The Effect And Possible Mechanism Of Hepatitis B Virus On Nonalcoholic Fatty Liver Disease

Chronic hepatitis B virus (HBV) infection is an important public health problem worldwide, with more than 350 million people being chronic carriers of the virus. Furthermore, about one million HBV carriers die of Hepatic failure, cirrhosis and hepatocellular carcinoma (HCC) per year. With the rising prevalence of Nonalcoholic fatty liver disease (NAFLD) in recently, Hepatic steatosis in persons with chronic viral hepatitis B become a common phenomenon. Therefore, studies on the relationship between Hepatic steatosis and HBV become more important. However, the association of chronic HBV infection and the emergence of NAFLD remains unclear. Epidemiology studies have showed that the prevalence of NAFLD in the persons with chronic viral hepatitis B was estimated to be between 3% and 76%. Several studies have suggested that HBV infection is associated with the development of hepatocellular steatosis and is a risk factor for fatty liver. However, other studies have showed that host characteristics such as obesity, Hypertriglyceridemia, type 2 diabetes and metabolism syndrome are important determinants of hepatic steatosis in chronic viral hepatitis B (CHB). Hepatic steatosis does not correlate with hepatitis B e antigen (HBeAg) status or HBV-DNA titer. Therefore, the relationship between HBV infection and hepatic steatosis remains to be further explored. In this study we investigated the effect and possible mechanism of Hepatitis B Virus on nonalcoholic fatty liver disease, and explored the relationship between the seven proteins that originate from the HBV genome, such as polymerase, surface, core, and HBx proteins and hepatic steatosis. The results may not only help to predict the risk of HBV infection on NAFLD and provide new insights into underlying molecular mechanisms involved in HBV replication and pathogenesis, but also provide rationale for the effective prevention and treatment of fatty liver. Which may have particular importance in the country with high prevalence of HBV infection.1.The results of a case-control study on the relationship between HBV infection and NAFLDMultivariate statistics method was adopted to explore the effect of HBV infection serum markers, serum models, HBV DNA titers and the interaction of virus factors and host characteristics on NAFLD. The results showed that: the total infection rate in cases with NAFLD was 84.16%, which was significantly higher than that (69.21%) in controls (P<0.05). After Adjustment of confounding variables, a positive significant association was observed in HBV total infection and NAFLD. Compared to non-HBV infection, HBV total infection had increased the risk of NAFLD (OR=3.15; 95%CI: 1.18~5.50). Among ten types of HBV infection serum models, there were six types of models such as the third type (only anti-HBc serum marker positive), the forth type(Anti-HBe and Anti-HBc positive),the fifth type(Anti-HBs, Anti-HBe and Anti-HBc positive),the sixth type(HBsAg and Anti-HBc positive), the seventh type(Anti-HBs and Anti-HBc positive) and the eighth type (HBsAg,Anti-HBe and Anti-HBc positive) closely correlated with NAFLD. As compared to non-HBV infection, the eighth type of HBV infection serum model with HBsAg, Anti-HBe and Anti-HBc serum markers positive had the highest risk of NAFLD (OR=6.47,95%CI: 2.90~14.44). HBV infection might also interact with host characteristics such as low levels of HDL-C to increase the risk of NAFLD (Pinteraction=0.034).2. The effect of HBV on proteins related to lipid metabolismThe previous results of Global proteomic analysis of HBV in the human hepatoblastoma HepG2 cell line have been identified six proteins such as apolipoprotein A-I(APOA1),apolipoprotein E(APOE) ,superoxide dismutase 2 (SOD2) NAd(P)H dehydrogenase, quinone 1 (NQO1), fatty acid binding protein 1(FABP1)and acetyl-Coenzyme A acetyltransferase 2(ACAT2), which were differentially expressed when using the criteria of 1.5-fold or greater( p<0.01).These proteins may play important roles in lipid metabolism and progression of hepatocellular steatosis. In this study, Western blot and real-time RT-PCR were used to confirm the changes of these six proteins related to lipid metabolism in HBV-expressing cells and control cells. We found that as compared to control cells (Repsal-1), ApoAI, APOE, SOD2 and NQO1 