丹参多酚酸盐对H_(2)O_(2)所致心肌细胞氧化应激损伤及PPARγ/Nrf2/HO-1通路的影响

Effects of Salvianolate on Oxidative Stress Injury of Cardiomyocytes Induced by H_(2)O_(2) and PPARγ/Nrf2/HO-1 Pathway

目的探讨丹参多酚酸盐(SAL)对过氧化氢(H_(2)O_(2))所致H9c2心肌细胞氧化应激损伤的影响及过氧化物酶体增殖子活化受体(PPARγ)/核转录因子E2相关因子2(Nrf2)/抗血红素加氧酶-1(HO-1)通路在其中的调控作用。方法以100μmol/L的H_(2)O_(2)干预对数生长期H9c2细胞4 h建立心肌细胞氧化损伤模型,设置H_(2)O_(2)组、SAL 50 mg/L组、SAL 100 mg/L组、SAL 200 mg/L组;另取对数生长期H9c2细胞作为正常组。药物干预24 h后,四唑盐(MTT)法和Annexin V-FITC/PI双染法分别检测细胞增殖和凋亡率,2,7-双乙酸二氯荧光(DCFH-DA)探针检测细胞活性氧(ROS)含量,生化分析法检测肌酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)漏出量,硫代巴比妥酸法检测丙二醛(MDA)含量,黄嘌呤氧化法、钼酸铵法分别检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,蛋白免疫印迹法(Western Blotting)检测PPARγ、Nrf2、HO-1、核转录因子-κB(NF-κB)、激活型半胱氨酸蛋白酶-3(Cleaved Caspase-3)蛋白表达。结果与H_(2)O_(2)组比较,SAL 50 mg/L组、SAL 100 mg/L组、SAL 200 mg/L组细胞增殖率升高且凋亡率降低,ROS含量和CK-MB、LDH漏出量降低,MDA含量降低且SOD、CAT活性升高,PPARγ、Nrf2、HO-1蛋白表达上调且NF-κB、Cleaved Caspase-3表达下调,差异均有统计学意义(P<0.05或P<0.01)。结论SAL对H_(2)O_(2)所致H9c2心肌细胞氧化应激损伤具有保护作用,其作用机制可能与激活PPARγ/Nrf2/HO-1通路有关。

Objective To explore effects of salvianolate(SAL)on oxidative stress injury of cardiomyocytes induced by H_(2)O_(2) and the regulatory effect of peroxisomal proliferator activated receptorγ(PPARγ)/nuclear transcription factor E2-related factor 2(Nrf2)/anti-heme oxygenase-1(HO-1)pathway.Methods H9c2 cells at logarithmic growth stage were treated with 100μmol/L H_(2)O_(2) for 4 h to establish myocardial oxidative damage model,and to set H_(2)O_(2) group,SAL 50 mg/L group,SAL 100 mg/L group.and SAL 200 mg/L group,H9c2 cells at logarithmic growth stage were taken as normal group.After 24 h of intervention,the rate of cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay and the rate of apoptosis was detected by Annexin V-FITC/PI double staining method.The content of reactive oxygen species(ROS)was detected by DCFH-DA probe.The leakage of creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH)were detected by biochemical analysis method.The content of malondialdehyde(MDA)was detected by thiobarbituric acid method.The activity of superoxide dismutase(SOD)was detected by xanthine oxidation method and the activity of catalase(CAT)was detected by ammonium molybdate method.The expressions of PPARγ,Nrf2,HO-1,nuclear transcription factor-κB(NF-κB),and Cleaved Caspase-3 were detected by Western Blotting.Results Compared with the H_(2)O_(2) group,the rate of cell proliferation increased,the rate of apoptosis decreased,the content of ROS decreased,the leakage of CK-MB and LDH decreased,the content of MDA content decreased,the activities of SOD and CAT increased,the expressions of PPARγ,Nrf2,HO-1 protein expression increased,NF-κB,Cleaved Caspase-3 expression decreased in the SAL 50 mg/L group,SAL 100 mg/L group,and SAL 200 mg/L group,and the differences were statistically significant(P<0.05 or P<0.01).Conclusion SAL could protect on oxidative stress injury of H9c2 cardiomyocytes induced by H_(2)O_(2),and its mechanism might be related to activation of PPARγ/Nrf2/HO-1 pathway.