Ferretti Emanuela, Tremblay Eric, Thibault Marie-Pier, Grynspan David, Burghardt Karolina M, Bettolli Marcos, Babakissa Corentin, Levy Emile, Beaulieu Jean-François
Research Consortium on Child Intestinal Inflammation, Division of Neonatology, Department of Pediatrics, University of Ottawa, Ottawa, Canada.
Research Consortium on Child Intestinal Inflammation, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada.
Nitric Oxide. 2017 Jun 1;66:53-61. doi: 10.1016/j.niox.2017.03.003. Epub 2017 Mar 14.
NO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine.
Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 μM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes.
In the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO.
These results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.
一氧化氮合酶2（NOS2）最近被确定为坏死性小肠结肠炎（NEC）早产儿肠道样本中过度表达最为明显的基因之一。NOS2广泛参与上皮细胞损伤/凋亡和宿主免疫防御过程，但其在未成熟人类肠黏膜炎症中的具体作用仍不清楚。有趣的是，诸如表皮生长因子（EGF）等预防NEC的因素，在无血清器官培养中可减轻孕中期人类小肠的炎症反应，而与NEC发生相关的药物，如非甾体抗炎药吲哚美辛（INDO），则对未成熟人类肠道产生多种有害影响。在本研究中，我们通过测定未成熟肠道中NOS2及其下游途径的表达谱，来研究NOS2在保护性和应激条件下调节肠道炎症反应的潜在作用。
使用Illumina全基因组微阵列、Ingenuity通路分析软件和定量PCR，研究在有无生理浓度的EGF（50 ng/ml）或1 μM INDO存在的情况下，培养48小时的孕中期人类肠道外植体的基因表达谱，以研究NOS2和NOS2通路相关基因的表达。
在未成熟肠道中，发现EGF可增加NOS2表达，而INDO则抑制其表达。生物信息学分析确定了差异调节的通路，其中NOS2在这些通路中发挥重要作用，包括瓜氨酸/精氨酸代谢、上皮细胞连接和氧化应激。在个体基因水平上，我们发现了许多瓜氨酸/精氨酸代谢途径中差异表达的基因，如ARG1、ARG2、GLS、OAT和OTC，它们对EGF和INDO有反应。紧密连接成分如CLDN1、CLDN2、CLDN7和OCN以及抗氧化标志物如DUOX2、GPX2、SOD2的基因表达也被发现受到EGF和INDO的差异调节。
这些结果表明，EGF的保护作用和INDO对未成熟肠道的有害影响可能是通过对NOS2的调节介导的。与这些作用相关的NOS2下游途径包括与NO产生相关的代谢、上皮屏障通透性和抗氧化剂表达。这些结果表明，NOS2可能是未成熟人类肠道炎症反应的调节因子，并可能为EGF的保护作用和INDO的有害作用提供机制基础。
