VitD/VDR通过NHE8-MUC2调控轴抑制溃疡性结肠炎肠粘膜细菌粘附的作用和机制研究

Inflammatory response induced by intestinal bacteria contacting to the epithelium is the triggering mechanism of ulcerative colitis (UC). The increase in bacteria adhesion caused by intestinal mucus decrease and lost is the precondition of bacteria adhesion. Previous studies have reported that vitamin D (VitD) and vitamin D receptor (VDR) signaling pathway might promote mucus secretion, but the potential mechanism remains unclear. Additionally, NHE8 has been reported to modulate mucin MUC2 secretion. Our preliminary results found that VitD could stimulate NHE8 expression, and promote MUC2 expression and secretion, as well as inhibit bacteria adhesion. As a result, we hypothesized that “VitD/VDR promotes MUC2 expression and inhibits bacteria adhesion to intestinal mucosa through modulating NHE8”. We would further illuminate the detailed mechanism that VitD/VDR inhibits bacteria adhesion through NHE8-MUC2 through NHE8-/- mice, gene silence or overexpression techniques in vivo and in vitro and colon organoid experiments. Meanwhile, we would uncover the potential mechanism of NHE8 regulating MUC2. Our prospective results could uncover a novel mechanism that VitD/VDR and NHE8 inhibiting intestinal bacteria adhesion, and provide new basis for UC prevention and therapy.

肠道内细菌与上皮细胞接触诱导机体炎症反应被认为是促进溃疡性结肠炎（UC）发生发展的重要机制。肠道粘液的减少和丢失导致细菌粘附增加，则是细菌与机体接触的重要前提。研究发现，维生素D（VitD）和维生素D受体（VDR）通路可促进肠道粘液分泌，但机制不清。另外，NHE8表达降低可下调粘蛋白MUC2的表达。课题组前期发现，VitD可同时上调NHE8和MUC2的表达，减少细菌粘附。因此我们提出“VitD/VDR通过上调NHE8的表达，进而促进MUC2的分泌，抑制肠粘膜细菌粘附”假说。本课题拟进一步通过NHE8-/-小鼠、基因干扰或过表达等研究手段，在体内、体外及结肠类器官培养实验中明确VitD/VDR通过NHE8-MUC2抑制细菌粘附的作用，同时阐明NHE8调控MUC2的具体机制。本项目将揭示VitD/VDR以及NHE8在抑制肠道细菌粘附中的新机制，为探寻UC有效的防治靶点提供依据。

近年来，大量的临床和基础研究表明维生素D（VD）缺乏以及VD受体（VDR）表达下调与溃疡性结肠炎（Ulcerative Colitis, UC）的发生与发展相关，提示VD/VDR通路在结肠炎中发挥重要保护作用，但是其相关作用机制尚不完全清楚。本研究采用VD缺乏小鼠及VDR-/-小鼠进行研究，VD缺乏小鼠在饮用DSS水后，其结肠长度显著短于对照饲料喂养的DSS结肠炎小鼠（p < 0.05）和帕立骨化醇提前治疗小鼠（p < 0.05）。VD缺乏会加重结肠炎小鼠肠道粘膜的损伤，帕立骨化醇治疗则可对此进行挽救。此外，qPCR检测发现，与对照组小鼠相比，VD缺乏小鼠结肠中NHE8（p < 0.001）、MUC2（p < 0.05）、FCGBP（p < 0.001）、CLCA1（p < 0.05）以及CLCA2（p < 0.001）的表达显著降低。此外，qPCR检测提示VDR-/-小鼠结肠粘液蛋白的表达较VDR+/+小鼠显著降低，VDR-/-结肠炎小鼠结肠粘液蛋白的表达也较VDR+/+结肠炎小鼠显著降低。AB-PAS染色、溶菌酶和嗜铬素A检测进一步明确VDR-/-小鼠中杯状细胞、潘氏细胞数量较VDR+/+对照小鼠显著降低，而神经内分泌细胞则无显著差异。体外实验中，PAS染色结果表明在siRNA沉默VDR表达后，LS174T细胞粘液分泌、MUC2的转录水平（p < 0.01）和上清液中MUC2蛋白含量（p < 0.001）均受到抑制。然而帕立骨化醇干预则显著上调了细胞（p < 0.001）和上清液中（p < 0.05）MUC2的表达。同样地，VDR过表达质粒转染LS174T细胞后也显著上调了MUC2 mRNA的表达（p < 0.001）和上清液中的分泌（p < 0.05）。机制方面，研究发现VDR siRNA干预后，LS174T细胞Notch信号通路部分配体和受体表达上调；NICD和Hes1的表达显著增加，且LY411,575不能显著刺激MUC2的表达。综上所诉，VD/VDR是溃疡性结肠炎中肠道粘膜屏障重要的保护因素，其保护机制与VD/VDR调节Notch信号通路进而影响MUC2的合成与分泌有关。这为临床上溃疡性结肠炎的治疗提供了新的靶点。

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