长托宁通过下调miR-217表达抑制缺氧/复氧诱导的大鼠心肌细胞H9C2损伤的机制研究

Penequinine hydrochloride inhibits hypoxia/reoxygenation-induced injury of rat cardiomyocyte H9C2 by down-regulating the expression of miR-217

目的:探讨长托宁(PHC)对缺氧/复氧(H/R)诱导的大鼠心肌细胞H9C2损伤的影响及作用机制。方法:体外建立H/R诱导的H9C2细胞损伤模型。不同浓度(12.5μg/L、25μg/L、50μg/L)的PHC作用于H/R诱导的H9C2细胞后,酶联免疫吸附法检测细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GSH-Px)水平,流式细胞术检测细胞凋亡,蛋白印迹(Western blotting)检测细胞中B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)蛋白表达,实时荧光定量PCR(qPCR)检测细胞中miR-217表达水平。转染miR-217抑制剂至H9C2,下调miR-217表达后,上述相同方法观察下调miR-217表达对H/R诱导的H9C2细胞中MDA含量、SOD和GSH-Px水平、细胞凋亡及Bcl-2和Bax蛋白表达的影响。结果:PHC作用H/R诱导的H9C2细胞后,细胞中MDA含量、细胞凋亡率、Bax蛋白表达及miR-217表达均降低(均P<0.05),而SOD和GSH-Px的活性及Bcl-2蛋白表达均升高(均P<0.05)。下调miR-217表达后,H/R诱导的H9C2细胞中MDA含量、细胞凋亡率及Bax蛋白表达均降低(均P<0.05),而SOD和GSH-Px的活性及Bcl-2蛋白表达均升高(均P<0.05)。miR-217过表达逆转了PHC对H/R诱导的H9C2细胞中MDA含量、SOD和GSH-Px活性及凋亡的影响。结论:PHC通过下调miR-217表达抑制H/R诱导的心肌细胞氧化应激和凋亡,保护细胞损伤。

Objective:To investigate the effects and mechanism of penequinine hydrochloride(PHC)on the injury of rat cardiomyocyte H9 C2 induced by hypoxia/reoxygenation(H/R).Methods:The H/R-induced H9 C2 cells injury model was established in vitro.After different concentrations(12.5μg/L,25μg/L,50μg/L)of PHC acted on H/R-induced H9 C2 cells,enzyme-linked immunosorbent assay was used to detect the expression levels of MDA,SOD and GSH-Px;flow cytometry was used to detect the cell apoptosis;Western blot was used to detect the protein expression levels of Bcl-2 and Bax;RT-qPCR was used to detect the expression level of miR-217.miR-217 inhibitor was transfected into H9 C2 cells,then the same methods as above were used to observe the effects of down-regulating miR-217 on the expression levels of MDA,SOD and GSH-Px,cell apoptosis,and the protein expression levels of Bcl-2 and Bax in H/R-induced H9 C2 cells.Results:After PHC acted on H/R-induced H9 C2 cells,the content of MDA,the apoptosis rate,the protein expression of Bax,and the expression of miR-217 decreased(P<0.05),while the activity of SOD and GSH-Px and the protein expression of Bcl-2 increased(P<0.05).After down-regulating miR-217 expression,the content of MDA in H/R-induced H9 C2 cells,the apoptosis rate,and the protein expression of Bax decreased(P<0.05),while the activity of SOD and GSH-Px and the protein expression of Bcl-2 increased(P<0.05).The overexpression of miR-217 reversed the effects of PHC on the expression levels of MDA,SOD and GSH-Px in H/R-induced H9 C2 cells and cells apoptosis.Conclusion:PHC protects against H/R-induced oxidative stress and apoptosis of cardiomyocytes by down-regulating miR-217 expression.