Antidepressant-like effects of Xiaochaihutang in a rat model of chronicunpredictable mild stress

Guang Yue Su a, Jing Yu Yang a, Fang Wang a, Jie Ma a, Kuo Zhang a, Ying Xu Dong a,Shao Jiang Song b, Xiu Mei Lu c, Chun Fu Wu a,n

a Department of Pharmacology, Shenyang Pharmaceutical University, Box 31, 103 Wenhua Road, 110016 Shenyang, PR Chinab Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 110016 Shenyang, PR Chinac Department of pharmaceutical analysis, Shenyang Pharmaceutical University, 110016 Shenyang, China

a r t i c l e i n f o

Article history:Received 30 August 2013Received in revised form14 November 2013Accepted 7 January 2014Available online 15 January 2014

Keywords:XiaochaihutangAntidepressantCUMSMonoamineNeurotrophin

Chemical compounds studied in this article:Liquiritin (PubChem CID: 503737)Ginsenoside Rg1 (PubChem CID: 441923)Baicalin (PubChem CID: 64982)Baicalein (PubChem CID: 5281605)Saikosaponin A (PubChem CID: 167928)Wogonin (PubChem CID: 5281703)Isoliquiritin (PubChem CID: 5318591)Glycyrrhizic acid (PubChem CID: 14982)Dopamine (PubChem CID: 681)5-Hydroxytryptamine (PubChem CID: 5202)

a b s t r a c t

Ethnopharmacological relevance: Xiaochaihutang (XCHT) has been used in China for thousands of years totreat “Shaoyang syndrome”, which involves depressive-like symptoms. However, few studies haveinvestigated its antidepressant effects and pharmacological mechanism of action. The present study wasdesigned to confirm the antidepressant effect of XCHT using a chronic unpredictable mild stress (CUMS)model and explore its potential mechanism of action by investigating the monoamine neurotransmitters(dopamine and 5-hydroxytryptamine) and neurotrophins (BDNF and NGF).Materials and methods: The CUMS model was established in rats, and the antidepressant effect of XCHT(0.6, 1.7 and 5 mg/kg/day, given by gastric gavage for 4 weeks) was investigated using the open field test(OFT), food consumption test and sucrose preference test. The concentrations of 5-HT and DA in thehippocampus were measured by high performance liquid chromatography with electrochemicaldetection (HPLC-ECD). The expressions of brain-derived neurotrophic factor (BDNF), nerve growth factor(NGF), and their receptors tyrosine receptor kinase B (TrkB) and tyrosine receptor kinase A (TrkA) in thehippocampus were measured by immunohistochemical staining analysis.Results: CUMS caused a significant decrease in OFT, food consumption and sucrose preference in rats,and these depression-like behaviors were significantly improved by XCHT (1.7 and 5 g/kg/day). Moreover,XCHT significantly increased the concentrations of 5-HT (0.6 and 5 g/kg/day) and DA (5 g/kg/day), andimproved the BDNF, NGF, TrkB and TrkA expressions in the hippocampus (1.7 and 5 g/kg/day), which wasreduced in CUMS rats.Conclusion: The results obtained suggested that XCHT may have therapeutic actions on depression-likebehavior induced by CUMS in rats possibly mediated by increasing the monoamine neurotransmitterconcentration and neurotrophin expression in the hippocampus.

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1. Introduction

Depression is one of the most common mental disorderscharacterized by feelings of sadness, and major depressive disorderis a primary cause of disability (Peet et al., 1998). The mechanism ofdepression is not yet fully understood, but several studies haveshown that chronic unpredictable mild stress (CUMS) may haveinhibitory effects on hippocampal 5-HT and DA neurotransmission(Ago et al., 2008; Li et al., 2009). In addition, clinical findings suggest

that depression is associated with a reduction in neurotrophin levelssuch as brain-derived neurotrophic factor (BDNF) and nerve growthfactor (NGF) in brain (Laske et al., 2010; Shi et al., 2010).

