基于组学策略研究EGCG在人源菌群小鼠结肠的代谢通路

The emerging research has spotlighted the interaction between dietary polyphenols and colonic flora. However,relatively little is know about the mechanism of the interaction due to the complexity of the intestinal flora and the diversity of its metabolism. This research aims to intervene the gut microbiota with epigallocatechin gallate (EGCG) in human-associated mice (HFA). Metabonomics of phenolics were analyzed in this intervention. Then, a possible metabolic pathway of EGCG was built, based on former studies, using molecular simulation. In the meanwhile, differentially expressed genes were determined by metatrascriptomics analysis. These genes were tested in the Kyoto Encyclopedia of Genes and Genomes (KEGG) to find a best math using approximate matching algorithm. In addition, key functional genes were marked by screening the functional genes. Based on the newly built pathway, BLAST, and existing research data, key flora were screened out among those that were up-regulated after EGCG intervention. The selected flora were separated by EGCG enriched and pure culture technology to investigated the interaction among gut flora, key genes, and metabolites. This interaction may further reveal how dietary polyphenol may affect human's health through gut microbiota.

膳食多酚与肠道菌群的相互作用及其对健康的影响已成为膳食与健康领域的热点问题。然而，由于肠道菌群的多样性和代谢的复杂性，导致我们对于膳食多酚在结肠与菌群互作知之甚少。本研究采用EGCG对人源菌群小鼠结肠菌群进行干涉，对酚类物质的代谢组学进行分析，然后通过分子模拟，结合前人研究结果构建EGCG的可能代谢途径；并用宏转录组学技术分析获得差异表达基因，采用近似匹配算法进行数据挖掘，在KEGG的代谢网络中寻找距离最近的最佳匹配，进而结合上调的表达基因的功能注释筛选关键功能基因，完成EGCG代谢通路草图的绘制。在此基础上，结合BLAST比对分析和原有研究基础，在受EGCG干涉后上调的菌群中，筛选可能的关键菌群，并通过EGCG体外富集和纯培养技术分离部分相关菌群进行验证，阐明结肠菌群-关键基因-代谢产物之间的相互关系，为进一步揭示多酚通过与肠道菌群互作影响人体健康的作用机制打下基础。

本研究通过构建HFA小鼠模型，分别饲喂普通纯化饲料和粗饲料，发现粗饲料饲养小鼠肠道菌群与人粪便菌群结构更相似。通过高脂纯化饲料构建HFA肥胖小鼠模型并采用EGCG膳食干预，发现EGCG以浓度依赖性方式促进脂肪代谢及肝脏损伤修复，并显著改变了肥胖小鼠肠道菌群结构，其中0.8%EGCG处理后，Peptostreptococcaceae科及 Romboutsia和Blautia属细菌明显增加。通过HPLC-MS分析代谢物差异，发现采用0.8%的EGCG干涉8周后，小鼠粪便中除了2.5min出现EGC物质峰外，在14-23.5min期间还出现了5-(3,5-dihydroxyphenyl)-γ-valerolactone 等7个不同的物质峰。肠道菌群主要介导EGCG最终转化成γ-戊内酯、戊酮酸、戊酸类等物质。.采用黑曲霉RAF106菌株对EGCG进行体外生物转化研究发现，酵母粉可显著促进EGCG生物转化，而葡萄糖起到显著抑制效果，这种结果可能是通过影响RAF106菌株单宁酶活性实现的，加入酵母粉24 h后，单宁酶活性是对照组的3.1倍，而葡萄糖加入后对其影响不大，继续发酵至48h和96h后，酶活则分别下降49.46%和32.67%。将RAF106与EGCG在YE培养液共孵育120h，代谢组测序分析发现EGCG被转化为6-Methoxyluteolin 3'-glucoside等6种黄酮类代谢物。经转录组分析发现，EGCG处理后共有1394个差异表达基因，其中上调653个，下调741个。利用KEGG分析其代谢或信号途径，发现这些差异基因主要与代谢、遗传信息加工、信号传导、细胞过程及发育系统密切相关。.研究发现EGCG诱导启动了黑曲霉cdc42-ste11-Fus3这条MAPK信号相关途径的表达，Rga1、Cdc42、Ste11、Fus3/MpkB、Ste50、Slt2/Spm1均明显上调，磷脂酰肌醇信号途径的Plc1、mTOR信号途径的NPRL2均下降。利用基因操作技术，获得MAPK激酶Fus3编码基因fus3敲除株Δfus3，通过与野生菌株生长及EGCG转化比较，证实EGCG是通过激活Fus3-MAPK级联通路调控黑曲霉生长，且该级联通路正向调控黑曲霉对茶多酚生物转化过程。