Urocortin对缺血大鼠心肌线粒体凋亡通路的影响

欧袁; 杨双强; 辛东

Urocortin对缺血大鼠心肌线粒体凋亡通路的影响

Effects of urocortin on mitochondria-dependent apoptotic pathway in ischemia rats

摘要

摘要: 目的: 研究Urocortin（UCN）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: 将24只Wistar大鼠随机分为3组，每组8只，即对照组(CON)、缺血/再灌注（I/R）组及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30 min后，于主动脉根部灌注4℃的St.Thomas-2心脏停搏液, 低温下使心脏停跳30 min，复灌K-H缓冲液90 min后，取左心室肌备检。UCN组：低温下使心脏停跳30 min后，复灌含UCN的、浓度为1×10-8 mol/L的K-H缓冲液90 min，取左心室肌备检。用蛋白免疫印迹（Western blot）法检测各组心肌细胞线粒体凋亡通路的标志性蛋白细胞色素C(CytoC)从线粒体的释放。用免疫组化染色法检测各组心肌细胞中Bcl-2和Caspase-3蛋白的表达。用原位末端转移酶标记法( TUNEL)检测心肌细胞的凋亡。结果: Western blot检测显示，CON组、I/R组和UCN组胞浆中CytoC的量分别为(0.56±0.05)、(1.29±0.02)和(0.88±0.07)，I/R组和UCN组胞浆中CytoC的含量明显高于CON组（P<0.05）；UCN组低于I/R组（P<0.05）。免疫组化染色结果显示，CON组、I/R组和UCN组Bcl-2蛋白的表达量，分别为(0.077±0.05)、(0.12±0.06)、(0.2±0.05),I/R组和UCN组高于CON组（P<0.05），UCN组高于I/R组（P<0.05）。CON组、I/R组和UCN组Caspase-3蛋白的表达量，分别为(0.15±0.22)、(0.36±0.15)和(0.24±0.20)。I/R组和UCN组高于CON组（P<0.05），UCN组低于I/R组（P<0.05）。各组心肌细胞的凋亡指数，分别为(2.29±0.67)、(12.81±0.62)和(6.77±1.12)，I/R组和UCN组高于CON组（P<0.05），UCN组低于I/R组（P<0.05）。结论: UCN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Caspase-3的激活，上调Bcl-2蛋白的表达，抑制线粒体通路的细胞凋亡有关。

Abstract: AIM: To investigate the effects of urocortin (UCN) on mitochondria-dependent apoptotic pathway. METHODS: Twenty four Wistar rats were randomly divided into three groups (n=8): control group (CON), ischemia/reperfusion (I/R) group and UCN group. The left ventricular samples in CON group were collected as pre-ischemia control through thoracotomy. After isolated heart Langendoff and Neely models were established, rat hearts in I/R group and UCN group were continuously perfused with Krebs-Henseleit (K-H) buffer solution for 30 min and then the rat hearts were arrested by cardioplegia, St.Thomas-2 solution (STH-2) for 30 min. Rat hearts in I/R group were reperfused with K-H buffer solution for 90 min and rat hearts in UCN group were reperfused with K-H buffer solution containing 10-8 mol/L urocortin for 90 min. Release of cytochrome C (Cyto C) protein into the cytosol was detected by Western blot. The expressions of Bcl-2 and caspase-3 protein were detected by immunohistochemical assay in cardiomyocyte, and the apoptosis index (AI) of cardiomyocyte was detected by TUNEL. RESULTS: Compared with that in CON group, expression of Bcl-2, caspase-3 and Cyto c protein in I/R and UCN groups significantly increased (P<0.05). Bcl-2 protein expression in UCN group was higher than in I/R group (0.2±0.05 vs.0.12±0.06, P<0.05). Expression of caspase-3 and Cyto C protein in UCN group was lower than in I/R group (0.24±0.70 vs.0.36±0.15, P<0.05, 0.88±0.07 vs.1.29±0.02, P<0.05). After reperfusion, AI in I/R group and UCN group was significantly higher than in CON group (P<0.05). Compared with I/R group, AI in UCN group increased (6.77±1.12 vs.12.81±0.62, P<0.05). CONCLUSION: UCN protects hearts against I/R injury through interfering with mitochondria-dependent apoptotic pathway.

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