Search

To explore the role and mechanism of the Fas/Fas ligand (FasL) system and its downstream signaling pathway related to the progression of alcoholic steatohepatitis and liver fibrosis.Eighteen C57BL/6J mice were randomly divided into three groups: controls; alcoholic steatohepatitis model, given four-weeks of a 4% ethanol-containing Lieber-DeCarli liquid diet; alcoholic steatohepatitis and liver fibrosis model, given the four-week alcohol diet followed by twice weekly intraperitoneal injections of carbon tetrachloride (5% olive oil solution; 2 mL/kg dose) during the fifth to eighth weeks. Mice in the model groups were sacrificed at the end of week 4 and 8, respectively, along with control mice for comparative analyses. Liver tissue sections were evaluated for hepatocellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The mRNA expression of Fas, FasL, cysteine aspartate-specific proteases 3 (caspase 3), and cytochrome P450 2E1 (CYP 2E1) in liver tissues was detected by reverse transcription (RT)-PCR, visualized by ethidium bromide staining, and normalized to the gray-value of GAPDH expression. The protein expression of Fas and caspase 3 were detected by western blotting (b-actin normalized), and of FasL and CYP 2E1 by immunohistochemistry staining. Intergroup differences and statistical significance were evaluated by single factor analysis of variance and the least squares difference-t test or the Kruskal-Wallis H test and the Mann-Whitney U test.The number of apoptotic cells in the liver sections was significantly higher in both model groups with alcoholic steatohepatitis (vs. controls) and the amount in the alcoholic steatohepatitis plus liver fibrosis model was significantly higher than that in the model with only alcoholic steatohepatitis. In addition, activation of Fas, FasL and its downstream signaling pathway showed an increasing trend with extent of liver injury. The hepatic mRNA (by RT-PCR) and protein (by western blotting) normalized expression levels in the controls, alcoholic steatohepatitis models, and alcoholic steatohepatitis plus liver fibrosis models were, respectively: Fas mRNA: 0.50+/-0.05, 0.61+/-0.10, 0.76+/-0.03 (H=12.137, P less than 0.05), protein: 0.52+/-0.14, 0.86+/-0.10, 0.99+/-0.09 (F=12.758, P less than 0.01); FasL mRNA: 0.31+/-0.03, 0.53+/-0.02, 1.02+/-0.04 (F=153.260, P less than 0.01); caspase 3 mRNA: 0.86+/-0.11, 0.85+/-0.05, 1.33+/-0.16 (F=8.740, P less than 0.01), protein: 0.40+/-0.03, 0.69+/-0.06, 1.02+/-0.10 (F=90.785, P less than 0.01); CYP 2E1 mRNA: 0.72+/-0.14, 1.00+/-0.15, 1.30+/-0.20 (H=4.713, P less than 0.01). The changes in hepatic FasL and CYP 2E1 expression detected by immunohistochemistry were consistent with the mRNA expression.Activation of Fas/FasL and its downstream signaling pathway, which induces hepatocellular apoptosis, contributes to the development of alcoholic steatohepatitis and liver fibrosis.

