HB-EGF and Intestinal Ischemia/Reperfusion

广泛的、长期的目标是利用肝素结合的EGF样生长因子(HB-EGF)的细胞保护能力来治疗肠缺血/再灌注(I/R)损伤。本研究的目的是确定HB-EGF减少I/R损伤后肠组织诱导型一氧化氮合酶(INOS)表达和活性氧(ROS)产生的途径。我们发现，在大鼠I/R缺血期给予HB-EGF显著减轻了肠组织损伤的程度，降低了死亡率；同时，I/R后iNOS的表达、一氧化氮的系统出现以及受影响肠道内ROS的产生显著减少。体外实验表明，HB-EGF可减少细胞因子诱导的人肠上皮(DLD-1)细胞诱导型一氧化氮合酶(INOS)的上调，并减少白细胞和大鼠肠上皮细胞产生ROS。我们的假设是，HB-EGF通过限制iNOS和ROS在这种情况下的表达增加来减少I/R后的组织损伤。为了验证这一假说，应用有两个特定的目的：(1)描述HB-EGF在在体肠I/R损伤模型中的保护作用；(2)阐明HB-EGF减轻I/R后iNOS和ROS增加的机制。研究设计和方法是为了阐明体内HB-EGF给药的剂量、途径和时间问题，并使用我们的肠道I/R动物模型来表征HB-EGF对肠I/R后iNOS、ROS、抗氧化酶和促炎细胞因子表达的影响。在AIM II中，将使用DLD-1细胞来确定HB-EGF是否改变iNOS mRNA的转录速率或其稳定性，以及在HB-EGF给药后是否发生核因子-kappaB的激活。人脐静脉内皮细胞将被用来确定HB-EGF是否影响黄嘌呤脱氢酶向氧化物型的转化，而人白细胞将被用来确定该肽对NAPDH氧化酶表达和功能的影响。这项工作与健康相关的是最终对HB-EGF在肠道I/R损伤患者中的使用进行临床试验，该提案中产生的数据将使我们获得关于HB-EGF肠道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.