Animals and treatment
Male C57BL/6 mice aged 6–8 weeks were used in this study. The animals were housed in random groups of six per cage, with food and water available ad libitum. The animals were maintained at a temperature of 24 ± 2 °C, a relative humidity of 50–60%, and under a 12-h light-dark cycle. All mice were given a commercial chow diet and allowed to adapt to the laboratory environment for at least 1 week before the experiments.
Anxiety-like behaviors were induced by an intraplantar injection of CFA (10 μL, 50% in saline, Sigma, St. Louis, MO, USA) into the plantar surface of the left hind paw of the mice. The same volume of saline (0.9%) was injected into the plantar surface of the left hind paw of control mice. SP and flumazenil (purity > 98%, Shanghai Pure One Biotechnology, China) were dissolved in 0.9% saline containing 1% dimethyl sulfoxide (DMSO). The doses of SP and flumazenil used in this study were based on previous studies [20, 21] and our preliminary tests. Animals were randomly distributed into five groups: a control group, a vehicle group, and three groups that received different doses of SP (2.0, 10.0, 50.0 mg/kg). Each group contained 12 mice. The SP groups were given intraperitoneal injections of SP after the CFA injection once a day for 14 consecutive days. The vehicle group was injected with an equal volume of 0.9% saline containing 1% DMSO at the same time. We also divided an additional cohort of mice into the following groups: control, vehicle, SP (50.0 mg/kg), and SP + flumazenil (10.0 mg/kg). Each group contained 12 mice, and the administration method was identical to the procedure described above.
Behavioral tests
All mice were subjected to the open field test (OFT) and the elevated plus maze (EPM) test, which were conducted as described in previous reports [22]. Each mouse was brought into the testing room 2 h before the tests. Mice were administered once 30 min before the behavioral test. The OFT was always performed before the EPM, but both tests were conducted on the same day.
OFT
The open field (JLBehv-LAM-4, Shanghai Jiliang Software, China) was a square arena (30 × 30 × 30 cm3) with clear Plexiglas walls and floor, and was placed inside an isolation chamber with dim illumination and a fan. For the testing, each mouse was placed in the center of the box and allowed to freely explore for 15 min. The exploratory behaviors of the mice were recorded using a camera fixed above the chamber. The total distance traveled and time spent in the central area were analyzed using a video-tracking system (MedAssociates, St. Albans, VT, USA).
EPM
The apparatus (DigBehv-EPMG, Shanghai Jiliang Software) consisted of two open arms (25 × 8 × 0.5 cm3) and two closed arms (25 × 8 × 12 cm3) that extended from a common central zone (8 × 8 cm2). The mice were exposed to gentle handling two times to eliminate nervousness. For each test, the individual mouse was placed in the central zone facing an open arm, and allowed to explore freely for 5 min while being videotaped using a camera fixed above the maze. The time spent in and the number of entries into the open and closed arms were analyzed with a video-tracking system (MedAssociates).
Enzyme-linked immunosorbent assay (ELISA)
Blood samples were obtained by eyeball extraction after the behavioral tests. Levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the plasma were evaluated using ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) following the manufacturer’s instructions.
Western blot analysis
The animals were sacrificed, and the tissue samples from the bilateral BLA amygdala were dissected from brain slices under an anatomical microscope immediately after the behavioral tests. Western blot analysis was performed as previously described [23]. BLA samples were homogenized in ice-cold RIPA lysis buffer containing phosphatase and protease inhibitors. The protein content of the collected samples was quantified using the BCA protein assay. Equal amounts of protein (30 μg) were analyzed using an SDS-PAGE gel and then electro-transferred onto PVDF membranes (Invitrogen, Carlsbad, CA, USA). The following primary antibodies were used: β-actin (1:10000, Sigma), TNF-α (1:500, Abcam, Cambridge, UK), IL-6 (1:500, Abcam), IL-1β (1:500, Abcam), GluA1 (1:1000, Abcam), GluN2A (1:1000, Abcam), GluN2B (1:1000, Abcam), PSD95 (1:1000, Abcam), GABA-T (1:1000, Abcam), GABAA α2 (1:1000, Abcam), GABAA γ2 (1:1000, Abcam), p-p38 (1:1000, Cell Signaling Technology, Danvers, MA, USA), p38 (1:1000, Cell Signaling Technology), p-JNK (1:1000, Cell Signaling Technology), JNK (1:1000, Cell Signaling Technology), NF-κB p65 (1:1000, Cell Signaling Technology). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit/anti-mouse IgG); densitometric western blot analysis was conducted using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ software (NIH, Bethesda, MD, USA). For data analysis, the band intensity of each blot was calculated as a ratio relative to that of β-actin. The intensity ratio of the control group was set at 100%, and the intensity for other treatment groups was expressed as percentages relative to the control group.
