TNF-α-ATX-LPA调控Slc26a3转运体参与溃疡性结肠炎上皮屏障的机制研究

TNF-α could destroy intestinal epithelial barrier by affecting the expression of tight junction (TJ) proteins in ulcerative colitis (UC), but the detailed pathogenesis could not be clarified yet. Our previous study found that overexpression TNF-α in TNF+/Δare transgenic mice could affect TJ proteins expression leading to increased intestinal epithelial permeability, whereas Slc26a3 (DRA) overexpression could affect TJ proteins expression and restore the TNF-α-induced decrease of epithelial barrier, which means TNF-α induced TJ disruption is in part through its effect on the expression of DRA. Further studies showed that ATX-LPA axis has a central role in the expression of DRA in DSS-induced colitis mice and the process of intestinal epithelial barrier reconstruction. Based on the present theories and our experiments, we pose a hypothesis that TNF-α regulates DRA via ATX-LPA axis therefore leading to intestinal epithelial barrier function change. Thus, we intend to demonstrate the role of TNF-α、ATX and LPA in intestinal epithelial barrier, and the mechanisms of TNF-α-ATX-LPA regulates the expression of DRA and TJ proteins in clinical and animal colitis model. Furthermore, the possible signal pathway and molecular mechanism in the process of TNF-α regulating DRA via ATX-LPA axis will be investigated through activating or blocking NF-κB pathway and the promoter activation analysis in cellular level. In this way, we want to illustrate the role and mechanisms of TNF-α-ATX-LPA-DRA involved in intestinal epithelial barrier in ulcerative colitis, to explore the potential intervention targets of UC.

溃疡性结肠炎(UC)中TNF-α可影响紧密连接蛋白表达破坏肠上皮屏障功能，但机制尚不清楚。本研究小组前期研究发现在TNF+/Δare转基因小鼠模型中TNF-α过表达可导致紧密连接蛋白表达异常、肠上皮通透性增加；而DRA过表达可影响紧密连接蛋白表达，部分恢复TNF-α引起的肠上皮屏障功能降低；进一步研究发现ATX-LPA轴在DSS诱导性小鼠结肠炎中对DRA的表达和上皮屏障重建有重要作用。结合前期研究和预实验结果，我们提出在UC中TNF-α经ATX-LPA轴调控DRA导致肠黏膜上皮屏障功能改变。本研究拟从临床和动物实验证实TNF-α、ATX和LPA调控DRA及紧密连接蛋白的表达，通过激活/阻断NF-κB途径和启动子激活在细胞水平进一步探讨TNF-α在ATX-LPA调控DRA过程中的信号通路和分子机制，阐明TNF-α-ATX-LPA-DRA在UC肠上皮屏障中的作用及意义，以期寻找潜在的干预靶点。

Autotaxin (ATX)是一种分泌酶，水解溶血磷脂酰胆碱(LPC)溶血磷脂酸(LPA)和胆碱。ATX与多种慢性炎症疾病有关，但对其在炎症性肠病(IBD)发展中的作用知之甚少。在这里，我们研究了ATX如何在结肠炎期间促进肠道炎症。我们发现溃疡性结肠炎(UC)患者急性活动期和DSS诱导结肠炎小鼠的肠道内ATX表达水平上调，这可能是由于包括巨噬细胞在内的炎症细胞浸润增加所致。有趣的是，ATX作用于小鼠和人肠上皮细胞Caco2BBE后，紧密连接发生紊乱，促进结肠炎发展。此外，ATX活性的抑制导致炎症细胞因子的产生减少且减轻结肠炎。这些发现提示ATX可能表现出强烈的促炎特性。重组小鼠ATX (rmATX)治疗增加了小鼠巨噬细胞细胞系RAW264.7和骨髓源性巨噬细胞(BMDM)中炎性细胞因子和酶的产生，而siRNA沉默ATX则减少了LPS刺激的促炎因子的产生。值得注意的是，我们发现在ATX处理的RAW264.7细胞和DSS处理的小鼠中，LPA2(一种LPA受体)的水平显著上调。siRNA沉默RAW264.7细胞中LPA2的基因，导致炎症细胞因子的产生减少。此外，腺病毒介导AdshLPA2递送到DSS治疗的小鼠可改善结肠炎。令人惊讶的是，ATX可诱导肠道炎症，而抑制mTOR可显著降低肠道炎症，ATX可通过激活mTOR通路在体内外抑制自噬。总之，我们的研究表明，ATX可能通过激活巨噬细胞的LPA2受体和抑制自噬上调mTOR来加重DSS诱导的结肠炎，这为治疗IBD提供了一个有希望的靶点。