宣降肺气法调控TRP介导免疫细胞分化干预PM2.5加重哮喘的机制研究

Dispersing and descending the lung qi is one of the main treatments for asthma, with great significance to syndrome differentiation and treatment in the more and more serious respiratory diseases resulting from enviromental problems nowadays. Based on the previous study, in conditions of asthma exacerbated by environment pollution such as PM2.5,etc., dispersing and descending the lung qi principle is characterized as the effect of adjusting cascade reaction of enviromental pollution-TRP channel-immune system, T-TRP channels are regulated to influence the T lymphoid cells differentiation and subsequently lead to immunoreaction of the proinflammatory cytokines, etc., thus the up and down movement of lung qi is maintained the common basis of diffusing the lung and relieving panting is built up. Research path: the dispersing and descending lung qi fomulas such as San’ao decoction, its derivative Wu’ao decoction and their effective substance group were selected, their interventional efficacy pattern on the asthma induced by PM2.5 was observed, as well as the T-TRPs expression in CD4+T cell ,the activation and function of the CD4+T immunocyte regulated by T-TRPs and the relationship between the proinflammatory cytokines and neuropeptide release in vitro and vivo. The animal and cell models with gene silencing and T-TRPs related gene knock out methods were used to verify the interactive relationship with the differentiation and function of CD4+T cell, the efficacy contribution of the dispersing and descending compatible principle and its main contributing substance were analyzed, according to the effective substance and metabolism analysis. The successful implementation of this research will disclose the scientific nature of treating exacerbated asthma by dispersing and descending lung qi, bring positive impact on the modernization of TCM theory.

宣降肺气法是治疗咳喘的主要治疗方法之一，对于现今日趋严重的环境问题所致呼吸病的辨治具有重要意义。在前期研究基础上，提出宣降肺气法干预环境污染物如PM2.5等诱导加重哮喘，具有调节环境污染物刺激-TRP离子通道-免疫系统级联反应的作用特征，通过TRP调控T淋巴细胞分化、以及随后促炎细胞因子等分泌，产生维持肺气升降的整体作用，是为宣肺平喘法的共性效应基础。研究路径：体内外分别观察宣降肺气代表方三拗汤及衍化方、方剂功效物质干预PM2.5诱导哮喘的效应特点、CD4+T淋巴细胞的T-TRPs的表达、经由T-TRPs调控的CD4+T分化和功能作用，以及促炎细胞因子分泌；以T-TRPs相关基因敲除及静默动物及细胞模型验证与免疫细胞分化及功能的关系；结合功效物质和代谢，分析宣降配伍的主要贡献物质。该项目的成功实施，将揭示宣降肺气治疗环境因素诱导加重哮喘的科学内涵，对中医治法理论现代化研究具有积极意义。

目的：探讨宣降肺气方药对空气污染物及病原物加重哮喘的干预作用，及经由调控TRPs的神经-免疫交互机制。.方法：体内外分别观察宣降肺气代表方剂三拗汤及加味方、麻杏石甘汤及组方部分等干预OVA诱导复合PM2.5暴露、RSV暴露等加重哮喘的效应，对肺和气道结构细胞、免疫细胞CD4+T中TRPs基因和蛋白表达，以及炎症细胞因子、神经因子和介质水平的影响，分析TRPs通道在神经-免疫交互中的作用；以trpa1,trpv1基因敲除小鼠及基因沉默细胞模型对TRPs通道活化功能和影响进行验证。.结果：空气污染物PM2.5暴露可诱导加重哮喘气道炎症、气道高反应性，肺组织及免疫细胞CD4+T细胞均有TRPs基因和蛋白高表达，同时TH2类细胞因子及神经因子和介质水平增高；trpa1、trpv1基因敲除模型则表型不显著。SO2衍生物、香烟烟雾（CS）等污染物及RSV也具有诱导加重哮喘模型的作用。宣降肺气组方配伍的代表性方剂三拗汤及加味方、麻杏石甘汤分别干预OVA诱导哮喘、咳嗽变异性哮喘模型及复合环境污染物PM2.5暴露加重哮喘模型、RSV暴露加重哮喘模型后，可缓解气道炎症、降低气道高反应性、改善肺组织病变，降低BALF中TH2相关炎症细胞因子IL-13、IL-4等水平、神经因子及介质SP、PGD2、NGF等水平，调控TRP介导免疫细胞分化。体外实验见PM2.5暴露加重哮喘模型的脾CD4+T细胞具有TRPA1高表达；麻黄甘草主要成分配伍、麻杏石甘汤及组方部分含药血清可降低IL-4、及IL-4复合RSV处理的16HBE中TRPA1、V1表达及钙离子内流，shRNA干扰技术构建TRPV1基因沉默细胞则钙离子浓度无明显变化。.结论：宣降肺气方药在致敏物、环境污染物、病原物等条件下，通过快速对组织结构细胞、免疫细胞的神经感觉通道的调控，以及随后的神经免疫交互调节，产生维持肺气升降的整体作用，是为宣肺平喘的共性基础。