The Role of PAF and TLR in NEC

描述（由申请人提供）：坏死性小肠结肠炎（NEC）是一种突然和破坏性的肠道炎症性坏死，10%的早产儿体重小于1500克。尽管这种复杂疾病的病因尚不清楚，但有证据表明，肠道缺血、配方喂养和细菌攻击可激活血小板活化因子（PAF）和其他炎症介质，导致肠道损伤。我们之前NIH资助的项目的结果表明，PAF受体被PAF激活导致肠上皮的一系列生理和/或病理变化，包括刺激经上皮电解质运输、细胞质酸化、caspase激活和DNA断裂。此外，内源性细菌刺激炎症级联，是新生儿NEC的先决条件。特定的细菌细胞壁产物刺激多种细胞类型上的toll受体（TLR），导致重要的下游事件。在健康的肠道环境中，粘膜屏障对细菌细胞壁产物的反应较差，部分原因是TLR表达低或不表达。然而，在新生儿早期发育和细菌定植过程中，肠道中TLR的表达及其调控尚不完全清楚。我们推测，在新生儿细菌定植的发育过程中，随着窒息应激的增加，肠道中存在TLR-4基因的异常表达，而PAF参与了这种异常上调。在TLR激活后，下游信号启动一系列事件，最终导致肠坏死和NEC的共同途径。利用一种新的NEC新生小鼠模型，该模型将允许对转基因动物和细胞培养研究进行测试，以提供额外的机制细节，该研究计划旨在1)表征肠道TLR表达的发育调节，并研究TLR在人类NEC和新生小鼠模型中的重要性；2)描述paf诱导的人类和啮齿动物肠上皮细胞中TLR表达的信号传导机制。3)在体内研究PAF对肠道TLR基因表达和蛋白合成的影响。拟议的研究将阐明PAF和TLR信号之间的相互作用，并阐明这些途径是否在新生儿NEC的发生中起潜在作用。

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is an abrupt and devastating inflammatory necrosis of the bowel affecting 10% of premature infants born weighing less than 1500 grams. Although the etiology of this complex disorder is poorly understood, the evidence suggests that intestinal ischemia, formula feeding, and bacterial challenge activates platelet activating factor (PAF) and other inflammatory mediators leading to intestinal injury. Results from our previous NIH funded project have shown that PAF receptor activation by PAF leads to a series of physiological and/or pathological changes in the intestinal epithelium, including the stimulation of transepithelial electrolyte transport, cytoplasmic acidification, caspase activation and DNA fragmentation. In addition, endogenous bacteria stimulate the inflammatory cascade and are a prerequisite for neonatal NEC. Specific bacterial cell wall products stimulate toll receptors (TLR's) on multiple cell types that result in significant downstream events. In a healthy intestinal environment, the mucosal barrier is poorly responsive to bacterial cell wall products, partly because of low or absent TLR expression. Nonetheless, in the neonate during early development and bacterial colonization, intestinal expression of TLR and regulation of this process is incompletely understood. We hypothesize that during the developmental acquisition of neonatal bacterial colonization with the addition of asphyxia stress, there is abnormal intestinal gene expression of TLR-4, and that PAF contributes to this abnormal upregulation. Following TLR activation, downstream signaling initiates a series of events that culminate in the final common pathway of intestinal necrosis and NEC. Utilizing a novel neonatal mouse model of NEC that will allow testing of genetically-altered animals and cell culture studies that will provide additional mechanistic detail, the research plan is designed to 1) characterize the developmental regulation of intestinal TLR expression and study the importance of TLR in human NEC and the neonatal mouse model, 2) delineate the signaling mechanisms responsible for PAF-induced TLR expression in intestinal epithelial cells from humans and rodents, and 3) investigate the role of PAF on intestinal TLR gene expression and protein synthesis in vivo. The proposed studies will elucidate the interactions between PAF and TLR signaling, and clarify whether these pathways play an underlying role in the initiation of neonatal NEC.