肠道菌群介导鞣花酸生物转化尿石素类物质的作用机理研究

Urolithins, metabolites produced from ellagic acid, are the key material basis for its physiological function in vivo. Previous studies showed that there are significant interindividual variability in urolithins produced by gut microbiota after ingestion of ellagic acid, which resulted in the individual differences in its physiological activities. In order to elucidate the biotransformation pathway of ellagic acid and the relationship between gut microbiota, C57BL/6J HFA mouse model of three metabotypes for urolithins will be created by transplanting human fecal flora of UM-A, UM-B and UM-0 to germ-free mice, and the bidirectional regulation process of intestinal flora and ellagic acids will be controlled in vivo. Intestinal microflora dynamic changes before and after feeding ellagic acid will be clarified using macrogenome sequencing, and the key gut microbiota will be found. The genes differentially expressed in three groups will be determined by RNA-seq and RT-PCR, and the metabolites in plasma and urine will be analyzed by HPLC-MS and NMR. The gut microbiota-the differentially expressed genes-metabolites correlation from the overall level will be explored. The ellagic acid metabolic pathway by gut microbiota of three phenotypes will also be studied in vitro. The key gut microbiota and metabolic pathways for urolithins production will be clarified. The results will enrich the theoretical basis of the interaction of polyphenols and gut microbiota.

尿石素是鞣花酸体内转化生成的主要生理活性物质，研究发现，肠道菌群转化尿石素的差异导致了鞣花酸生理活性的个体差异。为明确鞣花酸转化的关键肠道菌类群及其与鞣花酸的互作机制，本项目拟通过建立UM-A、UM-B和UM-0三种尿石素代谢表型的人源肠道菌群（HFA）小鼠模型，在体分析肠道菌群与鞣花酸转化的互作过程，采用宏基因组测序分析摄食鞣花酸前后不同代谢表型HFA小鼠肠道菌群的动态变化，明确转化鞣花酸的关键肠道菌群；利用转录组测序结合RT-PCR分析关键菌群的基因表达与尿石素转化的关系，结合血浆、尿液鞣花酸代谢产物分析，明确关键菌群转化鞣花酸的代谢途径；围绕肠道菌群结构-差异表达基因-代谢产物分析，结合鞣花酸代谢途径的体外验证实验，揭示鞣花酸机体代谢的关键肠道菌群及尿石素生成途径与差异机制。研究结果将丰富多酚类物质与肠道菌群互作的理论基础。

三种尿石素代谢型人群个体间尿石素类物质的生成能力及肠道菌群组成具有显著差异，UM-0型肠道菌群的丰富度及多样性均显著低于UM-A及UM-B，UM-A、UM-B及UM-0的F/B比值分别是48.67、48.72以及116.76。在属水平上，UM-A和UM-B的阿克曼氏菌、戈登氏菌属、克里斯滕森菌属、鞘氨醇杆菌、埃格特菌属、Paludicola的相对丰度显著高于UM-0。UM-A的Paludicola和Acetanaerobacterium的相对丰度显著高于UM-B。戈登氏菌属等12个菌属与尿石素总代谢量呈显著正相关；Moryella、克里斯滕森菌属等27个菌属与尿石素A代谢量呈显著正相关；Raoultibacter、光冈菌属等14个菌属与尿石素B代谢量呈显著正相关。成功构建了三种代谢型HFA小鼠模型，EA对三种代谢型HFA小鼠肠道菌群的富集作用具有显著差异，提高了疣微菌门在UM-A及UM-0小鼠的相对丰度、降低了厚壁菌门在UM-0的丰度。在属水平上，EA显著升高UM-A小鼠的阿克曼氏菌属、戈登氏菌属、Flavonifractor、埃格特菌属和乳杆菌属相对丰度，且与尿石素A含量呈显著正相关；显著提高UM-B小鼠的戈登氏菌属和乳杆菌属的相对丰度，以及UM-0小鼠的阿克曼氏菌属的相对丰度，推测上述菌属是EA代谢生成不同尿石素类物质关键菌属。KEGG通路分析表明EA主要影响UM-A小鼠DNA修复和重组蛋白、氨基酸相关酶、碳水化合物代谢、黄酮生物合成等；UM-B的脂肪酸生物合成、脂质合成蛋白和能量代谢等以及UM-0的脂质合成蛋白、脂多糖合成等代谢通路。利用UPLC-MS/MS定性定量分析HFA小鼠血浆、粪便及组织中EA代谢产物，结合体外培养实验，主要检测到9个不同的代谢转化产物，结合文献完成EA转化生成不同尿石素类代谢途径分析。EA对高脂饮食三种尿石素代谢型HFA小鼠的糖脂代谢、炎症和氧化应激水平及肠道菌群构成影响具有显著差异，与UM-B/0小鼠相比，对UM-A小鼠肥胖的干预效果明显，通过血清代谢组学和肠道菌群高通量测序数据关联分析初步揭示EA对三种代谢型HFA小鼠抗肥胖作用的差异机制。研究结果丰富多酚类化合物与肠道菌群互作个体差异的理论基础，为多酚类化合物的开发应用和精准营养提供理论基础。