腺苷预适应对再灌注诱导心肌细胞凋亡的影响(英文)

Effects of adenosine preconditioning on cardiomyocyte apoptosis induced by reperfusion

背景:缺血再灌注损伤除导致心肌细胞坏死,还可诱发细胞凋亡,而细胞凋亡可能是梗死早期心肌细胞死亡的主要方式,也可能是心肌梗死面积扩大的原因之一。目的:观察腺苷预适应在缺血再灌注的抗心肌细胞凋亡效应,探讨凋亡相关基因蛋白Bcl-2和Bax在其中的作用。设计:随机对照的动物实验。单位:三峡大学第一临床医学院湖北省宜昌市中心人民医院心内科。材料:选用健康雄性家兔36只,清洁级,体质量2.5￣3.0kg,由华中科技大学同济医学院实验动物学部提供。实验过程中对动物的处置符合动物伦理学标准。按随机数字表法将实验动物分为3组:对照组、腺苷预适应组和腺苷A1受体拮抗剂组,每组12只。方法:实验于2005-10/2006-10在三峡大学中心实验室完成。制备离体家兔心肌缺血再灌注模型。将动物麻醉后,肝素抗凝,行Langendorff逆行灌注。对照组:缺血40min,再灌注60min;灌注结束后将其中6只家兔用于确定心肌梗死范围,另外6只用作心肌细胞凋亡、基因表达和心肌组织超微病理的分析。腺苷预适应组:在缺血前30min给予腺苷10μmol/L持续灌流,其余同对照组。腺苷A1受体拮抗剂组:缺血前45min给予腺苷A1受体阻滞剂10mmol/L灌流15min后,与腺苷预适应组采取相同措施。主要观察指标:①观察缺血再灌注过程中3组动物的心率和血压。②用氯化三苯基四氮唑确定心肌梗死范围。③采用末端标记法和DNA琼脂糖凝胶电泳检测心肌细胞凋亡指数。④原位免疫组化检测凋亡相关蛋白Bcl-2和Bax的表达。结果:纳入的36只家兔全部进入结果分析。①心率和血压比较:在缺血再灌注过程中,心率和血压均持续下降,差异有显著性意义(P<0.01)。组间比较,差异无显著性意义(P>0.05)。②心肌梗死范围比较:对照组、腺苷预适应组和腺苷A1受体拮抗剂组缺血心肌范围比较,差异无显著性意义(P>0.05)。腺苷预适应组梗死范围小于对照组,差异有显著性意义(P<0.01),提示腺苷预适应可缩小家兔心肌梗死范围。腺苷A1受体拮抗剂组家兔心肌梗死范围大于腺苷预适应组,小于对照组,差异均有显著性意义(P<0.01),提示腺苷A1受体阻断剂可部分抑制腺苷的保护作用。③心肌细胞凋亡情况比较:在非缺血心肌组织未见凋亡细胞,梗死组织和梗死边缘缺血组织中可见凋亡细胞。对照组和腺苷A1受体拮抗剂组凋亡细胞多于腺苷预适应组;细胞凋亡指数高于对照组,差异有显著性意义(P<0.01)。④凋亡相关蛋白表达情况比较:腺苷预适应组Bax的吸光度值低于对照组,差异有显著性意义(P<0.01);腺苷A1受体拮抗剂组Bax吸光度值低于对照组,高于腺苷预适应组,差异均有显著性意义(P<0.01)。对照组Bcl-2/Bax比值低于腺苷预适应组,差异有显著性意义(P<0.01)。腺苷A1受体拮抗剂组Bcl-2/Bax比值高于对照组,差异有显著性意义(P<0.01);低于腺苷预适应组,差异有显著性意义(P<0.01)。结论:外源性腺苷可以抑制再灌注诱导的心肌细胞凋亡,且部分由腺苷A1受体介导;凋亡相关蛋白Bax表达下调在外源性腺苷的抗凋亡中扮演重要角色。

