Compounds and Drugs.

PEA (chemical structure is shown in Fig. 1; synthesized at the College of Pharmacy, Yanbian University, China) or fluoxetine (Enhua, Jiangsu, China) was suspended in 0.3% methyl cellulose (Loba–Chemie, Shanghai, China) and administered daily via gavage. MK886 (Han Xiang, Shanghai, China) was dissolved in sterile saline and administered daily via an intraperitoneal injection. All doses are presented as milligrams per kilogram body weight.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Structure of N-palmitoylethanolamide (PEA).

Animals.

Experimental procedures were approved by the Institutional Animal Care and Use Committee of China and were conducted in accordance with the Yanbian University of Health Guide for Care and Use of Laboratory Animals. Male Sprague-Dawley rats (180–200 g; the Experimental Animal Center, Yanbian University, Jilin, China) were housed individually in cages for 3 days to allow adaptation to the environment under the following controlled conditions: 12-hour light/12-hour dark cycle, free access to tap water and food pellets, and ambient temperature and relative humidity at 22 ± 2°C and 55% ± 5%, respectively.

Experimental Procedures.

All animals were randomly divided into one of the following two groups: a CUMS-treated group and a normal control (NC) group. The CUMS-treated rats were subjected to a series of variable stressors described by Jin et al. (2015). The animals were housed individually (330 × 215 × 200 mm) and subjected to the following stressors: 1) horizontal shock for 5 minutes (one shock/s); 2) swimming in 4°C water for 5 minutes; 3) swimming in 45°C water for 5 minutes; 4) food deprivation for 24 hours; 5) water deprivation for 24 hours; 6) exposure to wet bedding for 24 hours; 7) housing with other rats (five rats per cage) for 17 hours; 8) placement in an empty cage for 17 hours; 9) cage tilted at 45° for 24 hours; 10) foot shock for 1 minute (voltage 50 mV, frequency 0.05 Hz); 11) tail suspension for 1 minute; 12) foreign object placed in the cage for 6 hours; 13) light flashes for 10 minutes; and 14) reversed light/dark cycles (overnight illumination). The 14 stressors were scheduled semirandomly, with animals subjected to two different stressors daily. The rats in the CUMS group were exposed to the stressors for 35 days, and the same stressor was not administered on consecutive days to ensure that the rats were not able to anticipate the occurrence of specific stressors.

Untreated rats served as the NC group and were housed in a separate room in groups of five rats per cage (330 × 215 × 200 mm). Other than withdrawing food and water for 24 hours before the sugar preference experiment and withdrawing food for 6 hours before weighing, the rats were not subjected to additional stimuli. All behavioral tests were performed between 9:00 and 11:00 AM in a quiet, warm environment and were conducted in parallel for each experimental group. Biochemical parameters were measured on the last day of the experiment. After CUMS was induced for 7 days, the CUMS-treated rats were randomly divided into the following six groups (n = 10): 1) the CUMS control (vehicle) group; 2) 10-mg/kg fluoxetine group; 3) 2.5-mg/kg PEA group; 4) 5-mg/kg PEA group; 5) 10-mg/kg PEA group; and 6) 10-mg/kg PEA + 3-mg/kg MK886 group. Rats in these groups were treated with fluoxetine or PEA in 0.3% sodium carboxymethyl cellulose via gavage and MK886 via intraperitoneal injections daily for 28 days. The entire experimental period was 35 days (Fig. 2).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Experimental procedures. Animal grouping and treatments (A). Experimental procedures (B). Animals were exposed to CUMS for 35 days; drug treatments were administered for 28 days. Vehicle, PEA (2.5, 5, 10 mg/kg), fluoxetine (Fxt; 10 mg/kg), or MK886 (MK; 3 mg/kg) were administered for 28 days starting on day 8 after initiating the CUMS procedures.

Weight Monitoring.

The body weight gains reflect an animal’s appetite and digestive function. The body weights of the rats were recorded regularly once a week throughout the experimental period.

Sucrose Preference Test.

