Effects Of Metallo Thionein-2A On The Activity Expression Of MAPKs In Cardiomyocytes Under Hypoxia/Reoxygenation

ObjectiveIn this study,we focus on the activity expression changes of activate mitogen-activated protein kinases（MAPKs）and Caspase-3 under the hypoxia（H）,hypoxia/reoxygenation（H/R）processing in H9c2 cardiomyocytes,and a preliminary discussion on the control process role of Metallothionein2a（MT2A）in MAPKs signals.Methods1.The application of chemical hypoxia model revulsant-CoCl₂ processed H9c2 cells to establish H,H/R model,and observed the cell morphology with inverted microscope and PI staining.2.H9c2 cells were transfected by MT2A overexpression of adenovirus（Ad-MT2A-eGFP）and pure adenovirus（Ad-eGFP）in different virus drops degree and transfection time respectively,then detection virus transfection efficiency with fluorescent microscope.Adenovirus transfection experiment were divided into control group without transfection cells（Control）,Ad-MT2A-eGFP group and AdeGFP group,respectively under the normal oxygen,H,H/R processing（9 groups totally）.3.In order to further verify the MT2A role,before processing H9c2 cells in oxygen,H,H/R,we add exogenous Zn7-MT2A protein.The experiment was divided into control group（C）and the MT2A pretreatment group（T）,respectively under the normal oxygen,H,H/R processing（6 groups totally）.4.Under the above processing,Western blot tested the activity expression changes of MAPKs and Caspase-3 to clear myocardial ischemia/reperfusion injury in the effect on myocardial apoptosis and MAPKs pathway;Processing cells through endogenous expression MT2A or MT2A exogenous join,then Western blot detected MAPKs activity expression level change to explore the control process role of MT2A in p38/JNK/ERK signalsResults1.H Group,the apoptosis and cell vitality of H9c2 cells significantly increased than the control group,furthermore,the significant differences between H/R and H group.H group compared with control group,Caspase-3 activity of protein fragment expression increased（p<0.05）,H/R group compared with group H,Caspase-3 activity expressed higher（p<0.01）,indicating that CoCl₂ induced H,H/R model have been built successfully,and confirming that H and H/R processing can result in apoptosis of cardiomyocytes.2.Both Ad-MT2A-eGFP virus and Ad-eGFP virus were closely related to the drop degree and transfection time.The transfection efficiency can reach peak（94.4%,95.1%respectively）during the MOI of 1000 and the transfection time of 48h.3.CoCl₂ induced H,H/R could raise the activity expression of MAPKs in H9c2 myocardial cells.H group compared with control group,the expression of p-JNK,p-p38 increased（p<0.05）,while p-ERK expression has no obvious change.Furthermore H/R group compared with H group,all p-p38,p-ERK,p-JNK expressed higher（p<0.01）.Above results showed that compared with H group,H/R group can further increase activity expression of MAPKs.4.The endogenous overexpression of MT2A could inhibit the activity expression of JNK and p38 in H9c2 cardiomyocytes induced by H,H/R.Western blot results showed that control cells compared with Ad-eGFP cells,the expression of p-JNK、p-p38 were no statistical difference;compared with Ad-eGFP cells,Ad-MT2A-eGFP cells induced by H,H/R,expressed p-JNK,p-p38,which were significantly decreased（p<0.05）.5.Exogenous join MT2A could also inhibited MAPKs activity expression in H9c2 cardiomyocytes induced by H,H/R.MT2A treatment group compared with control group,under H,H/R processing,both p-JNK and p-p38 expression were significantly suppressed（p<0.01）.p-ERK in H processing,no difference with the control,while under the H/R processing,there were differences with the control（p<0.05）.ConclusionsMT2A could inhibit the activity of MAPKs under hypoxia and hypoxia/reoxygenation conditions,and played an important role in resistance to oxidative stress injure in myocardial cells.