MT2A Improves H9c2 Cardiomyocyte Damage Induced By Hypoxia/Reoxygenation

Objective:The amount of sick person with coronary artery illness is increasing little by little,and it is among most frequent fit questions health in the world.After developing AMI,early reperfusion using drug lysis and percutaneous interventional therapy is one of the most effective ways to rescue the damaged myocardium in time.However,this treatment method may aggravate myocardial insufficiency and myocardial cellula death,which may lessen the profitable ways of myocardial reperfusion.Myocardial ischemia-reperfusion injury are mainly relevant to the massive genaration of ROS,the promoting of autophagy and cell apoptosis.Metallothionein 2A(MT2A)can restrain the massive yield of free oxygen free radicals,and restrain autophagy and cell apoptosis.Therefore,MT2 A may protect cardiomyocytes from ischemia-reperfusion injury.In this study,we mainly analyzed the regulation of MT2 A on oxygen free radicals,autophagy and apoptosis of H9c2 cardiomyocytes under hypoxia/reoxygenation(H/R)conditions.Methods:H9c2 cardiomyocytes(Control),virus-laden H9c2 cardiomyocytes(Vector)and MT2 A gene overexpression lentiviral vector were used to transfect H9c2cardiomyocytes(MT2A).The test was segmented into Control、Vector and MT2 A group.They was treated under normoxia(N),hypoxia(H)and hypoxia/reoxygenation(H/R)conditions,then applying MTT method measures cytes activity,applying inverted fluorescence microscope and WB measure MT2 A,WB was applyed to measure the conveyed plane of relevant apoptotic factors,LC3-II and p62 autophagy factors,and DCFH-DA was applied to detect the conveyed plane of ROS.Results:The MT2 A gene overexpression lentiviral vector was successfully constructed,and then it was transfected into H9c2 cardiomyocytes and a stable transfected cell line was screened so that MT2 A can be overexpressed.And successfully used the H/R model to cultivate H9c2 cardiomyocytes to mimic myocardial ischemia reperfusion,to show the corresponding symptoms of ischemia-reperfusion injury.The consequences of reversed fluorescence microscope and WB demonstrated that the MT2 A gene over-expression lentiviral vector was successfully geted into H9c2 cardiomyocytes.WB test consequences demonstrated that under H/R conditions,contrasted with the Control and the Vector group,the relevant apoptosis factors of the MT2 A group were descended(p<0.01);the conveyed plane of p62 in the MT2 A group was obviously increased(p<0.001),the conveyed plane of LC3-II in the MT2 A group was descended(p<0.05).The consequences of the DCFH-DA test demonstrated that under H/R conditions,contrasted with the Control and the Vector group,the conveyed plane of ROS in the MT2 A group was obviously descended(p<0.001).The consequences of the MTT test demonstrated that under H/R conditions,contrasted with the Control and the Vector group,the cytes viability of the MT2 A group was obviously ascended(p<0.001).Conclusions: The results of this study indicate that under H/R conditions,overexpression of MT2 A can effectively protect H9c2 cardiomyocytes,by eliminating ROS,avoiding oxidative stress caused by the amassing of ROS,and restraining autophagy and apoptosis of H9c2 cardiomyocytes.And through the reduction of relevant apoptotic proteins to indicate the reduction of H9c2 cardiomyocyte programmed death,and the down-regulation of LC3-II and the up-regulation of p62 conveyed plane to indicate the restraint of H9c2 cardiomyocyte autophagy.In this case,it is indicated that MT2 A is a potential tool in the prevention or treatment of ischemic heart disease,but the relationship between these mechanisms in the protection of cardiomyocytes after MT2 A preconditioning is still unclear.In order to expound these relation,more and in-depth study is demanded.