白藜芦醇对缺氧心肌细胞的保护作用及机制研究

Protective effects of resveratrol on hypoxia myocardial cells and the mechanism research

目的:探讨白藜芦醇(resveratrol)是否可通过抑制内质网应激机制保护缺氧心肌细胞。方法:(1)H9c2心肌细胞随机分为对照组和缺氧组(1~8 h),Western blot法检测内质网应激(ERS)特征性分子伴侣葡萄糖调节蛋白78(GRP78)、葡萄糖调节蛋白94(GRP94)的表达以确定缺氧诱导ERS的时间。(2)培养H9c2心肌细胞建立缺氧模型,随机分为对照组、缺氧组、白藜芦醇+缺氧组、白藜芦醇组。Western blot检测缺氧对GRP 78,GRP 94,糖原合成酶激酶-3β(GSK-3β)的表达,观察白藜芦醇对缺氧所致GRP 78,GRP 94,GSK-3β磷酸化的影响;共聚焦显微镜观察白藜芦醇对缺氧所致线粒体通透性转移孔(mPTP)开放的影响;电镜观察白藜芦醇对缺氧所致心肌细胞超微结构的影响。结果:与对照组相比,缺氧3 h导致H9c2细胞GRP 78和GRP 94表达增加最多;白藜芦醇明显抑制缺氧所致GRP 78和GRP 94的表达及GSK-3β磷酸化水平。与对照组相比,缺氧组TMRE荧光强度明显减少,白藜芦醇明显抑制缺氧所致TMRE荧光强度的减少(即线粒体膜电位减少、mPTP开放)。与对照组相比,白藜芦醇明显减轻缺氧所致心肌细胞线粒体、内质网等超微结构的损伤。结论:白藜芦醇对缺氧所致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mPTP开放有关。

Objective ： To determine the cardioprotective effect of resveratrol on hypoxia myocardial cells by inhibiting the mechanism of endoplasmic retieulum stress （ERS）. Methods： H9c2 myocardial cells were randomly divided into control and hypoxia 1 - 8 h groups. The expressions of the ERS molecular markers GRP78 and GRP94 in H9c2 cardiomyocytes were measured by Western blotting to determine the time it took for hypoxia to induce ERS. Myocardial cells were exposed to hypoxia in the presence or absence of resveratrol and randomly divided into four groups： control group, hypoxia group, resveratrol plus hypoxia group, resveratrol group. The expressions of GRP78 and GRP94 and glycogen synthase kinase-3β （GSK-3β） after exposure to hypoxia were detected by Western blotting. Then, the effects of resveratrol on the expressions of GRP78, GRP94 and GSK-3β were determined. Mitoehondrial permeability transition pore （mPTP） was determined by laser scanning eonfocal microscopy. Lastly, the uhrastructural changes were observed by electron microscopy. Results： Compared with the control cells, 3 h of hypoxia treatment enhanced the expressions of GRP78 and GRP94 in the largest magnitude. Resveratrol significantly inhibited the enhanced expression of GRP78 and GRP94 as well as GSK-313 phosphorylation. Compared with the control cells, hypoxia significantly decreased TMRE fluorescence, indicating the mPTP reduction. Resveratrol prevented the loss of mitochondria membrane potential （MMP） induced by hypoxia. Compared with the control cells, resveratrol significantly reduced hypoxia-induced mitochondrial and endoplasmic reticulum ultrastructure injury. Conclusion： Resveratrol has cardioprotective effect on hypoxia injury in H9c2 cardiac cells. Its mechanism may be attributed to its inhibition of endoplasmic reticulum stress and inactivation of GSK-3β and mPTP.