• Authors:
  Zheng Gu
  • Liujie Chu
  • Yaqiong Han

View Affiliations / Copyright

Affiliations:Department of Pharmacy, The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453002, P.R. China

Copyright: ©Guet al.This is an open access article distributed under theterms ofCreative Commons Attribution License.
• Pages:3061-3064
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  Published online on:February 26, 2019
    https://doi.org/10.3892/etm.2019.7311
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Introduction

Depression is a common chronic neurological disease.It has the characteristics of high prevalence, easy recurrence,disability, low rate of visiting and low treatment rate. It hasbecome the fifth most serious disease affecting human health(1,2). The occurrence of depression is theresult of a combination of multiple factors, including heredity,personality, endocrine, living environment and disease. It iscurrently widely believed that the reduction in levels ofneurotransmitters (dopamine and serotonin) and neurotrophic factors(neuropeptide Y) is closely related to the occurrence of depression(3–5). Therefore, it has been found that thepurpose of alleviating or treating depression can be achieved byincreasing the levels of neurotransmitters and neurotrophicfactors. Resveratrol is a polyphenolic compound that is ubiquitousin plants. It has been found in modern pharmacological studies tohave multiple biological functions such as anti-inflammatory,anti-oxidation, antitumor, anti-thrombosis and anti-aging (6–10). Ourearly study found that resveratrol has antidepressant effects. Thisstudy analyzed its anti-depressant effects and possible mechanismsto provide a new approach to the treatment of depression.

Materials and methods

Experimental materials

A total of 50 SPF grade C57BL/6 mice (female, 6–8weeks old 20 g) were purchased from Shanghai Slac Laboratory AnimalCo., Ltd. (Shanghai, China); resveratrol was purchased from Sigma(USA); mouse dopamine (DA), serotonin (5-HT), brain-derived nervegrowth factor (BDNF) and ELISA test kit were purchased fromBioAssay Systems LLC (Hayward, CA, USA); Neuropeptide (NPY) andactin monoclonal antibodies were purchased from Santa Cruz,Biotechnology, Inc. (cat. nos. sc-133080 and sc-8432; Dallas, TX,USA); actin monoclonal antibody was purchased from Hangzhou HuaanBiological Co., Ltd. (Hangzhou, China); HRP-labled goat anti-mousesecondary antibody was purchased from Beijing Zhongshan JinqiaoBiological Co., Ltd. (cat. no. ZDR-5307; Beijing, China); RIPAtissue lysate was purchased from Beyotime Biotechnology Co., Ltd.(Nantong, China); chemiluminescence kit was purchased from HangzhouFude Biological Technology Co., Ltd. (Hangzhou, China); M200full-band microplate reader was purchased from Tecan Group, Ltd.(Mannedorf, Switzerland).

Construction and treatment ofdepression models

A model of depression was established using socialisolation combined with chronic unpredictable stress. All modeledmice were housed individually and randomized alternating stresstreatment was performed once a day for 21 days. The stressors wereas follows: i) fasting for 24 h; ii) water deprivation for 24 h;iii) tilting squirrel cage for 30° for 24 h; iv) clipping 1 cm ofthe root of mouse tail for 1 min; v) restraint with 9.5×3 cmfixator for 2 h; vi) continuously soaking in 17°C cold water for 3min; vii) continuously soaking in 40°C hot water for 3 min andviii) day alternates with night. Each stimulus was not usedcontinuously. After 21 days of stimulation, the mice were randomlydivided into the model group, the low-dose group, the medium-dosegroup, and the high-dose group, with 10 mice in each group, and 10mice of the same age were selected as controls. On the 11th dayafter stimulation, the low, medium and high dose groups wereintraperitoneally injected with resveratrol 10, 20 and 30 mg/kg,respectively. Both the control and model groups wereintraperitoneally injected with an equal volume of physiologicalsaline once a day. After 21 days of continuous treatment, theneurobehavioral changes of each group were analyzed by forcedswimming test, tail suspension experiment and sucrose consumptiontest.

The study was approved by the Ethics Committee ofThe Second Affiliated Hospital of Xinxiang Medical University(Xinxiang, China).

Suspension experiment

The experiment was carried out in a plexiglass blackbox. The distal end of the mouse tail was fixed on a crossbar 30 cmfrom the ground. The head was facing down, and inverted status wasmaintained. After 6 min, it was adapted for 2 min, and then theimmobility time was recorded. This result mainly reflected thedegree of acquired helplessness in mice.

Forced swimming experiment

Two hours after receiving the drug treatment, themice were placed in a glass cylinder of 30 cm in diameter and 25 cmin height, with a water depth of 15 cm; The water temperature wasmaintained at 24°C, and the mice were forced to soak for 6 min.Judging criteria: All mice showed only the head exposedhorizontally, floating, and the limbs were still. The immobilitytime was recorded 4 min after soaking.