The synthetic antidepressants produce therapeutic responses,while one-third patients remained treatment-resistant (Rush et al.,2006; Duman and Aghajanian, 2012; Hsu et al., 2012) and evensuffered adverse-effects such as cardiotoxicity, hypertensive crisis,sexual dysfunction, and sleep disorder (Khurana and Baudendistel,2003). Traditional Chinese Medicines (TCM) have less harmful sideeffects and are very safe when used therapeutically (Shi et al.,2006) and, therefore, one of the studies involving a new anti-depressant is focused on TCM prescription (Park et al., 2007).Experimental and case-based studies have investigated formula-tions containing Radix bupleuri, such as Chaihushugansan (Kimet al., 2005; Wang and Zhu, 2013), Xiaoyaosan (Luo et al., 2006;Dai et al., 2010; Zhou et al., 2011) and Chaihu-jia-longgu-muli-tang

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0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2014.01.006

Abbreviations: XCHT, Xiaochaihutang; CUMS, chronic unpredictable mild stress;DA, dopamine; 5-HT, 5-hydroxytryptamine; BDNF, brain-derived neurotrophicfactor; NGF, nerve growth factor; TrkB, tyrosine receptor kinase B; TrkA, tyrosinereceptor kinase A.

n Corresponding author. Tel./fax: þ86 24 23986339.E-mail addresses: wucf@syphu.edu.cn, chunfuw@gmail.com (C.F. Wu).

Journal of Ethnopharmacology 152 (2014) 217–226

(Zhu et al., 2006; Dong, 2013). However, there is a lack of studies ofXiaochaihutang (XCHT), a formulation in which Radix bupleuri isthe principal drug.

XCHT was described two thousand years ago in a manuscriptentitled “Shang Han Lun”, written by Zhang Zhongjing (150–219A.D.) during the Chinese Eastern Han Dynasty (Bao et al., 2004;Chen et al., 2006; Dai et al., 2008; Liu et al., 2008; Qin et al., 2010;Zhu et al., 2010). It contains the following 7 Chinese herbs:Chaihu (Radix bupleuri), Huangqin (Radix scutellariae), Renshen(Ginseng), Banxia (Pinellia tuber), Gancao (Radix glycyrrhizae),Shengjiang (Rhizoma zingiberis recens), and Dazao (Fructus juju-bae). Radix bupleuri is the chief active ingredient of XCHT, andXCHT is an effective TCM prescription used in the treatment ofdepressive disorders in clinical situations (Li and Gao, 1996).

The CUMS model is acknowledged as a good simulation ofdepression universally and, since the pharmacological mechanismof action of XCHT is not understood, we investigated the antide-pressant-like effect of XCHT in a CUMS model in rats, and evaluatedthe mechanism of action of XCHT by examining the levels ofmonoamine transmitters (5-HT and DA) and the expression of BDNFand NGF and their receptors TrkB and TrkA in the hippocampus inthe present study, respectively.

2. Materials and methods

2.1. Drugs

XCHT was composed of 12 g of Radix Bupleuri (Family: Umbelli-ferae; Latin name: Bupleurum chinense DC.), 9 g of Radix Scutellariae(Family: labiatae; Latin name: Scutellaria baicalensis Georgi), 9 g ofGinseng (Family: Araliaceae; Latin name: Panax ginseng C.A. Meyer),9 g of Rhizoma pinelliae [Family: Araceae; Latin name: Pinellia ternata(Thunb.) Breit.], 6 g of Radix glycyrrhizae (Family: Leguminosae; Latinname: Glycyrrhiza uralensis Fisch.), 6 g of Rhizoma zingiberis recens(Family: Zingiberaceae; Latin name: Zingiber officinale Rosc.) and4 pieces (about 9 g) of Fructus jujubae (Family: Rhamnaceae; Latinname: Ziziphus jujubaMill.). The crude drugs were purchased from theTongrentang Chinese Pharmaceutical Co. Ltd. (Beijing, China) and wereauthenticated by Professor Jincai Lu (School of Traditional ChineseMateria Medica, Shenyang Pharmaceutical University, China). Voucherspecimens were deposited in the Herbarium of Shenyang Pharma-ceutical University.

