肠粘膜屏障(IEB)是机体防御的重要屏障.前一课题里我们验证了肠胶质细胞(EGC)对IEB调控的关键作用,并发现生理及病理条件下诱导型一氧化氮合酶(iNOS)在EGC的IEB调控机制中扮演着不同角色.近来研究发现iNOS可能通过与亚硝基谷胱甘肽还原酶(GSNOR)协同形成双通道控制,来调节NO及其亚硝基化合物亚硝基谷胱甘肽(GSNO)合成与代谢,而GSNO为保护IEB的关键分子.本项目拟基于EGC-IEC共培养体系,通过干预EGC iNOS及GSNOR表达,对比观察生理及病理条件下EGC胞内NO-GSNO动态变化,验证iNOS-GSNOR双通道控制对EGC调控IEB的重要影响,进一步阐明EGC源iNOS在IEB调控中的作用;并以EGC源GSNO为切入点,利用体内外IEB损伤模型,通过Biotin-switch法及蛋白质组学技术,筛选IEB相关亚硝基化靶点蛋白,深入了解IEB调控分子机制.

Intestinal epithelial barrier (IEB) plays a central role in maintaining internal hemostasis of the body. The key influence of enteric glial cells (EGC) in IEB regulation has been demonstrated in our previous study. EGC-derived inductible nitric oxide synthase (iNOS) plays complicated roles in IEB regulation under various conditions. Recent study suggested that there may exist iNOS- S-nitrosoglutathione reductase (GSNOR) double gate control in manipulating the synthesis and metabolism of NO and its related molecule S-nitrosoglutathione (GSNO), the latter demonstrating the barrier-protective properties. Our aim is to investigate the role of iNOS-GSNOR double gate control in the metabolism of NO-GSNO in EGCs through interfering the expression and activities of two key molecules, prove the influence of iNOS-GSNOR in the mechanisms of EGC in IEB regulation, and further clarify the exact role of EGC-derived iNOS. Meanwhile, through GSNO administration in in vitro and in vivo IEB experiments, the targeted proteins with S-nitrosylation in intestinal epithelial cells will be investigated with biotin-switch technique and other corresponding proteomics techniques. The goal of our study is to reveal the underlying mechanisms of EGC in IEB regulation under various pathophysiological conditions, and search potential drugs with IEB protective effects by the detection of protein S-nitrosylation.