MOLECULAR BIOLOGY OF HEMORRHAGIC SHOCK

我们认为，了解失血性休克引起的器官损伤的关键之一是表征出血性休克中导致炎症变化的起始和促进的最早分子事件。大多数先前的研究已经检查了休克期复苏对参数或炎症和器官损伤的净效应。与以前的方法不同，我们将失血性休克的分子事件分为两个不同的阶段：休克阶段和复苏后阶段。在我们最过分的假设中，我们提出休克期间基因表达的变化调节复苏后的炎症变化，并导致直接的器官损伤和功能障碍。为了支持这一假设，我们已经证明，在休克早期，几个关键基因上调，包括诱导型一氧化氮合酶（iNOS）和环氧合酶-2（考克斯-2）。我们还证实了在休克过程中iNOS的上调激活了炎症信号通路，并导致终末器官损伤和复苏后功能障碍。我们总体假设的另一个主要方面是休克期的持续时间和严重程度决定了表型变化的程度，从而决定了复苏后炎症反应的强度。在最初两年半的资助中，各个项目产生的数据支持我们的假设。Project（Dr.Billiar）已经表明，在休克期间iNOS和考克斯-2上调，并且NF-κ B活化、IL-6表达和PMN流入在很大程度上依赖于iNOS。本课题旨在探讨休克时诱导型一氧化氮合酶上调的机制和复苏后一氧化氮介导的促炎信号转导机制。Bauer博士已经表明，iNOS和考克斯-2通过涉及PMN流入、IL-6和血管活性肠肽（VIP）的机制对休克诱导的肠道动力障碍负责。他将建立诱导型一氧化氮合酶和考克斯-2负责休克诱导的肠道动力障碍的机制。Fink博士（项目IV）是该中心的新成员，他带来了肠道粘膜屏障功能机制方面的专业知识。他的研究结果表明，粘膜中的iNOS上调导致粘膜损伤和屏障功能丧失。他将继续研究iNOS诱导粘膜损伤的机制。Pitt博士已经确定了休克期间肠系膜血管中NO和金属硫蛋白（MT）之间的关键相互作用。NO取代MT中的锌，Pitt博士将探讨NO在HS血管功能障碍中金属离子稳态中的作用。在我们的总体假设下，所有四个项目都研究共同的主要主题。休克期间iNOS的上调对器官功能和损伤具有重要影响。NO的作用部分是通过与超氧化物、考克斯-2和MT的相互作用来确定的。对共同主题的追求为合作和互动提供了许多机会。我们将一如既往，继续充分利用这些机会。根据我们迄今为止的进展，有充分的证据表明，我们已经在项目负责人之间建立了富有成效的协同作用。这不仅得益于共同的假设和主题，还得益于合作实验、联合会议和共享资源。

We propose that one of the keys to understanding organ injury resulting from hemorrhagic shock is to characterize the earliest molecular events leading to the initiation and promotion of inflammatory changes in hemorrhagic shock. Most previous studies have examined the net effect of shock phase resuscitation on parameters or inflammation and organ damage. In contrast to these previous approaches, we divide the molecular events of hemorrhagic shock into two distinct phases; the shock phase and the post-resuscitation phase. In our most over-arching hypothesis, we propose that changes in gene expression during shock regulate post-resuscitation inflammatory changes and contribute to direct organ damage and dysfunction. In support of this hypothesis, we have shown that several key genes are up-regulated early during shock, including the inducible nitric oxide synthase (iNOS) and cyclooxygenase- 2 (COX-2). We also have demonstrated that the up-regulation of iNOS during shock activates inflammatory signaling pathways and contributes to end-organ damage and dysfunction post-resuscitation. Another major aspect of our overall hypothesis is that the duration and severity of the shock phase determine the degree of phenotypic changes and hence the intensity of the inflammatory response following resuscitation. Data generated within the individual projects over the first two- and-a- half years of funding support our hypothesis. Project (Dr. Billiar) has shown that iNOS and COX-2 are up-regulated during shock and that NF- kappaB activation, IL-6 expression, and PMN influx are largely iNOS- dependent. This project will explore the mechanism of iNOS up- regulation during shock and the mechanisms of NO-mediated pro- inflammatory signaling post-resuscitation. Dr. Bauer has shown that both iNOS and COX-2 are responsible for shock-induced gut dysmotility through mechanisms involving PMN influx, IL-6, and vasoactive intestine peptide (VIP). He will establish the mechanisms by which iNOS and COX-2 are responsible for shock-induced gut dysmotility. Dr. Fink (Project IV) is a newcomer to the center, and brings expertise in the mechanisms of gut mucosal barrier function. His findings suggest that iNOS up-regulation in the mucosal leads to mucosal damage and loss of barrier function. He will pursue the mechanisms of iNOS-induced mucosal damage. Dr. Pitt has identified a key interaction between NO and metallothionine (MT) in mesenteric vessels during shock. NO displaces zinc from MT and Dr. Pitt will explore the role of NO in metal ion homeostasis in vascular dysfunction in HS. Under the umbrella of our overall hypothesis, all four projects study common major themes. iNOS up-regulation during shock has a major impact on organ function and damage. The actions of NO are determined, in part, through interaction with superoxide, COX-2, and MT. The pursuit of common themes presents many opportunities for collaboration and interaction. As in the past, we will continue to take full advantage of these opportunities. Based on our progress to date, there is ample evidence that we have established a productive synergy between the project leaders. This has been facilitated not only by common hypotheses and themes but also collaborative experiments, joint conferences, and shared resources.