An extract of XCHT was prepared by decocting the dried herbalmixture by macerating it in distilled water for 40 min, then boilingthree times (100 g/1000 ml the first time; 100 g/400 ml the secondtime; 100 g/400 ml the third time) for 30 min each time. The threedecoctions were mixed and filtered, then lyophilized and stored ina desiccator. The yield of powdered extract was about 25.2% (w/w).Forty four components, including saikosaponins, ginsenoside,baicalin, and liquiritin, were identified in this powder using theUPLC-MS/MS method described in a previous study by our group(Yang et al., 2012). Briefly, the analytes were qualified on a BEHC18 column (100 mm�2.1 mm, i.d. 1.8 μm) with the columntemperature maintained at 20 1C. The gradient mobile phasecomposed of acetonitrile (A) and 0.1% formic acid in water (B) ata flow rate of 0.2 ml/min was as follows: 5–25% A from 0 to 10 min,to 30% A for 5 min, to 40% A for 5 min, to 60% A for 5 min, 60––95%A for 2 min, and maintained 95% A for the final 3 min. MS analysiswas performed by an electrospray source operating in bothpositive and negative ion modes with the mass conditions asfollows: cone voltage was 25 V, and capillary voltage was 3.0 KV inpositive ion mode and 2.8 KV in negative ion mode. Nitrogen wasused as the desovation and cone gas with a flow rate of 500 and30 L/h at a temperature of 450 1C and source temperature was

120 1C. Collision gas is argon. MS data were collected in the fullscan mode from m/z 150 to 1500 amu.

Fluoxetine (FLU), used as a positive control, was obtained fromLilly S. A. (Alcobendas, Spain).

2.2. Animals

Male Sprague-Dawley rats weighing 180–200 g were suppliedby the Experimental Animal Centre of Shenyang PharmaceuticalUniversity. Animals were maintained on a 12-h light/dark cycle(lights on at 8:00 a.m., lights off at 8:00 p.m.) under controlledtemperature (2272 1C), and were given a standard diet and waterad libitum. They were allowed to acclimatize for 3 weeks withtraining for the sucrose preference test before starting the experi-ment. All experiments and procedures were carried out accordingto the Regulations of Experimental Animal Administration issuedby the State Committee of Science and Technology of China.

2.3. Groups and drug administration

Rats were randomly divided into two groups (12 for the controland 60 for the CUMS group). For the CUMS group, the animalswere isolated and treated simultaneously with CUMS. Two weekslater, the CUMS group was split into 5 treatment groups (proce-dure shown in Fig. 1), including the model group, XCHT (0.6, 1.7,and 5 g/kg) groups and FLU (15 mg/kg) group, and each groupcontained 12 rats. The Clinical Equivalency dose of XCHT was 5 g/kg/day. All drugs were dissolved in distilled water and given bygastric gavages once daily 30 min before the stress exposureduring the last 4 weeks.

2.4. CUMS procedure

The CUMS procedure was performed as described by Willneret al. (1987a), with a slight modification. Briefly, rats in the CUMSgroups were exposed to different stressors, namely, food depriva-tion, water deprivation, empty water bottles (after water depriva-tion), cage tilt, grouped housing, soiled cage, strobo-scobic lighting,restricted access to food (only give a small amount of food after fooddeprivation), 5-min cold swimming (at 15 1C), 1-min tail pinch (1 cmfrom the end of the tail), physical restraint, illumination and whitenoise. One of these stressors (in random order) was given every dayfor 6 weeks. The control group rats were left undisturbed except fornecessary procedures such as routine cage cleaning.

2.5. Sucrose preference test

As shown in Fig. 1, the sucrose preference test was carried outat the very end of the 6-week CUMS exposure. The test wasperformed as described previously with minor modifications.Briefly, 72 h before the test, rats were trained to adapt to 1%sucrose solution (w/v): two bottles of 1% sucrose solution wereplaced in each cage, and 24 h later the 1% sucrose in one bottle wasreplaced with tap water for 24 h. After the adaptation, rats weredeprived of water and food for 24 h. The sucrose preference testinvolved the rats being housed in individual cages and with free toaccess to two bottles containing 100 ml sucrose solution (1%, w/v)and 100 ml water. After 1 h, the volumes of consumed sucrosesolution and water were recorded and the sucrose preference wascalculated by the following formula:

Sucrose preference¼ sucrose consumptionwater consumptionþsucrose consumption

� 100%

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226218

2.6. Open-field test (OFT) and food consumption test

The OFT was carried out between 8:00 a.m. and 12:00 a.m. atthe end of 0, 2 and 6 weeks. The open-field apparatus consisted ofa square wooden arena (100 cm�100 cm�40 cm) with a blacksurface covering the inside walls. The floor of the wooden arenawas divided equally into 25 squares marked by black lines. In thetest, the rats were placed individually into the center of the arenaand allowed to explore freely. The number of crossings (squarescrossed with all paws) was recorded during a test period of 5 min.This apparatus was cleaned with a detergent and dried afteroccupancy by each rat (Pechlivanova et al., 2010).

