AhR与HIF-1α竞争性结合ARNT在肠胶质细胞调控肠粘膜屏障的作用机制研究

The intestinal epithelial barrier(IEB) is one of the important denfence barriers in the body. Our previous work revealed that both inducible nitric oxide synthase(iNOS) and S-nitrosoglutathione reductase(GSNOR) play important roles in the synthetize and metabolism of nitric oxide(NO) and S-Nitrosoglutathione(GSNO) as well as the modulation of IEB functions of enteric glial cell(EGC). Recent works showed that aryl hydrocarbon receptor(AhR) is involved in the iNOS-GSNOR double gate control as well as NO/GSNO synthetize and metabolism through competing with hypoxia-inducible factor-1α(HIF-1α) in binding AhR nuclear transporter(ARNT). We speculate that the competion and crosstalk between AhR/ARNT and ARNT/ HIF-1α may be the important molecule mechanism underlying the modulation of IEB function of EGC, and through the abovementioned pathway AhR agonists could enhance the capability of EGC in protecting IEB functions under normal and pathological stimuli. The influence of the competition and crosstalk between AhR/ARNT and ARNT/ HIF-1α pathways on IEB modulation of EGC under physiological and various pathological conditions will be evaluated based on intestinal epithelial cell(IEC)-EGC co-culture system.As well, the precise role of AhR pathways in the IEB modulation mechanism of EGCs will be investigated in different IEB dysfunction models in both in vitro and in vivo conditions.The present study will provide further clues in clarifying the exact role of EGCs in IEB regulation mechanisms.

肠粘膜屏障（IEB）是机体的重要防御屏障。前一课题已验证诱导型一氧化氮合酶（iNOS）及亚硝基谷胱甘肽还原酶（GSNOR）双通道控制机制在肠胶质细胞(EGC)对一氧化氮（NO）/亚硝基谷胱甘肽（GSNO）合成与代谢以及IEB调控的关键作用。近来研究显示，芳香烃受体（AhR）可通过与缺氧诱导因子-1α（HIF-1α）竞争性结合芳香烃受体核转位蛋白(ARNT)参与iNOS-GSNOR双通道控制以及NO/GSNO合成与代谢调控。我们推测AhR及HIF-1α与ARNT竞争性结合是影响EGC屏障调控功能重要机制，AhR激动剂通过抑制ARNT/HIF-1α异二聚体形成来增强EGC屏障保护功能。本研究拟基于体外EGC-IEC共培养体系，对比观察在生理及病理刺激下干预AhR与HIF-1α竞争性结合ARNT对EGC调控IEB功能的影响，同时利用体内外IEB损伤模型，进一步阐明AhR介导EGC调控IEB机制。

严重创伤、烧伤及大型手术所诱发的急性小肠缺血再灌注(IR)损伤是影响临床救治的重要问题之一。急性小肠IR损伤已成为临床诱发肠黏膜屏障（IEB）损伤的重要原因之一。我们前期研究已表明，肠道神经系统中最丰富的细胞，肠胶质细胞（EGC）与IEB的功能调控密切相关，但其机制还需要进一步阐明。.本研究设想，缺氧条件下EGC中芳香烃受体（AhR）被激活，并与缺氧诱导因子（HIF-1α）竞争性结合ARNT，增加AhR/ARNT转录活性，促进下游屏障保护性基因表达，并同时阻断HIF-1α/ARNT转录作用导致的肠屏障损伤。.本研究利用小鼠IR模型，发现在肠道缺血缺氧损伤条件下，EGC 中AhR的表达明显增加，说明缺氧刺激下EGC中AhR被明显激活。体外EGC细胞进行氧糖剥夺（OGD）刺激，HIF-1α、AhR及其活化标记CYP1A1的mRNA表达均明显升高。为了进一步研究AhR在IR条件下的作用，我们建立了EGC细胞特异性敲除AhR的小鼠模型，结果发现AhR 基因的缺失对正常情况及急性IR诱导的肠黏膜结构损伤以及肠黏膜屏障损伤并无明显影响；同时，我们利用体外EGC细胞干扰AhR后与肠上皮细胞（Caco-2）共培养，同样发现在常氧及OGD条件下敲低AhR并未显著改变肠上皮屏障; 此外，通过双荧光素酶报告基因检测，我们发现AhR与HIF-1α的竞争性结合在OGD条件下并没有发生显著改变。进一步的测序研究分析意外发现，敲低AhR引起多个与细胞迁移功能相关的基因变化最为显著增强；这也在后续的增殖实验中得到了验证：敲低AhR后明显促进了EGC的增殖及迁移能力，尤其是明显影响了离子通道TRPV4的活性。进一步的动物实验也出乎意料的验证了相同的结果。这提示我们，当小肠IR发生时，EGC中AhR的激活可能通过调控TRPV4的活性，从而影响EGC的增殖迁移能力。通过本项目，我们深入了解AhR介导EGC调控IEB以及小肠IR损伤的分子机制，为进一步阐明小肠IR损伤的调控机制、寻找治疗新靶点提供新依据。.