Pathogenesis of Experimental Necrotizing Enterocolitis

坏死性小肠结肠炎（NEC）是影响早产儿胃肠道的最常见和最致命的疾病。这种疾病的确切病因尚不清楚。唯一一致确定的NEC流行病学前兆是早产和肠内营养。我们之前已经发现急性NEC婴儿肠段诱导型一氧化氮（NO）合成酶（NOS-2） mRNA和蛋白上调。NOS-2与肠细胞凋亡和硝基酪氨酸免疫反应性共定位。NOS-2在急性炎症消退后肠造口关闭时下调。我们已经在新生大鼠中开发了一种可重复的肠道炎症模型，从而模拟了与人类NEC相关的条件。我们简单地用配方饲料喂养缺氧新生大鼠，从而模拟与人类NEC相关的条件。这些幼崽表现出NOS-2 mRNA上调、亚硝化应激、肠细胞凋亡增加和隐窝肠细胞增殖减少。与母乳喂养的大鼠相比，低氧配方喂养的大鼠上皮内淋巴细胞（IEL）分泌更多的tnf - α和IFN- γ。体外研究表明，IEL与大鼠肠上皮细胞系IEC-18共培养，在il -1 β存在的情况下，可诱导ifn - γ、tnf - α和NOS-2的产生，而ifn - γ抗体可消除这些作用。此外，过氧亚硝酸盐（ONOO）诱导IEC-6细胞凋亡，抑制细胞增殖。这些数据提示，NEC的肠道坏死可能是一氧化氮诱导的组织损伤与修复机制失衡的结果。围产期损伤引起的粘膜损伤导致细菌-上皮相互作用，IEL和固有层（LP）淋巴细胞局部释放ifn - γ和tnf - α等细胞因子。这些介质通过肠细胞或LP巨噬细胞产生NO和ONOO诱导NOS-2上调。NO或ONOO反过来促进进一步的组织损伤（肠细胞凋亡），同时抑制组织修复机制（肠细胞增殖），导致肠道屏障功能衰竭和NEC。为了验证我们的假设，我们提出以下目标：1)明确人类和实验性啮齿动物NEC中NOS-2上调的机制。II)明确NOS-2上调促进啮齿动物NEC肠道损伤和改变组织修复机制的机制。III)确定NO清除剂或NO产生抑制剂对实验性啮齿动物NEC发育的影响。

Necrotizing enterocolitis (NEC) is the most frequent and the most lethal disease that affects the gastrointestinal tract of the premature infant. The exact etiology of the disease is undefined. The only consistent identifiable epidemiological precursors for NEC are prematurity and enteral alimentation. We have previously shown upregulation of inducible nitric oxide (NO) synthase (NOS-2) mRNA and protein in intestinal segments from infants with acute NEC. NOS-2 colocalized with enterocyte apoptosis and nitrotyrosine immunoreactivity. NOS-2 was downregulated at the time of intestinal stoma closure when the acute inflammation had subsided. We have developed a reproducible model of gut inflammation in neonatal rats thereby simulating the conditions associated with human NEC. We simply formula-feed hypoxic neonatal rats thereby simulating the conditions associated with human NEC. These pups show NOS-2 mRNA upregulation, nitrosative stress, increased enterocyte apoptosis and decreased enterocyte proliferation in the crypts. Intraepithelial lymphocytes (IEL)from hypoxic formula-fed rats secrete more TNF-alpha and IFN- gamma compared to breast-fed rats. In vitro studies suggest that co- culture of IEL with the rat intestinal epithelial cell line IEC-18, in the presence of IL-1beta induces IFN-gamma, TNF-alpha and NOS-2 production which can be abrogated with antibody to IFN-gamma. Furthermore, peroxynitrite (ONOO) induces apoptosis and inhibits proliferation of IEC-6 cells. The data suggest that intestinal necrosis in NEC may be the result of NO-induced imbalance between tissue injury and repair mechanisms. Mucosal injury resulting from perinatal insults leads to bacterial-epithelial interactions, local release of cytokines such as IFN-gamma and TNF-alpha by IEL and lamina propria (LP) lymphocytes. These mediators induce NOS-2 upregulation with production of NO and ONOO by enterocytes or LP macrophages. NO or ONOO in turn promotes further tissue injury (enterocyte apoptosis) and concurrent inhibition of tissue repair mechanisms (enterocyte proliferation) leading to gut barrier failure and NEC. To test our hypothesis, we propose the following Aims: I) To define the mechanism of NOS-2 upregulation in human and experimental rodent NEC. II) To define the mechanisms by which NOS-2 upregulation promotes intestinal injury and alters tissue repair mechanisms in rodent NEC. III) To determine the effects of scavengers of NO or inhibitors of NO production on the development of experimental rodent NEC.