Then, 24 h after the final sucrose preference test, the foodconsumption test was carried out. In this test, 50 g food was givento each isolated rat and the food remaining was weighed after24 h, and the difference between the initial and final weight is thefood consumption for the rat. This method is based on Durcanet al. with a little modification (Durcan et al., 1988).

2.7. Measurement of 5-HT and DA levels in the hippocampus

Twenty four hours after completion of the final food consump-tion test, half of the rats in each group were used for tissue assaysof 5-HT and DA. The animals were decapitated and their brainsrapidly removed and dissected on an ice-chilled glass plate to obtainthe hippocampus according to Franklin and Paxinos (Franklin andHerberg, 1977; Paxinos et al., 1980). Each tissue sample was weighedand homogenized by sonication in 200 μl 0.4 M perchloric acid.The homogenate was kept on ice for 1 h, and then centrifuged at12,000 rpm (4 1C) for 20 min. The supernatant was preserved, andthe concentrations of 5-HT and DA were measured using highperformance liquid chromatography with electrochemical detection(HPLC-ECD) as described by Qi et al. (2008a) with minor modifica-tions. The mobile phase consisted of 85 mM citrate, 100 mM sodiumacetate, 0.9 mM octyl-sodium sulfate, 0.2 mM EDTA, and 12%methanol, pH 3.7. An LC-10A pump (Shimadzu, Kyoto, Japan) wasoperated at 1.0 ml/min. The detector (L-ECD-6A, Shimadzu) was setat þ0.60 V. External standard curves were used to quantify theamounts of 5-HT and DA in each sample, calculated using the areaunder the curve. The injection volume was 20 μl.

2.8. Evaluation of BDNF, NGF, TrkB and TrkA expressions in thehippocampus (immunohistochemistry)

The other half part of animals (n¼6) in each parallel groupwere used in this test. Rats were fully anesthetized with chloralhydrate (350 mg/kg, i.p.) at the end of the experiment, thentranscardially perfused with saline solution containing heparin(10 U/ml), followed by 4% paraformaldehyde dissolved in phos-phate buffer (0.1 M, pH 7.4). The brains were then removed fromthe skull, post-fixed for 3 h in the same medium and transferred toa 30% sucrose solution. Then, each brain was sectioned coronallyon a freezing microtome (AS-620, Shandon, Astmoor, UK) at athickness of 25 um for immunohistochemistry, all of the sectionsincludes the hippocampus area.

The sections were then rehydrated and blocked with normalgoat serum for 1 h. The blocked sections were incubated withrabbit polyclonal anti-BDNF antibody (1:60 dilution, Santa Cruz:sc-546), rabbit polyclonal anti-TrkB antibody (1:75 dilution, SantaCruz: sc-12), rabbit polyclonal anti-NGF antibody (1:400 dilution,abcam: ab66459), and rabbit monoclonal anti-TrkA antibody(1:100 dilution, abcam: ab76291) individually, and subsequentlyreacted with the biotinylated secondary antibody (goat polyclonalsecondary antibody to rabbit, abcam: ab136817), then washed andprotected with cover slips with mounting medium. Light micro-scope (Oly-pusBX 51, Japan) was used for capturing the image.Light levels were normalized to preset levels and the microscope,camera and software were background-corrected to ensure relia-bility of image acquisition. The immunoreactive staining densitywas quantified by Image-Pro Plus 6.0 and measured in integratedoptical density (IOD) (Dang et al., 2009b; Zuo et al., 2009; Zhaoet al., 2013).

2.9. Statistical analysis

Statistical analysis was carried out using SPSS 16.0 software forWindows (SPSS Inc., Chicago, IL, USA). All values are expressed asmean7SEM. Data were analyzed by an independent-sample T testin the sucrose preference test and OFT at 0 to 2 weeks. Data wereanalyzed by one-way ANOVA followed by Fisher0s LSD test in alltests after 2 weeks. The level of significance was set at Po0.05.

