基于噬菌体扰动研究小鼠结肠菌群的膳食调控机制

It will be a important direction of food nutrition to keeping health though dietary regulation of colonic flora. Phage is the shaper of the gut microbiota, however, kill-the-winner dynamics model of the relationship between the phage and bacteria is not exist in the health individual. Phage activity would be inactived by dietary polyphenols at lower concentrations, and this would be a key factor to protect the colonic microflora balance. In this study,colon dominant populations Enterococcus faecalis and Escherichia coli will be isolated and purified.Firstly, the phage activity would be test by polyphenols in vitro; Furthermore identification of phage, dual culture and qPCR would be used for analysis flora diversity and dominant populations quantities after the bacteira in mice faces attacted by phage. Then, the diversity of phages and gut flora in the colon will be analysised by the FLX454 sequencing technology after themice intake different concentrations of polyphenols, so as to prove dose-dependance of colon phages and dominant flora to tea polyphenols diet and its relevance. Relationship of tea polyphenols-phage-colonic microflora and the mechanism of the of colonic microflora regulation by phages though dietary regulation would be revealed.

结肠菌群的膳食调控将是未来食品营养的一个重要研究方向。噬菌体是结肠菌群结构的塑造者，但健康个体结肠中噬菌体-宿主细菌Kill-the-winner动力学模型并不常见。膳食多酚在较低浓度下可钝化噬菌体活性，极有可能是解释噬菌体结肠侵染悖论的关键因子。本研究通过结肠优势种群粪肠球菌和大肠杆菌噬菌体对宿主菌（纯培养）、小鼠粪便体外发酵（菌群多样性环境）以及小鼠结肠菌群（兼具菌群多样性和免疫环境）进行攻击实验，获得结肠环境膳食多酚干扰噬菌体侵染循环假说的实证证据；同时，采用FLX454测序获得的结肠噬菌体与细菌宏基因组深度测序数据分析噬菌体和结肠菌群结构变化，并结合qPCR技术验证优势种群的数量变化，明确噬菌体侵染受茶多酚采食影响的量效关系，获取噬菌体和结肠菌群结构之间的对应关系，在此基础上揭示茶多酚-噬菌体-结肠菌群三者的相关关系，阐明膳食多酚通过噬菌体调控结肠菌群结构的膳食调控机制。

噬菌体是肠道菌群重塑的重要影响因子，当膳食组分发生改变也会引起肠道噬菌体组成变化，从而影响结肠菌群结构，遗憾的是我们对膳食组分如何调控肠道噬菌体的作用机制知之甚少。本研究以茶多酚为研究对象，通过多组学技术手段，探究基于肠道噬菌体的变化研究茶多酚对肥胖小鼠脂质代谢的调控作用机制，对引导食品科学加工及国民健康饮食方式均具有重要的理论与现实意义。 .通过体外实验确定了肠道优势噬菌体的最佳感染复数和适宜侵染的pH值以及GTP的最适干预剂量1.0mg/mL。该浓度的GTP对SPF小鼠结肠Muribaculaceae、Akkermansia相对丰度分别下调23.82%和99.70%，对Faecalibacum和Bifidobacterium分别上调24.38%和89.78%；对噬菌体Salmonella-phage、Catovirus_CTV1 和Mycobacterium_phage_Misomonster分别上调98.64%、98.94%和98.36%，对Gordonia_phage_Yakult、Streptomyces_phage_Shawty 和 Staphylococcus_ phage_phiSP15-1分别下调99.99%、88.34%和87.97%。该浓度的GTP可显著改善肥胖小鼠的炎症水平，降低Firmicutes、Enterobacter和Siphovirida、Enterobacteria phage SfVe的相对丰度，增加Bifidobacterium choerinum和Bifidobacterium sp. AGR2158的相对丰度。结合肠道代谢组学分析发现，GTP可通过调控噬菌体，重塑肠道菌群结构从而产生不同代谢产物。.利用网络药理学分析发现Enterobacter cloacae phage和GTP可促进肥胖小鼠的脂质代谢的关键作用靶点是MAPK信号通路。Enterobacter cloacae phage有效降低HFD小鼠的体重及脂质重量并显著改善血脂炎症水平，显著降低了Enterococcaceae和增加了Akkermansiaceae等肠道细菌的相对丰度，并降低了ERK、P38、JNK和myc蛋白表达水平，揭示了GTP可能通过调控噬菌体进而改变结肠菌群结构，从而通过抑制MAPK炎症信号通路的激活，调控肥胖小鼠的炎症水平和脂质代谢。