Antiapoptotic Effects And Mechanisms Of Exenatide In The H9c2Cardiomyocytes With Oxidative Injury Induced By H₂O₂

Part I Effects of Exenatide in H9c2cardiomyocytes withoxidative injuryObjective: To detect the effects of GLP-1analogue （Exenatide，EX）pretreatment in H9c2cells after oxidative injury by H₂O₂.Methods: Cultured H9c2cells were divided into normal control group（NC group）, H₂O₂group （H₂O₂group） and four EX pretreatment groups（0.01,0.1,1,10nmol·L-1）. The expression of Glucagon-like peptide-1Receptor （GLP-1R） was observed under Laser Scanning ConfocalMicroscope. H9c2cells were pretreating with different doses of EX, thentreated with H₂O₂30min till cell viability （determined by MTT）, lactatedehydrogenase （LDH）, superoxide Dismutase （SOD）, maleic dialdehyde（MDA） and creatine kinase-MB （CK-MB）activity were determined.Results: GLP-1R was observed expressed on H9c2cell membrane.In H₂O₂group, cell viability reduced by50percent and SOD significantly reduced（6.68±0.25vs13.69±0.17），while LDH（196.48±2.84vs104.62±4.45）, MDA（293.06±37.79vs114.42±10.26） and CK-MB activity （13.88±1.79vs1.34±0.34）increased compared to NC group （P<0.05）. When the concentration of EX>0.1nmol·L-1,SOD activity increa sed, LDH, CK-MB and MDA decreased significantly comparing with H2O2group （P<0.05）.Conclusion: Exenatide showed effectively protective effects on H9c2cardiomyocytes from oxidative injury. Part Ⅱ Exenatide through PI3K/AKT signalingpathway reduced apoptosis in H9c2cardiomyocytes withoxidative injury induced by H₂O₂Objective: To investigate the anti-apoptotic effect of Exenatide onp-AKT（ser473） in H9c2cells post-oxidative injury.Methods: Cultured H9c2cells were divided into normal controlgroup （NC group）, H₂O₂group （H₂O₂group）,Exenatide pretreatmentgroup（EX,10nmol·L-1） and EX+LY294002pretreatment group（EX10nmol·L-1,LY15umol·L-1）. After200uM H₂O₂treating for6hours,the flowcytometry was performed to examine the apoptosis rates,and the expressions of p-AKT（ser473）,caspase-3in H9c2cells were examined byWestern-blot.The datas were analysed by SPSS17.0software.Results:（1）FCM: Comparing with NC group, the cardiomyocytesapoptosis were increased significantly in H₂O₂group（45.45±12.30vs11.43±5.69）（P<0.05）; To compare with H₂O₂group, the apoptosis rateswere significantly decreased in EX group（25.64±2.22vs45.45±12.30） （P<0.05）. However,the protective effect of Exenatide was attenuated inthe presence with LY294002,a inhibitor of PI3K（32.84±5.17vs25.64±2.22）（P>0.05）.（2） Western-blot（p-AKT（ser473））:As compared with NC group,the expressions of p-AKT（ser473）in protein level by Western-blot were significantly decreased in H₂O₂groups（0.35±0.021vs0.39±0.018）（P<0.05）; comparing with H₂O₂group, the expressions of p-AKT（ser473）protein were significantly increased in EX group（0.89±0.025vs0.35±0.021）（P<0.05）. The PI3k inhibitor LY29002decreasd the expression ofp-Akt（Ser473）（0.73±0.01vs0.89±0.025）（P<0.05）.（3） Western-blot（caspase-3）: As compared with NC group, the expressions of caspase-3in protein level by Western-blot were significantlyincreased in H₂O₂groups（0.66±0.033vs0.39±0.027）（P<0.05）; comparing with H₂O₂group, the expressions of caspase-3protein were significantly decreasd in EX group（0.35±0.03vs0.66±0.033）（P<0.05）. ThePI3k inhibitor LY29002increased the expression of caspase-3（0.62±0.025vs0.35±0.03）（P<0.05）.Conclusion: The data demonstrated that Exenatide protects against theoxidative stress-induced apoptosis through PI3-kinase/Akt signalingpathway.