ERK-dependent brain-derived neurotrophic factor regulation by hesperidin in mice exposed to chronic mild stress

Brain Research Bulletin 124 (2016) 40–47

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ERK-dependent brain-derived neurotrophic factor regulation by hesperidin in mice exposed to chronic mild stress Cheng-Fu Li a,∗ , Shao-Mei Chen a , Xue-Mei Chen a , Rong-Hao Mu b , Shuang-Shuang Wang b , Di Geng b , Qing Liu b , Li-Tao Yi b,∗ a b

Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361009, Fujian province, PR China Department of Chemical and Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian province, PR China

a r t i c l e

i n f o

Article history: Received 24 January 2016 Received in revised form 19 March 2016 Accepted 23 March 2016 Available online 24 March 2016 Keywords: Hesperidin Corticosterone Brain-derived neurotrophic factor Antidepressant Chronic mild stress Hemerocallis citrina

a b s t r a c t A previous study found that the antidepressant-like effects of ethanolic extracts from Hemerocallis citrina are predominantly related to the flavonoid, hesperidin. The study herein aimed to explore the antidepressant-like mechanism of hesperidin in mice induced by chronic mild stress (CMS). The results indicated that hesperidin reversed the reduction of sucrose preference and the elevation of immobility time in mice induced by CMS. In addition, the increase in serum corticosterone levels and decrease in hippocampal extracellular signal-regulated kinase (ERK) phosphorylation and brain-derived neurotrophic factor (BDNF) levels in CMS mice were also ameliorated by hesperidin treatment. In contrast, improvement by hesperidin was suppressed by pretreatment with ERK inhibitor SL327. Taken together, our findings confirmed the antidepressant-like effect of hesperidin and indicated that hesperidin-induced BDNF up-regulation was mediated in an ERK-dependent manner. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Depression is a serious mental health condition that requires medical treatment. The main medical treatment for depression is antidepressant medication. Despite numerous clinical studies, the molecular mechanisms underlying the therapeutic effects of antidepressants are not fully understood. Thus, research has focused on the mechanisms of antidepressants, from the monoamine hypothesis to other recent hypotheses (Berton and Nestler, 2006). The neurotrophic hypothesis, a new explanation in antidepressant treatment, has gained more attention because it demonstrates why clinically available antidepressants have a lag in action after starting treatment (Duman, 2014). Numerous studies have shown that brain-derived neurotrophic factor (BDNF), the most abundant neurotrophins in the central nervous system, played a vital role in the treatment of depression (Hill et al., 2012).

Abbreviations: BDNF, brain-derived neurotrophic factor; CREB, cAMP-response element binding protein; CMS, chronic mild stress; ERK, extracellular signalregulated kinase; FST, forced swimming test; GPCR, G-protein-coupled receptor; TrkB, tropomyosin receptor kinase B. ∗ Corresponding authors. E-mail addresses: [email protected] (C.-F. Li), [email protected], [email protected] (L.-T. Yi). http://dx.doi.org/10.1016/j.brainresbull.2016.03.016 0361-9230/© 2016 Elsevier Inc. All rights reserved.

Chronic mild stress (CMS), one model simulating the process of human depressive disorder, has been widely used to study the etiology of depression and evaluate the mechanism of antidepressants (Willner, 1997). During the CMS procedure, researchers firstly assess the sucrose preference of animals, a measure simulating assessment of anhedonia in depressed patients, to judge whether the model is successful or the antidepressants are effective (D’Aquila et al., 1997). Hesperidin (Fig. 1) is a flavanone glycoside extracted from Hemerocallis citrine, and its biological activities have recently been widely demonstrated in laboratory research. Hesperidin not only alleviates cancer and cardiovascular diseases (Roohbakhsh et al., 2015), but also improves central nervous system related disorders. For example, recent studies have shown that hesperidin ameliorated neurotoxicity and exerted neuroprotection (Chang et al., 2015; Justin Thenmozhi et al., 2015), improved cognition and preserved memory (Gaur and Kumar, 2010; Banji et al., 2014), as well as attenuated neuro-inflammatory reaction and reversed behavioral impairments (Li et al., 2015). Moreover, our and other previous study found that hesperidin was one of the most dominant constituents of H. citrine, and suggested that the antidepressant-like effects of H. citrine could be attributed to that of hesperidin (Yi et al., 2012; Du et al., 2014). Hesperidin decreased the immobility time in both the tail suspension test and forced swimming test (FST) (Donato et al., 2014,2015; El-Marasy et al., 2014), and inhibited

