肠胶质细胞介导的黏液屏障损伤在腹泻型IBS肠通透性改变的作用研究

Recent evidence indicated that mucus barrier injury may contribute to increased intestinal permeability in patients with diarrhea-predominant irritable bowel syndrome (IBS-D), however, the exact mechanism is unclear. The enteric glial cell(EGC), forming synaptic connections with goblet cells, may play an important role. This study was designed to explore the mechanism of the abnormal intestinal permeability in IBS-D at the level of EGC-mediated mucus barrier injury: ①Detect EGC alterations in the number, shape and TLR4 pathway protein expression in intestinal mucosa of IBS-D patients, and combined with intestinal mucus barrier assessment, to reveal the molecular mechanisms of EGC-induced mucus barrier injury; ② Establish the mice model of bowel dysfunction, using small interfering RNA (siRNA) technique, to confirm the role of TLR4-TRIF-RIP1-TAK1 pathway in the regulation of goblet cells secretion and its effect on the intestinal mucus barrier function in mice; ③ Culture the enteric glia in vitro, with intervention of LPS, TLR4 siRNA transinfection and TLR4 pathway antagonists, to study the electrophysiological activity of EGC. Stimulate the intestinal goblet cells or mice enema with interventioned EGC supernatant, and detect the shape and function of goblet cells and evaluate the completeness of intestinal mucus barrier in mice, to further clarify the mechanism of EGC in the modulation of intestinal mucus barrier function. In short, our study is meaningful to elucidate the mechanism of increased intestinal permeability in IBS-D, and find a new target for IBS treatment.

新近发现黏液屏障损伤可导致腹泻型肠易激综合征(IBS-D)患者肠通透性增高，但具体机制不清，与杯状细胞形成突触连接的肠胶质细胞(EGC)可能在其中发挥重要作用。本研究旨在从EGC调控肠黏液屏障层面探讨IBS-D肠通透性改变的潜在机制：①检测IBS-D患者肠黏膜EGC数目、形态及TLR4通路蛋白表达改变，结合黏液屏障评估，揭示EGC调控黏液屏障损伤的分子机制；②建立肠功能紊乱小鼠模型，结合siRNA技术，论证胶质细胞TLR4-TRIF-RIP1-TAK1通路对杯状细胞的调控及对黏液屏障的影响；③行EGC体外培养，采用LPS、TLR4 siRNA转染或通路蛋白拮抗剂干预，检测EGC电生理活性，将干预后EGC上清液刺激杯状细胞或导入小鼠肠道，检测杯状细胞形态功能及小鼠黏液屏障完整性，充分论证EGC调控肠黏液屏障的机制。本研究对阐明IBS-D肠通透性增高的机制，寻找IBS治疗新靶点具有深远意义。

肠黏液屏障是杯状细胞分泌的覆盖在黏膜内侧的凝胶型网状结构, 隔离上皮与肠腔内容物以起到保护作用。腹泻型肠易激综合征（Diarrhea-Irritable Bowel Syndrome, IBS-D）以腹痛或腹部不适伴排便异常为特征。肠黏液屏障功能的破坏和通透性的增加可能导致腹泻症状的发生。肠胶质细胞（Enteric glial cell，EGC）侵入基底膜并渗透到上皮细胞，通过旁分泌途径作用于肠上皮细胞。在本研究中，我们探讨了EGC是否能够通过直接或旁分泌途径调控杯状细胞，从而调节肠黏液屏障，参与IBS-D的发生。 .IBS-D患者中我们检测了肠黏液层结构、杯状细胞数量、分泌及超微结构和EGC表达及超微结构。IBS-D模型小鼠中，我们分别检测了TNBS、束缚应激及旋毛虫模型小鼠肠黏液层结构、杯状细胞数量、分泌及超微结构。为探索EGC对杯状细胞的可能影响，我们对CRL-2690和LS174T细胞系进行共培养，检测LS174T的muc2、tff3、WFDC2、SPDEF的表达。.透射电镜显示IBS-D患者结肠粘膜的上皮细胞排列整齐、形态正常、膜完整、细胞间隙紧密;粗面内质网丰富，扩张，内有絮状物；杯状细胞内黏液颗粒积累于顶端。与健康对照相比，AB-PAS和免疫荧光染色显示IBS-D患者杯状细胞内黏液颗粒致密，更易处在耗竭状态，而杯状细胞数量未有明显改变。黏液层主要蛋白MUC2表达减少，且与腹泻次数呈负相关趋势。与对照组小鼠相比，透射电镜显示TNBS模型小鼠杯状细胞胞质丰富，胞质内空泡变。黏液颗粒减少且集中于顶部，膜内可见浓缩絮状物，细胞间隙增宽，角蛋白减少。AB-PAS染色显示TNBS模型小鼠黏液层变薄，黏液更易耗竭；FISH显示TNBS模型小鼠肠黏液屏障通透性增加。束缚应激模型小鼠及旋毛虫模型小鼠同样可以观察到黏液层变薄。为了探讨EGC是否调控杯状细胞的功能，我们观察到IBS-D患者EGC标志物GFAP表达降低，胞体饱满，足突缩短。透射电镜未观察到EGC与杯状细胞的直接接触。CRL-2690和LS174T共培养后，可观察到LS174T的muc2、tff3、wfdc2的表达上调。.IBS-D患者肠黏液屏障存在异常。EGC可能通过旁分泌途径作用于杯状细胞调控肠黏液屏障。本研究对IBS-D的发病机制提供了新的思路。