先天性巨结肠伴小肠结肠炎特定细菌通过脂多糖-Toll样受体途径损伤肠粘膜屏障的致病机制研究

Hirschsprung-associated enterocolitis (HAEC) is a severe, life-threatening complication of Hirschsprung’s disease (HD). The HAEC occurrence rate is high with up to 40% of surgically treated HD patients developing HAEC after the treatment. In our previous study, we used the metagenomic approach to investigate the intestinal microbiota of HD and HAEC patients. This study found several bacterial strains specific to HD and HAEC cases. These results indicate that HAEC is closely related to distinctive changes in the gut microbiota. Recent studies showed that bacteria-derived lipopolysaccharide (LPS) and Toll like receptors(TLR4) signaling in mucosal barrier damage plays a crucial role in the pathogenesis of intestinal inflammatory disease. However, the associated pathogenesis of HAEC is unknown. This proposal seeks to explore LPSs from HD specific Bacteroides fragilis, and HAEC specific Veillonella parvula to investigate LPS-mediated inflammatory responses in mucosal epithelial cell models. We will investigate the connection between toll-like receptors（TLRs）binding affinity and the structure of specific LPS using Isothermal Titration Calorimetry (ITC) and Electron Ionization Gas Chromatography - Mass Spectrometry (EI GC-MS). The TLR4 signaling, along with other potential signaling pathways, will be examined using gene expression monitoring in LPS-stimulated wild-type and CRISPR/CAS9-assisted gene knock-out cell lines. Finally, the proinflammatory and epithelial barrier disruption capacity of the HAEC-related gut microbiota will be explored in vitro. Therefore this proposal will examine the HD and HAEC specific LPS-mediated effects on inflammatory reactions, fundamental cell functions and molecular signaling mechanisms in mucosal epithelial cells. The proposal will create a future basis and rationale for the development of novel therapeutic strategies targeting HAEC.

先天性巨结肠伴小肠结肠炎（HAEC）是先天性巨结肠症（HD）最严重且危及生命的并发症，即使经巨结肠根治术后，HAEC发生率仍高达40%，临床防治面临挑战。申请人在前期宏基因组学研究中发现HAEC患儿肠道以小韦荣球菌定殖为特征，而无肠炎HD以脆弱拟杆菌/柔嫩梭菌为主。已发现致病菌可通过其脂多糖(LPS)与肠粘膜上皮细胞内Toll样受体（TLR）结合，调节炎性反应并损伤肠粘膜屏障是炎症性肠道疾病重要发病机制，而HAEC相关机制研究尚未见报道。本课题拟提取纯化HAEC特异细菌的LPS，并与人结肠上皮细胞NCM460细胞株体外培养，以LPS饲喂无菌小鼠造模, 采用CRISPR/CAS9技术敲除肠上皮细胞内TLR等实验，探究HAEC特定细菌是否通过释放LPS并与TLR结合途径启动炎性因子释放及损伤肠粘膜屏障而致炎的分子信号通路，以确定HAEC致病分子机制和关键作用靶点，为其防治找到新途径。

儿童肠道疾病的发生发展常伴有肠道菌群紊乱，但不同疾病肠道菌群模式以及其致病机制至今未明。本研究探究了新生儿坏死性肠炎和迟发性脓毒血症中肠道菌群模式，并发现肠道菌群多样性降低，并且菌群在出生后随时间变化明显异常。我们基于前期研究基础，发现巨结肠伴小肠结肠炎特征性的小韦荣球菌可以通过释放LPS破坏肠道上皮细胞屏障并激活肠道巨噬细胞导致肠道炎症。我们还发现小分子代谢物衣康酸可以抑制小韦荣球菌来源的LPS对巨噬细胞的激活来抑制炎症的发展，具有一定的临床应用前景。我们项目的研究可以帮助我们更好理解肠道菌群在肠道疾病中的作用，也为临床中此类疾病的防治提供了创新性策略。