基于“肠道菌群-短链脂肪酸-肺部调节性T细胞”路径研究温肺化饮法治疗咳嗽变异性哮喘的分子机制

The method of “warming the lung to resolve fluid-retention" is a common traditional Chinese medicine treatment method for the pathogenesis of "phlegm and fluid incubating the lung", which follows Zhang Zhongjing's principle of "phlegm and fluid should be treated by warm medicine ". It is effective for treating multiple pulmonary diseases, but the mechanism of its action is not clear yet. The results of the two National Natural Science Foundation projects we completed earlier showed that the “warming yang to eliminate fluid method”(tend to warm the spleen), which also followed the principle of "phlegm and fluid should be treated by warm medicine ", took actions by multiple targets and links. The mechanism of eliminating pleural effusion (fluid-retention in anocoelia) in guinea pigs was revealed at molecular level. On this basis, according to the theory of "the lung and large intestine being interior-exteriorly related " in traditional Chinese medicine and "lung-gut axis" in modern medicine, we select cough variant asthma (phlegm and fluid incubating the lung) as the research carrier, and use the methods of advanced and mature 16s rRNA gene analysis, metagenomics, high performance liquid chromatography (HPLC) and flow cytometry to analyze the changes in the following five stratification planes : intestinal flora, short chain fatty acids, lung regulatory T cells, bronchial tissue cells morphology and symptoms and signs. It is of great scientific significance and clinical guiding value to intensively study the molecular mechanism of the treatment of “phlegm and fluid incubaing the lung“ by “warm the lung to resolve fluid-retention method”, in order to explain the scientific connotation of the principle of "warming medicine regulating method” in treatment of phlegm and fluid disease in an all-round and systematic way.

温肺化饮法是遵循张仲景“病痰饮者，当以温药和之”的治本原则制定的一种针对“痰饮伏肺”病机的常用治法，治疗多种肺系疾病疗效肯定，但其作用机制尚不清楚。我们前期完成的两项国家自然科学基金项目结果显示，同属“温药和之”原则指导下的温阳消饮法（侧重于温脾）通过多靶点、多环节调节，从分子水平揭示了其消除模型豚鼠胸腔积液（饮停胸腔）的作用机制。基于此，本项目根据中医“肺肠相合”和现代医学“肺-肠轴”理论，选择发病特点与“痰饮伏肺”有关的咳嗽变异性哮喘作为研究载体，采用先进、成熟的16SrRNA基因分析、宏基因组学、高效液相色谱和流式细胞检测等技术与方法，从肠道菌群-短链脂肪酸--肺部调节性T细胞--支气管组织细胞形态--症状体征变化五个层次，深入研究温肺化饮法治疗“痰饮伏肺”的分子作用机制，以期全面系统地阐释“温药和之”治本原则治疗痰饮病的科学内涵，具有重要的科学意义和临床指导价值。

-温肺化饮法是针对痰饮蕴肺引起的咳嗽、胸满、咯白痰的肺系病的一种治法。对该法作用机制的研究多围绕射干麻黄汤、小青龙汤展开，对同类治法的苓甘五味姜辛汤则研究较少。该方较前两方温肺化饮之力相似，却无麻黄之平喘、桂枝之助阳，故宜于寒饮在肺，以咳嗽为主偏虚者。本项目以“痰饮蕴肺”有关的咳嗽变异性哮喘为载体，苓甘五味姜辛汤为代表方，从肠道菌群-短链脂肪酸--肺部调节性T细胞—肺支气管组织形态--症状体征变化五个层次研究其分子机制。.研究发现，CVA模型豚鼠的咳嗽敏感性增加，温肺化饮法可减少其咳嗽敏感性，降低肺泡灌洗液中嗜酸性粒细胞百分比，减轻肺组织病理损伤。其机制可能与降低豚鼠肺组织TRPA1、TRPV1蛋白与TRPV1 mRNA表达有关。. 研究发现，CVA 模型豚鼠肺组织 Treg/Th17 免疫平衡失常、JAK1/STAT6 通路上调。该法升高肺组织Foxp3 mRNA及蛋白表达，下调Th17 特异性转录因子RORγtmRNA、JAK1 mRNA及蛋白、STAT6 mRNA及蛋白的表达，改善模型豚鼠上述免疫失常。以上可能为该法治疗 CVA 的机制之一。 . 研究发现，CVA模型豚鼠肠道内容物短链脂肪酸乙酸表达水平升高，其肺部特异受体GPR43 mRNA表达降低。温肺化饮法能降低乙酸表达水平，升高GPR43 mRNA表达水平。. 研究发现，CVA 模型豚鼠肠道菌群结构显示出差异，部分菌种丰度明显增加，另一部分菌种丰度显著降低，温肺化饮法则使其增加的这部分菌种丰度降低，而降低的这部分菌种丰度增加。.本研究从多个层面揭示了温肺化饮法治疗“痰饮蕴肺”的分子机制，为科学阐释该治法的有效性与作用机理提供了实验室依据，为临床拓展该治法的应用范围提供了参考。故该课题研究成果具有较高的学术价值和临床实用价值。