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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 open function of mitochondrial permeability transition pore (mPTP) in hypoxia and to explore the role of mPTP in the protection of hypoxia-induced cardiomyocyte apoptosis by salidroside. Methods Primary cultured cardiomyocytes were divided into control group, hypoxia group, 30μmol / L salidroside plus hypoxia group, 60μmol / L salidroside plus hypoxia group, 120μmol / L salidroside plus hypoxia Group and 25μmol / L cyclosporine A (CsA) + hypoxia group. The apoptosis of cardiomyocytes was detected by flow cytometry. The concentration of NAD + in cells was detected by NAD + kit. The expression of Bcl-2 protein and Cyto C in cytoplasm and mitochondria were detected by Western blot. Results Hypoxia induced a significant increase of cardiomyocyte apoptosis and a significant decrease of intracellular NAD + concentration. Salidroside pretreatment significantly inhibited hypoxia-induced cardiomyocyte apoptosis and increased intracellular NAD + concentration in a dose-dependent manner; mPTP opening inhibition CsA can significantly inhibit hypoxia-induced cardiomyocyte apoptosis and decreased NAD + concentration. Western blot results showed that salidroside and CsA pretreatment significantly inhibited hypoxia-induced Bcl-2 expression and cytoplasmic Cyto C expression, and increased mitochondrial Cyto C protein expression. Conclusion Salidroside can play an anti-apoptotic role in hypoxic cell model by inhibiting the opening of mPTP in cardiomyocytes.