Animals and experimental groups
Fourty-eight male C57BL/6 mice (20-25g, 6-8 weeks old) were obtained from the Experimental Animal Department of the General Hospital of Northern Theater Command. After acclimating for one week, all mice were randomly divided into six groups (n=8/group): control, 12 h, 24 h, 48 h, 72 h and 1 w after low-intensity blast exposure. All mice were kept in a room, maintained at a temperature of 20±2°C and humidity of 55%–65%, and given unrestricted access to food and water. Animal welfare and experimental design were approved by the Ethics Committee of the General Hospital of Northern Theater Command.
Blast-exposure induced lung injury
A precise model of lung blast injury was used as previously described [10]. Briefly, mice were anesthetized by abdominally injecting 2% pentobarbital sodium (1.5 ml/kg). After stabilizing the device with screws, mice were placed on rubber pads with 10 regular holes atop the device. The pressure pump was connected to the bottom of the device and was continuously pressurized until 5 layers of 18mm thick aluminum film bursts. The compressed air rapidly expanded from the blasting port at high speed, forming shock waves that impacted the chest of the mice. The pressure detected by a pressure sensor was transmitted through a data cable and recorded by a computer. The mice after detonation fall into the prepared soft woven bag to avoid secondary impact damage. The overpressure value of the shock wave at the instant of blasting was 115.8±10.4 per square inch (PSI). The mice in control group underwent identical procedures as blast groups only without blast exposure. After blast exposure, mice were removed from the woven bag and returned to the original cag.
Samples collection and pathological examine
All mice were intraperitoneally anesthetized with 2% sodium pentobarbital (1.5 ml/kg), and lung samples were collected 12 h, 24 h, 48 h, 72 h and 1 w after blast exposure, respectively. After blast exposure, a segment of the lungs was removed and dried in a culture dish for 24 h at 37℃. The dry weight was calculated, and Evans blue was extracted using 1 ml formamide for 24 h at room temperature. The amount of Evans blue in the lungs was quantified by measuring the absorbance at 620nm in an ELISA plate reader (Imark, Bio-Rad, USA). The concentrations of the dye were calculated from a standard curve. The results are presented as the amount of Evans blue in mg/g of tissue. For the histological analysis, lung samples were fixed in 10% formaldehyde at room temperature, processed, and embedded in paraffin blocks using a Leica Microsystem tissue processor (ASP 300S, Germany). For histological staining, 4-μm-thick sections were sliced using a Leica Microsystem microtome (Model RM 2265, Germany), which were stained with hematoxylin and eosin (H&E).
Protein extraction
Lung sample was grinded by liquid nitrogen into cell powder and then transferred to a 5-mL centrifuge tube. After that, four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) were added to the cell powder, followed by sonication three times on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000g at 4°C for 10 min. Finally, the supernatant was collected and the protein concentration was determined with the BCA kit according to the manufacturer’s instructions.
Trypsin digestion
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion.
TMT/iTRAQ labeling
After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Briefly, one unit of TMT/iTRAQ reagent was thawed and reconstituted in acetonitrile. Peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.
HPLC fractionation
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6mm ID, 250mm length). Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, peptides were combined into 18 fractions and dried by vacuum centrifuging.
LC-MS/MS analysis
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
Database search
The resulting MS/MS data was processed using Maxquant search engine (v.1.5.2.8). Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Mass tolerance for precursor ions was set at 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set at 0.02 Da. Carbamidomethyl of Cys was specified as fixed modification and oxidation on Met was specified as variable modifications. FDR was adjusted to < 1% and a minimum score for peptides was set > 40.
Bioinformatics
Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/). Firstly, Converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan soft would be used to annotated protein’s GO functional based on protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component and molecular function. Identified proteins domain functional description were annotated by InterProScan (a sequence analysis application) based on protein sequence alignment method, and the InterPro domain database was used. InterPro (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information about protein families, domains and functional sites, and makes it freely available to the public via Web-based interfaces and services. Central to the database are diagnostic models, known as signatures, against which protein sequences can be searched to determine their potential function. InterPro has utility in the large-scale analysis of whole genomes and meta-genomes, as well as in characterizing individual protein sequences.
Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathway. Firstly, using KEGG online service tools KAAS to annotate protein’s KEGG database description. Then mapping the annotation result on the KEGG pathway database using KEGG online service tools KEGG mapper. There, we used wolfpsort a subcellular localization predication soft to predict subcellular localization. Wolfpsort is an updated version of PSORT/PSORT II for the prediction of eukaryotic sequences. Special for protokaryon species, Subcellular localization prediction soft CELLO was used.
Proteins were classified by GO annotation into three categories: biological process, cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p-value < 0.05 is considered significant. Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website. For each category proteins, InterPro (a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites) database was researched and a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. Protein domains with a p-value < 0.05 were considered significant.
For further hierarchical clustering based on different protein functional classification (such as: GO, Domain, Pathway, Complex). We first collated all the categories obtained after enrichment along with their P values, and then filtered for those categories which were at least enriched in one of the clusters with P value <0.05. This filtered P value matrix was transformed by the function x = −log10 (P value). Finally these x values were z-transformed for each functional category. These z scores were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. Cluster membership was visualized by a heat map using the “heatmap.2” function from the “gplots” R-package.
All differentially expressed protein database accession or sequence were searched against the STRING database version 10.5 for protein-protein interactions. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confidence score >0.7 (high confidence). Interaction network from STRING was visualized in R package “networkD3”.
Western blotting
Western blotting was performed as described previously [24]. Lung tissues were lysed in complete RIPA buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride [PMSF] and 1× protease inhibitor cocktail [Roche]) and homogenized using a Sonic Dismembrator 100 (Fisher Scientific). The protein concentration of the tissue homogenates was measured using a Bio-Rad Protein Assay, and equal amounts of soluble protein were separated on 10% polyacrylamide gels, transferred onto a nitrocellulose membrane, and followed by routine western blot analysis. Primary antibody: TNF-α (1:2000, ab8348, Abcam, UK), IL-10(1:2000, ab9969, Abcam, UK), IL-1β (1:2000, sc-7884, Santa Cruz Biotechnology, Inc., USA), TGFβ1, Ahsg (1:1000, ab187051, Abcam, UK),Sema7a (1:1000, ab23578, Abcam, UK), Scgb1 (1:1000, ab40873, Abcam, UK), CD11a (1:1000, ab186873, Abcam, UK), Rac2 (1:1000, ab2244, Abcam, UK), PKCα (1:1000, ab32376, Abcam, UK), Mpo (1:1000, ab208670, Abcam, UK), Ncf1 (1:1000, ab795, Abcam, UK), H2-Ab1(1:1000, ab63567, Abcam, UK), Calr (1:1000, ab2907, Abcam, UK), Lbp (1:1000, ab233524, Abcam, UK), Hsp90ab1 (1:1000, ab53497, Abcam, UK) , CD74 (1:1000, ab202844, Abcam, UK), Ltf (1:1000, ab10110, Abcam, UK), Ankrd17 (1:1000, ab85726, Abcam, UK). Secondary antibody was goat anti-mouse secondary antibody (HRP) (1:4000 mouse IgG, ab6789, Abcam, UK), goat anti-rabbit secondary antibody (HRP) (1:4000, ab6721, Abcam, UK) and goat anti-rat secondary antibody (HRP) (1:2000, ab7097, Abcam, UK). Proteins were visualized using a ClarityTM Western ECL Substrate (170-5061; Bio-Rad Laboratories, Inc., USA) and a Tanon 5200 Full automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).
Statistical analysis
Statistical analysis was performed using SPSS 20.0 statistical software (IBM Corp., Armonk, NY, USA). All data were expressed as means ± SEM. Statistical comparisons were made by student’s t test for two groups and a one-way ANOVA test followed by Tukey test for multiple comparisons. Differences were considered significant at p < 0.05 for all analyses.