The Effect Of P66^Shc On Sodium Arsenite-mediated Oxidative Stress In H9C2 Cells

With the rapid development of China’s economy,the incidence of cardiovascular disease is high and on an upward trend.Cardiovascular disease with high morbidity,mortality,disability and high relapse rates,has become the second killer threatening human health,only next to the malignant tumor.Cardiovascular disease’s forming and developing are because of all sorts of dangerous factors.A long-term intake of arsenic induces heart disease,hypertension,atherosclerosis and other cardiovascular diseases.Arsenic cause myocardial damage by oxidative stress,and the adaptor protein p66Shc plays an important role in the oxidative stress in cells.However,the function of p66Shc in sodium arsenite induced oxidative stress has not been reported.In this study,we demonstrated that p66Shc mediated H9c2 rat myocardial cells oxidative stress induced by sodium arsenite.Cell viability was decreased by sodium arsenite treatment which was a dose-dependent manner.Detection of apoptosis in H9c2 cells by Hoechst33258 dyes,we found treatment with the sodium arsenite resulted in dose-dependent increase apoptosis rate.And the ROS level was increased after sodium arsenite treatment.These results indicated that oxidative damage might be the mechanism of cell apoptosis induced by sodium arsenite treatment.The adaptor protein p66sh,played a very important role in oxidative stress response.The results of western blotting showed that after treatment the ratio of phosphorylated-p66Shc and p66Shc was significantly increased,as well as the total of p66Shc.We also studied the subcellular localization of p66Shc after arsenic treatment in H9c2 cells by confocal imaging.Cells were labeled by anti-p66Shc monoclonal antibodies,and costained with the Mito Tracker.This experiment showed that the adaptor protein p66Shc was translocated into mitochondria and nuclei after arsenic treatment.These results showed that p66Shc might be functional in arsenic-mediated oxidative stress in H9c2 Cells.To further clarify the function of p66Shc in arsenic-mediated oxidative stress in H9c2 Cells,we carried out knock down and overexpresson experiments.Western blot results showed that compared to control siRNA,p66Shc siRNA could decrease the expression of p66Shc in the presence of arsenic.Meanwhile,knockdown of p66Shc decreased intracellular ROS levels after arsenic treatment.To overexpress p66Shc we constructed eukaryotic expression vectors of wild type and mutant p66Shc.Overexpression of wild type p66Shc increased intracellular ROS levels in sodium arsenite-exposed H9c2 cells.However,overexpression of mutant p66Shc decreased it.Taken together,our results suggested that p66Shc was involved in the activation of ROS responses induced by sodium arsenite in H9c2 cells.p66Shc could phosphorylate transcription factor FOX03a through AKT dependent and independent pathways.Phosphorylation of FOXO3a inhibited its activity,thus inhibited the expression of antioxidant enzyme genes,then increased ROS levels.So we detected the phosphorylation of FOX03a andAKT after arsenic treatment.We found the ratio of phospho-AKT and AKT was increased.Phospho-FOXO3a and FOXO3a was the same.These results indicated that oxidative stress induced by arsenic treatment might be through p66Shc phosphorylating AKT and FOX03a.In conclusion,sodium arsenite treatment increased p66Shc expression and its phosphorylation.Then p66Shc translocated from cytoplasm to mitochondria,increased intracellular ROS level.Meanwhile,oxidative stress induced by arsenic treatment might be through p66shc phosphorylating AKT and FOXO3a.