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危重的外科疾病中，如创伤、烧伤、出血、败血症和大规模外科手术，导致腔内有毒物质和细菌转移到血流中。由于急性上皮屏障功能障碍的确切机制尚不清楚，保护屏障完整性的有效治疗方法有限，导致肠漏的危重手术患者死亡。在之前的资金支持期间，我们已经证实，紧密连接(TJ)中occludin和ZO-1以及黏附连接(AJ)中E-cadherin的表达减少在增加肠上皮细胞旁通透性方面起着重要作用，并发现了一种新的机制，即细胞多胺通过调节编码这些细胞间连接蛋白的基因的转录。然而，关键手术条件下TJs和AJs转录后调控的确切机制在很大程度上仍不清楚，这是这一竞争性更新应用的重点。我们的初步结果表明：a)RNA结合蛋白(RBP)Hur与occludin mRNA结合，而Hur沉默抑制occludin翻译；b)Hur结合亲和力在临界手术应激下显著降低；c)细胞多胺减少不仅抑制Hur与occludin mRNA的结合，还诱导翻译抑制物Tiar的细胞质水平。基于这些观察，我们假设1)RBPs、HUR和TIAR在关键手术条件下通过调节TJ mRNAs的稳定性和翻译在调节肠上皮屏障功能中发挥重要作用；2)细胞多胺通过改变HUR和TIAR的活性来调节阻滞素的翻译。通过组织特异性的基因修饰策略，确定Hur和TIAR在关键手术应激时TJ表达和屏障功能障碍中的作用；2)确定HUR和TIAR在TJ蛋白表达转录后调控中的确切作用；以及3)探讨多胺调节手术应激过程中阻滞素翻译的机制。这些特定目的的完成将提供关于TJ转录后调控的新信息，并产生一种新的模型，在该模型中，阻滞素的翻译由手术应激过程中的多胺调节。希望我们的发现将确定可用于保护外科危重疾病患者的上皮屏障的因素和机制。 与公共卫生相关：肠道通透性增加通常发生在各种严重的外科疾病中，如创伤、热损伤、休克、败血症和大规模外科手术，导致管腔有毒物质和细菌转移到血流中。由于这种急性上皮屏障功能障碍的确切机制尚不清楚，保护该屏障完整性的有效治疗方法有限，导致患有肠漏的危重外科患者死亡。该项目的完成将确定危重外科疾病中屏障功能障碍的发病机制，并为开发新的治疗方法以保护外科危重疾病患者的肠道屏障提供基本基础。

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.