protect against acute ischemia-hypoxia-induced liver injury. PMID:27872855

  5. Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2.

    PubMed

    Shu, Nan; Hu, Mengyue; Liu, Can; Zhang, Mian; Ling, Zhaoli; Zhang, Ji; Xu, Ping; Zhong, Zeyu; Chen, Yang; Liu, Li; Liu, Xiaodong

    2016-10-01

    1. Atorvastatin is frequently prescribed for lowering blood cholesterol and for prevention of events associated with cardiovascular disease. The aim of this study was to investigate the pharmacokinetics of atorvastatin in diabetic rats. 2. Diabetes was induced in rats by combination of high-fat diet and low-dose streptozotocin (35 mg/kg). Plasma concentrations of atorvastatin following oral (10 mg/kg) and intravenous (2 mg/kg) administrations to rats were measured by LC-MS. Metabolism and uptake of atorvastatin in primary hepatocytes of experimental rats were assessed. Protein expressions and activities of hepatic Cyp3a and Oatp2 were further investigated. 3. Clearances of atorvastatin in diabetic rats following oral and intravenous administrations were remarkably increased, leading to marked decreases in area-under-the-plasma concentration-time curve (AUC). The estimated oral and systematic clearances of atorvastatin in diabetic rats were 4.5-fold and 2.0-fold of control rats, respectively. Metabolism and uptake of atorvastatin in primary hepatocytes isolated from diabetic rats were significantly increased, which were consistent with the up-regulated protein expressions and activities of hepatic Cyp3a and Oatp2. 4. All these results demonstrated that the plasma exposure of atorvastatin was significantly decreased in diabetic rats, which was partly due to the up-regulated activities and expressions of both hepatic Cyp3a and Oatp2.

  6. Effect of a lignan-enriched fructus schisandrae extract on hepatic glutathione status in rats: protection against carbon tetrachloride toxicity.

    PubMed

    Ko, K M; Ip, S P; Poon, M K; Wu, S S; Che, C T; Ng, K H; Kong, Y C

    1995-04-01

    The effect of a lignan-enriched extract of the fruits of Schisandra chinensis (FS) on hepatic glutathione (GSH) status was examined in both control and carbon tetrachloride (CCl4)-treated rats. FS treatment caused a dose-dependent enhancement in hepatic GSH status, as evidenced by significant increases in hepatic GSH level and activities of hepatic glucose-6-phosphate and glutathione reductase (GRD), as well as a decreased susceptibility of hepatic tissue homogenates to in vitro peroxide-induced GSH depletion. The beneficial effect of FS treatment on hepatic GSH status became more evident after CCl4 challenge. Pretreating rats with FS extract at increasing daily doses ranged from 0.2 to 3.2 g/kg for 3 days caused a dose-dependent protection against the CCl4-induced impairment in hepatic GSH status. The enhancement in hepatic GSH status was associated with corresponding decreases in tissue malondialdehyde levels and plasma alanine aminotransferases activities, indicating a significant reduction in the extent of oxidative hepatocellular damage. Our results indicate that the molecular mechanism of hepatoprotection afforded by FS pretreatment may involve the facilitation of GSH regeneration via the GRD-catalyzed and NADPH-mediated reaction.

  7. 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.

  8. Protective effect of Piper betle leaf extract against cadmium-induced oxidative stress and hepatic dysfunction in rats.

    PubMed

    Milton Prabu, S; Muthumani, M; Shagirtha, K

    2012-04-01

    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.

  9. Myricetin Increases Hepatic Peroxisome Proliferator-Activated Receptor α Protein Expression and Decreases Plasma Lipids and Adiposity in Rats

    PubMed Central

    Chang, Chia Ju; Tzeng, Thing-Fong; Liou, Shorong-Shii; Chang, Yuan-Shiun; Liu, I-Min

    2012-01-01

    The aim of this study was to investigate the antiobesity and antihyperlipidaemic effects of myricetin. Myricetin exhibited a significant concentration-dependent decrease in the intracellular accumulation of triglyceride in 3T3-L1 adipocytes. The high-fat diet (HFD)-fed rats were dosed orally with myricetin or fenofibrate, once daily for eight weeks. Myricetin (300 mg kg−1 per day) displayed similar characteristics to fenofibrate (100 mg kg−1 per day) in reducing lowered body weight (BW) gain, visceral fat-pad weights and plasma lipid levels of HFD-fed rats. Myricetin also reduced the hepatic triglyceride and cholesterol contents, as well as lowered hepatic lipid droplets accumulation and epididymal adipocyte size in HFD-fed rats. Myricetin and fenofibrate reversed the HFD-induced down-regulation of the hepatic peroxisome proliferator activated receptor (PPAR)α. HFD-induced decreases of the hepatic protein level of acyl-CoA oxidase and cytochrome P450 isoform 4A1 were up-regulated by myricetin and fenofibrate. The elevated expressions of hepatic sterol regulatory element binding proteins (SREBPs) of HFD-fed rats were lowered by myricetin and fenofibrate. These results suggest that myricetin suppressed BW gain and body fat accumulation by increasing the fatty acid oxidation, which was likely mediated via up-regulation of PPARα and down-regulation of SREBP expressions in the liver of HFD-fed rats. PMID:22474525

  10. 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

  11. Increased hepatic expression of nitric oxide synthase type II in cirrhotic rats

    PubMed Central

    Wang, Hai; Chen, Xiao-Ping; Qiu, Fa-Zu

    2004-01-01

    AIM: To determine the role and effect of nitric oxide synthase type II (NOS II) in cirrhotic rats. METHODS: Expression of NOS II mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO. Double-blind method was applied during the experiment. RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portal-venous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis. Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA. CONCLUSION: Increased hepatic expression of NOS II is one of the important causes of hepatic cirrhosis and portal hypertension. PMID:15222038

  12. Dietary quercetin supplementation increases serum antioxidant capacity and alters hepatic gene expression profile in rats.