PI3K-Akt-eNOS信号通路在三磷酸腺苷后处理减轻兔心肌缺血再灌注损伤中的作用

ATP Post Conditioning of PI3K-Akt-eNOS Signaling Pathway Reducing the Myocardial Ischemia Reperfusion Injury in Experimental Rabbits

目的:探讨磷酯酰肌醇-3激酶/蛋白激酶B/内皮型一氧化氮合酶(PI3K-Akt-eNOS)信号通路在三磷酸腺苷(ATP)后处理减轻兔心肌缺血再灌注损伤中的作用。方法:建立兔心肌缺血再灌注模型,随机将动物分为4组:即缺血再灌注组(对照组)、ATP后处理组、磷酯酰肌醇-3激酶抑制剂(Wortmannin)+ATP后处理组(Wortmannin+ATP组)、线粒体ATP依赖性钾通道抑制剂5-羟葵酸(5-HD)+ATP后处理组(5-HD+ATP组)。再灌注结束后,苏木素伊红染色法观察心肌病理组织变化、TUNEL法检测心肌细胞凋亡指数,免疫组化方法检测心肌Bcl-2、Bax蛋白表达、Bcl-2/Bax比值及磷酸化eNOS(p-eNOS)蛋白表达。结果 :ATP后处理组心肌细胞核变小及细胞间质水肿程度较对照组明显减轻,Wortmannin+ATP组和5-HD+ATP组心肌细胞核大小及细胞间质水肿程度与对照组相似;ATP后处理组与对照组比较,心肌细胞凋亡指数明显减低,抗凋亡因子Bcl-2表达及Bcl-2/Bax比值升高,差异均有统计学意义(P<0.01);Wortmannin+ATP组和5-HD+ATP组心肌细胞凋亡指数、Bcl-2/Bax比值与对照组比较差异无统计学意义(P>0.05);p-eNOS蛋白阳性表达ATP后处理组较对照组和Wortmannin+ATP组显著增高,差异有统计学意义(P<0.01),与5-HD+ATP组比较差异无统计学意义(P>0.05)。结论 :ATP后处理可能通过激活PI3K-Akt-eNOS信号通路最终作用于线粒体ATP依赖性钾通道,减少细胞凋亡,减轻兔缺血再灌注损伤。

Objective： To explore the effect of adenosine-5＇-triphosphate （ATP） post conditioning of PI3-kinase-Akt-endothelial nitric oxide synthase （PI3K-Akt-eNOS） signaling pathway reducing the myocardial ischemia reperfusion injury （IRI） in experimental rabbits. Methods： The IRI model was established in New Zealand white male rabbits and the animals were divided into 4 groups. （1） Control group, the rabbits with ischemia reperfusion （IR）, （2） ATP group, IR rabbits received ATP post conditioning, （3） Wortmannin＋ATP group, IR rabbits were treated with PI3-kinase inhibitor wortmannin and ATP post conditioning, （4） 5-HD＋ATP group, IR rabbits were treated with mitochondria ATP -dependent K＋ channel （mitoKATP ） pathway inhibitor 5-HD and ATP post conditioning. The myocardial pathological changes were observed by HE staining, the myocardial cell apoptosis was determined by TUNEL method, the protein expressions of Bcl-2, Bax, the ratio of Bcl-2/ Bax and p-eNOS were examined by immuno-histochemistry method. Results： Compared with Control group, the ATP group showed less smaller of myocardial cell nuclear and tissue edema, reduced apoptosis index and increased ratio of Bcl-2/Bax, all P〈0.01. In both Wortmannin＋ATP group and 5-HD＋ATP group, the size of myocardial cell nuclear and the condition of tissue edema were similar to Control group, the myocardial cell apoptosis index and the ratio of Bcl-2/Bax were similar to Control group, P〉0.05. Compared with Control group and Wortmannin＋ATP group, the p-eNOS protein positive expression was significantly increased in ATP group, P〈0.01 and it was similar to 5-HD＋ATP group, P〉0.05. Conclusion： ATP post conditioning may activate PI3K-Akt-eNOS signaling pathway, work on mitoKATP pathway, to reduce cell apoptosis and IRI in experimental rabbits.