Sucrose consumption test

Each group of mice was fasted and water-deprived for24 h, then 100 ml 30 mmol/l sucrose solution and 100 ml purifiedwater were returned to the squirrel cage at 10:00 a.m. the nextday. After the mice were allowed to drink for 1 h, the consumptionof sucrose solution and water was measured. The sucrose partialitywas then calculated according to the following formula. Sucrosepartiality (%) = sucrose consumption/total liquid volume ×100%.

Analysis of DA, 5-HT and BDNF levelsby ELISA

After 24 h of the last administration, the mice weresacrificed. Brain tissue was taken, and 100 mg was weighed.Physiological saline (200 µl) was added, and the brain tissue washomogenized by a homogenizer, then centrifuged at 12,000 × g and4°C for 10 min. The supernatant was taken, the levels of DA, 5-HTand BDNF were detected according to the manufacturer's instructionsof ELISA.

Western blot analysis of NPY proteinexpression levels

The mice were sacrificed 24 h after the lastadministration; brain tissue was taken, and 100 mg was weighed.Tissue lysate (200 µl) was added, and homogenized by homogenizer;allowed to stand on ice for 30 min, and centrifuged at 12,000 × gfor 30 min at 4°C. Loading buffer 5X (50 µl, 60 mM Tris-HCl pH6.8,2% SDS, 0.1% bromophenol blue, 25% glycerol; 14 mMβ-mercaptoethanol) was added to the supernatant, boiled for 20 minin a metal bath, then SDS-PAGE was performed. Afterelectrophoresis, the gel transferred to PVDF membrane, blocked with5% skim milk powder for 1 h, and then incubated overnight with theprimary antibody diluted with 5% skim milk powder (dilution,1:1,000) at 4°C; then washed with PBST 3 times the next day, 5 mineach time; after incubated with HRP-labeled goat anti-mousesecondary antibody diluted with 5% skim milk powder (dilution,1:2,000) for 2 h at room temperature, wash 3 times with PBST, 5 minafter each time, the chemiluminescence substract was applied to thePVDF membrane to develop color, and actin was used as an internalreference. Image J software (National Institute of Mental Health)was used to perform quantitative analysis.

Statistical analysis

All data were analyzed by SPSS 17.0 statisticalsoftware (SPSS, Inc., Chicago, IL, USA). The measurement data wereexpressed by mean ± standard deviation (mean ± SD), and themeasurement data were compared by Chi-square test. ANOVA was usedfor comparison of multiple groups with LSD test. P<0.05indicated that the difference was statistically significant.

Results

Comparison of behavioral indicatorsafter treatment in each group of mice

Compared with the control group, the immobility timeof the mice in the model group was significantly prolonged(P<0.05) in the tail suspension and forced swimming experiment,while the consumption of syrup water was significantly decreased at24 h, with a statistical difference (P<0.05). Compared with themodel group, the immobility time of the tail suspension experimentand the forced swimming experiment in the low-dose group wassignificantly shortened, while the 24-h sucrose consumption wassignificantly increased, and the difference was statisticallysignificant (P<0.05). Compared with the low-dose group, theimmobility time of the suspension experiment and forced swimmingexperiment in the middle dose group was significantly reduced,while the 24 h syrup consumption was significantly increased, thedifference was statistically significant (P<0.05). Compared withthe middle dose group, the immobility time of the tail suspensionexperiment and forced swimming experiment in the high dose groupwas significantly reduced, while the 24-h sucrose consumption wassignificantly increased, and the difference was statisticallysignificant (P<0.05) (TableI).

Table I. Comparison of behavioral indicatorsafter treatment in each group of mice.

Table I.

Comparison of behavioral indicatorsafter treatment in each group of mice.

Groups	Tail suspensionexperiment(s)	Forced swimmingexperiment(s)	Sucrose consumptionexperiment(s)
Control	89.32±12.21	79.33±14.20	21.43±6.17
Model	271.43±20.19a	194.74±21.09a	8.43±3.18a
Low-dose	221.33±17.17b	170.32±18.39b	17.11±4.21b
Medium-dose	174.21±14.12b,c	141.30±16.33b,c	14.20±3.99b,c
High-dose	130.39±15.18b–d	129.43±16.01b–d	11.98±3.02b–d

a P<0.05 compared with the control group

b P<0.05 compared with the model group

c P<0.05 compared with the low-dose group

d P<0.05 compared with the medium-dose group.

Comparison of DA, 5-HT and BDNF levelsin each group of mice after treatment

We further analyzed the effects of resveratrol onneurotransmitter levels in the mice brain tissue. The ELISA resultsshowed that the levels of DA, 5-HT and BDNF in the brain tissue ofthe model group were significantly downregulated compared with thecontrol group, and the difference was statistically significant(P<0.05). After treatment with low-dose resveratrol, the levelsof DA, 5-HT and BDNF in brain tissue were significantly higher thanthose in the model group, and the difference was statisticallysignificant (P<0.05). Compared with the low-dose group, thelevels of DA, 5-HT and BDNF in the brain tissue of mice treatedwith medium dose of resveratrol were significantly increased(P<0.05). Compared with the middle dose group, the levels of DA,5-HT and BDNF in the brain of the high dose group weresignificantly increased, and the difference was statisticallysignificant (P<0.05) (TableII).