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Fig. 1. The chemical structure of hesperidin.

serum corticosterone levels in CMS mice (Cai et al., 2013). Furthermore, one study by Donato et al. (2014) indicated that hesperidin increased hippocampal BDNF levels in normal mice; however, it was unclear whether hesperidin could regulate BDNF expression in stress-induced mice, as depression is a stress related mental disorder. In addition, it was also unclear how BDNF expression was regulated. Therefore, our present study aimed to explore the antidepressant-like effect of hesperidin and a possible mechanism of the antidepressant-like effect in the hippocampus of CMS mice. 2. Materials and methods 2.1. Animals Male ICR mice (24 ± 2 g; 5 weeks old) were purchased from the Shanghai Slac Animal Center, PR China. Five animals were housed per cage (320 × 180 × 160 mm) with a normal 12-h/12-h light/dark schedule with the lights on at 07:00 a.m. The animals were allowed one week to acclimatize to the housing conditions before the beginning of the experiments. Ambient temperature and relative humidity were maintained at 22 ± 2 ◦ C and 55 ± 5%, respectively, and the animals were given standard chow and water ad libitum for the duration of the study. All procedures were approved and performed in accordance with the published guidelines of the China Council on Animal Care (Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the State Council on 31 October, 1988 and promulgated by Decree No. 2 of the State Science and Technology Commission on 14 November, 1988). 2.2. Chemicals and reagents Hesperidin (purity >98% by HPLC) was purchased from Shanxi Huike Botanical Development Co., Ltd. (Xi’an, P.R. China). Fluoxetine was purchased from Sigma-Aldrich (St. Louis, USA). SL327 was purchased from Medchem Express (Princeton, USA). BDNF antibody (#sc-546) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). ERK1/2 (#4695S), phospho-ERK1/2 (#4370S) and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology (Beverly, USA). 2.3. Drug administration To confirm the antidepressant-like effects of hesperidin, mice were randomly divided into five groups (n = 10): the control-vehicle group (saline), the CMS-vehicle group (saline), the CMS-hesperidin group (25, 50 mg/kg), and the CMS-fluoxetine group (20 mg/kg). Drug was administered by oral gavage in a volume of 10 ml/kg body

weight. The repeated drug treatment was performed once daily for the last 3 weeks of the CMS procedure. To evaluate the role of ERK signaling in the antidepressantlike effect of hesperidin, mice were randomly divided into five groups (n = 10): the control-vehicle group (saline), the CMS-vehicle group (saline), the CMS-hesperidin group (25 mg/kg), the CMSSL327 group (30 mg/kg), the CMS-hesperidin combined SL327 group (25 + 30 mg/kg). Saline/hesperidin were administered by oral gavage, and saline/SL327 was intraperitoneally injected in a volume of 10 ml/kg body weight. The drug treatment was repeated once daily for the last 3 weeks of the CMS procedure (Fig. 2). 2.4. CMS procedure The mice were firstly trained to consume a sucrose solution before CMS. The baseline sucrose intake tests were performed five times (once every three days). Based on sucrose consumption during the five training tests, the animals were randomly distributed into two matched groups (stressed and control mice). The CMS procedure was performed according to the traditional method described by Willner et al. (1987), with some modifications (Willner et al., 1987). Briefly, the weekly stress regime consisted of food and water deprivation, exposure to an empty bottle, exposure to a soiled cage, light/dark succession every 2 h, space reduction, 45◦ cage tilt, overnight illumination, and predator sounds (Table 1). Mice were group-housed during the CMS procedure, except during the sucrose preference test. All stressors were applied individually and continuously both day and night. The control animals were housed in a separate room and had no contact with the stressed groups. To prevent habituation and ensure unpredictability of the stressors, all stressors were randomly scheduled over a one-week period and repeated throughout the six-week experiment. Based on their sucrose preference following four weeks of CMS, both stressed and control mice were divided into matched subgroups. 2.5. Sucrose preference test The sucrose preference test was conducted at the end of four and seven weeks of CMS exposure (1 h after the last drug treatment). Briefly, before the sucrose preference test, the mice were trained to adapt to the sucrose solution (1%, w/v) using two bottles of sucrose solution 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 test was conducted at 9:30 a.m. The mice were housed in individual cages and had free access to two bottles, one containing sucrose solution and the other containing water. After 24 h, the volumes of

