The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis
The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis
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DOI：
10.1016/s0022-3468(97)90194-9
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发表时间：
1997-02-01
影响因子：
2.4
通讯作者：
Rowe, M
中科院分区：
文献类型：
--
作者：
Ford, H;Watkins, S;Rowe, M
目的：炎症细胞因子在坏死性小肠结肠炎（MEG）发病机制中的作用仍不清楚。在患有 NEC 的婴儿中检测到白细胞介素 (IL)-6 和肿瘤坏死因子 (TNF)-α 水平升高，据报道患有临床脓毒症的新生儿中一氧化氮 (NO) 水平升高。然而，NO 或细胞因子的细胞来源尚不清楚。作者推测，细胞因子诱导的局部肠道产生 NO 可能通过诱导细胞凋亡（程序性细胞死亡）或肠上皮细胞坏死，从而导致 NEC 肠坏死的发病机制。我们检查了接受 NEC 手术切除的婴儿肠道中炎性细胞因子和 NO 的水平，以及人诱导型 NO 合酶 (NOS-2) 在发炎肠道中的细胞定位。方法：我们比较了 15 名因 NEC 接受肠切除术的患者，以及 6 名因回肠闭锁或结构、胎粪性腹膜炎、肠套叠或盲肠穿孔而接受肠切除术的婴儿（年龄相近）（对照）。 NEC 的诊断经过组织学证实。通过 Northern 印迹和原位杂交检查手术标本的代表性片段中 NOS-2 的信使 RNA (mRNA)。由于 Northern 印迹法无法检测到 mRNA，因此通过聚合酶链式反应 (PCR) 测量细胞因子 mRNA。通过原位杂交和免疫组织化学确定NO产生位点。使用原位 DNA 链断裂延伸 (TUNEL) 测量细胞凋亡。评估硝基酪氨酸免疫反应性以确定 NO 是否通过过氧亚硝酸盐形成介导细胞损伤。结果：几乎所有 NEC 患者中均检测到 NOS-2 的信使 RNA，除了一名仅接受近端转向空肠造口术且在该部位没有 NEC 组织学证据的婴儿外。在对照患者中 NOS-2 mRNA 的检测频率较低。原位杂交和免疫组织化学显示，肠上皮细胞是 NEC 患者肠道中 NOS-2 活性的主要来源。在患有 NEC 的婴儿的顶端绒毛肠细胞中观察到广泛的细胞凋亡，并且与硝基酪氨酸染色相关。 NOS-2 活性在造口闭合时显着降低，但在死于疾病进展的婴儿中仍保持升高状态。 PCR 显示肠道中不同的细胞因子 mRNA 表达。 NEC 和对照中的转化生长因子 (TGF)-β 表达几乎相同。然而，10 名 NEC 患者中有 9 名存在干扰素 (IFN)-γ，但仅 6 名对照患者中的 1 名存在。结论：数据显示NEC婴儿肠壁肠细胞大量产生NO，并通过形成过氧亚硝酸盐导致顶绒毛肠细胞凋亡。版权所有 (C) 1997 W.B.桑德斯公司。
Purpose: The role of inflammatory cytokines in the pathogenesis of necrotizing enterocolitis (MEG) is still undefined. Elevated levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha have been measured in infants with NEC, white elevated levels of nitric oxide (NO) have been reported in newborn infants with clinical sepsis. However, the cellular source of the NO or cytokines is unknown. The authors hypothesized that local intestinal production of NO induced by cytokines may contribute to the pathogenesis of bowel necrosis in NEC by inducing apoptosis (programmed cell death) or necrosis of the enterocytes. We examined the levels of inflammatory cytokines and NO in the intestine of infants undergoing surgical resection for NEC, and the cellular localization of human inducible NO synthase (NOS-2) in the inflamed gut. Methods: We compared 15 patients undergoing bowel resection for NEC, with six infants (of similar age) undergoing intestinal resection for ileal atresia or structure, meconium peritonitis, intussusception, or cecal perforation (control). Diagnosis df NEC was confirmed histologically. Representative segments of the surgical specimen were examined for messenger RNA (mRNA) for NOS-2 by Northern blotting and in situ hybridization. Cytokine mRNA was measured by polymerase chain reaction (PCR) because mRNA could not be detected by Northern blotting. The site of NO production was determined by in situ hybridization and immunohistochemistry. Apoptosis was measured using in situ DNA strand break extension (TUNEL). Nitrotyrosine immunoreactivity was assessed to determine if NO mediates cellular injury via peroxynitrite formation. Results: Messenger RNA for NOS-2 was detected in nearly all patients with NEC except for one infant who underwent proximal diverting jejunostomy alone, and who did not have histological evidence of NEC at ti;at site. NOS-2 mRNA was detected less frequently in control patients. In situ hybridization and immunohistochemistry showed that the enterocytes were the predominant source of NOS-2 activity in the intestine of NEC patients. Extensive apoptosis was seen in enterocytes in the apical villi of infants with NEC, and correlated with nitrotyrosine staining. NOS-2 activity was markedly diminished at the time of stoma closure, but remained elevated in infants who died from progressive disease. PCR showed variable cytokine mRNA expression in the intestine. Transforming growth factor (TGF)-beta expression was nearly indentical in NEC and control. However, interferon (IFN)-gamma was present in 9 of 10 NEC, but only in one of six control patients. Conclusion: The data show that NO is produced in large quantity by enterocytes in the intestinal wall of infants with NEC and leads to apoptosis of enterocytes in apical villi through peroxynitrite formation. Copyright (C) 1997 by W.B. Saunders Company.