Male CD-1 mice (n=48; weight, 35–40 g; age, 10–14 weeks) were obtained from the animal facility at the Chongqing Medical University (Chongqing, China). The animals were housed under standard laboratory conditions (12 h light/dark cycle; temperature, 23±1°C; humidity, 45–55%), in a single cage with a shelter, and food and water was available ad libitum. The present study was approved by the Ethics Committee of the Chongqing Medical University and all experiments were conducted in accordance with the National Institutes of Health (Bethesda, MD, USA) Guidelines for Animal Research (Guide for the Care and Use of Laboratory Animals).

The mice were randomly divided into three groups: A control group (Con; n=16) treated with 1 mg/kg sterile saline, an LPS-induced acute inflammatory reaction group (AIR; n=16) treated with 0.83 mg/kg LPS, and an LPS-induced depressive-like behavior group (Dep; n=16) treated with 0.83 mg/kg LPS. For the AIR and Dep mice, LPS (L-3129, serotype 055:B5; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline and intraperitoneally injected at a dose of 0.83 mg/kg to induce acute inflammatory reaction (11) and depressive-like behavior (12).

Prior to experimentation, the mice were placed in the testing room for 30 min to allow adaptation. The animals were then individually placed in the center of the open field area (44.5×44.5×45 cm) and after 30 sec adaptation, a 6 min period of free exploration was recorded using a Sony DCR-SR45E digital camera (Sony, Tokyo, Japan). The total distance was recorded during the last 5 min. Following each trial, the apparatus was wiped and cleaned with 80% alcohol. The videos of the test were analyzed using a Smart video-tracking system (PanLab Harvard Apparatus, Holliston, MA, USA).

An FST was conducted as previously described (13) with minor modifications. Briefly, the mice were individually placed into transparent glass cylinders (diameter, 15 cm; height, 30 cm) containing 15 cm of water at 23±1°C. Each mouse was required to swim for 6 min, and the total duration of immobility was recorded during the last 5 min of testing. Immobility was defined as the absence of active, escape-oriented behaviors, such as swimming, jumping, rearing, sniffing, or diving (14). The water was replaced with fresh water between each test. Following completion of each test, the mouse was gently removed from the cylinder, dried, and returned to its cage.

The TST is one of the most widely-used models for assessing antidepressant-like activity in mice. The test was conducted as previously described (15) with minor modifications. Briefly, each mouse was suspended by its tail using adhesive tape placed 2 cm from the tip of the tail in an acoustically and visually-isolated suspension box (22×21×33 cm) for 6 min, and the duration of immobility was recorded during the last 5 min of testing. The mice were regarded as immobile only when they hung passively or stayed completely motionless.

Our previous studies established a highly effective protein sample preparation protocol (16,17). Briefly, 48 mice (n=16) were decapitated under 100% diethyl ether [Chongqing Chuandong Chemical (Group) Co., Ltd., Chongqing, China] anesthesia. PFC tissue samples were rapidly dissected from the brain and subsequently frozen in liquid nitrogen. The tissue samples of each experimental group were stored at −80°C until use.

The PFC tissue samples from each experimental group (n=10) were removed from the liquid nitrogen and suspended in 2 ml acetone solution supplemented with 0.2% (w/v) dithiothreitol (DTT) and 10% (w/v) trichloroacetic acid. Following tissue homogenization (Eppendorf, Hamburg, Germany), the cell suspension was incubated at −20°C overnight prior to centrifugation at 35,000 × g for 30 min at 4°C. The supernatant was decanted, and the cell pellet was suspended in 2 ml pre-cooled acetone solution supplemented with 0.2% (w/v) DTT, and incubated at −20°C for 1 h, prior to being centrifuged at 35,000 × g for 30 min at 4°C. The supernatant was then decanted, and the cell pellet was dried in a fuming cupboard. Each sample was dissolved in 2 ml 2-D lysis buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 65 mM DTT, 0.3 mg/ml EDTA, 35 µg/ml phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin, 0.5 µg/ml leupeptin, and 0.5% v/v CA, and centrifuged at 40,000 × g for 60 min at 15°C. The protein concentration was calculated using a Bradford Protein Assay kit (Bio-Rad Laboratories, Inc.). Bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) was used as a standard control.

2-DE was conducted as previously described (18). Isoelectric focusing (IEF) was performed using 17 cm ReadyStrip™ IPG Strips (pH 3–10; Bio-Rad Laboratories, Inc.), and 350 µl rehydration buffer containing 0.1 mg protein were loaded onto the strips. Each strip was rehydrated for 12 h (at 30 V) using 350 µl rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.2% Bio-Lyte, and 0.001% bromophenol blue (Bio-Rad Laboratories, Inc.) (19). IEF was performed on a Protean IEF cell (Bio-Rad Laboratories, Inc.) as follows: 50 V for 12 h, 250 V for 30 min, 1,000 V for 1 h, 1,000–10,000 V over a 5 h step-up period, and 10,000 V for 6 h. Following IEF, the IPG strips were equilibrated in the reduction buffer containing 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% glycerol, and 2% SDS. The first equilibration was performed using equilibration buffer supplemented with 2% DTT, and then the second equilibration was performed using equilibration buffer supplemented with 2.5% indole-3-acetic acid. The second dimension was performed by running the strips on 1-mm thick 10% SDS-PAGE using a Protean^® IIG xi Multi-Cell (Bio-Rad Laboratories, Inc.). Six gels (three gels per group) were simultaneously run at 12.5 mA/gel for 30 min and then 25 mA/gel for 5.0–5.5 h at 20°C. The protein spots were visualized by silver staining as previously described by Yan et al (20).

