The Protective Role of Fistein in Oxidative Damage Induced by HBV Replication and Its Mechanism

Abstract: This study was aimed to investigate the protective role of sirtuins activator fisetin in oxidative damage induced by hepatitis B virus (HBV) replication and its mechanism. Huh-7 cells were transfected with HBV expressing plasmid pCH9/3091 or treated with n-acetyl-cysteine (NAC). The mitochondria and cytosolic reactive oxygen species (ROS) levels were measured by MitoSOXTM Red reagent and DCFH-DA probe assay， respectively.The malondialdehyde (MDA) level was detected by MDA assay kit. The superoxide dismutase 1 (SOD1) and SOD2 protein levels were measured by Western blot. Furthermore， the Huh-7 cells transfected with pCH9/3091 were treated with fisetin， then the levels of ROS， MDA， SOD1 and SOD2 were determined. In addition， the Huh-7 cells transfected with pCH9/3091 were treated with fisetin or simultaneously silencing the SOD2 expression.The phosphorylated Histone H2AX (γ-H2AX) formation was analyzed by cyto-immunofluorescence and Western blot. Under the condition of oxidative stress， the effect of fisetin and SOD2 silencing to the cell viability of HBV expressing cells was measured by MTS [3-(4，5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium， inner salt] assay. Moreover， the levels of total mitogen-activated protein kinases (MAPKs) and phosphorylated MAPKs were detected by Western blot. The results showed that HBV replication significantly increased the ROS and MDA levels， and significantly decreased the SOD1 and SOD2 protein levels in Huh-7 cells. NAC decreased the ROS and MDA levels in HBV expressing cells. Fisetin attenuated the increase of ROS and MDA levels， and the decrease of SOD1 and SOD2 protein levels. Moreover， fisetin inhibited the enhancement of γ-H2AX formation in HBV replication cells. SOD2 silencing attenuated the inhibitory role of fisetin in γ-H2AX formation in HBV replication cells. Furthermore， under the oxidative stress condition， fisetin significantly attenuated the effect of HBV replication on cell viability. SOD2 silencing decreased the effect of fisetin on the viability of HBV replication cells. In addition， fisetin significantly antagonized the promotion effect of HBV expression to the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38). SOD2 silencing attenuated the inhibitory role of fisetin in the phosphorylation of JNK and p38. These results indicated that fisetin could attenuated the oxidative stress response induced by HBV replication possibly through enhancing the expression of SOD2. Moreover， fisetin could decrease the oxidative damage possibly by inhibiting the activation of JNK and p38， finally play a protection role in HBV replication cell.

CSTR: 32200.14.cjcb.2017.03.0006