Immunofluorescence staining
After the behavioral studies, the mice were anesthetized using pentobarbital sodium and perfused with sterile saline, followed by 4% polyformaldehyde. The separated brains were dehydrated with a sucrose gradient, 20 and 30% (w/v) sucrose in 0.1 M phosphate buffered saline (PBS) at 4 °C overnight, respectively. 20 μm-thick BLA sections were cut on a cryostat (Leica Microsystems). All sections were washed with 0.3% Triton X-100 PBS and blocked (10% goat serum, 0.1% Triton X-100 in PBS) for 2 h at 4 °C. Then, the slices were incubated with goat anti-Iba1 (1:1000, Abcam) in blocking solution overnight at 4 °C, followed by incubation with mouse anti-rabbit IgG Alexa Fluor 594 (1:200, Invitrogen) and mouse anti-goat IgG (1:200, Invitrogen) for 2 h at room temperature. All antibodies were diluted in PBS with 0.1% Triton X-100 and 2% bovine serum albumin. Nuclei were counterstained using Hoechst 33258. Slices were then coverslipped with 50% glycerin, and stained samples were photographed and analyzed using a FluoView FV1000 microscope (Olympus, Tokyo, Japan).
Determination of glutamate and GABA levels
Mice were anesthetized with sodium pentobarbital and mounted on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). A microdialysis probe (CMA7 model, Carnegie Medicine, Stockholm, Sweden) was implanted in the left BLA nucleus (coordinates: − 1.45 mm anterior to the bregma, ± 2.5 mm lateral from the midline, and 4.3 mm beneath the surface of the skull). On the day of the experiment, the probe was perfused with artificial cerebrospinal fluid at a flow rate of 1.5 μL/min using a CMA/100 pump (Carnegie Medicine, Stockholm, Sweden). After the probe trial of the behavioral tests, following a 30-min equilibration period, dialysate samples were collected every 15 min for 60 min and stored immediately at − 80 °C. Only mice with correctly implanted probes were included in the data analysis.
Levels of glutamate and GABA in the BLA were detected by reverse-phase high-performance liquid chromatography (HPLC, Agilent Technologies 1260 Infinity, Santa Clara, CA, USA) according to previously reported methods [24, 25]. 2, 4- dinitrofluorobenzene (DNFB) was used for pre-column derivatization. Microdialysate samples (50 μL) were mixed with 50 μL 0.5 mol/L NaHCO3 solution and 100 μL DNFB for 1 h at 60 °C. Then, 300 μL phosphate buffer (pH 7.0) was added to stop the reaction. The resulting products were analyzed using a UV detector at an absorbance of 360 nm. The mobile phase was KH2PO3 buffer (0.05 mol/L, pH 6.0)-acetonitrile-H2O (84:8:8, v/v/v) at a flow rate of 1.0 mL/min. A Thermo TC-C18 column (4.6 × 250 mm2; particle size: 5 mm) was used. The concentrations were calculated using LCsolution software (Shimadzu, Kyoto, Japan) based on standard samples.
Molecular docking analysis
Docking analyses of SP with GABA-T (PDB code: 1OHW), GABAA receptor (GABAAR) (PDB code: 6HUP), N-methyl-D-aspartate (NMDA) receptor (NMDAR) (PDB code: 4PE5), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) (PDB code: 6QKC) were performed using the Glide module of Maestro 11.9 [26, 27]. All protein structures were downloaded from the Protein Data Bank (http://www.rcsb.org) and prepared using the Protein Preparation Wizard Workflow in the Schrodinger suite [26]. This involved the addition of hydrogen atoms to the protein, the assignment of bond orders, and the deletion of unnecessary water molecules. Moreover, H-bonds were optimized, and in the end, restrained minimization was performed wherein the heavy atoms were converged to root mean square deviation 0.3 Å. SP and the original crystal ligands were sketched in a 3D format and prepared for docking using the Ligand Preparation Application in the Schrodinger suite. The Receptor Grid Generation Workflow was used to define a grid around the bound co-crystallized ligand, and the grid was then used for docking SP in the ligand-binding site. Extra Precision (XP) mode was used for the docking analyses. For the validation of the docking parameters, the co-crystal ligand was re-docked at the catalytic site of the protein. The ligand interaction tool was used to view the interaction diagram of the ligands with the residues at the active site of the target protein.
Statistical analysis
Results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis of multiple groups was performed using one-way analysis of variance (ANOVA) in Microsoft Excel and Prism (GraphPad, San Diego, CA, USA). In all cases, p < 0.05 was considered to be statistically significant.