甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制

The protective mechanism of differential isomers of glycyrrhetinic acid on cisplatin-induced H9c2 cardiomyocyte injury

目的 探究甘草次酸(GA)的差向异构体18α-GA和18β-GA对顺铂(CDDP)诱导H9c2心肌细胞损伤的保护机制。方法 采用CCK-8法检测细胞活力,筛选CDDP诱导细胞损伤的浓度,18α-GA和18β-GA安全浓度以及18α-GA与18β-GA改善CDDP致心肌细胞活力下降的有效浓度;将细胞分为对照组、CDDP组、18α-GA组和18β-GA组,使用Hoechst染色检测各组细胞凋亡情况;流式细胞术检测活性氧(ROS)水平;Mito-Tracker Red CMXRos染色评估线粒体活性;Western blot法检测各组细胞剪切化胱天蛋白酶3(C-Caspase3)、B-淋巴细胞瘤基因-2(Bcl-2)、Bcl-2相关X蛋白(Bax)以及细胞色素C(Cyt-c)的蛋白表达。结果 CCK-8实验结果显示,CDDP在20μmol/L时可使H9c2心肌细胞活力显著下降(P<0.01);18α-GA和18β-GA浓度小于100μmol/L时,对心肌细胞无明显影响,且50和100μmol/L时均可改善CDDP导致的心肌细胞活力的降低(P<0.01)。与对照组相比,CDDP组细胞凋亡明显,ROS水平升高,心肌细胞内线粒体足长度和具有网状结构的线粒体降低,C-Caspase3、Cyt-c的蛋白表达升高,Bcl-2/Bax的蛋白表达降低(P<0.05);与CDDP组相比,18α-GA组和18β-GA组细胞凋亡情况改善,ROS水平降低,心肌细胞内线粒体足长度和具有网状结构的线粒体增加,C-Caspase3、Cyt-c的蛋白表达水平降低,Bcl-2/Bax的蛋白表达水平升高(P<0.05)。结论 18α-GA和18β-GA可通过抑制ROS水平以及保护线粒体功能减少CDDP诱导的H9c2心肌细胞凋亡。

Objective To explore the protective mechanism of 18α-glycyrrhetinic acid(GA) and 18β-GA,the differential isomers of glycyrrhetinic acid,on cisplatin(CDDP)-induced H9c2 cardiomyocyte injury.Methods CCK-8 assay was used to detect cell viability and the concentration of CDDP-induced cell injury.The safe doses of 18 a-GA and 18β-GA,and the effective concentrations of 18 a-GA and 18β-GA to improve the decrease of cardiomyocyte viability induced by CDDP were screened.The cells were divided into the control group,the CDDP group,the 18 a-GA group and the18β-GA group.Hoechst staining was used to detect apoptosis in each group.The level of reactive oxygen species(ROS) was detected by DCFH-DA.Mito-Tracker Red CMXRos staining was used to assess mitochondrial activity.The expressions of C-Caspase3,B-lymphocytoma gene-2(Bcl-2),Bcl-2 associated X protein(Bax) and cytochrome C(Cyt-c) were detected by Western blot assay in each group.Results The results of CCK-8 assay showed that CDDP at 20 μmol/L significantly decreased the viability of H9c2 cardiomyocytes(P <0.01).When the concentrations of 18α-GA and 18β-GA were less than 100 μmol/L,there were no significant effects on cardiomyocytes,and 50 and 100 μmol/L could improve the decrease of cardiomyocyte viability caused by CDDP(P<0.01).Compared with the control group,the apoptosis was obvious in the CDDP group,the ROS level was increased,the mitochondrial foot length and mitochondria with reticular structure were decreased,the protein expression levels of C-Caspase3 and Cyt-c were increased,and the protein expression of Bcl-2/Bax was decreased(P <0.05).Compared with the CDDP group,cell apoptosis was improved in the 18α-GA group and the 18β-GA group,ROS level decreased,mitochondrial foot length and mitochondria with reticular structure increased,protein expression levels of C-Caspase3 and Cyt-c decreased,and protein expression levels of Bcl-2/Bax increased(P <0.05).Conclusion 18α-GA and 18β-GA can reduce the apoptosis of H9c2 cardiomyocytes induced by CDDP by inhibiting ROS level and protecting mitochondrial function.