The Effect And Mechanism Of Hepatitis B Virus X Protein On The Expression Of High Mobility Group Protein B1 And Generation Of Reactive Oxygen Species

The infection of Hepatitis B virus(HBV)is one of major reasons causing many kinds of liver diseases.Of the four proteins encoded by HBV,hepatitis B virus X protein(HBX)is encoded by the shortest open reading frame in HBV-DNA,and which plays an important role in the process of HBV infection.High mobility group box-1 protein(HMGB1)and reactive oxygen species-ROSs(Ros)play an important role in the prevention of exogenous biological infection and autologous cell carcinogenesis.It is of great clinical and social significance to study the molecular mechanism of HBx promoting hepatocyte regulation of HMGB1 and ROS and to better understand the pathogenesis of hepatitis B virus and improve the prognosis of hepatitis B patients.ObjectiveHBx protein plays an important role in the process of HBV infection.This study focuses on the regulatory effects of HBx overexpression on HMGB1 and reactive oxygen species(ROS)in human normal hepatocytes(LO2)and the role of NF-?B pathway in this process.MethodsWe first constructed swp-n4flag-HBx plasmid,identified it by double enzyme digestion and PCR,transfected 293T cells,and packaged to produce retrovirus.Then we use lentiviral vector to overexperss the HBx protein in LO2 cells(a normal hepatocyte cell line).Detected the expression of HBx protein by Western-blot to confirm the HBx-overexpressed cells were constructed successfully.Stimulated cells with ROS activator(rotenone)and NF-?B activator(LPS).Finally,detected the HMGB1 protein and NF-?B signaling protein by Western blot,detected the generation of ROS by DCFH-DA(ROS detector)staining.ResultsChapter I:(1)Amplicating HBx Gene by PCR:We got a 500bp band(Consistent with the theoretical prediction value of 486bp);(2)Enzyme digestion identification:We got a 500bp band(Consistent with the theoretical prediction value of 486bp);(3)Sequencing:the results were completely consistent with the HBx sequence reported by Genebank,indicating that the construction of swp-n4flag-HBx plasmid was successful;(4)Lentivirus detection:T-band color development by the colloidal gold test paper,and the virus titer of swp-HBx-flag was 3×10~7Tu/mL by the flow cytometer detection;(5)Western-blot:The results show that,the expression of protein in HBx-over group was significantly higher than that in mock group(P<0.05).Chapter II:(1)The expression of HMGB1 protein in HBx-over group was significantly lower than that in mock group(P<0.05);(2)Comparison between HBx-over group and mock group,the lower fluorescence intensity indicates that the amount of intracellular ROS is low.(3)Comparison between HBx-over group and mock group,under the same expression of NF-?B and I?B protein(P<0.05),the phosphorylation level was decreased,indicating that the activation of NF-?B signaling pathway was decreased(P<0.05).There was no significant difference in the expression of NF-?B and I?B protein(P>0.05),but the expression of p-NF-?B and p-I?B protein decreased significantly(P<0.05).Chapter III:(1)The fluorescence intensity of both HBx-over group and mock group increased with the increase of ROS concentration when the cells were treated with ROS stimulator,which indicated that the stimulator had played an important role,Western-blot results showed that the expression of HMGB1 was increased,and the phosphorylation levels of p-NF-?B and I?Ba were increased.(2)When LPS was used as a stimulant of NF-?B,the phosphorylation level of p-NF-?B and I?B was increased,and the expression of HMGB1 was up-regulated in both HBx overexpression cells and control cells.The above two results show that there is a mutual regulation between NF-?B and HMGB1.Conclusion1.In this study,HBx gene was cloned successfully,and LO2 cells were transduced by HBx lentivirus.The cells were screened by multiple purine mycin and detected by Western blot.It is proved that we have successfully constructed HBx overexpression cell lines(HBx-over)and reference cell lines(mock).2.The expression of HMGB1 and the generation of ROS in LO2 cell lines with overexpression of HBx were significantly lower than those in the control group.These results suggest that HBx inhibits the expression of HMGB1 and the generation of ROS.3.Overexpression of HBx inhibits the expression of HMGB1 and the generation of ROS via NF-?B signaling pathway.