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Fig. 2. The experimental protocol. Mice were acclimated for one week and trained with sucrose solution for five times in the next two weeks (once every three days). Then the CMS procedure was performed until scarification. First sucrose preference test (SPT) was conducted on day 28 and finished on day 29. The treatment of hesperidin (and) SL327 was subsequently administrated daily starting on day 29 and lasting until the day performing FST. Second SPT was conducted on day 49 and finished on day 50. FST was performed on day 51. Finally, the mice were sacrificed by decapitation on day 52.

Table 1 CMS procedure.

the consumed sucrose solution and water were recorded. The CMS procedure was also performed during the sucrose preference test. 2.6. FST The FST was performed after 24 h of the last sucrose preference as described in detail elsewhere, with some modification (Bourin et al., 2004). Briefly, mice were individually placed in a glass cylinder (20 cm in height, 14 cm in diameter) filled with 10-cm high water (25 ± 2 ◦ C). All animals were forced to swim for 6 min, and the duration of immobility was recorded during the final 4 min interval of the test. The immobility period was regarded as the time spent by the mouse floating in the water without struggling and making only those movements necessary to keep its head above the water. The test sessions were recorded by a video camera and scored by an observer blind to treatment. The CMS procedure was paused from the drug treatment (1 h prior to FST) to the completion of the FST. 2.7. Blood and tissue sampling Animals were sacrificed by decapitation one day after the FST. To avoid fluctuations in hormone levels due to circadian rhythms, the animals were bled at 10:00 a.m.–12:00 p.m. on the day of sacrifice. The brain region of hippocampus was isolated immediately, and then stored at −80 ◦ C for future measurement of BDNF and ERK.

2.9. Western blot Each hippocampus sample was randomly selected and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH7.4), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM trichostatin A, phosphatase inhibitor cocktail. The homogenates were centrifuged at 14000g for 20 min at 4 ◦ C, and the supernatants were collected. The protein concentration was determined by a BCA assay. The total proteins were separated by SDS-PAGE and were transferred to a PVDF membrane. Following blocking in 3% BSA/TBST at room temperature for 1 h, the membranes were incubated with the appropriate primary antibodies at 4 ◦ C overnight (anti-BDNF: 1:500, anti-ERK: 1:1000, anti-pERK: 1:1000, anti-GAPDH: 1:5000). After the membranes were washed three times with TBST, the membranes were incubated with an HRP-labeled secondary antibody (1:4000). The blots were washed again for three times with TBST buffer and the immunoreactive bands were detected using an enhanced chemiluminescence method. The results were normalized using GAPDH expression as the internal standard. 2.10. Statistical analysis Data are presented as the mean ± S.E.M. and analyzed by oneway or two-way ANOVA followed by post-hoc Newman-Keuls tests. A value of P < 0.05 was considered statistically significant.

2.8. Serum corticosterone assay 3. Results Blood was collected on ice and separated in a refrigerated centrifuge at 4 ◦ C (4000g for 10 min). The serum was stored at −20 ◦ C until assays were performed. Serum corticosterone levels were measured using an enzyme immunoassay kit (Enzo Life Sciences, USA).

3.1. Effects of hesperidin on behaviors induced by CMS The results of the sucrose preference test and FST at week 7 are presented in Fig. 3. CMS induced a reduction of sucrose

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Fig. 3. Effects of hesperidin and fluoxetine on sucrose preference (A) and immobility time (B) in mice. Values represent means ± SEM. n = 10 per group. ## P < 0.01 vs. control group. * P < 0.05 and ** P < 0.01 vs. CMS vehicle group.

