Animals
SD rats (Male, 180-220g; Beijing Vitonlihua Experimental Animal Technology Co. Ltd, Beijing, China) were initially housed in groups in a temperature-controlled environment under a 12/12 h light/dark cycle. Food and water were freely available in the whole experiment except for rats kept under a deprivation procedure. This study was approved by the Animal Care and Use Committee of the eighth affiliated hospital of Sun Yat-sen University. All experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals (Chinese Council).
Drug and treatment
Rats were randomly assigned to four groups (n=7): control, CUMS, CUMS + CUR, CUMS + CUR + SR18292 (PGC-1α inhibitor). The CUR groups received daily gavage of 100 mg/kg CUR (suspended in 0.5% Tween 80, purchased from Sigma Chemical Co., USA) for 6weeks at 60 min prior to CUMS. Rats received SR18292 (dissolved in DMSO, purchased from Macklin, Shanghai, China) via intraperitoneal injection at a dose of 40mg/kg every day in the last week for a total of seven injections. The doses of CUR and SR18292 were based on previous studies [15,16].
At the end of six weeks, behavioral tests were carried out, and the rats were sacrificed under anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). Blood samples and the hippocampus were collected in our study.
CUMS procedure
Rats in the control group were housed in groups of 3-4 per cage in a separate room while rats in the CUMS groups were housed individually and received random unpredictable stress for 6 consecutive weeks. Stress stimuli included: cage tilting for 24 h; damp bedding for 24 h; fasting for 24 h; water deprivation for 24 h, finally with 1 h an empty bottle; light–dark-cycle reversal (12 h/12 h), behavior restriction for 2 h; 30 min noise, 5 min tail pinch. Rats received one of these stressors per day and same stressor was not applied in 2 consecutive days.
Sucrose preference test (SPT)
The SPT was performed as our previous study [15]. Before the SPT test, all the rats were housed individually and provided two bottles containing 1% sucrose solution for 48h to habituate them to the taste of sucrose. After 14h of water deprivation, two preweighted bottles with one containing 1% sucrose solution and another containing tap water were given to each rat. Then after 1h, the bottles were weighed again, and the weight difference in each bottle was considered the rat intake. The sucrose preference was measured as a percentage of the consumed 1% sucrose solution relative to the total amount of liquid intake.
Open-field test (OFT)
The test was performed in a square arena consisted of a 76×76 cm gray wooden box with 42 cm high boundary walls with the floor divided into 25 equal squares by black lines. Each rat was placed into the center of the open field and allowed to move freely for 5min. The apparatus was cleaned with ethanol and water prior to each test session to remove olfactory cues. The number of crossing and rearing was recorded by the observer blind to the treatment condition of the animal to assess locomotor activity and exploratory behavior.
Forced Swimming Test (FST)
The FST was performed as previously described [15]. Before the FST test, each rat was placed in a plastic drum (45 cm height, 25 cm diameter) containing approximately 35 cm of water (24 ± 1 °C) for a 15-min pretest. After swimming, rats were dried with towels and placed back in their home cage. After 24h, the rats were exposed to the same experimental conditions outlined above for a 5-min FST. Water was changed before each trial. Immobility time was scored by an experienced observer blind to the experiment design and was defined as floating passively and only making slight movements to keep the head above water.
Novelty-Suppressed Feeding Test (NSFT)
The SPT was performed as our previous study [15]. Before NSFT, all the rats were food-deprived for 24 h in their home cages. A small amount of food was placed on a piece of white paper (10 × 10 cm) in the center of an open field (75 × 75 × 40 cm). The rats were allowed to explore the open field for 8 min. The latency time was recorded in our study, defined as the time it took for each rat to approach and take the first bite of the food. Immediately afterwards, the animals were transferred to their home cages and were provided the same amount of food as in the open field. Total food intake for the next 5 min in each cage was weighed to avoid the influence of the animals’ appetite.
Western blotting analysis
Total protein was prepared from the hippocampus and the Bradford method was used to determine the protein concentration. Samples were loaded on a precast 12% SDS-PAGE gels with 50 μg of protein in each lane. The proteins in the gels were transferred to a PVDF membrane and then blocked for 1 h in 5% nonfat dry milk in TBS-T (25 mM Tris, 150 mM NaCl, pH 7.5, 0.05% Tween-20). The following antibodies and concentrations were used overnight at a temperature of 4 °C: PGC-1α (ab106814, 1:1000, Abcam), ERRα (#13826, 1:1000, Cell Signaling Technology), FNDC5 (23995-1-AP, 1:1000; Proteintech), BDNF (28205-1-AP 1:1000; Proteintech), β-actin (8046S, 1:1000, Cell Signaling Technology). The membrane was then probed with an HRP conjugated secondary antibody for 40 min. Finally, the film signal was digitally scanned and quantified using Image-Pro Plus 6.0, and values were normalized to β-actin as an internal standard.
