Na Fu

Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice.

Objective. Hepatic oxidative stress plays a key role in the development of non-alcoholic steatohepatitis (NASH). However, the protective effects of antioxidants on NASH are largely unknown. The aim of this study was to elucidate the effect and mechanism of antioxidants on NASH in mice. Material and methods. C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 10 days or 3 weeks to induce steatohepatitis. Antioxidants (vitamin E, ABT, or vitamin E plus ABT) were supplemented in mice fed a MCD diet, respectively. The effect of antioxidants on oxidative stress and apoptosis was assessed, and activation of adiponectin and expressions of inflammatory factors, apoptosis-related genes, and fibrosis-related genes were assayed. Results. MCD feeding in mice showed increasing serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels, and progressive hepatic injury including hepatic steatosis and inflammatory infiltration. Administration of antioxidants vitamin E and/or ABT significantly lowered serum ALAT and ASAT levels (p<0.001) and ameliorated hepatic steatosis and necroinflammation. These effects were associated with repressed hepatic lipid peroxides through reducing hepatic MDA content and enhancing hepatic superoxide dismutase (SOD) activity; down-regulated inflammatory factor COX-2, lowered activity of NF-κB, up-regulated anti-apoptotic gene Bcl-2, and down-regulated pro-apoptotic gene Bax suppressed expression of the fibrotic genes TGF-β1 and MMP2. Moreover, expression of the anti-inflammatory factor adiponectin was also induced by vitamin E or ABT. A combination of vitamin E and ABT showed an additive effect on preventing liver injury. Conclusions. The present study provides morphological and molecular biological evidence for the protective role of the antioxidant vitamins E and ABT in ameliorating oxidative stress, hepatic apoptosis, and necroinflammation in experimental nutritional steatohepatitis.

Rosiglitazone prevents nutritional fibrosis and steatohepatitis in mice

Objective. Currently, no agent has been confirmed as preventing the fibrosing progression of non-alcoholic steatohepatitis (NASH). In this study, rosiglitazone was used in the clinical treatment of insulin resistance in patients with type 2 diabetes mellitus. However, its protective effect on non-alchoholic fibrosing steatohepatitis is not clear. The study aimed to elucidate the effect and the mechanism of rosiglitazone in inhibiting nutrition-related fibrosis in mice. Methods. C57BL6/J mice were fed a high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce hepatic fibrosis, and rosiglitazone was given in the treated group. The effect of rosiglitazone was assessed by comparing the severity of hepatic fibrosis in liver sections, the activation of hepatic stellate cells (HSCs) and the expression of TGF-β1 and connective tissue growth factor (CTGF). Results. At week 8, MCD-diet-induced fibrosing NASH models showed increased serum ALT and AST levels, severe hepatic steatosis, and infiltration of inflammation and fibrosis which, associated with down-regulated PPARγ mRNA and protein expression, up-regulated α-SMA protein expression and enhanced TGF-β1, CTGF mRNA and protein expression. Rosiglitazone significantly lowered serum ALT and AST and it reduced MCD-induced fibrosis by repressing levels of α-SMA protein expression and pro-fibrosis factors TGF-β1 and CTGF. It also restored expression of PPARγ. Conclusions. The present study provides clear morphological and molecular biological evidence of the protective role of rosiglitazone in ameliorating nutritional fibrosing steatohepatitis. Rosiglitazone may ameliorate hepatic fibrosis by activating PPARγ, which can inhibit HSC activation and suppress TGF-β1 and CTGF expression.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

ObjectiveHeme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice.MethodsC57BL/6J mice were fed with methionine-choline deficient (MCD) diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin), HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX) and/or adenovirus carrying HO-1 gene (Ad-HO-1) were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot.ResultsHepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1), inhibited cytochrome c (Cyt-c) release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes.ConclusionsThe present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

Nonalcoholic fibrosing steatohepatitis is a uniform process that occurs throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been shown to be involved in the biological processes, but the role and molecular mechanism of miRNAs in NAFLD are not entirely clear. In this study, we observed a significant reduction in the expression of miR-130a-3p in livers of a mouse model with fibrosis induced by a methionine–choline-deficient diet, of NAFLD patients, and in activated hepatic stellate cells (HSCs). A dual-luciferase activity assay confirmed that transforming growth factor-beta receptors (TGFBRs) 1 and 2 were both the target genes of miR-130a-3p. The hepatic expression of TGFBR1 and TGFBR2 was significantly increased. Moreover, the overexpression of miR-130a-3p in HSCs inhibited HSC activation and proliferation, concomitant with the decreased expression of TGFBR1, TGFBR2, Smad2, Smad3, matrix metalloproteinase-2 (MMP-2), MMP-9, type I collagen (Col-1), and Col-4. In addition, the overexpression of miR-130a-3p promoted HSC apoptosis by inducing the expression of caspase-dependent apoptosis genes. Transfection with si-TGFBR1 and si-TGFBR2 revealed effects on HSC function that were consistent with those of miR-130a-3p. TGFBR1 and TGFBR2 rescued the miR-130a-3p-mediated reductions in the mRNA and protein expression levels of Smad2, Smad3, Col-1, and Col-4. In conclusion, our findings suggest that miR-130a-3p might play a critical role in negatively regulating HSC activation and proliferation in the progression of nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2 via the TGF-β/SMAD signaling pathway.

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

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.

