MOLECULAR MECHANISMS OF GUT BARRIER DYSFUNCTION

肠道屏障功能障碍的分子机制

• 批准号: 6343031
• 负责人: RUSSELL L DELUDE
• 金额: $ 27.06万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343031
• 关键词: ADP ribosylation; adenosine triphosphate; bacterial disease; cell line; cytokine; cytoskeletal proteins; endotoxins; enzyme activity; gastrointestinal absorption /transport; gastrointestinal epithelium; gastrointestinal function; gene targeting; genetic regulation; genetic transcription; genetically modified animals; inflammatory bowel diseases; intestinal mucosa; laboratory mouse; laboratory rat; mitochondria; molecular dynamics; nitric oxide; nitric oxide synthase; phosphorylation; pulmonary respiration

Although the mechanisms promoting intestinal injury in sepsis and endotoxemia remain to be elucidated, extensive data obtained by our group as well as others suggest that one important factor is probably over- production of the pluripotent mediator, nitric oxide (NO-). Although some clues exist in the literature, the mechanisms whereby NO-modulates intestinal epithelial barrier function in inflammatory conditions, such as sepsis or inflammatory bowel disease, are poorly understood. Accordingly, the goal of the studies proposed herein is to improve our understanding of the fundamental cellular and molecular mechanisms underlying alterations in intestinal epithelial permeability induced by NO- and/or other related reactive nitrogen intermediates (RNIs). The project has been organized under four Specific Aims. Aim 1: Engineer a tetracycline-regulated expression plasmid to permit controlled transcriptional regulation of inducible nitric oxide synthase (iNOS) gene expression in a well- differentiated enterocytic cell-line, Caco-2, in order to test the hypothesis that excessive endogenous generation of NO- is sufficient (even in the absence of other pro-inflammatory mediators) to increase intestinal epithelial permeability. Aim 2: Using (i) Cytokine-stimulated cultured enterocytes (Caco-2 and T84 cells), (ii) the engineered cell line described under Aim 1, (iii) an in vivo a model system for monitoring gut mucosal respiration in rats; and (iv) mucosal samples from endotoxemic wild-type or iNOS "knock-out" mice, test the hypothesis that up-regulation of NO production in the intestinal epithelium leads to cellular ATP depletion on the basis of mitochondrial dysfunction and/or activation of the enzyme, poly(ADP)-ribose polymerase. Aim 3: The cells lines described under Aim 2 will be used to test the hypothesis that exogenously supplied or endogenously produced NO- promotes the phosphorylation or dephosphorylation of key cytoskeletal proteins and that NO-mediated alterations in protein tyrosine phosphorylation or dephosphorylation of key cytoskeletal proteins and that NO-mediated alterations in protein tyrosine phosphorylation lead to changes in cytoskeletal integrity and epithelial permeability. Aim 4: Test the hypothesis that exogenously supplied or endogenously produced NO-promotes mono(ADP)-ribosylation; and identify the NO-sensitive elements responsible for this phenomenon. The proposed experiments will provide powerful new tools (e.g., the Caco-2 cell line expressing iNOS in a Tc-regulated fashion) for studying the effects of NO- on epithelial function. In addition, the proposed studies should open up fruitful lines of investigation regarding the fundamental mechanisms [e.g., mono(ADP)-ribosylation of cytoskeletal proteins] underlying the regulation of intestinal epithelial permeability under physiologic and pathophysiologic conditions.

尽管败血症和肠道损伤的促进机制 内毒素血症仍有待阐明，我们小组获得了大量数据 以及其他人表明，一个重要因素可能是过度 多能介质一氧化氮 (NO-) 的产生。虽然有些 文献中存在线索，NO 调节的机制 炎症条件下的肠上皮屏障功能，例如 败血症或炎症性肠病，人们知之甚少。因此， 本文提出的研究的目的是提高我们对 改变背后的基本细胞和分子机制 NO-和/或其他相关物质诱导的肠上皮通透性 活性氮中间体（RNI）。该项目已组织实施 四个具体目标。目标 1：设计四环素调节的 表达质粒允许受控转录调节 诱导型一氧化氮合酶（iNOS）基因在良好的表达 分化的肠细胞系 Caco-2，以测试 假设过量的内源性 NO- 生成就足够了（甚至 在没有其他促炎介质的情况下）以增加肠道 上皮通透性。目标 2：使用 (i) 细胞因子刺激的培养物 肠上皮细胞（Caco-2 和 T84 细胞），(ii) 工程细胞系 目标 1 (iii) 中描述的用于监测肠道的体内模型系统 大鼠粘膜呼吸； (iv) 内毒素血症的粘膜样本 野生型或 iNOS“敲除”小鼠，检验上调的假设 肠上皮中 NO 的产生导致细胞 ATP 基于线粒体功能障碍和/或激活的耗竭 酶，聚（ADP）-核糖聚合酶。目标 3：描述的细胞系 目标 2 将用于检验外源供给的假设 或内源产生的NO-促进磷酸化或 关键细胞骨架蛋白的去磷酸化以及 NO 介导的去磷酸化 蛋白质酪氨酸磷酸化或去磷酸化的改变 关键的细胞骨架蛋白以及 NO 介导的蛋白质改变 酪氨酸磷酸化导致细胞骨架完整性的变化 上皮通透性。目标 4：检验外生假设 供应的或内源产生的NO-促进单(ADP)-核糖基化；和 确定造成这种现象的 NO 敏感元件。 拟议的实验将提供强大的新工具（例如 Caco-2 以 Tc 调节方式表达 iNOS 的细胞系）用于研究 NO-对上皮功能的影响。此外，拟议的研究 应就根本问题开辟富有成果的调查路线 机制[例如细胞骨架蛋白的单(ADP)-核糖基化] 肠上皮通透性调节的基础 生理和病理生理条件。