Urocortin后处理抑制线粒体凋亡通路保护心肌缺血再灌注损伤

Urocortin-postconditioning protects heart from ischemia-reperfusion injury through interfering mitochondria-dependent apoptotic pathway

目的:研究Urocortin(UCN)后处理对缺血大鼠心肌线粒体凋亡通路的影响,探讨其心肌保护的可能机制。方法:24只Wistar大鼠随机分为3组,每组8只,分为对照组(CON),缺血再灌注组(I/R),Urocortin(UCN)组。CON组大鼠麻醉后直接开胸取左心室肌作缺血前对照。I/R组,建立离体心脏Langendorff-Neely模型,灌注K-H缓冲液30min后,主动脉根部灌注4℃St.Thomas-2心脏停搏液,低温下心脏停跳30min,复灌K-H缓冲液90min后,取左心室肌备检。UCN组,低温下心脏停跳30min后,复灌含Urocortin浓度为10-8mol/L的K-H缓冲液90min,取左心室肌备检。蛋白免疫印迹(Western blot)法检测各组心肌细胞线粒体凋亡通路的标志性蛋白CytoC从线粒体的释放,免疫组化法检测各组心肌细胞Caspase-3蛋白的表达。原位末端转移酶标记法(TUNEL)检测心肌细胞凋亡。结果:Western blot检测显示CON组,I/R组,UCN组胞浆中CytoC量分别为0.56±0.05,1.29±0.02,0.88±0.07,I/R组,UCN组高于CON组(P<0.05),UCN组低于I/R组(P<0.05);免疫组化显示CON组,I/R组,UCN组Caspase-3蛋白表达量分别为0.15±0.22,0.36±0.15,0.24±0.20,I/R组,UCN组高于CON组(P<0.05),UCN组低于I/R组(P<0.05);各组心肌细胞凋亡指数分别为2.29±0.67,12.81±0.62,6.77±1.12,I/R组,UCN组高于CON组(P<0.05),UCN组低于I/R组(P<0.05)。Caspase-3蛋白表达与胞浆中CytoC含量呈正相关(r=0.775,P<0.05);心肌组织中心肌细胞凋亡指数与胞浆中CytoC含量呈正相关(r=0.865,P<0.05)。结论:Urocortin后处理能够抑制细胞凋亡,其机制可能与抑制线粒体释放CytoC和随后的Caspase-3的激活,抑制线粒体通路的细胞凋亡有关。

Objective：To investigate the effects of Urocortin-postconditioning on mitochondria-dependentapoptotic pathway.Methods： 24 Wistar rats were randomly divided into 3 groups（n=8）：Control group（CON）,I/R group（I/R）,Urocortin group（UCN）.The left ventricular samples in the Control group were collected as pre-ischemia controls through thoracotomy.After isolated heart Langendoff and Neely models were established,the rat hearts in the I/R group and the UCN group were continuously perfused with Krebs-Henseleit （K-H）buffersolution for 30 min,then the rat hearts were arrested by cardioplegia,St.Thomas-2 solution（STH-2）for 30min.After that,the rat hearts in the I/R group were reperfused with K-H buffer solution for 90 min.The rat hearts in the UCN group were reperfused with K-H buffer solution containing 10-8 mol/L Urocortin for 90 min.The releasing of Cyto C protein into cytosol was detected by Western blot.The expression of Caspase-3 protein in cardiomyocyte was detected by immunohistochemical assay.The apoptosis index （AI）of cardiomyocyte was detected by TUNEL.Results：Compared with Control group,the expression ofCaspase-3 and Cyto Cprotein in the I/R group and the UCN group were significantly increased（P0.05）.Caspase-3 and Cyto C protein in the UCN group were lowly expressed than those in the I/R group（0.24±0.70 vs 0.36±0.15,P0.05,0.88±0.07 vs 1.29±0.02,P0.05）.After reperfusion, apoptosis index（AI）in the I/R group and UCN group were significantly higher than that in the Control group（P0.05）;Compared with I/R group group,AI in UCN group was decreased increased（6.77±1.12 vs 12.81±0.62,P0.05）.The expression of Caspase-3 protein had a positive correlation with Cyto C protein in cytosol（r=0.775,P0.05）,and the apoptosis index（AI）of cardiomyocyte had a positive correlation with Cyto Cprotein in cytosol（r=0.865,P0.05）。Conclusion：Urocortin protect hearts from ischemic-reperfusion injury through interfering mitochondria-dependent apoptotic pathway.