The antidepressant effects of hesperidin on chronic unpredictable mild stress-induced mice

European Journal of Pharmacology 853 (2019) 236–246

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The antidepressant effects of hesperidin on chronic unpredictable mild stress-induced mice

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Huiling Fua,b,1, Li Liue,1, Yue Tongb, Yuanjie Lib, Xia Zhangb, Xiaojuan Gaob, Jingjiao Yongb, Jianjun Zhaob, Dong Xiaob, Kuishen Wenb, Hanqing Wanga,b,c,d,∗ a

The First People's Hospital of Yinchuan, Ningxia Medical University, Ningxia, 750004, China College of Pharmacy, Ningxia Medical University, Ningxia, 750004, China c Ningxia Research Center of Modern Hui Medicine Engineering and Technology, Ningxia Medical University, Yinchuan, 750004, China d Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education (Ningxia Medical University), Yinchuan, 750004, China e Guangdong Key Laboratory for Research and Development of Natural Drugs, School of Pharmacy, Guangdong Medical University, Dongguan, 523808, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Hesperidin Chronic unpredictable mild stress (CUMS) Inflammation HMGB1/RAGE/NF-κB pathway BDNF/TrkB pathway

Hesperidin, a kind of citrus bioflavonoid distributed in foods including grapefruits, oranges and lemons, has many pharmacological activities. This study was aimed to evaluate the anti-depressant-like effect of hesperidin on chronic unpredictable mild stress (CUMS)-induced mice. Depressive-like behavior was detected by the sucrose preference test (SPT), tail suspension test (TST) and forced swimming test (FST). A 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to assess the cell viability of corticosterone-induced PC12 cells. The serum, hippocampal and cell supernatant concentrations of interleukin (IL)1β, IL-6 and tumor necrosis factor (TNF)-α were determined using enzyme-linked immunosorbent assay (ELISA) commercial kits. Furthermore, the protein expression levels of high-mobility group box 1 protein (HMGB1), receptor for advanced glycation end-products (RAGE)/NF-κB and brain-derived neurotrophic factor (BDNF)/ tropomyosin-related kinase B (TrkB) pathway in the hippocampus and corticosterone-induced PC12 cells were detected by Western blot. Our results showed that hesperidin (100, 200 mg/kg) significantly relieved depressivelike behaviors, including decreased sucrose consumption in sucrose preference test (SPT), immobility in the forced swimming test (FST), tail suspension test, and locomotor activity in the open field test (OFT). Hesperidin reduced inflammatory cytokine levels by attenuating the HMGB1/RAGE/NF-κB signaling pathway and BDNF/ TrkB pathway both in vivo and in vitro. In conclusion, hesperidin possessed efficient neuroprotective effects on depression, which was associated with neuroinflammation mediated by the HMGB1/RAGE/NF-κB and BDNF/ TrkB pathways.

1. Introduction As one of the most frequently occurring psychiatric diseases, clinical depression is featured by guilty conscience, physiological disorder, cognitive dysfunction and even suicidal tendency (Lenze et al., 2015). According to the World Health Organization, depression may become the second most prevalent disabling disease following heart disease by 2020 (Zu et al., 2017). Numerous lines of evidence have demonstrated that the pathophysiological symptoms of depression include abnormal neuroplasticity, deficient neurotrophic factors, and an excessive inflammatory response (Zhao et al., 2017). The chronic unpredictable mild stress (CUMS) model is widely used to mimic depressive behavior in rodents (Ayuob et al., 2017). CUMS contributes to endogenous

depression that is implicated in neuropsychiatric disorders, including behavioural, biochemical and neurochemical derangements (Adebesin et al., 2017). Growing evidence has proposed that neuronal loss and cellular atrophy are mediated by neuroinflammation during the pathogenesis of depression (Qiao et al., 2016). Thus, it is generally believed that antidepressants exhibit ameliorated effects on depressive-like behavior by suppressing inflammatory mediators and promoting neurotrophic factors. For instance, brain-derived neurotrophic factor (BDNF), a representative member of the neurotrophic factor family, governs the physiological functions of the frontal lobe and hippocampal tissues by regulating neuroplasticity (Tao et al., 2016). It has been shown that the serum BDNF levels of depressive patients are lower than those of

