基于PKA/eNOS通路的艾灸激活TRPV1调脂通脉机制研究

Moxibustion consists of treatment of warm stimulation to the body. The existing research confirms that the mechanism of warming and promoting therapeutic effect from moxibustion is related to transient receptor potential vanilloid subfamily1 (TRPV1) .Although the subsequent effects of activating TRPV1 mechanism are unclear, hyperlipidemia treated effectively by moxibustion therapy is selected as object in this project . We plan to observe the influence of moxibustion stimulation at different temperature(38℃,45℃) on blood lipids and the structure and function of thoracic aortic endothelium in rats and mice model of hyperlipidemia. The aim is to analysise the relevance between the moxibustion-stimulation associated with TRPV 1 activation and the regulation of blood lipids and improvement of the vascular endothelial function. On this basis, the TRPV1 gene knockout (TRPV1-/-) mice are selected as comparison. We will also detect the endothelial function related factors,endothelial nitric oxide synthase (eNOS), protein kinase A (PKA) and the expression of dorsal root ganglion TRPV1mRNA of mice by using ELISA method, immunofluorescence and real-time Polymerase Chain Reaction (PCR) method,in order to study the mechanism that the TRPV1 actived by moxibustion through PKA/eNOS signal pathsis to regulate blood lipids and cleare the blood ,providing the experimental basis and theoretical guidance for further studies.

艾灸是通过对机体的温热刺激发挥其治疗作用，已有的研究证实艾灸的温通效应机制与瞬时受体电位香草酸亚型1（TRPV1）相关，但激活TRPV1后的相应作用机制尚不明确。本项目选用高脂血症这一艾灸治疗的有效病种，观察38℃和45℃两种不同温度艾灸热刺激对高脂血症模型大鼠、小鼠血脂及胸主动脉内皮形态结构、功能的影响，分析艾灸激活TRPV1调节血脂与改善内皮功能之间的联系；在此基础上，选用瞬时受体电位香草酸亚型1敲除（TRPV1-/-）小鼠做对照，采用ELISA法、免疫荧光法及实时荧光定量PCR法检测内皮功能相关因子、内皮型一氧化氮合酶（eNOS）、蛋白激酶A（PKA）及小鼠背根神经节TRPV1mRNA的表达，研究艾灸激活TRPV1通过PKA/eNOS信号通路调脂通脉的效应机制，从而为艾灸温通效应机制的深入研究提供实验基础和理论指导。

本项目首先确定38℃、45℃这两种温度艾灸热刺激对高脂血症模型大鼠血脂及胸主动脉内皮形态结构的影响，确认艾灸温度的不同所造成的调节血脂、改善内皮功能作用效应上的差异。其次观察艾灸热刺激激活TRPV1对高脂血症模型大鼠血清NO、eNOS以及内皮功能相关因子（CGRP、ET-1）的影响，确定艾灸调脂通脉作用与激活TRPV1的联系。最后研究艾灸对高脂血症模型小鼠PKA/eNOS信号通路的影响，分析艾灸调脂抗氧化扩血管的作用与激活TRPV1之间的联系。在此基础上，观察艾灸热刺激对小鼠背根神经节(DRG) TRPV1mRNA表达的影响，并利用TRPV1-/-小鼠作对照，进一步明确艾灸调脂通脉作用机制与激活TRPV1及PKA/eNOS信号通路的相关。. 本项目的研究过程和结果如下：课题组首先通过成熟的生化及形态学方法，明确45℃艾灸组热刺激对高脂血症模型大鼠血脂的调整优于38℃艾灸组，胸主动脉内皮HE染色结果也同样表明这一差异。其次明确45℃艾灸具有明显的调节血脂，保护血管内皮的作用，其调脂保护内皮的作用机制，与调节NO、eNOS、ET-1、CGRP、OX-LDL相关; 通过免疫组化法发现艾灸激活TRPV1调脂通脉与PKA，eNOS相关。在此基础上，观察艾灸热刺激对小鼠背根神经节(DRG) TRPV1mRNA表达的影响，并利用TRPV1-/-小鼠作对照，明确激活TRPV1与PKA/eNOS信号通路的相关，进一步探讨艾灸调脂通脉作用的TRPV1调控机制。. 基于本项目的研究，我们认为艾灸激活TRPV1调脂通脉，与PKA/eNOS通路有关，为艾灸温通效应机制的深入研究提供实验基础。.