BMP通过NRAGE信号诱导肠粘膜屏障损伤的作用及机制研究

Intestinal epithelial barrier (IEB) is an important defense barrier. Our previous study has found that the activation of BMP signaling pathway is closely related to the damage of IEB functions. However, the mechanisms remain unclear. Recent study shows that NRAGE may play an important role in the activation of NF-κB by BMP signaling pathway, and its up-regulation excerts negative effects on the expression and distribution of the E-cadherin. Based on the preliminary study, we hypothesize that BMP distribupted the expression and distribution of the key adherens junctions and tight junction proteins of IEB through NRAGE may be the key mechanism through which BMP mediated IEB founction damage. This study will further detect NF-κB activation and its effects on structure and function of IEB by the administration of BMP in the NRAGE knockout mice model and in IEC using SiRNA blocking NRAGE expression, in vivo and vitro, meanwhile we will design a small molecular peptides which based on NRAGE protein functional areas (repeat domain) to detect its effects on the NF-κB activation and the protection on intestinal epithelial barrier in ischemical reperfusion injury mice. This project will be carried out to analyze the possible mechanism of NRAGE involved in the IEB injury and to find a novel therapeutic target for IEB dysfunction.

肠黏膜屏障（IEB）是机体防御的重要屏障，在前期研究中，我们首次发现骨形态蛋白（BMP）信号通路的激活与IEB功能损伤密切相关，但其机制不清楚，近来研究表明：NRAGE在BMP激活NF-κB信号通路中可能扮演着关键角色，同时NRAGE的上调可干预细胞粘附分子E-cadherin的表达与分布。由此我们推测：NRAGE通过激活NF-κB来干预IEB关键粘附分子表达与分布可能是BMP介导IEB损伤的关键机制。本研究拟采用NRAGE kockout 鼠模型及利用SiRNA阻断肠上皮细胞中NRAGE，通过给予或激活BMP观察对NF-κB激活状况及肠粘膜结构及功能的影响，并利用NRAGE的功能区（重复区域）设计小分子的多肽，进一步明确在缺血再灌注条件下其对IEB的保护作用，初步阐明BMP通过NRAGE信号诱导肠粘膜屏障损伤的机制，为IEB功能障碍的治疗寻找新的靶点。

目的探讨神经生长因子受体作用黑色素瘤抗原基因同源蛋白(neurotrophin receptor-interacting MAGE homolog,NRAGE)是否参与肠缺血再灌注(ischemia and reperfusion,I/R)损伤的过程及其对肠上皮细胞凋亡及occludin蛋白的影响。方法制备SD大鼠小肠I/R损伤模型,取肠组织行免疫组化染色,观察NRAGE蛋白的表达变化;体外肠上皮永生化细胞株IEC-6给予缺氧及复氧后观察细胞NRAGE的表达变化;将NRAGE过表达慢病毒(Lv-NRAGE组)、NRAGE干扰表达慢病毒(sh-NRAGE组)及对照慢病毒(Lv-control组)感染IEC-6细胞,并设置不加慢病毒的对照组(NC组),Western blot及RT-PCR观察各组细胞NRAGE蛋白及mRNA的表达变化,流式细胞术观察各组IEC-6细胞的凋亡情况,Western blot观察感染慢病毒后紧密连接蛋白occludin蛋白的表达变化。结果 I/R损伤后肠黏膜上皮细胞NRAGE表达较sham组升高(P<0.01);体外IEC-6细胞缺氧6h后NRAGE蛋白的表达增加(P<0.01),NRAGE mRNA表达升高(P<0.01)。感染慢病毒48h后,相比于Lvcontrol组,Lv-NRAGE组早期凋亡率增加(P<0.01),occludin蛋白表达降低(P<0.01),sh-NRAGE组早期凋亡率降低(P<0.01),occludin蛋白表达增高(P<0.001)。结论 NRAGE参与了I/R损伤过程,并促进肠上皮细胞凋亡,降低occludin蛋白的表达。