益心解毒方对大鼠心肌细胞内活性氧水平及信号转导通路的影响

摘要

Objective Rat myocardial cells had been used to research inhibiting effect of Yixin Jiedu formula medicated serum on reactive oxygen species ( ROS) level and the change of signal transduction pathways related molecules in myocardial cells induced by An -giotensinⅡ(AngⅡ).Methods Rat H9c2 cardiac muscle cells in logarithmic phase were detected after the synchronization .Experi-ment were divided into normal group:none modeling stimulation(no intervention),model group (10 -6mol/L AngⅡstimulated 24h, cul-tured with 1%serum of normal rats), Yixin Jiedu Formula group(10-6mol/L AngII stimulated 24h, cultured with 1% Yixin Jiedu For-mula contained serum), Diphenylene iodide (DPI) in the control group(10 -6mol/L AngII stimulation, 10umol/L DPI intervention 24h). H9c2 cardiac muscle cells ROS level was detected by DCFH -DA method.Change of signal transduction pathways related molecules in myocardial cells were detected by Westernblot method .Results We found that ROS level in myocardial cells induced by AngⅡimproved significantly, and Yixin Jiedu formula medicated serum had great inhibiting effect on the improved ROS level .Compared with model group,the lower dosage treatment group showed the most significant change (P<0.05).After 24h cultivation with AngII, the angiotensin receptor (AT1 receptor), signal transduction element and expression levels of activation about transcription 3 (STAT3) protein, matrix metalloproteinases (MMP9) protein at signal transduction pathway of low dose group was lower than the model group , with statistically sig-nificant difference (P<0.01,P<0.05).Conclusion The experiment had been showed that Yixin Jiedu formula could reduce the ROS level to inhibit the excessive activation of the sympathetic nervous system and the renin -angiotensin system ( RAAS) caused by AngⅡ, reduce the AT1 receptor expression level , and inhibit the role of STAT3, and finally delay the process of myocardial hypertrophy , playing its role in myocardial protection during the process of myocardial hypertrophy ;at the same time , inhibit the activity of MMPs to reduce my-ocardial remodeling .%目的 研究益心解毒方含药血清抑制血管紧张素Ⅱ( AngⅡ)诱导的大鼠心肌细胞内活性氧( ROS)水平及信号转导通路相关分子的变化. 方法 取对数生长期的大鼠H9 c2心肌细胞,同步化后进行实验,实验分为正常组:无造模刺激,无干预;模型组:10 -6 mol/L的AngⅡ刺激24h,1%正常大鼠血清干预;益心解毒方组:10 -6 mol/L的AngⅡ刺激24h,1%益心解毒方含药血清干预;二亚苯基碘( DPI)对照组:10 -6 mol/L的AngⅡ刺激,10μmol/L的NADPH氧化酶抑制剂DPI干预24h,采用乙酰乙酸双氯荧光素(DCFH-DA)法测定H9c2心肌细胞ROS水平;Western blot法测定各组H9c2心肌细胞信号转导通路相关分子的变化. 结果 研究发现AngⅡ诱导的H9 c2心肌细胞中ROS水平显著提高,而益心解毒方含药血清对增高的ROS水平都有明显的抑制作用,与模型组相比,低剂量组作用最为显著(P<0.05);在给予AngⅡ培养24h后,低剂量组信号转导通路上的血管紧张素受体(AT1受体)、信号转导子和转录激活子3(STAT3)蛋白、基质金属蛋白酶(MMP9)蛋白表达水平低于模型组,差异有统计学意义(P<0.01,P<0.05).结论 益心解毒方通过降低心肌ROS水平;抑制AngⅡ引起的交感神经系统、肾素-血管紧张素系统( RAAS)过度激活、降低AT1受体的表达水平,抑制STAT3的作用而延缓心肌肥厚的过程,发挥其在心肌肥厚过程中的心肌保护作用;同时抑制MMPs的活性,减缓心肌重构.