research article nobiletin ameliorates the deficits in...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 359682 11 pageshttpdxdoiorg1011552013359682

Research ArticleNobiletin Ameliorates the Deficits in Hippocampal BDNF TrkBand Synapsin I Induced by Chronic Unpredictable Mild Stress

Jing Li1 Ying Zhou2 Bin-Bin Liu1 Qing Liu1 Di Geng1 Lian-Jin Weng1 and Li-Tao Yi1

1 Department of Chemical and Pharmaceutical Engineering College of Chemical Engineering Huaqiao UniversityXiamen Fujian 361021 China

2Department of Biotechnology and Bioengineering College of Chemical Engineering Huaqiao University XiamenFujian 361021 China

Correspondence should be addressed to Li-Tao Yi litaoyiyahoocom

Received 3 November 2012 Revised 14 February 2013 Accepted 14 February 2013

Academic Editor David Mischoulon

Copyright copy 2013 Jing Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Our previous study has demonstrated that nobiletin could reverse the behavioral alterations in stressedmice Howeverthe relation of its antidepressant-like actionwith neurotrophicmolecular expression remains unknownThis study aimed to explorethe antidepressant-like mechanism of nobiletin related to the neurotrophic system in rats exposed to chronic unpredictable mildstress (CUMS) Methods Depressive-like anhedonia (assessed by sucrose preference) and serum corticosterone secretion wereevaluated in the CUMS followed by brain-derived neurotrophic factor (BDNF) its tropomyosin-related kinase receptor B (TrkB)and the downstream target synapsin I expressions in the hippocampus Results Anhedonia which occurred within week 2 wasrapidly ameliorated by nobiletin While fluoxetine needed additional 2 weeks to improve the anhedonia In addition nobiletinadministration for 5 weeks significantly ameliorated CUMS-induced increase in serum corticosterone levels Furthermore we alsofound that CUMS-induced deficits of hippocampal BDNF TrkB and synapsin I were ameliorated by nobiletin Conclusions Takentogether these findings suggest that nobiletin produces rapidly acting antidepressant-like responses in the CUMS and imply thatBDNF-TrkB pathway may play an important role in the antidepressant-like effect of nobiletin

1 Introduction

Depression one of themajor causes of disabilityworldwide isamood or affective disorder caused bymany factors from thepsychological social and environmental to the genetic andmetabolics [1] Although clinical and experimental researcheshave provided some insight into the pathophysiologicalprocesses that may be occurring in the depression at presentthe molecular correlates underpinning these abnormalitiesare not fully understood [2] Neurotrophins are a family ofproteins that play multiple roles in regulating neural survivaldevelopment function and plasticity [3] Limiting quantitiesof neurotrophins control the numbers of surviving neuronsto ensure a match between neurons and the requirementfor a suitable density of target innervation [4] Beyond thepromotion of neural function the family is also a powerfulmodulator in hippocampal-dependent learning andmemory[5] Brain-derived neurotrophic factor (BDNF) one of the

most widely distributed neurotrophins after binding withand activating tropomyosin-related kinase receptor B (TrkB)is involved in the pathophysiology and treatment of depres-sion [6 7]They play a critical role in the modulation of somefunctions such as neurotransmitter release and postsynapticresponses to neurotransmitters which are closely relatedto the antidepressant therapy [8] Either reduced BDNFavailability or decreased expression of TrkB receptor couldreduce BDNF-TrkB signaling in animals [9] In contrasttreatment with antidepressants could upregulate BDNF oractivate TrkB receptor in the brain in rodents [10 11]Additionally in the animal model of depression resultsdemonstrate that antidepressant efficacy is mediated at leastin part through an elevation of BDNF levels or BDNF-TrkBsignaling in the hippocampus [12 13] On the contrary intransgenic animals with decreased brain BDNF levels orinhibited BDNF-TrkB signaling antidepressant agents fail toexert behavioral responses [14 15] Collectively the findings

2 Evidence-Based Complementary and Alternative Medicine

reviewed earlier strongly suggest that the behavioral effectsof antidepressants require functional BDNF signaling in thebrain

