尼可地尔通过磷脂酰肌醇-3激酶通路对缺氧/复氧心肌细胞抗凋亡作用的研究

Research of anti-apoptosis effect of nicorandil to hypoxia/re-oxygenation cardiomyocytes through phosphatidylinositol 3-kinase pathway

目的探讨尼可地尔通过磷脂酰肌醇-3激酶(PI3K)通路对大鼠缺氧/复氧(H/R)心肌细胞发挥抗凋亡作用的机制。方法原代培养SD乳鼠的心肌细胞,建立H/R模型,实验随机分为4组,Con组:在正常培养箱中孵育;H/R组:在三气培养箱和正常培养箱先后孵育;Nic组:孵育前加入尼可地尔50μmol/L;Nic+Ly294002组:孵育前加入尼可地尔50μmol/L和Ly29400220μmol/L。采用比色法检测心肌细胞乳酸脱氢酶(LDH)漏出率,CCK-8法检测心肌细胞存活率,流式细胞仪检测心肌细胞凋亡率,蛋白质印迹法(Western blot)检测淋巴细胞瘤-2(Bcl-2)、Bcl-2相关x蛋白(Bax)及磷酸化的蛋白激酶B(p-Akt)表达水平。结果H/R组、Nic组和Nic+Ly294002组心肌细胞凋亡率、LDH漏出率和Bax表达水平均高于Con组;Nic组和Nic+Ly294002组上述指标均低于H/R组;Nic组上述指标均低于Nic+Ly294002组(P<0.05)。H/R组、Nic组和Nic+Ly294002组心肌细胞存活率和Bcl-2表达水平均低于Con组;Nic组和Nic+Ly294002组上述指标均高于H/R组;Nic组上述指标均高于Nic+Ly294002组(P<0.05)。Nic组心肌细胞p-Akt表达水平高于Con组和H/R组,Nic+Ly294002组低于Con组、H/R组和Nic组(P<0.05)。结论尼可地尔可阻止H/R引起的心肌细胞凋亡,其作用的发挥与PI3K信号通路关系密切。

Objective To explore anti-apoptosis mechanism of nicorandil to hypoxia/re-oxygenation(H/R)cardiomyocytes of rats through phosphatidylinositol 3-kinase(PI3K)pathway.Methods Primary cultured cardiomyocytes of SD suckling rat were established H/R model,and they were divided into 4 groups randomly.Con group:incubated in normal incubator.H/R group:incubated in three gas incubator and normal incubator successively.Nic group:added nicorandil 50μmol/L before incubation.Nic+Ly294002:add 50μmol/L nicorandil and Ly29400220μmol/L before incubation.Leakage rate of lactate dehydrogenase(LDH)was detected and evaluated by colorimetry,survival rate of cardiomyocytes was detected and evaluated by CCK-8 method,apoptosis rate of cardiomyocytes was detected by flow cytometry,and the expression levels of apoptotic protein lymphoma-2(Bcl-2),Bcl-2 related x protein(Bax)and phosphorylated protein kinase B(p-Akt)were detected by Western blotting.Results Apoptosis rate of cardiomyocyte,leakage rate of LDH and expression level of Bax in H/R group,Nic group and Nic+Ly294002 group were higher than those in Con group,above indexes in Nic group and Nic+Ly294002 group were lower than those in H/R group,and above indexes in Nic group were lower than those in Nic+Ly294002 group(P<0.05).Survival rate of cardiomyocytes and expression level of Bcl-2 in H/R group,Nic group and Nic+Ly294002 group were lower than those in Con group,above indexes in Nic group and Nic+Ly294002 group were higher than those in H/R group,and above indexes in Nic group were higher than those in Nic+Ly294002 group(P<0.05).Expression level of p-Akt in Nic group was higher than that in Con group and H/R group,above index in Nic+Ly294002 group was lower than that in Con group,H/R group and Nic group(P<0.05).Conclusion Nicorandil can prevent the apoptosis of cardiomyocyte induced by H/R,which is closely related to PI3K signaling pathway.