丹皮酚对缺氧复氧诱导H9c2心肌细胞损伤的保护作用机制研究

The protective mechanism of paeonol on H9c2 cardiomyocyte injury induced by hypoxia and reoxygenation

目的 评估丹皮酚对H9c2细胞内活性氧(ROS)水平调节作用并探讨其潜在机制。方法通过缺氧/复氧诱导方法建立心肌缺血/再灌注损伤细胞模型。根据H9c2细胞是否用丹皮酚(10μmol/L)预处理分为空白对照组、模型组、治疗组;根据乳腺癌易感基因1(BRCA1)-siRNA或control-siRNA转染H9c2细胞分为实验组和阴性对照组。通过流式细胞检测仪检测细胞内ROS水平,使用商业试剂盒检测总抗氧化能酶(T-AOC)和超氧化物歧化酶(SOD)活性,实时荧光定量聚合酶链式反应法和蛋白质印迹评估BRCA1和Nrf 2转录因子表达水平;使用RNA干扰技术验证丹皮酚可能通过BRCA1调控细胞内ROS水平。结果 治疗组细胞中ROS的水平明显低于模型组,SOD和T-AOC水平明显高于模型组,BRCA1和Nrf 2转录因子mRNA表达水平明显高于模型组(P <0.05)。使用siRNA阻断BRCA1表达后实验组ROS水平明显高于阴性对照组,Nrf 2转录因子、谷胱甘肽S-转移酶(GST)mRNA和T-AOC水平明显低于阴性对照组(t=5.862、7.630、5.706、5.914,P <0.05)。结论 丹皮酚可显著抑制H9c2细胞内ROS水平,提高细胞抗氧化能力,其可能作用机制是通过BRCA1基因依赖的Nrf 2抗氧化信号通路。

Objective To evaluate the regulatory effects of paeonol on reactive oxygen species(ROS) in H9c2 cells and to explore its underlying mechanism. Methods The cell model of myocardial ischemia/reperfusion injury was established by hypoxia/reoxygenation in culture medium. According to whether H9c2 cells were pretreated with paeonol(10 μmol/L), they were divided into blank control group, model group and treatment group. H9c2 cells were separated into negative control group and experimental group based on being transfected with either BRCA1-siRNA or control-siRNA. ROS levels were detected by flow cytometry, and commercial kits were used to evaluate the total antioxidant capacity(T-AOC) and the activity of superoxide dismutase(SOD). Nrf 2 transcription factor and breast cancer type 1(BRCA1) gene expression were determined by real-time fluorescence quantitative polymerase chain reaction and Western blotting. RNA interference techniques were used to demonstrate that paeonol could regulate intracellular ROS levels by BRCA1 gene. Results Compared with the model group,the level of ROS in the treatment group was significantly reduced;the activity of SOD and T-AOC,the expression of Nrf 2 transcription factor and BRCA1 mRNA were significantly higher(P < 0.05).After BRCA1expression was blocked with siRNA, the ROS level in the experimental group was significantly higher than that in the negative control group, the level of Nrf 2 transcription factor,glutathione S-transferase(GST) mRNA and T-AOC were significantly lower(t=5.862, 7.630, 5.706,5.914;P < 0.05). Conclusion Paeonol can significantly inhibit the level of reactive oxygen species in H9c2cells and improve the antioxidant capacity of cells. Its possible mechanism is through the BRCA1 gene dependent Nrf 2 antioxidant pathway.