Fig. 1. Animal groups, schematic representation of the experimental procedure and behavioral test. At the start, rats were randomly divided into two groups: control group(n¼12) and CUMS group (n¼60). All rats in the CUMS group were subjected to a variety of CUMS during 6 weeks; whereas animals in the control group remainedundisturbed. After 2 weeks, the CUMS group was separated into 5 treatment groups: model group (CUMSþdistilled water), XCHT (CUMSþXCHT 0.6 g/kg, CUMSþXCHT1.7 g/kg, and CUMSþXCHT 5 g/kg) groups and fluoxetine (15 mg/kg) group (n¼12 each group). Sucrose preference was measured at the end of every week. Open field test(OFT) was measured before stress (0 week), before drug administration (2 week), and at the end of the experiment (6 week). Food consumption was measured at the end ofthe experiment. For analysis of 5-HT and DA, half part of rats (n¼6) in each group were sacrificed on the final day of the 6-week period. For BDNF, NGF and their receptors,the other half part of animals (n¼6) in each parallel group were sacrificed and used.

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226 219

3. Results

3.1. Effect of XCHT on the sucrose preference on CUMS rats

Fig. 2 summarizes all sucrose preference test results at 0–6weeks in CUMS. The independent-sample T test showed that therewas no significant difference between the control and CUMS groupafter 1 week CUMS. After 2 weeks of CUMS, the sucrose preferencewas significantly reduced in the CUMS group (Po0.001) comparedwith the control group, indicating that the model was success-fully established. One-way ANOVA showed an up-regulation of thesucrose preference 3 weeks after XCHT treatment (5 g/kg, Po0.05)and 4 weeks after XCHT (1.7 and 5 g/kg, Po0.05) or FLU (15 mg/kg, Po0.001) treatment. The sucrose preference of the modelgroup remained significantly reduced compared with the controlgroup during the last 4 weeks (Po0.01, Po0.05, Po0.01 andPo0.001).

3.2. Effects of XCHT on the OFT in CUMS rats

As shown in Fig. 3, the effect of CUMS on general activity in therats and the effect of XCHT on the models were examined in the OFT.The independent-sample T test showed that 2-week CUMS leads toa significant decrease in crossing numbers in the OFT (Po0.01). Theone-way ANOVA showed that treatment for 4 weeks, with XCHT(1.7 g/kg) or FLU (15 mg/kg), the reduced number of crossings ofthe CUMS group was significantly reversed in the OFT (Po0.05),compared with the model group.

3.3. Effects of XCHT on food consumption in CUMS rats

The effects of XCHT and FLU on food consumption by the CUMSrats are shown in Fig. 4. The one-way ANOVA showed a marked groupeffect on the food consumption. Post hoc comparison revealed thatCUMS produced a reduction in food consumption after a 6-week stresscompared with the control group (Po0.05). Administration of XCHT(1.7 and 5 g/kg) or FLU (15 mg/kg) increased the food consumption(Po0.001, Po0.001 and Po0.05) compared with the model group.

3.4. Effects of XCHT on 5-TH and DA levels in the hippocampusin CUMS rats

The effect of XCHT on 5-HT and DA levels in the hippocampusin CUMS rats is shown in Fig. 5. CUMS significantly reduced 5-HT

Fig. 2. Effects of CUMS on rats and the therapeutic effect of XCHT in the sucrosepreference test. The values are expressed as mean7SEM. For statistical significancenPo0.05, nnPo0.01, nnnPo0.001 compared with the control group; #Po0.05,##Po0.01, ###Po0.001 compared with the model group.

Fig. 3. Effects of CUMS on rats and the therapeutic effect of XCHT in the open fieldtest. The values are expressed as mean7SEM. For statistical significance nPo0.05,nnPo0.01 compared with the control group; #Po0.05 compared with themodel group.

Fig. 4. Effects of CUMS and XCHT on the food consumption in CUMS rats. Thevalues are expressed as mean7SEM. For statistical significance nPo0.05 comparedwith the control group; #Po0.05, ###Po0.001 compared with the model group.

Fig. 5. Effects of XCHT on 5-TH and DA levels in the hippocampus in CUMS rats. Thevalues are expressed as mean7SEM. For statistical significance nPo0.05 comparedwith the control group; #Po0.05 compared with the model group.

