再灌注损伤救援激酶信号转导通路在后处理抗再灌注心肌凋亡中的作用

Reperfusion injury salvage kinase pathway mediates the antiapoptotic effect of postconditioning following myocardial ischemia/reperfusion

目的:目前对后处理保护机制的研究还较孤立,缺乏上下游调节机制的联系。通过建立在体大鼠心肌缺血再灌注模型,探讨后处理在整体实验下的抗凋亡效应,以及这种心肌保护作用是否通过再灌注损伤救援激酶(reperfusion injury salvage kinase,RISK)信号转导通路来实现。方法:实验于2006-07/2007-05在首都医科大学病理生理学教研室完成。健康成年雄性SD大鼠32只,均分至以下4组:①心肌缺血再灌注组:结扎冠状动脉左前降支(缺血)45min,再灌注4h。②后处理组:在再灌注前,给予10s再灌、10s缺血,连续3个循环。③PI3K抑制剂组:在再灌注前5min经颈静脉注射磷脂酰激醇3激酶(PI3K)抑制剂LY-294002(0.3mg/kg),余同②组。④MAPKK抑制剂组:在再灌注前5min经颈静脉注射丝裂原激活蛋白激酶激酶(MAPKK)抑制剂PD98059(0.3mg/kg),余同②组。再灌注结束后取左室缺血区,使用Western blotting法检测各组心肌组织中凋亡指标Bax,Bcl-2,Caspase-3表达量变化及RISK通路中Akt,P-ERK1/2及eNOS表达量变化。结果:32只大鼠均进入结果分析。与心肌缺血再灌注组相比,后处理组心肌凋亡显著减轻,Akt、P-ERK1/2及eNOS含量显著升高;PI3K抑制剂组心肌凋亡较后处理组严重,Akt、eNOS含量降低;MAPKK抑制剂组心肌凋亡较后处理组严重,ERK1/2含量降低。结论:后处理对再灌注心肌存在明显的抗凋亡作用,该作用可能与RISK信号转导通路的激活有关。

AIM: Studies on protective mechanisms of postconditioning are simple, and lack of association between upstream and downstream regulatory mechanism. This study was designed to elucidate the effects of postconditioning on ischemia/reperfusion cardiac apoptosis in vivo and the role of reperfusion injury salvage kinase pathway in the pro/anti-apoptotic effect of postconditioning. METHODS: The experiment was performed at the Department of Pathophysiology of Capital Medical University from July 2006 to May 2007. Thirty-two healthy adult male SD rats were assigned randomly into 4 groups. ①Rats of the ischemia/reperfusion group received left anterior descending coronary artery deligation for 45 minutes and reperfusion for 4 hours. ②Rats of the postconditioning group received reperfusion for 10 s and ischemia for 10 s for successively 3 cycles before reperfusion. ③Rats of the phosphatidylinositol 3-kinase inhibitor group were infused with phosphatidylinositol 3-kinase inhibitor LY-294002 (0.3 mg/kg) via cervical vein 5 minutes before reperfusion, and other interventions were the same as that of the postconditioning group. ④Rats of the mitogen-activated protein kinase kinase inhibitor group were infused with mitogen-activated protein kinase kinase inhibitor PD 98059 (0.3 mg/kg) via cervical vein 5 minutes before reperfusion, and other interventions were the same as that of the postconditioning group. At the end of reperfusion, the ventricular tissue at risk was excised, and the levels of Bax, Bcl-2, Caspase-3, Akt, P-ERK 1/2 and eNOS were analyzed by Western blotting. RESULTS: Thirty-two rats were involved in the result analysis. Compared with the ischemia/reperfusion group, postconditioning group exerted a significant anti-apoptotic effect, and the levels of Akt, P-ERK 1/2 and eNOS were significantly increased in the postconditioning group. Myocardial apoptosis was more severe in the phosphatidylinositol 3-kinase inhibitor group than the postconditioning group, and the levels of Akt and eNOS were decreased. Myocardial apoptosis was more severe in the mitogen-activated protein kinase kinase inhibitor group than the postconditioning group, and the level of ERK 1/2 was reduced. CONCLUSION: These results implicate the involvement of reperfusion injury salvage kinase pathway in the reduction of ischemia/reperfusion cardiac apoptosis of postconditioning.