CREB signaling pathway

CREB signaling pathway

Psychiatry Research 243 (2016) 135–142 Contents lists available at ScienceDirect Psychiatry Research journal homepage: www.elsevier.com/locate/psych...

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Psychiatry Research 243 (2016) 135–142

Contents lists available at ScienceDirect

Psychiatry Research journal homepage: www.elsevier.com/locate/psychres

Schisandra chinensis produces the antidepressant-like effects in repeated corticosterone-induced mice via the BDNF/TrkB/CREB signaling pathway Tingxu Yan a, Mengjie Xu a, Shutong Wan a, Mengshi Wang a, Bo Wu a, Feng Xiao a, Kaishun Bi b, Ying Jia c,n a b c

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China School of Functional Food and wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 5 January 2016 Received in revised form 16 May 2016 Accepted 23 June 2016 Available online 23 June 2016

The present study aimed to examine the antidepressant-like effects and the possible mechanisms of Schisandra chinensis on depressive-like behavior induced by repeated corticosterone injections in mice. Here we evaluated the effect of an ethanol extract of the dried fruit of S. chinensis (EESC) on BDNF/TrkB/ CREB signaling in the hippocampus and the prefrontal cortex. Three weeks of corticosterone injections in mice resulted in depressive-like behavior, as indicated by the significant decrease in sucrose consumption and increase the immobility time in the forced swim test, but without any influence on the locomotor activity. Further, there was a significant increase in serum corticosterone level and a significant downregulation of BDNF/TrkB/CREB signaling pathway in the hippocampus and prefrontal cortex in CORT-treated mice. Treatment of mice with EESC (600 mg/kg) significantly ameliorated all the behavioral and biochemical changes induced by corticosterone. Moreover, pharmacological inhibition of BDNF signaling by K252a abolished entirely the antidepressant-like effect triggered by chronic EESC treatment. These results suggest that EESC produces an antidepressant-like effect in CORT-induced depression in mice, which is possibly mediated, at least in part, by rectifying the stress-based hypothalamic–pituitary– adrenal (HPA) axis dysfunction paradigm and upregulation of BDNF/TrkB/CREB signaling pathway. & 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Antidepressant-like effect Schisandra chinensis Corticosterone HPA axis BDNF/TrkB/CREB signaling

1. Introduction Major depression has been recognized as long as human records have existed, and research shows that it continues to be a widespread major threat to public health, which afflicts one in six people at some point in life, also it is one of the main causes of human suffering and the leading global cause of years of life lived with disability (Ingram, 2016). It is generally assumed that multiple mechanisms are responsible for the development of depression. Despite recent progress in understanding the molecular, cellular, and circuit-level correlates of depression, the biological mechanisms that causally underlie this disease are still unclear. Previous studies have shown a causal relationship between the incidence of major depressive disorders and the dysregulation of hypothalamic-pituitary-adrenal (HPA) axis (Ali et al., 2015; Mizuki et al., 2014). The HPA axis is activated in response to stress, which n

Corresponding author. E-mail address: [email protected] (Y. Jia).

http://dx.doi.org/10.1016/j.psychres.2016.06.037 0165-1781/& 2016 Elsevier Ireland Ltd. All rights reserved.

results in an overproduction of glucocorticoid hormones in the circulating blood. High concentration of blood glucocorticoids is reportedly maintained in patients with depression as compared to healthy controls due to a dysfunction in the feedback mechanism (Chen et al., 2016; Li et al., 2015b). Stimulation and sustained action of the HPA axis are attenuated via the negative feedback action of circulating glucocorticoid following exogenous corticosterone (CORT) administration, and this is closely associated with the development of psychosomatic disorders, which produce serious changes in affective behavior that are indicative of, or consistent with depressive-like symptoms (Lee et al., 2015). These findings suggested that a chronic corticosterone treated rodent model is suitable for evaluating the efficacy of potential antidepressant candidates and to explore the mechanism of action of antidepressants. In addition to hyperactivity of the HPA axis, neurotrophin dysfunction is also involved in the pathogenesis of depression. Brain-derived neurotrophic factor (BDNF) is one of the most extensively investigated targets with respect to brain plasticity.

