The Role Of Fas/FasL In Alcoholic Hepatitis And Related Fibrosis In Mice

Objective Alcoholic liver disease (ALD) is a progressive liver injurycaused by chronic consumption of alcohol, ranging from simple fatty liver toalcoholic hepatitis, hepatic fibrosis and cirrhosis[1]. Alcoholic hepatitis (AH)/alcoholic liver fibrosis (ALF), mainly characterized by hepatic steatosis andnecrotic inflammation, plays an important role in the progressive course ofAH or ALF to liver cirrhosis, and even to hepatocellular carcinoma. Apoptosis,one of the features of liver injury, is a programmed cell death induced byactivation of the extrinsic and intrinsic apoptotic pathway, and futher causehepatic inflammation and fibrosis. Fas/Fas Ligand (FasL) system is one of thedominant genes involved in cell apoptosis. Our previous study found thatFas/FasL played a key role in development of non-alcoholic steatohepatitisand fibrosis. However, it is unclear that the role and mechanism of Fas/FasLpromotes progression of alcoholic hepatitis and liver fibrosis. Therefore, thereare no targeted drug and gene therapy on Fas, FasL and related genes for ALDtreatment. We aimed to explore the role and mechanism of Fas/FasL systemand the downstream signal pathway in progression of alcoholic hepatitis andrelated fibrosis, then to further clarify the pathogenesis of alcoholic liverdisease. It might provide reliable evidences for target therapy of alcoholicliver disease.Methods C57BL/6J mice were fed with4%ethanol-containingLieber-DeCarli liquid diet and combined with a small amount of carbontetrachloride intraperitoneal injection to induce alcoholic hepatitis and liverfibrosis. Control mice were fed with the same volume of a control liquid dietprepared by replacing ethanol in the Lieber-DeCarli liquid diet with isocaloricmaltose. Animals were sacrificed in the fourth and eighth weeks, respectively.Blood and liver samples were collected. A part of livers were fixed in4%formalin, the rest were snap-frozen in lipid nitrogen followed by storage at -80°C in a freezer until required. Serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) levels were measured by enzymatic methodusing an automatic biochemical analyzer (Olympus UA2700, Japan)according to the manufacturer’s instructions. Haematoxylin and eosin (HE)stained liver sections were used to observe the degree of liver steatosis,inflammation and fibrosis, while Masson stained ones were graded for fibrosis.Hepatocelluler apoptosis was evaluated by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay. The mRNA and proteinexpressions of Fas, FasL, cysteine aspartate-specific proteases3(caspase3)and cytochrome P4502E1(CYP2E1) were detected by RT-PCR, Westernblot assay and immunohistochemistry stain, respectively. All data areexpressed as mean±standard deviation (SD). Statistical analysis on the datawas performed by one-way analysis of variance (ANOVA), with the leastsignificant difference-t (LSD-t) test for comparison among groups using SPSS13.0, and P-value below0.05was considered significant.Results Following4%ethanol-containing Lieber-DeCarli liquid diet andcombined with a small amount of carbon tetrachloride intraperitonealinjection, mice rapidly and consistently developed a pattern of alcoholichepatitis (at week4) and liver fibrosis (at week8) with lower mortality. Thisexperimental model was useful for alcoholic liver fibrosis study. Withalcoholic hepatitis and liver fibrosis development, apoptotic cells in the liversections were increased and apoptosis related gene expression was enhanced.In Control group, alcoholic hepatitis group and alcoholic liver fibrosis group,mice apoptosis related gene expression level increased in turn (P <0.05).1. General condition of experimental mice: In control groups, mice wereactive with glossy fur, normal diet; while, in alcoholic hepatitis group andalcoholic liver fibrosis group mice were inactive with sparse fur, eating lessand inelastic skin.2. The examination of biochemical markers of serum: Mice in alcoholicliver fibrosis group showed significantly higher serum ALT and AST levelscompared with the mice in control or alcoholic hepatitis group (P <0.05). (Table2)3. Routine pathologic examinations: With HE and Masson stain, the liverhistology is normal in control mice. The liver section from mice fed with4%ethanol-containing Lieber-DeCarli liquid diet exhibited mild-moderate livermacrovesicular steatosis and hydropic degeneration, lobular spot or focalhepatocyte necrosis and inflammatory infiltration at week4; while markedlyimproved spot or focal hepatocyte necrosis and inflammatory infiltration atweek8, which also showed piecemeal hepatocyte necrosis, fibrosis locatedaround central vein and sinus，portal area enlarge，fibrosis tissue extend intolobular，thenbridging fibrosis developed.4. Analysis of the expression of Fas/FasL system and its downstreamrelated genes in alcoholic hepatitis/fibrosis mice: the hepatic mRNA andprotein expression levels of Fas, FasL, caspase3and CYP2E1in alcoholicliver fibrosis group were significant higher than those in control and alcoholichepatitis group. In the immunohistochemical stained liver sections, FasL,TNF-α and CYP2E1positive cells were located near the necroinflammationfoci around the central vein and the number of the positive cells was relatedwith severity of inflammatory and fibrosis.Conclusions Activation of Fas/FasL and its downstream signaltransduction pathway plays a key role in alcoholic hepatitis and relatedfibrosis in which hepatocelluar apoptosis was involved.