花青素-3-葡萄糖苷干预过氧化氢诱导的H9c2细胞损伤的作用机制

Protective Effect of Anthocyanin-3-glucoside in H9c2 Cells Injury Induced by Hydrogen Peroxide

目的:探究花青素-3-葡萄糖苷干预过氧化氢(H_(2)O_(2))诱导的H9c2细胞损伤的作用机制。方法:将H9c2细胞暴露于不同浓度H_(2)O_(2)中4、8、12 h诱导细胞毒性,同时以不同浓度花青素-3-葡萄糖苷处理细胞,并设置阳性对照组(1.5 mg/mL益气活血颗粒),采用噻唑蓝(MTT)法检测细胞存活率,Hoechst 33342染色和流式细胞术检测细胞凋亡,实时荧光定量聚合酶链式反应(PCR)和蛋白免疫印迹法检测细胞中解偶联蛋白2(UCP2)表达,二氢乙锭(DHE)染色检测活性氧(ROS)含量,蛋白免疫印迹法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及切割后半胱天冬酶3(Cleaved Caspase-3)的表达。结果:600μmol/L的H_(2)O_(2)处理4 h为最佳造模条件,50.00μmol/L为花青素-3-葡萄糖苷最适药物干预浓度。与对照组比较,H_(2)O_(2)组细胞凋亡率、Bax/Bcl-2及Cleaved Caspase-3表达明显升高,UCP2 mRNA及蛋白表达明显降低,差异均有统计学意义(P<0.05);与H_(2)O_(2)组比较,H_(2)O_(2)+花青素-3-葡萄糖苷组和H_(2)O_(2)+益气活血颗粒组细胞凋亡率、Bax/Bcl-2及Cleaved Caspase-3表达明显降低,UCP2 mRNA及蛋白表达明显升高,差异均有统计学意义(P<0.05)。shRNA-3可明显降低细胞中UCP2蛋白表达,与H_(2)O_(2)组比较,H_(2)O_(2)+shRNA-3组UCP2、Bcl-2蛋白表达明显降低,Bax、Cleaved Caspase-3蛋白表达及ROS含量明显升高,差异均有统计学意义(P<0.05);与H_(2)O_(2)+shRNA-3组比较,花青素-3-葡萄糖苷处理后UCP2、Bcl-2蛋白表达明显升高,Bax、Cleaved Caspase-3蛋白表达ROS含量明显降低,差异均有统计学意义(P<0.05)。结论:花青素-3-葡萄糖苷对H_(2)O_(2)诱导的H9c2细胞凋亡有显著的保护作用,其机制可能与上调UCP2表达,从而降低Bax/Bcl-2、Cleaved Caspase-3表达,清除ROS有关。

Objective:To explore the mechanism of anthocyanin-3-glucoside in H9c2 cells injury induced by hydrogen peroxide(H_(2)O_(2)).Methods:H9c2 cells were exposed to different concentrations of H_(2)O_(2) for 4,8,and 12 h to induce cytotoxicity.At the same time,they were treated with different concentrations of anthocyanin-3-glucoside.Positive control group(1.5 mg/mL Yiqi Huoxue Granules)was set up.The survival rate of cells was detected by methyl thiazolyl tetrazolium(MTT).Apoptosis was detected by Hoechst 33342 staining and flow cytometry.The expression of uncoupling protein 2(UCP2)in cells was detected by rea-l time fluorescent quantitative PCR and Western blot.The contents of reactive oxygen species(ROS)were detected by dihydroethidium(DHE)staining.The expressions of B-cell lymphoma-2(Bc-l 2),Bc-l 2 associated X protein(Bax),and cleaved cysteine aspartase-3(Cleaved Caspase-3)were detected by Western Blot.Results:600μmol/L H_(2)O_(2) treatment for 4 h was the best modeling condition,and 50μmol/L anthocyanin-3-glucoside was the optimal drug intervention concentration.Compared with control group,apoptosis rate,Bax/Bc-l 2,and Cleaved Caspase-3 were significantly increased in H_(2)O_(2) group,while expressions of UCP2 mRNA and protein were significantly decreased(P<0.05).Compared with H_(2)O_(2) group,apoptosis rate,Bax/Bc-l 2,and Cleaved Caspase-3 were significantly decreased in H_(2)O_(2)+anthocyanin-3-glucoside group and H_(2)O_(2)+Yiqi Huoxue granule group,while expressions of UCP2 mRNA and protein were significantly increased(P<0.05).The shRNA-3 could significantly reduce the expression of UCP2 in cells.Compared with H_(2)O_(2) group,the expressions of UCP2 and Bc-l 2 were significantly decreased in H_(2)O_(2)+shRNA-3 group,expressions of Bax and Cleaved caspase-3,and ROS contents were significantly increased(P<0.05).Compared with H_(2)O_(2)+shRNA-3 group,expressions of UCP2 and Bc-l 2 were significantly increased after anthocyanin-3-glucoside treatment,expressions of Bax and Cleaved Caspase-3,and ROS contents were significantly decreased(P<0.05).Conclusion:Anthocyanin-3-glucoside shows significant protective effect in H9c2 cells apoptosis induced by H_(2)O_(2).The mechanism may be related to up-regulating UCP2 expression,reducing Bax/Bc-l 2 and Cleaved Caspase-3,and eli_(min)ating ROS.