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Corresponding author. College of Pharmacy, Ningxia Medical University, Ningxia, China. E-mail address: [email protected] (H. Wang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ejphar.2019.03.035 Received 23 July 2018; Received in revised form 14 March 2019; Accepted 22 March 2019 Available online 28 March 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

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healthy people (Kerling et al., 2017). A number of studies confirmed that the expression of BDNF and its downstream regulator TrkB were both suppressed in the brain tissues of depressive mice, stimulating multiple signaling cascades and impairing cellular survival rate (Franklin et al., 2018b). Hence, the BDNF/TrkB pathway is essential to the progression of CUMS-related depression. In addition, chronic stressrelated cellular inflammation accelerates the generation of danger-associated molecular pattern (DAMP) molecules including high mobility group box 1 protein (HMGB1). Generally, HMGB1 is a nuclear nonhistone DNA-binding protein that modulates the transcription of inflammatory cytokines. HMGB1 binds to receptors for advanced glycation end products (RAGE) and then leads to NF-κB activation, which consequently triggers the inflammatory cascade (Plazyo et al., 2016). It was demonstrated that the chronic recruitment of HMGB1/RAGE resulted in chronic stress-induced depressive-like behavior (Franklin et al., 2018a). Thus, anti-depressants might exert their effects by mediating inflammatory responses through the HMGB1/RAGE/NF-κB and BDNF/TrkB signaling pathways. Hesperidin (4′-methoxy-7-O-rutinosyl-3′, 5-dihydroxyflavanone) is a natural flavanone glycoside mainly distributed in citrus fruits (Liu et al., 2016). According to previous experimental studies, hesperidin produced a variety of pharmacological effects, such as promoting memory, increasing neurogenesis, and inhibiting inflammation (Yang et al., 2011b). Previous studies also suggested that the treatment with hesperidin exerted antidepressant-like effects on rodents (Feng et al., 2016). However, the underlying mechanisms are complex and remain to be explored. Therefore, the present study was carried out to investigate whether hesperidin treatment ameliorated CUMS-induced depression by suppressing inflammatory processes through the BDNF/ TrkB and HMGB1/RAGE/NF-κB signaling pathways.

Fig. 1. Schematic representation of the experimental procedure for chronic unpredictable mild stress (CUMS) and treatments.

2.3. Chronic unpredictable mild stress (CUMS) experimental design and drug treatment All animals were randomly split into the following eight groups: control (No-CUMS) group, fluoxetine (20 mg/kg) group, hesperidin (100 mg/kg) group, hesperidin (200 mg/kg) group, vehicle (CUMS or model) group, CUMS + fluoxetine (20 mg/kg) group, CUMS + hesperidin (100 mg/kg) group, and CUMS + hesperidin (200 mg/kg) group (n = 10). The fluoxetine (20 mg/kg) group, hesperidin (100 mg/kg) group and hesperidin (200 mg/kg) group were only used for behavioural tests. The CUMS schedule was carried out according to a previously described method (Luo et al., 2008). The overall experimental procedure is illustrated in Fig. 1. The mice, except for the control mice, were subjected to CUMS challenge for six weeks. The detailed arrangement of CUMS challenge was presented in Table 1. From the fourth week, the mice received the relevant drug treatment orally three times a week for 3 weeks (during 14:00 p.m. to 15:30 p.m. on Monday, Wednesday and Friday of each week). Simultaneously, the control (No-CUMS) group and the vehicle (CUMS) group were administered with an equal volume of normal saline. One h after final drug administration, the SPT was performed. The TST, FST, and OFT were performed on days 42, 43, and 44, respectively. Two h after the

