Hepatitis B Virus Involved In Hepatic Steatosis Via Effect On Apolipoprotein And NQO1Expression

Infection of human hepatitis B virus (HBV) resulted in acute and chronichepatitis, and closely related to cirrhosis and hepatocellular carcinoma (HCC).Recently, with the increasing prevalence of Nonalcoholic fatty liver disease (NAFLD),hepatic steatosis along with chronic viral hepatitis B in persons become a commonphenomenon. Therefore, studies on the interaction between hepatic steatosis and HBVbecome more and more important. However, the exact association of HBV infectionand hepatic steatosis remains elusive. Previous studies from our laboratory haveshown that the protein level of Apolipoprotein is marked affected and NQO1issignificantly lower by using a proteomic approach and MALDI-TOF/TOF MSanalysis. These proteins may play important roles in lipid metabolism and progressionof hepatic steatosis. And in that basis, we investigated the effect of HBV protein(LHBs、MHBs、HBs、HBc、HBe、HBpol、HBx) on Apolipoprotein (Apo) expression,and further investigated the mechanism of HBx involvement in hepatic steatosis viaNQO1repression. These data may suggested a mechanism for HBV-associatedpathogenesis and provide theoretical basis for the effective prevention and treatmentof fatty liver.The first part of this study is to investigate whether HBV affected transcriptionApo genes. HBsAg and HBeAg were measured by ELISA kit to confirmed HBVreplication and expression on stable HBV-producing hepatoma cell line and transienttransfection cells. Realtime RT-PCR to examine the16Apo genes transcriptional levelof HepG2.2.15and HepG2cells. And further confirmed by transient transfectionassay. The results showed that the transcriptional level of ApoAI，ApoAII，ApoAV，ApoB，ApoCIII，ApoE，ApoF，ApoH，ApoJ，ApoL1and ApoM is marked lowerin HepG2.2.15cells, while ApoD is significantly higher compared with that in control cell line. Besides, the effect of HBV on Apo genes transcriptional level istime-dependent.In the second part of this study, AdEasy XL System was used to constructrecombinant adenovirus Ad-LHBs、Ad-MHBs、Ad-HBs、Ad-HBc、Ad-HBe、Ad-HBpol、Ad-HBx and control adenovirus Ad-GFP. Realtime R-PCR and westernblot was used to explore the relationship between seven of HBV proteins and Apogenes. The results showed that recombinant adenovirus were successfully constructed,which could effectively infect target cells and express corresponding proteins. Ascompared to Ad-GFP infected Huh7and HepG2cells, ApoAII, ApoF and ApoH wasdown-regulated by HBs, MHBs and HBpol, respectively, tested by real time RT-PCR,which was coincident wih HBV level. The protein level of ApoAII, ApoF and ApoHwere further detected by western blot, the results indicated that ApoAII wasdown-regulated by HBs, which was coincident wih transcriptional level. While ApoFand ApoH was up-regulated by MHBs and HBpol, the exact mechanism was unclear,we consider there are transcription, post-transcription, translation and post-translationlevels involved in these course.In the third part of this study is to elucidate the mechanism of HBV, expeciallyHBx induced hepatic steatosis via NQO1repression. The amount of intracellular ROSlevel and superoxide anion were detected, the results indicated that HBx-mediatedNQO1expression could increase the generation of intracellular ROS and superoxide.The intracellular GSH levels were measured and indicated that high levels of ROScould exhaust cellular antioxidant enzymes. To investigate whether conditions ofoxidative stress induced by HBx could predispose the cells to apoptosis,Annexin-V/PI dual staining for flow cytometric analysis was used to measure an earlyevent of apoptosis. The results showed that HBx expression increased apoptoticsusceptibility of the cells. To address directly whether HBx impairs cellular defensesagainst exogenous pro-oxidant stimuli, cells were exposed to increasingconcentrations of H2O2and cell viability measured. It caused much more cell death inHBx-expressing Ad-HBx cells than the control Ad-GFP cells. In addition,HBx-induced Δψm loss and ATP depletion is reversible by NQO1over-expression or DAC treatment.