Mechanism of hepatocyte apoptosis in rats with liver fibrosis induced by lipogenic methionine-choline-deficient diet

Abstract

To clarify the effects of endoplasmic reticulum stress (ER stress) and mitogen-activated protein kinase (MAPK) on hepatocyte apoptosis in rats with non-alcoholic fatty liver fibrosis induced by methionine-choline-deficient diet (MCDD). Nonalcoholic steatohepatitis with advanced fibrosis was induced in rats by giving a MCDD for 10 weeks (group M). A methionine-choline-control diet (MCCD) instead of MCDD was given for the last 2 weeks to the experimental group (group R). Steatosis, fibrosis and inflammation were determined by tissue staining. The activation of hepatic stellate cells and oxidative stress were determined by immunostaining, immunoblotting or real time-PCR (RT-PCR), respectively. Hepatocyte apoptosis was determined by TUNEL staining. Expressions of glucose-regulated protein 78 (GRP78), caspase-12, caspase-7, cleaved caspase-7, caspase-3, cleaved caspase-3, and caspase-9 were evaluated to clarify the presence of ER stress. Expressions of c-Jun, ERK1/2, p-ERK1/2 were evaluated to clarify the states of MAPK signaling. Changing the diet from MCDD to MCCD triggered the reduction of fat in hepatocytes, a decrease in inflammatory response, oxidative stress, and fibrosis. The protein expressions of ERP78, caspase-12, caspase-7, and cleaved caspase-7 were increased significantly in group M compared with normal control group (group N, P < 0.05 or P < 0.01), the mRNA expressions of ERP78, caspase-12, and caspase-7 were also increased significantly in group M compared with group N (3.03 ± 0.41 vs 2.12 ± 0.37, 1.86 ± 0.36 vs 0.78 ± 0.20, and 2.38 ± 0.19 vs 1.84 ± 0.13, respectively, P < 0.05 or P < 0.01), while they recovered immediately in group R. In contrast, the protein levels of caspase-3, cleaved caspase-3 and mRNA expressions of caspase-3 and caspase-9 revealed no significant differences in three groups (P > 0.05). The mRNA expressions of c-Jun and protein levels of ERK1 and p-ERK1 were increased significantly in group M compared with group N (P < 0.01), while they recovered immediately after changing the diet from MCDD to MCCD. ER stress plays a role in the development and regression of non-alcoholic fatty liver fibrosis induced by MCDD, however, ER stress-related caspase-12 pathway may not be the main mechanism of hepatic apoptosis, and MAPK signaling may play an important role in hepatic apoptosis in the model.