2. Materials and methods 2.1. Reagents Hesperidin (CAS number:520-26-3) was purchased from SigmaAldrich (Saint Louis, MO, USA). Fluoxetine (Flu, CAS number：5491089-3) was provided by Xiansheng Drug Store (Nanjing, China). Commercial Enzyme-linked immunosorbent assay (ELISA) kits for IL1β, IL-6 and TNF-α were supplied by Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China). BCA protein concentration assay kit was obtained from Elabscience Biotech. Co. Ltd. (Wuhan, Hubei, China). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT), corticosterone and DMSO were produced by Sigma (St. Louis, USA). Highglucose Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from GIBCO-BRL (Grand Island, USA). The corresponding primary antibodies including anti-HMGB1(#6893), antiRAGE(#6996), anti-p-IκBα(#2859), anti-IκBα(#4812), anti-peNFeκB (#3039), antieNFeκB(#8242), anti-BDNF(#4201), anti-TrkB(#4603) and anti-GAPDH(#5174) antibodies were purchased from Cell Signaling Technology (Danvers, USA). Anti-p-TrkB(#bs-3732R) antibody was provided by Bioss Biological Technology Co., Ltd (Beijing, China).

Table 1 Chronic unpredictable mild stress (CUMS) procedure. Monday 9:00 11:00 Tuesday 9:00 9:40 Wednesday 9:00 10:00 Thursday 9:00 15:00 Friday 9:00 9:40 Saturday 9:00 10:00 Sunday 9:00 15:00

2.2. Animals ICR male mice (6–8 weeks old) purchased from the Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China) were used in the present study. The mice were maintained under a 12 h light/dark cycle in 24–25 °C temperature conditions with free access to food and water. The experiments were approved by the Institutional Animal Care and Use Committee at Ningxia Medical University (permission number IACUC-20160711).

Closed light Remove food and water, 20 h cold–wet cage (200 ml water (4 °C)/cage) Change dry cage, restore food and water, and 40 min of case shaking (200 rpm) Stop case shaking, continuous light for 24 h Closed light, record animal weight 24 h of tilted cage (45°), and remove water Stop tilted cage(45°), restore water, and change to 5 mice/cage Change to single cage, remove food Restore food, 40 min of case shaking (200 rpm) Stop case shaking, 20 h hot–wet cage (200 mL water (45 °C)/cage) Change dry cage 24 h of tilted cage (45°), and remove water Stop tilted cage(45°), restore water, and change to 5 mice/cage Change to single cage, continuous light for 20 h

The mice were exposed to these stressors according to this schedule and repeated every week, kept on 6 weeks. 237

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MTT (5 mg/ml, Sigma) solution for another 4 h. After incubation, the culture medium was removed, and 150 μl dimethyl sulfoxide (DMSO) was added. Absorbance values were detected at a test wavelength of 570 nm using a microplate spectrophotometer. Experiments were conducted in triplicate. Data were evaluated as the percentage of each group average absorbance to control group absorbance. Cell viability is represented as:

last behavioural test, the animals were killed, and blood samples were harvested and centrifuged at 3000 rpm for 10 min. The serum and brain tissue of each mouse were collected and stored at −80 °C for further investigation. Only the control (No-CUMS) group, vehicle (CUMS or model) group, CUMS + fluoxetine (20 mg/kg) group, CUMS + hesperidin (100 mg/kg) group, and CUMS + hesperidin (200 mg/kg) group were applied for the ELISA and western blot analyses.

Cell viability (%) = (A

2.4. Sucrose preference test (SPT) The behavioural tests were performed after the last CUMS exposure. The SPT is usually used to evaluate rodent behavior associated with a human clinical depressive symptom by assessing the ability to seek out pleasure. This test was conducted as described previously (Tang et al., 2015b). After the deprivation of water for 12 h, each mouse was simultaneously presented with two premeasured bottles complemented with water or 1% sucrose solution (w/v) for 6 h. Then the fluid intake was recorded and the bottles were exchanged their place for another 6 h. The sucrose preference was defined as follows: sucrose preference = sucrose consumption/(sucrose consumption + water consumption) × 100%.

Treated

/A

Control)

× 100%

2.10. Experimental design for PC12 cells The PC12 cells were pre-treated with hesperidin (10, 20, and 40 μM) and fluoxetine (10 μM) for 2 h, and then the cells were stimulated with corticosterone (800 μM) for 24 h. The cells and cell supernatants were harvested for other experiments. 2.11. Analysis of pro-inflammatory cytokines in serum, hippocampus and cell supernatant The levels of pro-inflammatory mediators, such as IL-1β, IL-6, and TNF-α in serum, hippocampus of CUMS-induced mice and cell supernatant of corticosterone-induced PC12 cells were determined with commercial enzyme-linked immunosorbent assay (ELISA) kits in accordance with the manufacturer's instructions.

