基于NO-cGMP-PKG信号通路对VSMC舒张的调节作用探索“按之则热气至”的靶器官效应机制

The mechanism of “Tui-na” Manipulation is hotspot and difficulty research in this field, and Pressing Manipulation is the key of research, because Pressing Manipulation is one of the most basic manipulation, and most manipulations contain press action. The yellow emperor's Canon has the say "After pressing, It Heat Up" to interpretation Promoting Flow of Qi & Blood and Warming Channel for Dispelling Cold effect of Pressing Manipulation, and use the case of CHD Angina Pectoris of Congealing Cold with Blood Stasis and Qi Stagnation pathogenesis was relieve through Acupressing Shenshu Point produces heat. The key factor for its pathogenesis is Coronary Spasm, myocardial ischemia hypoxia. Induced coronary artery smooth muscle relaxation can effectively alleviate myocardial ischemia hypoxia, the NO-cGMP-PKG signaling pathways play an important role in regulating vasodilation. This project take this as the breakthrough point to explore the possible mechanism of target organ effect of "After pressing, It Heat Up". As the Model of Congealing Cold with Blood Stasis and Qi Stagnation CHD Rat as the subjects. And using the laser doppler blood flow analysis, infrared temperature measurement technology, immunohistochemistry and Western blot techniques. And acupressing the Shenshu point to observation the changes of points local temperature, electrocardiogram, heart microcirculation after myocardial ischemia, and organization form. And the changes of VSMC intracytoplasmic NO, cGMP, PKG content. As well as the changes of NO-cGMP-PKG signaling pathways downstream link regulatory proteins or phosphorylation status.

推拿手法作用机制一直是推拿领域里研究的热点和难点，按法是最基本手法之一，大部分手法都含有按压的动作，故按法是手法机制研究的重点手法。内经中有“按之则热气至”之说，以按压心俞穴产生热气至缓解寒凝气滞血瘀病机的冠心病心绞痛来阐释按法的温经散寒行气活血的作用。其病机的关键是冠脉痉挛，心肌缺血缺氧。诱导冠脉平滑肌舒张可有效的缓解心肌缺血缺氧，NO-cGMP-PKG信号通路在调节血管舒张方面有重要作用。本项目以此为切入点，以寒凝气滞血瘀型冠心病大鼠模型为受试对象，运用激光多普勒血流分析、红外测温技术、免疫组化、Western-blot 等技术，通过按压心俞穴，观察穴位局部温度、心肌缺血后心电图、心脏微循环和组织形态的变化，以及VSMC胞浆内NO、cGMP、PKG含量变化和NO-cGMP-PKG信号通路下游环节中各调节蛋白含量或磷酸化状态及相关的变化，探索“按之热气至”的靶器官效应可能机制。

本项目基于中医经典著作《黄帝内经》中有关推拿按法治病机制的论述—“按之则热气至”，即按压心俞穴产生热气至缓解寒凝气滞血瘀病机的冠心病心绞痛，进而来阐释推拿按法的温经散寒行气活血的作用。本课题研究先以“按之则热气至”理论为源头做文献分析，分析“按之则热气至”的理论内涵和临床应用原理。然后以兔的心肌缺血再灌注损伤模型为载体，研究按压心俞对心肌的保护效应及其效应机制。将48只兔随机分为正常组、模型组、假手术组、按压心俞组，按压心俞组在再灌注前按压心俞穴7.5 min。观察各组的心电、心肌损伤的血清指标及心肌缺血区的腺苷、RISK通路蛋白和心肌保护蛋白HSP70的变化，结果显示按压心俞穴能促使缺血区心肌产生大量腺苷增强RISK通路激活，进而发挥心肌保护效应。再以异丙肾上腺素诱导冠脉痉挛导致的心肌缺血缺氧的大鼠模型为载体，观察按压心俞穴对心肌缺血缺氧的改善和对冠脉舒张的调节效应，以及调节舒张效应的机制。先将60只大鼠分为正常组、模型组、按法组、西药组，按法组造模成功后进行按压操作，按压两侧心俞穴7天（1次/天），观察各组的心电、血清CK-MB、cTn-I和心肌细胞凋亡变化。结果显示按压心俞穴能改善心肌缺血缺氧导致的损伤。再用PKG抑制剂阻断NO-cGMP-PKG信号通路，观察按压心俞穴改善心肌损伤与此信号通路的关系。将60只大鼠分为正常组、模型组、按法组、模型+抑制剂组、模型+抑制剂+按法组，观察各组的心电、血清NO和ET指标、冠脉平滑肌PKG表达和Ca2+量的变化。结果显示按压心俞穴可以调节冠脉舒张，并通过激活PKG，调节冠脉平滑肌胞内的钙离子浓度来舒张血管平滑肌。由此可知，“按之则热气至”的效应机制是由按压心俞穴产生心肌保护效应和抗心肌损伤效应，其按压心俞产生的靶器官效应与抗心肌损伤的RISK通路和舒展冠脉的NO-cGMP-PKG通路有关。