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Moreover, BDNF has been proposed to participate in stress response by modifying the HPA axis activity (Nowacka et al., 2015). Reduced BDNF expression by adverse stressors contributes to structural anomalies and functional impairment in the central nervous system (CNS). After binding with and activating tropomyosin-related kinase receptor B (TrkB), BDNF is thought to underlie the pathophysiology and treatment of depression. Further, BDNF increases phosphorylation of cAMP response element binding protein (CREB) via TrkB (Liu et al., 2015b). And CREB is involved in social isolation stress-induced emotional deficits (Li et al., 2015a). BDNF–TrkB signaling plays a critical role in the modulation of several functions, such as neurotransmitter release and postsynaptic responses to neurotransmitters, which are closely related to antidepressant therapy (Yi et al., 2014). Due to a prevalent belief that “natural is better,” a significant amount of public interest in antidepressant development has focused on plant material or natural products extracted from plant sources. Schisandra chinensis (Turcz.) Baill., as a traditional Chinese medicine, is a functional food, and is extensively used in the clinic with the functions of inducing astringency, replenishing and promoting the production of body fluid and tonifying the kidney to relieve mental strain (Ahn et al., 2015; Chan, 2012). And the most reported are lignans, an antioxidant component of S. chinensis (Chiu et al., 2002; Li et al., 1996). Based on the previous studies of our group, we found that lignans could ameliorate learning and memory deficits, cognitive declines, exert sedative and hypnotic effects, and mitigate other neurodegenerative symptoms (Li et al., 2014; Mao et al., 2015; Yan et al., 2016; Zhang et al., 2014; Zhao et al., 2016). Moreover, the results of numerous studies implicate the involvement of the HPA axis and CNS in the effects exerted by Schizandra preparations (Panossian and Wikman, 2008). However, the mechanisms of the above effects for S. chinensis are still unclear. The aim of the present study was to investigate the antidepressant-like effect of an ethanol extract of the dried fruit of S. chinensis (EESC) in mice that were repeatedly exposed to exogenous corticosterone. Firstly, we examined the effect of EESC on depressive-like behavior and BDNF, TrkB and CREB protein expression in the hippocampus and prefrontal cortex of CORT-treated mice; secondly, we used K252a, an inhibitor of the BDNF receptor TrkB, to further investigate the direct link between BDNF/ TrkB/CREB signaling and the antidepressant-like effect of EESC in mice following repeated corticosterone administration.

2. Materials and methods 2.1. Animals Adult male Kunming mice (4 weeks, weighing 20 72 g) were purchased from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). All of them were maintained under standard laboratory conditions of constant temperature (23 71 °C), relative humidity (50 710%) and a 12 h light/dark cycle (light from 7:00 a.m. to 7:00 p.m.) with food and water available ad libitum and were allowed to habituate to the novel environment for 1 week prior to use in experiments. The experiment was carried out in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals and was approved by the Animal Care Committee of Shenyang Pharmaceutical University (protocol No.: SYPU-IACUC2015C-1211-101). Every effort was made to minimize the number of animals used and any pain and discomfort experienced by the subjects.