Serum corticosterone (ng/ml)

3.3. Effects of hesperidin on hippocampal ERK phosphorylation and BDNF expression induced by CMS

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Fig. 4. Effects of hesperidin and fluoxetine on serum corticosterone concentrations in mice. Values represent means ± SEM. n = 10 per group. ## P < 0.01 vs. control group. ** P < 0.01 vs. CMS vehicle group.

preference [F(1,18) = 10.37, p < 0.01] and an elevation of immobility time [F(1,18) = 13.23, p < 0.01] in mice. One-way ANOVA revealed a significant change in sucrose preference [F(3,36) = 6.30, p < 0.01] and immobility time [F(3,36) = 6.09, p < 0.01] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin (25, 50 mg/kg) and fluoxetine (20 mg/kg) could significantly reverse the CMS-induced decreased sucrose preference (p < 0.01, p < 0.01, p < 0.01, respectively) and increased immobility time (p < 0.01, p < 0.01, p < 0.01, respectively) in mice.

3.2. Effects of hesperidin on serum corticosterone levels induced by CMS The results of serum corticosterone levels are presented in Fig. 4. CMS induced a significant elevation of serum corticosterone levels [F(1,18) = 16.22, p < 0.01]. One-way ANOVA revealed a significant change [F(3,36) = 7.55, p < 0.01] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin (25, 50 mg/kg) and fluoxetine (20 mg/kg) could significantly reverse the CMS-induced increased corticosterone levels (p < 0.01, p < 0.01, p < 0.01, respectively) in mice.

As shown in Fig. 5A, CMS induced a significant reduction of ERK phosphorylation in the hippocampus [F(1,8) = 7.10, p < 0.05]. One-way ANOVA revealed a significant change [F(3,16) = 4.92, p < 0.05] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin (25, 50 mg/kg) and fluoxetine (20 mg/kg) could significantly reverse the CMS-induced decreased ERK phosphorylation (p < 0.05, p < 0.05, p < 0.05, respectively) in the hippocampus. The results of hippocampal BDNF expression are presented in Fig. 5B. CMS induced a significant reduction of BDNF expression in the hippocampus [F(1,6) = 16.49, p < 0.01] of mice. One-way ANOVA revealed a significant change [F(3,12) = 11.10, p < 0.01] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin (25, 50 mg/kg) and fluoxetine (20 mg/kg) could significantly reverse the CMS-induced decreased BDNF expression (p < 0.01, p < 0.01, p < 0.01, respectively) in the hippocampus.

3.4. Effects of SL327 on hesperidin-mediated behaviors induced by CMS To investigate the involvement of the ERK signaling pathway in the antidepressant-like effects of hesperidin, an inhibitor of ERK, SL327 was injected with or without hesperidin administration. Two-way ANOVA indicated a significant effect of hesperidin on the sucrose preference [F(1,36) = 4.52, p < 0.05]. However, the effect of SL327 [F(1,36) = 2.76, p > 0.05] and the interaction of hesperidin × SL327 [F(1,36) = 3.06, p > 0.05] were not significant. Additionally, only the interaction [F(1,36) = 4.34, p < 0.05] of the immobility time was found in the FST. The effects of hesperidin [F(1,36) = 2.83, p < 0.05] and SL327 [F(1,36) = 3.36, p > 0.05] were not significant. Consistent with the results above, CMS induced a reduction of sucrose preference [F(1,18) = 10.45, p < 0.01] and an elevation of immobility time [F(1,18) = 7.95, p < 0.05] (Fig. 6). One-way ANOVA revealed a significant change of sucrose preference [F(3,36) = 3.44, p < 0.05] and immobility time [F(3,36) = 3.51, p < 0.05] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin reversed the CMS-induced abnormality (p < 0.05, p < 0.05, respectively). However, these effects were prevent by SL237 injection (p < 0.05, p < 0.05, respectively).

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Fig. 5. Effects of hesperidin and fluoxetine on hippocampal ERK phosphorylation (A) and BDNF expression (B) in mice. Values represent means ± SEM. n = 4-5 per group. # P < 0.05 and ## P < 0.01 vs. control group. * P < 0.05 and ** P < 0.01 vs. CMS vehicle group.

