S1P-fluid therapy to reduce hemorrhagic shock & intoxication-induced injury

关于标准液体复苏方案为什么如此有效，人们在理解上存在一个根本性的差距 酒精中毒创伤患者合并失血性休克的情况较差，占所有 创伤案例。这一缺口的持续存在是一个重要的问题，因为在它被填补之前， 预防全身炎症和创伤后持续低血压的有效治疗方法的发展 患者将继续失败。这项研究的长期目标是开发有效的治疗方法 减少微血管通透性升高和水肿。此应用程序的总体目标是确定 生物活性磷脂鞘氨醇-1-磷酸(S1P)对酒精诱导的改善作用 微血管通透性升高。中心假设是S1P减少酒精/损伤诱导的 通过激活S1P受体-1(S1P1R)导致微血管高通透性，导致最初的连接修复，以及 更长期的有利于生存的信号，从而促进健康的内皮屏障。建议的理由是 研究表明，它将提供从酒精中毒相关模型中获得的重要细节 以及制定有效的液体复苏策略所需的出血性损伤。指导原则 强劲的初步数据，中心假设将以三个具体目标进行检验：目标1是确定 酒精中毒合并出血后S1P治疗的最佳剂量和时机窗 令人震惊。目的2是测试选择性S1P1R激动剂疗法以改善酒精/休克诱导的微血管 高通透性和低血压。目标3是确定S1P诱导、RhoA介导的程度 连接增强和细胞存活信号有助于改善内皮屏障功能障碍。这些 AIMS将利用已建立的酒精中毒和失血性休克联合的清醒大鼠模型 和复苏术。实验终点将是S1P或S1P1R激动剂改善的程度 低血压，肠道微血管渗漏，肠道屏障损伤。体内研究将得到补充 通过研究S1P或S1P1R激动剂改善培养微血管屏障完整性的能力 酒精暴露内皮细胞单层。药理拮抗剂或特定的siRNA敲除将 用于评估S1P1R的作用。活体显微镜、激光共聚焦显微镜、 生化分析将用于研究微血管内皮细胞连接结构和存活率 单个毛细血管后小静脉内皮细胞内的信号。拟议研究的贡献将是 意义重大，因为它将提供来自啮齿动物模型的概念证据，即酒精诱导的 微血管通透性升高是目前临床干预缺乏成功的原因之一 抢救酒精中毒失血性休克患者。这项拟议的研究具有创新性，因为它 通过研究一种治疗方法来改善现状，代表着与现状的实质性背离 酒精中毒引起的微血管高通透性是稳定血压的关键途径。

There is a fundamental gap in understanding pertaining to why standard fluid resuscitation protocols work so poorly in alcohol-intoxicated trauma victims with hemorrhagic shock, representing a significant portion of all trauma cases. Continued existence of this gap represents an important problem, because until it is filled, the development of effective therapies to prevent systemic inflammation and prolonged hypotension in trauma patients will continue to be unsuccessful. The long-term goal of this study is to develop effective therapies to reduce elevated microvascular permeability and edema. The overall objective in this application is to determine the effectiveness of the bioactive lipid sphingosine-1-phosphate (S1P) for ameliorating alcohol-induced elevated microvascular permeability. The central hypothesis is that S1P reduces alcohol/injury-induced microvascular hyperpermeability by activating S1P receptor-1 (S1P1R), causing initial junctional repair, and longer term pro-survival signals, thereby promoting a healthy endothelial barrier. The rationale for the proposed research is that it will provide important details obtained from a relevant model of combined alcohol intoxication and hemorrhagic injury that will be needed for development of effective fluid resuscitation strategies. Guided by strong preliminary data, the central hypothesis will be tested with three specific aims: Aim 1 is to determine the optimal dose and window of opportunity for S1P therapy after combined alcohol intoxication and hemorrhagic shock. Aim 2 is to test selective S1P1R agonist therapies to ameliorate alcohol/shock-induced microvascular hyperpermeability and hypotension. Aim 3 is to determine the extent to which S1P-induced, RhoA-mediated junctional enhancement and cell survival signals contribute to ameliorate endothelial barrier dysfunction. These aims will utilize an established conscious rat model of combined alcohol intoxication and hemorrhagic shock and resuscitation. The experimental endpoints will be the extent to which S1P or S1P1R agonists ameliorate hypotension, microvascular leakage in the gut, and gut barrier injury. The in vivo studies will be complemented by an investigation of the ability of S1P or S1P1R agonists to improve barrier integrity of cultured microvascular endothelial cell monolayers exposed to alcohol. Pharmacologic antagonists or specific siRNA knockdown will be used to assess the role of the S1P1R. A combination of intravital microscopy, laser confocal microscopy, and biochemical analyses will be used to study microvascular endothelial junctional architecture and survival signals in individual endothelial cells of postcapillary venules. The contribution of the proposed research will be significant because it will provide proof of concept from a rodent model that alcohol-induced amplifications of elevated microvascular permeability contribute to the lack of success with current clinical interventions used to resuscitate alcohol-intoxicated hemorrhagic shock patients. The proposed research is innovative because it represents a substantive departure from the status quo by investigating a therapeutic approach to ameliorate alcohol intoxication-induced microvascular hyperpermeability as the key approach to stabilize blood pressure.