CRF调控肥大细胞打破肠道内毒素耐受在肠易激综合征发病中的机制

Irritable bowel syndrome (IBS) is a common functional disorder of the intestine with an increasing morbidity. The pathogenesis of IBS is not well understood. The intestinal epithelial endotoxin tolerance is defined as a reduced capacity of the host to respond to lipopolysaccharide (LPS) activation following a first exposure to this stimulus. Yet, factors and mechanisms in breaching the established endotoxin tolerance are to be further understood. One of the major consequences of the disturbance of intestinal epithelial endotoxin tolerance is to induce the epithelial barrier dysfunction, which is proposed to contribute to the pathogenesis of IBS. Published data indicate that psychological stress plays an important role in the intestinal epithelial barrier dysfunction. The corticotropin releasing factor (CRF) is an important mediator in the stress-induced pathophysiological changes in the body. Mast cells were found involved in the stress induced epithelial barrier dysfunction as reported by several research groups. To date, the underlying mechanisms by which psychological stress induced intestinal epithelial barrier dysfunction are to be further investigated. By integrating the information of psychological stress-induced intestinal barrier dysfunction and the concept of intestinal endotoxin tolerance, we hypothesize that the psychological stress-derived CRF may be involved in breaching the established intestinal endotoxin tolerance that further induce the barrier dysfunction via mast cells activation which might be one of the primary pathogenesis of IBS. Based on our preliminary studies, we will further investigate that: 1) The expression and the changes of distribution of CRF receptors and TLR4 in patients' intestinal mucosa mast cells with IBS. Mast cells avtivation in the patients' intestinal mucosa will also be elucidated; 2) The role of CRF in breaching the established intestinal endotoxin tolerance that further induce the barrier dysfunction by challenging the mice with water avoid stress; 3) The precise molecular mechanism that CRF modulates the expression of TLR4 in mast cells which may further breach the intestinal endotoxin tolerance by employing the transwell cell culturing system. This study may provide a better understanding of the role of breaching endotoxin tolerance in the intestinal barrier dysfunction and may help develop strategies to prevent and treat IBS.

肠易激综合征（IBS）是常见功能性疾病，肠道屏障功能障碍在其发病机制中具有重要作用，而应激因素是其发病的关键诱导因素。正常肠道黏膜可针对细菌主要组分脂多糖（LPS）产生耐受，应激反应因子促肾上腺皮质激素释放因子（CRF）可能能够打破肠道内毒素耐受导致肠道屏障功能障碍。肥大细胞表达CRF受体及TLR4，TLR4可针对LPS刺激产生反应,我们推测CRF可通过调控肥大细胞打破肠道内毒素耐受，进一步导致肠屏障功能障碍。我们前期研究显示应激可增加小鼠肠道黏膜TLR4表达，再次暴露于LPS时肥大细胞TLR4表达下调。本研究进一步探讨：1）IBS患者肠道黏膜肥大细胞CRF受体、TLR-4表达和分布变化及肥大细胞活化状态；2 ）CRF在打破肠道LPS耐受导致屏障功能障碍过程中肥大细胞的作用；3）CRF调控肥大细胞TLR-4表达并进一步打破肠道内毒素耐受的分子机制。本研究有望为IBS的治疗提供理论基础。

肠易激综合征（irritable bowel syndrome, IBS）是消化内科最常见的慢性复发性胃肠道疾病，其全球发病率为1.1%到29.2%。应激反应中释放的促肾上腺皮质激素释放因子（corticotropin releasing factor，CRF）是IBS病理生理过程中最重要的效应因子，肥大细胞是肠黏膜免疫系统中重要的固有免疫细胞，肠道屏障功能障碍是IBS发病的关键环节。正常生理条件下，肥大细胞保持着对LPS的内毒素耐受。那么，CRF是否能够调控肥大细胞打破其对LPS的内毒素耐受，进而引起肠道屏障功能障碍，通过什么机制导致肠道屏障功能障碍，是本研究旨在探讨的问题。本研究通过体内体外实验证实：（1）IBS小鼠结肠黏膜CRF-R1和CRF-R2 2种受体均发生上调；肠上皮细胞及肥大细胞上TLR4表达上调。（2）CRF可以通过两种途径激活肥大细胞：一是与肥大细胞上的CRF-R1和CRF-R2结合，直接引起肥大细胞脱颗粒，导致MCP和TNF-α释放。二是与肥大细胞上的CRF-R1结合，通过细胞内的Erk1/2信号转导途径，上调肥大细胞表面TLR4的表达；同时，下调了对LPS-TLR4-NFκB通路有抑制作用的细胞因子信号抑制蛋白SCOS-1和SCOS-3的表达，最终导致肥大细胞对LPS的内毒素耐受被打破，LPS-TLR4途径的细胞因子IL-1β、IL-6、IL-13和TNF-α释放。（3）CRF可调控肥大细胞导致IBS小鼠肠黏膜透性增高，这种作用是通过CRF激活肥大细胞所释放的细胞因子IL-1β，IL-6，IL-13，MCP和TNF-α通过p38 MAPK途径抑制结肠上皮细胞中Trek1的表达实现的。（4）结肠上皮细胞表达Trek1，体外将T84细胞的Trek1基因进行沉默可导致肠上皮屏障的通透性增高，再加入重组的rTrek1蛋白则可恢复肠上皮屏障功能。提示Trek1对维持正常的肠道屏障功能有重要的作用。本研究按期完成实验计划,发表相关论文13篇，其中SCI索引论文5篇,中华及核心期刊论文8篇。培养博士生4名,硕士生2名。