白藜芦醇对H9c2心肌细胞抗氧化应激损伤的内质网应激机制研究

Cardioprotective effect of resveratrol on H_2O_2-induced oxidative injury via inhibiting endoplasmic reticulum stress in H9c2 cardiac cells

目的:研究白藜芦醇(resveratrol)对H9c2心肌细胞抗氧化应激损伤的"内质网-线粒体对话"保护作用并探讨其可能的机制。方法:培养H9c2心肌细胞建立氧化应激模型,随机分为4组:正常组(对照组)、模型组(H2O2组)、白藜芦醇+H2O2组、白藜芦醇组。Western blot测定H2O2对内质网应激(ERS)特征性分子伴侣,即葡萄糖调节蛋白78(GRP 78)、葡萄糖调节蛋白94(GRP 94)的表达;观察白藜芦醇对氧化应激所致GRP 78、GRP 94、糖原合成酶激酶-3β(GSK-3β)磷酸化的影响;共聚焦显微镜观察白藜芦醇对氧化应激所致线粒体通透性转移孔(m PTP)开放的影响;电镜观察白藜芦醇对氧化应激所致心肌细胞超微结构的影响。结果:与正常组相比,H2O2(600μmol·L-1)引起H9c2心肌细胞氧化损伤并使GRP 78,GRP 94表达明显增加;白藜芦醇(10μmol·L-1)明显抑制氧化应激所致GRP 78,GRP 94的表达及GSK-3β磷酸化水平。与正常组相比,模型组TMRE荧光强度明显减少,白藜芦醇明显抑制氧化应激所致TMRE荧光强度的减少(即线粒体膜电位减少、m PTP开放)。白藜芦醇明显减少氧化应激所致心肌细胞线粒体、内质网超微结构的损伤。结论:白藜芦醇对氧化应激所致H9c2心肌细胞损伤具有保护作用,该作用可能与抑制ERS使GSK-3β失活,进而阻止m PTP开放有关。

Objective： To determine the cardioprotective effect of resveratrol on H2O2-induced oxidative injury in H9c2 cardiac cells and the underlying mechanism. Methods： The model of oxidative stress injury was induced by H2O2 in H9c2 cardiac ceils. Cells were exposed to H2O2 in the presence or absence of resveratrol. The expression of the endoplasmic reticulum stress molecular makers GRP 78 and GRP 94 after exposure to H2O2 was detected by Western blotting. Then, the effect of resveratrol on the expression of GRP 78 and GRP 94 and the phosphorylation of glycogen synthase kinase-3β （GSK-3β） was determined. Third, rnitochondrial permeability transition pore （mPTP） was determined by laser scanning confocal microscopy. Lastly, the ultrastructural changes were observed by electron microscopy. Results： Compared to normal cells, H2O2 （600μmol·L-1） enhanced the expression of GRP 78 and GRP 94, and significantly decreased TMRE fluorescence （ mPTP reduction）. Resveratrol inhibited the enhanced expression of GRP 78 and GRP 94 as well as GSK-3β phosphorylation, and prevented the loss of mitochondria membrane potential （MMP） induced by H2O2. These results showed that resveratrol significantly reduced oxidative stress-induced mitochondrial and endoplasmic reticulum ultrastructure injury. Conclusion： Resveratrol has the cardioprotective effect on oxidative injury induced by H2O2 in H9c2 cardiac cells. Its mecha- nism may be attributed to its inhibition of endoplasmic reticulum stress, and inactivation of GSK-3β and mPTP.