Novel Mechanisms of NSAID-induced GI Toxicity

描述（由申请人提供）：非甾体抗炎药（NSAID）是全球最广泛使用的缓解炎症和疼痛的药物之一。然而，NSAID在大部分使用它们的人中引起不良胃肠道（GI）副作用，其中许多人需要住院治疗才能恢复。传统上，NSAID诱导的GI毒性与环氧化酶（考克斯）酶，特别是考克斯-1的抑制有关.然而，最近的证据表明，考克斯抑制不能解释胃肠道中观察到的全部毒性作用。此外，考克斯-2特异性NSAID的不良心血管作用的发现证明了确定NSAID引起GI毒性的机制的重要性。因此，我们建议调查潜在的抑制恢复非甾体类抗炎药，非甾体类抗炎药对胃肠道上皮细胞的负面影响之一的分子机制。胃肠道上皮细胞形成了一个屏障，将胃肠道的内部环境与胃肠道内腔的恶劣条件隔开。修复是无细胞增殖的过程，通过该过程，通过邻近细胞向伤口中的扩散和迁移来修复上皮屏障中的伤口或间隙。具有致溃疡潜力的NSAID抑制肠上皮细胞中的细胞迁移。我们的研究小组已经表明，这种抑制与钙蛋白酶蛋白酶的表达和活性的降低以及膜电位（Em）的去极化有关。在这些数据的基础上，我们假设，NSAID损害肠上皮细胞迁移和抑制钙蛋白酶活性，通过一个信号通路，是高度敏感的药物诱导的Em的变化。因此，在特定目标1中，我们将通过测量细胞粘附的数量和分布以及特定粘附组分的表达和转换速率来确定NSAID是否损害细胞接触的去粘附。在具体目标2中，我们将通过测量EGF途径的表达和激活、PIP水平和钙蛋白酶活性以及膜定位来确定NSAID或Em去极化是否通过破坏EGF刺激的钙蛋白酶磷酸化或破坏PIP平衡来调节钙蛋白酶活性。在具体目标3中，我们将通过确定NSAID对细胞迁移、钙蛋白酶和Kv通道表达以及钙蛋白酶参与NSAID治疗大鼠膜定位复合物的影响来扩展我们的体外实验。这些创新的实验有可能确定的分子机制，通过NSAID降低钙蛋白酶活性，除了确定钙蛋白酶活性的减少对细胞的脱粘过程中在体外和体内迁移的影响。该项目将提供适用于其他组织的肠细胞伤口愈合过程的基本信息，记录NSAID对这些过程的影响，并深入了解NSAID如何影响其他组织的愈合过程。这项研究也有很大的潜力，有助于开发这类能够改善疼痛而没有现有药物的不良副作用的新药。 公共卫生相关性：广泛使用非甾体类抗炎药（NSAID）缓解炎症和疼痛，由于这些药物对胃肠道的常见毒性作用，对患者构成重大医疗风险。我们的研究旨在阐明这些毒性作用发生的新机制，以消除与使用非甾体抗炎药相关的副作用，并提高未来此类药物的安全性。这项研究可能有助于减少或消除每年因NSAID诱导的胃肠道毒性引起的医疗并发症而造成的数十亿美元的损失。

DESCRIPTION (provided by applicant): Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs worldwide for the relief of inflammation and pain. However, NSAIDs cause adverse gastrointestinal (GI) side-effects in a large proportion of people who use them, many of whom require hospitalization to recover. Traditionally, NSAID- induced GI toxicity was associated with the inhibition of cyclooxygenase (COX) enzymes, particularly COX-1. Recent evidence however, indicates that COX inhibition does not account for the full range of toxic effects observed in the GI tract. Additionally, the discovery of the adverse cardiovascular effects of COX-2 specific NSAIDs has demonstrated the importance of identifying mechanisms through which NSAIDs cause GI toxicity. Accordingly, we propose to investigate the molecular mechanisms underlying inhibition of restitution by NSAIDs, one of the negative effects that NSAIDs have on GI epithelia. GI epithelia form a barrier that separates the internal milieu from the harsh conditions of the lumen of the GI. Restitution is the cell proliferation-free process by which wounds or gaps in the epithelial barrier are repaired by the spreading and migration of neighboring cells into the wound. NSAIDs with ulcerogenic potential inhibit cell migration in intestinal epithelial cells. Our research group has shown that this inhibition is associated with a decrease in the expression and activity of calpain proteases and depolarization of membrane potential (Em). On the basis of these data, we hypothesize that NSAIDs impair intestinal epithelial cell migration and inhibit calpain activity through a signaling pathway that is highly sensitive to drug-induced changes in Em. Therefore, in Specific Aim 1 we will determine if NSAIDs impair de-adhesion of cell contacts by measuring the number and distribution of cellular adhesions as well as the expression and rate of turnover of specific adhesion components. In Specific Aim 2, we will determine if NSAIDs or Em depolarization modulate calpain activity through disruption of EGF-stimulated phosphorylation of calpains or disruption of PIP balance by measuring expression and activation of the EGF pathway, PIP levels, and calpain activity and localization to the membrane. In Specific Aim 3, we will extend our in vitro experiments by determining the effects of NSAIDs on cell migration, calpain and Kv channel expression, and calpain participation in membrane localized complexes in rats treated with NSAIDs. These innovative experiments have the potential to identify molecular mechanisms through which NSAIDs decrease calpain activity, in addition to determining the effects of reductions in calpain activity on cellular de-adhesion during migration both in vitro and in vivo. This project will provide basic information on wound healing processes in intestinal cells that are applicable to other tissues, document the effects of NSAIDs on these processes, and give insight into how NSAIDs may affect the healing process in other tissues. The research also has great potential for aiding in the development of new drugs in this class capable of ameliorating pain without the adverse side effects of current drugs. PUBLIC HEALTH RELEVANCE: The widespread use of nonsteroidal anti-inflammatory drugs (NSAIDs) for the alleviation of inflammation and pain poses a major medical risk to patients due to the common toxic effects these drugs have on the gastrointestinal tract. Our research is designed to elucidate novel mechanisms through which these toxic effects occur in order to eliminate the side effects associated with NSAID use and enhance the safety of future drugs of this class. This research may help reduce or eliminate the loss of billions of dollars per year due to medical complications resulting from NSAID-induced GI toxicity.