益心解毒方对NOX2亚基和NOX4亚基过表达及小干扰RNA引起的心肌细胞还原型辅酶Ⅱ氧化酶活性变化的机制研究

Mechanism of Yixin Jiedu Formula on the Overexpression of NOX2 and NOX4 and the Myocardial NADPH Ⅱ Oxidase Activity Changes Induced by RNAi

目的观察益心解毒方含药血清对NOX2、NOX4亚基过表达和小干扰RNA(RNAi)导致的H9C2心肌细胞还原型辅酶Ⅱ(NADPH)氧化酶活性的变化,探讨益心解毒方的作用机制。方法采用正常雄性Sprague-Dawley大鼠16只,构建针对NOX2、NOX4亚基的过表达质粒和RNAi质粒。采用转染试剂瞬时转染H9C2心肌细胞,流式细胞术检测转染率,转染24 h后分组(正常H9C2细胞组、过表达组、H9C2转阴性对照质粒组、RNAi质粒组、益心解毒方高剂量组、益心解毒方中剂量组、益心解毒方低剂量组)给予不同剂量的含药血清干预,24 h后检测NADPH氧化酶活性。结果转染NOX2亚基过表达质粒的各组NADPH氧化酶活性比较,差异有统计学意义(P<0.05)。其中,过表达组、益心解毒方高剂量组、益心解毒方低剂量组与正常H9C2细胞组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);H9C2转阴性对照质粒组、RNAi质粒组、益心解毒方中剂量组与过表达组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);RNAi质粒组、益心解毒方高剂量组、益心解毒方低剂量组与H9C2转阴性对照质粒组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方高剂量组、益心解毒方低剂量组与RNAi质粒组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方中剂量组与益心解毒方高剂量组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方低剂量组与益心解毒方中剂量组NADPH氧化酶活性比较,差异有统计学意义(P<0.05)。转染NOX4亚基过表达质粒的各组NADPH氧化酶活性比较,差异有统计学意义(P<0.05)。其中,过表达组、益心解毒方高剂量组、益心解毒方低剂量组与正常H9C2细胞组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);H9C2转阴性对照质粒组、RNAi质粒组、益心解毒方中剂量组、益心解毒方低剂量组与过表达组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);RNAi质粒组、益心解毒方高剂量组、益心解毒方低剂量组与H9C2转阴性对照质粒组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方高剂量组、益心解毒方低剂量组与RNAi质粒组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方中剂量组与益心解毒方高剂量组NADPH氧化酶活性比较,差异有统计学意义(P<0.05);益心解毒方低剂量组与益心解毒方中剂量组NADPH氧化酶活性比较,差异有统计学意义(P<0.05)。结论 NOX2、NOX4亚基的过表达可以提高NADPH氧化酶活性,而RNAi表达可以沉默NOX2、NOX4亚基,从而降低NADPH氧化酶活性,益心解毒方不会直接影响NADPH氧化酶活性。实验研究表明干预和抑制NOX2、NOX4亚基的NADPH氧化酶活性调控环节,降低心肌活性氧(ROS)水平,保护心肌细胞,改善心功能,可能是益心解毒方发挥药效的重要机制。

Objective To observe the influence of Yixin Jiedu formula on NOX2 and NOX4 overexpression and H9C2 myocardial NADPH Ⅱ oxidase activity changes induced by RNAi and investigate its mechanism. Methods We selected 16 normal male Sprague- Dawley rats and constructed the overexpression plasmids of NOX2 and NOX4 and RNAi plasmids. Transfection reagents were employed to conduct transient transfection of H9C2 myocardial cells, flow cytometry was undertaken to examine transfection efficiency. At 24 hours after transfection, the subjects were divided into normal H9C2 cell group,overexpression group,H9C2 negative- turning control plasmid group, RNAi plasmid group, high- dose Yixin Jiedu formula group,middle- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group. All the groups were intervened with different doses of drug serum,and NADPH oxidase activity was detected 24 hours after drug administration. Results The groups in which overexpression plasmids of NOX2 was transfected were significantly different in NADPH oxidase activity（ P〈0. 05）; overexpression group,high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity（ P〈0. 05）; H9C2 negative- turning control plasmid group,RNAi plasmid group, middle- dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity（ P〈0. 05）; RNAi plasmid group,high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from H9C2 negative- turning control plasmid group in NADPH oxidase activity（ P〈0. 05）; high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from RNAi plasmid group in NADPH oxidase activity（ P〈0. 05）; middle- dose Yixin Jiedu formula group and high- dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity（ P〈0. 05）; low- dose Yixin Jiedu formula group and middle- dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity（ P〈0. 05）. The groups in which overexpression plasmids of NOX4 was transfected were significantly different in NADPH oxidase activity（ P〈0. 05）;overexpression group,high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from normal H9C2 cell group in NADPH oxidase activity（ P〈0. 05）; H9C2 negative- turning control plasmid group,RNAi plasmid group,middle- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from overexpression group in NADPH oxidase activity（ P〈0. 05）; RNAi plasmid group,high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from H9C2 negative- turning group in NADPH oxidase activity（ P〈0. 05）; high- dose Yixin Jiedu formula group and low- dose Yixin Jiedu formula group were significantly different from RNAi plasmid group in NADPH oxidase activity（ P〈0. 05）; middle- dose Yixin Jiedu formula group and high- dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity（ P〈0. 05）; low- dose Yixin Jiedu formula group and middle- dose Yixin Jiedu formula group were significantly different in NADPH oxidase activity（ P〈0. 05）. Conclusion The overexpression of NOX2 and NOX4 can significantly improve NADPH oxidase activity,and RNAi expression can silence NOX2 and NOX4,thereby significantly reducing NADPH oxidase activity. Yixin Jiedu formula doesn＇t directly influence NADPH oxidase activity. The study shows the important mechanism of Yixin Jiedu formula may be that it can intervene and inhibit NADPH oxidase activity of NOX2 and NOX4,reduce the ROS level of myocardial muscle,protect myocardial cell and improve cardiac function.