红景天苷抑制低氧诱导的心肌细胞凋亡机理的实验研究

The Experimental Study on Mechanism of Salidraside Inhibiting Cardiomyocyte Apoptosis Induced By Hypoxia

目的观察低氧状态下心肌细胞线粒体通透性转换孔(mPTP)的开放功能变化,探讨mPTP在红景天苷保护低氧诱导的心肌细胞凋亡中的作用。方法原代培养心肌细胞,分为对照组、低氧组、30μmol/L红景天苷+低氧组、60μmol/L红景天苷+低氧组、120μmol/L红景天苷+低氧组及25μmol/L环孢素A(CsA)+低氧组。采用流式细胞术检测心肌细胞早期凋亡,NAD+检测试剂盒检测细胞内NAD+浓度,Western blot检测抗凋亡蛋白Bcl-2的表达及胞浆和线粒体细胞色素C(Cyto C)的表达。结果低氧导致心肌细胞凋亡显著增加,细胞内NAD+浓度显著下降;红景天苷预处理可剂量依赖性地显著抑制低氧诱导的心肌细胞凋亡,升高细胞内NAD+浓度;mPTP开放抑制剂CsA能显著抑制低氧诱导的心肌细胞凋亡及NAD+浓度的下降。Western blot结果表明,红景天苷及CsA预处理能明显抑制低氧诱导的心肌细胞Bcl-2表达下调及胞浆Cyto C表达水平的升高,并可提高线粒体Cyto C蛋白表达水平。结论红景天苷可通过抑制心肌细胞mPTP的开放在低氧细胞模型中发挥抗凋亡作用。

Objective To investigate the effects of hypoxia on the opening function of mPTP and explore its role in salidroside induced anti-apoptosis effects in hypoxia-treated cardiomyocyte.Methods The cultured primary rat cardiomyocytes were divided into 6 groups： control,hypoxia,30 μmol/L salidroside ＋ hypoxia,60 μmol/L salidroside ＋ hypoxia,120 μmol/L salidroside ＋ hypoxia and 25 μmol/L CsA ＋ hypoxia group.Early cell apoptosis were detected with flow cytometry.NAD＋ concentration,the opening marker of mPTP,was measured with NAD＋ assay kit.The protein expression of Bcl-2 and Cyto C in cytoplasma and mitochondrial were detected with Western blot.Results Hypoxia increased the apoptotic rate in cardiomyocytes and decreased the concentration of NAD＋ in cytoplasma.Pretreatment with salidroside inhibited hypoxia-induced apoptosis significantly and increased NAD＋ concentration in dose-dependent manner.CsA,the inhibitor of mPTP opening,also inhibited the apoptosis and decrease of NAD＋ concentration induced by hypoxia significantly.Western blot results showed that salidroside and CsA obviously inhibited the decrease of Bcl-2 expression and increase of Cyto C expression in cytoplasma,and reversed the decrease of Cyto C expression in mitochondrial induced by hypoxia.Conclusion Salidroside protects cardiomyocyte apoptosis induced by hypoxia through inhibiting the opening function of mPTP.