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确定导致失血性休克炎症变化的最早分子事件的特征。以前的大多数研究都考察了休克期复苏对参数或炎症和器官损伤的净影响。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。在我们最全面的假设中，我们认为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。为了支持这一假说，我们已经证明了几个关键基因在休克早期上调，包括诱导型一氧化氮合酶(INOS)和环氧合酶-2(COX-2)。我们还证明，休克时iNOS的上调激活了炎症信号通路，并导致复苏后的终末器官损伤和功能障碍。我们总体假设的另一个主要方面是，休克阶段的持续时间和严重程度决定了复苏后表型变化的程度，从而决定了炎症反应的强度。在最初两年半的资助中，各个项目中产生的数据支持了我们的假设。Billiar博士的研究表明，休克时iNOS和COX-2表达上调，而核因子-kappaB的激活、IL-6的表达和PMN的内流在很大程度上依赖iNOS。本项目将探讨休克时iNOS上调的机制，以及复苏后NO介导的促炎信号的机制。Bauer博士已经证明，iNOS和COX-2都通过涉及PMN内流、IL-6和血管活性肠肽(VIP)的机制与休克诱导的肠道运动障碍有关。他将建立诱导型一氧化氮合酶和环氧合酶-2导致休克引起的肠道运动障碍的机制。芬克博士(项目IV)是该中心的新人，他带来了肠道粘膜屏障功能机制方面的专业知识。他的发现表明，iNOS在粘膜中的上调会导致粘膜损伤和屏障功能的丧失。他将探讨iNOS诱导的粘膜损伤的机制。皮特博士已经确定了休克时肠系膜血管中NO和金属硫氨酸(MT)之间的关键相互作用。一氧化氮取代了MT中的锌，皮特博士将探索一氧化氮在金属离子动态平衡中在HS血管功能障碍中的作用。在我们总体假设的保护伞下，所有四个项目都研究共同的主要主题。休克时iNOS上调对器官功能和损伤有重要影响。NO的作用部分是通过与超氧化物、COX-2和MT的相互作用来确定的。对共同主题的追求提供了许多合作和互动的机会。一如既往，我们将继续充分利用这些机会。根据我们迄今的进展，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效应。这不仅得益于共同的假设和主题，而且还得益于合作实验、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.