HB-EGF and Intestinal Ischemia/Reperfusion

肝素结合表皮生长因子样生长因子（HB-EGF）具有细胞保护作用，可用于肠缺血/再灌注损伤的治疗。 本研究的目的是确定HB-EGF降低I/R损伤后肠内诱导型一氧化氮合酶（iNOS）表达和活性氧（ROS）产生的方式。我们已经表明，在大鼠I/R发作的缺血期给予HB-EGF可显著减轻肠组织损伤的程度并降低死亡率;同时，I/R后iNOS的表达、一氧化氮的全身性出现和受影响肠内ROS的产生也显著降低。 使用体外研究，我们已经证明用HB-EGF处理降低了人肠上皮（DLD- 1）细胞中细胞因子诱导的iNOS上调，并减少了白细胞和大鼠肠上皮细胞产生的ROS。 我们的假设是HB-EGF通过限制在这种情况下发生的iNOS和ROS的表达增加来减少I/R后的组织损伤。 为了验证这一假设，本申请有两个具体的目的：（I）描述HB-EGF在肠I/R损伤的体内模型中的保护作用，和（II）阐明HB-EGF减弱I/R后iNOS和ROS增加的机制。本研究的设计和方法是为了阐明HB-EGF在体内给药的剂量、途径和时机，并使用我们的肠道I/R动物模型来表征HB-EGF对I/R后肠道iNOS、ROS、抗氧化酶和促炎细胞因子表达的影响。 在Aim II中，DLD-1细胞将用于确定HB-EGF是否改变iNOSmRNA转录速率或其稳定性，以及HB-EGF给药后是否发生NF- kappaB激活。 人脐静脉内皮细胞将用于确定HB-EGF是否影响黄嘌呤脱氢酶转化为氧化酶形式，人白细胞将用于确定肽对NAPDH氧化酶表达和功能的影响。这项工作的健康相关性是最终在肠I/R损伤患者中使用HB-EGF进行临床试验，该提案中产生的数据将使我们能够获得有关HB-EGF肠细胞保护机制的重要信息。

The broad, long term objectives are to utilize the cytoprotective abilities of heparin-binding EGF-like growth factor (HB-EGF) in the treatment of intestinal ischemia/reperfusion (I/R) injury. The goal of the present study is to determine the means by which HB-EGF decreases expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS) within the intestine following I/R injury. We have shown that administration of HB-EGF during the ischemic phase of an I/R episode in rats substantially attenuates the extent of intestinal tissue damage and decreases mortality; as well, the post-I/R expression of iNOS, systemic appearance of nitric oxide, and generation of ROS within the affected intestine are significantly decreased. Using in vitro studies, we have demonstrated that treatment with HB-EGF decreases cytokine- induced upregulation of iNOS in human intestinal epithelial (DLD- 1) cells, and reduces generation of ROS by leukocytes and rat intestinal epithelial cells. Our hypothesis is that HB-EGF decreases tissue injury following I/R by limiting the increased expression of iNOS and ROS that occur in this setting. To test this hypothesis, the application has two specific aims: (I) To delineate the protective effects of HB-EGF in an in vivo model of intestinal I/R injury, and (II) To elucidate the mechanisms by which HB-EGF attenuates the post-I/R increase in iNOS and ROS. The research design and methods are to clarify issues regarding the dose, route, and timing of HB-EGF administration in vivo, and to use our animal model of intestinal I/R to characterize the effects of HB-EGF on expression of iNOS, ROS, antioxidant enzymes and pro-inflammatory cytokines by the intestine post-I/R. In Aim II, DLD-1 cells will be used to determine if HB-EGF alters the rate of iNOS mRNA transcription or its stability, and if NF- kappaB activation occurs following HB-EGF administration. Human umbilical vein endothelial cells will be used to determine if HB- EGF affects the conversion of xanthine dehydrogenase to the oxidase form, and human leukocytes will be used to determine the effects of the peptide on NAPDH oxidase expression and function. The health relatedness of this work is to eventually perform clinical trials of the use of HB-EGF in patients with intestinal I/R injury, and the data generated in this proposal will allow us to obtain important information regarding the mechanisms of HB- EGF intestinal cytoprotection.