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226220

and DA concentrations in the hippocampus compared with that incontrol animals (Po0.05). Administration of 0.6 g/kg XCHT wasable to reverse the effects of CUMS on 5-HT (Po0.05), while itfailed to influence the DA concentrations. FLU and 5 g/kg XCHTwere both able to reverse the effects of CUMS on DA (Po0.05),

3.5. Effects of XCHT on BDNF and TrkB expressions in thehippocampus in CUMS rats

The effects of XCHT and FLU on BDNF expression in thehippocampus are shown in Fig. 6. It was found that the BDNFprotein level was significantly reduced in the model group com-pared with the control group (Po0.05). Administration of XCHT (5 g/kg) or FLU (15 mg/kg) increased the expression of BDNF (Po0.05)compared with the model group.

A post hoc comparison also revealed that CUMS produceda reduction in the expression of TrkB (Fig. 7) after 6-week CUMS(Po0.05). Administration of XCHT (5 g/kg) or FLU (15 mg/kg)increased the expression of TrkB (Po0.05) compared with themodel group.

3.6. Effects of XCHT on NGF and TrkA expression in the hippocampusin CUMS rats

The effect of XCHT treatment on NGF protein levels in thehippocampus in CUMS rats is shown in Fig. 8. CUMS exposuresignificantly reduced the NGF protein levels in the hippocampus,compared with the control group (Po0.01). XCHT 1.7 and 5 g/kgsignificantly increased the NGF protein levels in the hippocampus ofrats exposed to CUMS (Po0.05 and Po0.01 respectively). Treatment

Fig. 6. Effects of XCHT on BDNF expression in the hippocampus of CUMS rats. Immunohistochemical analysis of BDNF proteins was performed on sections of rat brain of thecontrol group, CUMSþdistilled water group, CUMSþFLU group, CUMSþXCHT 0.6, 1.7, and 5 g/kg groups. Positive cells are represented as brown spots. Bar¼50 μm. The levelof staining density was quantified by Image-Pro Plus 6.0 and presented as mean7SEM. For statistical significance, *Po0.05 compared with the control group; #Po0.05compared with the model group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226 221

with FLU (15 mg/kg) significantly increased the NGF protein levels inthe hippocampus in CUMS rats (Po0.001).

CUMS exposure also significantly reduced the TrkA proteinlevels in the hippocampus (Po0.001), compared with the controlgroup as shown in Fig. 9. After 4-week treatment, XCHT (1.7 g/kg)significantly increased the TrkA protein levels in the hippocampusin CUMS rats (Po0.01), but the other two doses and the FLU hadno significant effect on the TrkA protein levels.

4. Discussion

The results obtained in the studies indicated that chronic stress isa key factor in the development and acceleration of affective disorderslike depression and anxiety (Lee et al., 2002), and CUMS may play

a prominent role in the etiology of depressive illness (Willner et al.,1987b; Ye et al., 2011). CUMS is considered as an effective method ofproducing an animal model of depression (Willner, 1986; Zhang et al.,2010) because this model closely mimics the behavioral and physio-logical symptoms of clinical depression which can be reversed byantidepressant agents (Willner et al., 1987b; Willner, 1997). Therefore,the CUMS model is a good simulation of clinical depression. In thepresent study, we demonstrated that CUMS leads to depressionsyndrome by inducing changes in behavior including sucrose pre-ference, general activity and food consumption. A reduction in sucrosepreference and crossing numbers was observed after a 2-week periodof CUMS, which reflects some aspects of refractory depression andloss of interest (Luo et al., 2008), indicating that the depression modelwas successfully established. Treatment for XCHT for 4-weeks sig-nificantly suppressed these behavioral changes, suggesting that XCHT

Fig. 7. Effects of XCHT on TrkB expression in the hippocampus of CUMS rats. Immunohistochemical analysis of TrkB proteins was performed on sections of rat brain of thecontrol group, CUMSþdistilled water group, CUMSþFLU group, CUMSþXCHT 0.6, 1.7, and 5 g/kg groups. Positive cells are represented as brown spots. Bar¼50 μm. The levelof staining density was quantified by Image-Pro Plus 6.0 and presented as mean7SEM. For statistical significance, nPo0.05 compared with the control group; #Po0.05compared with the model group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226222

has a therapeutic effect on the depression behavior in the ratmodel used.