2.5. Tail suspension test (TST) TST is a commonly used test for assaying potential antidepressant treatments in mice. The TST method was conducted according to previously described (Huang et al., 2018). Generally, the mouse was individually suspended 50 cm from the bottom for 6 min using adhesive tape at 1 cm from tail tip. The immobile duration was recorded within the last 4 min.

2.12. Western blot analysis

The FST protocol was performed in a quiet room as described in previous literature (Tang et al., 2015a). Each mouse was individually placed in a transparent polypropylene tank (40 cm height × 30 cm diameter) filled with 25 ± 1 °C water at 25 cm high height. The animals were forced to swim in the tank for 6 min and the immobility duration in the last 5 min was recorded. The immobility was defined when the mouse floated with limited movements to keep its head above water. The apparatus was cleaned after each use, while mice were dried immediately and returned to their home cages after the swimming test.

The hippocampus samples and PC12 cells were homogenized and lysed in ice-cold RIPA buffer solution. After centrifugation, the protein concentration of the supernatant was measured using a BCA kit (Beyotime, Nanjing, China). Then, the samples were subjected to SDSpolyacrylamide gel electrophoresis and transferred onto PVDF membranes. The PVDF membrane was blocked with 5% skim milk and treated with the corresponding primary antibodies (1:1000) overnight at 4 °C. The blot was washed using Tris-buffered saline-containing Tween 20 (TBST), and the PVDF membrane was incubated with horseradish peroxidase-conjugated secondary antibody at room temperature. The immunoblot was scanned using the BIO-RAD ChemiDoc XRS system and enhanced chemiluminescence (ECL) reagent (Beyotime, Nanjing, China).

2.7. Open field test (OFT)

2.13. Statistical analysis

OFT analysis was carried out in accordance with previous procedure (Ma et al., 2013). Each mouse was individually placed in the center of the well-illuminated (∼300 lux) transparent apparatus (40 × 40 × 15 cm) facing the wall and allowed to explore for 6 min. The apparatus was cleaned after occupancy of every mouse. The number of crossings in the final 5 min was counted by two experienced observers who were unaware of the specifics of the experiment.

All results were presented as the mean ± standard deviation (S.D.). The data were analysed by one-way analysis of variance (ANOVA) with Tukey's post hoc test or two-way ANOVA with Bonferroni post hoc test by Graphpad 6.0. A P value less than 0.05 was regarded as significant different.

2.6. Forced swimming test (FST)

3. Results

2.8. Culture of PC12 cells

3.1. The effects of hesperidin on CUMS-induced depression-related behaviors

PC12 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) complemented with 10% foetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C filled with 95% air and 5% CO2.

3.1.1. Sucrose consumption The change in sucrose consumption was the pivotal index to evaluate anhedonia in mice. As illustrated in Fig. 2A, non-CUMS treatment did not influence the sucrose preference of mice in the hesperidin or fluoxetine groups. CUMS challenge notably reduced the percentage of sucrose consumption compared with that of the control group without CUMS stimulation, whereas mice treated with hesperidin (100 and 200 mg/kg) showed an increased percentage of sucrose solution consumption. The sucrose intake was also increased after the administration of fluoxetine (20 mg/kg). A two-way ANOVA revealed significant

2.9. MTT assay Cell viability was evaluated using the MTT assay. PC12 cells were incubated with different concentrations of hesperidin (5, 10, 20, 40, 80, and 160 μM) for 2 h. Then the cells were stimulated with corticosterone (800 μM) for 24 h. Next, the cells in each well were exposed to a 20 μl 238

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Fig. 2. Effects of hesperidin on sucrose preference test (SPT) (A), forced swimming tests (FST) (B), tail suspension tests (C), open field test (OFT) (D). Data were expressed as mean ± S.D. (n = 8). ##P < 0.01 vs. the Control (No-CUMS) group; **P < 0.01 vs. the Vehicle chronic unpredictable mild stress (CUMS) group; & P < 0.05 and &&P < 0.01 vs. the control (No-CUMS) group.

