Mitochondrial Dysfunction, Permeability Transition Pore, and Acute Pancreatitis

描述(由申请人提供)： 关键词：急性胰腺炎，胰腺腺泡细胞，线粒体，通透性转变孔，溶酶体，组织酶，自噬，胰酶，空泡。目的：急性胰腺炎是一种潜在的致命性胰腺外分泌疾病，其发病机制尚不清楚，目前尚无特效治疗方法。腺泡内胰酶原的激活和大空泡的形成是胰腺炎早期的关键病理反应。我们最近的研究表明，急性胰腺炎导致自噬的严重损害，自噬是主要的细胞降解、溶酶体驱动的过程。我们发现，自噬损伤是由有缺陷的、低效的溶酶体蛋白降解引起的，并介导了腺泡细胞中空泡和胰酶的积累。另一方面，我们发现线粒体功能障碍，表现为线粒体膜电位(DYM)的丧失，是急性胰腺炎模型中的常见反应。线粒体去极化的主要机制是通过打开线粒体膜上的非选择性通道--通透性转换孔(PTP)。这项建议的总体目标是确定溶酶体和线粒体这两个关键细胞器的功能障碍是否有关联，即依赖PTP的线粒体去极化是否介导了胰腺炎的溶酶体功能障碍、自噬损伤、腺泡细胞空泡化和胰蛋白酶原激活。我们将使用实验性的体内和体外胰腺炎模型来确定PTP介导的DYM和线粒体活性氧物种(ROS)的变化，以及PTP和线粒体ROS在上述病理反应中的作用。为此，我们将应用遗传学(基因敲除小鼠)、分子(转基因)和药理学方法。我们的假设认为，PTP开放，导致DYM丢失和线粒体ROS下降，介导了急性胰腺炎的关键病理反应。此外，线粒体功能障碍介导了胰腺炎中有缺陷的溶酶体蛋白的降解和自噬，潜在的腺泡细胞空泡化和胰蛋白酶原激活。PTP介导的线粒体ROS减少是连接胰腺炎线粒体和溶酶体功能障碍的一个重要机制。基于我们已发表的关于这一应用的初步数据，我们认为PTP失活是治疗或减轻急性胰腺炎严重程度的一种有前途的新治疗策略。研究计划：我们建议的具体目标是：(1)。观察PTP对急性胰腺炎大鼠体内、外DYM和ROS的影响。(2)。在体内和体外胰腺炎模型中，确定PTP在溶酶体功能障碍和自噬受损、腺泡细胞空泡化和胰蛋白酶原激活中的作用。在体外胰腺炎模型中，确定线粒体ROS在溶酶体功能障碍、自噬受损、腺泡细胞空泡化和胰蛋白酶原激活中的作用。(4)。确定药物抑制PTP对急性胰腺炎病理反应的影响。 公共卫生相关性：急性胰腺炎是一种潜在的致命的胰腺外分泌疾病，其发病机制尚不清楚，也没有开发出具体的治疗方法。这种疾病在VA患者中很常见。我们最近的研究表明，急性胰腺炎会导致线粒体和溶酶体这两个关键细胞器的严重功能障碍。这项建议将使用小鼠的实验模型和分离的细胞来确定这些功能障碍的分子机制，它们之间的联系，以及它们在胰腺炎关键病理反应中的作用(如胰腺中积累的大空泡和消化酶的提前激活)。此外，基于这一机制，我们提出了一种新的治疗策略来纠正线粒体和溶酶体功能障碍，从而治疗或减轻胰腺炎的严重程度。

DESCRIPTION (provided by applicant): MITOCHONDRIAL DYSFUNCTION, PERMEABILITY TRANSITION PORE, AND ACUTE PANCREATITIS Key words: acute pancreatitis, pancreatic acinar cell, mitochondria, permeability transition pore, lysosomes, cathepsin, autophagy, trypsin, vacuoles. Aims: Acute pancreatitis is a potentially fatal disease of exocrine pancreas, the pathogenesis of which remains obscure and specific treatments for which do not exist. Intra-acinar trypsinogen activation and formation of large vacuoles are key early pathologic responses of pancreatitis. Our recent studies revealed that acute pancreatitis causes profound impairment of autophagy, the main cellular degradative, lysosome-driven process. We showed that autophagy impairment results from defective, inefficient lysosomal protein degradation and mediates the accumulation of vacuoles and trypsin in acinar cells. On the other hand, we found that mitochondrial dysfunction, manifest by loss of the mitochondrial membrane potential (DYm), is a common response in models of acute pancreatitis. The principal mechanism of mitochondrial depolarization is through opening of permeability transition pore (PTP), a non-selective channel in the mitochondrial membrane. The overall goal of this proposal is to determine whether the dysfunctions of 2 critical organelles, the lysosomes and mitochondria, are linked; that is, whether PTP-dependent mitochondrial depolarization mediates lysosomal dysfunction, autophagy impairment, acinar cell vacuolization and trypsinogen activation in pancreatitis. We will use experimental in vivo and in vitro models of pancreatitis to determine PTP-mediated changes in DYm and mitochondrial reactive oxygen species (ROS), and the roles of PTP and mitochondrial ROS in the above-referred pathologic responses. For this purpose, we will apply genetic (knockout mice), molecular (transfections), and pharmacologic approaches. Our hypothesis states that PTP opening, which results in DYm loss and drop in mitochondrial ROS, mediates key pathologic responses of acute pancreatitis. Further, mitochondrial dysfunction mediates the defective lysosomal protein degradation and autophagy in pancreatitis, underlying acinar cell vacuolization and trypsinogen activation. An important mechanism linking the mitochondrial and lysosomal dysfunctions in pancreatitis is the PTP-mediated decrease in mitochondrial ROS. Based on our published and preliminary data for this application, we propose that PTP inactivation represents a promising novel therapeutic strategy to treat or mitigate the severity of acute pancreatitis. Research Plan: Specific Aims of our proposal are: (1). Determine the effect of PTP on DYm and ROS in in vivo and in vitro models of acute pancreatitis. (2). Determine the role of PTP in lysosomal dysfunction and impaired autophagy, underlying acinar cell vacuolization and trypsinogen activation in in vivo and in vitro models of pancreatitis.(3). Determine the role of mitochondrial ROS in lysosomal dysfunction, impaired autophagy, acinar cell vacuolization and trypsinogen activation in the in vitro model of pancreatitis. (4). Determine the effects of pharmacologic PTP inhibition on pathologic responses of acute pancreatitis. PUBLIC HEALTH RELEVANCE: Acute pancreatitis is a potentially fatal disease of exocrine pancreas, the pathogenesis of which remains unknown and specific treatments for which have not been developed. This disease is common in VA patient population. Our recent studies revealed that acute pancreatitis causes profound dysfunction of 2 critical cellular organelles, the mitochondria and the lysosomes. This proposal will use experimental models in mice and isolated cells to determine the molecular mechanisms of these dysfunctions, the link between them, and their roles in key pathologic responses of pancreatitis (such as accumulation of large vacuoles in pancreas and the premature, intrapancreatic activation of digestive enzymes). Further, based on the mechanism, we propose a novel therapeutic strategy to correct the mitochondrial and lysosomal dysfunctions and thus to treat or mitigate the severity of pancreatitis.