Blast exposure induced acute lung injury
According to the HE results, infiltration of inflammatory cells, and destruction of the alveolar architecture were observed in the the model group mice (Fig. 1). This result indicated that blast could induce acute lung injury, and the model could be uesd for further proteome study.
Quantitative Proteomic Analysis Of Differentially Expressed Protein
For global proteome analysis, the fold-change cutoff was set at 1.2 (models vs. controls); we identified 6498 proteins in lung tissues after blast exposure. Among these, there were 132 proteins were upregulated, and 104 proteins were downregulated as compared to sham control (Supplementary Table S1). The 236 differentially expressed proteins were divided into four quantitative categories according to blast vs. control ratios: 0 < Ratio < 1/1.3, 48 proteins, 1/1.3 < Ratio < 1/1.2, 56 proteins, 1.2 < Ratio < 1.3, 61 proteins and Ratio > 1.3, 71 proteins (Fig. 2).
Protein Annotation And Functional Classification Of Differentially Expressed Protein
To understand the proteins identified and quantified, we annotated the functions and characteristics of these proteins from the aspects of Gene Ontology (GO), Protein domain, KEGG pathway, COG functional classification and sub-cellular structural localization (Supplementary Table S2). From biological process, cellular component, and molecular function results, the biological role of proteins were explained by GO secondary annotation classification. Among them, 84 proteins participate in cellular process; 85 proteins distribute in cell; and 89 proteins are binding proteins. Furthermore, we used software to localize, predict and classify differentially expressed proteins. Cytoplasm (27.27%) and mitochondria (18.94%) exist the maximum amount of differential protein. COG/KOG function classification were used to show the clusters of orthologous groups of proteins (Fig. 3).
Functional Enrichment Of Differentially Expressed Proteins
In order to to find out whether the differentially expressed proteins had significant enrichment trends in some functional types, enrichment analysis of GO classification, KEGG pathway and protein domain were conducted. 20 of the most significant enrichment classifications are presented in the bubble chart. In the biological process category, the upregulated proteins were highly enriched in terms such as positive regulation of cytokine production, regulation of carbohydrate metabolic process, acute-phase response. The enrichment analysis of the cellular component category showed that proteins related to extracellular space, mitochondrial protein complex, mitochondrial membrane part and so on. According to the molecular function enrichment results, we found they are take part in NADH dehydrogenase (ubiquinone) activity, peptidase inhibitor activity, cyclic-nucleotide phosphodiesterase activity, serine-type endopeptidase inhibitor activity and so on (Fig. 4A-C). KEGG pathway enrichment, which is an information network that connects known intermolecular interactions, was further conducted to elucidate the biological functions of proteins. We found the changes of vitamin digestion and absorption, Asthma, oxidative phosphorylation and so on (Fig. 4D). In addition, protein domain, which refers to some components that occur repeatedly in different protein molecules were shown in Fig. 4E.
Analysis Of Functional Enrichment Cluster
Cluster analysis were performed to find the function correlations of the different proteins. Heat maps, which analyzed by clustering based on GO classification, KEGG pathway and protein domain enrichment, were shown in Fig. 5 (A. biological processes; B. cell composition; C. molecular function; D. KEGG pathway; E. Protein domains).
Changes Of Oxidative Phosphorylation Pathway
Based on the protein interaction network, we pay attention to oxidative phosphorylation pathway. The expression levels of NADH ubiquinone oxidoreductase subunits (NDUFV, NDUFA, NDUFB), Cyclooxygenase 2 inhibitors (COX2), Cytochrome c reductase Cytochrome c1 (cyt1), and F-type ATPase (OSCP) were upregulated according to KEGG pathway enrichment (Fig. 6A).
In order to further indentified the role of oxidative phosphorylation in blast-induced lung injury, western blot was used to detected the changes of related proteins. As shown by the western blot results (Fig. 6B), the blast at 12 hrs post injury led to a significant increase of complex I NDUFV1, NDUFA4, NDUFB3, NDUFB5, NDUFB6 subunit protein and COX2 in mice exposed to blast as compared to control.
Taken together, these results are in agreement with proteomics and bioinformatics data demonstrating oxidative phosphorylation involved with primary blast-induced lung injury