Mouse macrophage RAW264.7 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) at 37°C in incubator with 5% CO₂. The ET group was cultured as follows: RAW264.7 cells (5×10⁵/ml) were cultured in DMEM containing 10 ng/ml LPS (Sigma-Aldrich; Merck KGaA) at 37°C for 24 h, followed by 100 ng/ml LPS for 4 h at 37°C. The LPS group was cultured as follows: RAW264.7 (5×10⁵/ml) cells were cultured in DMEM with 100 ng/ml LPS for 4 h at 37°C. Subsequently, the RAW264.7 cells of the 2 groups were harvested for analysis.

The concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 in the cell supernatant of the ET and LPS groups was measured using ELISA kits (TNF-α, cat. no. EK0527; IL-6, cat. no EK0411; IL-10, cat. no. EK04167; Wuhan Boster Biological Technology, Ltd.) according to the manufacturer's instructions. The assay was performed in triplicate, and the absorbance in each well was measured with a microplate reader at 450 nm.

RAW264.7 cells in the ET and LPS groups were treated with 100 ng/ml LPS for 4 or 24 h as aforementioned, then washed twice with PBS and centrifuged at 1,000 × g for 3 min at room temperature. The cell precipitate was resuspended in PBS and adjusted to a concentration of 1×10⁶/ml. Cells were stained with fluorescein isothiocyanate tagged monoclonal anti-CD-16/32 kit (cat. no. 561728, BD Biosciences Pharmingen), diluted with PBS, for 30 min at room temperature in the dark in accordance with the manufacturer's protocol, then washed two times with PBS and resuspended in PBS. Analysis was performed using the BD FACSVerse system flow cytometer and BD FACSuite version 1.0 software (BD Biosciences).

Lysis buffer containing 8 M carbamide (Gibco; Thermo Fisher Scientific, Inc.), 30 mM HEPES (Wuhan Boster Biological Technology, Ltd.), 1 mM PMSF (Amresco, LLC), 2 mM EDTA (Amresco, LLC), and 10 mM DTT (Promega Corporation) was added to cells and centrifuged at 20,000 × g and 4°C for 30 min to collect the supernatant. Protein concentrations were determined using the Bradford method. For digestion, each sample was reduced with 10 mM dithiothreitol for 60 min at 56°C and alkylated using 55 mM iodoacetamide for 60 min at room temperature in the dark. The proteins were digested with 1 µg/µl trypsin at a weight ratio of 1:30 (trypsin:protein) overnight at 37°C. Tryptic peptides were lyophilized and resuspended in 0.5 M Triethylammonium bicarbonate. Following trypsin digestion, each iTRAQ reagent was dissolved in isopropanol and added to the appropriate peptide mixture. A total of 3 biological replicates of the ET group were labelled with iTRAQ tags 113, 114 and 115, respectively. Similarly, 3 biological replicates of the LPS group were labelled with iTRAQ tags 118, 117 and 116, respectively. The labelled peptide mixtures were incubated at room temperature for 2 h and obtained by vacuum-drying. Then, peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA), and analyzed using a mass spectrometer (TripleTOF 6600; SCIEX, Framingham, MA, USA). The instrument parameters were as follows: Mass range, 350–2,000 m/z for time-of-flight mass spectrometry (TOF MS) and 100–1,500 m/z for TOF MS/MS; dynamic exclusion, 12.0 sec. Mass spectra raw data were analyzed with ProteinPilotä software (version 5.0; SCIEX). The parameters for the analysis were set as follows: Cys alkylation, iodoacetamide; digestion, trypsin; species, Mus musculus. Peptides were identified using a false discovery rate of <1%. Proteins were considered differentially expressed if they differed in at least 2 of the 3 biological replicates. The criteria of P<0.05 and fold change (ET/LPS)>1.2 or <0.833 were selected to identify up- and down-regulated proteins.

Functional enrichment analysis for the differentially expressed proteins was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID version 6.7; david.ncifcrf.gov) for Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/kegg/) pathway analysis. P<0.05 was selected as the cut-off criterion. A protein-protein interaction network was constructed using the inBio Map database (www.intomics.com/inbio/map). Key genes located in the center of the network were subsequently verified.