The monoamine theory of depression has been around formore than 50 years (Marije aan het Rot et al., 2009). Severalstudies have shown that chronic stress produces changes in theconcentrations of neurotransmitters in CUMS-induced depressionmodels, mainly exhibited as a significant reduction in the levels of5-HT and DA (Pardon et al., 2004; Julio-Pieper et al., 2012), and areduced levels of brain 5-HT and DA have been proposed to be akey pathogenic factor in depression (Holmes et al., 2003). In thepresent study, the CUMS procedure markedly reduced both 5-HTand DA levels in the hippocampus. Furthermore, XCHT adminis-tration significantly restored these changes and led to markedincrease in 5-HT and DA concentrations. This data suggested thatXCHT affects the concentrations of monoamine transmitters. In

fact, our previous study found that the main constituents of XCHTinclude flavonoids, flavonoid glycosides and saponins, such asskullcapflavon II and Baicalin (Yang et al., 2012). In addition,flavonoids in Scutellariae Radix have been reported to produceeffective anti-depression possibly through the DA system (Wanget al., 2010). Thus, in the present study, the potential mechanism ofXCHT as an antidepressant in the CUMS model may be related tothe changes in 5-HT and DA, and the flavonoids may play animportant role in these effects.

In recent years, studies on depression have paid special attentionto the hippocampus, which is structurally and functionally affected bystress responses and the regulation of mood (Luo et al., 2008; Qi et al.,2008b). The neurotrophic hypothesis of depression suggests thatneurotrophic factors, especially BDNF, might mediate the clinicaleffects of antidepressants and are involved in synaptic plasticity,

Fig. 8. Effects of XCHT on NGF expression in the hippocampus of CUMS rats. Immunohistochemical analysis of TrkB proteins was performed on sections of rat brain of thecontrol group, CUMSþdistilled water group, CUMSþFLU group, CUMSþXCHT 0.6, 1.7, and 5 g/kg groups. Positive cells are represented as brown spots. Bar¼50 μm. The levelof staining density was quantified by Image-Pro Plus 6.0 and presented as mean7SEM. For statistical significance, nPo0.05, nnPo0.01 compared with the control group;#Po0.05, ##Po0.01, ###Po0.001 compared with the model group. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.).

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226 223

neuronal circuit formation, and neuronal survival (Nibuya et al., 1995;Smith et al., 1995; Karege et al., 2005). The hippocampus has beenreported to contain a large number of BDNF. In experimental studies,it has been found that a decrease in BDNF expression inthe hippocampus of animals exposed to CUMS can be reversed byantidepressant treatment (Xu et al., 2006; Li et al., 2007a; Monteggiaet al., 2007). Accordingly, BDNF may be an important factor in thedevelopment and treatment of depression, and our study investigatedthe BDNF level in the hippocampus in CUMS rats and found thatCUMS exposure reduces BDNF protein expression in the hippocam-pus, while a 4-week treatment of XCHT significantly reverses theCUMS-induced changes in BDNF protein.

BDNF exerts its influence chiefly by signaling through itsspecific high-affinity receptor tyrosine kinase B (TrkB) (Barbacid,1994; Tsai, 2004). It has been reported that BDNF binds to TrkBreceptors and activates PI3K/Akt signaling, resulting in the

phosphorylation and inhibition of GSK-3β that has an antidepres-sant effect (Nakagawara et al., 1994; Li et al., 2007b). A trend inreduced TrkB protein levels was found in the hippocampus of ratswith CUMS in our present study. Treatment with XCHT reversedthis change, resulting in an increase in TrkB protein level in thehippocampus. These results indicate that the antidepressant effectof XCHT might involve regulating the signal pathway that isdownstream of BDNF and TrkB. This research is the first reportdemonstrating the effect of XCHT on TrkB levels in hippocampus.