Quantitative real-time PCR (qPCR)
Total RNA was isolated from the hippocampal homogenates using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. The mRNA expression of PGC-1α, ERRα, FNDC5, BDNF, Bax, Bcl-xl and Bcl-2 was detected. Quantitative real-time PCR was performed on a Bio-Rad Cx96 Detection System (Biorad, USA) using an SYBR green PCR kit (Applied Biosystems, USA) and gene-specific primers. Oligonucleotide primers specific for rats are listed in Table 1. The 5 ng cDNA samples received 40 cycles of amplification. Each cDNA was tested in triplicate. Relative mRNA expression levels were normalized to β-actin as an internal standard.
Table 1: Primer sequences used for the qPCR analysis.
Gene
|
Sense primer (5′–3′)
|
Antisense primer (5–3′)
|
Amplicon length (bp)
|
PGC-1α
|
5’-GAACCATGCAAACCACACCC-3’
|
5’-GGAGGGTCATCGTTTGTGGT-3’
|
162
|
ERRα
|
5’-AACCTGAGAAGCTGTACGCC-3’
|
5’-CCATCCACACACTCTGCAGT-3’
|
186
|
FNDC5
|
5’-TATATCGTCCACGTGCAGGC-3’
|
5’-ACGACGATGATCAGCACCTC-3’
|
179
|
BDNF
|
5’-TACCTGGATGCCGCAAACAT-3’
|
5’-CGACATGTCCACTGCAGTCT-3’
|
135
|
Bax
|
5’-GAACCATCATGGGCTGGACA-3’
|
5’-GTGAGTGAGGCAGTGAGGAC-3’
|
157
|
Bcl-xl
|
5’-AGGCTGGCGATGAGTTTGAA-3’
|
5’-AGAAGAAGGCCACAATGCGA-3’
|
159
|
Bcl-2
|
5’-GAACTGGGGGAGGATTGTGG-3’
|
5’-CATCCCAGCCTCCGTTATCC-3’
|
164
|
β-Actin
|
5’-CCACCATGTACCCAGGCATT-3’
|
5’-CGGACTCATCGTACTCCTGC-3’
|
189
|
Nissl staining
The Nissl staining was performed as previously described [26]. Formaldehyde-fixed specimens were embedded in paraffin and cut into 4-μm-thick sections that were deparaffinized with xylene and rehydrated in a graded series of alcohol. Samples were treated with Nissl staining solution for 5 min. Damaged neurons were shrunken or contained vacuoles. Normal neurons had a relatively large, full soma, and round, large nuclei. We calculate the average intensities or cell counts from the same sections in seven rats per group with Image-Pro Plus 7.0. Investigators were blinded to the experimental groups.
Immunofluorescence analysis
The Immunofluorescence analysis was performed according to the previous study [9]. Formaldehyde-fixed specimens were embedded in an optimal cutting temperature compound (SAKURA, USA), and cut in sections using a microtome. After washing three times using PBS, the tissues were blocked with 10% goat serum (Solarbio, China) and 0.3% Triton X-100 (Solarbio, China) in PBS at 37°C for 2 h. Then the tissue were incubated with primary antibodies (PGC-1α, ab106814, 1:300, Abcam) overnight at 4°C. After washing with PBS, the tissues were incubated with secondary antibodies (4412, 1:1000, Cell Signaling Technology) for 1 h and with 4′,6-diamidino-2-phenylindole (Solarbio, China) for 5 min. Fluorescence was observed using a fluorescence microscope. The results were analyzed using Image-Pro Plus software. Investigators were blinded to the experimental groups.
Bromodeoxyuridine treatment
BrdU (100 mg/kg) was injected intraperitoneally once daily for 3 consecutive days before the brain slice collection. After washing in 0.1M borate buffer (pH=8.5) for 30 min, the thirty-μm-thick coronal sections containing dentate gyrus (DG) were collected and pretreated with HCl at 37 °C for 30 min, and then incubated with 3% BSA for 1 h. Then the sections were incubated with the antibody for BrdU, followed by Alexa Fluor secondary antibody. Photomicrographs were obtained with a FluoView FV1000 microscope.
Statistical Analysis
All statistical procedures were performed using Statistical Package for the Social Science (SPSS) 24.0. Data were expressed as mean±SD. All the data were analyzed statistically by one-way analysis of variance (ANOVA) with Tukey post hoc multiple comparisons. P<0.05 was considered as statistically significant.