胡黄连苦苷Ⅱ通过下调microRNA-1表达抑制H_2O_2诱导的心肌细胞凋亡

Effect of Picroside Ⅱ on Expression of microRNA-1 in H_2O_2-induced H9c2 Cardiomyocytes Damage

目的:研究胡黄连苦苷Ⅱ(picrosideⅡ)对过氧化氢(H_2O_2)诱导的大鼠H9c2心肌细胞凋亡中微小RNA-1(microRNA-1,miR-1)及下游靶基因抗凋亡因子B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)表达的影响,探讨胡黄连苦苷Ⅱ的心肌保护作用及其机制。方法:将H9c2心肌细胞随机分为正常组,模型组(H_2O_2,200μmol·L^(-1)),胡黄连苦苷Ⅱ(50,100,200μmol·L^(-1))+H_2O_2(200μmol·L^(-1))组,胡黄连苦苷Ⅱ200μmol·L^(-1)组。胡黄连苦苷Ⅱ与心肌细胞孵育6 h后,加入H_2O_2继续培养2 h。药物处理结束后,噻唑蓝(methyl thiazolyl tetrazolium,MTT)比色法检测细胞存活率,比色法测定细胞培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,4',6-二脒基-2-苯基吲哚(destination access point identifier,DAPI)染色和细胞凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3 (cysteinyl aspartate specific proteinase-3,Caspase-3)检测评估细胞凋亡,实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中Caspase-3,Bcl-2,miR-1 mRNA的表达,蛋白免疫印迹法(Western blot)检测细胞抗凋亡因子Bcl-2蛋白的表达。结果:与正常组相比,H_2O_2能显著降低细胞生存率,增加细胞凋亡率,且Caspase-3活性和mRNA表达均增加(P <0. 01),同时伴有miR-1 mRNA表达水平的上调及其下游靶基因抗凋亡因子Bcl-2 mRNA表达的下调(P <0. 01)。与模型组比较,胡黄连苦苷Ⅱ预处理可明显升高细胞存活率,明显降低LDH活性,降低细胞凋亡率,明显降低Caspase-3活性和mRNA表达(P <0. 05,P <0. 01),并使Bcl-2 mRNA表达升高(P <0. 05,P <0. 01),Western blot结果表明,胡黄连苦苷Ⅱ能明显下调其下游靶基因抗凋亡因子Bcl-2蛋白表达(P <0. 05,P <0. 01)。结论:胡黄连苦苷Ⅱ对H_2O_2诱导的H9c2心肌细胞凋亡具有保护作用,其机制与下调miR-1的表达进而上调其靶基因Bcl-2的表达有关。

Objective: To study the effect of picroside Ⅱ on the expression of microRNA-1( miR-1) in the H2O2-induced H9 c2 cardiomyocytes damage,in order to explore the mechanism of picroside Ⅱ in protecting H9 c2 cardiomyocytes from oxidative stress. Method: H9 c2 cardiomyocytes were divided into 6 groups: control group,model group( H2O2200 μmol·L-1),picroside Ⅱ( 50,100,200 μmol·L-1) + H2O2( 200 μmol·L-1)group and picroside Ⅱ( 200 μmol·L-1) group. Picroside Ⅱ group was incubated with picroside Ⅱ for 6 h and then cultured with H2O2 for 2 h. At the end of drugs treatment,the cell viability and the cellular damage of cardiomyocytes were respectively assessed by 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) and lactate dehydrogenase( LDH) assays. 4’,6-diamidino-2-phenylindole( DAPI) staining and cysteinyl aspartate specific proteinase-3( Caspase-3) test were used to evaluate cell apoptosis. The mRNA expressions of Caspase-3,B-cell lymphoma-2( Bcl-2) and miR-1 were measured by Real-time polymerase chain reaction( Realtime PCR). The protein expression of Bcl-2 was detected by Western blot. Result: Compared with the control group,H2O2 could significantly decrease the cell viability and increase the rate of apoptosis,up-regulate mRNA expression of Caspase-3 and miR-1,and down-regulate expression of Bcl-2 in H9 c2 cells( P < 0. 01). Compared with the model group,the cell survival rate was significantly increased by pretreating with Picroside Ⅱ for 6 h( P < 0. 05,P < 0. 01). Picroside Ⅱ not only decreased the rate of apoptosis and up-regulated mRNA expression of Bcl-2,but also down-regulated mRNA expressions of Caspase-3 and miR-1( P < 0. 05,P < 0. 01). Western blot showed that picroside Ⅱ can significantly enhance the expression of Bcl-2( P < 0. 05, P < 0. 01).Conclusion: Picroside Ⅱ has a protective effect on H9 c2 cells from H2O2-induced cardiomyocyte injury by downregulating mRNA-1 expression and up-regulating the expression of the downstream Bcl-2.