Pathogenesis of Experimental Necrotizing Enterocolitis

实验性坏死性小肠结肠炎的发病机制

• 批准号: 6845322
• 负责人: HENRI R FORD
• 金额: $ 38.08万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845322
• 关键词: apoptosis; cellular pathology; clinical research; disease /disorder etiology; disease /disorder model; enzyme activity; enzyme mechanism; free radical scavengers; gastrointestinal epithelium; human subject; immunocytochemistry; infant human (0-1 year); inflammation; intestine disorder; laboratory rat; necrosis; necrotizing enterocolitis; newborn animals; nitric oxide; nitric oxide synthase; oxidative stress; patient oriented research; polymerase chain reaction; protein biosynthesis; western blottings

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.

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。该病的确切病因尚未确定。 NEC唯一一致的可识别的流行病学前兆是早产和肠内营养。 我们先前已经显示了诱导型一氧化氮（NO）合酶（NOS-2）的mRNA和蛋白质在急性NEC的婴儿肠段上调。 NOS-2与肠上皮细胞凋亡和硝基酪氨酸免疫反应共定位。 NOS-2在急性炎症消退后肠吻合口关闭时表达下调。我们已经开发了一种可重复的新生大鼠肠道炎症模型，从而模拟与人类NEC相关的条件。 我们简单地配方饲料缺氧新生大鼠，从而模拟与人类NEC相关的条件。 这些幼崽显示NOS-2 mRNA上调，亚硝化应激，肠上皮细胞凋亡增加，肠上皮细胞增殖减少。 与母乳喂养的大鼠相比，缺氧配方奶喂养的大鼠的上皮内淋巴细胞（IEL）分泌更多的TNF-α和IFN-γ。 体外研究表明，在IL-1 β存在下，IEL与大鼠肠上皮细胞系IEC-18的共培养诱导IFN-γ、TNF-α和NOS-2的产生，其可被IFN-γ抗体消除。过氧亚硝基阴离子（ONOO）可诱导IEC-6细胞凋亡并抑制其增殖。 这些数据表明，肠坏死的NEC可能是一氧化氮诱导的组织损伤和修复机制之间的失衡的结果。由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）淋巴细胞局部释放细胞因子如IFN-γ和TNF-α。 这些介质通过肠细胞或LP巨噬细胞诱导NOS-2上调，产生NO和ONOO。 NO或ONOO反过来促进进一步的组织损伤（肠细胞凋亡）和同时抑制组织修复机制（肠细胞增殖），导致肠屏障衰竭和NEC。 为了验证我们的假设，我们提出了以下目的：I）确定NOS-2在人类和实验啮齿动物NEC中上调的机制。II）确定NOS-2上调促进啮齿动物NEC中肠损伤和改变组织修复机制的机制。III）确定NO清除剂或NO产生抑制剂对实验啮齿动物NEC发展的影响。