PERK通路在吗啡保护心肌H9c2细胞过程中的作用

目的:探讨吗啡(morphine)是否通过PERK通路降低内质网应激,阻止线粒体膜通透性转换孔(mPTP)开放,从而保护氧化应激损伤的心肌细胞。方法:体外培养大鼠心肌H9c2细胞,用H₂O₂建立氧化应激模型,随机分为对照组、H₂O₂组、H₂O₂+morphine组、H₂O₂+morphine+PERK通路抑制剂GSK2656157组、morphine组和GSK2656157组。免疫组化法检测吗啡对氧化应激引起葡萄糖调节蛋白(GRP)78和GRP94表达的影响;Western blot法检测PERK信号通路相关蛋白的水平;利用共聚焦显微镜观察吗啡对氧化应激所致mPTP开放及内质网的影响;乳酸脱氢酶(LDH)和MTT试剂盒分别检测细胞毒性和细胞活力。结果:与对照组相比,H₂O₂组GRP78和GRP94蛋白为强阳性表达,棕黄色颗粒明显增加,吗啡明显抑制此过程。与对照组相比,不...

Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM:To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore(mPTP)opening.METHODS:Rat myocardial H9 c2 cells were cultured to establish an oxidative stress model,and then randomly divided into control group,H2O2 group,H2O2+morphine group,H2O2+morphine+PERK pathway inhibitor GSK2656157 group,morphine group and GSK2656157 group.Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein(GRP)78 and GRP94 induced by oxidative stress.The protein levels of PERK signaling pathway-related molecules were determined by Western blot.Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress.Cellular toxicity was detected by lactate dehydrogenase(LDH)kit and cell viability was measured by MTT assay.RESULTS:Compared with control group,GRP78 and GRP94 proteins in H2O2 group were strongly expressed,and the brown-yellow particles were significantly increased,but morphine significantly inhibited this process.Compared with control group,the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations,among which 2μmol/L had the most significant effect(P<0.05).Oxidative stress significantly increased the protein levels of GRP78,GRP94,p-PERK and CHOP,but significantly decreased p-GSK-3βlevel.These changes were inhibited by morphine,and the effects of morphine were further enhanced by GSK2656157(P<0.05).Compared with control group,oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red.Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced,mPTP was open,and endoplasmic reticulum was damaged,while GSK2656157 further enhanced the effect of morphine(P<0.05).Compared with control group,H2O2 significantly increased cellular toxicity and decreased the cell viability.Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine(P<0.05).CONCLUSION:Morphine protects cardiac H9 c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3βinactivation.