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Fig. 6. SL327 (30 mg/kg) abolishes the antidepressant-like effects of hesperidin (25 mg/kg) in both sucrose preference test (A) and forced swimming test (B). Values represent means ± SEM. n = 10 per group. # P < 0.05 and ## P < 0.01 vs. control group. * P < 0.05 vs. CMS vehicle group. & P < 0.05 vs. CMS-hesperidin group.

3.5. Effects of SL327 on hesperidin-BDNF expression induced by CMS Firstly, we measured ERK phosphorylation after hesperidin and SL327 treatment (Fig. 7A). Two-way ANOVA indicated that the effect of SL327 [F(1,16) = 13.83, p < 0.01] and the interaction [F(1,16) = 12.44, p < 0.01] but not the effect of hesperidin [F(1,16) = 3.01, p < 0.05] were significant. Also, we found that CMS induced a significant reduction of ERK phosphorylation in the hippocampus [F(1,8) = 13.07, p < 0.01]. One-way ANOVA revealed a significant change [F(3,16) = 9.76, p < 0.01] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin reversed the CMS-induced decreased ERK phosphorylation (p < 0.05) in the hippocampus. However, this increase was prevented by SL237 injection (p < 0.05). Next, as shown in Fig. 7B, two-way ANOVA found that the effect of hesperidin [F(1,14) = 11.68, p < 0.01], the effect of SL327 [F(1,14) = 5.60, p < 0.05] and the interaction [F(1,14) = 16.71, p < 0.01] were significant. CMS induced a significant reduction of BDNF expression in the hippocampus [F(1,6) = 21.15, p < 0.01]. One-

way ANOVA revealed a significant change [F(3,14) = 10.40, p < 0.01] among the groups. Newman-Keuls post-hoc test indicated that treatment with hesperidin reversed the CMS-induced decreased BDNF expression (p < 0.01) in the hippocampus. However, this increase was prevented by SL237 injection (p < 0.01).

4. Discussion In the present study, we found that continuous stress resulted in a decreased sucrose preference in mice, indicating that CMS mice appeared the anhedonia. However, this reduction was ameliorated by chronic administration with hesperidin for three weeks. Subsequently, we adopted the FST to evaluate the effects of CMS and hesperidin. In parallel to the change in sucrose preference, hesperidin also reversed the increased immobility time induced by CMS. Taken together, these two behavioral tests verified that hesperidin possessed antidepressant-like action. Our present study indicated the antidepressant-like effects of hesperidin at 25 mg/kg and 50 mg/kg. Notably, in other studies, a lower dose of hesperidin was used to evaluate its antidepressant-

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Fig. 7. SL327 (30 mg/kg) abolishes the effects of hesperidin (25 mg/kg) on ERK phosphorylation (A) and BDNF expression (B) in the hippocampus. Values represent means ± SEM. n = 4-5 per group. ## P < 0.01 vs. control group. * P < 0.05 and ** P < 0.01 vs. CMS vehicle group. & P < 0.05 and && P < 0.01 vs. CMS-hesperidin group.

