Résumé
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.
Résumé
Neonatal necrotizing enterocolitis (NEC) is the most common neonatal gastrointestinal emergency,and its pathogenesis is still not clear.In recent years,intestinal flora has been keenly concerned about its pivotal role in the progress of NEC pathology.At the technical level,the popularity and in-depth development of high throughput sequencing,fluorescence in situ hybridization and polymerase chain reaction-denaturing gradient gel electrophoresis are also favorable conditions for further study of the correlation between NEC and intestinal flora.Now,the relationship between the pathological development of NEC and intestinal flora was reviewed.
Résumé
Objective To analyze the biological activity of bioactive peptides in human breast milk and to find the polypeptides so as to investigate the preventive and therapeutic effects of breast milk-derived bioactive peptides on neonatal necrotizing enterocolitis(NEC).Methods Six mothers who gave birth to preterm neonates were enrolled in this study and 5 mL of their breast milk secreted within 2-5 postnatal days were collected for 6 times and blended subsequently.Bioactive peptides from maternal milk of the preterm infants were separated by ultrafiltration and analyzed by using tandem mass spectrometry.Polypeptides possibly with biological function were screened out by using bioinformatics software and the protein function cluster online analysis software was used to predict the polypeptides associated with infection according to the biological function of their precursor proteins.The ATCC25922,an Escherichia coli strain commonly associated with infection in NEC and drug solution (sulbactarr/cefoperazone) were used to conduct the drug susceptibility testing and bactericidal kinetics testing,so as to verify the antibacterial effects of bioactive peptides in the breast milk.Results Four thousand three hundred and eleven peptides contained in breast milk were identified successfully,of which 1 370 were non-differential peptides,and 188 peptides possibly with biological activity and 11 peptides were associated with infection.The peptide compound in the breast milk had antimicrobial activity and bactericidal power against Escherichia coli.Conclusions The active peptide compounds in the breast milk have antimicrobial activity,which play an important role in the prevention of NEC.Finding the true antimicrobial peptides with in vivo and in vitro biological activity by using antimicrobial spectrum test is expected.