HBx Sensitizes HL-7702 Cells To Oxidative Stress-induced Apoptosis Via Enhancing The Opening Of MPTP

Background and Aim: HBV X protein?HBx?,a viral protein encoded by HBV,is essential for HBV replication and it acts as a key factor in the development of inflammatory injury and hepatocarcinoma.However,the mechanism and details of HBx in HBV-induced liver cell injury remain unclear.A number of studies demonstrated that the specific function of HBx is linked to its interaction with mitochondrion.Our previous study had reported the finding of a new HBx-interacting protein,cytochrome c oxidase subunit III?COX??,and demonstrated that the co-locolization of HBx and COX? is crucial for mitochondrial dysfunction.In this research,we make a further investigation of the mechanism of HBx-induced mitochondrial damage and pay attention to the effect of HBx in modulating mitochondrial permeability transition pore?MPTP?,therefore offer a new sight of the mechanism in HBx-induced apoptosis in HL-7702 cells under the condition of oxidative stress.Methods: We constructed a new pcDNA3.1-mic-his plasmids which expressed HBx-flag protein,and established another two recombinant plasmids: p GEM-HBV,which express a greater-than-genome-length c DNA of wild-type HBV?ayw?and p GEM-HBV-?HBx,which is identical to HBx-deficient mutant HBV.Cultured HL-7702 cells were transiently transfected with the four recombinant plasmids to build the four research groups: 1?HL-7702/pc DNA3.1;2?HL-7702/HBV;3?HL-7702/HBV-?HBx;4?HL-7702/HBx.Antibodies of flag fusion protein and HBc Ag were detected by Western Blot for the representative of the expression of HBx-flag and p HBV/p HBV-?HBx.Activities of cytochrome c oxidase,level of cell ATP and mitochondrial membrane potential were investigated in transfected cells.Then,the four groups of transfected cells treated with or without Cs A,a specific inhibitor of MPTP,were loaded with calcium sensitive dye Fluo-4 to monitor the occurrence cytosolic calcium levels.Next,transfected cells with or without H₂O₂ treatment were incubated with DCFH-DA and fluorescence was measured by confocal laser scanning microscopy to measure intracellular ROS levels.Furthermore,transfected cells?HL7702/pc DNA3.1 and HL7702/HBx,?were exposed to H₂O₂ with or without treatment of Cs A,and the distribution of Bax and were examined by Western blot and immunofluorescence.Meanwhile,the expression of cleaved caspase3 and PARP were measured by Western Blot.Finally,the levels of apoptosis of these cells were evaluated by flow cytometric analysis.Results: Our studies demonstrated that HBx reduced COX activity to decrease cell ATP levels and modulate mitochondrial membrane potential,which contributed to the opening of MPTP and increased the level of cytosolic calcium in HL-7702 cells.While using Cs A,a MPTP specific inhibitor,the concentration of cytosolic calcium of HL-7702/HBx cells is reduced to a level similar to HL-7702/pc DNA3.1 cells.Similarly,HL-7702/HBV cells also exhibited a reduction of fluorescence intensity with Cs A to the extent of HL-7702/HBV-?HBx cells.Interestingly,there wasn' an apparent change in HL-7702/HBx cells compared with the control group without treatment of H₂O₂,while a significant increase of fluorescence intensity were observed in HL-7702/HBV cells compared with those in HL-7702/HBV-?HBx cells.However,although an increased ROS levels were observed in all the four groups when exposed to H₂O₂,much more DCFH-DA-positive cells were found in H₂O₂-exposed HL-7702/HBx cells and HL-7702/-HBV cells than control groups under the same condition.In addition,when exposed to H₂O₂ treatment,HL-7702/HBx cells has shown a visible co-localization of Bax and mitochondria than the control group,and this translocation of Bax was blocked by treatment of Cs A.In addition,Western Blot analysis revealed that the whole protein levels of Bax didn't show an apparent change in each group.But,the expression of Bax protein exhibited a significant decrease in cytosol and increase in mitochondrion in HL-7702/HBx cells treated with H₂O₂.Treatment with Cs A blocked the imbalance of Bax in cytosol and mitochondrion.Moreover,the expression of cleaved-caspase3 and cleaved-PARP were dramatically increased as a consequence of the release of cytosolic cytochrome c in H₂O₂-exposed HL-7702/HBx cells.Finally,a significant increase of apoptosis levels were found in H₂O₂-exposed HL-7702/HBx cells by flow cytometric analysis.Conclusions: Our study in HL-7702 cells reported a possible approach for HBx to induce mitochondrion dysfunction in normal liver cells.Meanwhile,HBx has shown to enhanced the susceptibility of normal hepatocytes to oxidative stress-induced apoptosis and this effect is associated with the ability of HBx in modulating MPTP,which is considered as the major cause of Bax translocation from mitochondrion to cytosolic.