like effects. For example, Donato et al. found that hesperidin at 0.1 mg/kg possessed antidepressant-like effects in mice after treatment (Donato et al., 2014,2015). Also, another study found that hesperidin at higher doses (25–100 mg/kg in rats, equivalent to 35–140 mg/kg in mice) produced antidepressant-like effects in diabetic rats (El-Marasy et al., 2014). However, also another study found that at 40 mg/kg, hesperidin did not exert antidepressantlike effects (Cai et al., 2013). In our preliminary study, we did not observe a positive effect of hesperidin at doses ranging from 1 mg/kg to 10 mg/kg. We think this discrepancy could be attributed to the animal species, animal strains and stress procedures used. Therefore, we selected 25 mg/kg and 50 mg/kg in the present study. Next, we measured serum corticosterone levels in mice exposed to CMS. It is generally accepted that neuroendocrine hyperactivity is involved in the pathophysiology of depression (Martinac et al., 2014; Rhebergen et al., 2015) the brain region of hippocampus and cause the impairment of cognition and memory. In our present study, we found that CMS increased serum corticosterone levels, and hesperidin reversed this elevation. These results imply that hesperidin can restore the hyperactivity of neuroendocrine function. BDNF plays a vital role in the process of cognition and memory. After binding with high affinity to the tropomyosin receptor kinase B (TrkB) receptor, BDNF can modulate neurotransmission and enhance synaptic function in a variety of mechanisms (Bjorkholm and Monteggia, 2015). Thus, BDNF is considered to be involved in psychiatric diseases and may serve as a therapeutic target for the treatment of these disorders (Autry and Monteggia, 2012). Monteggia et al. demonstrated that female mice with BDNF knockout had decreased sucrose preference in the sucrose preference test and increased immobility time in the FST after chronic stress. Both male and female mice attenuated the actions of the antidepressant desipramine in the FST (Monteggia et al., 2007). Another study showed that the deletion of TrkB restricted the proliferation of newborn neurons, and these TrkB knockout mice lacked positive response to antidepressants fluoxetine and imipramine in the FST (Li et al., 2008). These studies provided direct evidence that alterations in BDNF contribute to stress regulation and antidepressant treatment (Duman and Li, 2012). To investigate the neurotrophic mechanism in the antidepressant-like effects of hesperidin, we

subsequently measured BDNF expression in the hippocampus. Accumulating evidence has shown that various treatments can increase the BDNF levels both in depressed patients and stressinduced animals (Bus et al., 2015; Yi et al., 2015; Yu et al., 2015), although there are some controversial reports about the relationship between depression and BDNF in clinical studies (Basterzi et al., 2009; Tsuchimine et al., 2015). As a key target for depression treatment, BDNF can recognize and bind to its receptor to activate down-stream signaling pathways and lead to promotion of the neuronal proliferation and survival (Duman, 2009; Mullen et al., 2012). The results herein indicated that hesperidin attenuated the reduction of BDNF expression in the hippocampus induced by CMS, suggesting the potential role of BDNF involvement in the antidepressant-like effects of hesperidin. These results corroborated those of El-Marasy et al. (2014) and Donato et al. (2014), who demonstrated that hesperidin elevated BDNF levels in the entire brain and hippocampus in rodents. Because BDNF is involved in synaptic growth and promotes neurogenesis (Choi et al., 2009; Waterhouse et al., 2012), the antidepressant-like effect of hesperidin may be mediated, at least in part through enhanced neurogenesis in CMS mice. Moreover, in parallel with the finding that BDNF in the hippocampus was required for clinical antidepressant efficacy (Adachi et al., 2008), our study also found that fluoxetine increased BDNF levels in this brain region. Numerous signaling pathways can undoubtedly promote the expression of BDNF. In the present study, we focused on the ERK signaling pathway, as a growing number of studies indicated that ERK signaling played an important role in the central regulation (Menard et al., 2015). First, numerous studies have suggested that the ERK signaling pathway controls the neural plasticity, survival and apoptosis (Wiegert and Bading, 2011; Zhu et al., 2015). In addition, the function of ERK is partly accomplished via cAMP-response element binding protein (CREB)-dependent gene expression. Briefly, ERK phosphorylation activates its downstream CREB phosphorylation, and the activation of CREB in turn opens the transcription of target genes including BDNF to alter animals’ behaviors and promote neurogenesis. Moreover, the blockade of ERK signaling by its inhibitors resulted in abnormal behaviors and abolished antidepressant response (Duman et al., 2007; Zhang et al., 2012). In accordance with these previous studies, we found that

