Biomarker Study Of Neonatal Hypoxic-ischemic Encephalopathy Based On ITRAQ

Objective Isobaric tags for relative and absolute quantification(iTRAQ)was used to screen differentially expressed proteins in children with mild,moderate,and severe hypoxic-ischemic encephalopathy(HIE)and healthy neonates.A portion of the differentially expressed proteins with further research value were verified by polymerase chain reaction(PCR)and Western blotting methods(Western Blot).To explore its clinical significance and provide new evidence for the discovery of HIE biomarkers.Methods 1.A total of 57 full-term newborn infants with HIE(27 cases with mild HIE,16 cases with moderate HIE,and 14 cases with severe HIE)who were hospitalized in the neonatal intensive care unit of Soochow University Children's Hospital between October 2015 and October 2017 and met HIE's diagnostic and indexing criteria(Designed by the Neonatology Group of the Pediatric Society of the Chinese Medical Association in 2004)were collected.They were all collected blood samples on admission(5.19±2.12 hours after birth)and before treatment.Sixteen healthy full-term neonates born at the municipal hospital of Suzhou during the same period who matched gender,birth weight,and delivery patterns with the case group were chosen as normal controls.Their blood collection time was 6.46±1.79 hours after birth.2.Four cases were randomly selected from the normal control group,the mild,moderate,and severe HIE groups.The plasma differential proteins between HIE group and normal control group were screened by iTRAQ method,and bioinformatics analysis was performed to screen HIE potential organisms.3.Based on the results of proteomics,blood samples from the remaining normal and experimental groups were selected.PCR and Western Blot methods were used to select differentially expressed proteins with further research value for verification.Results 1.In the comparison between the mild HIE group and the control group,69 differential proteins were found,of which 18 proteins were up-regulated and 51 proteins were down-regulated.Among the up-regulated proteins,Haptoglobin(HP)increased most significantly,with a multiple of 8.44 compared with the normal control group.In the comparison between moderate HIE group and normal control group,115 differential proteins were screened,of which 27 proteins were up-regulated and 88 proteins were down-regulated.In the comparison between the severe HIE group and the normal control group,133 differential proteins were screened,of which 14 proteins were up-regulated and 119 proteins were down-regulated.2.There were 51 differential proteins in the mild HIE group and the control group,the moderate HIE group and the control group,and the severe HIE group and the control group.Among them,the protein S100A8 was up-regulated in all three groups of HIE.As the HIE progressed,the fold-to-fold increase was observed.Compared with the normal control group,apolipoprotein M(APOM)was down-regulated in the light,medium,and heavy HIE groups.With the progression of HIE,the multiples of the apolipoprotein M(APOM)decreased.Compared with the normal control group,apolipoprotein E(APOE)was up-regulated in mild HIE and down-regulated in moderate and severe HIE.3.Bioinformatics analysis: 3.1 Gene ontology(GO)functional analysis and enrichment results: We performed enrichment analysis of the biological processes involved in the differential proteins in the mild HIE group and the normal control group.The most significant ones were the injury response,acute inflammatory reaction,and inflammatory reaction.Significant rankings for differential protein cell localization enrichment were extracellular space,extracellular organelles,and exosomes.Phenidase modulators,heparin binding,and glycosaminoglycan binding were the most important factors in the analysis of molecular enrichment of differential proteins.We performed enrichment analysis of the biological processes involved in the differential proteins in the moderate HIE group and the normal control group.The top rankings for significance were injury response,acute inflammatory reaction,and inflammatory reaction.Significant rankings for differential protein cell localization enrichment were extracellular space,extracellular organelles,and exosomes.Glycosaminoglycan binding,phenidase modulatorsand,and heparin binding were the most important factors in the analysis of molecular enrichment of differential proteins.We performed enrichment analysis of the biological processes involved in the differential proteins in the moderate HIE group and the normal control group.The top rankings for significance were acute inflammatory response,protein activation cascade,and injury response.Significant rankings for differential protein cell localization enrichment were extracellular region,exosomes,and extracellular vesicles.Peptidase modulator activity,serine-type endopeptidase activity,and serine hydrolase activity were the most important factors in the analysis of molecular enrichment of differential proteins.3.2 KEGG(Kyoto Encyclopedia of Genes and Genomes)Pathway Analysis and Enrichment Results: We performed an enrichment analysis of the KEGG pathways involved in the differential proteins of the mild HIE group and the normal control group,with the first in significance being the complement and coagulation cascades.We performed enrichment analysis of the major KEGG pathways involved in the differential proteins in the moderate HIE group and the normal control group,the most significant being the complement and coagulation cascades.We performed an enrichment analysis of the KEGG pathway involved in the differential proteins in the severe HIE group and the normal control group,and the significance of the complement and coagulation cascades was significantly higher than that of the other.4.Verification results: PCR validation results showed that Compared with the normal control group,the expression of HP in mild,moderate,and severe HIE increased,and the increase in mild HIE was the largest.The difference was statistically significant.Compared with the normal control group,the expression of S100A8 protein in the mild,moderate and severe HIE group increased,and with the increase of HIE expression,the differences were statistically significant.Western Blot showed that compared with the normal control group,HP was elevated in mild,moderate,and severe HIE,and the differences were statistically significant,and the degree of increase in mild HIE was significantly higher than that in moderate HIE and severe HIE.Conclusions 1.For the first time,we screened the statistically significant plasma differentially expressed protein profile in neonates with HIE and healthy neonates by the iTRAQ method.2.A significant increase in HP in the differential proteins may indicate the occurrence of mild HIE in newborns.The S100A8 protein has the potential to become a biomarker of neonatal HIE.3.The KEGG pathway analysis and enrichment results suggest that the complement and coagulation cascades occupy an important position in the development of neonatal HIE,which may provide new ideas for exploring the pathogenesis of neonatal HIE.