目的 分析新生大鼠坏死性小肠结肠炎(necrotizing enterocolitis,NEC)肠道内容物代谢轮廓的变化和特征,探讨潜在的NEC生物标志物.方法 新生3日龄大鼠均采用特殊配方奶(15 g雀巢早产儿奶粉加入75 ml Pat-Ag幼犬专用奶中配制而成)喂养,随机分为对照组(n=8)及NEC组(n=12),NEC组以3次/d,连续3d进行4℃冷刺激10 min +95％ N2缺氧10 min诱导建立.造模结束后处死所有大鼠,回结肠部位的肠道内容物以灌流方式取出,冻干后进行基于超高效液相色谱-组合型四级杆Orbitrap质谱仪(UHPLC-QE-MS)的非靶向代谢物检测,并用SIMCA软件对代谢物测定结果进行多元变量统计分析;回肠组织切片进行苏木精-伊红染色并行病理评分.结果 NEC组病理评分显著高于对照组[(3.13±0.83)分比(0.25±0.46)分,P＜0.001],正离子扫描(ESI+)模式下正交偏最小二乘判别分析(OPLS-DA)模型R2(x)=0.604,R2(y)=0.583,Q2=0.960,负离子扫描(ESI-)模式下OPLS-DA模型R2(x) =0.828,R2(y) =0.999,Q2 =0.713,对照组和NEC组大鼠肠内容物代谢轮廓存在显著差异.对照组和NEC组之间存在48个差异代谢物,其中ESI-模式筛选出22个差异代谢物,包括L-别异亮氨酸(+221％)、D-苯丙氨酸(+230％)、L-组氨酸(+284％)、黄嘌呤(+207％)、谷氨酰亮氨酸(+246％)、阿洛糖(-70％)、肉豆蔻酸(-57％)和十五烷酸(-35％)等;ESI+模式下筛选出26个差异代谢物,包括鸟氨酸(+268％)、D-亮氨酸(+176％)、L-异亮氨酸(+213％)、乙酰胆碱(+195％)、烟酰胺腺嘌呤二核苷酸(+199％)、瓜氨酸(+158％)、胞嘧啶(-58％)、黄尿酸(-64％)等.差异代谢物进行京都基因与基因组百科全书(KEGG)分析可映射到33条不同的代谢通路,MetaboAnalyst软件的通路富集分析和通路拓扑分析表明精氨酸和脯氨酸代谢通路、组氨酸代谢通路和谷胱甘肽代谢通路在NEC组出现较大扰动.结论 NEC大鼠肠内容物代谢轮廓显著偏离于正常大鼠,以氨基酸蓄积为主要特征,主要涉及精氨酸、脯氨酸、组氨酸和谷胱甘肽代谢通路.检测肠内容物代谢组尤其是氨基酸代谢组对NEC诊断可能有重要意义,改善肠道微环境可能是防治NEC的关键策略.“,”Objective To study the change and characterization of metabolic profile of intestinal contents of the neonatal rats with necrotizing enterocolitis (NEC) using metabolomics approach,in order to figure out potential biomarkers of NEC.Method Twenty rats with three-postnatal day-old fed with special formula were assigned to control group (n =8) and NEC group (n =12) randomly.Experimental NEC of rats in NEC group were induced by exposing to cold stimulation at 4 degrees Celsius for 10 minutes and to hypoxia at 95％ nitrogen for 10 minutes,three times a day for three consecutive days.All the rats were sacrificed after model preparation.Segments of the ileum of all the rats were collected for hematoxylin-eosin staining and subsequent pathological damage evaluation.The intestinal contents of the ileum and colon were collected by perfusion,followed by lyophilization and analyzed by UHPLC-QE-MS in order to conduct the non-target metabolomic determination.The information of the metabolites determined was calculated by multivariable analysis using SIMCA software.Result The pathological damage scores of NEC group were higher than those of the control group [(3.13 ± 0.83) vs.(0.25 ± 0.46),P ＜ 0.001].The results of orthogonal partial least squares discriminant analysis (OPLS-DA) model showed that in the ESI + mode,R2(x) =0.604,R2(y) =0.583,Q2 =0.960,while in the ESI-mode,the OPLS-DA model R2(x) =0.828,R2(y) =0.999,and Q2 =0.713,indicating that there is a significant difference in the intestinal content metabolic profile between the control group and the NEC group.Forty-eight differential metabolites related to NEC were identified.In ESI-mode,there were 22 differential metabolites,including L-isoisoleucine (+ 221％) and D-phenylalanine (+ 230％),L-histidine (+ 284％),xanthine (+ 207％),glutamyl leucine (+ 246％),allose (-70％),myristic acid (-57％) and pentadecanoic acid (-35％).What is more,in the ESI + mode,26 other differential metabolites were identified,including ornithine (+ 268％),D-leucine (+ 176％),L-iso Leucine (+ 213％),acetylcholine (+ 195％),nicotinamide adenine dinucleotide (+ 199％),citrulline (+ 158％),cytosine (-58％),xanthoic acid (-64％).These metabolites were reflected to 33 different metabolic pathways in KEGG databases.The pathway enrichment analysis and pathway topology analysis with MetaboAnalyst indicated that the arginine and proline metabolic pathways,histidine metabolic pathways,and glutathione metabolic pathways were the top altered pathways in the condition of NEC.Conclusion The metabolic profile of intestinal contents in NEC rats was significantly different from that in normal rats,which was characterized by amino acid accumulation,mainly involving the metabolic pathways of arginine,proline,histidine and glutathione.The detection of intestinal contents metabolic profile,especially amino acid metabolize group may be of great significance for the diagnosis of NEC,and improving intestinal microenvironment may be the key strategy for the prevention and treatment of NEC.