Intestinal Perfusion and Permeability in Sepsis

描述(由申请人提供)： 在多器官功能障碍综合征(MODS)中，受影响最明显的实质器官是肺、肝、肾和肠道。这些器官的正常功能依赖于组成不同的隔室的建立和维护，这些隔室由一片片上皮细胞排列。这一过程中的一个基本要素是相邻上皮细胞之间形成紧密连接(TJ)。TJ作为一个受调控的半透屏障，限制了溶质在相邻细胞之间的细胞旁途径上的被动扩散。因此，TJ的屏障功能是必要的，以防止存在于由上皮定义的两个隔室之间的浓度梯度的消散。 人类MODS的组织病理学非常平淡；大量细胞死亡，无论是由于坏死还是细胞凋亡，几乎肯定不是MODS的原因。相反，MODS发展的最后一步可能是由于控制不良的全身炎症反应的有害影响，导致多个器官中广泛存在的实质细胞功能障碍。因此，一个极未被探索的研究领域可以用这个问题来概括：炎症反应如何导致实质细胞功能障碍？基于我们在上一轮资助中的工作，我们假设MODS的结果至少部分是由于一氧化氮(NO)依赖的TJ蛋白表达和亚细胞定位的扰动。为了验证这一假设，我们建议在从整个动物到培养细胞或亚细胞部分的整合水平上研究炎症诱导的上皮屏障功能和TJ形成的变化。在体外，我们将使用Caco-2(人肠细胞样单分子层)作为还原模型系统，重点研究肠上皮通透性的变化。然而，在体内，在使用小鼠和大鼠的研究中，我们不仅将评估肠道上皮屏障功能的变化，还将评估肝脏和肺的上皮屏障功能的变化。在一系列6个特定的目标中，我们将检验这些特定的假设：1)小鼠脓毒症导致TJ结构和功能的改变，其机制依赖于NO、ROS和/或ONOO-的形成；2)MODS死亡患者上皮TJ的结构紊乱；3)TJ蛋白ZO-1的转录减少是导致炎症或非炎症诱导的上皮屏障功能改变的关键步骤；4)细胞因子或NO诱导的事件损害了培养的Caco-2细胞中关键TJ蛋白claudin-1和occludin的正确包装和靶向；5)3-磷酸甘油醛脱氢酶翻译后修饰导致上皮屏障功能障碍；6)Na，K-ATPase功能改变导致炎症诱导的上皮屏障功能紊乱。

DESCRIPTION (provided by applicant): The parenchymal organs that are most prominently affected in the multiple organ dysfunction syndrome (MODS) are the lungs, liver, kidneys and gut. The normal functioning of these organs depends on the establishment and maintenance of compositionally distinct compartments that are lined by sheets of epithelial cells. An essential element in this process is the formation of tight junctions (TJs) between adjacent epithelial cells. The TJ acts as a regulated semi-permeable barrier that limits the passive diffusion of solutes across the paracellular pathway between adjacent cells. Thus, the barrier function of the TJ is necessary to prevent dissipation of the concentration gradients that exist between the two compartments defined by the epithelium. The histopathology of MODS in humans is remarkably bland; massive cell death, whether due to necrosis or apoptosis, is almost certainly not the cause of MODS. Rather, the final step in the development of MODS is probably the widespread dysfunction of parenchymal cells in multiple organs as a result of the deleterious effects of a poorly controlled systemic inflammatory response. Thus, a hugely under-explored area of research can be summarized by this question: How does the inflammatory response lead to parenchymal cell dysfunction? Based on our work during the previous cycle of funding, we hypothesize that MODS results, at least in part, from nitric oxide (NO)- dependent perturbations in the expression and subcellular localization of TJ proteins. To test this hypothesis, we propose to study inflammation-induced alterations in epithelial barrier function and TJ formation at levels of integration ranging from whole animals to cultured cells or subcellular fractions. In vitro, we will focus on changes in intestinal epithelial permeability using Caco-2 (human enterocyte-like) monolayers as a reductionist model system. In vivo, however, in studies using mice and rats, we will evaluate changes in epithelial barrier function not only in the gut, but also in the liver and lung as well. In a series of 6 Specific Aims, we will test these Specific Hypotheses: 1) sepsis in mice leads to alterations in TJ structure and function via mechanisms that depend on the formation of NO., reactive oxygen species (ROS), and/or ONOO-; 2) the structure of epithelial TJs is deranged in patients dying with MODS; 3) decreased transcription of the TJ protein, ZO-1, is a critical step leading to inflammation- or NO-induced alterations in epithelial barrier function; 4) cytokine- or NO-induced events impair the proper packaging and targeting of the key TJ proteins, claudin-1 and occludin, in cultured Caco-2 cells; 5) post-translational modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contributes to epithelial barrier dysfunction; 6) alterations in the function of Na+,K+-ATPase contribute to inflammation-induced derangements in epithelial barrier function.