Intestinal Perfusion and Permeability in Sepsis

脓毒症的肠道灌注和渗透性

• 批准号: 7089011
• 负责人: Mitchell P. Fink
• 金额: $ 33.86万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7089011
• 关键词: DNA binding protein; cell component structure /function; cytokine; free radical oxygen; gastrointestinal epithelium; gene expression; glyceraldehyde 3 phosphate dehydrogenase; human tissue; inflammation; laboratory mouse; laboratory rat; liver cells; membrane permeability; multiple organ failure; nitric oxide; polymerase chain reaction; posttranslational modifications; protein localization; septic shock; septicemia; sodium potassium exchanging ATPase; tight junctions; tissue /cell culture

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.

描述（由申请人提供）： 多器官功能障碍综合征 (MODS) 中受影响最严重的实质器官是肺、肝、肾和肠道。这些器官的正常功能取决于组成不同的区室的建立和维持，这些区室排列有上皮细胞片。此过程中的一个基本要素是相邻上皮细胞之间紧密连接 (TJ) 的形成。 TJ 充当受调节的半渗透屏障，限制溶质在相邻细胞之间的细胞旁通路上的被动扩散。因此，TJ 的屏障功能对于防止上皮定义的两个区室之间存在的浓度梯度消散是必要的。 人类 MODS 的组织病理学非常温和。大量细胞死亡，无论是由于坏死还是凋亡，几乎可以肯定不是 MODS 的原因。相反，MODS 发展的最后一步可能是由于全身炎症反应控制不良造成的有害影响，导致多个器官的实质细胞广泛功能障碍。因此，一个尚未充分探索的研究领域可以通过这个问题来总结：炎症反应如何导致实质细胞功能障碍？根据我们在上一个资助周期中的工作，我们假设 MODS 至少部分是由于 TJ 蛋白的表达和亚细胞定位中一氧化氮 (NO) 依赖性扰动造成的。为了检验这一假设，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）单层作为还原模型系统来关注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，还将评估肝脏和肺部的上皮屏障功能的变化。在一系列 6 个具体目标中，我们将测试这些具体假设：1) 小鼠败血症通过依赖于 NO、活性氧 (ROS) 和/或 ONOO 形成的机制导致 TJ 结构和功能的改变-; 2）死于MODS的患者上皮TJ结构紊乱； 3) TJ蛋白ZO-1的转录减少是导致炎症或NO诱导的上皮屏障功能改变的关键步骤； 4) 细胞因子或NO诱导的事件会损害培养的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（GAPDH）的翻译后修饰导致上皮屏障功能障碍； 6) Na+,K+-ATP酶功能的改变导致炎症诱导的上皮屏障功能紊乱。