To elucidate the effect of targeted gene modulation of peroxisome proliferator activated receptor gamma (PPARg) on hepatocellular apoptosis in nutritional fibrotic steatohepatitis in mice. C57BL/6J mice were fed with high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPARg (Ad-PPARg), adenovirus-beta-galactosidase (Ad-LacZ), Ad-PPARg plus PPARg agonist rosiglitazone, or PPARg antagonist 2-chloro-5-nitro- benzanilide (GW9662), respectively. H and E stain was performed for observation of hepatocellular apoptosis, hepatic steatosis, inflammation and fibrosis in the liver sections. The expression levels of mRNA and protein of PPARg and apoptosis related genes, Fas, Fas Ligand (FasL), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine-containing aspartate-specific proteases-3 (caspase-3) were detected by real-time RT-PCR and Western blot assay, respectively.Mice fed with MCD diet for 8 weeks showed severe hepatic injury including steatosis, hepatocellular apoptosis, inflammatory infiltration and fibrosis, concomitancy with enhanced expression of pro-apoptosis genes, Fas, FasL, Bax and caspase-3 and increased expression of anti-apoptosis gene Bcl-2, by comparing with the control group. The mRNA expression levels of these genes were 3.59+/-0.35 vs 1.11+/-0.37, 4.37+/-1.03 vs 1.09+/-0.33, 4.27+/-0.48 vs 1.03+/-0.10, 4.93+/-0.67 vs 1.12+/-0.24 and 3.95+/-0.34 vs 1.20+/-0.19, and LSD-t values were 2.49, 3.28, 3.25, 3.80 and 2.75, as compared with the control group, P is less than 0.01; the protein expression levels were 1.96+/-0.07 vs 0.45+/-0.07, 0.53+/-0.07 vs 0.22+/-0.02, 1.32+/-0.06 vs 0.59+/-0.03, 1.51+/-0.23 vs 0.36+/-0.09 and 0.57+/-0.01 vs 0.29+/-0.01, and LSD-t values were 1.51, 0.31, 0.73, 1.14 and 0.28, P is less than 0.01. Administration of PPARg agonist rosiglitazone and/or Ad-PPARg significantly ameliorated hepatic steatosis, hepatocellular apoptosis, necro inflammation and fibrosis. These effects were associated with repressed expression of pro-apoptosis genes and up-regulated expression of anti-apoptosis gene. After rosiglitazone treatment, the mRNA expression levels were 3.78+/-0.58, 3.66+/-0.83, 3.04+/-0.37, 2.54+/-0.62 and 4.42+/-0.42, and LSD-t values were 0.18, 0.71, 1.23, 2.39 and 0.46, as compared with MCD group, the P values were 0.627, 0.241, less than 0.01, less than 0.01 and 0.278, the protein expression levels were 1.06+/-0.03, 0.30+/-0.01, 0.70+/-0.05, 1.19+/-0.30 and 0.90+/-0.01, and LSD-t values were 0.90, 0.23, 0.62, 0.31 and 0.34, the P values were less than 0.01, less than 0.01, less than 0.01, 0.122, less than 0.01. After Ad-PPARg treatment, the mRNA expression levels were 2.31+/-0.16, 2.71+/-0.23, 2.52+/-0.27, 1.79+/-0.32 and 5.97+/-0.72, and LSD-t values were 1.28, 1.66, 1.75, 3.13 and 2.02, as compared with MCD group, P is less than 0.05; the protein expression levels were 1.73+/-0.07, 0.43+/-0.04, 1.01+/-0.08, 1.31+/-0.10 and 1.56+/-0.04, and LSD-t values were 0.23, 0.10, 0.30, 0.20 and 0.99, with P values equal 0.009, 0.01, less than 0.01, 0.322 and less than 0.01.This study provided evidences for the protective role of activation and overexpression of PPARg in ameliorating hepatocellular apoptosis in mice with hepatic fibrosing steatohepatitis.

Our previous study indicated that the death receptor Fas played a key role on hepatocyte apoptosis in nutritional steatohepatitis in mice. This study aimed to explore whether Fas mutation accelerated hepatic steatosis and inflammatory infiltration in methionine-choline deficient (MCD) diet feeding mice.Mice homozygous for the lymphoproliferation spontaneous mutation (C57BL/6J-Faslpr) and wild type C57BL/6J mice were fed with MCD diet for three weeks to induce non-alcoholic steatohepatitis (NASH). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) levels were detected by an Olympus AU5400 automatic chemical analyzer. The role of Fas gene mutation on NASH was assessed by comparing the severity of hepatic steatosis and inflammation in the liver sections, the mRNA and protein expressions of hepatic inflammatory and fibrogenesis related factors, proliferating cell nuclear antigen (PCNA) and transforming growth factor beta 1 (TGFb1).The serum ALT levels of the wild type and Faslpr mice fed with MCD were significant higher than that of the control mice (126.33+/-10.50 U/L vs (25.00+/-10.14) U/L, (160.33+/-48.29) U/L vs (18.33+/-9.08) U/L, with the LSD-t value 12.02, 5.08 respectively, the P value0.001, 0.007 respectively. The serum ALT levels showed no significant difference between the Faslpr and wild type mice fed with MCD, with the LSD-t value 1.19, the P value 0.229. The serum AST, TG and TC levels showed neithere significant difference among the four groups. MCD diet induced hepatic steatosis and inflammatory infiltration in both of the wild type and Faslpr mice. Especially, severer hepatic injury was observed in Faslpr mice as compared with wild type mice. The mRNA expression levels of cell proliferation factor PCNA and fibrogenesis growth factor TGF b1 in wild type mice fed with MCD were significantly higher than that of the control mice (2.84+/-0.73, 2.77+/-0.54 vs 1.31+/-0.18, 0.89+/-0.18), with the LSD-t value 4.99, 8.08 respectively, the P value 0.001,0.001 respectively. The mRNA expression levels of PCNA and TGFb1 in Faslpr mice fed with MCD were significantly higher than that of the Faslpr control mice and the wild type mice fed with MCD (5.57+/-1.13, 5.73+/-0.89 vs 1.04+/-0.16, 0.85+/-0.11 and 2.84+/-0.73, 2.77+/-0.54), with the LSD-t value 10.15, 13.19 and 5.33, 6.91 respectively, the P value0.001. The protein expressions levels of PCNA and TGFb1 were concordant with the mRNA.Faslpr promoted hepatic steatosis and inflammatory infiltration in mice fed with MCD diet, which might associated with excessive release of cell proliferative, inflammatory and fibrogenesis factors.

