This study was obeyed the Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University.
Animals and treatment
Total of 40 male C57BL/6 J mice (six-week-old) were purchased from Huafukang Company. Every mouse was fed adaptively under a normal 12 h light/dark cycle at 23 ± 2 °C (humidity 45%–55%) for 2 weeks before experiments began. During the study, the mice were given food and water every day. The mice were randomly divided into five groups (n = 8 per group):
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(a)
Control;
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(b)
Control+ 10 mg/kg PB;
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(c)
Chronic unpredictable mild stress (CUMS);
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(d)
CUMS+ 10 mg/kg PB;
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(e)
CUMS+ 10 mL/kg imipramine hydrochloride (IMI);
The CUMS experiments were performed for 6 weeks. At the 4th week, the PB was administrated once a day for 3 weeks by oral gavage. The IMI treatment was served as a positive control, the IMI were administrated every day during the UCSM experiments by oral gavage. The UCSM and SPT, depressive-like behavioral assessment were carried out in the order presented in Fig. 1A.
Chronic unpredictable mild stress (CUMS)
To explore the effects of PB on depression-like behaviors in animals, the CUMS-induced mouse model was used in this study. The control group did not receive any of the chronic unpredictable mild stresses experiment, whereas CUMS-induced mice received single housed and subjected to one of the following stressors once every day in a randomized fashion: (I) lake of food for 24 h, (II) lake of water for 24 h (III) lighting for 24 h, (IV) cage tilt (45°), (V) the damp environment (per 100 g padded add 200 ml of water), (VI) exposed to the external environment, (VII) the light light/dark cycle, (VIII) alternately suspension for 10 min, (IX) exposed to an empty bottle, (X) clip tail (1 min, 1 cm in from the tail to start), (XI) oscillation for 5 min, (XII) white noise. Mice were subjected to different stressors daily for 42 days in an unpredictable way. Finally, the mice were sacrificed at the 43th day. The mice anesthetized with sodium pentobarbital (100 mg/kg) and fentanyl (0.05 mg/kg). The brains were quickly removed, washed with phosphatic buffer solution (PBS), and post-fixed in 4% paraformaldehyde overnight at 4 °C for further experiments.
Sucrose preference test (SPT)
Anhedonia is the inability to experience pleasure from rewarding or enjoyable activities, which is a core symptom of depression in humans, and can be assessed by SPT assay (Liu et al. 2018). In this experiment, weight was weighed every 7 days for 6 weeks. In addition, SPT was administered every 7 days before and after induction in CUMS. First, the mice were placed in separate cages, each adapted to a 1% sucrose solution (w/v). After 24 h of exposure to two bottles of sucrose solution, a bottle of 1% sucrose solution in each cage was replaced with a bottle of water and exposed for another 24 h. Then, all mice were deprived of water and food for 24 h, and SPT was administered for 12 h. During this time, the mice were free to use two bottles, one contained a 1% sucrose solution and the other was only water. The location of the bottle was changed during the 6-h test to avoid possible side effects. The sucrose preference was calculated as follows: SPT (%) = ((sucrose intake (g)/(sucrose intake (g) + water intake(g))) × 100.
Forced swim test (FST)
The FST is a model of depressive-like behavior (Yankelevitch-Yahav et al. 2015). To allow adaptation to the environment, all mice were taken by a 6 min FST. The mice in each group were placed in a water container 25 cm high and 10 cm in diameter (10 cm deep; 23 ± 2 °C). The total 6-min process included the first 2-min unmeasured swim and the subsequent 4-min swim. The immobility time of 4 min after swimming was recorded by the software tracking analysis system (Xinsoft Information Technology, China). The latency to abandon is thought to be the time which ranged from the time that the animal was introduced into the pool to the time it first stopped completely (at least 2 s).
Tail suspension test (TST)
The TST assay is a widely used models for assessing antidepressant-like activity in animal models (Cryan et al. 2005). In this study, the TST assay was performed as a previous reference described (Jiang et al. 2017b). Each mouse was hung 50 cm away from the ground for 6 min, with its tail attached to the hook (about 2 cm from the end). Calculate the immobility time for the last 4 min. The mice are considered to be motionless only when they are completely motionless.
Open field test (OFT)
The OFT experiment was a test of anxiety-related behavior and locomotion and performed as described previously (Wu et al. 2019; Belzung and Griebel 2001). In short, the instrument is divided into 12 equal squares. The rats were then placed in the middle of the field and allowed to explore their surroundings Finally, the numbers of square crossings, rearing and grooming scores within 5 min of free activity were recorded.
Measurement of MDA and SOD
The hippocampus from mice were homogenized (10% w/v) with PBS and centrifuged for collecting supernatants. The levels of MDA and the activity of SOD in the supernatant were respectively measured by MDA Assay Kit (Abnova, USA) and SOD Assay Kit (Abnova, USA), according to the manufacturer’s instructions.
