Regulation of Intestinal Paracellular Permeability

描述（由申请人提供）：肠上皮紧密连接（TJ）屏障缺陷是炎症性肠病（IBD）和肠道其他炎症性疾病的关键致病因素。有缺陷的TJ屏障允许细菌的肠渗透增加， 诱导炎症反应的抗原。然而，目前还没有靶向肠TJ屏障的治疗剂。此外，导致肠TJ屏障增强的机制仍然知之甚少。该提案的主要目标是引入和开发一种新的治疗剂，其靶向肠TJ屏障，可以快速推进临床使用;并确定调节肠TJ屏障收紧的新型细胞内机制。在我们的初步研究中，我们筛选了超过20种益生菌菌株（广泛用于商业）以鉴定单一菌株，嗜酸乳杆菌（LA），其在IBD动物模型中具有显著的肠道TJ屏障增强特性和治疗功效。本申请的主要目的是研究LA的肠TJ屏障增强作用并确定LA在IBD动物模型中的治疗功效。 基于我们的初步研究，我们提出了一个新的假设，即LA增强肠道TJ屏障是由一个核苷酸结合寡聚化结构域蛋白1（Nod 1）信号转导途径激活occludin基因调控的。封闭蛋白是一种跨膜蛋白，在肠道TJ屏障调节中起重要作用。在这项拨款申请中，我们挑战了公认的科学范式：1）Nod 1是一种细胞质模式识别受体，2）Nod 1的主要细胞靶点是NF-κB的激活。基于我们的初步数据显示，LA诱导的快速胞质顶端膜易位的Nod 1在肠上皮细胞在体外和体内，我们假设，LA诱导的增强肠TJ屏障调节顶端膜易位的Nod 1。我们还假设LA激活Nod 1信号转导通路导致NF-κB的抑制（而不是激活），NF-κB是诱导TJ开放的关键促炎介质，p38激酶通路激活，其信号为occludin基因激活和occludin依赖性增强肠道TJ屏障功能。提出的具体目标是：1）描述Nod 1在LA诱导的肠上皮TJ屏障增强中的作用; 2）描述LA诱导的occludin基因和肠TJ屏障调节的机制; 3）描述LA在IBD动物模型中的治疗效果。拟议研究的成功完成将提供必要的关键临床前数据，以支持 我们未来的临床试验将检查LA在IBD患者肠道屏障收紧以及预防和治疗IBD中的治疗效果。

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.