白藜芦醇通过MCU抑制mPTP开放减轻H9c2细胞氧化应激损伤

Resveratrol alleviates oxidative stress injury by inhibiting the mPTP opening through MCU in H9c2 cells

目的观察白藜芦醇(resveratrol)保护心肌细胞减轻氧化应激损伤的过程是否与线粒体通透性转换孔(mPTP)和线粒体钙离子单向转运蛋白(MCU)相关,并探讨可能的机制。方法H9c2心肌细胞常规培养,随机分为对照组、过氧化氢组、白藜芦醇+过氧化氢组、MCU抑制剂(钌红)+白藜芦醇+过氧化氢组。微板法检测细胞外液中乳酸脱氢酶(LDH)的含量;Western印迹法检测内质网应激分子伴侣葡萄糖调节蛋白(GRP)78、GRP94和MCU蛋白表达;激光扫描共聚焦显微镜观察细胞内钙离子(Ca^(2+))、活性氧(ROS)和mPTP变化。结果与对照组相比,过氧化氢显著增加细胞外液中LDH的含量、GRP78、GRP94和MCU蛋白表达、细胞内Ca^(2+)和ROS含量,显著减少mPTP特异性探针TMRE的荧光强度(均P<0.05)。白藜芦醇明显抑制过氧化氢引起的变化,而钌红显著增强白藜芦醇的作用(均P<0.05)。结论白藜芦醇通过MCU减少细胞内Ca^(2+)超载和ROS生成进而抑制mPTP开放,减轻氧化应激引起的内质网应激损伤,保护心肌细胞。

Objective To investigate whether the cardioprotective effect of resveratrol against oxidative stress was related to mitochondrial permeability transition pore(mPTP) and mitochondrial calcium uniporter(MCU) and explore the possible mechanism.Methods H9c2 cardiomyocytes were routinely cultured,and were randomly divided into control group,hydrogen peroxide group,resveratrol+hydrogen peroxide group,and MCU inhibitor(ruthenium red)+ resveratrol + hydrogen peroxide group.The content of lactic dehydrogenase(LDH) in extracellular fluid was detected by microplate method;the expressions of endoplasmic reticulum stress chaperone glucose regulatory protein(GRP)78,GRP94 and MCU protein were detected by Western blot method;the changes of intracellular Ca~(2+),reactive oxygen species(ROS) and mPTP were observed by laser scanning confocal microscope.Results Compared with the control group,hydrogen peroxide significantly increased the content of LDH in extracellular fluid,the expressions of GRP78,GRP94,MCU and the contents of intracellular Ca^(2+) and ROS,and significantly decreased the mPTP specific fluorescence intensity of TMRE(all P<0.05).Resveratrol significantly inhibited the changes caused by hydrogen peroxide,while ruthenium red significantly enhanced the effect of resveratrol(all P<0.05).Conclusions Resveratrol could reduce intracellular Ca^(2+) overload and ROS generation through MCU,thereby inhibiting the mPTP opening,reducing endoplasmic reticulum stress injury caused by oxidative stress and playing a cardioprotective role.