钙激活氯离子通道ANO1在结肠炎粘膜损伤修复及肠动力紊乱中的作用

The significant increase in the incidence of colon cancer has been demonstrated in patients with moderate to severe ulcerative colitis, which seriously endangers human life and health. The pathophysiological mechanisms of ulcerative colitis remain unclear. Anoctamin-1(ANO1) is expressed in intestinal epithelial cells and interstitial cells of Cajal (ICC), which play an important role in intestinal epithelial secretory function and slow wave respectively. Recent studies have reported, ANO1 activation can induce cell proliferation, migration, and it also has been involved in inflammation by regulating TNF-α and interleukin. Our previous studies showed that ANO1 was significantly increased in both epithelium and submucosal tissue in the colon of colitis model compared with control. ANO1 blocker tannic acid significantly decreased the inflammatory response and alleviated diarrhea in colitis. On the basis of these results, mucosal injury related colitis rat model will be established to (1) test the ANO1 expression in epithelium and to study the high expression of ANO1 in intestinal mucosal injury and repair; (2) test ANO1 expression in ICC, to clarify the role of ANO1 in bowel motility disorder of colitis through studying the effects of ANO1 on ICC electrophysiological characteristics. Combined with in vivo and in vitro studies, we try to explore the effectiveness of ANO1 becoming a new target for colitis therapy.

中重度溃疡性结肠炎患者结肠癌罹患率显著增加，严重危害生命健康，且溃疡性结肠炎病理机制尚未阐明。钙激活氯离子通道Anoctamin1(ANO1)在肠上皮细胞与Cajal间质细胞(ICC)中表达，分别在肠内皮分泌及慢波电位产生中发挥重要作用。近期研究发现, ANO1可诱导细胞增殖迁移，并通过干扰TNF-α与白细胞介素等细胞因子参与炎症反应调控，本课题前期研究表明，结肠炎大鼠结肠上皮层与肌层ANO1表达较对照组显著增加, ANO1抑制剂鞣酸可降低炎症反应, 改善结肠炎大鼠腹泻症状，在此基础上，本项目建立粘膜损伤致结肠炎大鼠模型, (1)检测ANO1在结肠上皮层表达特征，考察其在结肠炎大鼠结肠粘膜损伤修复中的作用；(2)检测结肠ICC中ANO1表达特征，通过研究ANO1对ICC电生理特征的作用阐明其在结肠炎大鼠肠动力紊乱中的作用及相关机制。结合体内外研究，探讨ANO1成为结肠炎治疗新靶标的有效性。

肠上皮屏障由多种因素共同构建而成，包括分泌屏障和肠上皮细胞运输相关屏障(比如黏液蛋白、抗菌肽、IgA)、上皮细胞凋亡/增殖、细胞间连接等。肠上皮紧密连接是指两个相邻细胞的细胞膜连接形成一个屏障，阻止分子通过并阻止膜蛋白的移动。肠粘膜上皮细胞的增殖与分化是一个动态过程，依赖于上皮细胞凋亡与增殖的平衡。过度的凋亡发生在病理状态下，如在炎性肠病（IBD）中，就存在上皮细胞过度凋亡。Ca2+激活的Cl-通道跨膜成员16A (TMEM16A，也称为anoctamin-1, ANO1)在2008年被新鉴定为Ca2+激活的Cl-通道。ANO1在肠上皮细胞中表达，控制Cl−在细胞膜两侧的运输，间接协助粘液的分泌和运输。ANO1已被证明与许多疾病有关，包括癌症、高血压和囊性纤维化，而ANO1的激活也与轮状病毒毒素引起的腹泻有关。然而，ANO1在肠上皮的表达和功能目前还存在争议。有研究表明，ANO1是肠内ATP依赖性黏液分泌所必需的，也有研究发现TMEM16A不参与致电性钙激活阴离子转运和黏液稳态。本项目探索了ANO1在生理状态下和结肠炎状态下表达的变化，在离体细胞水平检测了ANO1对肠上皮细胞凋亡和紧密连接屏障功能的影响，细胞水平的研究可以避免肠道细菌、肠粘液等因素的潜在干扰。采用脂多糖(LPS) 孵育大鼠肠上皮细胞IEC-6诱导离体细胞屏障损伤，研究ANO1的下调和过表达对细胞凋亡和紧密连接屏障的影响。通过实时定量聚合酶链反应、Western Blotting和免疫荧光分析分别检测相应的mRNA和蛋白表达变化。研究发现，ANO1表达可被LPS显著促进，核转录因子κB（NF-κB）和Th1、Th2细胞因子均与ANO1表达有关。低剂量和高剂量LPS分别调节紧密连接(肌球蛋白轻链激酶（MLCK）表达)和细胞凋亡依赖性细胞屏障功能障碍。ANO1通过激活ERK1/MLCK信号通路而加重低剂量LPS预处理的IEC-6细胞细胞屏障功能障碍; ANO1通过激活ERK/Bcl-2/Bax抑制高剂量LPS预处理的IEC-6细胞凋亡。本研究结果提示了在肠道炎症中ANO1表达的复杂调控机制，可能为肠上皮屏障损伤的治疗提供潜在的治疗策略，并为今后ANO1在正常和炎症性肠道疾病体内表达和功能的研究奠定基础。