Without CUMS stimulation, notable decreases in immobility time were observed in the hesperidin (100 mg/kg, 200 mg/kg)- and fluoxetine (20 mg/kg)-treated groups versus the CUMS-induced group. Two-way ANOVA revealed significant differences between the hesperidin (100 mg/kg) and CUMS (F (1, 28) = 111.3, P < 0.01), hesperidin (200 mg/kg) and CUMS (F (1, 28) = 56.05, P < 0.01), and fluoxetine (20 mg/kg) and CUMS treatment groups (F (1, 28) = 16.96, P < 0.01) in the FST. The analytical data revealed that hesperidin might ameliorate depressive-like behavior in CUMS-stimulated mice.

differences for the hesperidin (100 mg/kg) × CUMS interaction (F (1, 28) = 26.65, P < 0.01), hesperidin (200 mg/kg) × CUMS interaction (F (1, 28) = 38.11, P < 0.01), and Fluoxetine × CUMS interaction (F (1, 28) = 103.6, P < 0.01).

3.1.2. The effects of hesperidin on the TST and FST An immobile posture in the TST and FST reflects a condition of despair or helplessness. As shown in Fig. 2B, exposure to CUMS prolonged the immobility time in the FST compared with that in control group without CUMS induction. The administrations of hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) reduced immobility during the FST compared with that seen for mice in the vehicle groups both with CUMS induction or without CUMS induction. Two-way ANOVA revealed significant differences for the hesperidin (100 mg/ kg) × CUMS interaction (F (1, 28) = 44.68, P < 0.01), hesperidin (200 mg/kg) × CUMS interaction (F (1, 28) = 53.93, P < 0.01), and Fluoxetine × CUMS interaction (F (1, 28) = 109.2, P < 0.01) in TST. Similarly, the presence of CUMS in the vehicle group contributed to a notable increase in TST immobility time compared with that in the control group without CUMS. By contrast, the immobility time was obviously reduced in the TST with treatment with hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) under CUMS conditions.

3.1.3. Effects of hesperidin on the OFT As shown in Fig. 2D, decreased locomotor activity was revealed in chronically stressed mice compared with the locomotor activity in control mice without CUMS challenge. Treatment with hesperidin and fluoxetine did not significantly alter the number of crossings in nonCUMS conditions. With CUMS induction, the treatments with hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) relieved the reduction of locomotor activity as evidenced by an increased crossing number compared with that in the vehicle group. Two-way ANOVA revealed significant differences for the hesperidin (100 mg/ kg) × CUMS interaction (F (1, 28) = 23.72, P < 0.01), hesperidin (200 mg/kg) × CUMS interaction (F (1, 28) = 29.16, P < 0.01), and 239

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Fig. 3. Effects of hesperidin on pro-inflammatory cytokines in serum of CUMS-induced mice. Data were expressed as mean ± S.D. (n = 8). ## P < 0.01 vs. the control; *P < 0.05 and **P < 0.01 vs. the CUMS group.

#

P < 0.05 and

Fig. 5, the elevated protein levels of HMGB1, RAGE, peNFeκBp65 and p-IκBα were observed in the CUMS-exposed group. However, hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) administrations notably inhibited the expressions of HMGB1, RAGE as well as the phosphorylation of NF-κBp65, IκBα.

Fluoxetine × CUMS interaction (F (1, 28) = 134.7, P < 0.01). The results proved that hesperidin was capable of attenuating CUMS-induced changes in locomotor activity. 3.2. Effects of hesperidin on pro-inflammatory cytokines in mice To evaluate the anti-inflammatory effects of hesperidin, the levels of pro-inflammatory cytokines in serum and the hippocampus were measured using ELISA kits. As presented in Figs. 3–4, CUMS stimulated TNF-α, IL-1β and IL-6 in both serum and the hippocampus. In contrast, the treatments with hesperidin (100 mg/kg, 200 mg/kg) and fluoxetine (20 mg/kg) dramatically decreased the concentrations of IL-1β and IL-6 in serum. The administrations of hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) also significantly inhibited the TNF-α content in serum compared with that in the model group. Furthermore, treatment with hesperidin (100 mg/kg, 200 mg/kg) and fluoxetine (20 mg/kg) markedly reduced hippocampal levels of IL1β and TNF-α. It was noteworthy that hesperidin (200 mg/kg) administration exhibited more efficiency than hesperidin (100 mg/kg) administration on IL-1β inhibition (P < 0.05). Treatment with hesperidin (100 and 200 mg/kg) and fluoxetine (20 mg/kg) evidently inhibited IL6 secretion in the hippocampus. Our data suggested that hesperidin could suppress the inflammatory condition caused by CUMS stimulation in serum and hippocampus.