2.2. Preparations of extract of Schisandra chinensis and chemicals The fruits of S. chinensis were purchased from the TCM shop of Tongrentang (Shenyang, China) and identified by Professor Ying Jia (Department of Pharmacognosy, Shenyang Pharmaceutical University) according to the guidelines of the Chinese Pharmacopoeia (2015). Then, the fruits of S. chinensis were exhaustively extracted with 95% ethanol at reflux for 2 h 3 times. After concentration in a vacuum, the residue was suspended in 0.5% sodium carboxymethycellulose (CMC-Na) at a certain concentration of 600 mg/kg or 300 mg/kg. Corticosterone was purchased from Tokyo Chemical Industry (Tokyo, Japan). K252a was purchased from Santa Cruz Biotechnology (California, USA). Fluoxetine hydrochloride as a positive control drug was obtained from Melone Pharmaceutical Co. (Dalian, China). ELISA kits of CORT (LY 201,524), BDNF (LY 201,581), pTrkB (LY 201,510), TrkB (LY 201,509), pCREB (LY 201,674) and CREB (LY 201,530) were purchased from Liyu Bioengineering Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade. 2.3. UPLC–Q-TOF/MS analysis of EESC The chemical composition of EESC was analyzed by using a Waters-UPLC-Q-TOF/MS with an ultraviolet/visible detector (UV/ vis) coupled to an ion trap mass spectrometer with an ESI interface. The separation was achieved on an HSS T3 Column (100 mm  2.1 mm, 1.8 ??m). The elution conditions showed in Table 1. The chromatogram was recorded at 216 nm. Mass analyses were performed using an ESI interface in the positive ion mode. Data were performed with Masslynx V4.1 software. As shown in Fig. 1 and Table 2, fourteen lignans were tentatively identified by the full scan on the positive ion mode of MS/MS analysis. Eight main compounds (1, 4, 7, 8, 11, 12, 13 and 14) of those lignans were identified with the retention time and UV spectra of the reference substance. 2.4. Experimental design and treatment Animals were randomly divided into eight groups with 8 mice in each: Control group, Vehicle group, Corticosterone only, Fluoxetine (10 mg/kg), EESC (300 mg/kg), EESC (600 mg/kg), EESC (300 mg/kg) þK252a and EESC (600 mg/kg)þ K252a. Corticosterone (dissolved in saline containing 0.1% dimethyl sulfoxide (DMSO) and 0.1% Tween-80) was injected subcutaneously at 40 mg/kg in a volume of 1 ml/kg as this dose reliably increases depression-like behavior in mice without altering nonspecific motor activity (Fenton et al., 2015). Fluoxetine (10 mg/kg, suspended in 0.5% CMC-Na) (Cai et al., 2015), EESC (300 mg/kg) and EESC (600 mg/kg) was administrated by gavage 30 min prior to the corticosterone injection. K252a (dissolved in 0.1% DMSO in saline) was injected i.p. in a volume of 10 ml/kg before 30 min of gavage Table 1 The elution conditions of UPLC-Q-TOF/MS for analysis of EESC. (A: Water; B: Acetonitrile). Time (min)

A (%)

B (%)

0 6 8 10 11 14 16 18

57 48 40 38 38 34 30 0

43 52 60 62 62 66 70 100

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Fig. 1. UPLC chromatogram of S.C. (1) schisandrin; (2) gomisin D; (3) gomisin J; (4) schisandrol B; (5) tigloylgomisin H; (6) angeloylgomisin H; (7) schisantherin A; (8) schisantherin B; (9) Schisanhenol; (10) gomisin E; (11) deoxyschizandrin; (12) gomisin N; (13) Schisandrin B; (14) Schisandrin C.

Table 2 Tentative identification of fourteen compounds from EESC by using UPLC-Q-TOF/ MS according to Retention Time and Major Fragments. Peak TR (min) MS(m/z) 1

4.00

2

4.60

3 4 5

4.74 5.04 6.08

6

6.81

7 8 9 10 11

9.01 9.04 9.37 10.44 12.51

12 13 14

14.00 14.38 14.94

Major fragments (m/z)

433.2227[M þH] þ , 415.2125[M þ H  H2O] þ 530.2152 531.2224[Mþ H] þ , 548.2492[M þNH4] þ 388.1886 389.1964[M þH] þ 416.1835 399.1804[Mþ H  H2O] þ 500.2410 501.2477[MþH] þ , 483.2390[M þH  H2O] þ 500.2410 501.2484[M þ H] þ , 483.2390[M þH  H2O] þ 536.2046 537.2094[M þ H] þ 514.2203 515.2275[M þ H] þ 402.2042 403.2116[M þ H] þ 514.2203 515.2275[M þ H] þ 416.2199 417.2284[M þH] þ , 439.2108[M þ Na] þ 400.1886 401.1962[M þ H] þ 400.1886 401.1962[M þ H] þ 384.1573 385.1651[M þ H] þ , 407.1471[M þ Na] þ 432.2148

Chemical compounds Schisandrin Gomisin D Gomisin J Schisandrol B Tigloylgomisin H Angeloylgomisin H Schisantherin A Schisantherin B Schisanhenol Gomisin E Deoxyschizandrin Gomisin N Schisandrin B Schisandrin C

