基于TRP离子通道调控的麻杏配伍方稳定气道功能的机制研究

Formulas with the compatibilible pair of Ephedra and Almond are the commonly used ones for opening lung to send down the ascending qi and releasing asthma.Based on the previous study, it modifies the nerve-immune interaction TRPA1, TRPV1 channel, reduces incoming extracellular information, and prevents recruitment of inflammatory cells and cellular structure interaction, environmental mitigation, inflammatory stimuli and oxidative stress-related proteins to maintain a stable airway function, lung compliance and to improve the body's defensive ability.Research path:Taking San'ao Decoction and Maxing Shigan Decoction, which are the representative formulas treating asthma by opening lung and clearing lung, as an example, it activates small molecule chemicals TRPA1,TRPV1, reduces the impact of calcium influx, associates the level of neuropeptide, inflammatory mediators, chemokines and cytokines expression to reduce inflammatory pathology, the biological characterization of airway reactivity and maintain the airway stability; it also takes TRPA1 and TRPV1 gene deletion and selective antagonist to compare the relationship between the expression and function in vitro cell structure, to explain the contribution declared down lung airway function and stability of substance and metabolic characteristics, combining features in different environments to provide more comprehensive in-depth knowledge of the scientific connotation of treating asthma by opening lung in different compatibility environment.Finally,it takes the advantage of the treatment of traditional Chinese medicine under the condition of increasingly serious environmental problems.

麻杏宣降肺气是平喘止咳方的典型配伍，具有抑制气道炎症和气道高反应的作用。在前期研究基础上对环境刺激物造成气道受损的治疗机理进行探索，提出麻杏配伍方剂是在损伤性因素袭肺时，通过调控神经-免疫交互应答的环境感觉离子通道TRPA1、TRPV1，减少细胞外信息传入，形成神经-免疫共调节，发挥维持气道功能稳定、提高肺顺应性的机体防御作用。研究路径：以麻杏配伍宣肺和清肺平喘代表方剂三拗汤、麻杏石甘汤为例，体内外分别观察其调控小分子化学物质对TRPA1、TRPV1活化，降低钙离子内流、神经肽释放和炎症介质表达情况，并用TRPA1、TRPV1基因敲除动物及细胞模型反证该通道与效应的关系，评价其改善气道炎症、降低气道高反应从而维持气道稳定的作用；结合功效物质和代谢特点，比较不同配伍环境的共性和差异，更加全面深入地认知中医宣降肺气平喘的科学内涵，发挥在环境问题日趋严重情况下的中医治疗优势。

目的：探讨麻杏配伍代表方宣降肺气干预哮喘气道变应性炎症及气道高反应性的TRPs离子通道调控机制。.方法：建立环境污染物（如PM2.5,TMA）诱导加重型小鼠哮喘模型；以3张麻杏配伍经典方剂为代表分别干预OVA模型及PM2.5诱导加重型小鼠哮喘模型，综合评价其改善气道炎症、降低气道反应性、调控细胞因子和神经递质水平的作用；qPCR，WB检测小鼠肺TRPA1、TRPV1基因和蛋白表达，并与TRPA1,TRPV1 基因敲除小鼠比较，以及体外检测IL-4/TRPA1激动剂致细胞钙离子内流、免疫磁珠法分选CD4+细胞中TRPA1、V1基因与蛋白表达验证该通道与效应的关系；UPLC-TOFMS检测麻杏配伍代表方代谢组和分布特点。.结果：(1) PM2.5、TMA复合OVA均可诱导加重哮喘气道变应性炎症及气道高反应性，包括加剧外周血和BALF中EOS等炎细胞量增高、RI和Cdyn显著变化、严重肺损伤，IL-4,IL-13,SP,PDG2和NGF水平升高，更高的TRPA1, TRPV1等基因和蛋白表达（与单纯OVA组比P<0.01-0.05）。(2)麻杏配伍代表方对PM2.5复合OVA诱导加重小鼠哮喘模型具有降低气道炎症和气道阻力的共性作用，显著降低BALF中EOS及白细胞总数与气道阻力、改善肺病理（与模型组比P<0.01-0.05）。(3)麻杏配伍代表方明显改善小鼠哮喘气道变应性炎症与气道高反应性，调控TRPA1、V1基因和蛋白表达，降低BALF中细胞因子IL-13、IL-4，神经递质SP、PGD2、NGF等水平（与模型组比较P<0.01-0.05）；TRPA1-/- 、TRPV1-/- 小鼠BALF 中炎细胞及气道阻力与空白组比则未见统计学差异。(4)该通道具有调控免疫-神经关联作用，分选小鼠模型脾淋巴细胞CD4+细胞显示TRPA1、V1基因和蛋白高表达（与对照组比P<0.01）；IL-4/TRPA1激动剂可致细胞钙离子内流升高、并受到麻黄等药味有效成分配伍调控（与模型组比较P<0.01-0.05）。(5)UPLC-TQ/MS分析见麻杏配伍方呈现整体复杂网络调控的特点，且有效成分具有一定的肺组织富集特点。.结论:麻杏配伍方经由TRPA1、TRPV1的神经-免疫调控是抑制气道炎症、降低气道反应性、稳定气道功能的重要机制。