The Role of FXR on the Intestinal Barrier in Acute Injury.

项目总结/摘要 该提案的目标是作为外科医生科学家通过以下方式成为独立调查员 完成拟议的研究项目和职业发展的监督下，一个优秀的 胃肠道和FXR信号专家的指导团队。所描述的职业发展将提供 在我设计实验方法和评估研究结果时，我有基本的洞察力。该奖项还将 支持在肠道生理学领域建立强有力的出版记录所需的保护时间 并为竞争性R级提交开发初步数据。 法尼醇X受体（FXR）是影响肠上皮屏障的关键胆汁酸受体。而 我们的初步数据显示，FXR的激活似乎对肠慢性损伤模型有益 在急性炎性损伤中，FXR活化是有害的。相比之下，FXR敲除（KO）动物， 保护免受急性损伤。在这个建议中，我们将定义FXR在肠屏障功能中的作用， 急性损伤。我们假设FXR激活通过上调小分子的表达而损害肠屏障功能。 异二聚体蛋白（SHP），同时干扰EGFR信号传导，导致紧密连接破坏。我们将 我们将通过以下具体目标来检验这一假设：1）确定参与FXR调节的机制 肠上皮屏障功能，和2）测试组织特异性FXR抑制对肠屏障功能的影响。 使用体内急性损伤模型。 我们将在体外和体外实验中确定FXR及其下游效应物SHP在肠屏障功能中的作用。 vivo.为了探索这一点，我们将使用来自野生型、FXR KO、SHO KO和EGFR的肠源性单层细胞， 显性阴性“天鹅绒”小鼠，以确定对肠屏障和紧密连接蛋白的影响 无论是否受伤我们将使用qPCR和Western印迹分析来确定FXR和EGFR信号激活。 是的。我们将使用来自转基因小鼠的骨髓来源的培养物来确定巨噬细胞的作用。 动物这些体外研究结果将在体内进行测试，使用LPS注射模型的急性损伤，利用FXR （全身、巨噬细胞特异性和巨噬细胞特异性）和SHP KO小鼠。盲肠结扎和穿孔将 用于检测FXR途径对疾病进展的药理学操作。 该项目将开始建立FXR调节肠上皮屏障在以下情况下的作用： 急性损伤。从长远来看，这些发现将为预防和治疗的发展提供必要的见解。 肠源性脓毒症、炎性肠病或其他GI病理的治疗策略。本研究 有可能通过更好的治疗，改善患有影响肠道完整性疾病的患者的预后。 了解FXR介导的肠屏障功能的机制。

Project Summary/Abstract The goal of this proposal is to advance as a surgeon-scientist to become an independent investigator through completion of the proposed research project and career development under the oversight of an outstanding mentoring team of gastrointestinal and FXR signaling experts. The career development described will provide foundational insight as I design experimental approaches and assess study outcomes. This award will also support the protected time needed to establish a strong publication record in the field of intestinal physiology and develop preliminary data for a competitive R level submission. The farnesoid X receptor (FXR) is a key bile acid receptor that influences the intestinal epithelial barrier. While activation of FXR seems to be beneficial in chronic injury models of the intestine, our preliminary data show that in acute, inflammatory injury, FXR activation is deleterious. FXR knock-out (KO) animals, in contrast, are protected from acute injury. In this proposal, we will define the role of FXR in intestinal barrier function during acute injury. We hypothesize that FXR activation compromises intestinal barrier function by upregulating small heterodimer protein (SHP), while interfering with EGFR signaling, leading to tight junction disruption. We will test this hypothesis with the following specific aims: 1) Determine the mechanisms involved in FXR regulating intestinal epithelial barrier function, and 2) Test the effect of tissue-specific FXR inhibition on the intestinal bar- rier using acute injury models in vivo. We will determine the role of FXR and its downstream effector SHP in intestinal barrier function in vitro and in vivo. To explore this, we will use enteroid-derived monolayers from wild type, FXR KO, SHO KO, and EGFR dominant-negative ‘Velvet’ mice to determine the effects on the intestinal barrier and tight junctional proteins with and without injury. We will determine FXR and EGFR signal activation using qPCR and Western blot anal- yses. We will determine the role of macrophages using bone marrow-derived culture from genetically-modified animals. These in vitro findings will be tested in vivo using an LPS-injection model of acute injury, utilizing FXR (whole body, intestinal-specific, and macrophage-specific) and SHP KO mice. Cecal ligation and puncture will be used to test pharmacologic manipulation of the FXR pathway on disease progression. This project will begin to establish the role of FXR regulation of the intestinal epithelial barrier in the setting of acute injury. Long term, these findings will provide needed insight into the development of preventative and therapeutic strategies in gut origin sepsis, inflammatory bowel disease, or other GI pathologies. This research has the potential to improve the outcomes of patients with diseases that affect intestinal integrity through better understanding of the mechanisms of FXR-mediated intestinal barrier function.