基于“菌-证-方”相应研究不同中医证型泄泻形成及方剂干预的机制

The prescription made in accordance with the symptoms is the key for an objective diagnosis and treatment by Traditional Chinese Medicine (TCM). An objective diagnosis, which is the base of TCM modernization, relies on the use of modern biotechnology in revealing the accordance of prescription/syndrome and identifying the quantitative indexes of syndromes. Intestinal microbiota plays a crucial role in keeping human healthy and changes under different disease conditions. There is a close correlation between diarrhea and the imbalance of intestinal microbiota. As such, we hypothesize that intestinal microbiota influences the syndrome of diarrhea, which determines the corresponding prescription. We propose to establish different animal models of diarrhea with different syndromes following the TCM classification and then treat animals using each corresponding prescription. The dynamic changes of intestinal microbiota during the development of diarrhea with or without the treatment by the corresponding prescription in each animal model will be studied by microbial culture with an attempt to reveal the characteristics of intestinal microbiota. Next generation high-throughput sequencing then will be used to determine the composition and characteristics of intestinal flora and the abundance of different species at different sites of the intestine system. We aim to ultimately build each specific intestinal flora spectrum and find out the characteristic intestinal flora corresponding to each specific syndrome of diarrhea. The current project will reveal the mechanisms underlying the development of diarrhea syndromes and the efficacy of the prescriptions and help develop a useful system of intestinal flora and new strategies for objective diagnosis and TCM treatment of diarrhea with different syndromes, thereby facilitating the modern use of TCM in treating diarrhea.

方证相应是中医诊疗客观化的基础，中医诊断客观化是中医现代化的关键，有赖现代生物技术研究方证相应机制、寻找证的量化指标。疾病及方剂疗效与肠道菌群有关。肠道存在疾病相关特征微生物种类，泄泻与肠道菌群失调有关。我们提出“菌-证-方相应”的假说。本项目拟根据中医对泄泻的分型，建立不同中医证型泄泻动物模型，并通过对应方剂干预，运用微生物培养技术研究各中医证型泄泻形成及方剂干预的肠道菌群动态变化，揭示泄泻形成及方剂干预的肠道菌群动态特征；采用第三代高通量测序检测不同泄泻形成及方剂干预后肠道内容物和黏膜的菌群，分析各中医证型泄泻形成及方剂干预的肠道菌群种类及其丰度变化，探明肠道不同部位的菌群特征，进一步建立肠道特征菌群谱，明确与中医证型泄泻相关的肠道菌群。研究结果有利于揭示中医证型泄泻形成及方剂疗效的机制，建立中医证型泄泻客观化诊疗的肠道菌群体系，为中医证型客观化诊疗提供新的方法和思路，促进中医现代化。

方证相应是中医诊疗客观化的基础，中医诊疗客观化是中医现代化的关键，有赖现代生物技术寻找证的量化指标。泄泻及方剂疗效与肠道菌群有关，存在特征肠道菌群，可作为生物标记物。本项目根据《泄泻中医诊疗专家共识意见(2017)》，从中医证型形成相关因素及方证相应入手，根据泄泻证型的特点，采用相应条件优化建立了脾气亏虚证泄泻、肠道湿热证泄泻、寒湿困脾证泄泻、食滞胃肠证泄泻、肾阳亏虚证泄泻及肝气乘脾证泄泻6种证型泄泻的小鼠模型；分别应用专家共识推荐的参苓白术散、葛根芩连汤、藿香正气散、保和丸、四神丸及痛泻要方进行了干预验证；在各证型泄泻造模及方剂干预后，分别采集肠道内容物及黏膜，提取宏基因组，利用16S rRNA第三代高通量测序检测，分析获得了中医证型泄泻及方剂干预的小鼠肠道优势菌群和特征菌群；研究了肠道菌群与中医证型泄泻及方剂干预机体相关特征指标的关联；进一步研究了肠道菌群代谢产物与证型泄泻及方剂干预的关系；首次建立了肠道微生物活度的研究方法，并研究了6种证型泄泻造模及方剂干预的肠道微生物活度；考究了性别的肠道菌群特征；运用肠道菌群失调与泄泻的关系探讨了“同病异治”的机制。结果发现肠道菌群存在性别二态性；不同证型泄泻及方剂干预分别存在肠道优势菌群和特征菌群；肠道内容物和黏膜的菌群不同；肠道菌群对“同病异治”机制的阐明具有很好的价值，为肠道菌群作为中医诊疗客观化指标研究提供了重要实验依据。同时，本项目还研究了造模各因素对肠道菌群的可能影响，为肠道菌群的中医临床诊疗应用提供了思路。项目发表论文23篇，其中SCI收录12篇（中科院二区5篇）；学术交流18人次，学术报告3次；毕业博士生3名、硕士生4名。研究结果有利于揭示中医证型泄泻形成及方剂疗效的机制，建立中医证型泄泻客观化诊疗的肠道菌群体系，为中医证型客观化诊疗提供新的方法和思路，促进中医现代化。