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hesperidin restored the reduction of ERK phosphorylation in CMS, and this increase was abolished by SL327, an inhibitor of ERK. Furthermore, the improvement in behavior and BDNF expression was also blocked by SL327. These results suggest that hesperidin exerts antidepressant-like action and regulates BDNF expression in an ERK-dependent manner. Similar to our study, another report indicated that hesperidin promoted the phosphorylation of ERK to up-regulate the expression of heme oxygenase-1 (Chen et al., 2010), an endogenous anti-oxidative enzyme activated by the antidepressant desipramine (Lin et al., 2012). Moreover, the authors also found that PD184161, another ERK inhibitor, inhibited ERK phosphorylation and the downstream target gene expression induced by hesperidin (Chen et al., 2010). ERK can be activated by multiple G-protein-coupled receptors (GPCRs), including serotonergic and noradrenergic receptors, and these receptors are activated by serotonin and noradrenaline, respectively (Berumen et al., 2012; Lorton and Bellinger, 2015). Study has shown that noradrenaline activated BDNF by the GPCRPKA/ERK/PI3K-CREB signaling pathway (Chen et al., 2007). Our previous study showed that the antidepressant-like effects of Hemerocallis citrina were dependent on the serotonergic and noradrenergic receptors as well as the elevation of serotonin and noradrenaline levels in hippocampus (Gu et al., 2012). Considering that hesperidin is the main constituent of Hemerocallis citrina, we speculate that hesperidin may increase the levels of serotonin and noradrenaline, which in turn activate serotonergic and noradrenergic receptors. The activated monoaminergic receptors up-regulate ERK phosphorylation and finally promote the expression of BDNF. Of course, ERK can be activated by many receptors and signaling pathways, including BDNF-dependent TrkB activation. Thus, ERK and BDNF co-regulate one another such that ERK stimulates the expression of BDNF, and BDNF activates the ERK signaling pathway (Mattson et al., 2004). Various studies have shown that after binding TrkB, BDNF activates its downstream intracellular signaling cascades such as PLC/PKC, AMPK/ACC, NF␬B, PI3K/Akt and Ras/ERK (Sandhya et al., 2013). The latter two signaling pathways are considered to inhibit neuronal apoptosis as well as promote neuronal proliferation and differentiation (Jiang et al., 2012; Koskimaki et al., 2015). Several studies also found that the BDNF-ERK signaling pathway was involved in the antidepressant-like effects of alarin, ginsenoside Rg1 and oleanolic acid (Jiang et al., 2012; Wang et al., 2014; Yi et al., 2014). Therefore, if the ERK signaling pathway is blocked by SL327, the number of neurons may be decreased and in turn reduces the synthesis of BDNF. Therefore, considering the complexity of ERK regulation, further studies are required to elucidate the detailed mechanism of hesperidin. Based on the results from behavioral and molecular experiments, the effects of hesperidin (25, 50 mg/kg) are similar to that of fluoxetine (20 mg/kg), as there was no difference between the effects of fluoxetine and hesperidin on behaviors, ERK and BDNF. However, it is hard for us to draw a confident conclusion that the efficiency of hesperidin is equal to that of fluoxetine, since fluoxetine was evaluated in large-scale clinical trials and clinical applications; however, hesperidin has not yet been approved for clinical trials.

5. Conclusion In summary, we verified that hesperidin, the main constituent of Hemerocallis citrina, produced antidepressant-like effects and increased the levels of hippocampal p-ERK and BDNF in CMS mice. All alterations observed in hesperidin-treated mice were abolished by SL327, an ERK inhibitor. Taken together, it can be presumed that the ERK-BDNF signaling pathway participates in the antidepressant-like effects of hesperidin.