It has been reported that the aqueous extract of BupleuriRadixhe (the main drug of XCHT) exerts its antidepressant effectsthrough actions on BDNF activation, and leading to a stimulationof the PI3K/Akt/GSK-3β signaling pathway which is associatedwith depression (Rowe et al., 2007; Bandaru et al., 2010; Seo et al.,2012). In previous studies, we found that XCHT contains constitu-ents of ginseng saponins (Yang et al., 2012) and the activity of

Fig. 9. Effects of XCHT on TrkA expression in the hippocampus of CUMS rats. Immunohistochemical analysis of TrkB proteins was performed on sections of rat brain of the controlgroup, CUMSþdistilled water group, CUMSþFLU group, CUMSþXCHT 0.6, 1.7, and 5 g/kg groups. Positive cells are represented as brown spots. Bar¼50 μm. The level of stainingdensity was quantified by Image-Pro Plus 6.0 and presented as mean7SEM. For statistical significance, nPo0.05, nnPo0.01, nnnPo0.01 compared with the control group; ##Po0.01compared with the model group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

G.Y. Su et al. / Journal of Ethnopharmacology 152 (2014) 217–226224

ginseng total saponins in antidepression may be mediated throughenhancing BDNF expression in the hippocampus (Dang et al.,2009a). Thus, we would speculate that the antidepressant effectsof XCHT might be related to the increased BDNF signaling path-way, and components of Bupleuri Radix and ginseng saponins mayplay a crucial role in this effect.

Nerve growth factor (NGF) is also a neurotrophin which plays animportant role in neuronal survival and function. The antidepressantpotential for NGF has been established by demonstrating that itreduces swim test immobility in the Flinders Sensitive Line (FSL) ratmodel of depression (Overstreet et al., 2010). In addition, NGF is alsoinvolved in the pathophysiology of stress-related mood disorderswhich is supported by reports that NGF expression is reduced byexposure to stress (Purves and Njå, 1976). Furthermore, antidepressanttreatments or electroconvulsive shock therapy increases the expres-sion and concentrations of NGF in the brain (Alfonso et al., 2006;Hellweg et al., 2008). In our studies, in the CUMS group, the NGF levelin the hippocampus was significantly reduced compared with that inthe control group. The XCHT treatment had a different effect, and theNGF protein level was significantly increased after a 4-week treatmentwith XCHT and was almost equal to that in the control group.

Changes in the expression of NGF affect the basal activity ofneurons expressing NGF-receptors (Badowska-Szalewska et al., 2011)while it has been reported that antidepressants, such as amitriptyline,act as a TrkA agonist and possess marked neurotrophic activity (Janget al., 2009). In our research, the TrkA protein level was reduced in theCUMS rat brain, which indicated that stress might affect the expres-sion of TrkA. However, administration of XCHT significantly reversedthese effects in the CUMS model. These results indicate that themechanism of the antidepressant effect of XCHTmay be also related toNGF and its receptor TrkA.

It has been reported that neurotrophin is able to promote thesurvival of dopaminergic and serotonergic neurons (Spencer et al.,1995). Based on all the results in our present study, it appears that themechanisms underlying the antidepressant-like effects of XCHT mightbe via regulation of the expression of BDNF and NGF in CUMS rats.BDNF and NGF conjugate with their receptors (TrkB and TrkA) andincrease the functions of neurons by stimulating the downstreampathways. Monoaminergic neurons0 survival is increased, and mono-amine neurotransmitter (5-HT and DA) synthesis is further increasedby neurons and causes the antidepressant effect observed. However,the exact target of the antidepressant effect of XCHT remains unknownand further research is needed. Our group will try to answer thesequestions in future studies.

5. Conclusion

CUMS produces a series of abnormalities relevant to depression. Inour study, treatment with XCHT significantly improved thedepression-like behaviors including general activities, food consump-tion and sucrose preference in the CUMS rat model. Moreover, XCHTalso significantly increased the concentrations of 5-HT and DA,improved the BDNF and NGF pathway (reflected by increased TrkBand TrkA) in the hippocampus. Based on our results, we demonstratedthat XCHT had therapeutic effects on the CUMS-induced depressionmodel in rats, and these effects might be related to simultaneousalterations in monoamine transmitters and neurotrophic factors.Collectively, all of these findings suggest that XCHT deserves furtherinvestigation as a potential antidepressant.

Acknowledgments

This research was supported by the Key Project of NationalNatural Science Foundation of China, P.R. China (81130071), and

the National Key Scientific Project for New Drug Discovery andDevelopment, P.R. China (2010ZX09401).

Appendix A. Supplementary materials

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jep.2014.01.006.

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