探索IBD中肠道菌群调控肠道干细胞的机制及在肠黏膜修复中的意义

Their incidence of inflammatory bowel disease (IBD) is increasing worldwide, and the diseases remain incurable. Our recent studies suggest that fecal microbiota transplant (FMT) is efficacious treating IBD by not only suppressing inflammation conditions but also enhancing mucosal repair, suggesting that gut microbiome can influence the proliferation of intestinal stem cells (ISC). By integrating the pervious findings of the applicant in the fields of stem cell and gut microbiota (eight publications including Cancer Research, and one book chapter), we hypothesize that the gut microbiota of IBD impact ISC proliferation and mucosal healing by indirectly shifting metabolism and/or by indirectly regulating inflammatory cytokines. This proposed project will integrate shotgun metagenomics sequencing, multiplexed bead-based immunoassay and enteroscopy, unraveling the links in gut microbiome, circulating inflammatory cytokines and mucosal epithelium integrity. On this basis, microbiome-phenotype triangulation (MPT) method will be performed to identify the key bacterial species and that are associated with ISC proliferation activity. Finally, the identified microbes will be isolated, and their metabolites as well as the FMT-shifted inflammatory cytokines will be independently transplanted into germ-free zebrafish to examine their specific effects on influencing on ISC proliferation. This project will explore the gut microbial species in the IBD patients which influence ISC, and reveal whether these bacteria affect the proliferation of ISC through metabolites and/or by regulating inflammatory cytokines. The expected results aim to provide new evidences about the mechanisms responsible for the regulation of ISC by gut microbiota, and FMT's effect in treating IBD.

炎症性肠病(IBD)发病率逐年递增，尚无治愈方法。本项目组近期研究发现，粪菌移植（FMT）在抑制IBD炎症的同时促进了肠黏膜的修复，提示菌群对肠道干细胞有重要的调节作用。结合申请人在干细胞和微生态领域的研究基础（发表Cancer Research等国际期刊8篇），本项目旨在探索IBD患者在炎症活跃期（FMT前）和缓解期（FMT后）肠道菌群的差异，是否通过调节炎症细胞因子对肠道干细胞的增殖产生影响。本项目拟结合宏基因组学、高通量多因子检测和内镜检查，揭示IBD肠道菌群和炎症细胞因子之间的变化关系，以及其在肠黏膜修复中的作用。在此基础上,通过微生物-表型三角校正法聚焦影响肠道干细胞的关键菌种，应用无菌斑马鱼模型验证所识别的菌种是否通过代谢产物直接、和/或通过调节炎症细胞因子间接对肠道干细胞的增殖产生影响。本研究可望进一步揭示肠道菌群调控肠道干细胞的机制，并为FMT治疗IBD提供新的理论依据。

本项目围绕所提出的关键科学假说，即“IBD患者从炎症活跃期到缓解期肠道菌群和代谢的协同变化，通过诱导肠道干细胞加速增殖促进肠黏膜修复和肠稳态恢复”，开展了包括临床数据分析、多组学分析、动物实验、细胞实验、分子实验等一系列研究，系统性地探索了肠道菌群-胆汁酸代谢轴通过干预肠道上皮细胞、隐窝干细胞、免疫细胞，恢复肠道稳态并促进黏膜修复的相关机制，主要发现如下：.1.单一供体、低频次粪菌移植（FMT）可有效治疗轻、中、重度溃疡性结肠炎 (UC); FMT应答良好和长期缓解的UC病人肠道中M2型巨噬细胞在治疗后显著增加，并且伴随着多种次级胆汁酸（如石胆酸LCA）水平的升高。.2.英夫利昔单抗（IFX）和全肠内营养（EEN）治疗儿童CD的有效性与肠道菌群和胆汁酸代谢的协同变化有关，产胆盐水解酶的细菌（如Clostridium clusters IV）显著增加，造成多种次级胆汁酸（如别胆酸ACA，猪胆酸HCA，石胆酸LCA，6-酮石胆酸6-ketoLCA，熊去氧胆酸UDCA）的水平升高并且与疾病活动严重程在治疗后降低相关性。.3.HCA可以有效缓解DSS诱导的小鼠结肠炎；LCA和ACA可以在体外促进人肠道上皮细胞的增殖，增加小鼠肠道类器官中干细胞的数量和隐窝干细胞标志物Lgr5, Sox2, CD44, Axin2的水平。ACA和LCA的作用或通过代谢重编程促进能量代谢。.4.ACA抑制M1型巨噬细胞极化，LCA促进巨噬细胞M2型极化，这些作用可以抑制IBD炎症反应并促进肠黏膜修复。.5.CD患者的胆汁酸代谢异常和焦虑抑郁状态显著关联。粪便中猪去氧胆酸HDCA、脱氧胆酸12-DHCA的含量与抑郁程度呈负相关，牛磺脱氧胆酸TDCA、牛磺石胆酸TLCA、以及牛磺酸β-鼠胆酸TβMCA的含量与焦虑程度呈正相关；血清中脱氧胆酸7-DHCA的含量与抑郁程度呈正相关。..以上这些发现揭示了肠道菌群-胆汁酸轴在IBD治疗中发挥的重要作用，为进一步发掘IBD治疗的新靶标提供了有价值的数据。