Digestive Enzymes and Microvascular Inflammation in Shock

描述(申请人提供)：生理性休克和多器官衰竭是最重要的医学问题之一，死亡率很高。休克伴随着强大的炎症级联反应，但对于炎症的触发机制尚无共识。我们的长期目标是确定休克时炎症的来源，并开发新的干预措施，将炎症和多器官衰竭降至最低。我们最近开发了一系列新的研究，用于确定肠道中的胰腺消化酶是休克的关键因素。作为正常消化的一部分，这一家族的酶通常局限于肠腔。作为正常消化的一部分，消化酶的浓度相对较高，能够在几个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。但在休克条件下，同样的消化酶从肠腔运输到肠壁。一旦进入壁内，它们就会启动自动消化过程，将胰酶和炎性消化产物释放到中央循环中，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性介质的产生，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，通常由刷状边缘上皮提供的保护屏障被破坏，并允许先前存在的消化酶进入肠壁的间质组织。消化酶通过多种途径进入中央循环，通过对膜受体的酶促裂解引起微血管炎症和主要的细胞功能障碍，如紧密连接蛋白胞外区的裂解，上皮和内皮通透性的不可逆性升高，或胰岛素受体胞外结合部位的裂解和胰岛素抵抗。阻断胰酶和暂时防止肠腔内的消化有助于预防炎症并降低因多器官衰竭而导致的死亡率。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的活性和从肠腔沿多条途径进入外周微循环的运输，以及相关的微血管炎症反应水平。2.用肠腔酶阻滞法测定胰腺消化酶在失血性休克后炎性介质和细胞毒介质产生及长期存活中的作用。3.在休克早期，测定与紧密连接失败和关键细胞功能丧失相关的蛋白水解酶对细胞外受体的切割水平。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，这可能具有临床实用价值。

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.