Regulation of Intestinal Paracellular Permeability

描述(申请人提供)：肠道上皮紧密连接(TJ)屏障缺陷是炎症性肠病(IBD)和其他肠道炎症条件的关键致病因素。有缺陷的TJ屏障允许细菌在肠道中渗透 引起炎症反应的抗原。然而，目前还没有可用的治疗药物来靶向肠道TJ屏障。此外，导致肠道TJ屏障增强的机制仍然知之甚少。这项建议的主要目标是引入和开发一种针对肠道TJ屏障的新治疗剂，该药物可以迅速推进用于临床；并确定调节肠道TJ屏障收紧的新的细胞内机制。在我们的初步研究中，我们筛选了20多株益生菌(商业上广泛使用)来鉴定单一菌株，嗜酸乳杆菌(LA)，它在IBD动物模型中具有显著的肠道TJ屏障增强特性和治疗效果。该应用的总体目标是研究LA对肠道TJ屏障的增强作用，并确定LA在IBD动物模型中的治疗效果。在前期研究的基础上，我们提出了一个新的假说，即LA对肠道TJ屏障的增强是由occludin基因激活的核苷酸结合的寡聚化结构域蛋白1(Nod1)信号转导途径调节的。Occludin是一种跨膜蛋白，在肠道TJ屏障调节中起核心作用。在这次拨款申请中，我们挑战了现有的科学范式：1)Nod1是细胞质模式识别受体；2)Nod1的主要细胞靶点是激活NF-κB。根据我们的初步数据显示，在体外和体内，LA诱导肠上皮细胞中Nod1迅速从细胞质到顶膜转位，我们假设LA诱导的肠TJ屏障的增强是由Nod1的顶膜转位调节的。我们还假设，Nod1信号转导通路的LA激活导致了诱导TJ开放的关键促炎介质NF-κB的抑制(而不是激活)，以及p38激酶途径的激活，p38激酶通路发出occludin基因激活和occludin依赖的增强肠道TJ屏障功能的信号。本研究的目的是：1)阐明Nod1在LA诱导的肠上皮TJ屏障增强中的作用；2)阐明LA对occludin基因和肠TJ屏障的调节机制；3)阐明LA对IBD动物模型的治疗效果。拟议研究的成功完成将提供关键的临床前数据，以支持 我们未来的临床试验将检验LA在IBD患者肠道屏障收紧以及预防和治疗IBD方面的疗效。

DESCRIPTION (provided by applicant): Defective intestinal epithelial tight junction (TJ) barrier is a key pathogenic factor of inflammatory bowel disease (IBD) and other inflammatory conditions of the gut. The defective TJ barrier allows increased intestinal permeation of bacterial antigens that induce inflammatory response. However, there are no currently available therapeutic agents that target the intestinal TJ barrier. Moreover, the mechanisms that lead to an enhancement of intestinal TJ barrier remain poorly understood. The major goals of this proposal are to introduce and develop a new therapeutic agent that targets the intestinal TJ barrier, which can be rapidly advanced for clinical usage; and to identify novel intracellular mechanisms that regulate the tightening of the intestinal TJ barrier. In our preliminary studies, we screened over 20 probiotic bacterial strains (that are widely used commercially) to identify a single strain, Lactobacillus acidophilus (LA), which has a remarkable intestinal TJ barrier augmenting properties and therapeutic efficacy in animal models of IBD. The over-arching goals of this application are to investigate the intestinal TJ barrier enhancing effects of LA and to determine the therapeutic efficacy of LA in animal models of IBD. Based on our preliminary studies, we advance a novel hypothesis that LA enhancement of intestinal TJ barrier is regulated by a nucleotide-binding oligomerization domain - containing protein 1 (Nod1) signal transduction pathway activation of occludin gene. Occludin is a trans-membrane protein which plays a central role in intestinal TJ barrier regulation. In this grant application, we challenge to well-established scientific paradigms 1) that Nod1 is a cytoplasmic pattern recognition receptor and 2) that primary cellular target of Nod1 is the activation of NF-κB. Based on our preliminary data showing that LA induces a rapid cytoplasmic-to-apical membrane translocation of Nod1 in intestinal epithelial cells in-vitro and in-vivo, we hypothesize that LA- induced augmentation of intestinal TJ barrier is regulated by apical membrane translocation of Nod1. We also hypothesize that LA activation of Nod1 signal transduction pathway leads to a suppression (not activation) of NF-κB, a key pro-inflammatory mediator that induces the TJ opening, and an activation of p38 kinase pathway, which signals occludin gene activation and occludin-dependent enhancement in intestinal TJ barrier function. The proposed specific aims are to: 1) delineate the role of Nod1 in LA-induced augmentation of intestinal epithelial TJ barrier; 2) delineate the mechanism of LA-induced regulation of occludin gene and intestinal TJ barrier; and 3) delineate the therapeutic efficacy of LA in animal models of IBD. The successful completion of the proposed studies will provide the critical pre-clinical data necessary to support our future clinical trials that will examine the therapeutic efficacy of LA in intestinal barrier tightening in IBD patients and also in the prevention and treatment of IBD.