(Brachman et al., 2015). The whole experimental procedure is shown in Fig. 3. 2.5. Sucrose preference test The sucrose preference test represents the anhedonia-like behavioral change and is a behavioral paradigm detecting antidepressant effects (Jin et al., 2015). The sucrose preference test was performed at the beginning of the whole procedure and the end of 3-week corticosterone exposure. Briefly, before the test, the mice were trained to adapt to the sucrose solution (1%, w/v): two bottles of sucrose solution were placed in each cage for 24 h, and then one bottle of sucrose solution was replaced with water for 24 h. After the adaptation, the mice were deprived of water and food for 24 h. The mice were housed in individual cages and had free access to two bottles containing sucrose solution and water, respectively. After 24 h, the volumes of the consumed sucrose solution and water were recorded. Food was withdrawn during the sucrose preference test (Xing et al., 2015). The sucrose preference was calculated as a ratio of the amount of sucrose solution to that of total solution: Sucrose preference (%)¼sucrose consumption/(water consumptionþ sucrose consumption)*100%. 2.6. Locomotor activity test As certain behavioral tasks like the forced swim test can be affected by changes in locomotor activity, the open field test was used to assess locomotor activity. In order to rule out the possibility that the alteration in the immobility time in the forced swim test was due to interference of the locomotor activity, spontaneous locomotor activity of each mouse was observed in an open filed experimental video analysis system (ZS-ZFT, Huaibei Zhenghua Bio-Apparatus Co. Ltd, China). The apparatus was placed in a darkened and sound attenuated testing room. The total path of Spontaneous locomotive was evaluated over a 5 min period (Prut and Belzung, 2003). After each testing session, the enclosures were thoroughly cleaned with 70% ethanol. 2.7. Forced swim test

Fig. 2. The timelines of the whole experiment design.

administration (Kumar et al., 2015). The dose of EESC was chosen based on the results of the forced swim test in the preliminary experiment as shown in Fig. 2. Control mice only received the same volume of 0.5% CMC-Na solution without corticosterion. Mice in the vehicle group received only vehicle (0.1% DMSO and 0.1% Tween-80 saline solution) without corticosterone for the same period. The repeated drug treatment was performed once daily and continuously for 21 days on the other six groups

The forced swim test was carried out 48 h after the sucrose preference test according to the method of Porsolt (Porsolt et al., 1977). Mice were individually placed in a glass cylinder (25 cm high and 10 cm in diameter) containing 10 cm of water maintained at 23–25 °C and were left there for 6 min. A mouse was regarded immobile when it remained floating on the water, making only small movements to keep its head above the water surface. The total duration of immobility was recorded during the last 4 min of a 6 min test session.

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Fig. 3. Effects of different doses of EESC on FST in mice. The data represented the values of mean 7 S.E.M. from 8 mice/group. **po 0.01 vs. Control group.

2.8. Biochemical sample collection Mice were killed under deep anesthesia immediately after the behavioral tests. Blood samples were collected immediately after decapitation and centrifuged at 3000 rpm and 4 °C for 15 min to isolate serum. Serum was stored at  20 °C until use. The entire hippocampus and prefrontal cortex were rapidly removed, and the samples were stored at  80 °C until further estimations. 2.9. Determination of serum corticosterone level Serum corticosterone level was measured using a commercially customized ELISA kit according to the manufacturer's protocol. Briefly, 50 μl of sample and standard solutions were added to the already precoated antibody plate provided with the kit and incubated for 30 min at 37 °C. The reaction was terminated and followed by washing, 50 μl of the TMB color reagent was added and incubated for 20 min without shaking. The reaction was stopped by adding 50 μl of stop solution and absorbance was read at 450 nm using a microplate reader (Varioskan flash, Thermoscientific, USA). 2.10. Determination of BDNF, pTrkB, TrkB, pCREB and CREB BDNF, pTrkB, TrkB, pCREB and CREB concentrations of hippocampus and prefrontal cortex determination were performed using the ELISA method, according to the mouse BDNF, pTrkB, TrkB, pCREB and CREB ELISA kits manufacturer's instructions, respectively. Optical density was obtained at 450 nm using a microplate reader (Varioskan flash, Thermoscientific, USA) within 15 min of stop solution addition. 2.11. Statistical analysis All data were analyzed using one-way ANOVA, followed by Tukey HSD post-hoc test when significant main effects were indicated. All analyses were two-tailed and *p o0.05 was considered significant a priori.

