Molecular Mechanism Of Neonatal Acute Bilirubin Encephalopathy Based On Quantitative Proteomics Of MV/E From Cerebrospinal Fluid

Background and Objective:1.Severe hyperbilirubinemia may induce acute bilirubin encephalopathy in neonates,leading to neurological dysfunction,but the basic mechanism has not been fully elucidated.2.Based on iTRAQ quantitative proteomics method,this study explored the neuro-proteomic characteristics of extracellular micro-vesicles/exosomes in cerebrospinal fluid,providing a new entry point for elucidation of pathogenesis and prevention of neonatal bilirubin brain injury.Materials and Methods:1.Term infants with severe hyperbilirubinemia and acute bilirubin encephalopathy who were admitted to neonatology department of the First People's Hospital of Chenzhou on January 1,2017 and January 1,2020 were selected as the research objects.2.Cerebrospinal fluid was obtained by lumbar puncture and vesicles/exosomes were separated by centrifugation and precipitation with reagent.NanosightNS300,electron microscope and Western blotting were used to characterize and verify the physical characteristics,morphology and molecular markers of vesicles/exosomes,respectively.3.Vesicles/exosomes proteins were extracted,reduced,alkylated and enzymolyzed,and the iTRAQ and high pH UPLC were used for peptide segment labeling and peptide segment separation,and liquid chromatography tandem mass spectrometry was used for identifying differential proteins.4.Cluster analysis of protein expression,GO and KEGG pathway and protein-protein interaction network bioinformatics analysis were performed for the differentially expressed proteins.5.Western blotting was selected for four different proteins(S100A9,S100A7,DEFA1 and LTF)to preliminarily verify the quantitative proteomics results.6.One-way ANOVA and Bonferroni's Post Hoc test was applied for statistical analysis by IBM SPSS 20.0 software.Results:1.The size distribution of extracellular vesicles/exosomes was 70-200nm,and the peak concentration was at 103nm.There was no statistically significant difference in the mean of particle size and concentration among the groups.2.Vesicular structures of different.sizes had been observed by electron microscope,and the expressions of vesicle/exosome molecular markers of CD9 and CD63 had been verified by Western blotting.3.A total of 182,834 MS/MS spectra,5,913 matching peptides,634 total proteins and 291 differential proteins were identified in extracellular vesicles/exosomes.4.The cluster analysis of differentially expressed proteins showed that the protein expression patterns of each group were different.5.The GO annotation and functional enrichment of differential proteins were mainly in the cell components of "extracellular region","extracellular region part"and "membrane",and they were mainly involved in the biological process of"immune system process" and "metabolic process","Response to Stimulus" and exercise the molecular functions of "structural molecule activity","antioxidative activity" and "Molecular function regulator".6.The KEGG Pathways of differential proteins were mainly enriched in the Ras Signaling Pathway,PI3K-Akt signaling Pathway,MAPK signaling Pathway,and Cytokine-Cytokine receptor interaction,and Rap1 signaling Pathway.7.Interaction network analysis of differential proteins showed that differential proteins,such as SAA1,APP,C4B,C5,DEFA1,LTF,CRP,S100A7,S100A9 and LBP,were correlated with each other and were related to the immune system and inflammatory process.8.Compared with the control group,the expression levels of LTF and DEFA1 in the acute bilirubin encephalopathy group were significantly up-regulated,while the expression levels of S100A7 and S100A9 were significantly down-regulated.These results were consistent with the results of mass spectrometry.Conclusions:1.Extracellular vesicles/exosomes can be successfully extracted from the cerebrospinal fluid of neonatal acute bilirubin encephalopathy,and there are differentially expressed proteins in these nano-vesicles.2.Bioinformatics analysis of differential proteins in extracellular vesicles/exosomes shows that bilirubin brain damage might be related to immune and inflammatory processes.3.Extracellular vesicles/exosomes and the differential proteins they contain have potential value as early diagnostic markers and therapeutic targets for neonatal bilirubin brain injury.