Cell lysates were obtained using a lysis buffer containing phosphatase inhibitor and the protease inhibitor phenylmethanesulfonyl fluoride (100 mM; cat. no. KGP250; Nanjing KeyGen Biotech Co., Ltd.) and the protein concentration in the lysates determined by Bradford assay. A total of 100 µg lysate was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane at 250 mA for 1 h. The PVDF membranes were blocked with 5% FBS for 1.5 h at 37°C and incubated overnight at 4°C with rabbit anti-mouse antibodies against high mobility group (HMG)A1 (1:10,000; cat. no. ab129153; Abcam), HMGA2 (1:1,000; cat. no. D1A7; Cell Signaling Technology, Inc.), HMGB1 (1:10,000; cat. no. ab79823; Abcam), HMGB2 (1:10,000; cat. no. ab124670; Abcam) and β-actin (1:1,000; cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.). The PVDF membranes were washed three times with TBST (0.1% Tween) and incubated with goat anti-rabbit Immunoglobulin G-HRP (1:1,000; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) for 1.5 h at 37°C, followed by the 3,3′-diaminobenzidine (EMD Millipore) method at room temperature for 15 sec. PVDF membranes were subjected to densitometry analysis (chemiDox.XRS+; Bio-Rad Laboratories, Inc.), then the image was analyzed using Quantity One 4.0 software (Bio-Rad Laboratories, Inc.).

Data are expressed as the mean ± standard error of the mean. SPSS version 21.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. Statistical analysis was performed using an independent sample Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To determine the concentrations of cytokines in the ET and LPS groups, the supernatants of the cells were analyzed by ELISA. As indicated in Fig. 1A and B, TNF-α and IL-6 were significantly downregulated in the ET group when compared with the LPS group (P<0.05). The expression of IL-10 in the ET group was higher when compared with in the LPS group, but the difference was not statistically significant (P>0.05; Fig. 1C). These results indicated that the ET model had been successfully constructed.

CD16/32 is the cell surface marker that is induced in M1-polarized cells. Based on the theory that ET skews cell polarization into an M2-like phenotype, and the number of M1-polarized cells will not continue to increase (8), cell surface marker analysis was performed to determine the expression level of CD16/32 by flow cytometry. As shown in Fig. 2, following stimulation with high-dose LPS for 4 h, CD16/32 was induced in the ET group compared with the LPS group or untreated sample (P<0.05; Fig. 2C and D), but following stimulation with LPS for 24 h, significant downregulation of this marker was observed in the ET group compared with the LPS group (P<0.05; Fig. 2E and F). The results indicated that there were more M1-polarized cells in the ET group compared with in the LPS group at the early stage (4 h), and the ET model was successfully constructed.

A total of 3 biological replicates were performed for the ET and LPS groups (n=3). A total of 235 different proteins were identified by iTRAQ, and there were 63 differentially expressed proteins identified in at least 2 of the experiments. Of the 63 differentially expressed proteins, 36 upregulated proteins with >1.2-fold difference and 27 downregulated proteins with <0.833-fold difference were selected between the ET and LPS groups (Fig. 3). Specific upregulated proteins are presented in Table I and downregulated proteins are presented in Table II.

The 63 differentially expressed proteins were analyzed by DAVID to study their biological processes, molecular functions and cellular component. The biological processes of these proteins were mainly involved in the growth and development of organ tissues, the regulation of biological quality and responses to external stimuli (Fig. 4). The molecular functions analysis demonstrated that these proteins were mainly involved in ‘oxidoreductase activation’, ‘receptor binding’, ‘DNA binding’ and ‘antioxidant activity’ (Fig. 5). In the cellular component analysis, the differentially expressed proteins were mainly located in ‘chromatin’, the ‘extracellular space’, ‘membrane-bounded vesicles’ and ‘cytoplasmic vesicles’ (Fig. 6). According to KEGG pathway analysis, the 63 differentially expressed proteins were involved in 27 signaling pathways (P<0.05). The top 10 signaling pathways included TLR signaling pathway, nuclear factor (NF)-κB signaling pathway, TNF signaling pathway and antigen presentation processes (Table III).

The inBio Map database was used to construct a protein-protein interaction network (Fig. 7). From the 63 differential proteins, 4 key proteins were located at the core of network. According to the bioinformatics analysis, HMGA1/2 and HMGB1/2 were selected for further study.

To validate the 4 key proteins, western blot analysis was performed. The expression of HMGA1 and HMGA2 in the ET group was higher compared with the LPS group at 4 h following high-dose LPS stimulation, while HMGB1 and HMGB2 exhibited the opposite trend in expression under the same conditions (Fig. 8). These results were consistent with the findings from iTRAQ analysis.