were down-regulated and FABP1 was up-regulated in the HBV-expressing cells (RHBV-3) tested by Western blotting and real-time RT-PCR analysis. The results were consistent with those obtained from the previous proteomic analysis. ACAT2 was not found to be up-regulated in the HBV-expressing cells tested by Western blotting and real-time RT-PCR, which was not consistent with that obtained from the previous proteomic analysis. To strengthen the link between HBV and lipid metabolism proteins expression, Western blotting and real-time RT-PCR were used to analyze the six proteins expression in HepG2 cells,Huh7 cells and YY cells transient transfected with a complete HBV genom(epREP-HBV) or control plasmid (pRepsal). We found that as compared to HepG2 cells, Huh7 cells and YY cells transfected with control plasmid, SOD2 and NQO1 were down-regulated and FABP1 was up-regulated in the HepG2 cells, Huh7 cells and YY cells transfected with a complete HBV genome tested by Western blotting and real-time RT-PCR analysis. The results were consistent with those obtained from the previous proteomic analysis. ApoAI was down-regulated in HepG2 transfected with a complete HBV genome HBV tested by Western blotting, which was not verified by real-time RT-PCR analysis. ACAT2 was up-regulated in HepG2, Huh7 and YY cells transfected with a complete HBV genome HBV tested by real-time RT-PCR analysis, which was not verified by Western blotting. Those results suggested that HBV can regulate the expression of lipid metabolism proteins, especially the proteins such as SOD2, FABP1 and NQO1 which may be more closely related to HBV.Those results above provided new insights into underlying molecular mechanisms of HBV involved in hepatocellular steatosis.3. The relationship between the seven proteins that originate from the HBV genome and SOD2, NQO1 and FABP1AdEasy XL System was used to construct recombinant Adenovirus such as Ad-X, Ad-S(small HBV surface protein),Ad-MS (middle HBV surface protein), Ad-LS(large HBV surface protein), Ad-Core(HBV core protein),Ad-E(HBV e protein),Ad-P (HBV polymerase protein) and control Adenovirus(Ad0).Western blot and real time RT-PCR was used to explore the relationship between seven proteins that originate from the HBV genome and six proteins related with lipid metabolism. The results have showed that we have successfully constructed the recombinant Adenovirus, which could effectively infect target cells and express corresponding protein in target cells respectively. As compared to Ad0 infected HepG2 cells, SOD2 and NQO1 were down-regulated and FABP1 was up-regulated in Ad-X infected HepG2 cells tested by western blot and real time RT-PCR analysis. The expression level of FABP1 detected by western blot and real-time RT-PCR were higher in Ad-S infected HepG2 cells than that in Ad0 infected HepG2 cells. The expression level of SOD2 and NQO1 detected by real-time RT-PCR were lower in Ad-S infected HepG2 cells than that in Ad0 infected HepG2 cells, which was not verified by western blot. The expression level of SOD2 detected by western blot was lower in Ad-MS infected HepG2 cells than that in Ad0 infected HepG2 cells, which was not verified by real-time RT-PCR.The expression level of FABP1 detected by western blot was higher in Ad-LS infected HepG2 cells than that in Ad0 infected HepG2 cells, which was not verified by real-time RT-PCR. The expression levels of three genes detected by western blot in Ad-P infected HepG2 cells were not different from that in Ad0 infected cells, but the expression levels of SOD2 and NQO1 detected by real time RT-PCR were lower in Ad-P infected cells than that in Ad0 infected cells. The expression levels of three genes detected by western blot and real-time RT-PCR in Ad-CORE infected HepG2 cells were not different from that in Ad0 infected cells.The expression of SOD2 detected by western blot and real-time RT-PCR and the expression level of NQO1 detected by real-time RT-PCR in Ad-E infected cells were lower than that in Ad0 infected cells.The results suggested that although all of the seven proteins encoded by HBV can regulate the expression level of those three lipid metabolism protein, HBV x protein and small HBV surface protein were closely associated with the changes of those three lipid metabolism proteins. Therefore, HBx and small HBV surface protein may be involved in the development of hepatic steatosis.