The Effect Of 17β-Estradiol On Oxidative Stress-induced Apoptosis And The Regulatory Mechanism In Myocardiac H9c2 Cells

Backgound:Ischemic heart disease with subsequent myocardial infarction and congestive heart failure is the leading cause of mortality.The incidence of ischemic heart disease is significantly lower in women than in men until menopause,after which the cardiovascular risk of women accelerates to equal that of men.This observation suggests a possibility that female sex hormones,such as estrogen,may have a favorable cardiovascular role.There is evidence from epidemiological,animal,and in vitro studies that estrogen may be cardioprotective.However,the mechanisms involved in this process,are poorly understood.It has been show that estradiol has important role on development, growth and differentiation of organism.It can affect proliferation and apoptosis of cells by modulating several signaling pathway,including transforming growth factor-β(TGF-β)signaling.There are several studies on the interaction between E₂ and TGF-βsignaling in tumor cells,but few in myocardial cells.Oxidative stress is the common pathophysiologic mechanism of myocardial hypertrophy, apoptosis and remodeling caused by miscellaneous heart diseases.It is indicated that estradiol has somewhat anti-oxidative effect.So we observed the effect of estradiol on oxidative stress and apoptosis of myocardial cell and on modulating TGF-βsignaling pathway,in order to explore the mechanism of myocardial cell apoptosis modulated by estradiol and its biological significance.Objective:To explore the mechanism of apoptosis modulated by estradiol in myocardial cells and its biological significant by observing the effect of estradiol on oxidative stress and apoptosis of myocardial cells induced by H₂O₂ and its modulate effect on TGF-βsignaling pathway. Methods:Use H9c2 cells,a clonal line derived from embryonic rat heart,as the cell model.1)To observe the effect on cell viability,apoptosis and generation of reactive oxygen speciesk(ROS)in H9c2 cells stimulated by H₂O₂ and the protective effect of estradiol on myocardial cells by means of MTT assay and flow cytometry.Expression of ERαmRNA and ERβmRNA「in H9C2 cells was detected by RT-PCR.2)Real-time RT-PCR and Western blot were performed to examine The expression of TGF-β1,TβRâ… and TβRâ…¡in H9c2 cells treated with 200riM E2 for different hours.The level of phosphorylation of Smad2/3 was detected by western blot.Results:1)MTT assay shows that exogenous H₂O₂ induces decreasing of cell viability of H9C2 cells in a dose-dependent manner(P＜0.05),and 100μM H₂O₂ treating for 4 hours was chosen as inducing condition for the following experiments.Various concentrations of E₂ alone has no effect on the cell viability of H9c2 cells(P＞0.05).Exogenous H₂O₂ increasing the apoptosis rate and ROS generation.While H9c2 cells pretreated with 200nM E₂ for 24 hours significantly attenuate the cell viability injury caused by H₂O₂.Time-phase analysis shows that the maximal protective effect achieved at 16 hours,and last for 24 hours.E₂ inhibited the cell viability injury caused by H₂O₂ in a dose dependent manner.So we choose 200nM E₂ treating for 24 hours as the intervention treatment in the follow experiment.2)Exogenous H₂O₂ may induce increased apoptosis and ROS generation of H9c2 cells,there is a significant difference compared with control group.E₂ pretreatment can significantly extenuate apoptosis and ROS generation of H9C2 cells induced by H₂O₂(P＜0.05).3)Determination of estradiol receptor expression of H9C2 cells by RT-PCR finds that H9C2 cells only express ERβ,almost express no ERα,and suggests that anti-apoptosis effect of estradiol on H9C2 cells may be mediated by ERβ.4)Detection of Realtime RT-PCR and western blot finds that H9C2 cells pretreated by estradiol for 8 hours may downregulate the expression of TβRâ… and TβRâ…¡mRNA,and downregulate of protein expression occurred at 16 hours.But it has no effect on the expression of TGF-β1.ER blocker ICI182,780 can block the binding of E₂ and ER,and can delete the downregulation effect of E₂ on TβRâ… and TβRâ…¡.5)Western blot exanmination shows that the p-smad2/3 level in H9c2 cells decreased pretreated by estradiol for 16 hours H9c2 cells.Conclusion:1)Oxidative stress caused by exogenous H₂O₂ can increase the ROS generation,damage the cell viability and induce apoptosis of H9c2 cells. Exogenous E2 alone has no effect on cell viability and apoptosis of H9C2 cells, but it can decrease the sensitivity of H9C2 cells to H₂O₂,hence,protect cells against apoptosis caused by oxidative stress.2)Exogenous E₂ has downregulation effect on TβRâ… and TβRâ…¡of H9c2 cells,blocking ER may delete this effect of estradiol。H9c2 cells express ERβonly,suggests that E₂ may exert modulating effect on TGF-βpathway through ERβ。...