缺血后处理和CGS-21680后处理对兔心肌缺血再灌注后细胞凋亡的影响

Effect of Ischemic Postconditioning and CGS-21680 Postconditioning on Cardiomyocyte Apoptosis in Rabbit Hearts Following Ischemia-Reperfusion

目的:观察缺血后处理和选择性腺苷A2a受体激动剂CGS-21680后处理对兔缺血再灌注损伤心肌的影响,并探讨其作用机制。方法:健康新西兰大耳白兔36只,随机分成三组:缺血再灌注组(I/R组)、缺血后处理组(IPO组)、CGS-21680后处理组,每组12只。建立兔心肌I/R模型,于再灌注末颈静脉取血测定血清肌酸激酶同工酶(CK-MB);采用伊文思蓝和NBT染色后计算各组兔心肌梗死范围;原位末端标记(TUNEL)法检测各组兔心肌细胞凋亡;实时荧光定量RT-PCR检测各组心肌组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA和Bcl-2mR-NA的表达。结果:(1)与I/R组比较,IPO组和CGS-21680后处理组CK-MB水平显著下降,心肌梗死范围分别减少34.5%、31.2%。(2)TUNEL法结果:IPO组和CGS-21680组的细胞凋亡指数(AI)分别为9.57±1.34%和11.31±0.97%,与IR组(18.32±1.75%)相比明显降低并有显著差异(P<0.01)。(3)荧光定量RT-PCR结果:与IR组比较,IPO组和CGS-21680组的促凋亡基因Caspase-3mRNA的表达明显下调,抗凋亡基因Bcl-2mRNA的表达显著上调,P均<0.05。结论:缺血后处理和CGS-21680后处理可减少心肌梗死范围,减少细胞凋亡,保护I/R损伤心肌,其机制可能与下调促凋亡基因Caspase-3和上调抗凋亡基因Bcl-2的表达有关。

Objective:To explore the effects of ischemic postconditioning and adenosine A2a receptor agonist CGS-21680 pharmacological postconditioning on myocardial ischemia reperfusion(IR)injury in rabbits and its mechanism.Method:Thirty-six New Zealand white male rabbits were randomly divided into three groups(n=12 each):IR group,ischemic postconditioning(IPO)group and CGS-21680 postconditioning group.The model of myocardial ischemia-reperfusion was established.Serum biochemical markers creatine kinases MB(CK-MB)of three groups were evaluated at the end of 3h reperfusion.Ischemic and infarct areas were measured by Evans blue and NBT staining.The terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)method was used to detect apoptotic changes.The expression of caspase-3 and Bcl-2 gene in myocardial tissue was detected by real-time quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Results:(1)Compared with IR group,the level of CK-MB reduced markedly in IPO group and CGS-21680 group,myocardial infarct size decreased 34.5%,31.2% respectively.(2)TUNEL method showed that apoptosis indexes in IPO group and CGS-21680 group were 9.57±1.34% and 11.31±0.97% respectively,and compared with IR group(18.32±1.75%)they were significantly decreased(P<0.01).(3)The results of real-time RT-PCR showed that the expression of caspase-3 mRNA was down-regulated,while Bcl-2 mRNA was up-regulated between IPO group and CGS-21680 group(P<0.05).Conclusion:Ischemic postconditioning and CGS-21680 postconditioning have a protective effect on myocardial ischemia reperfusion injury by significantly up-regulating the expression of Bcl-2 mRNA and down-regulating the expression of caspase-3 mRNA,inhibiting myocardial apoptosis.