The sucrose preference test is used to assess the anhedonia of animals and indicates an animal’s reward sensitivity and depressive state (Remus et al., 2015). Initially, the rats were acclimated to a sucrose solution for 3 days before the test. On the 1st day, food and water were withheld for 24 hours; on the 2nd day, rats were exposed to a 1% sucrose solution (w/v) for 24 hours. On the 3rd day, a bottle of the 1% sucrose solution and a bottle of tap water were presented to each rat at random positions for 24 hours. During the test, all rats were exposed to the 1% sucrose solution (w/v) and tap water presented at random positions for 4 hours, followed by food and water deprivation for 24 hours. The bottles containing tap water or the sucrose solution were prepared 24 hours before the sucrose preference test and were stored in the same room to avoid leakage. The sucrose preference was calculated using the following formula: sucrose preference percentage (%) = sucrose consumption (g)/water consumption (g) + sucrose consumption (g) × 100.

Open-Field Test.

The open-field test (OFT) is commonly used to investigate the exploratory behavior and spontaneous activity of experimental animals (Jin et al., 2015). The OFT was performed using the TM-Vision behavioral experiment system (Tai-Meng, China). The OFT-100 experimental apparatus consisted of a nontransparent plastic container (625 × 740 × 510 mm) with an experimental area of 525 × 525 × 415 mm. The entire experimental process was recorded using a computer-controlled camera. The experimental area was divided into four active parts, namely, the sides, corners, perimeter, and center. Each rat was individually placed in the center of the open-field apparatus and allowed to explore its surroundings while the following parameters were recorded for 3 minutes: immobility time, locomotor time, and distance traveled. The walls and floor surfaces were thoroughly cleaned with 10% ethanol between the tests to remove possible biases caused by odor cues from previous rats.

Western Blot Analysis.

Nuclear proteins were extracted from rat brain tissues using a nuclear protein extraction kit (Beyotime, China), and the protein concentrations were detected using a commercially available BCA protein assay kit (Beyotime). Initially, 20 μg of the nuclear protein sample in lane marker loading buffer (Cwbiotech Company) were denatured at 100°C for 8 minutes. Brain lysates were separated on 10% Tris-glycine SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (0.45 μm pores; Millipore, Germany) using a wet electroblotting approach. The membrane was blocked with 5% (w/v) fat-free milk in PBS for 2 hours at room temperature and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-PPAR-α (1:250; Sigma-Aldrich) and monoclonal mouse anti-β-actin (1:500; Sigma-Aldrich) as an internal loading control. All antibodies were diluted in Tris-buffered saline solution containing 0.1% Tween-20 (TBST). After thorough washes with TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG, 1:750, and goat anti-mouse IgG, 1:1000; Sigma-Aldrich) for 2 hours at room temperature, and then, the blots were developed using an enhanced chemiluminescence detection kit (ECL Plus; Beyotime) for 20 seconds and visualized using the lane one-dimensional gel analysis system (Beijing Sage Creation Science, Beijing, China). For densitometry analyses, the bands were scanned and quantified using lane gel analysis software (Beijing Sage Creation Science), and the results were expressed as the ratio of target protein to β-actin immunoreactivity, namely, the relative density value of PPARα/β-actin (the ratio of integrated density value, IDV).

Total RNA Extraction and Reverse Transcription–Polymerase Chain Reaction Analysis.

Total RNA was extracted from the rat hippocampi, purified using an RNAiso Plus reagent kit (Takara, Japan) according to the manufacturer’s instructions, and quantified using a Micro-UV spectrophotometer (Q5000; Huayunante Technology). First-strand complementary DNAs (cDNAs) were synthesized from 2 μg of total RNA using a Primescript RT-PCR kit (Takara) according to the manufacturer’s instructions, and these cDNAs were used as the template for the reverse transcription–polymerase chain reaction analysis. The cDNA templates were then amplified by polymerase chain reaction in a 20 μl total reaction volume using a StepOne real-time PCR system (Applied Biosystems, CA). The primer sequences (Invitrogen) were as follows: PPARα, 5′-AGTGCCTGTCCGTCGGGAT-3′ (forward) and 5′-GCTAGTCTTTCCTGCGAGTATG-3′ (reverse) (222 bp). As an internal control, the GAPDH cDNA was coamplified using the primer sequences 5′-GTGCCAGCCTCGTCTCATAG-3′ (forward) and 5′-CTTGCCGTGGGTAGAGTCAT-3′ (reverse) (184 bp). The amplification was performed with the following parameters: preheating at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 1 minute. The reaction was terminated by a final extension step at 72°C for 10 minutes. The PCR products were separated on 2.5% agarose gels, and the density of each band was analyzed using a one-dimensional gel analysis system (Sage Creation Science). The expression of the gene of interest was normalized to GAPDH. The relative integrated density value was computed as PPARα/GAPDH. The threshold cycle (CT) of PPARα production was determined and normalized to the GAPDH internal standard. The samples obtained from four independent tissues were used to analyze relative gene expression with the 2^−ΔΔCT method.

