Animals
Sprague Dawley (SD) rats (male; approximately 6 to eight weeks old; 200 ± 20 g) were obtained from the Experimental Animal Center of Air Force Military Medical University (Xi’an, China). The animals were reared under conditions of 25 ± 2°C, light/dark cycle = 12 hours, and ad libitum access to water and food. The animal experiments were carried out acording to The National Institutes of Health guidelines for care and use of Laboratory Animals and were approved by the relevant ethics committee.
Grouping and treatment
Rats (n = 60) were assigned randomly to six groups: Control group, CUMS group, CUMS + Cur (50 mg/kg) group, CUMS + Cur (100 mg/kg) group, CUMS + Fluoxetine (Flu, antidepression drug) (20 mg/kg) group, and CUMS + Glycyrrhizic Acid (Gly, HMGB1 inhibitor) (10 mg/kg) group, with ten rats in each group. Rats in the control group were maintained under normal conditions. Rats in CUMS groups were subjected to CUMS according to the protocol of a previous study [34–36], and were scheduled randomly and changed weekly (Table 1). Cur was dissolved cocoa buffer at different concentrations; the other drugs were dissolved in water. All drugs were administered once per day between 08:00 and 09:00 h. The experimental procedure is shown in Fig. 1A. After a 7-day adaption period, all rats, except the control group, were exposed to CUMS for 28 days. Drugs were administrated to the specific groups and behavior tests were assessed 2 weeks after drug administration (between 09:00 and 12:00 h).
Table 1
Chronic unpredictable mild stress (CUMS)-related stressors
Stressor | Duration |
Restraint stress | 8 hours |
Noise | 4 hours |
Cold water-immersion | 5 mins |
Tail clamp | 10 mins |
Light/dark cycle reversal | 12 hours |
The cage is tilted | 12 hours |
The cage is soiled | 12 hours |
Sucrose preference test (SPT)
A previously described protocol was used to carry out the SPT [37]. Before the test, all rats were acclimated to a 1%, w/v sucrose solution for 24 h as a training period. After 23 h of deprivation of food and water, the rats were fed with water and 1% sucrose solution at the same time. The consumption of water and sucrose solution in the following 1 h were recorded. According to the ratio of sucrose solution consumption to total liquid, the sugar solution preference was obtained.
Forced swimming test (FST)
A previously described protocol was used to carry out the FST, with a minor change [38]. In brief, individually, the rats were forced to swim in a glass cylinder (50 cm height ⋅ 20 cm diameter) containing water (30 cm height, 24 ± 2°C) for ten mins. A trained technician, who was blinded to the groups, recorded the rat’s immobility time during the last 4 mins, which was defined as the time that rats relaxed with no movement except keep their head above water.
Tail suspension test (TST)
A previously described protocol was used to carry out the TST, with a minor change [5]. Rats were suspended individually via their tails using adhesive tape (approximately 50 cm above the floor). The test lasted for 6 mins. The immobility time was recorded during the last 4 mins by a trained technician who was blinded to groups, which was defined as the time that rat relaxed without any struggling movemet.
RNA extraction and qRT-PCR
Total RNA extraction from the hippocampus was performed using the Trizol reagent (Bioteke, Beijing, China) according to the manufacturer’s instructions. Complementary DNA was reverse transcribed using a PrimeScript RT Reagent Kit (Bioteke). Quantitative real-time PCR was performed using a 2 × Power Taq PCR MasterMix (Bioteke) with Actb (β-actin) as the internal control. All primers used in PCR were provided by Sangon Biotech (Shanghai, China) Co., Ltd and listed below:
Hmgb1 (forward: TGA AGA TAT GGC AAA GGC; reverse: GGC GGT ACT CAG AAC AGA); Il1b (forward: TTC AAA TCT CAC AGC AGC AT; reverse: CAC GGG CAA GAC ATA GGT AG); Il6 (forward: AAC TCC ATC TGC CCT TCA; reverse: CTG TTG TGG GTG GTA TCC TC); Tnfa (forward: CGG AAA GCA TGA TCC GAG AT; reverse: AGA CAG AAG AGC GTG GTG GC); Actb (forward: ACG TTG ACA TCC GTA AAG AC; reverse: TAG GAG CCA GGG CAG TAA).
Determination of oxidative stress markers and inflammatory cytokines in the hippocampus
To measure the oxidative stress markers and inflammatory cytokines, rat hippocampi were excised, homogenized in 1.0% KCl, centrifuged at 4°C for 10 min, and the supernatant retained. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of pyruvate dehydrogenase (PDH), superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), IL-1β, IL-6, and TNF-α, following the manufacturer's protocols.
Immunofluorescence staining
After the behavioral tests, intracardial perfusion was used to sacrifice the rats. The hippocampi were excised and a cryostat (CM1800, Leica, Wetzlar, Germany) was used to prepare 20 µm longitudinal sections. Then, according to a previously described method, immunofluorescence staining was carried out [38]. The sections were visualized under a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan). The following primary antibodies were used: rabbit anti-IL-1β (1:100, Bioworld Technology, Bloomington, MN, USA), rabbit anti-HMBG1 (1:100, Bioworld), and rabbit anti-TNF-α (1:200, Bioworld).
Western blotting
Samples of the hippocampus were cut into small pieces and homogenized in icecold Radioimmunoprecipitation assay (RIPA) buffer. The samples were centrifuged and a bicinchoninic acid (BCA) kit (Wanlei Biotechnology, Xi’an, China) was used to determine the protein concentration in the supernatant. The protein samples were subjected to 12% Tris-glycine SDS-PAGE, electrotransferred to polyvinylidene fluoride membranes, blocked, and incubated with antibodies against HMGB1 (1:500, Bioworld), TLR4 (1:500, Bioworld), NF- κB (1:500, Bioworld), phosphorylated (p)-NF-kB (1:500, Biorbyt, Cambridge, UK), and β-actin (1:5000, Bioworld) at 4°C overnight. The membranes were then incubated for 2 h at room temperature with secondary antibodies (1:5000, Bioworld). A ChemiScope 6200 (Clinx Science Instruments, Shanghai, China) captured the signal from the immunoreactive proteins, and the band levels were quantified using the Clinx Image Analysis software.
Statistical analysis
All data are shown as the mean ± the standard error of the mean (SEM). The data were analyzed using one way analysis of variance (ANOVA) together with Student-Newman-Keuls (SNK-q) test in SPSS 22.0 IBM Corp., Armonk, NY, USA). Statistical significance was indicated by P < 0.05.