Experimental Study On The Role Of Cell Apoptosis And Regulatory Mechanism In Nonalcoholic Steatohepatitis

ObjectiveWith the increasing of obesity and metabolic disorder-related diseases, nonalcoholic fatty liver disease(NAFLD) is highly common in many countries included China, and up to date it has become the second prevalent liver disease after viral hepatitis in China. NAFLD has been a very challenging health problem. NASH is the key stage in the spectrum of NAFLD which ranges from simple fatty liver disease to liver cirrhosis, but the patholoagical injury mechanism of NASH remain unclear and clinical therapies to the patients are lacking. Cellular apoptosis as a major pattern of cell death in the physiological and pathological conditions should take part in the mechanism of many liver injuries included NASH. In this study, our aim is to study hepatocyte apoptosis, the relationship between the cell apoptosis and disease severity included degree of fatty degeneration, inflammation and fibrosis in the established experimental NASH model of rats. Meanwhile, the expression of apoptosis regulated proteins and their mRNAs were investigated qualitatively and quantitatively Through the exploration of cellular apoptosis and its mechanism in the initiation and progression of NASH, we try to provide experimental evidence for pathogenesis of NASH and new potential therapeutic strategy for NASH.MethodsThe experimental NASH model of rat was established with the high-fat diet.The pathological estimation included the severity of fatty degeneration, inflammation, and fibrosis was performed according to the Scoring System for NAFLD designed by the Pathology Committee of NASH Clinical Research Network (NASH-CRN). The cellular apoptosis and proliferation in experimental NASH liver tissuses were detected by TdT-mediated x-dUTP nick end labling (TUNEL) and PCNA immunostaining. The levels of serological alanine aminotransferase (ALT) ,aspartate aminotransferaseherat (AST)and TNF-αwere detected by enzyme linked immunosorbent assay (ELISA) and biochemistry test.The expression of apoptotic molecules including Fas, FasL, caspase-8, Cyt-C and caspase-3, Bax, Bcl-2 and NF-κB, and oxidative stress molecule CYP2E1 in liver tissues were examined on both of protein and or mRNA levels by using immunohistochemistry and in situ hybridization. Results1. The pathological changes of NASH rat liver tissue shows mixed macrovesicle and microvesicle steatosis, ballooning and apoptotic bodies of hepatocytes, lobular inflammation and lipogranulomas, and sinusoidal cell proliferation. Liver fibrosis occurred after high-fat induced for twelve weeks.2. TUNEL-positive cells markedly increased at 2th week compared to controls and reached the peak at 8th week, which is significantly correlated with the severity of steatosis,and hepatic fibrosis(P<0.01) and the serum levels of ALT(P<0.01), AST(P<0.05) and TNF-α(P<0.01), respectively. PCNA-positive proliferative cells were less in the early stage of NASH model(2th week and 4th week)than those in controls, but obviously increased from 6th week to 12 th week.3. Compared with control rats, Fas and FasL mRNA positive cells in the NASH liver tissues increased obviously at 6th week, while Fas and FasL proteins significantly increased at 8th week. Caspase-8 positive cells were located in the cytoplasm of hepatocytes and presented upregulated expression at 8th week. Caspase-3 postive cells started to increase at 2th week, and significantly increased at 6th week, especially in severe steatosis areas. Cytochrome C and CYP2E1-positive cells which mainly located in acinar zone 3 increased in number at 2th week, ,and the up-regulated expression was maintained with the development of experimental NASH model.4. The expression of Bax and Bcl-2 proteins were mainly located in cytoplasm of hepatocytes. In the early stage of NASH model, the number of Bax positive cells were more than Bcl-2, but both proteins approach to parallel at the late stage.5.The expression of NF-κB protein was located in cytoplasm/nucleus of hepatocytes in NASH liver tissues and increased at 6 th week. The semi-quentitative examination showed the up-regulated expression was closely correlated with the degree of cell apoptosis and proliferation(P<0.01).Conclusions1. We have successfully established experimental NASH model of rat whose pathological characteristic was very similar to the human nonalcoholic steatohepatitis, which provides a rational experimental model for the exploration of pathogenesis and novel treatment of NASH.2.The results showed that cellular apoptosis is one of the major parterns of cell injury in the pathological process of NASH. A positive correlation was found between cell apoptosis and severity of steatosis and fibrosis. In the early period of NASH ,hepatocyte apoptosis is more predominant than cell proliferation, but both parterns co-existed in the late period of NASH model,which suggested that anti-apoptotic therapy might be a novelly interventional strategy for NASH treatment.3.Both cell death ways through receptor- and mitochondria- mediated apoptosis way might contribute to the initiation and progression of NASH. Furthermore, the mitochondria mediated apoptosis way is predominate at early stage,and receptor mediated way through Fas and FasL also participate in regulation of the cell apoptosis in the late period of NASH.4. Oxidative stress exists in the whole process of NASH , which might trigger cell apoptosis. This result suggested anti-oxidative stress therapy which has been used on clinic might ameliorate liver injury caused by NASH through suppressing cell apoptosis and other mechnisms.