Determination of ROS level
The levels of ROS in hippocampus were evaluated by using 2, 7-dichlorofluorescindiacetate (DCFDA) Kit (Sigma, China). Briefly, hippocampus homogenate was washed with PBS, mixed with DCFDA (dissolved with methanol) and then incubated in water for 15 min at 37 °C. After that, the samples were stained by DAPI. ROS images were measured using a DCFH-DA fluorescence probe under a fluorescence microscope with a magnification of 400×. Fluorescence intensity was calculated using a fluorometer at 488 nm excitation wavelength and 525 nm emission wavelength (Hemmati et al. 2018).
Terminal-deoxynucleoitidyl Transferase mediated Nick end labeling (TUNEL) assay
The cell apoptosis was detected by FragEL™ DNA Fragmentation Dectection Kit (Merck-Calbiochem, USA). The hippocampus from mice were embedded in paraffin, and cut into 5-μm slices. After deparaffinization, the tissue sections were incubated with proteinase K for 25 min and incubated with H2O2 in methanol (8 min). Next, the sections were placed in the TUNEL reaction mixture and incubated in darkness for 60 min. After washing by PBS, the sections were incubated with streptavidin-horseradish peroxidase (HRP) conjugate, and DAB was served as chromogen substrate. The nuclear staining of cell in intense and brown was TUNEL-positive.
Immunohistochemistry assay
The hippocampus from mice after deparaffinization were incubated in citrate buffer solution, and then were blocked with 10% goat serum. After that, slides incubated with the Anti-Cleaved Caspase-3 antibody (dilution:1:300; Abcam, USA) at 4 °C overnight. Then, the sections were stained with DAB chromogen substrate in the dark. Finally, the images were recorded by a microscope.
Measurement of TNF-α, IL-1β, TGF-β and IL-10
The concentrations of TNF-α, IL-1β, TGF-β and IL-10 in hippocampus from mice were measured by Mouse IL-1β ELISA Kits (MULTI SCIENCES, China), Mouse TNF-α ELISA Kits (MULTI SCIENCES, China), Mouse IL-10 ELISA Kits (MULTI SCIENCES, China) or Mouse TGF-β ELISA Kits (MULTI SCIENCES, China).
Quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from hippocampus in mice using TRIzol reagent (Takara, Japan). And then the cDNA was synthesized by Prime-Script™ RT regent Kits (Takara, Japan). The mRNA expression levels were measured using qRT-PCR by SYBR Premix Ex Taq (Takara, Japan). The critical threshold cycle (Ct) value was determined and the relative quantification data were calculated with the 2-ΔΔCt method, the GAPDH was served as a reference. The primers were as follows: IL-1β-forward: 5′-GAGTCTGCACAGTTCCCCAA-3′, IL-1β-reverse: 5′-TGTCCCGACCATTGCTGTTT-3′; TNF-α-forward: 5′-CGTC AGCCGATTTGCCATTT-3′, TNF-α-reverse: 5′-CTCCCTCAGGGGTGTCCTTA-3′; TGF-β-forward: 5′-GAACTCGCTTTGTCTCCA-3′, TGF-β-reverse: 5′-TACAGTCGCAGTATAACCTCA-3′; IL-10-forward: 5′-TCTCCGAGATGCCTTCAGCAGA-3′, IL-10-reverse: 5′-TCAGACAAGGCTTGGCAACCCA-3′; GAPDH-forward: 5′-ATGGGGAAGGTGAAGGT-3′, GAPDH-reverse: 5′-AAGCTTCCCGTTCTCAG-3′.
Western blot
The protein from the tissues of mice were lysed with RIPA lysis buffer (Beyotime, China). Then, the protein concentrations were detected by a BCA protein kit (Beyotime, China). Next, equal proteins from different groups were separated by SDS-poluacrylamide gel electrophoresis, transferred onto a PVDF membrane. After washed by TBST buffer for three times, the membrane was incubated with first antibodies: Nrf2 antibody (dilution: 1:400; Abcam, USA), HO-1 antibody (dilution: 1:400; Abcam, USA), Bcl-2 antibody (dilution: 1:800; Proteintech, USA), Bax antibody (dilution: 1:800; Proteintech, USA), p-NF-kB antibody (dilution: 1:400; Abcam, USA), NF-kB antibody (dilution: 1:400; Abcam, USA), GAPDH antibody (dilution: 1:10000; Proteintech, USA) overnight at 4 °C, followed by incubation with the appropriate secondary antibodies (dilution: 1:5000; Beyotime, China) for 2 h. The GAPDH was served as a loading control.
Data analysis
All Data were expressed as mean ± standard deviation (SD) and analyzed with were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA by GraphPad Prism 7.0. P < 0.05 was considered significant.
