Digestive Enzymes and Microvascular Inflammation in Shock

描述（申请人提供）：生理性休克和多器官功能衰竭是最重要的医学问题之一，死亡率高。一个强大的炎症级联伴随着休克，但没有共识的触发机制的炎症。我们的长期目标是确定休克中炎症的起源，并开发新的干预措施，以尽量减少炎症和多器官功能衰竭。我们最近开展了一项新的研究，以确定肠道中的胰腺消化酶在休克中起着关键作用。作为正常消化的一部分，这类酶通常局限于肠腔。作为正常消化的一部分，消化酶的浓度相对较高，能够在数小时内降解大多数生物分子和整个组织。在正常情况下，消化酶被粘膜屏障保留在肠腔内。但在休克的情况下，同样的消化酶从肠腔转移到肠壁。一旦进入肠壁，它们就会启动一个自动消化过程，释放胰腺酶和炎症性消化产物进入中央循环，在那里它们会导致细胞损伤和多器官衰竭。我们获得的初步证据表明，阻断肠腔内的消化酶可显著减少炎症介质的产生，并显著提高严重休克后的存活率。我们的假设是，在失血性休克中，通常由刷状缘上皮提供的保护屏障受到损害，从而允许先前存在的消化酶进入肠壁间质组织。消化酶通过多种途径进入中枢循环，通过酶促膜受体的分裂引起微血管炎症和主要的细胞功能障碍，例如，紧密连接蛋白的胞外结构域的分裂和上皮和内皮通透性的不可逆升高或胰岛素受体的胞外结合位点的分裂和胰岛素抵抗。阻断胰酶和暂时阻止肠腔内的消化有助于预防炎症和减少多器官衰竭引起的死亡率。因此，我们建议研究以下三个重要的具体目标：1。测定失血性休克时胰腺消化酶从肠腔沿多种途径进入外周微循环的活性和转运以及相关微血管炎症反应的水平。2. 通过肠腔内酶阻断测定胰腺消化酶在产生炎症和细胞毒性介质以及失血性休克后长期生存中的作用。3. 测量与休克早期紧密连接失败和关键细胞功能丧失相关的蛋白酶的细胞外受体切割水平。这些研究将确定休克中强大的细胞和器官损伤机制的起源机制。我们将测试一种新的干预形式，以对抗休克的高死亡率，可能具有临床应用价值。公共卫生相关性：本研究的总体目标是开发一种新的干预措施，以对抗休克和多器官衰竭时的炎症和细胞损伤，这是死亡率最重要的问题之一。这项工作是基于一个新的假设，即在休克中，胰腺消化酶在炎症、细胞功能障碍和器官衰竭的发展中起着核心作用。这些异常强大的消化酶通常作为正常消化的一部分保留在肠腔内，它们被粘膜屏障阻止进入肠壁。但在肠缺血和休克的情况下，粘膜屏障变得可渗透，酶通过粘膜上皮进入肠壁，并从那里进入其他组织室，在那里它们迅速自动消化健康组织。我们提供的初步证据表明，在肠腔的消化酶的封锁是非常有效的，以减少各种形式的休克炎症。但对胰腺消化酶在休克条件下的转运和作用的细节知之甚少，以便优化消化酶的阻断。在这个项目中，我们将定量测定出血性休克中消化酶通过肠上皮进入肠壁、进入淋巴管、腹膜液和周围微循环的运输。我们将确定肠腔内消化酶的阻断在多大程度上降低休克死亡率。此外，我们将研究消化蛋白酶裂解受体形式的休克急性细胞功能障碍的新假设，例如，紧密连接蛋白的裂解是由于胰岛素受体细胞外结构域的裂解导致上皮或内皮通透性不可逆增加或胰岛素抵抗的机制。本研究将为开发一种通过胰消化酶短暂阻断治疗多器官功能衰竭的新方法提供重要信息。

DESCRIPTION (provided by applicant): Physiological shock and multi-organ failure is one of the most important medical problems with high mortality. A powerful inflammatory cascade accompanies shock, but there is no consensus for the trigger mechanisms of the inflammation. It is our long-term objective to identify the origin of the inflammation in shock and develop new interventions to minimize the inflammation and multi-organ failure. We recently developed a new line of research that has served to identify pancreatic digestive enzymes in the intestine as key players in shock. This family of enzymes is usually restricted to the lumen of the intestine as part of normal digestion. The digestive enzymes are present in comparatively high concentrations as part of normal digestion, capable to degrade most biological molecules and entire tissues within hours. Under normal circumstances, the digestive enzymes are retained within the lumen of the intestine by the mucosal barrier. But under conditions of shock, the same digestive enzymes are transported from the lumen into the wall of the intestine. Once inside the wall, they initiate an auto-digestion process with release of pancreatic enzymes as well as inflammatory digestive products into the central circulation where they cause cell injury and multi-organ failure. We obtained preliminary evidence to indicate that blockade of the digestive enzymes in the lumen of the intestine dramatically reduces the production of inflammatory mediators and significantly improves survival after severe forms of shock. It is our hypothesis that in hemorrhagic shock the protective barrier normally provided by the brush border epithelium is compromised and allows access of preexisting digestive enzymes into interstitial tissue in the wall of the intestine. The digestive enzymes are carried through multiple pathways into the central circulation where they cause microvascular inflammation and major cell dysfunctions by enzymatic cleavage of membrane receptors, e.g. cleavage of the extracellular domain of tight junction proteins and irreversible