3.4. Effects of hesperidin on the BDNF/TrkB signaling pathway in CUMSinduced mice

3.3. Effects of hesperidin on the HMGB1/RAGE/NF-κB signaling pathway in the hippocampus of CUMS-induced mice

As shown in Fig. 8, stimulation with corticosterone contributed to elevated levels of IL-1β, IL-6 and TNF-α in the supernatant. The treatments with hesperidin (20 and 40 μM) and fluoxetine (10 μM) effectively decreased the concentrations of IL-1β and IL-6. Moreover, the incubations with hesperidin (10, 20, and 40 μM) and fluoxetine (10 μM) suppressed TNF-α generation compared with that in corticosterone

Next, the BDNF/TrkB signaling pathway was examined. As described in Fig. 6, decreased levels of BDNF and p-TrkB were observed in the CUMS-exposed group. However, hesperidin (200 mg/kg), hesperidin (100 mg/kg) and fluoxetine (20 mg/kg) administrations remarkably restored BDNF expression and TrkB phosphorylation. 3.5. Effects of hesperidin on cell viability in PC12 cells As shown in Fig. 7, the MTT assay results revealed that corticosterone challenge obviously reduced the viability of PC12 cells compared with that in the control cells. However, this change in viability was recovered by hesperidin (10, 20, and 40 μM) treatment to different degrees. 3.6. Effects of hesperidin on pro-inflammatory cytokines in PC12 cells

To explore the intracellular signaling pathway responsible for the anti-depressant effect of hesperidin on CUMS-induced depression, the HMGB1/RAGE/NF-κB signaling pathway was examined. As shown in

Fig. 4. Effects of hesperidin on pro-inflammatory cytokines in hippocampus of CUMS-induced mice. Data were expressed as mean ± S.D. (n = 8). #P < 0.05 and ## P < 0.01 vs. the control；*P < 0.05 and **P < 0.01 vs. the CUMS group；@P < 0.05 vs. the CUMS + hesperidin(100 mg/kg) group. 240

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Fig. 5. Effects of hesperidin on high-mobility group box 1 protein (HMGB1)/receptor for advanced glycation end-products (RAGE)/NF-κB signaling pathway in hippocampus of chronic unpredictable mild stress (CUMS)-induced mice. Data were expressed as mean ± S.D. (n = 3). #P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the CUMS group.

hesperidin (10, 20 and 40 μM) and fluoxetine (10 μM) incubations dramatically inhibited the expressions of HMGB1, RAGE and the phosphorylations of NF-κBp65, IκBα. Of note, hesperidin (40 μM) treatments were more potent than those of hesperidin (10 μM) treatments in the downregulations of HMGB1, RAGE and peNFeκBp65 (P < 0.05).

group.

3.7. Effects of hesperidin on the HMGB1/RAGE/NF-κB signaling pathway in PC12 cells To characterize the intracellular pathway responsible for the ameliorated effect of hesperidin in corticosterone-induced PC12 cells, the HMGB1/RAGE/NF-κB signaling pathway was tested. As shown in Fig. 9, the increased protein levels of HMGB1, RAGE, peNFeκBp65 and p-IκBα were seen in cells induced with corticosterone. In contrast, 241

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Fig. 6. Effects of hesperidin on brain-derived neurotrophic factor(BDNF)/tropomyosin-related kinase B(TrkB) signaling pathway in hippocampus of CUMS-induced mice. Data were expressed as mean ± S.D. (n = 3). #P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the CUMS group.

