TLR4 signaling in the pathogenesis of surgical necrotizing enterocolitis

描述（由申请人提供）：本提案的长期目标是了解外科坏死性小肠结肠炎（NEC）的发病机制，NEC是影响应激早产儿的最常见和最致命的胃肠道疾病。虽然最初影响肠道，但NEC患者可能会迅速发展为全身性脓毒症和多系统器官衰竭，并死于严重的脓毒性休克。目前的想法表明，NEC的发展反映了全身应激的影响，导致肠道屏障的破坏。这导致细菌和脂多糖（LPS）的易位，脂多糖（LPS）在免疫细胞和肠上皮细胞上被Toll样受体4（TLR 4）识别。我们现在提出LPS激活TLR 4导致脓毒症和进一步的肠损伤，这是NEC的特征。损伤的肠粘膜的愈合通常通过上皮恢复发生，其中健康的肠细胞向裸露的粘膜迁移。我们已经开发了一种模拟人类NEC的啮齿动物模型，并表明与对照组相比，NEC动物的肠道恢复明显受损。引人注目的是，与野生型同窝出生的小鼠相比，TLR 4突变的小鼠NEC的严重程度显著降低。在体外，肠细胞迁移受RhoA和整联蛋白的调节，用LPS处理肠细胞通过激活RhoA和增加细胞-基质粘附而显着抑制肠细胞迁移。我们现在假设TLR 4通过激活RhoA和整合素以及受损的肠道恢复在NEC的发病机制中起关键作用。为了验证这一假设，我们提出：目的1研究LPS对肠上皮细胞TLR 4表达和活性的影响。目的2.研究LPS对RhoA活化和肠上皮细胞迁移的影响是否需要TLR 4信号通路，并确定其信号通路。目的3.通过对RhoA活性和肠上皮细胞迁移的影响，确定TLR 4在体内诱导坏死性小肠结肠炎中是否是必需的。通过检验TLR 4信号在NEC发病机制中起关键作用的假设，我们建议了解导致这种毁灭性疾病发展的主要步骤，并为这些脆弱的患者确定新的治疗策略。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the pathogenesis of surgical necrotizing enterocolitis (NEC), the most frequent and lethal gastrointestinal disorder affecting the stressed, preterm infant. Although initially affecting the intestine, patients with NEC may rapidly develop systemic sepsis and multi-system organ failure and death from overwhelming septic shock. Current thinking indicates that the development of NEC reflects the effects of systemic stress causing a breakdown of the intestinal barrier. This leads to the translocation of bacteria and lipopolysaccharide (LPS), which is recognized on immune cells and enterocytes by Toll Like Receptor 4 (TLR4). We now propose that activation of TLR4 by LPS leads to sepsis and further intestinal injury, characteristic of NEC. Healing of the injured intestinal mucosa typically occurs through epithelial restitution, in which healthy enterocytes migrate towards the denuded mucosa. We have developed a rodent model that mimics human NEC, and have shown that intestinal restitution is significantly impaired in animals with NEC compared with controls. Strikingly, mice with mutations in TLR4 have significantly reduced severity of NEC as compared to wild-type littermates. In vitro, enterocyte migration is regulated by RhoA and integrins, and treatment of enterocytes with LPS leads to a significant inhibition in enterocyte migration through the activation of RhoA and increased cell-matrix adhesion. We now hypothesize that TLR4 plays a critical role in the pathogenesis of NEC through the activation of RhoA and integrins and impaired intestinal restitution. To test this hypothesis, we propose: Aim 1 To assess the effects of LPS on the expression and activity of TLR4 in enterocytes in vitro and in vivo. Aim 2.To determine whether TLR4 signaling is necessary for the effects of LPS on RhoA activation and enterocyte migration and to determine the signaling pathways involved. Aim 3.To determine whether TLR4 is required for the induction of necrotizing enterocolitis in vivo through effects on RhoA activity and enterocyte migration. By testing the hypothesis that TLR4 signaling plays a critical role in the pathogenesis of NEC, we propose to understand the principal steps that lead to the development of this devastating illness, and to identify novel treatment strategies for these fragile patients.