BACKGROUND: Ischemia/reperfusion injury can cause the necrosis of cardiomyocyte, and it can also induce cell apoptosis. However, cell apoptosis may be the main death type of cardiomyocyte at the early stage of infarction, and it may be one of causes for expanding myocardial infarction area. OBJECTIVE: The goal of this study was to observe the anti-apoptotic effect of adenosine (ADO) preconditioning on cardiomyocytes during the ischemia/reperfusion, and to investigate the role of apoptosis-related gene protein Bcl-2 and Bax. DESIGN: A randomized controlled animal experiment. SETTING: First College of Clinical Medical Science, China Three Gorges University&Department of Cardiology, Yichang Central People's Hospital. MATERIALS: Thirty-six healthy male rabbits of clean grade, weighing 2.5 to 3.0 kg, were provided by Laboratory Animal Department, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. All rabbits were divided into 3 groups according to random number table. There were 12 animals in either control, ADO or ADO+DPCPX (an adenosine A1 receptor antagonist). METHODS: This experiment was carried out in the Central Laboratory, China Three Gorges University between October 2005 and October 2006. Ex vivo rabbit myocardial ischemia/reperfusion models were prepared. After being anesthetized, the rabbits were performed anticoagulation with heparin and carried out Langendorff retroperfusion. In the control group, the hearts of animals subjected to 40 minutes of ischemia and 60 minutes of reperfusion. Six of them were used for determining myocardial infarct size after reperfusion, another six for cardiomyocyte apoptosis, gene expression and ultrastructural analysis of myocardium. In the ADO group: The ADO hearts were continuously infused with 10 μmol/L of adenosine 30 minutes before ischemia, and operated according to the requirement of control group. In the DPCPX group: the isolated hearts of animals were infused for 15 minutes with 10 mmol/L of DPCPX 45 minutes before ischemia, and operated in accordance with ADO group. MAIN OUTCOME MEASURES: ① The heart rate and blood pressure of animals in 3 groups were measured during ischemia/reperfusion process. ② The infarct size was determined by triphenyltetrazolium chloride(TTC) staining. ③ The apoptotic index of cardiomyocytes was detected by histological TUNEL staining and DNA ladder on agarose gel electrophoresis.④ Apoptosis-related protein Bcl-2 and Bax expressions were detected by in situ immunohistochemical staining. RESULTS: Thirty-six rabbits were enrolled in the final analysis. ① Comparison of heart rate and blood pressure: During the process of ischemia/reperfusion, both heart rate and blood pressure were persistently decreased significantly (P < 0.01). There were no significant differences in two indexes between any two groups (P > 0.05). ②Comparison of myocardial infarct size: There were no significant differences in myocardial infarct size among the control group, ADO group and DPCPX group (P > 0.05). The myocardial infarct size of rabbits in the ADO group was significantly smaller than that in the control group. It suggested that ADO preconditioning could contract the myocardial infarct size of rabbits. The myocardial infarct size of rabbits in the ADO+DPCPX group was significantly larger than that in the ADO group, but significantly smaller than that in the control group (P < 0.01). It suggested that DPCPX could partially inhibit the protective effect of ADO. ③ Comparison of apoptosis of cardiomyocytes: Apoptotic cells were not found in the non-ischemic myocardial tissue, but found in the infarct tissue and infarct edge ischemic tissue. Apoptotic cells in the control group and ADO+DPCPX group were significantly more than those in the ADO group. Apoptotic index in the control group and ADO+DPCPX group was significantly higher than that in the control group, respectively (P < 0.01). ④ Comparison of apoptosis-related protein expression: The absorbance of Bax in the ADO group was significantly lower than that in the control group (P < 0.01). The absorbance of Bax in the ADO+ DPCPX group was significantly lower than that in the control group, but significantly higher than that in the ADO group (P < 0.01). The value of Bcl-2/Bax in the control group was significantly lower than that in the ADO group (P < 0.01). The value of Bcl-2/Bax in the ADO+ DPCPX group was significantly higher than that in the control group, but significantly higher than that in the ADO group (P < 0.01). CONCLUSION: Exogenous ADO inhibits reperfusion-induced apoptosis of cardiomyocytes, which is partially mediated by DPCPX; Down-regulation of apoptosis-related Bax protein plays an important role in the anti-apoptotic effect of exogenous ADO.