Background Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced liver injury. However, its role on liver fibrosis remains unclear. This study aims to elucidate the effect and the mechanism of HO-1 in nutritional fibrosing steatohepatitis in mice. Methods Male C57BL/6J mice were fed with a methionine-choline deficient (MCD) diet for eight weeks to induce hepatic fibrosis. 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. Liver injury was assessed by serum ALT, AST levels and histological examination; hepatic lipid peroxides levels were determined; the expression levels of several fibrogenic related genes were assayed by real-time quantitative PCR and Western blot. Results MCD feeding mice showed progressive hepatic injury including hepatic steatosis, inflammatory infiltration and fibrosis. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver injury. This effect was associated with the up-regulation of HO-1, reduction of hepatic lipid peroxides levels, down-regulation of inflammatory factors tumor necrosis factor-alpha, interleukin-6 and suppressor of cytokine signaling-1 as well as the pro-fibrotic genes alpha-smooth muscle actin, transforming growth factor-β1, matrix metallopeptidase-2 and matrix metallopeptidase-9. A contrary effect was observed in mice treated with ZnPP-IX. Conclusions The present study provided the evidence for the protective role of HO-1 in ameliorating MCD diet-induced fibrosing steatohepatitis. Modulation of HO-1 expression might serve as a therapeutic approach for fibrotic steatohepatitis.

To investigate the potential role of heme oxygenase-1 on preventing non-alcoholic steatohepatitis (NASH) in mice.Experimental models of NASH were established by feeding male C57BL/6J mice with choline-methionine deficient diet (MCD) for four weeks. Control animals were fed with choline-methionine supplemented diet. The treatment groups were fed with MCD diet combined with HO-1 inducer hemin or inhibitor zinc protoporphyrin IX (ZnPP-IX). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by enzymic method with automatic biochemistry analyzer. The degree of hepatic steatosis, inflammation and fibrosis were examined under HE staining. The hepatic mRNA and protein expressions of HO-1, TNFalpha and IL-6 were analyzed by RT-PCR and Western blot respectively. MCD fed mice showed increased serum ALT and AST levels and moderate to severe hepatic steatosis with inflammatory infiltration, hepatic spot or focal necrosis, light portal and sinus hepaticus fibrosis in the liver sections, which associated with enhanced expression of HO-1, TNFalpha and IL-6 mRNA and protein (1.13+/-0.11, 1.74+/-0.05; 0.20+/-0.01, 1.92+/-0.10; 0.58+/-0.02, 2.06+/-0.05 vs 0.43+/-0.02, 0.75+/-0.05; 0.08+/-0.00, 0.59+/-0.02; 0.22+/-0.01, 0.91+/-0.02). Administration of hemin significantly decreased serum ALT and AST levels and attenuated hepatic steatosis and necroinflammation which associated with up-regulation of antioxidative gene HO-1 and down-regulation of pro-inflammatory cytokines TNFalpha and IL-6 (P0.01). A contrary effect on serum aminotransferase levels and liver histopathology was observed in mice injected with ZnPP-IX (P0.01).The effect was associated with suppressed HO-1 expression and increased TNFaLPHA and IL-6 expression. The data provided a biochemical, morphological and molecular biological evidence for the protective role of HO-1 in ameliorating hepatic steatosis, necroinflammation in experimental nutritional steatohepatitis.