DIDS对十字孢碱诱导心肌细胞凋亡中磷脂酰肌醇3激酶/蛋白激酶B信号转导的影响

The Effect of DIDS on Phosphatidylinositol 3'-Kinase/Proteinase B Signal Transduction of Staurosporine-Induced Cardiomyocyte Apoptosis

目的探讨氯离子通道阻断剂DIDS对十字孢碱诱导心肌细胞凋亡与磷脂酰肌醇3激酶/蛋白激酶B信号及其下游分子一氧化氮合酶/一氧化氮的关系。方法实验分为对照组、十字孢碱组、DIDS组、LY294002(特异性磷脂酰肌醇3激酶抑制剂)组和L-NAME(非特异性一氧化氮合酶抑制剂)组。在十字孢碱诱导心肌细胞凋亡模型上,观察DIDS对心肌细胞存活率、凋亡和磷脂酰肌醇3激酶/蛋白激酶B及其下游分子一氧化氮合酶/一氧化氮的影响。结果与十字孢碱组比,DIDS明显改善了细胞存活率,提高了细胞磷酸化蛋白激酶B活性2.1倍(P<0.01),增加了一氧化氮合酶和磷酸化一氧化氮合酶的水平和一氧化氮水平(P<0.01);LY294002预处理完全抑制了磷酸化蛋白激酶B、一氧化氮合酶和磷酸化一氧化氮合酶水平的升高及升高的一氧化氮,完全阻断了DIDS的抗细胞凋亡作用;L-NAME预处理也使升高的一氧化氮水平下降,但仅部分阻断了DIDS的细胞保护作用。结论DIDS通过激活磷脂酰肌醇3激酶/蛋白激酶B信号通路发挥其抑制十字孢碱诱导的心肌细胞凋亡作用。

Aim To explore the effect of chloride channel blocker DIDS on cell signaling pathway phosphatidylinositol 3＇-kinase/proteinase B （PI3K/Akt） and its downstream molecules endothelial nitric oxide synthase （eNOS） and nitric oxide （NO） in staurosporine （STS）-treated cardiomyocyte apoptosis. Methods Neonatal rat cardiomyocytes were exposed to STS in the presence or absence of DIDS. Cell viability, apoptosis and expressions of Akt, phospho-Akt （ p-Akt）, eNOS, phospho-eNOS （p-eNOS） and NO production were determined. Results DIDS markedly improved cell viability on STS-exposed eardiomyoeytes. DIDS resulted in a 2.1-fold increase of p-Akt over control levels, prevented the reduction in eNOS expression and phospho-eNOS levels induced by STS and significantly increased NO production （ all P 〈0.01}. Pretreatment with LY294002, a selective PI3K inhibitor, abolished DIDS-indueed increases in p-Akt, eNOS, p-eNOS and NO production, and completely abrogated the DIDS-induced anti-apoptotie effect （P 〈 0.01 ）. Pretreatment with L-NAME, a non-selective NOS inhibitor similarly inhibited the increased NO but only partly abolished protective effects of DIDS （ P 〈 0.05 ）. Conclusion DIDS inhibits STS-induced eardiomyocyte apoptosis via activating PI3K/Akt signaling pathway.