4. Discussion It was experimentally verified that depressive patients exhibited high concentrations of inflammatory cytokines (Kim et al., 2017). The present study revealed that hesperidin effectively alleviated the depressive-like behaviours of CUMS-induced mice. Hesperidin could suppress the inflammatory cytokine generation and oxidative stress in diabetic depressive rats (El-Marasy et al., 2014). Large numbers of literature demonstrated that hesperidin exhibited anti-inflammatory properties in numerous disorders. It was indicated that hesperidin suppressed Lipopolysaccharide(LPS)-induced neuroinflammation via miRNA-132 cascade (Li et al., 2016). The anti-depressant activity of hesperidin was closely associated with its neuroprotective and anti-inflammatory effects (Antunes et al., 2016). Thus, it was assumed that hesperidin could relieve the depression caused by CUMS challenge. As the mechanism responsible for depression was complex (Han et al., 2013) and the anti-depressant effect of hesperidin was not fully understood, the current work was to explore the potential mechanism of hesperidin on CUMS-induced depression in mice. Increasing evidence has revealed that stress, especially chronic stress, plays an essential role in the pathophysiology of depression. The CUMS depression model is established by stimulating animals with chronic randomizing different low-intensity stress factors to induce a depressive state. This model can mimic the clinical symptoms of depressed patients and is widely used in screening drugs with anti-depressant activity (Yang et al., 2011a). Fluoxetine can cause various adverse effects, such as weight loss, dry mouth, dizziness, sweating, nausea and suicidal tendency (Magni et al., 2013). Thus, it is necessary to seek novel compounds for the treatment of depression. However, there are limited reports on the adverse effects of hesperidin. Furthermore, it was demonstrated that hesperidin could attenuate insulin resistance, lipid accumulation,

Fig. 7. Effects of hesperidin on cell viability in PC12 cell. Data were expressed as mean ± S.D. (n = 3). #P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the corticosterone group.

3.8. Effects of hesperidin on the BDNF/TrkB signaling pathway in PC12 cells As described in Fig. 10, the suppressed expressions of BDNF and pTrkB were observed in cells induced with corticosterone. However, hesperidin (10, 20 and 40 μM) and fluoxetine (10 μM) administrations dramatically restored the changes in BDNF expression. In addition, the treatments with hesperidin (10, 20 and 40 μM) and fluoxetine (10 μM) promoted TrkB phosphorylation.

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Fig. 8. Effects of hesperidin on pro-inflammatory cytokines in the supernatant of corticosterone-induced PC12 cells. Data were expressed as mean ± S.D. (n = 6). # P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the corticosterone group.

Fig. 9. Effects of hesperidin on HMGB1/RAGE/NF-κB signaling pathway in corticosterone-induced PC12 cells. Data were expressed as mean ± S.D. (n = 3). # P < 0.05 and ##P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the corticosterone group; @P < 0.05 vs. the corticosterone + hesperidin (10 μM) group. 243

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Fig. 10. Effects of hesperidin on BDNF/TrkB signaling pathway in corticosterone-induced PC12 cells. Data were expressed as mean ± S.D. (n = 3). #P < 0.05 and ## P < 0.01 vs. the control group; *P < 0.05 and **P < 0.01 vs. the corticosterone group.

hippocampus of mice in the hesperidin (100 and 200 mg/kg)- and fluoxetine (positive control)-treated groups were significantly decreased compared with those in the serum and hippocampus of CUMSinduced group mice. Hesperidin (10, 20 and 40 μM) treatment also inhibited the production of inflammatory cytokines in corticosteroneinduced PC12 cells. The results demonstrated that hesperidin could suppress the generation of pro-inflammatory cytokines in CUMS-induced mice and corticosterone-induced PC12 cells. Neurotrophic factors are proteins that regulate neural circuit development and function in the mammalian brain, affecting neurogenesis, synaptic plasticity and depression. Brain-derived neurotrophic factor (BDNF) is a key regulator of neurotrophic factors. The level of BDNF is abnormally low in depressed patients and can be notably upregulated by anti-depressants. The pharmacological effect of BDNF is regulated by the specific receptor TrkB (Yang et al., 2014). Long-lasting BDNF activation can disturb the balance of hormones and aggravate depression (Yuluğ et al., 2009). The decreased protein levels of BDNF and p-TrkB were dramatically restored with hesperidin administration in our research, which revealed that hesperidin exerted efficient neuroprotective effects on depression. Mounting evidence has demonstrated that the promotion of immune system is involved in the pathogenesis of depression (Mcafoose and Baune, 2009). HMGB1 is expressed by various cell types and is involved in numerous inflammatory disorders. Once triggered by stress, HMGB1 is released and leads to translational modification (Musumeci et al., 2014). HMGB1 functions as a DAMP molecule and activates inflammatory signaling by combining with its primary receptor RAGE. It was confirmed that HMGB1 was required for the treatment of CUMSinduced depression (Wang et al., 2018b). With the treatment of glycyrrhizic acid, the selective inhibitor of HMGB1, previous investigators confirmed that HMGB1 prevented the kynurenine pathway in the development of depressive-like behaviours (Wang et al., 2018a). Glycyrrhizic acid also downregulated the generation of pro-inflammatory cytokines and relieved LPS-induced depression (Wu et al., 2015). HMGB1-RAGE signaling consequently causes the activation of NF-κB, resulting in the augmentation of inflammatory conditions during focal

