基于TRPV1通道探讨白芥子涂方穴位经皮给药促透和免疫增效的机制

Asthma is presently incurable, and there are severe side effects when glucocorticoids or broncholilator agents are administered for controlling the symptoms with the disease in western medicine. Baijiezi Tufang, a traditional Chinese medicine, is widely and effectly used for clinical prevention and treatment of the common winter diseases of asthma and bronchitis by acupuncture point percutaneous administration in the summer. However, the exact mechanism of action of the medication remains unclear, which limits its development in clinical application. In the present study, one of the action mechanisms of the medication was supposed to enhance the permeation of drugs and improve the immunity function through activation of TRPV1 channel in the acupuncture point skin. The enhancement of permeation and improvement of immunity function were confirmed in our previous study. In this project, comparation of TRPV1 expression between acupoint and non-acupoint was carried out with immunohistochemical methods combining with CLSM and RT-PCR. After the activation of TRPV1,skin barrier was researched by measuring the value of TWEL, the permeability of drugs was comparatively evaluated with TRPV1+/+ and TRPV1-/- C57BL/6 mice,and both of the skin and body immunity function were evaluated from organ, cell, and cytokine. The above hypothesis of permeation enhancement and immunity function improvement through the activation of TRPV1 channel will be confirmed. The results will elucidate the possible mechanism underlying the medication from neurobiology, immunology, and percutaneous delivery, which will promote the elucidation of acupuncture percutaneous delivery, benefit the new research approach to external treatment for internal diseases, and set an example for how to use modern molecular biology technology to study the Traditional Chinese Medicine.

哮喘尚无有效的根治方法和药物，西医糖皮质激素及支气管扩张药对症治疗具有严重副作用，而中医白芥子凃方穴位经皮给药防治哮喘具有广泛临床实践基础及确切的疗效，但目前其经皮给药的作用机理尚未明确，限制了该特色疗法的发展。我们推测其作用机理之一可能是通过激活皮肤TRPV1通道而促进药物渗透，同时发挥免疫增效作用。本项目在前期证实穴位对白芥子凃方经皮给药具有促渗和免疫增效作用的基础上，利用免疫组化、激光共聚焦，结合RT-PCR，对穴位与非穴位的TRPV1表达进行比较，并研究该通道激活后皮肤屏障功能及渗透特性，同时比较皮肤免疫启动和机体免疫应答，验证TRPV1激活可促进白芥子凃方经穴渗透且发挥免疫增效作用的假设。研究结果以神经生物学、免疫学和经皮给药制剂理论相结合阐明白芥子凃方给药防治哮喘的作用机理，启发和推动穴位经皮给药的机理探讨，为中医内病外治提供研究思路，并为利用现代技术研究传统中医理论提供范例。

白芥子凃方穴位经皮给药防治哮喘具有广泛临床实践基础及确切的疗效，但其经皮给药药防治哮喘的作用机理尚未明确。我们推测其作用机理之一是通过激活皮肤TRPV1通道而促进药物渗透，同时发挥免疫增效作用。主要研究内容为：（1）白芥子涂方经穴位与非穴位渗透特性的比较研究；（2）穴位与非穴位TRPV1表达的比较研究；（3）白芥子涂方中有效成分激活TRPV1通道的研究；（4）白芥子涂方穴位与非穴位经皮给药的皮肤免疫启动研究；（5）白芥子涂方穴位与非穴位经皮给药的机体免疫调控的比较研究；白芥子涂方穴位与非穴位经皮给药的TRPV1通道激活与药物渗透特性及免疫相关性的探讨。取得的重要结果和关键数据为：（1）以芥子涂方中芥子碱和延胡索乙素为指标，白芥子涂方经穴位给药具有比非穴位显著增加的渗透特性。芥子碱在穴位的累积渗透量和皮肤滞留量分别是非穴位的4.15及2.73倍。延胡索乙素穴位为非穴位的2.89及1.38倍。（2）通过免疫荧光、RT-qPCR及Western-blot研究，证实穴位TRPV1表达显著高于非穴位。（3）白芥子涂方作用于皮肤后，显著上调了穴位皮肤中TRPV1的表达；且通过体外培养背根神经节的Ca2+内流实验进一步证实芥子碱可部分激动TRPV1。（4）通过RT-qPCR证明穴位的免疫神经肽P物质受体和CGRP显著高于非穴位，且芥子涂方作用后穴位P物质受体和CGRP显著升高，启动皮肤免疫和增强经皮肤的免疫作用。（5）在体实验证实穴位皮肤中TRPV1通道被辣椒素激活后，促进了芥子碱和延胡索乙素的经皮渗透，白芥子涂方穴位经皮给药的渗透性增加与穴位高表达TRPV1通道相关。在哮喘小鼠模型中，芥子涂方穴位作用后，较非穴位给药组，芥子涂方显著改善肺组织病理、IgE、Th1/Th2 及Treg/Th17指标；并显著降低哮喘小鼠肺组织中的TRPV1蛋白及SP、CGRP神经肽表达，芥子涂方穴位给药对机体的免疫调控与调节TRPV1的表达相关。科学意义：以TRPV1通道为新的切入点阐明了白芥子涂方穴位经皮给药促渗和调节机体免疫从而预防和治疗哮喘的新机制，为其临床应用提供了客观科学的内涵。