基于整体观（代谢组学结合肠道微生态学）黄连解毒汤“减毒增效”配伍机制的研究

Huang-lian-jie-du decoction is a very famous and classic decoction which has been used to treat damp-heat syndrome patient for more than one thousand years, but the compatibility mechanism of it is still not very clearly. Especially, why Coptis and Evodia used together can increase the effect and reduce the toxicity. Preliminary study has found there was a relation between the compatibility mechanism and the intestinal microflora. Metabonomics in combination with intestinal micro ecology is a new method to study of Traditional Chinese Medicine compatibility mechanism. Previous studies have found that there was a significantly different in metabonomics behaviors and intestinal micro ecology behaviors between damp-heat syndrome disease model control group and intervened model group with Huang-lian-jie-du decoction. Therefore, based on previous research results, through the breakthrough point of "disorder" and "balance" of gut microflora, and based on the combination of "metabonomics" and "microbial molecular ecology techniques (16S rDNA)", the compatibility mechanism of Huang-lian-jie-du decoction will be research in this project. UPLC-MS and GC-MS will be used to achieve the data of the urine, faeces and intestinal contents metabolomics, and the technology of denaturing gradient gel electrophoresis based on the 16S rRNA molecular ecology will be used to get the intestinal micro ecology characteristics. Through principal components analysis, partial least squares discrimination analysis, and pattern recognition, the metabolites and functional bacteria which are covariation and correlation each other will be found. Then, the correlative metabolites and functional bacteria will be identified by the high resolution mass spectrometry, cloning and sequencing technology, respectively. The biological basis which is related to the intestinal microflora "imbalance", "balance" and Huang-lian-jie-du decoction will be achieved. Based on the biological basis, an evaluation system which can be used to evaluate the efficacy and toxicity of Huang-lian-jie-du decoction will be built up. With the using of the evaluation system, the efficacy and toxicity of the different combination of Huang-lian-jie-du decoction on model animal will be evaluated. So as to clarify the reason of Coptis and Evodia are used at the same time can increase the effect and reduce the toxicity and to the compatibility mechanism of Huang-lian-jie-du decoction. At the end, the new method of based on metabonomics in combination with intestinal micro ecology to study the TCM compatibility mechanism would be developed successfully.

本课题以清热解毒且其药效与肠道菌群代谢密切相关的经典名方黄连解毒汤为模型方剂，以肠道微生态的"失调"与"平衡"为切入点，采用基于16S rDNA的微生物分子生态学技术结合代谢组学技术，对该方"减毒增效"配伍机制进行研究。前期研究发现温病湿热证模型对照组和黄连解毒汤干预组的代谢组学行为及肠道中梭菌类菌群行为明显不同。因此，本课题拟采集模型动物尿液、粪及肠内容物的代谢物谱和微生物谱，结合模式识别及偏最小二乘回归法分析，发现相关联及共变化的代谢物与功能菌，并利用高分辨质谱鉴定代谢组，利用克隆及测序技术鉴定功能菌，探明肠道微生态结构与宿主代谢的共变化特征，肠道微生态失衡、平衡与黄连解毒汤配伍相关的生物学基础，构建毒性及疗效评价体系，评价黄连解毒汤不同拆方对模型动物干预中和后的疗效及毒性，阐明其"减毒增效"配伍机制，并最终建立基于整体观（代谢组学结合肠道微生态学）的中药复方配伍机制研究的新方法。

黄连解毒汤是清热解毒的经典名方，其君药味黄连。黄连解毒汤的配伍“增效减毒”的机制有待深入研究。本课题围绕黄连解毒汤及处方中的君药-黄连的调节肠道菌群、调节“肠道菌群-宿主”代谢相互作用开展系列研究。1）利用通用引物和特定引物，建立了基于16S rRNA的肠道优势菌群和特定菌属的PCR-DGGE分析技术，实现了肠道微生物的多样性特征分析；2）利用GC-MS建立了黄连或黄连解毒汤长期干预后的大鼠尿样、血样非靶标代谢组学分析方法；3）利用600 MHz的NMR技术建立了大鼠尿、粪样的非靶标代谢学分析方法；4）利用LC-MS-MS技术建立了大鼠尿、粪、血清中的氨基酸代谢、核酸代谢、单胺类神经递质等37种内源性代谢物的靶标定量代谢组学；5）利用LC-MS-MS技术实现了肠道菌群共代谢物定量代谢组学方法，可用于测定大鼠血清、尿液、粪便中的11种肠道菌群-宿主共代谢产物，实现了三维立体定量研究肠道菌群失平衡状态下的肠道菌群定量代谢组学特征；6）利用SIMCA-P+，Quantity One，Matlab等软件结合PCA、PLS-DA、OPLS-DA、层次聚类分析的化学计量学分析，实现了对代谢组学和PCR-DGGE数据进行生物意义挖掘；以皮尔森相关系数为原理，利用R语言编写相关程序，计算NMR变化代谢物与DGGE变化条带的相关系数值，并统计p<0.05的相关代谢物和条带的关系，实现代谢组学特征与肠道微生物组学特征的关联分析。重要结果：黄连大剂量或黄连解毒汤干预后，宿主及其肠道菌群的代谢都发生了明显的变化，黄连解毒汤的配伍“减毒增效”机制与其调节肠道菌群结构及肠道菌群与宿主的共代谢相关。如经黄连干预后大鼠尿液中葡萄糖、胆碱、甲胺和支链脂肪酸等物质的代谢水平都显著升高，而丙酮酸、组氨酸、马尿酸则呈下降趋势。大鼠粪便中葡萄糖和胆碱也呈现显著升高水平，该现象说明宿主及其肠道菌群的能量代谢均受到干扰；同时发现甘油磷脂代谢，苯丙氨酸代谢，缬氨酸、亮氨酸和异亮氨酸生物合成代谢等5个通路均受到干扰。这就说明黄连及黄连解毒汤可通过调节“宿主-菌群”之间的共代谢发挥功效（药理作用及毒性），并且其肠道菌群结构的变化情况与粪样和尿样代谢波动情况具有一致性。本研究显示代谢物组学结合肠道微生物组学整体观的研究与肠道菌群密切相关的中药复方或单味中药的功效及其机制是一个崭新的研究新方法。