The Relationship Between Expression Of Nrf2 And Hepatocyte Apoptosis In Nonalcoholic Fatty Live Disease In Mice

Background and ObjectiveNonalcoholic fatty liver disease(NAFLD) is emerging as one of the most common causes of abnormal liver function, which encompasses progressive stages of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). NAFLD involves the accumulation of triglycerides in hepatocytes, necrosis, and apoptosis of these cells, without long history of drinking, accompanied by inflammatory and fibrogenic responses within the liver, potentially leading to cryptogenic cirrhosis or hepatocarcinoma. Studies have shown that apoptosis of liver cells may play a role in promoting the development of NAFLD. NF-E2-related Factor 2(Nrf2) as a newly discovered transcription factor, exists in almost all cells, which plays an important role in the progress of anti-oxidative stress, anti-inflammatory, anti-apoptosis in liver cells by regulating antioxidant enzymes and the phase II detoxification enzyme, such as NADPH quinone oxidoreductasel(NAD (P) H quinine oxidoreductasel,NQO1) and so on. Therefore, Nrf2 may be very important in the clinical treatment of NAFLD in the future. Its roles and significances are worthy of further study. Still the relationships between Nrf2 and hepatoeyte apoptosis, cell proliferation have not been reported. We establish the mice models with high fat diet, and then test the expression of Nrf2, NQO1, hepatocyte apoptosis and cell proliferation (Ki-67) during different phases of NAFLD process. The aim is to investigate what impact the Nrf2 and hepatocyte apoptosis have on the liver cells during the progression of NAFLD, including their relationship between each other, and finally to provide a theoretical basis for clinical treatment of NAFLD.Methods24 mice were divided randomly into three groups with 8 mice in each group: control group been fed 8 weeks with normal food, high-fat diet group been fed 8 weeks(nonalcoholic fatty liver group, NAFL group), and 12 weeks(nonalcoholic steatohepatitis group,NASH group)with high fat. Each group shared their own animal cases where temperature was 22-26℃and they were free to drink, eat. All mice were sacrificed at the end of 8 or 12 weeks. The mice were anesthetized by celio-injection of 10% cholral hydrate solution(0.3ml/100g weight). The livers were picked off and were fixed by 10% formalin solution for the purpose of pathologic slices for light microscope. Hepatic steatosis, inflammation were graded by routine HE staining of liver sections. Hepatocyte apoptosis was tested by TUNEL method. Immunohistochemical staining was used to detect the expression of Nrf2, NQO1 and Ki-67 in the liver.Results1. There were obvious pathologic changes in the liver tissue from the high-fat diet group. Light to middle fatty liver was observed in the NAFL group. At the end of 12th week the liver developed into steatohepatitis (NASH group). The fatty change and inflammation of liver in the model groups aggravated and presented time-dependent manner (p<0.05).2. Samples from high-fat diet group exhibited more NQO1-positive cells, TUNEL-positive cells and Nrf2-positive cells than that from the control group (p<0.05). Also compared with NAFL group, the number of these cells were more higher in NASH group (p<0.05).3. The expression of Ki-67 in the NASH group was higher than that from the control and NAFL group, however, there were no significant difference between the control group and NAFL group(p>0.05).4. Hepatocyte apoptosis in the liver tissue was positively correlated with hepatocyte steatosis and inflammation of hepatic tissues(r=0.731, r=0.786, p<0.01), and the expression of Nrf2 was also positively correlated with hepatocyte steatosis and inflammation of hepatic tissues (r=0.948, r=0.850, p<0.01). Nrf2 expression was positively correlated with the hepatic apoptosis, Ki-67 and NQO1 in the mouse(r=0.754, r=0.640, r= 0.805,p<0.01).Conclusion1. With the development of NAFLD, the fatty change and inflammation of liver in the model groups aggravated and presented time-dependent manner. Hepatocyte apoptosis and the expression of Nrf2 increased, and the degree of hepatocyte apoptosis and the Nrf2 expression is positively correlated with hepatocyte steatosis and inflammation of hepatic tissues, which suggests that apoptosis and Nrf2 may play an important role in promoting the progression of NAFLD.2. There is a positive correlation between hepatocyte apoptosis and Nrf2, which means Nrf2 may take part in the hepatocyte apoptosis and may have an effect on apoptosis.3. During the progress of NAFLD, the expression of NQO1 is increased, and Nrf2 is closely correlated with NQO1, which suggests that Nrf2 may play the role of oxidative stress through the phaseⅡdetoxification enzyme NQO1.4. Also the expression of Ki-67 is increased, and Nrf2 is closely correlated with Ki-67, which suggests that Nrf2 may have effect on the proliferation of hepatocyte.