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）： 在多器官功能障碍综合征（MOD）中受到最大影响的实质器官是肺，肝，肾脏和肠道。这些器官的正常功能取决于由上皮细胞衬里的构图不同隔室的建立和维护。此过程中的一个重要元素是形成相邻上皮细胞之间紧密连接（TJ）的形成。 TJ充当一个受调节的半渗透屏障，限制了溶质在相邻细胞之间的细胞细胞途径上的被动扩散。因此，TJ的屏障功能对于防止上皮定义的两个隔室之间存在的浓度梯度的耗散是必要的。 Mod在人类中的组织病理学非常平淡。无论是由于坏死还是凋亡引起的大规模细胞死亡，几乎肯定不是MOD的原因。相反，MOD的发展的最后一步可能是由于系统性炎症反应不良的有害作用，多个器官中实质细胞的广泛功能障碍。因此，可以通过这个问题来总结一个大量探索的研究领域：炎症反应如何导致实质细胞功能障碍？基于我们在上一个资金周期中的工作，我们假设MOD至少部分是由于一氧化氮（NO） - 依赖性扰动TJ蛋白的表达和亚细胞定位。为了检验这一假设，我们建议研究炎症诱导的上皮屏障功能的改变和TJ形成，从整个动物到培养细胞或亚细胞分数的整合水平。在体外，我们将专注于使用CACO-2（人类肠肠状）单层作为还原主义模型系统的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，我们将不仅在肠道中而且在肝脏和肺中评估上皮屏障功能的变化。在一系列的6个特定目标中，我们将测试这些特定的假设：1）小鼠中的败血症会导致TJ结构和功能的改变，这些机制取决于NO。，反应性氧（ROS）和/或Onoo-的形成； 2）在死于mod的患者中，上皮TJ的结构被扰乱； 3）TJ蛋白ZO-1的转录减少是导致炎症或无诱导的上皮屏障功能改变的关键步骤； 4）在培养的Caco-2细胞中，细胞因子或无诱导的事件会损害关键TJ蛋白Claudin-1和occludin的正确包装和靶向； 5）3-磷酸甘油醛-3-磷酸脱氢酶（GAPDH）的翻译后修饰有助于上皮屏障功能障碍； 6）Na+，K+-ATPase功能的改变有助于炎症引起的上皮屏障功能的扰动。