Early Enterocyte Injury in Neonatal Necrotizing Enterocolitis

描述（由申请人提供）：坏死性小肠结肠炎（NEC）是一种病因不明、危及生命的炎症性肠病，约10%的出生体重<1500克的早产儿会受到影响。血小板活化因子（Platelet activating factor， PAF）是一种磷脂细胞间和细胞内的介质，在临床研究中显示，在一些患者出现临床症状前几天，血小板活化因子（Platelet activating factor， PAF）就会升高，这表明它是疾病发展的关键因素。因此，抑制PAF的早期作用可以限制NEC的临床发病率。然而，paf诱导肠道损伤的确切机制尚不清楚。本提案的目的是研究PAF直接对肠上皮细胞的早期作用，以确定预防NEC发展的治疗干预措施。该提案的初步数据表明，PAF直接对肠上皮细胞有四种潜在的关联，即PAF诱导的细胞内酸化，以及PAF诱导的Cl-通道激活，增加的游离细胞内[Ca2+]I和凋亡。我假设PAF通过Ca2+依赖的Cl-通道诱导细胞内酸中毒，PAF诱导的细胞内酸中毒导致肠上皮细胞凋亡，导致肠屏障功能受损。各种Cl-通道已被证明除了Cl-外还能传导碳酸氢盐，从而改变细胞内ph值。细胞内酸中毒与其他细胞类型的凋亡有关。肠上皮细胞凋亡可导致肠通透性增加、细菌跨肠上皮易位、炎症和NEC。因此，paf诱导的肠粘膜Cl-通道的激活和由此产生的酸中毒可能是上皮细胞凋亡的重要早期触发因素，导致肠屏障功能受损。这个建议的目的是…确定PAF诱导肠上皮细胞酸中毒的机制探讨PAF诱导的酸中毒与细胞凋亡的具体关系。3. 研究PAF对肠屏障功能的早期影响。实验设计采用描述良好的大鼠小肠上皮细胞系IEC-6、人类胎儿小肠细胞系H4（人类未成熟小肠对NEC具有独特易感性的模型）和断奶前大鼠肠道外植体。这些研究将开始回答关键问题“NEC肠道损伤周期的开始是什么？”和“PAF在初始肠上皮细胞损伤中的作用是什么？”由于与Cl-通道激活相关的肠上皮细胞凋亡之前没有被描述过，了解这一机制可能为治疗或预防高危婴儿NEC提供一种新的方法。坏死性小肠结肠炎是一种知之甚少的、危及早产儿生命的炎症性肠病，其中20-30%的患者死亡，幸存者面临严重的肠道和神经发育后果的风险。血小板活化因子（PAF）被认为在这种疾病的发展中起关键作用。本提案将研究PAF如何引起肠道损伤，以便开发一种治疗方法来限制易感婴儿坏死性小肠结肠炎。

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is a life-threatening inflammatory bowel disorder of unknown cause that affects approximately 10% of premature infants born <1500gm. Platelet activating factor (PAF) is a phospholipid inter and intra-cellular mediator which has been shown in clinical studies to rise several days before the onset of clinical symptoms in some patients, suggesting that it is a critical factor in the development of the disease. Thus inhibition of the early effects of PAF could limit the clinical morbidity of NEC. However, the exact mechanisms of PAF-induced intestinal injury are poorly understood. The objective of this proposal is to investigate the early effects of PAF directly on intestinal epithelial cells in order to identify a therapeutic intervention to prevent development of NEC. Preliminary data for this proposal demonstrate four potentially linked, early effects of PAF directly on intestinal epithelial cells -PAF-induced intracellular acidification, along with PAF-induced Cl- channel activation, increased free intracellular [Ca2+]I, and apoptosis. I hypothesize that PAF induces intracellular acidosis via a Ca2+ dependent Cl- channel and that PAF induced intracellular acidosis results in apoptosis of intestinal epithelial cells leading to compromised intestinal barrier function. Various Cl- channels have been shown to conduct bicarbonate in addition to Cl-, thereby altering intracellular pH. Intracellular acidosis has been linked to apoptosis in other cell types. Apoptosis of intestinal epithelial cells may lead to increased intestinal permeability, bacterial translocation across the intestinal epithelium, inflammation, and NEC. Thus, PAF-induced activation of an intestinal mucosal Cl- channel and resultant acidosis may be an important early trigger of epithelial cell apoptosis resulting in compromise of intestinal barrier function. This proposal is designed to 1. Determine the mechanism of PAF induced acidosis in intestinal epithelial cells 2. Investigate the specific relationship between PAF induced acidosis and apoptosis. 3. Examine the early effects of PAF on intestinal barrier function. The experimental design uses the well described rat small intestinal epithelial cell line IEC-6, the human fetal small intestinal cell line H4 which is a model of the human immature small intestine uniquely susceptible to NEC, and pre-weaned rat intestinal explants. These studies will begin to answer the critical questions "What begins the cycle of intestinal injury in NEC?" and "What is the role of PAF in initial intestinal epithelial cellular injury?" Since apoptosis of intestinal epithelial cells associated with Cl- channel activation has not been previously described, understanding this mechanism may provide a novel approach to treating or preventing NEC in at risk infants. PUBLIC HEALTH RELEVANCE Necrotizing enterocolitis is a poorly understood, life threatening inflammatory bowel disease of premature infants in which 20-30% of patients die and survivors are at risk for significant intestinal and neurodevelopmental consequences. Platelet activating factor (PAF) is believed to play a critical role in the development of this disease. This proposal will investigate how PAF causes intestinal injury, in order to develop a treatment to limit necrotizing enterocolitis in vulnerable infants.