丹参酮ⅡA抗心肌细胞氧化应激作用及抗增殖蛋白功能研究

Anti-oxidative Effect of TanshinoneⅡA and Prohibitin in Cardiomyocytes

摘要:

目的: 观察丹参酮Ⅱ_A对过氧化氢诱导的心肌细胞凋亡的保护作用及其对抗增殖蛋白(prohibitin,PHB)表达的影响,研究PHB蛋白在心肌细胞氧化应激中的作用。 方法: 实验组分为正常对照组、氧化应激组、丹参酮Ⅱ_A高、中、低剂量组、siRNA组、siRNA+氧化应激组、siRNA阴性对照组。采用差数贴壁法体外分离培养新生乳鼠心肌细胞,以200 μmol ·L^-1过氧化氢作用2 h 模拟心肌细胞氧化应激损伤,心肌细胞在氧化应激前给予丹参酮Ⅱ_A(1×10^-4,5×10^-5,1×10^-5 mol ·L^-1)预先干预24 h。采用PHB特异性的siRNA干扰心肌细胞PHB蛋白表达,流式细胞仪检测细胞凋亡率、 i及心肌细胞线粒体膜电位的变化,Western blotting 检测PHB蛋白表达水平。 结果: 与正常对照组比较,氧化应激组心肌细胞经200 μmol ·L^-1过氧化氢作用2 h后,PHB蛋白表达显著增加,细胞内游离钙浓度、凋亡率显著增高,线粒体膜电位显著降低。与氧化应激组比较,丹参酮Ⅱ_A可呈浓度依赖性显著降低细胞内游离钙浓度及细胞凋亡率,增加线粒体膜电位,降低PHB蛋白表达水平。与siRNA阴性对照组比较,siRNA组心肌细胞内PHB蛋白表达可被显著抑制,且细胞内游离钙浓度和细胞凋亡率显著增加,线粒体膜电位显著降低。 结论: PHB蛋白在心肌细胞氧化应激中可代偿性增加,对心肌细胞具有保护作用。丹参酮Ⅱ_A可减少氧化应激损伤心肌细胞内PHB蛋白表达,其机制可能与丹参酮Ⅱ_A减轻心肌细胞氧化应激损伤从而减少心肌细胞自身代偿性作用有关。

Abstract:

Objective: To study the anti-apoptotic mechanism of tanshinoneⅡ_A and the function of prohibitin (PHB) on myocardial cell apoptosis induced by hydrogen peroxide(H₂O₂). Method: there were six groups of myocardial cells in the study: normal control group, oxidative stress group, tanshinoneⅡ_A group, siRNA group, siRNA+oxidative stress group, siRNA control group. Primary cultured neonate rat myocardial cells were cultured in medium with 200 μmol ·L^-1 hydrogen peroxide, and the medium was supplemented with tanshinoneⅡ_A (1×10^-4,5×10^-5,1×10^-5 mol ·L^-1) in advance for 24 hours. PHB in myocardial cells was knocked down by small interfering RNA (siRNA) interference, and the expression level of PHB was determined by western blotting analysis. Flow cytometric analysis was used to detect apoptosis rate, i and mitochondrial membrane potential(MMP). Result: H₂O₂-mediated cell apoptosis resulted in activation of PHB, increasing of i, and decreasing of mitochondrial membrane potential. Tanshinone IIA profoundly prohibited myocardial cells apoptosis induced by hydrogen peroxide, and decreased i, and increased mitochondrial membrane potential in a dose dependent manner. Specific silence of PHB by siRNA down-regulated the expression level of PHB and increased of apoptosis rate and i, and decreased mitochondrial membrane potential. Conclusion: The results demonstrated that the activation of PHB is compensatory increased in myocardial cells against oxidative stress. tanshinone Ⅱ_A could reduce the expression of PHB by attenuate the injury induced by oxidative stress.