3. Results 3.1. Effects of EESC on the sucrose preference test As shown in Fig. 4A, there was no difference in sucrose preference among animal groups before corticosterone treatment. Fig. 4B presents the effects of EESC on the sucrose preference test in 3 weeks corticosterone-induced mice. One–way ANOVA showed significant differences between animal groups. Subsequent group comparisons revealed that the sucrose preference in CORT group was significantly lower than that in normal control group (p o0.01). Treatment with EESC (300 mg/kg), EESC (600 mg/kg) and fluoxetine (10 mg/kg) increased the percentage of sucrose consumption compared with CORT group (p o 0.05, p o0.01 and p o0.01, respectively). Besides, we also observed that the CORT-

Fig. 4. Effects of EESC administration for base-line (A), and after 3 weeks treatment (B) on the sucrose preference in the SPT. The data represented the values of mean7 S.E.M. from 8 mice/group. **p o 0.01 vs. Control group. #p o0.05 and ##p o0.01 vs. CORT group.

induced decrease in sucrose preference was not respectively reversed by the administration of EESC (300 mg/kg) and EESC (600 mg/kg) in the K252a treatment groups compared with CORT group. In summary, the intervention of K252a blocked the reversion of EESC in increase the sucrose preference of mice exposed to corticosterone. 3.2. Effects of EESC on open field test The effects of EESC on locomotor activity were shown in Table 3. All treatments showed no significant alterations to the total

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Table 3 Influence of administration of EESC, Fluoxetine or K252a on the locomotor activity in mice (total travel distance over a 5 min period). Groups

Locomotion length (cm/5 min)

Control Vehicle CORT Fluoxetine (10 mg/kg) SC (300 mg/kg) SC (600 mg/kg) SC (300 mg/kg)þ K252a SC (600 mg/kg)þ K252a

10647 45 11867 92 993 732 10057 23 985 781 1034 7 67 990 748 899 787

Fig. 6. Effects of EESC administration on the serum corticosterone level. The data represented the values of mean7 S.E.M. from 8 mice/group. **p o0.01 vs. Control group. #po 0.05 and ##p o 0.01 vs. CORT group.

pTrkB/TrkB (Fig. 7B) and pCREB/CREB (Fig. 7C) compared with the control group (p o0.01, p o0.01, p o0.01, respectively). EESC (600 mg/kg) and fluoxetine (10 mg/kg) treatments significantly increased the BDNF, pTrkB/TrkB and pCREB/CREB levels compared with the CORT group (p o0.01 or p o0.05). However, the increases reversed by K252a administration. 3.6. Effects of EESC on BDNF, pTrkB/TrkB and pCREB/CREB levels in prefrontal cortex Fig. 5. Effects of EESC administration on the immobility time in the FST. The data represented the values of mean 7S.E.M. from 8 mice/group. **p o 0.01 vs. Control group. ##p o 0.01 vs. CORT group.

path of spontaneous locomotive activity. 3.3. Effects of EESC on the forced swim test After receiving the corticosterone procedure, a significant increase of immobility duration in CORT-treated mice was observed when compared to the control mice (p o0.01) (Fig. 5). Chronic administration of EESC (600 mg/kg) and fluoxetine (10 mg/kg) (p o0.01, p o0.01) decreased the immobility time significantly. However, this antidepressant effect of EESC was completely blocked by the injection of the BDNF-TrkB receptor antagonist K252a. 3.4. Effects of EESC on serum corticosterone level As shown in Fig. 6, corticosterone injection induced a significant increase in the serum corticosterone level compared with the control group (p o0.01). After 21 days of treatment, EESC (600 mg/kg) and fluoxetine (10 mg/kg) significantly decreased serum corticosterone levels compared with the CORT group (p o0.05, p o0.01). But these decreases were increased by K252a treatment in corticosterone-induced mice. 3.5. Effects of EESC on BDNF, pTrkB/TrkB and pCREB/CREB levels in hippocampus The effects of EESC and fluoxetine on BDNF, pTrkB/TrkB and pCREB/CREB levels in hippocampus in CORT-induced mice were exhibited in Fig. 7. The results revealed that corticosterone induced a significant reduction in the expression of BDNF (Fig. 7A),

