毒蕈碱乙酰胆碱M2受体在迷走神经介导针刺改善心肌缺血过程中的机制分析

As a non-direct vagus nerve stimulating modality, acupuncture may enhance the reduced vagal activity resulted from myocardial ischemia (MI) and thereby result in preventive benefits on the ischemic myocardium. However, there is no systematic analysis about the involvement of the vagal motor centers in medulla and their efferent pathways, and the role of muscarinic acetylcholine receptor M2 (M2AChR) in mediation of acupuncture's effects. This application aims to, based on our previous work and by means of Physiology, Biochemistry, Immunohistochemistry and Molecular Biology etc, record the extracellular activities of the nucleus ambiguous, measure the alterations of acetylcholine and M2AChR in nucleus ambiguous, nodose ganglion in particular the ischemic myocardium, examine the expression of Bad and bcl-2 in the downstream intracellular signaling pathway mediated by M2AChR, and analyze the influence of M2 receptor on acupuncture effects by deleting this gene. The C57BL/6J strain mice and M2AChR knockout mice are used in the study. The acute myocardial ischemia (AMI) model is made by ligating the anterior descending branch of left coronary artery. It is expected that this work helps systematically clarify the role of the medulla-nodose ganglion-cardial vagal nerve pathway in the treatment of MI by acupuncture "Neiguan" acupoint, elucidate the M2 mediated molecular mechanism underlying acupuncture's benefits for MI and provide novel theoretical substrates for the prevention and treatment of cardiovascular diseases by acupuncture.

作为一种非直接刺激迷走神经的体表介入疗法，针刺可加强心肌缺血时减弱的迷走神经活性，发挥对缺血心肌的保护作用。然而，在这一过程中，有关延脑内迷走运动中枢及其下行通路如何通过毒蕈碱乙酰胆碱M2受体发挥作用，目前尚无系统性报道。本申请项目拟在既往研究的基础上，以C57BL/6J和M2受体基因敲除动物（M2KO）为载体，以结扎左冠状动脉前降支制造心肌缺血为动物模型，采用生理、生化、免疫组化以及分子生物学等技术，分别观察心肌缺血及电针"内关"穴后，延脑疑核神经元的放电活动、疑核和结状神经节特别是缺血心肌内乙酰胆碱及M2受体的变化、M2受体介导的细胞内信号转导通路下游分子Bad和bcl-2的表达水平，及敲除M2受体后对针刺效应的影响，以系统分析延脑-结状神经节-心迷走通路参与针刺改善心肌缺血作用，阐明M2受体介导的缺血心肌保护的分子机制，为心血管病防治的基础研究提供重要的理论依据。

缺血性心脏病是一种临床常见病，严重影响人们的身体健康。研究表明，针刺对心肌缺血具有良好治疗作用。鉴于急性心肌缺血通常表现为交感神经系统活动增强，迷走神经系统活动减弱，我们假定，针刺通过提高迷走神经活性改善心肌缺血。本项目以C57BL/6J小鼠和毒蕈碱乙酰胆碱M2受体基因敲除小鼠为载体，结扎左冠状动脉前降支及异丙肾上腺素腹腔注射制造心肌缺血模型，采用电生理、免疫组化及分子生物学等技术，观察延脑疑核、结状神经节和心室肌在心肌缺血后的变化以及电针“内关”穴的影响。结果显示：1. 心肌缺血后，心率明显加快，RMSSD与HF显著下降，LF/HF明显升高，电针可逆转上述变化；2. M2受体拮抗剂阿托品可部分阻断电针效应；3. 电针能增强心肌缺血引起的疑核神经元放电活动减弱，上调M2受体表达水平；4. 电针能提高缺血心肌M2受体及其下游分子bcl-2的基因及蛋白表达水平，降低Bad和caspase3的表达水平; 5. 电针可降低结状神经节内乙酰胆碱含量；6. M2受体基因敲除后，针刺效应部分减弱。上述证据提示，针刺可能通过延脑-结状神经节-心迷走通路改善心肌缺血。本研究为从分子水平阐明针刺的缺血心肌保护机制，为心血管病防治的基础研究提供重要的理论依据。