PAR2-MLC介导粘膜屏障功能的改变在PI-IBS肠道免疫激活中的作用

PI-IBS as an important subtype of IBS, the pathogenesis of it is not clarified yet, currently, lots of studies found that intestinal mucosal immunity activation might be an important mechanism in it. PAR-2 is present particularly on the intestinal epithelial cells. Activation of PAR-2 in the intestinal epithelial cells can increase myosin light chain kinase (MLCK) activity through phosphorylation of MLC, which is associated with opening of tight junction and junction protein deformations that favour macromolecule passage and bacterial translocation. The intercellular structural proteins (claudins and occludins) are anchored to the cytoskeleton actinomyosin apical ring through intermediary proteins. Destroy of intestinal mucosal barrier function might activate mucosal immunity. The phosphorylation /dephosphorylation of myosin light chain is controlled by MLCK and myosin phosphatase (MP). Whether this PAR-2 mediated changes of intestinal mucosal barrier function contributed to the intestinal mucosal immunity activation and finally induced visceral hypersensitivity in PI-IBS has not been defined. This study is based on the PAR2-MLC mediated changes of intestinal mucosal barrier function, to interfere several key points in the PAR2-MLC pathway with antagonist of PAR-2, MLCK, and tight junction, to observe the structures of tight juction proteins in the mucosal barrier with electron microscope, to quantitate several key proteins (claudins, occludins, ZO-1) in this pathway with western blot, to evaluate the visceral sensitivity, to observe those immune cells (mast cell, ECs, T cells) in the intestinal mucosal immunity activation with the changes of the mucosal barrier function, and finally to clarify the relationship with the visceral sensitivity in the development of PI-IBS. This study will explore the upper pathway of the intestinal mucosal immunity activation to clarify the mechanism of PI-IBS, and also may suggest some clues for the new prevention and threatment methods of PI-IBS.

PI-IBS作为IBS重要的一类亚型其发病机制尚不明确，目前研究发现肠道粘膜免疫激活是其重要发病机制之一。肠上皮细胞表面的PAR-2活化后可通过MLC磷酸化途径使肠上皮细胞间通透性增加，肠道粘膜屏障功能的破坏能激活粘膜免疫，PI-IBS中是否存在PAR-2介导的肠道屏障功能的改变从而导致PI-IBS持续的粘膜免疫激活影响内脏敏感性尚不明确。本研究将从PAR-2介导的肠道粘膜屏障功能的改变出发，通过体内及体外试验来干预PAR2-MLC通路中的相关环节，电镜观察粘膜屏障紧密连接蛋白结构改变、流式细胞术检测粘膜免疫细胞及免疫因子的改变、对相关蛋白进行定量检测、评价内脏敏感性，观察肠道粘膜通透性改变后的粘膜免疫激活状态的变化，以及最终对PI-IBS内脏敏感性的影响。本研究将通过对肠道粘膜免疫激活的上游途径的探索，进一步阐明PI-IBS的发病机制，同时也为PI-IBS新的治疗手段提供理论依据。

肠易激综合征（irritable bowel syndrome, IBS）是消化科常见的功能性疾病，其机制尚未完全阐明。近年来研究表明IBS肠道粘膜中存在肠道通透性及肠道粘膜免疫激活，免疫细胞和免疫因子的改变。PAR2是蛋白酶激活受体（protease-activated receptor, PAR）家族的一个亚型，主要表达于肠上皮细胞，它通过G蛋白偶联途径，激活肌球蛋白轻链激酶（myosin light chain kinase， MLCK），使肌球蛋白收缩，肠道紧密连接开放，肠道粘膜通透性增加，肠腔内细菌或者大分子物质进入粘膜下激活肠道粘膜免疫，导致肠粘膜微炎症。本研究主要探索PAR2-MLC通路在介导IBS肠道粘膜屏障功能改变中的作用。本研究通过对PAR2-MLC通路上的关键靶点进行激动及抑制后，观察PI-IBS模型小鼠的肠道通透性及肠道免疫的改变，发现激活PAR2或MLCK后肠道通透性增加，肠道免疫激活，模型小鼠内脏敏感性增高，而阻断PAR2或抑制MLCK后肠道通透性降低，肠道免疫抑制，模型小鼠内脏敏感性降低。通过本研究阐明了PAR2-MLC通路在PI-IBS发病中的重要作用，并通过干预通路中的重要靶点降低了PI-IBS的内脏敏感性，为今后研发治疗IBS的药物提供了很好的思路。