熊果酸通过抑制自噬对血清剥夺诱导的H9c2细胞起抗凋亡作用

Anti-apoptotic effect of ursolic acid by inhibiting serum deprivation-induced autophagy in H9c2 cells

目的冠状动脉粥样硬化心脏病(coronary atherosclerotic cardiopathy)造成的心肌缺血(myocardial ischemia)严重威胁人类生命安全,本研究从细胞水平阐述了熊果酸(ursolic acid,UA)对血清剥夺心肌细胞自噬和凋亡的作用。方法体外培养大鼠H9c2心肌细胞。分为对照组、血清剥夺组、熊果酸处理组、自噬抑制剂3-甲基腺嘌呤(3-MA)预处理联合熊果酸组、雷帕霉素预处理联合熊果酸组。流式细胞术检测细胞凋亡情况;蛋白免疫印迹检测自噬相关蛋白LC3、Beclin1、ATG5以及凋亡相关蛋白cleaved-caspase3、Bc L-2、Bax的表达。结果10μmol/L熊果酸为处理H9c2细胞的最适浓度;血清剥夺能造成H9c2细胞LC3 II/I比值、Beclin1和ATG5表达增加(P<0.01),熊果酸处理后能降低其表达(P<0.05);血清剥夺造成H9c2细胞凋亡率增加(P<0.001),熊果酸处理后能降低凋亡率(P<0.01),3-MA预处理组能进一步降低凋亡率(P<0.001),而雷帕霉素预处理组凋亡率上升(P<0.001);血清剥夺能造成H9c2细胞cleaved-caspase3表达量、Bax/Bc L-2比值升高(P<0.001),熊果酸处理后抑制了血清剥夺造成的cleaved-caspase3的表达量和Bax/Bc L-2比值升高(P<0.01),3-MA预处理组能进一步降低cleaved-caspase3的表达量和Bax/Bc L-2比值(P<0.001),雷帕霉素预处理组Bax/Bc L-2比值回升(P<0.05)。结论熊果酸对血清剥夺H9c2细胞的抗凋亡作用与抑制自噬有关。

Objective Myocardial ischemia caused by coronary atherosclerotic cardiopathy is a serious disease that can cause death.This study describes the effects of ursolic acid on autophagy and apoptosis caused by serum deprivation of cardiomyocytes at the cellular level.Methods Rat H9 c2 cardiomyocytes were cultured in vitro.The cells were divided into the control group,serum deprivation group,ursolic acid treatment group,autophagy inhibitor 3-methyladenine(3-MA)pretreatment combined with ursolic acid group,and rapamycin pretreatment combined with ursolic acid group.Apoptosis was detected using flow cytometry.Expression of the autophagy-related proteins LC3,Beclin1,and ATG5,and the apoptosis-related proteins cleaved-caspase3,Bc L-2,and Bax,were detected by western blotting.Results10μM ursolic acid was the optimal concentration for H9 c2 cells.Serum deprivation increased the expression of Beclin1 and ATG5 as well as the ratio of LC3 II/I in H9 c2 cells(P<0.01),and these levels decreased after ursolic acid treatment(P<0.05).Serum deprivation caused an increase in the apoptosis rate of H9 c2 cells(P<0.001),and ursolic acid treatment reduced the rate of apoptosis(P<0.01).The 3-MA pretreatment group had a further reduced apoptosis rate(P<0.001),while the rapamycin pretreatment group had an increased apoptosis rate(P<0.001).Serum deprivation increased the expression of cleaved-caspase3 and the ratio of Bax/Bc L-2(P<0.001).Following treatment with ursolic acid,the expression of cleaved-caspase3 and the ratio of Bax/Bc L-2 was decreased(P<0.01),and pretreatment with 3-MA further reduced the expression of cleaved-caspase3 and the ratio of Bax/Bc L-2(P<0.001).The ratio of Bax/Bc L-2 was increased in the rapamycin pretreatment group(P<0.05).Conclusions The anti-apoptotic effect of ursolic acid on serum-deprived H9 c2 cells is related to the inhibition of autophagy.