Immature intestinal NF-kB regulation, probiotics, and necrotizing enterocolitis

描述(由申请人提供)：坏死性小肠结肠炎(NEC)是一种原因不明的危及生命的炎症性肠道疾病，影响大约10%出生的早产儿。早产是最大的风险因素，而不是任何特定的侮辱，这表明肠道不成熟是一个根本问题。然而，不成熟导致NEC的确切方面却知之甚少。我发现，未成熟和成熟肠上皮细胞(IEC)发育差异的一个关键点是核因子kappaB的调节。针对RFA-HD-07-08《预防和治疗坏死性小肠结肠炎的新方法》，我建议检验一种假设，即肠道上皮细胞核因子-kB调节的内在不成熟导致过度的炎症反应，使早产儿易患NEC。此外，我假设益生菌分泌的可溶性因子可以调节核因子-kB的调节，保护肠道屏障功能，从而减轻炎症，保护NEC。该建议的初步数据表明，与成熟的IEC相比，未成熟的IEC在调节抑制性kappaBα和A20关键的NF-kB下调蛋白方面存在差异。此外，我们的初步数据表明，在动物模型中，益生菌的分泌产物可以降低NF-kB的活性，并对NEC具有保护作用。益生菌是一种细菌，除了其固有的营养价值外，还具有有益的健康影响。最近的临床试验表明，益生菌可能对NEC有一定的保护作用。本研究旨在1.评估在未成熟的IEC中，通过IkBA和A20下调NF-kB信号的可能机制。2.在没有引入活体的动物模型中，测定益生菌植物乳杆菌、嗜酸乳杆菌和婴儿双歧杆菌分泌的细菌产物预防坏死性小肠结肠炎的能力。实验设计采用了两种未成熟的IEC-IEC模型，分别来自断奶前的小鼠和人胚胎小肠细胞系H4。将使用常规和无菌小鼠。对于疾病模型，将使用已建立的NEC的Caplan大鼠模型。这项建议将产生有关肠道不成熟方面的重要信息，这些方面有助于NEC，并与了解任何拟议治疗的效果有关。此外，了解无微生物的益生菌条件培养液的潜在作用可能允许一种手段，在没有活体风险的情况下管理益生菌的有益影响，从而为治疗或预防高危婴儿的NEC提供一种新的方法。公共卫生相关性：坏死性小肠结肠炎是一种知之甚少、威胁生命的早产儿炎症性肠病。虽然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，但目前还没有已知的具体治疗方法。这项建议将调查肠道不成熟的哪些方面导致这种疾病，并确定某些有益或益生菌分泌的因素是否可以影响这些方面，从而降低易受坏死性小肠结肠炎影响的婴儿的易感性。

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. Prematurity is the greatest risk factor rather than any particular insult, suggesting that intestinal immaturity is a fundamental issue. However, the exact aspects of immaturity contributing to NEC are poorly understood. I have found that a key point of developmental difference between immature and mature intestinal epithelial cells (IEC) is regulation of nuclear factor kappaB. In response to RFA-HD-07-08 "New Approaches for the prevention and treatment of necrotizing enterocolitis," I propose to test the hypothesis that an intrinsic immaturity of intestinal epithelial cell NF-kB regulation leads to an exaggerated inflammatory response predisposing the preterm infant to NEC. Furthermore I hypothesize that soluble factors secreted by probiotic bacteria can modulate NF-kB regulation and preserve intestinal barrier function, thus decreasing inflammation and protecting against NEC. Preliminary data for this proposal demonstrate that compared to mature IEC, immature IEC have differences in regulation of both inhibitory kappaB alpha and A20 - key NF-kB down-regulatory proteins. Furthermore, our preliminary data demonstrate that secreted products from probiotic bacteria can decrease NF-kB activity and protect against NEC in an animal model. Probiotics are bacteria which have beneficial health effects beyond their inherent nutritive value. Recent clinical trials suggest that probiotics may confer some protection against NEC. This proposal is designed to 1. Assess possible mechanisms behind deficient down-regulation of NF-kB signaling via IkBa and A20 in immature IEC. 2. Determine the ability of secreted bacterial products from the probiotic organisms Lactobacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium infantis to protect against necrotizing enterocolitis in an animal model without the introduction of live organisms. The experimental design uses two models of immature IEC - IEC isolated from pre-weaned mice and the human fetal small intestinal cell line H4. Both conventional and germ-free mice will be used. To model disease, the well established Caplan rat model of NEC will be used. This proposal will yield important information regarding aspects of intestinal immaturity which contribute to NEC and which are relevant to understanding the effect of any proposed treatment. Furthermore understanding the potential role of microbe free probiotic conditioned media may allow a means to administer the beneficial effects of probiotics without the risk of live organisms, thus providing 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. Although 20-30% of patients die and survivors are at risk for significant intestinal and neurodevelopmental consequences, there is no known specific treatment. This proposal will investigate what aspects of intestinal immaturity contribute to this disease and determine if factors secreted by certain beneficial or probiotic bacteria can influence these aspects, thus decreasing the susceptibility of vulnerable infants to necrotizing enterocolitis.