Activation of Fas/FasL and its downstream signaling pathway promotes development of alcoholic steatohepatitis and liver fibrosis in mice

Abstract

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.