RESUMO
Objective: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 300 μmol/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%, P=0.027), L-leucine (+ 232.7%, P=0.015), N-acetylneuraminic acid (-41.6%, P=0.014), and so forth.These metabolites were associated with 34 metabolic pathways.Among them, such 6 pathways as arginine biosynthesis, arginine and proline metabolism were the most disturbed pathways affected by NEC.There were 15 diffe-rential metabolites in between NEC+ DSLNT group and NEC group, which included D-mannose (-73.5%, P=0.032), xanthine (-63.4%, P=0.008), linoleic acid (+ 137.9%, P=0.047), nicotinamide adenine dinucleotide (+ 278.2%, P=0.005), and so forth.These metabolites were mapped to 7 metabolic pathways, among them, linoleic acid metabolism pathway was the most relevant differential pathway affected by DSLNT.There were 8 overlapped meta-bolites in both comparison strategies, and the variation trend of these overlapped metabolites in the NEC group was significantly reversed by DSLNT supplementation. Conclusions:DSLNT could significantly attenuate the NEC pathological damage caused by hypoxia/cold stress in neonatal rats.This protective effect is associated with the improvement of the metabolic profile of intestinal contents caused by NEC and the modulation of the linoleic acid metabolic pathway.The early preventive supplementation of DSLNT is of great significance in maintaining neonatal intestinal homeostasis and preventing the process of NEC.
RESUMO
Objective:To study the expression of zonula occludens-1(ZO-1) in neonates with necrotizing enterocolitis (NEC) and to explore the effects of disialyllacto-N-tetraose (DSLNT), a compound extracted from human milk, on the intestinal barriers in rat model of NEC.Methods:(1) Human study: From Feb 2013 to Dec 2020, the pathological samples of ileum tissue from 21 neonates (12 patients with NEC and 9 with intestinal atresia) from Pathology Department of our hospital were collected. The expressions of ZO-1 in these samples were examined using immunohistochemistry (IHC) method. (2) Animal study: A total of 28 newborn rats were randomly assigned into control group ( n=8), NEC group ( n=10) and DSLNT+NEC group ( n=10). Experimental NEC model was established based on hypoxia (95%N 2 10 min) /cold exposure (4 ℃ 10 min) three times a day for consecutive 3 days. DSLNT+NEC group were fed with formula+DLSNT (300 μmol/L) during hypoxia/cold exposure. All the surviving rats were sacrificed at the end of the experiment (72 h) and the terminal ileum tissues were collected. Hematoxylin-Eosin (HE) staining was used to evaluate tissue damage and Western blotting was used to determine the expressions of ZO-1. (3) Cellular study: Bacterial lipopolysaccharide (LPS) was used to establish a cellular inflammation model in human intestinal epithelial cell lines (Caco-2) and DSLNT (300 μmol/L) was applied to this model. Thiazolyl blue assay was used to examine cell viabilities and immunofluorescence assay was used to detect ZO-1 expression. The effects of DSLNT on cell growth and tight junctions of Caco-2 cells were analyzed. Results:(1)Human study: The villi of mucous layer of the lesion were damaged in NEC patients. ZO-1 expressions at the epithelial junction of NEC patients were decreased compared with intestinal atresia patients and non-lesion intestines of NEC patients. (2)Animal study: Apical extrusion, necrosis and shedding of epithelial cell were seen at the lesions in NEC group. The expression of ZO-1 in NEC group was significantly lower than the control group and DSNLT+NEC group ( P<0.05).DSNLT+NEC group had higher survival rates (8/10 vs. 6/10) and lower ileum inflammatory pathological scores [2.0(1.0, 2.8) vs. 3.5(3.0, 4.0)] than NEC group. (3) Cellular study: Caco-2 cells exposed to LPS showed inhibited cell growth and decreased ZO-1 immunofluorescence staining. Caco-2 cells in the DSLNT+LPS group showed better viability than LPS group and were comparable with the control group. The expression of ZO-1 was significantly increased in the DSLNT+LPS group. Conclusions:Tight junction injury of the intestinal epithelial cell is an important characteristic of NEC. ZO-1 is a potential target for the prevention and treatment of NEC. DSLNT may protect the neonatal intestines by modulating the expression of ZO-1 and keeping tight junction integrity.