Determination of ACTH and CORT Levels.

ELISAs were used to detect serum ACTH and CORT levels. The steps described below were performed according to the instructions provided with the rat ACTH and CORT ELISA kits (Shanghai Enzyme linked Biotechnology, China). We added 50 μl of the standard to the standard well and added 10 μl of the test sample and 40 μl of the sample diluent to the test well. We then added 100 μl of the HRP-conjugated reagent to each well, covered it with an adhesive strip, and incubated the plate at 37°C for 60 minutes. After the last washing step, we removed the remaining wash solution and then added 50 μl each of the chromogens A and B to each well. We gently mixed it and incubated it for 15 minutes at 37°C, followed by adding 50 μl of stop solution. The color in the wells changed from blue to yellow. We read the optical density (OD) at 450 nm using a microplate reader (LIUYI, Beijing, China) within 15 minutes. The serum ACTH and CORT concentrations are reported in picograms per milliliter and nanograms per milliliter, respectively.

Determination of T-AOC, GSH-PX, T-SOD and MDA Levels.

The T-AOC and MDA levels and the GSH-PX and total superoxide dismutase (T-SOD) activities in the supernatants of the hippocampal homogenates were determined using commercial analysis kits (Nanjing Jiancheng, China) according to the manufacturer’s instructions. Many endogenous antioxidants reduce Fe³⁺ to Fe²⁺, the latter of which forms a stable compound with phenanthroline; therefore, T-AOC levels were measured using a colorimetric method with a spectrophotometer at the visible wavelength of 520 nm (V-5800; METASH, China). A T-AOC unit in tissue is defined as follows: at 37°C, each milligram of tissue protein per minute increases the OD value of the reaction system by 0.01. T-AOC levels are reported as units per milligram of tissue protein. GSH-PX promotes the reaction of hydrogen peroxide (H₂O₂) with reduced glutathione to produce H₂O and oxidized glutathione (GSSG); therefore, the activity of GSH-PX is determined by the rate of its enzymatic reaction. GSH-PX activity is reported as units per milligram of protein. The xanthine oxidase method was used for the T-SOD assays. Superoxide radicals (O₂⁻) are generated by xanthine and the xanthine oxidase reaction system, which oxidizes hydroxylamine to form nitrite. This reaction produces a purplish red color via the chromogenic agent, and the absorbance is measured with a spectrophotometer at visible wavelengths. T-SOD activity is presented as units per milligram of protein. MDA levels were determined using the thiobarbituric acid (TBA) method. MDA levels in lipid peroxidation degradation products are condensed with TBA to form red products, resulting in a maximal absorption peak at 532 nm, and its OD value was measured using a colorimetric method at a wavelength of 532 nm. The MDA level is reported as nanomoles per milligram tissue protein.

Determination of BDNF and GDNF Levels.

The hippocampal BDNF and GDNF concentrations were determined using ELISAs according to the manufacturer’s instructions (Sbjbio, China). Briefly, the following steps were performed. Fifty microliters of each of the standards were added to respective wells at concentrations of 90, 60, 30, 15, and 7.5 ng/l. We then added 10 μl of the test sample and 40 μl of the diluent to the test well. After an incubation and washes, we added 50 μl of the HRP-conjugated reagent. We then added 50 μl each of chromogen A and chromogen B and incubated the plate in the dark for 15 minutes. We added 50 μl of stop solution to stop the reaction. We read the OD at 450 nm using a microplate reader (LIUYI) within 15 minutes. The BDNF and GDNF concentrations are presented as picograms per milligram of brain tissue.

Hippocampal Weight and the Hippocampus Index.

On the last day of the experiment, the rats were anesthetized with 10% chloral hydrate (4 ml/kg; Chaoyang, Beijing, China) and sacrificed by decapitation. After rapidly removing and weighing the whole brain of each rat, the bilateral hippocampi were immediately dissected on an ice-cold table. The hippocampus index (the ratio of hippocampus weight to whole brain weight) was calculated using the following formula: the hippocampus index (%) = hippocampus weight (mg)/whole brain weight (mg) × 100%.