oxidative stress and the inflammatory process (Omar et al., 2016; Sun et al., 2017b). It is commonly acknowledged that fluoxetine exerts an anti-depressive effect via the selective suppression of serotonin reuptake. Nevertheless, a large number of studies have recently shown that fluoxetine attenuates neuroinflammation through various processes, including anti-oxidative defence, PPAR inhibition, NLRP3 inflammasome inhibition and the enhancement of neurotrophic factors (Alcocer-Gómez et al., 2017; Song et al., 2018a, 2018b). Although numerous pieces of evidence indicated the potential anti-inflammatory effect of fluoxetine on the inflammatory process in depressive aetiology, there were still limited reports providing an overall view of the relationship between selective serotonin reuptake inhibitors and neuroinflammation. We hypothesized that CUMS-induced depression was an inflammatory disorder, and amelioration of the inflammatory reaction caused by fluoxetine might be due to the attenuation of depression. This result might also be attributed to the anti-inflammatory property of fluoxetine itself. Behavioural experiments are commonly applied to assess whether drugs exert anti-depressant properties. A decreased partiality for sugar indicates a lack of appetite and is regarded as an indicator of depression (Masi and Brovedani, 2011). The OFT, FST and TST usually serve as depression-associated indices because of their reliability (Deng et al., 2015). According to the data obtained from the behavioural tests including the SPT, OFT, TST and FST in the current study, hesperidin treatment could substantially attenuate the behavioural deficiencies in CUMS-induced mice. It has been extensively acknowledged that psychosocial stressors may contribute to depression via the inflammatory process (André et al., 2014). It was proposed that pro-inflammatory compounds affect neuronal plasticity, which functions as the physiological foundation for learning and memory during the progression of major depression (Cantone et al., 2017). Additionally, IL-6 is implicated in neurogenesis, neuro-mediation and synaptic plasticity (Sun et al., 2017a). TNF-α plays a crucial role in the cerebral immune response and is considered as a biomarker for depressive disorder (Zhang et al., 2019). In our research, the concentrations of IL-1β, TNF-α and IL-6 in the serum and 244

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ischaemia (Wang et al., 2010a). NF-κB is a major transcription factor governing the transcription of chemokines and inflammatory mediators. Previous studies have illustrated that NF-κB participates in the pathogenesis of inflammation-mediated nerve inflammatory dysfunctions including depression (Dantzer, 2012). The HMGB1/RAGE/NF-κB pathway has been indicated to be required for the treatment of status epilepticus by inhibiting P-glycoprotein expression (Yuan et al., 2017). The inhibitions of HMGB1, RAGE and NF-κB were also efficient for controlling permanent focal ischaemia (Wang et al., 2010b). Our results suggested that hesperidin significantly ameliorated the damage in PC12 cells induced by corticosterone via the increased expressions of the HMGB1/RAGE/NF-κB and BDNF/TrkB pathways. The present study revealed that hesperidin exerted an anti-depressive effect on CUMS-induced mice, which mainly resulted from the modulation of neuroinflammation associated with the HMGB1/RAGE/ NF-κB and BDNF/TrkB signaling pathways. These findings suggested that hesperidin might be used as a neuroprotective agent against depression and contributed to the further understanding of depressive pathogenesis.

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