elevation of epithelial and endothelial permeability or cleavage of the extracellular binding site of the insulin receptor and insulin resistance. Blockade of the pancreatic enzymes and temporary prevention of digestion in the lumen of the intestine serves to prevent inflammation and reduces mortality due to multi-organ failure. Thus we propose to investigate the following three important Specific Aims: 1. Determine in hemorrhagic shock the activity and transport of the pancreatic digestive enzymes from the lumen of the intestine along multiple pathways into the peripheral microcirculation and the level of the associated microvascular inflammatory reaction. 2. Determine by enzyme blockade in the lumen of the intestine the role of pancreatic digestive enzymes in generation of inflammatory and cytotoxic mediators and in long-term survival after hemorrhagic shock. 3. Measure the level of extracellular receptor cleavage by proteases associated with failure of tight junctions and loss of key cell functions during the early stage of shock. These studies will determine the mechanisms for the origin of the powerful cell and organ injury mechanisms in shock. We will test a new form of intervention against the high mortality in shock that may have clinical utility. PUBLIC HEALTH RELEVANCE: The overall objective of this research is to develop a new intervention against inflammation and cell injury in shock and multi-organ failure, one of the most important problems in terms of mortality. The work is based on the new hypothesis, that in shock the pancreatic digestive enzymes play a central role in the development of inflammation, cell dysfunction and organ failure. These extraordinary powerful digestive enzymes are usually retained in the lumen of the intestine as part of normal digestion and they are prevented from entry into the wall of the intestine by the mucosal barrier. But under conditions of ischemia in the intestine and shock, the mucosal barrier becomes permeable and enzymes pass across the mucosal epithelium into the wall of the intestine and from there into other tissue compartments where they rapidly auto-digest healthy tissue. We provide preliminary evidence that blockade of digestive enzymes in the lumen of the intestine is highly effective to reduce inflammation in diverse forms of shock. But little is known about the details of the transport and the action of pancreatic digestive enzymes under shock conditions in order to optimize blockade of the digestive enzymes. In this project we will determine quantitatively with a new microzymographic technique in hemorrhagic shock the transport of digestive enzymes across the intestinal epithelium into the wall of the intestine, into lymphatics, the peritoneal fluid and into the peripheral microcirculation. We will determine to what degree blockade of the digestive enzymes in the lumen of the intestine reduces mortality in shock. Furthermore we will investigate a new hypothesis for the acute cell dysfunctions in shock in form of receptor cleavage by digestive proteases, e.g. cleavage of tight junction proteins as a mechanisms to cause irreversible increase in epithelial or endothelial permeability or insulin resistance due to cleavage of the extracellular domain of insulin receptors. This research will provide essential information for development of a new intervention against multi-organ failure by transient blockade of the pancreatic digestive enzymes.