NHE2转运体在阿司匹林相关性肠病中对肠上皮屏障作用的机制研究

The incidence of aspirin-associated enteropathy (AAE) is increasing year by year, and there is still a lack of effective prevention and treatments. The contact between aspirin enteric-coated tablets and intestinal epithelial cells is an independent risk factor. Increased paracellular permeability (PP) of intestinal epithelial cells caused by abnormal expression and localization of tight junction protein ZO-1 is an important mechanism at early onset. What molecules on the surface of intestinal epithelial cells are affected by aspirin, leading to abnormal ZO-1 and increased PP? So far, it had not been investigated. PP is regulated by sodium/hydrogen exchanger (NHE). Our previous studies have confirmed that NHE2 is the dominant NHE on the apical membrane (contacting intestinal lumen contents) of intestinal epithelial cells and the inhibition of NHE2 activity can reduce PP, suggesting that the increase of NHE2 activity may lead to the increase of PP. Our preliminary experiments confirmed that aspirin significantly enhanced the expression and activity of NHE2 in intestinal epithelial cells. However, the relationship between NHE2 and PP in AAE and the specific mechanisms of its influence on PP are not clear. Therefore, this study intends to study the effects of NHE2 on ZO-1 and PP in AAE in vitro and in vivo, and to explore the possible signaling pathway and molecular mechanism of NHE2 regulating ZO-1 at cellular level, in order to clarify the role and significance of NHE2 in AAE, and to provide new ideas and basis for intervention of AAE.

阿司匹林相关性肠病（AAE）的发病率逐年上升，尚缺乏有效防治措施。阿司匹林肠溶片与肠上皮细胞接触是其独立危险因素。紧密连接蛋白ZO-1表达、定位异常导致肠上皮细胞旁路通透性（PP）增高是发病早期的重要机制。阿司匹林通过肠上皮细胞表面何种分子导致ZO-1异常、PP增高？目前无研究。PP受钠/氢交换体（NHE）调节。我们前期研究证实NHE2是肠上皮细胞表面顶膜侧（接触肠腔内容物）的优势NHE，抑制其活性可降低PP，提示NHE2活性增高可能导致PP增加。我们预实验证实阿司匹林显著增强肠上皮细胞NHE2的表达和活性。但在AAE中NHE2与PP的关系及其影响PP的具体机制未明。因此，本研究拟从体内外实验研究NHE2在AAE中对ZO-1及PP的影响，在细胞水平探讨NHE2调节ZO-1的可能信号通路和分子机制，以阐明NHE2在AAE中影响肠上皮屏障的作用及意义，为干预AAE提供新思路和依据。

阿司匹林被广泛、长期地用于治疗缺血性心脑血管疾病，最常见的副作用是胃、肠道黏膜损害。近年来阿，阿司匹林相关性肠病（aspirin associated enteropathy，AAE）的发生率增高，表现为小肠黏膜炎症，可并发出血和梗阻，给有心脑基础病的患者带来致命性的危险，是临床亟需解决的问题。然而，目前AAE尚缺乏有效治疗措施。由于缺乏理想的动物模型，关于AAE的发病机制的研究尚处于起步阶段。.本课题组经过大量实验和探索，成功地在小鼠上建立了AAE的慢病动物模型。经阿司匹林干预的小鼠出现了体重下降、摄食量减少、粪便隐血阳性、显著的小肠缩短、绒毛萎缩、隐窝拉长等变化，小肠炎症因子（IL-1、IL-6、TNF-α）的表达显著增高，该模型体现了慢性肠炎的特点。经阿司匹林干预的小鼠小肠对大分子物质FD4的通透性显著增加，与临床上观察到阿司匹林服用者的小肠上皮通透性增加的表现一致。我们还找到AAE发病的首个步骤——氧化应激损伤的依据，如氧化应激指标ROS、nhe4表达增强，抗氧化基因CAT、Sod2、Gpx1、Gpx4表达下降，线粒体结构紊乱。该模型补充了目前国际上缺乏小型动物慢病模型的不足，为后续该病的深入研究打下了良好的基础。.本课题的研究证实了阿司匹林通过下调紧密连接蛋白ZO-1、occludin的表达导致肠上皮细胞旁路通透性增加在 AAE 中的作用，找到了阿司匹林致病的关键分子ZO-1、occludin。.用肠上皮细胞建立体外模型，我们发现酸性阿司匹林溶液下调紧密连接蛋白ZO-1、occludin的表达，导致肠上皮细胞旁路通透性增加；而中性阿司匹林溶液却不影响紧密连接蛋白ZO-1、occludin的表达，也不会导致肠上皮细胞旁路通透性增加。此外，酸性阿司匹林溶液上调肠上皮细胞的酸调节蛋白NHE2的表达，NHE2促进细胞内H+排出细胞外，而中性阿司匹林溶液不上调NHE2的表达。抑制NHE2的功能后，细胞内H+的排出减少，加重了阿司匹林所致的氧化应激。以上结果提示阿司匹林的致病作用与其酸性有关，细胞内的酸性环境可加重氧化应激，参与致病。.综上，本课题建立了AAE慢病动物模型，证实了AAE的致病机制——氧化应激和肠上皮通透性增加，确立了相关分子ZO-1和occludin，发现了药物酸性和肠上皮细胞内酸性环境参与阿司匹林致病，为进一步探索AAE的防治策略提供了新思路。