Western blot analysis

On the 16th day after CFA injection (Fig. 1a), at 30 min after the administration of FMNT in CFA + FMNT and FMNT groups, all mice were anesthetized with 4% isoflurane and then decapitated. Coronary slices (300 μm) of their extracted brains were obtained by Vibratome, and the bilateral BLA were isolated under anatomical microscope. Western blot analysis was performed as detailed in Liu et al. [27]. The BLA sample was dissociated via sonication in RIPA lysis buffer containing phosphatase and protease inhibitors. The protein content of the collected samples was quantified using the BCA Protein Assay Kit. Equal amounts of protein (40 μg) were dispersed on SDS-PAGE gels then electro-transferred to PVDF membranes (Invitrogen). The latter were in turn probed with antibodies after incubation for 1.5 h in 5% non-fat milk. The antibodies used are Anti-β-actin (1:50000; A5316) purchased from Sigma (St. Louis, MO, USA); Anti-Iba-1 (1: 1:1000; ab178847), anti-GluN2B (1:1000; ab65783), anti-phosphorylated GluN2B at the S1303 site (p-GluN2B-S1303; 1:1000; ab81271), anti-GluA1 (1:1000; ab31232), anti-PSD95 (1:1000; ab2723), and anti-GABA_Aα2 (1:1000; ab72445) from Abcam (Cambridge, UK); Anti-GluN2A (1:1000; ab1555), anti-phosphorylated GluA1 at the S845 site (p-GluA1-S845; 1:1000; ab5849), and anti-phosphorylated GluA1 at the S831 site (p-GluA1-S831; 1:1000; ab5847) from Millipore (Billerica, MA, USA); Anti-NF-κB p65 (1:750; AF0874) from Affinity Biosciences (USA); Anti-GABA_Aγ2 (1:500; BS6858) from Bioworld (St. Louis Park, MN, USA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): glial fibrillary acidic protein (GFAP; 1:1000; #3670), anti-phosphorylated GluN2B at the T1472 site (p-GluN2B-T1472; 1:1000; #4208 s), anti-cAMP-response element binding protein (CREB; 1:1000; #9197), and anti-phosphorylated CREB (p-CREB; 1:1000; #9198). The membranes were further incubated in media containing horseradish peroxidase-conjugated secondary antibodies (anti-rabbit/anti-mouse IgG for the primary antibodies, Santa Cruz, CA, USA). All of the chemicals and reagents were commercially available with standard biochemical quality. Densitometric analysis of Western-blot was conducted using a ChemiDoc XRS (Bio-Rad, Hercules, CA) and quantified using Image J software (NIH, Bethesda, Maryland), according to the instructions. For data analysis, the band intensity of each blot was calculated as a ratio, using β-actin as reference. The intensity ratio for the control group was set at 100%, and the intensity ratios of other treatment groups were expressed as relative percentages.

FMNT relieved anxiety-like behaviors in mice injected with CFA. a Schedule showing the experimental procedure. b Representative traces in OF test during a period of 15 min. Behavioral tests were performed on Day 14. c-e In OF test, administration with FMNT (25 mg/kg) for 8 days significantly increased the time (c) and distance (d) spent in the central area but had no effects on the total traveled distance (e). f Representative traces in EPM test during a period of 5 min. g-i FMNT treatment reversed the time spent in open arms (g) and closed arms (h). However, total arm entries had no difference among four groups (i). n = 7 per group. ^*p < 0.05, ^**p < 0.01 vs. control; ^#p < 0.05 vs. CFA