The effects of EESC and fluoxetine on BDNF, pTrkB/TrkB and pCREB/CREB levels in prefrontal cortex in CORT-induced mice were exhibited in Fig. 8. There was a significant decrease of BDNF (Fig. 8A), pTrkB/TrkB (Fig. 8B) and pCREB/CREB (Fig. 8C) observed in the corticosterone treated mice compared with the normal control mice. EESC (300 mg/kg or 600 mg/kg) and fluoxetine (10 mg/kg) increased these reductions induced by corticosterone (p o0.01 or po 0.05). These effects were completely blocked by K252a.

4. Discussion In rodents, accumulated evidence has indicated that repeated corticosterone injections induce behavioral and neurochemical aspects of depression. Chronic corticosterone injections reduced sucrose consumption and increased the immobility time on the forced swimming test and the tail suspension test (Mao et al., 2014; Pazini et al., 2015). Furthermore, corticosterone treatment produced adult neurogenesis deficit in the hippocampus of rodents by reducing the levels of hippocampal and cerebrocortical brain-derived neurotrophic factor (Alfarez et al., 2009; Yogesh Dwivedi and Pandey, 2006). These findings suggest that repeated corticosterone injections may mimic the behavioral and neurochemical alterations associated with depression and it is a wellvalidated method to cause depression, and the model has face and predictive validity (Iijima et al., 2010; Zhao et al., 2008). Herein, mice with 3 consecutive weeks of corticosterone administration exhibited the behavioral deficits, including decreased sucrose preference, prolonged immobility time in FST. These behavioral changes of CORT-treated mice suggested that this model of depression was successfully established. Anhedonia, a core symptom of human major depression, is

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Fig. 7. Effects of EESC administration on BDNF (A), pTrkB/TrkB (B) and pCREB/CREB (C) levels in hippocampus. The data represented the values of mean 7 S.E.M. from 8 mice/group. **p o 0.01 vs. Control group. ##p o0.01 vs. CORT group.

modeled by inducing a decrease in responsiveness to rewards, as reflected by a reduced consumption or preference for sucrose solutions (Hsiao and Smith, 1995). The sucrose preference test represents the anhedonia-like behavioral change and is a behavioral

Fig. 8. Effects of EESC administration on BDNF (A), pTrkB/TrkB (B) and pCREB/CREB (C) levels in prefrontal cortex. The data represented the values of mean 7S.E.M. from 8 mice/group. **po 0.01 vs. Control group. #po 0.05 and ##p o 0.01 vs. CORT group.

paradigm detecting antidepressant effects in depression (Kant and Bauman, 1993). We found that corticosterone treatment could indeed reduce the preference for sucrose, which is consistent with

