结肠菌群介导大豆异黄酮生物转化S-雌马酚的作用机理研究

The soy isoflavone is activated to S-equol exclusively by colonic flora. S-equol shows a wide range of potential health benefits by possessing estrogenic activities and anti-oxidant properties. However, 40-70% of healthy adult persons aren’t able to produce S-equol following ingestion of soy isoflavone. The metabolism mechanism between soy isoflavone and colonic flora is poorly understood because of the diversity and complexity of intestinal flora. In this study, C57BL/6J HFA mice model will be constructed by planting equol producer stool suspension and then soy isoflavone will be used to interfere with the colonic flora of model mice. The up-regulated dominant bacteria are supposed to be screened out by analyzing change rules of colonic flora structure using metagenome high-throughput sequencing technique. The key functional genes modulated in the process of soy isoflavone metabolism are planned to be screened out by functional annotation of differentially expressed genes by using macro transcriptome RNA-Seq technology. The pathway of biotransformation from soy isoflavone to S-equol will be confirmed based on associated analysis of metabolites group and functional genes. On this basis, the related bacteria will be isolated for confirmation through vitro enrichment and pure culture step by step. The interference relations among colonic flora, key functional genes and metabolism products will be clarified and the mechanism of biotransformation of soy isoflavone to S-equol will be revealed. This project will provide a theoretical basis for supporting human health by studying the interaction mechanism between dietary composition and colonic flora.

S-雌马酚（Eq）是大豆异黄酮（SI）经结肠菌群代谢产生的微生物转化物，其类雌激素活性及抗氧化作用介导了广泛的健康生物学效应，但40-70%人群不能将SI有效生物转化为Eq。 由于肠道菌群的多样性和复杂性，导致我们对SI在结肠与菌群互作代谢机制知之甚少。本课题拟采用Eq产生者粪便构建C57BL/6J HFA模型小鼠，用SI对模型小鼠结肠菌群进行干涉，利用宏基因组高通量测序技术对结肠菌群结构变化规律进行分析，筛选获得上调的优势菌群种类；利用宏转录组RNA-Seq技术获得差异表达基因，通过功能注释筛选SI代谢过程中的关键功能基因；结合代谢物组关联分析推定SI在小鼠结肠代谢Eq的通路；在此基础，通过体外富集和纯培养技术分离相关菌群进行逐步验证，阐明SI代谢过程中结肠菌群-关键基因-代谢产物间的相互关系，揭示结肠菌群调控SI生物转化Eq的作用机理，为膳食与肠道菌群互作影响健康作用机制研究奠定基础。

膳食摄入大豆异黄酮与人体健康有密切关系。但由于结肠菌群结构和功能的差异，只有 30-60%的人群能够对大豆异黄酮进行有效生物转化。为了探究大豆异黄酮在肠道内的生物转化机理，本研究利用抗生素干预及粪菌移植技术，构建了人源菌群小鼠模型，利用组学技术结合代谢产物分析，对大豆异黄酮代谢通路进行了关联分析，在此基础通过体外富集和纯培养技术分离产雌马酚菌株，对代谢途径进一步验证。研究结果表明：1）大豆异黄酮转化为S-雌马酚过程中，拟杆菌门（Bacteroidetes）、拟杆菌科（Bacteroidaceae）和普雷沃氏菌科（Prevotellaceae）、拟杆菌属（Bacteroides）和普雷沃氏菌属（Prevotella）、Akkermansia和Adlercreutzia是关键生物转化菌属。2）中剂量大豆异黄酮能显著增加Clostridium的相对丰度，从而增加碳水化合物代谢中EC 2.7.1.40和EC 2.3.1.9的分泌。作为碳代谢的辅助途径，碳水化合物代谢中关键调控因子的分泌促进了碳代谢中EC 5.1.99.1和EC 1.3.5.4的分泌，从而影响雌马酚的产生。通过宏转录组学验证得知，EC：2.7.1.40确定能作为关键辅助酶影响大豆异黄酮生物转化。3）在两种不同益生元干预下，EC：2.7.1.40被发现同时具有上调和下调基因作用，且复配的合生元可以显著提高低产小鼠血浆中雌马酚的浓度。4）经体外选择性分离和纯培养，得到一株雌马酚产生菌Y11，此菌株属于Slackia属，菌株含有表达二氢大豆苷元还原酶、四氢大豆苷元还原酶的基因，对大豆异黄酮的代谢转化率达到56%。以上研究对进一步“塑造”结肠菌群，调控大豆异黄酮的生物转化过程，提高其在机体的生物可利用度，促进人体健康具有重要意义。