Following visualization, images were scanned using an Epson 10000XL scanner (Epson Co., Ltd. Beijing, China) at an optical resolution of 300 dpi. Image analysis and spot detection were conducted using PDQuest 8.0.1 (Bio-Rad Laboratories, Inc.) with Gaussian spot modeling. For quantitative comparison of the spots across the gels, replicate images of the gels were created. To correct for the variability in silver staining, the individual spot volumes were normalized by dividing the optical density (OD) of each spot value by the sum the total OD of all spots in the respective gels. This method controlled for differences in sample loading and color intensities among the gels. Automated and manual spot matching were also performed. Integrated intensities demonstrating a ≥1.5-fold change were applied to determine the statistical differences in protein expression between the two groups (17).

Protein samples were separated by 2-DE and visualized with silver staining. In-gel protein digestion was performed as previously described by Zhou et al (21) with minor modifications. The protein spots of interest were excised from the gels and destained. Following reduction and alkylation, the gel sections were digested overnight with Sequencing Grade Modified Trypsin (Promega Corporation, Madison, WI, USA). The digested peptides were extracted using 100 µl 60% CAN (Merck Millipore, Darmstadt, Germany) supplemented with 0.1% TFA (Merck Millipore) and concentrated in a SpeedVac^® vacuum concentrator (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The peptides were re-dissolved in a matrix solution and spotted on a MALDI target plate (Applied Biosystems Life Technologies, Foster City, CA, USA). The peptides were then analyzed using the 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems Life Technologies) in default mode.

To investigate the differential protein expression in the PFC, two proteins, creatine kinase B-type (CKB) and dihydropyrimidinase-related protein 3 (DPYSL3) were selected for western blotting. β-tubulin was used as a loading control (1:1,000). The PFC samples from each group (n=6) were homogenized using a standard lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin (Beyotime Institute of Biotechnology, Haimen, China), prior to protein extraction. A bicinchoninic acid assay was used to analyze the protein concentrations. A total of 30 µg protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked using 5% (w/v) skimmed milk for 1 h at room temperature, and then incubated overnight at 4°C with anti-CKB rabbit monoclonal antibody (1:3,000; cat. no. ab92452; Abcam, Cambridge, UK) and anti-DPYSL3 rabbit monoclonal antibody (1:1,000; cat. no. ab126787; Abcam). The membranes (Merck Millipore) were washed in Tris-buffered saline with 0.05% Tween 20 containing 150 mM NaCl, 0.05% Tween 20, and 10 mM Tris-HCl (pH 7.5), and incubated with their respective horseradish peroxidase-conjugated secondary antibodies (1:10,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 2 h at room temperature. The membranes were visualized using enhanced chemiluminescence and exposed to autoradiography films. Band intensity was determined using Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc.).

Statistical analyses were conducted using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). The results from the western blotting were analyzed using a one-way analysis of variance followed by a Bonferroni's test. Body weight and behavioral test results were compared using the Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.

LPS-induced changes in body weight and mobility were measured 6 and 24 h post-injection by assessing changes in body weight loss and OFT (22). The weights of the Dep mice were significantly reduced, as compared with their weights prior to injection (Fig. 1A), and the AIR mice exhibited a significant decrease in the total distance travelled in the OFT test, as compared with the control group (Fig. 1B).

The Dep mice exhibited a significant increase in immobility in the TST and FST conducted 24 h post-LPS injection (Fig. 1C and D). This was the time point when typical acute sickness behavior was no longer apparent (22). These results suggest that acute activation of the peripheral innate immune system by LPS induces depressive-like symptoms in mice that are not biased by acute sickness behaviors.

Differentially expressed protein identification by 2-DE and MALDI-TOF-MS/MS were conducted to examine whether PFC protein expression was altered by LPS. Wide-range pH 3–10 strips were used to investigate the differential protein expression in the AIR and Dep mice, as compared with the Con mice. A total of ~1,780 protein spots were visualized by silver staining (Fig. 2). To identify the differential proteins in the 2-DE gels, a 1.5-fold change was used as a threshold (Table I). The functional classification of differentially expressed proteins was energy metabolism, neurogenesis, cytoskeleton, signal transducer, redox homeostasis, nuclein metabolism and molecular chaperones (Table I).

CKB and DPYSL3 were selected for analysis by western blot analysis. The expression levels of CKB and DPYSL3 were significantly increased in the Dep mice, as compared with the Con and AIR mice (Fig. 3).