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the previous studies (Kott et al., 2016). The present findings revealed that 3 weeks of EESC (300 mg/kg or 600 mg/kg) treatment by daily gavage on CORT-injected mice could reverse the decreased sucrose preference, and reversed the anhedonic-like behavior, suggesting antidepressant-like actions of EESC. The forced swimming test is a classical behavioral model in rodents for assessing pharmacological antidepressant activity by measuring the persistence time of immobility as described accordingly (Detke et al., 1995). In accordance with previous findings (Colich et al., 2015), corticosterone could significantly increase the immobility time in the forced swimming test, and this phenomenon was reversed by EESC (600 mg/kg) and fluoxetine (10 mg/kg). Taken together, all above results suggested that EESC exerted antidepressant-like effect in the CORT-induced depression mice. It is well-evidenced that the HPA axis consists of a complex set of interactions between the hypothalamus, pituitary gland and adrenal cortex, which mediates the stress response through the actions of glucocorticoids (cortisol in humans, sheep, and guinea pigs, and corticosterone in rodents) (Xiong and Zhang, 2013). Chronically elevated levels of glucocorticoids can affect neurogenesis and dendrite growth in the hippocampus, which in turn contribute to a dysregulation of the HPA axis, as under normal conditions the hippocampus controls the negative feedback of the HPA axis (Demuyser et al., 2015). In line with the previous reports (Li et al., 2015c), we observed that a notable elevation of serum corticosterone level in mice followed by chronic corticosterone injection, which means that exogenous corticosterone infusion produced a similar effect with hyperactivity of HPA axis. EESC (600 mg/kg) and fluoxetine (10 mg/kg) significantly decreased the serum corticosterone level almost to the normal status. That demonstrated the antidepressant-like effect of EESC, and suggested the mechanism was possibly involved in the reduction of serum corticosterone level and accompanied by alterations in the HPA axis. Although neurotrophic factors are critical signaling molecules for nervous system development, they continue to play an important role in the survival, function and adaptive plasticity of neurons in the adult brain (Duman and Voleti, 2012). BDNF is one of the most extensively investigated targets with respect to brain plasticity, survival and differentiation in both the periphery and central nervous system (Sousa et al., 2015). According to the hypothesis of neurotrophins in depression, the expression of BDNF in the hippocampus was decreased, followed by exogenous corticosterone treatment, but it was significantly increased in adrenalectomy rats, which disclosed that the expression level of BDNF in the hippocampus is possibly regulated by glucocorticords (Wang et al., 2014b). A postmortem study reported that the expression of BDNF was decreased in the prefrontal cortex and the hippocampus of suicide subjects with depression, while a similar reduction in BDNF was not observed in antidepressant-treated subjects (Karege et al., 2005), therefore we chose the hippocampus and the prefrontal cortex as the targets of investigation region. TrkB, a member of the Trk family of receptor tyrosine kinases, is a highaffinity receptor for BDNF and it could mediate the cellular actions of BDNF (De Vry et al., 2016). Decreased levels of BDNF and TrkB were both shown in the hippocampus of suicide and depressed patients (Autry and Monteggia, 2012). As a transmembrane receptor with an intracellular tyrosine kinase domain, TrkB upregulates or downregulates many second messengers that lead to the phosphorylation of transcriptional factors such as CREB. CREB is a regulator of gene expression and is involved in many processes that are associated with neuroprotection. CREB was found to play an important role in the pathophysiology of neuropsychiatric disorders as well as in the response of psychotropic drugs (Mlyniec and Nowak, 2015). A reduction in CREB level was observed in animal models of depression. Moreover, postmortem studies

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showed a significant decrease of CREB in the cortex (Yamada et al., 2003). All together indicated that BDNF mitigates depressive symptoms mainly by binding to TrkB, leading to autophosphorylation of TrkB tyrosine residues, and activation of downstream signaling molecules, including the phosphorylate CREB (Lin et al., 2014). In this study, we found that EESC (600 mg/kg) administration upregulated the BDNF/TrkB/CREB signaling pathway. Further, both antidepressant-like effects and enhanced BDNF/ TrkB/CREB signaling were completely blocked by K252a. The K252 family of alkaloid toxins are protein kinase inhibitors, and K252a has a long history of being used to antagonize Trk receptors, with particular affinity for TrkB (Pinnock et al., 2010). Treatment of K252a was found to abolish the antidepressant-like effect of BDNF and the antidepressant-like effects of antidepressant in the forced swim test and animal model of depression (Liu et al., 2015a; Wang et al., 2014a). Taken together, in line with the previous studies, these results indicated that activation of BDNF/TrkB/CREB signaling is required for antidepressant actions of EESC. In conclusion, our data provide new information with regard to the antidepressant-like effect of EESC in corticosterone-induced mice. Furthermore, we observed that EESC could regulate the HPA axis and the BDNF/TrkB/CREB signaling following chronic administration. On the contrary, pharmacological inhibition of BDNF signaling by K252a abolished entirely the antidepressant-like effect of chronic EESC treatment, revealing that BDNF/TrkB/CREB signaling is involved in the effect. Although additional studies are required to further investigate in detail of the interaction between HPA axis and BDNF/TrkB/CREB downstream signaling pathway.

Conflict of interest statement The authors have no conflict of interest to declare.

Acknowledgements This research was supported by National Natural Science Foundation of China (No. 81573580) and Natural Science Foundation of Liaoning Province of China (No. 2014020076).

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