肠道寄生菌群通过调节胆汁酸代谢促进2型糖尿病致病的分子生物学机制研究

Owing to high throughput second generation sequencing technique, metagenomics studies on relevant of intestinal microbiome with human diseases have been soared in recent years. Type 2 Diabetes Mellitus (T2DM) is suggested to relate with intestinal dysbiosis. Whereas, latest studies reveal the role of antidiabetic medicines in regulating intestinal microbiome symbiosis. Our previous work, based on a randomized, multicenter, interventional clinical study, has investigated the metagenomics changes of Acarbose treatment could bring to T2DM patients intestinal microbiome. To our surprise, substantial taxonomy and functional alteration in microbiome were found after use of Acarbose but not use of Glipizide. Strong relevant of clinical benefits, bile acids profile in particular, to microbiome changes were considered to a previous unappreciated mechanism underlying the hypoglycemic effects as well as metabolic benefits of Acarbose. Besides, the taxa found increased in T2DM intestinal microbiome in previous reports were decreased by treatment, suggesting their roles in pathogenesis of T2DM. And these taxa are significantly related to secondary bile acid metabolism. To test the above hypothesis, we designed to set up a proof of principle study on 1, Diet induced obesity(DIO) mice with Acarbose treatment after antibiotics treatment or FXR ablation. 2, DIO mouse transplanted with the two candidate T2DM pathogens: Clostridium Bolteae, Bilophila Wadsworthia after antibiotics treatment with or without FXR signaling. By phenotyping above mouse models with metabolic features as well as by means of metabolome, metagenomics, biochemistry and molecular biology to identify potential molecular pathway mediated by intestinal bacteria to regulate host glucose metabolism via bile acid signaling. Finally, we will replenish the expression of FXR in an organ specific way to delineate the molecular pathway of bacterial/bile acid signaling in promoting T2DM.

宏基因组学研究发现人体众多疾病与肠道寄生菌群失衡有关，2型糖尿病（T2DM）位列其一。最新报道提示口服降糖药物可能会改变肠道菌群组分。因此现有数据无法定位真正T2DM"致病菌”. 本课题前期工作利用随机入组未用药的初发T2DM患者进行临床多中心干预研究，结合宏基因组结果具体描绘口服降糖药物对肠道菌群的影响并找到几株候选致病菌种，并提出肠道菌群的变化可能通过胆汁酸代谢调节宿主糖代谢。为验证候选致病菌及此机制假设，我们将利用广谱抗生素进行肠道除菌结合胆汁酸受体FXR敲除高脂高糖喂养小鼠模型，验证肠道寄生菌通过胆汁酸信号介导口服降糖药的疗效以及T2DM致病机制。于FXR敲除小鼠肠道内种植候选菌种鲍氏梭菌,沃氏嗜胆菌，通过器官特异恢复FXR的表达观察两株菌改变小鼠表型及相关分子信号是否为特定器官胆汁酸信号所调控。通过本课题望能确定1-2株糖T2DM致病菌，阐明肠菌-胆汁酸-T2DM的病理生理机制。

在不依赖体外培养技术的情况下宏基因组研究进展神速，发现数以万计的肠道内微生物种群与多种人类疾病相关，包括自身免疫疾病，肿瘤，精神疾患以及包括2型糖尿病在内的代谢性疾病。肠道共生菌群可能主要通过 宿主的共代谢产物：氨基酸，短链脂肪酸及次级胆汁酸；通过维持宿主肠道内氧化还原状态梯度，控制致病细菌的致炎和毒力因子（脂多糖，LPS）产生而参与宿主肠道黏膜屏障维持及与宿主模式识别受体结合。然而，目前大量的发现和假设都基于相关分析，亟待因果关系研究证明。因此，在国家自然基金委的支持下，本课题开展了“肠道寄生菌群通过调节胆汁酸代谢促进2型糖尿病致病的分子生物学机制研究”。. 本课题基于临床RCT 研究，静脉葡萄糖钳夹实验以及多组学分析，首先在人体层面证实传统口服降糖药改造菌群胆汁酸代谢可能在糖尿病治疗中发挥重要作用。发现次级胆汁酸在总胆汁酸池中的比例可能是调控机体胆汁酸信号的重要因素，可能参β细胞葡萄糖刺激的早期相反应。在动物水平，我们先后用抗生素处理模拟无菌小鼠，以及胆汁酸受体敲除小鼠模型, 证实利用抗生素或口服降糖药阿卡波糖，可能通过重塑菌群胆汁酸信号而激活肝脏、胰腺胆汁酸信号同时抑制肠道胆汁酸信号，减缓β细胞在营养应激下的代偿性增生，减轻高胰岛素血症，提高胰岛素敏感性，抑制肝糖输出，最终改善血糖代谢。我们的研究最终证实了肠道菌群在营养过剩及压力下，如何诱导β细胞功能失代偿，肝脏糖脂代谢以及肝肠循环失调，参与糖尿病的发生，同时也提供了有效的2型糖尿病干预的新靶点。该课题不仅在理解肠道菌群参与宿主代谢调控的病理生理机制方面进行了有意义的探索，也为2型糖尿病治疗靶点开发提供了崭新的视角，为医疗健康大数据研究做了有意义的探索；为开展类似糖尿病这样的慢性非传染性疾病的的精准治疗提供了很好的应用范例及可参考的方法。. 与此同时，我们提供并分析了fxr敲除高糖高脂喂养小鼠的胰岛RNA测序数据，db/db小鼠抗生素使用前后，阿卡波糖治疗后的肠道菌群数据，肝、肠、血、粪的胆汁酸谱的数据。提供了中国人初发2型糖尿病患者基线和治疗后的肠道菌群宏基因组测序数据，以及血粪胆汁酸谱的数据库。相关结果发表SCI论文5篇，专利2项，培养硕士\博士研究生共5名。