Effect Of Hepatitis B Virus On Nuclear Erythroid2-related Factor2

Background and Aim:Hepatitis B infection is distributes all over the world. There is a high prevalence of HBV infection in China, and the rate of HBV infection is about7.18%, which means93million people have infected.Chronic HBV infection can lead to liver inflammation and fibrosis, even develop to liver cirrhosis or hepatocellular carcinoma, which is a great harm on patient health and life.Reactive oxygen species is one of the most important mechanisms on hepatocellular inflammation, fibrosis, even liver cirrhosis and hepatocellular carcinoma. Viral protein expressed by HBV infected liver cells can generate radicals and reactive oxygen species (ROS) which form through electron leakage from the mitochondrial respiratory chain. Oxidative stress contributes to lipid peroxidation and DNA damage. One of the most important protective mechanisms is Nrf2/ARE pathway, which regulates phase Ⅱ detoxifying enzyme genes and antioxidant-responsive genes, including HO-1, NQO1, GST, and GCS. Nrf2regulating proteins protect liver cells against harmful chemicals and their potentially damaging metabolites. A group of clinical researches about alcoholic liver disease, nonalcoholic steatohepatitis-related disease and drug induced disease demonstrates that Nrf2plays an important role on the protection of hepatocytes. But only a few researches focus on the role of Nrf2during HBV infection, and the mechanism that how hepatitis B virus affects Nrf2is not clear. In this study, plasmid HBV was transfected into HepG2cells. We detect the impact of HBV on the expression of Nrf2mRNA, and protein in the liver cells and chronic hepatitis B (CHB) patient liver tissues. It can afford the experimental evidence for the pathogenesis of CHB.Methods:1. Plasmid transfection:We planted HepG2cells in six-well plates before transfection, until60%-70%cells merging, put pHBV or pcDNA plasmids into cells, changed for10%of fetal bovine serum medium after4hours, harvested after48hours for the next experiment.2. RT-qPCR:Transfected cells were extracted total RNA.We measured the concentration of RNA by UV spectrophotometer, took2μg RNA to make cDNA on the basis of reverse transcription, the products were augmented by Realtime quantitative PCR.3. Western blot:We detected Nrf2protein expression by Western blot method, using β-actin as a reference.4. Immunohistochemistry method:We used immunohistochemistry method to detect Nrf2protein expression in the CHB patient liver tissues.Results:1. By real time quantitative PCR method, we found the relative expression levels of Nrf2mRNA between the pcDNA plasmid transfected cells and the untransfected cells is similar. This shows that HBV does not stimulate the transcription of Nrf2mRNA.2. By western blot method, we found the cells transfected with plasmid pHBV expressed HBx protein. Non-transfection cells and that of transfected pcDNA plasmid did not express HBx protein. It shows that HBV transfected into HepG2cells successfully. And we found that the Nrf2protein expression in pHBV transfected cells was much higher than in the untransfected cells or in the cells transfected with pcDNA plasmid. This shows HBV up-regulated the expression of Nrf2protein.3. By the Immunohistochemistry method, we found that the Nrf2protein expression of the patients with CHB is significantly higher than normal group and the negative control group. The expression of Nrf2in mild inflammation and necrosis of liver tissue is the highest, followed by the moderate inflammation and necrosis of liver tissue, and expression of Nrf2in severe inflammation and necrosis of liver tissue is the lowest.This shows that HBV infection can up-regulate the expression of Nrf2protein. But persistent HBV infection can down-regulate the expression of Nrf2protein.Conclusion:1. HBV expression plasmid was transfected to HepG2cells successfully. HBV promotes the expression of Nrf2protein, but HBV does not stimulate the transcription of Nrf2mRNA. HBV may regulate Nrf2protein expression after the transcription level.2. HBV can up-regulate the expression of Nrf2protein in CHB patients liver tissue, but persistent HBV infection can down-regulate the expression of Nrf2protein. This may be one of the mechanism that liver inflammation and necrosis continues to progress.