TARGETING THE TRANSIENT RECEPTOR POTENTIAL CHANNELS TO IMPROVE BOWEL DYSFUNCTION

 描述(由申请人提供)：胃肠道(GI)手术后的术后肠梗阻(POI)会导致严重的患者发病率和住院时间延长。POI是一个主要的健康问题，也是医疗保健系统的重大负担，据估计，美国每年的医疗保健系统费用在7.5亿至10亿美元之间。目前，还没有一种治疗方法能完全成功地减少POI对患者和卫生保健系统的重大影响。最近的研究表明，肠道操作和手术创伤可激活炎性巨噬细胞(MΦ)，并释放一氧化氮(NO)等炎性介质，从而抑制POI中的肠平滑肌细胞。因此，抑制免疫功能被认为是治疗POI的一种有前途的手段。然而，抑制肠道免疫系统可能会引起严重的副作用，因为肠道免疫系统承载着整个免疫系统的近70%，对免疫平衡至关重要。肠道MΦ是天然免疫系统中高度异质性和动态的种群。初步研究表明，F4/80+/CD2 0 6+肠道抗炎M2 MΦ专一表达瞬时受体电位香草酸4通道，是组织损伤和炎症的分子感受器。在POI小鼠模型中，激活TRPV4可产生肠道收缩反应并改善胃肠道转运。目前的建议旨在阐明肠道M2 MΦ中TRPV4激活的细胞和分子机制。钙离子成像和膜片钳研究将用于研究TRPV4在肠道MΦ中的功能。通过在致死性照射的TRPV4+/+和TRPV4-/-小鼠体内相互移植骨髓构建嵌合小鼠，以及使用巨噬细胞缺陷的OP/OP小鼠和在CD11b+肠道MΦ中表达人白喉毒素受体的转基因小鼠，将确定骨髓来源的MΦ在TRPV4介导的收缩反应中的需求。药理学和遗传消融研究将用于测试是否需要TLR4介导的5-羟色胺从粘膜嗜铬细胞和/或肥大细胞释放，以增强细菌脂多糖(LPS)增强TRPV4介导的肠道收缩反应。更重要的是，将建立POI的小鼠模型，以研究选择性激活TRPV4是否可以在体内改善或逆转POI。这些研究的成功完成将促进我们对以前未知的TRPV4和肠道MΦ在调节胃肠动力中的作用的理解。更重要的是，通过直接针对肠道天然免疫系统和TRPV4通道，拟议的研究将为未来治疗POI的临床研究定义新的治疗靶点。

 DESCRIPTION (provided by applicant): Postoperative ileus (POI) following gastrointestinal (GI) surgery leads to significant patient morbidity and prolonged hospitalizations. POI is a major health problem and a significant burden on the health care system, which is estimated at $750 million to $1 billion per year in the United States. Currently, no treatments have been completely successful in reducing POI's significant impacts on patients and health care system. Recent studies have demonstrated that intestinal manipulation and surgical trauma activate inflammatory macrophages (MΦ) and release inflammatory mediators such as nitric oxide (NO) to inhibit intestinal smooth muscle cells in POI. Therefore, suppression of the immune function has been considered as a promising means to treat POI. However, inhibition of the intestinal immune system could elicit severe side effects because intestinal immune system hosts near 70% of the entire immune system and is critical to immune homeostasis. Intestinal MΦ are a highly heterogeneous and dynamic population in the innate immune system. Preliminary studies show that transient receptor potential vanilloid 4 (TRPV4) channel, a molecular sensor of tissue damage and inflammation, is exclusively expressed by the F4/80+/CD206+ intestinal anti-inflammatory M2 MΦ. Activation of TRPV4 produces an intestinal contractile response and improves GI transit in a mouse model of POI. The current proposal aims to elucidate the cellular and molecular mechanisms underlying the activation of TRPV4 in the intestinal M2 MΦ. Ca2+ imaging and patch-clamp studies will be used to study the function of TRPV4 in intestinal MΦ. The requirement of bone marrow (BM)-derived MΦ in TRPV4-mediated contractile response will be determined by constructing chimeric mice by reciprocal BM transplantation in lethally irradiated Trpv4+/+ and Trpv4-/- mice as well as using macrophage-deficient op/op mice and transgenic mice expressing the human diphtheria toxin receptor (DTR) in CD11b+ intestinal MΦ (itgam-DTR). Pharmacological and genetic ablation studies will be used to test if TLR4-mediated release of serotonin from mucosal enterochromaffin cells and/or mast cells is required for potentiation of TRPV4-mediated intestinal contractile response by bacterial lipopolysaccharides (LPS). More importantly, a mouse model of POI will be generated to investigate if selective activation of TRPV4 can improve or reverse POI in vivo. Successful completion of these studies will advance our understanding of the previously unrecognized role of TRPV4 and intestinal MΦ in regulating GI motility. More importantly, the proposed studies will define novel therapeutic targets for future clinical studies for the treatment of POI by directly targeting both intestinal innate immune system and the TRPV4 channels.