肠屏障功能紊乱在术后肠麻痹发病学中的作用及与Toll样受体信号通路的关系

Postoperative ileus (POI) is a clinically common ailment. Although some cases are mild with a benign finale, serious paralytic ileus may occur in severe cases, causing a variety of complications. With mechanism still being elusive, these cases are intricate and resistant to clinical intervention; hence the call for special attention, funding and investigation. Our privious study showed that the inflammation and high permeability in the intestinal mucosa are involved in the development of POI. In this project, by using POI model in C57BL mice（wild type,and TLR4-/-、JNK -/- types）, focusing the study on the possible cause and effect chain “the Postoperative ileus-Abnormal gut barrier and intestinal mucosal permeability-Translocation of the intestinal bacteria/ endotoxin-Inflammation-Aggravation of the postoperative ileus”, we plan to investigate the intestinal barrier function, in particular, the changes in intestinal permeability, alterations in intestinal flora and in gut-associated lymphoid tissue (GALT),and their roles and the related mechanisms in the progress of postoperative ileus, by in vivo and animal isolated-organ experiments. Moreover, the characteristics of gastrointestinal motility disorders, inflammatory reactions, and intestinal barrier malfunctions in the POI models of different mice will be compared, then the possible signal transduction pathways that may be involved in POI progression will be analyzed. The purpose of the proposed project is to make further efforts to elucidate the pathogenesis of POI and its related factors, as well as to provoke further clues for the development of new prevention and therapeutic strategies of POI.

术后肠麻痹(postoperative ileus，POI)临床常见，部分严重者常出现各种并发症，其机制不明，成为临床预防和治疗的重点和难点。课题组前期研究提示，手术引发的炎症反应及肠黏膜通透性增高是POI的主要发病学环节。本项目聚焦于“术后肠麻痹-肠屏障异常/肠黏膜通透性变化-肠道细菌/内毒素移位-炎症反应-加重术后肠麻痹”这一可能的因果连锁反应，采用C57BL小鼠野生型和TLR4、JNK基因敲除型，通过在体和离体动物实验，动态观测POI小鼠肠屏障功能，尤其肠黏膜通透性、肠道菌群、肠道相关淋巴组织（GALT）及免疫功能的变化, 揭示肠屏障功能紊乱在POI发病中的作用和可能的机制；通过比较不同类型小鼠胃肠运动紊乱、肠黏膜通透性改变、炎症反应等方面的特点，明确肠屏障改变引发/促进POI可能涉及的信号通路及其作用。研究将为进一步阐明POI的发病机制提供客观依据，为POI的临床防治提供新的思路。

术后肠麻痹（Postoperative ileus, POI）时肠屏障的变化特点以及相关机制仍不清楚。已知炎症反应参与POI的发生发展，而Toll样受体4（Toll like receptor 4，TLR4）、JNK等信号通路参与了炎症反应的调控。本实验主要探讨肠屏障在POI中的变化，以及炎症反应及相关信号通路在其中的作用。实验通过标准化小肠干扰手术在野生型（Wild type, WT）和TLR4基因敲除（TLR4 knock out, tlr4-/-）、JNK基因敲除（JNK-/-）C57BL小鼠中诱导POI模型。结果揭示：WT-POI小鼠的肠屏障结构和功能包括肠道机械屏障、免疫屏障、生物屏障、化学屏障都出现一定程度的改变，变化特征主要有结肠杯状细胞数量减少，肠道紧密连接蛋白Claudin-2表达增加，肠道派氏结中CD4 + / CD8 +细胞比值降低，肠道绿色荧光蛋白标记的大肠杆菌易位，以及血浆脂多糖水平升高。这些变化在tlr4-/--POI小鼠中都有不同程度的改善。此外，WT-POI小鼠肠道组织中的促炎因子白细胞介素-6、单核细胞趋化蛋白-1、丝裂原活化的蛋白激酶（MAPK）pp38和pJNK的表达水平以及血浆中的TNF-α都显著性升高，而这些指标在tlr4-/-- POI小鼠中则未见明显改变。总之，本研究工作历时四年多，主要研究结果揭示：POI 时肠屏障功能，尤其肠黏膜通透性、肠道菌群、肠道免疫功能等发生改变，这些变化与炎症反应密切相关，进而在POI 发生发展中起有重要作用。在POI的发病过程中，TLR4以及炎症相关信号通路如MAPKp38、JNK、NF-KB等都有参与，针对炎症、应激进行干预对POI均有一定防治作用。这些研究结果为阐明POI 的发病机制提供了客观实验依据，为POI 的临床防治提供了新的思路。