同型半胱氨酸影响实验性结肠炎肠粘膜通透性的作用机制

Intestinal epithelial barrier dysfunction is one of the key pathogenesis in inflammatory bowel disease (IBD), which is characterized by the change of intestinal permeability caused by the damage of the tight junction. Homocysteine (Hcy) exhibited potential aggravating effect on the intestinal injury, whether Hcy is involved in the regulation of intestinal mucosal permeability in IBD was not yet clear, and no relevant research was reported. Our previous study has shown a significant correlation of the plasma levels of Hcy and the intestinal permeability in IBD, the expression of myosin light chain kinase (MLCK) was increased significantly in the animal colitis model, which showed the correlation with the plasma Hcy levels. According to the effect of the vascular endothelial cell permeability of Hcy, on the establishment of experimental colitis and Caco-2 cell models, we are aimed to observe the effect of Hcy on intestinal permeability in vivo and in vitro. By using ELISA, RT-PCR, Western Blot and immunofluorescence histochemistry method, focus on the key protein in MLCK which regulating the intestinal permeability, try to investigate the mechanism of Hcy on the expression of MLCK, provide experimental data that decreasing the plasma Hcy levels could improve intestinal permeability and mucosal healing in IBD.

肠粘膜上皮屏障损害是炎症性肠病(IBD)发病机制中的关键环节之一,主要特征表现为细胞连接破坏以及由此引起的肠粘膜通透性改变,同型半胱氨酸(Hcy)具有加重IBD肠道炎症损伤的潜在作用。但Hcy是否影响并参与IBD中肠粘膜通透性的调节，国内外目前尚无相关研究。我们以前的研究发现，IBD中Hcy水平与肠粘膜通透性有明显相关，结肠炎模型中肌球蛋白轻链激酶（MLCK）表达增高，与Hcy水平呈现良好相关。由于Hcy被证实可直接影响血管内皮细胞屏障的通透性，因此拟在建立实验性结肠炎模型和Caco-2细胞模型基础上，从整体和细胞水平观察Hcy对肠粘膜通透性的影响特点。同时以调控肠粘膜通透性的关键蛋白MLCK为标靶，采用ELISA，RT-PCR，Western Blot及免疫荧光组织化学方法，阐明Hcy影响MLCK表达的作用机制，为临床降低IBD中Hcy水平，改善肠粘膜通透性和促进粘膜愈合提供实验依据。

炎症性肠病(IBD)的病因和发病机制尚未完全阐明，近年来我国IBD发病率有明显增高趋势，由于IBD药物治疗疗程长，病情易复发，严重影响患者生活质量，可导致严重并发症，因此，探讨IBD发病机制具有重要临床意义。研究表明，肠粘膜上皮屏障损害是IBD发病机制中的关键环节之一,主要特征表现为细胞连接破坏以及由此引起的肠粘膜通透性改变,同型半胱氨酸(Hcy)作为致病因素具有加重IBD肠道炎症损伤的潜在作用。为阐明Hcy影响并参与IBD中肠粘膜通透性的调节作用机制，本课题以实验性结肠炎大鼠模型和Caco-2细胞模型为基础，通过加入不同浓度Hcy处理，从整体和细胞水平研究了Hcy影响肠黏膜通透性结构和功能改变的特点。在上述研究基础上，进一步观察和研究了Hcy对实验性结肠炎模型和Caco-2细胞模型中MLCK和上游MEK/ERK信号通路相关蛋白（Claudin-1，Occludin，ZO-1）表达的影响，阐明了Hcy调控MLCK影响肠黏膜通透性的具体机制。因此，本研究证明了Hcy具有增加实验性结肠炎大鼠肠黏膜通透性和Caco-2细胞通透性的作用，作用机制与Hcy影响MEK/ERK信号通路，调控MLCK改变肠黏膜通透性有关。本课题的科学意义在于从整体和细胞水平证明了Hcy增加了IBD中肠黏膜通透性以及具体调控机制，有助于更新IBD病理生理机制的理解和IBD治疗策略的改变，为进一步通过临床措施降低IBD中Hcy水平，改善IBD患者肠黏膜通透性，达到促进肠道黏膜的愈合目标，提高IBD的临床疗效，具有重要的应用前景。