Table II. Comparison of DA, 5-HT and BDNF levelsin each group of mice after treatment.

Table II.

Comparison of DA, 5-HT and BDNF levelsin each group of mice after treatment.

Groups	DA(ng/g)	5-HT(ng/g)	BDNF(ng/g)
Control	654.39±30.19	719.25±43.12	21.43±6.17
Model	198.25±21.44a	300.18±21.44a	8.43±3.18a
Low-dose	298.33±23.56b	381.24±25.44b	17.11±4.21b
Medium-dose	357.19±28.14b,c	430.29±28.10b,c	14.20±3.99b,c
High-dose	490.99±30.23b–d	551.33±32.55b–d	11.98±3.02b–d

a P<0.05 compared with the control group

b P<0.05 compared with the model group.

c P<0.05 compared with the low-dose group

d P<0.05 compared with the medium-dose group.

Comparison of NPY levels in braintissue of each group of mice

The expression levels of total NPY in the braintissue of each group of mice were analyzed by western blotanalysis. The results are shown inFig.1. Compared with the control group, the NPY levels of the modelgroup were significantly downregulated, and the difference wasstatistically significant (P<0.05). After treatment withresveratrol, the level of NPY in the brain tissue of mice increasedgradually, and in a dose-dependent manner. The comparison betweenthe levels of NPY in the brain tissue of the low-dose, medium-doseand high-dose groups were significantly different (P<0.05).

Figure 1. Comparison of NPY levels in braintissue of each group of mice.

Discussion

With the gradual increase of social and workpressure, depression, a chronic neurological disease, seriouslythreatens human cognition. Its incidence is increasing year byyear. Epidemiological analysis shows that the incidence ofdifferent depression in young individuals is more significant andhas become a social problem (11).Therefore, screening for highly effective and low-toxic drugs is aprerequisite for the treatment of depression. Our previousinvitro studies showed that after treatment of neurons withresveratrol, the level of dopamine was significantly increased. Thestudy showed that the levels of DA, 5-HT and BDNF in patients withdepression were significantly reduced, which is one of the maincauses of depression. Therefore, we speculated whether resveratrolhas the effect of relieving depression.

DA and 5-HT are monoamine neurotransmitters in thebrain. In the pathogenesis of depression, the DA and 5-HThypotheses are recognized. Studies also showed that the level of DAand 5-HT decreased in the hypothalamic tissue of patients withdepression, indicating that depression is closely related to thelow function of DA and 5-HT (12,13). Thelevels of DA and 5-HT in the brain tissue of the mouse model ofdepression established in this study were significantly lower thanthose in normal mice, indicating that DA and 5-HT play an importantrole in the pathogenesis of depression. After treatment withresveratrol, the levels of DA and 5-HT increased gradually and in adose-dependent manner. This result suggests that resveratrol mayhave the effect of treating depression. BDNF is a member of theneurotrophic factor family, which has the effect of promotingproliferation and differentiation of neurons, promoting neuronalsurvival and development, altering neuronal morphology in thebrain, increasing synaptic terminal density, and promotingdendritic and axon growth (14). NPYis a known neuroendocrine polypeptide that plays an importantregulatory role in neuronal excitability (15). The current study indicated that BDNFlevels and brain NPY were significantly decreased in patients withdepression. This result is consistent with the results obtained inthe mouse model of this study. After treatment with resveratrol,the level of BDNY increased with the increase of resveratrolconcentration, indicating that resveratrol may increase theactivity of neurons by increasing brain-derived neurotrophicfactor, thus achieving anti-depressant effect.

Behavioral analysis of mice showed that the behaviorof depression in mice after resveratrol treatment was significantlyrelieved, indicating that resveratrol has a role in alleviatingdepression. Early studies on resveratrol have shown that they haveimportant biological functions such as anti-inflammatory, antitumorand anti-aging. In addition, this study revealed a newanti-depressant biological function of resveratrol, which expandedthe clinical use of resveratrol.

In summary, resveratrol can significantly increasethe levels of DA, 5-HT, BDNF and NPY in the brain to achieve theeffect of treating depression.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the presentstudy are available from the corresponding author on reasonablerequest.

Authors' contributions

ZG conceived the study and drafted the manuscript.ZG and LC were responsible for construction and treatment of thedepression models. ZG and YH helped with the sucrose consumptiontest. All authors have read and approved the final manuscript.

Ethics approval and consent toparticipate

The study was approved by the Ethics Committee ofThe Second Affiliated Hospital of Xinxiang Medical University(Xinxiang, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competinginterests.

References