双唾液酸乳糖-N-四糖改善肠道代谢微环境稳态抑制新生大鼠坏死性小肠结肠炎病理进程  

Disialyllacto-N-tetraose improves intestinal homeostasis of metabolic microenvironment to prevent the pathological development of necrotizing enterocolitis in neonatal rats

作　　者：张雯婷 闫竞宇[2] 壮文军[3] 姜春红 屠文娟[3] Zhang Wenting;Yan Jingyu;Zhuang Wenjun;Jiang Chunhong;Tu Wenjuan(Department of Pharmacy,Changzhou Children′s Hospital Affiliated to Nantong University,Changzhou 213003,China;Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China;Department of Neonatology,Changzhou Children′s Hospital Affiliated to Nantong University,Changzhou 213003,China;Graduate School,Medical School,Nantong University,Nantong 226000,China)

机构地区：[1]南通大学附属常州儿童医院药学部,常州213003 [2]中国科学院大连化学物理研究所分析化学分离科学重点实验室,大连116023 [3]南通大学附属常州儿童医院新生儿科,常州213003 [4]南通大学医学院研究生院,南通226000

出　　处：《中华实用儿科临床杂志》2022年第5期371-376,共6页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(81801493);中国博士后科学基金第70批面上资助(2021M700546);江苏省卫生健康委科研课题(H2018008);常州市科技计划项目(CJ20210161);常州市卫生健康青苗人才工程(CZQM2020105)。

摘　　要：目的探讨双唾液酸乳糖-N-四糖(DSLNT)对坏死性小肠结肠炎(NEC)新生大鼠肠道内容物低分子量代谢谱的影响,探索其对新生儿肠道的保护作用方式。方法新生SD大鼠按随机数表法随机分为对照组、NEC组和NEC+DSLNT组,大鼠均采用特殊配方奶人工喂养,NEC组和NEC+DSLNT组以3次/d的频率进行缺氧(950 mL/L氮气,10 min)/冷刺激(4℃,10 min)、连续3 d诱导新生大鼠NEC模型,NEC+DSLNT组在特殊配方奶中添加300μmol/L DSLNT。造模72 h时处死所有存活大鼠,采集回结肠部位肠内容物进行基于超高效液相色谱-组合型四级杆Orbitrap质谱仪(UHPLC-QE-MS)的非靶向代谢组检测,末端回肠行苏木精-伊红染色。代谢组数据用SIMCA 14.1软件进行多元变量统计分析。以正交偏最小二乘分析(OPLS-DA)模型的变量投影重要度(VIP)值>1和t检验中P<0.05筛选两两比较的组间差异代谢物。结果DSLNT降低NEC发生率和NEC大鼠回肠组织病理学评分[3.0(2.0,3.0)分比1.0(1.0,2.0)分,P<0.01],并可有效抑制炎症浸润。基于UHPLC-QE-MS代谢组检测结果建立的OPLS-DA模型能较好地对NEC组和对照组、NEC+DSLNT组和NEC组实现分离。NEC组和对照组之间有64个差异代谢物(OPLS-DA模型的VIP值>1且P<0.05),包括二十二碳六烯酸(+288.0%,P=0.028)、黄嘌呤(+372.1%,P=0.007)、L-精氨酸(+233.1%,P=0.027)、L-亮氨酸(+232.7%,P=0.015)、N-乙酰神经氨酸(-41.6%,P=0.014)等,这些代谢物可映射到34条不同的代谢通路,其中精氨酸生物合成、精氨酸和脯氨酸代谢等6条代谢通路为NEC主要扰动的代谢通路。NEC+DSLNT组与NEC组之间存在15种差异代谢物,包括D-甘露糖(-73.5%,P=0.032)、黄嘌呤(-63.4%,P=0.008)、亚油酸(+137.9%,P=0.047)、烟酰胺腺嘌呤二核苷酸(+278.2%,P=0.005)等,这些差异代谢物可映射到7条代谢通路,其中亚油酸代谢为DSLNT主要影响的差异代谢通路。两种比对策略中重合的差异代谢物数量为8个,其在NEC中的变化趋势在DSLObjective To investigate the effects of disialyllacto-N-tetraose(DSLNT)on low molecular weight metabolic profile of intestinal contents in neonatal rats with necrotizing enterocolitis(NEC),in an attempt to explore the protective mechanism of DLSNT on intestinal tract of neonates.Methods Immediately after birth,SD rats were randomly divided into the control group,the NEC group and the NEC+DSLNT group according to random number tale method.All rats were hand-fed by special formula milk.Rats in the NEC group and NEC+DSLNT group were exposed to hypoxia(950 mL/L nitrogen,10 min,thrice per day)and cold stress(4℃,10 min,thrice per day)for continuous 3 days to establish rodent NEC model.Rats in the NEC+DSLNT group were hand-fed with special formula containing 300μmol/L DSLNT.All rats were sacrificed after 72 h,and intestinal contents were collected from ileum and colon,followed by untargeted metabolomic determination with the ultrahigh-performance liquid chromatography Q extractive mass spectrometry(UHPLC-QE-MS)method.The terminal ileum was examined by hematoxylin-eosin staining.The metabolome data were analyzed with multivariable analysis using SIMCA 14.1.The metabolites that met both variable importance in the projection(VIP)>1 in the orthogonal partial least squares analysis(OPLS-DA)model and P<0.05 in the t-test were screened as differential metabolites between groups.Results DSLNT reduced the incidence of NEC and pathological scores of ileum tissue from neonatal rats with NEC[3.0(2.0,3.0)scores vs.1.0(1.0,2.0)scores,P<0.01],and also significantly suppressed inflammatory infiltration.OPLS-DA model based on the metabolome data determined by UHPLC-QE-MS could perform effective discrimination between the NEC group and the control group,as well as the NEC+DSLNT group and the NEC group.There were 64 differential metabolites between the NEC group and the control group(VIP value>1 and P<0.05 for the OPLS-DA model).These metabolites included docosahexaenoic acid(+288.0%,P=0.028),xanthine(+372.1%,P=0.007),L-arginine(+233.1

关 键 词：坏死性小肠结肠炎 代谢组学 人乳寡糖 双唾液酸乳糖-N-四糖 

分 类 号：R722.1[医药卫生—儿科]