Hsp70 Participates In OGD/R Injury By Regulating P38MAPK Signaling-mediated Calcium Overload And Apoptosis Of Cardiomyocytes

Part one:p38MAPK signal participates in OGD/R injury bymediating calcium overload and apoptosis of cardiomyocytesObjective To investigate the function of p38MAPK signaling pathway in the OGD/R induced calcium overload and apoptosis of cardiomyocytes.Methods The neonatal rat primary cardiomyocytes were randomly divided into 3 groups:control group(group C),oxygen-glucose deprivation/reoxygenation group(group OGD/R),SB203580 pretreatment group(group OGD/R+SB).The cells were treated with oxygen-glucose deprivation for 6h,followed by reoxygenation for 2h in group OGD/R;group OGD/R+SB were pre-treated with SB203580(10?mol/L)for lh before OGD.After reoxygenation,the cell morphology was observed under an inverted microscope.Cell viability,the release rate of lactate dehydrogenase(LDH)in the supernatant,intracellular calcium concentration,apoptosis rate of cardiomyocytes,the relative expression of p-p38MAPK,p-STAT3,SERCA2,Cleaved-caspase 3 protein,the IL-6 and IL-1? mRNA expression levels were detected.Results When compared with group C,the release rate of LDH,intracellular calcium concentration,the ratio of apoptosis and the relative expression of p-p38MAPK,Cleaved-caspase 3 protein,IL-6 and IL-1? mRNA were obviously increased(P<0.01),the cell viability and the relative expression of p-STAT3,SERCA2 proteins was markedly decreased in group OGD/R(P<0.01),the cytoplasm retraction,cell membrane refraction rate decreased,cell necrosis,and cell quantity decreased were observed.When compared with the OGD/R group,the release rate of LDH,intracellular calcium concentration,the ratio of apoptosis and the p-p38MAPK,Cleaved-caspase 3 proteins,the IL-6 and IL-1?mRNA was significantly reduced(P<0.05),the cell viability and the relative expression of p-STAT3,SERCA2 proteins was markedly increased in the group OGD/R+SB(P<0.05).The condition of cell necrosis,cytoplasm shrinkage and the decline of cell membrane refractive index caused by OGD/R injury can be partly reversed by SB203580.Conclusion Activation of p38MAPK induces calcium overload and apoptosis of cardiomyocytes after OGD/R injury,which is related to the down-regulation of SERCA2 and p-STAT3 signaling.Part two:Hsp70 participates in OGD/R injury by regulatingp38MAPK signaling-mediated calcium overload and apoptosis of cardiomyocytesObjective To further investigate whether Hsp70 participates in the OGD/R induced calcium overload and apoptosis of cardiomyocytes by regulating the p38MAPK signaling pathway,we reduced the expressionof Hsp70 by gene silencing.Methods The neonatal rat primary cardiomyocytes were randomly divided into 4 groups:negative control-shRNA group(group NC),NC-shRNA+oxygen-glucose deprivation/reoxygenation group(group NC+OGD/R),Hsp70-shRNA group(Hsp70shRNA+OGD/R),Hsp70shRNA and precondition group(group Hsp70shRNA+ODG/R+SB).After reoxygenation,cell morphology was observed under an inverted microscope.Cell-viability,the release rate of LDH in the supernatant,the intracellular calcium concentration,the apoptosis rate of cells,the relative expression of Hsp70,p-p38MAPK,p-STAT3,SERCA2,Cleaved-caspase 3 proteins were detected.Results When compared with group NC+OGD/R,the LDH release,intracellular calcium concentration,the ratio of apoptosis and the relative expression of p-p38MAPK,Cleaved-caspase 3 protein was obviously increased(P<0.05),the cell viability and the Hsp70,p-STAT3 and SERCA2 proteins were markedly decreased in group Hsp70shRNA+OGD/R(P<0.05),the cytoplasm retraction,cell membrane refraction rate decreased,cell necrosis,and quantity decreased were observed.When compared with the Hsp70shRNA+OGD/R group,calcium overload,cell apoptosis and the activation of p-38MAPK can be partly reversed in group Hsp70shRNA+ODG/R+SB(P<0.05);cell viability and the expression of p-STAT3 and SERCA2 proteins were raised in group Hsp70shRNA+ODG/R+SB(P<0.01).Conclusion Hsp70 participates in OGD/R injury by regulating p38MAPK signaling mediated calcium overload and apoptosis of cardiomyocytes.