毛蕊花糖苷对缺氧/复氧诱导心肌细胞损伤的影响

Effects of Verbascoside on Hypoxia/Reoxygenation Induced Cardiomyocyte Injury

目的观察毛蕊花糖苷对缺氧/复氧(H/R)诱导的心肌细胞损伤的影响及可能机制。方法采用H/R诱导大鼠心肌细胞H9C2建立心肌缺血再灌注损伤模型,采用不同剂量毛蕊花糖苷处理细胞;利用脂质体转染技术将微小RNA-455-5p(miR-455-5p)寡核苷酸模拟物(miR-455-5p mimics)及阴性对照mimics NC序列(miR-NC)分别转染至心肌细胞后构建模型;miR-455-5p特异性寡核苷酸抑制剂(anti-miR-455-5p)及其阴性对照序列(anti-miR-NC)分别转染至心肌细胞后使用80μg/L毛蕊花糖苷处理细胞,之后构建模型。采用试剂盒检测丙二醛(MDA)水平与超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性;流式细胞术检测细胞凋亡率;逆转录定量聚合酶链式反应(RT-qPCR)检测miR-455-5p表达;免疫印迹法(Western Blot)检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-Caspase 3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved-Caspase 9)蛋白表达。结果毛蕊花糖苷可降低H/R诱导的心肌细胞MDA水平、凋亡率和Cleaved-Caspase 3、Cleaved-Caspase 9蛋白水平(P<0.05),升高miR-455-5p表达和SOD、GSH-Px活性(P<0.05),且呈剂量依赖性;转染miR-455-5p mimics后H/R诱导的心肌细胞MDA水平、凋亡率和Cleaved-Caspase 3、Cleaved-Caspase 9蛋白水平降低(P<0.05),SOD、GSH-Px活性升高(P<0.05);转染anti-miR-455-5p可逆转毛蕊花糖苷对H/R诱导的心肌细胞氧化应激及凋亡的抑制作用。结论毛蕊花糖苷通过上调miR-455-5p表达,抑制心肌细胞氧化应激及细胞凋亡,从而减轻H/R诱导的心肌细胞损伤。

Objective To explore the effect of verbascoside on cardiomyocyte injury induced by hypoxia/reoxygenation(H/R)and its possible mechanism.Methods The myocardial ischemia-reperfusion injury model was established by H/R-induced rat cardiomyocytes H9C2,and the cardiomyocytes were treated with different doses of verbascoside.The microRNA-455-5p(miR-455-5p)oligonucleotide mimics(miR-455-5p mimics)and the negative control mimic NC sequence(miR-NC)were transfected into cardiomyocytes by lipofection technology to construct models.The miR-455-5p-specific oligonucleotide inhibitor(anti-miR-455-5p)and its negative control sequence(anti-miR-NC)were transfected into cardiomyocytes,and the cardiomyocytes were treated with 80μg/L verbascoside,then the model was constructed.The level of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by kits;apoptosis rate was detected by flow cytometry.The expression of miR-455-5p was detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR)method.The protein expression levels of activated cysteine-containing aspartate proteolytic enzyme 3(Cleaved-Caspase 3)and activated cysteine-containing aspartate proteolytic enzyme 9(Cleaved-Caspase 9)were detected by Western Blot.Results Verbascoside could decrease the level of MDA,apoptosis rate,the protein levels of Cleaved-Caspase 3 and Cleaved-Caspase 9 in H/R-induced cardiomyocytes(P<0.05),and increase the expression of miR-455-5p and the activities of SOD and GSH-Px(P<0.05).and it was a dose-dependent manner.After transfection of miR-455-5p mimics,the level of MDA in H/R-induced cardiomyocytes was decreased(P<0.05),the apoptosis rate and the protein levels of Cleaved-Caspase 3,and Cleaved-Caspase 9 were decreased(P<0.05),while the activities of SOD and GSH-Px were increased(P<0.05).Transfection of anti-miR-455-5p could reverse the inhibitory effect of verbascoside on H/R-induced oxidative stress and apoptosis of cardiomyocytes.Conclusion Verbascoside could inhibit the oxidative stress and apoptosis in cardiomyocytes by up-regulating the expression of miR-455-5p,thereby reducing H/R-induced cardiomyocyte injury.