Surgical Studies of Gut Permeability

描述（由申请人提供）：肠上皮屏障的破坏通常发生在各种严重外科疾病中，如创伤、烧伤、出血、脓毒症和大规模外科手术，导致管腔有毒物质和细菌移位至血流中。由于急性上皮屏障功能障碍的确切机制仍然不清楚，有效的治疗，以保持屏障的完整性是有限的，导致死亡的危重手术患者与肠漏。在之前的资助期间，我们已经确定紧密连接（TJ）中occludin和ZO-1以及粘附连接（AJ）中E-cadherin的表达降低在肠上皮细胞旁通透性增加中起重要作用，并确定了细胞多胺调节编码这些细胞间连接蛋白的基因转录的新机制。然而，在关键手术条件下TJ和AJs的转录后调控的确切机制在很大程度上仍然未知，并且是这种竞争性更新应用的焦点。我们的初步结果表明：a）RNA结合蛋白（RBP）HuR与闭合蛋白mRNA结合，而HuR沉默抑制闭合蛋白翻译; B）HuR结合亲和力在临界手术应激中显著降低;和c）减少细胞多胺不仅抑制HuR与闭合蛋白mRNA结合，而且诱导翻译阻遏物TIAR的细胞质水平。基于这些观察结果，我们假设1）RBP HuR和TIAR通过调节关键手术条件下TJ mRNA的稳定性和翻译在肠上皮屏障功能的调节中起重要作用，以及2）细胞多胺通过改变HuR和TIAR活性来调节闭合蛋白翻译。提出了三个具体的目标来验证假设：1）通过使用组织特异性遗传修饰策略来确定HuR和TIAR在临界手术应激期间TJ表达和屏障功能障碍中的作用; 2）确定HuR和TIAR在TJ蛋白表达的转录后调节中的确切作用; 3）研究多胺在手术应激期间调节闭合蛋白翻译的机制。这些特定目标的完成将提供新的信息，关于转录后调控的TJ，也产生一个新的模型，其中occludin翻译调节多胺在手术应激。希望我们的研究结果将确定可用于保护严重外科疾病患者上皮屏障的因素和机制。 公共卫生相关性：肠通透性增加通常发生在各种严重外科疾病期间，例如创伤、热损伤、休克、脓毒症和大规模外科手术，导致管腔有毒物质和细菌移位到血流中。由于这种急性上皮屏障功能障碍的确切机制仍然不清楚，有效的治疗，以保持屏障的完整性是有限的，导致死亡的危重手术患者与肠漏。该项目的完成将确定关键手术条件下屏障功能障碍的发病机制，并为开发新的治疗方法提供基础，以保护患有严重外科疾病的患者的肠道屏障。

DESCRIPTION (provided by applicant): Disruption of the gut epithelial barrier occurs commonly in various critical surgical conditions such as trauma, burns, hemorrhage, sepsis, and massive surgical operations, leading to the translocation of luminal toxic substances and bacteria to the blood stream. Since the exact mechanism underlying the acute epithelial barrier dysfunction remains obscure, effective therapies to preserve the integrity of the barrier are limited, contributing to death in critically surgical patients with leaky gut. During previous funding period, we have established that decreased expression of occludin and ZO-1 in the tight junction (TJ) and E-cadherin in the adherens junction (AJ) plays an important role in increased intestinal epithelial paracellular permeability, and have identified a novel mechanism through which cellular polyamines regulate transcription of the genes encoding these intercellular junctional proteins. However, the exact mechanism underlying posttranscriptional regulation of TJs and AJs in critical surgical conditions remains largely unknown and is the focus of this competitively renewal application. Our preliminary results indicate a) RNA-binding protein (RBP) HuR binds to the occludin mRNA, while HuR silencing represses occludin translation; b) HuR- binding affinity decreases significantly in critical surgical stress; and c) decreasing cellular polyamines not only inhibits HuR-binding to occludin mRNA but also induces cytoplasmic levels of the translational repressor TIAR. Based on these observations, we HYPOTHESIZE that 1) RBPs HuR and TIAR play an important role in the regulation of the intestinal epithelial barrier function by modulating the stability and translation of TJ mRNAs in critical surgical conditions and 2) cellular polyamines regulate occludin translation by altering HuR and TIAR activities. Three specific aims are proposed to test the hypotheses: 1) to determine the roles of HuR and TIAR in TJ expression and barrier dysfunction during critical surgical stress by using tissue-specific genetic modification strategy; 2) to define the exact roles of HuR and TIAR in the posttranscriptional regulation of TJ protein expression; and 3) to investigate the mechanism by which polyamines regulate occludin translation during surgical stress. Completion of these specific aims will provide novel information regarding the posttranscriptional regulation of TJs and also yield a novel model in which occludin translation is regulated by polyamines during surgical stress. It is hoped that our findings will identify factors and mechanisms that can be used to protect the epithelial barrier in patients with critical surgical illnesses. PUBLIC HEALTH RELEVANCE: Increased gut permeability occurs commonly during various critical surgical disorders such as trauma, thermal injury, shock, sepsis, and massive surgical operations, leading to the translocation of luminal toxic substances and bacteria to the blood stream. Since the exact mechanism underlying this acute epithelial barrier dysfunction remains obscure, effective therapies to preserve the integrity of the barrier are limited, contributing to death in critically surgical patients with leaky gut. Completion of this project will identify the pathogenesis of the barrier dysfunction in critical surgical conditions and provide a fundamental base for development of new therapies to protect the gut barrier in patients with critical surgical illnesses.