通过TLR4/NF-κB途径探讨护心方对心肌梗死大鼠心脏交感神经重构的作用及机制研究

Recent studies have found that myocardial infarction resulted in cardiac sympathetic remodeling, which were related to ventricular arrhythmias and sudden cardiac death. Nerve growth factor and Sema 3A which were activated by TLR4/NF-κB pathway played an important role in sympathetic remodeling. Sympathetic remodeling may become a new target for treating ventricular arrhythmias after myocardial infarction. Huxin Formula was created by well-known CM doctor Prof. DENG Tie-Tao and had marked effects in treating coronary heart disease. Recent our study showed that Huxin Formula had the effects of inhibiting TLR4/NF-κB pathway. We speculated that Huxin Formula could reduce the incidence of ventricular arrhythmias after myocardial infarction through regulating sympathetic remodeling. We will establish myocardial infarction in rats, and then investigate the outcomes of Huxin Formula on cardiac sympathetic remodeling through the animal and cellular study, combing with molecular biology and tissue morphology analysis. The purpose of this study is to provide a theory and a new treatment for ventricular arrhythmias after myocardial infarction.

近年研究发现心肌梗死后存在心脏交感神经重构，与心梗后室性心律失常和心脏性猝死有关。心肌梗死后TLR4/NF-κB信号通路激活引起的心脏神经生长因子和信号素3A平衡改变，在心肌梗死后交感神经重构中起重要作用。交感神经重构可能成为心肌梗死后室性心律失常治疗的新靶点。护心方由著名的国医大师邓铁涛教授创制，对于冠心病具有良好治疗效果。近期我们的研究显示护心方抑制TLR4/NF-κB信号通路。由这些临床经验以及基础研究推断，中医方剂护心方可以对心肌梗死后交感神经重构起到调节作用，减少室性心律失常的发生。本项目拟建立大鼠心肌梗死模型，通过在体和细胞水平研究，结合分子生物学和组织形态学观察，探讨护心方对心肌梗死大鼠心脏交感神经重构的影响及其作用机制，为心梗后室性心律失常的防治提供理论依据和新的治疗方法。

近年研究发现心肌梗死（MI）后TLR4/NF-κB信号通路激活引起的心脏神经生长因子（NGF）和信号素3A（Sema3A）改变，在交感神经重构中起重要作用。本研究观察护心方对心肌梗死后交感神经重构以及室性心律失常的影响并探讨其机制。. 通过RT-PCR、Western blot和ELISA方法，检测护心方干预LPS孵育的H9c2细胞中p65以及NGF和Sema3A的表达，细胞外液IL-1、IL-6、TNF-α含量。结果显示，与LPS组相比，中剂量、高剂量护心方能抑制LPS诱导H9c2细胞p65、NGF的mRNA和蛋白表达，而促进Sema3A的mRNA和蛋白表达。低剂量、中剂量、高剂量护心方均能抑制LPS诱导H9c2细胞炎症因子的分泌。显示护心方可以通过减少p65的激活而达到对心肌细胞的保护作用。. 构建大鼠MI模型，采用免疫组化、心脏超声、心脏程序性刺激、RT-PCR、Western blot以及ELISA方法，观察护心方对MI大鼠心脏交感神经重构的影响及室性心律失常诱发的作用。结果显示，与MI组相比较，中剂量、高剂量护心方能减少MI大鼠室性心律失常诱发，但对心脏功能无明显影响。护心方干预后，MI边缘GAP43和TH阳性交感神经纤维密度明显降低，提示中剂量、高剂量护心方对MI后交感神经重构具有抑制作用。护心方明显降低血清炎症因子水平。RT-PCR和Western blot结果表明，中剂量、高剂量护心方抑制MI后p65、NGF、p75NTR的mRNA和蛋白表达；上调MI后Sema3A、Neuropilin-1的mRNA和蛋白表达，说明护心方通过TLR4/NF-κB信号通路，调节NGF/p75NTR和Sema3A/Neuropilin-1表达，抑制心梗后交感神经重构。. 本研究证实护心方能够抑制心肌梗死后心脏交感神经重构，减少室性心律失常发生，降低血清炎症因子的水平，具有心梗治疗的临床应用前景。