持续内质网应激导致的上皮-间充质转化：炎症性肠病肠壁纤维化的新机制

Luminal stenosis secondary to intestinal fibrosis is the leading cause for the irreversiblity and need for surgery in patients with chronic inflammatory bowel disease (IBD), especially Crohn''s disease (CD). New evidence indicates that epithelial-mesenchymal transition (EMT) plays an important role in the pathogenesis of IBD intestinal fibrosis, however, the exact mechanism for EMT is still unclear. Based on previous work, we will further explore the role of epithelial endoplasmic reticulum stress (ERS) in the pathogenesis and progress in IBD. In this proposal, we proposed the hypothesis that "sustained epithelial ERS is probably a novel mechanism for EMT and intestinal fibrosis in IBD". RNA interference, in vivo cell tracing, and colon organ culture will be used based on in vitro cell culture model, animal model of TNBS-induced chronic colitis and fibrosis, and tissue from clinical CD patients in the current project. The role of ERS activation or inhibition in epithelial cell phenotype, EMT process and tissue fibrosis will be explored subsequently. In addition, the influence of infliximab (IFX) on ERS-induced EMT process would be examined. We hope that the result of the current study would provide a new therapeutic target for the prevention and early treatment of IBD-related intestinal fibrosis, and also provide rationale for the early usage of biological therapy (the top-down approach) in IBD patients.

肠壁纤维化所致的肠腔狭窄是炎症性肠病（IBD）病变不可逆和手术治疗的首位病因。有证据表明上皮-间充质转化（EMT）在IBD肠壁纤维化过程中起到重要作用，但导致肠道上皮EMT的病因尚不清楚。本项目拟在前期青年基金项目的基础上，继续深入探讨肠上皮内质网应激（ERS）在IBD发生和疾病进展中的作用，提出“肠道上皮细胞持续ERS是导致上皮-间充质转化和肠壁纤维化的新机制”这一假说。项目采用体外细胞培养模型、TNBS诱导的肠壁慢性炎症和纤维化动物模型，以及IBD患者的临床标本等，结合RNA干扰、活体细胞示踪技术、临床结肠标本组织块培养等手段，探讨在肠道上皮细胞激活或阻断ERS过程后对上皮细胞表型、EMT过程和组织纤维化的影响。并初步探讨生物制剂肿瘤坏死因子单抗（IFX）对这一过程的干预作用和效果。研究结果将为临床IBD肠壁纤维化的预防和早期干预提供新的靶点和早期降阶梯治疗治疗理论依据。

肠壁纤维化和肠腔狭窄是IBD病变不可逆和手术治疗的首位病因。迄今为止，IBD时的肠壁纤维化尚无有效治疗手段，多数针对IBD的治疗药物均无法逆转已发生的肠壁纤维化反应。明确炎症性肠病肠壁纤维化的机理，并预防和延缓这一过程，对改善IBD自然病程和降低手术率具有重要意义。有证据表明上皮-间充质转化（EMT）在组织纤维化过程中起到重要作用，但具体机理和防治措施并不清楚。本项目主要探讨肠腔共生菌群变化、上皮内质网应激（ERS）和上皮EMT过程在肠道纤维化过程中的作用。研究发现：（1）在临床UC患者结肠标本纤维性狭窄部位和TNBS诱导的肠道纤维化模型中，存在ERS过程的过度活化，表现为GRP78、ATF6β、XBP1和p-eIF2α表达增加，提示ERS参与了肠道纤维化过程；采用ERS保护剂TUCDA可改善TNBS诱导的肠道纤维化；进一步分析表明，通过可溶性膳食纤维果胶和短链脂肪酸丁酸钠改善肠道菌群紊乱，可以减轻肠道ERS和纤维化过程；（2）采用pVillin-Cre-EGFP转基因小鼠的分析表明，上皮-间充质转化（EMT）在肠道纤维化过程中起到重要作用；可溶性膳食纤维可减轻肠道纤维化和EMT过程；这一过程与改善照射后小鼠肠道菌群改变，增加肠腔短链脂肪酸水平有关；（3）采用IL-10基因敲除小鼠模型，发现短链脂肪酸丁酸盐可以减轻肠道炎症、炎症因子水平和炎症细胞浸润，而丁酸盐的作用与调节肠道菌群有关，尤其是可以减少IgA包裹的致肠炎菌数量。.研究结果表明，肠道菌群紊乱导致持续内质网应激，是IBD肠道纤维化的机制之一；改善肠道菌群紊乱，可以有效减轻肠道的持续ERS和EMT过程，从而减轻肠道纤维化。可溶性膳食纤维及其代谢产物丁酸盐，可以改善病理状态下的肠道菌群紊乱，尤其是减少IgA包裹的致肠炎菌数量。研究结果可为临床应用可溶性膳食纤维防治肠道纤维化提供理论依据。