The Role Of Sirtuin3(SIRT3) In Oxidative Stress Mediated By Hepatitis B Virus X Protein Expression

Objective To study the role of Sirtuin3(SIRT3) in oxidative stress mediated by hepatitis B virus X protein expression and its effect on HBV replication process.Methods 1. Cellular ROS level in HBV-expressing cells or in stably expressing HBV cells were determined by both carboxy-H2 DCFDA staining methods and flow cytometry. 2. Cellular ROS level in HBx overexpressing or expressing HBV genome with HBx mutation cells were determined by both immunofluorescence and carboxy-H2 DCFDA staining methods. 3. SIRT3 m RNA and protein level in HBV or HBx expressing cells were analyzed by q RT-PCR and western blot respectively. And further study on HBV/HBx regulating promoter activity of SIRT3 was detected by dual luciferase reporter system. 4. SIRT3 and HBx overexpressing efficiency were measured by western blot. Cellular ROS level in both HBx-expressing and SIRT3-overexpressing cells were determined by both carboxy-H2 DCFDA staining methods and flow cytometry, and cells GSH/GSSG ratio were detected by GSH/GSSG detection kits. 5. γ-H2 AX formation level and AP sites number in HBx-expressing cells or in both HBx-expressing and SIRT3-overexpressing cells were detected by immunofluorescence, western blot and DNA damage quantitative kit respectively. Cell survival and cell apoptosis in HBx-expressing cells or in both HBx-expressing and SIRT3-overexpressing cells were analyzed by MTS and flow cytometry under the condition of oxidative stress respectively. 6. HBV replication level in HBV stably expressing cells, treating with oxidant H2O2 or antioxidant NAC was detected by both real-time PCR and southern blot. The role of SIRT3 against HBx promoting HBV replication was detected by both real-time PCR and southern blot.Results 1. Hepatitis B virus replication induces oxidative stress. 2. HBx could directly increase the ROS level. 3. HBV replication, especially HBx expression, could downregulate SIRT3 expression. 4. SIRT3 attenuates the oxidative stress induced by HBV replication.5. SIRT3 increases resistance of HBx-overexpressing cell to oxidant damage. 6. SIRT3 attenuctes increased HBV replication induced by ROS.Conclusion These data suggest HBx expression induces oxidative stress, which promotes cellular oxidative damage and viral replication during HBV pathogenesis. Mitochondrial protein SIRT3 protected HBx expressing-cells from oxidative damage and inhibited HBV replication possibly by decreased cellular ROS level.