Conflict of interest The authors report no conflict of interest. Contributors Authors C.-F. Li and L.-T. Yi designed the study and wrote the protocol. Authors C.-F. Li, S.-M. Chen, X.-M. Chen, R.-H. Mu, and S.S. Wang conducted the experiment. Authors R.-H. Mu, C.-F. Li and Q. Liu managed the literature searches and analyses. Authors R.H. Mu and D. Geng undertook the statistical analysis. Authors C.-F. Li and L.-T. Yi wrote the first draft of the manuscript. All authors contributed to and have approved the sfinal manuscript. Acknowledgements The project was supported by grants from the Science Research Foundation of ministry of Health & United Fujian Provincial Health and Education Project for Tacking the Key Research (WKJ-FJ31), the Youth Research Foundation of Health Department of Fujian Province (2012-2-108), and the Outstanding Youth Scientific Research Training Program in Colleges and Universities of Fujian Province (JA14015). References Adachi, M., Barrot, M., Autry, A.E., Theobald, D., Monteggia, L.M., 2008. Selective loss of brain-derived neurotrophic factor in the dentate gyrus attenuates antidepressant efficacy. Biol. Psychiatry 63, 642–649. Autry, A.E., Monteggia, L.M., 2012. Brain-derived neurotrophic factor and neuropsychiatric disorders. Pharmacol. Rev. 64, 238–258. Banji, O.J., Banji, D., Ch, K., 2014. Curcumin and hesperidin improve cognition by suppressing mitochondrial dysfunction and apoptosis induced by d-galactose in rat brain. Food Chem. Toxicol. 74, 51–59. Basterzi, A.D., Yazici, K., Aslan, E., Delialioglu, N., Tasdelen, B., Tot Acar, S., et al., 2009. Effects of fluoxetine and venlafaxine on serum brain derived neurotrophic factor levels in depressed patients. Prog. Neuropsychopharmacol. Biol. Psychiatry 33, 281–285. Berton, O., Nestler, E.J., 2006. New approaches to antidepressant drug discovery: beyond monoamines. Nat. Rev. Neurosci. 7, 137–151. Berumen, L.C., Rodriguez, A., Miledi, R., Garcia-Alcocer, G., 2012. Serotonin receptors in hippocampus. Sci. World J. 2012, 823493. Bjorkholm, C., Monteggia, L.M., 2015. BDNF—a key transducer of antidepressant effects. Neuropharmacology 102, 72–79. Bourin, M., Mocaer, E., Porsolt, R., 2004. Antidepressant-like activity of S 20098 (agomelatine) in the forced swimming test in rodents: involvement of melatonin and serotonin receptors. J. Psychiatry Neurosci. 29, 126–133. Bus, B.A., Molendijk, M.L., Tendolkar, I., Penninx, B.W., Prickaerts, J., Elzinga, B.M., et al., 2015. Chronic depression is associated with a pronounced decrease in serum brain-derived neurotrophic factor over time. Mol. Psychiatry 20, 602–608. Cai, L., Li, R., Wu, Q.Q., Wu, T.N., 2013. Effect of hesperidin on behavior and HPA axis of rat model of chronic stress-induced depression. Zhongguo Zhong Yao Za Zhi 38, 229–233. Chang, C.Y., Lin, T.Y., Lu, C.W., Huang, S.K., Wang, Y.C., Chou, S.S., et al., 2015. Hesperidin inhibits glutamate release and exerts neuroprotection against excitotoxicity induced by kainic acid in the hippocampus of rats. Neurotoxicology 50, 157–169. Chen, M.J., Nguyen, T.V., Pike, C.J., Russo-Neustadt, A.A., 2007. Norepinephrine induces BDNF and activates the PI-3K and MAPK cascades in embryonic hippocampal neurons. Cell Signal. 19, 114–128. Chen, M., Gu, H., Ye, Y., Lin, B., Sun, L., Deng, W., et al., 2010. Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes. Food Chem. Toxicol. 48, 2980–2987. Choi, S.H., Li, Y., Parada, L.F., Sisodia, S.S., 2009. Regulation of hippocampal progenitor cell survival, proliferation and dendritic development by BDNF. Mol. Neurodegener. 4, 52. D’Aquila, P.S., Newton, J., Willner, P., 1997. Diurnal variation in the effect of chronic mild stress on sucrose intake and preference. Physiol. Behav. 62, 421–426. Donato, F., de Gomes, M.G., Goes, A.T., Filho, C.B., Del Fabbro, L., Antunes, M.S., et al., 2014. Hesperidin exerts antidepressant-like effects in acute and chronic treatments in mice: possible role of l-arginine-NO-cGMP pathway and BDNF levels. Brain Res. Bull. 104, 19–26. Donato, F., Borges Filho, C., Giacomeli, R., Alvater, E.E., Del Fabbro, L., Antunes Mda, S., et al., 2015. Evidence for the involvement of potassium channel inhibition in the antidepressant-like effects of hesperidin in the tail suspension test in mice. J. Med. Food 18, 818–823.

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