LncRNA-ATB/microRNA-200a/β-catenin regulatory axis involved in the progression of HCV-related hepatic fibrosis

OBJECTIVE(S) Long noncoding RNAs (lncRNAs)-activated by transforming growth factor beta (lncRNA-ATB) is known to be involved in the invasion of hepatocellular carcinoma by regulating target genes of miR-200a. However, the role and molecular mechanisms of lncRNA-ATB/miR-200a in HCV-related liver fibrosis remains unclear. In this study, we examined the expression of lncRNA-ATB/miR-200a, and their target gene β-Catenin in liver tissues of HCV patients and hepatic stellate cells (HSCs) to elucidate the possible role of lncRNA-ATB/miR-200a axis in HSC activation and development of liver fibrosis. MATERIALS AND METHODS Liver tissues were obtained by biopsy or surgery from eighteen HCV patients with severe liver fibrosis and six healthy subjects (control). Conditioned media (CM) from cultured HepG2-CORE cells (HepG2 cells stably expressing HCV core protein) were used to treat LX-2 cells. The binding sites between lncRNA-ATB/miR-200a and β-catenin were predicted and then verified by a dual luciferase reporter assay. The effect of lncRNA-ATB/miR-200a/β-catenin on HSC activation was assessed by examining the expression of alpha-smooth muscle actin (α-SMA) and collagen type 1 alpha 1 (Col1A1) in HSCs. Further, the regulatory role of lncRNA-ATB on HSC activation and miR-200a/β-catenin expression was assessed by using siRNA-mediated knockdown of lncRNA-ATB. RESULTS LncRNA-ATB was up-regulated in fibrotic liver tissues and activated LX-2 cells treated with CM from HepG2-CORE cells. Dual luciferase reporter assays confirmed that lncRNA-ATB contained common binding sites for miR-200a and β-catenin. Decreased expression of miR-200a and increased expression of β-catenin were observed in liver tissues of patients with HCV-related hepatic fibrosis and activated HSCs. Knockdown of lncRNA-ATB could down-regulate β-catenin expression by up-regulating the endogenous miR-200a and suppress the activation of LX-2 cells. CONCLUSION LncRNA-ATB/miR-200a/β-catenin regulatory axis likely contributed to the development of liver fibrosis in HCV patients. Knockdown of lncRNA-ATB might be a novel therapeutic target for HCV-related liver fibrosis.

Role of Oxidative Stress in Hepatitis C Virus Induced Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Hepatitis C virus (HCV) infection is the predominant cause of chronic liver diseases and HCC, particularly in Western countries. Multiple molecular mechanisms are involved in the development and progression of HCV-related HCC, of which oxidative stress plays a pivotal role. HCV infection induces overproduction of reactive oxygen species (ROS) and impairs the function of endogenous antioxidants. Excessive amount of ROS directly damages DNA, lipids and proteins. Meanwhile, ROS indirectly activates a series of signaling cascades, and modulates the activity of many transcription factors, resulting in altered expression of genes that control cell survival, proliferation, angiogenesis, invasion and metastasis. In this review, we aim to summarize the possible molecular mechanisms underlying the link between the oxidative stress and hepatocarcinogenesis in HCV-infected individuals, in order to facilitate discovery of possible approaches or interventional targets for HCV-related HCC.

Involvement of the Interleukin-23/Interleukin-17 Axis in Chronic Hepatitis C Virus Infection and Its Treatment Responses.

Interleukin-23 (IL-23) and its downstream factor IL-17 are the key cytokines involved in immune and inflammatory response in chronic liver diseases. This study aimed to investigate the role and molecular mechanisms of the IL-23/Th17 axis in chronic hepatitis C virus (HCV) infection, and the efficacy of IL-23/Th17 modulation in response to anti-HCV therapy. Sixty-six HCV-infected patients and 20 healthy controls were enrolled. The patients received PegIFNa-2a and ribavirin therapy for at least 48 weeks. The plasma level of IL-23 and the number of IL-17A-, IFN-γ-, and IL-21-producing peripheral blood mononuclear cells (PBMCs) at baseline and 12, 24, and 48 weeks following treatment were determined. The mRNA level of Th17 immune-associated molecules in PBMCs was evaluated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) following treatment with IL-23 agonist or antagonist. Our data showed that, compared to healthy controls, HCV-infected patients had an increased plasma level of IL-23 and increased frequencies of IL-17A- and IFN-γ-producing PBMCs, whereas the HCV patients exhibited a reduced number of IL-21-producing PBMCs. However, the baseline frequencies of IL-21-producing PBMCs were markedly higher in HCV patients who achieved rapid virological response (RVR) than those without RVR. Additionally, the mRNA expressions of IL-21, IFN-γ, myxovirus resistance protein A (MxA), and suppressor of cytokine signaling 3 (SOCS3) were significantly upregulated in PBMCs, while FoxP3 expression was suppressed by IL-23 agonist. Thus, the IL-23/Th17 axis plays an important role in development of chronic HCV infection and antiviral response. IL-23 may enhance the antiviral activity of interferon-based therapy by modulating the expression of Th17 cells-associated molecules in HCV-infected patients.

Peroxisome proliferator-activated receptor α, a potential therapeutic target for alcoholic liver disease.

Alcoholic liver injury represents a progressive process with a range of consequences including hepatic steatosis, steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Targeting key molecular regulators involved in the development of alcoholic liver injury may be of great value in the prevention of liver injury. Peroxisome proliferator-activated receptor α (PPARα) plays a pivotal role in modulation of hepatic lipid metabolism, oxidative stress, inflammatory response and fibrogenesis. As such, PPARα may be a potential therapeutic target for the treatment of alcoholic liver disease.