Stress is an important precipitant factor in depressionand the changes in various body systems that occurred indepression are similar to those observed in response to stress[16] Chronic unpredictable mild stress (CUMS) the mostpromising rodent model for depression is widely used forpreclinical testing and screening of antidepressants [17] Inthe CUMS paradigm animals are subjected to a variety ofmild stressors presented intermittently for prolonged periodsof time which mimic chronic stressful life events and resultin a behavioral deficit anhedonia a core symptom of humandepression [18] Anhedonia is monitored by a reduction insucrose preference which can be restored by therapeuticallyeffective drugs for the treatment of depression [11]

Nobiletin a dietary constituent of flavonoids isolatedfrom citrus fruits has been reported to upregulate synaptictransmission and improve memory impairment in rodents[19 20] Our previous study found that nobiletin adminis-tration significantly reduced the immobility time in both thetail suspension test (TST) and forced swimming test (FST)without accompanying changes in locomotor activity in theopen-field test (OFT) in mice [21] Furthermore we alsodemonstrated that the antidepressant-like effect of nobiletinwas mediated by monoaminergic systems in this studyHowever until now the neurotrophic molecular expressionunderlying the antidepressant-like effect of nobiletin remainsunknown

Therefore considering that hippocampus is critical forstress learning and memory processes in depression and inthe antidepressant response to pharmacotherapy [22ndash24] theaim of the present study was to evaluate the effects of dailynobiletin treatment on sucrose preference and correspondingchanges in BDNF TrkB receptor and the downstream targetsynapsin I in the hippocampus after CUMS

2 Materials and Methods

21 Animals Male Sprague-Dawley rats (260ndash300 g) werepurchased from Laboratory Animal Centre Fujian MedicalUniversity Fujian Province China Animals were single-housed under a normal 12-h12-h lightdark cycle with thelights on at 0700 AM Ambient temperature and relativehumidity were maintained at 22 plusmn 2∘C and at 55 plusmn 5 andanimals were given a standard chow and water ad libitum forthe duration of the study except when the CUMS procedureor sucrose preference test was required The animals wereallowed to acclimatize to these conditions for 1 week beforeany experimental procedure was initiated At the beginningof the experiments the average body weight of rats wasapproximately 310 g All procedures were performed in accor-dance with the published guidelines of the China Council onAnimal Care (regulations for the Administration of Affairsconcerning experimental animals approved by the StateCouncil on October 31 1988 and promulgated by Decreeno 2 of the State Science and Technology Commission onNovember 14 1988)

22 Chemicals and Reagents Nobiletin (purity gt98 byHPLC) was obtained from Shanxi Huike Botanical Devel-opment Co Ltd (Xirsquoan China) Fluoxetine hydrochloridewas purchased from Changzhou Siyao Pharmaceuticals CoLtd (Changzhou China) All primers used in this studywere designed and synthesized by Sangon Biotech Co Ltd(Shanghai China)The anti-BDNF (Catalog number sc-546detecting mature BDNF) and anti-TrkB (Catalog numbersc-12 detecting full-length TrkB) antibody and the respec-tive secondary antibodies were purchased from Santa CruzBiotechnology Inc (Santa Cruz USA) The antisynapsin I(Catalog numberAB1543 detecting synapsin I) antibodywaspurchased from Millipore Corporation (Billerica USA) Theanti-GAPDH (Catalog number KC-5G5 detecting GAPDH)antibody was purchased from Kangcheng Biotech (Shang-hai China) Trizol reagent was purchased from Invitrogen(Carlsbad USA) Reverse transcriptase moloney murineleukemia Virus (M-MLV) used for cDNA synthesis wasfrom Promega Corporation (Madison USA) All otherreagents used in polymerase chain reaction (PCR) andwestern blot were purchased from Sangon Biotech Co Ltd(Shanghai China)

23 CUMS Procedure The CUMS procedure was performedaccording to the traditional method described by Willner etal [17] with some modifications Briefly the weekly stressregime consisted of food and water deprivation stroboscopicillumination (150 flashesmin) white noise lightdark suc-cession every 2 h overnight illumination 45∘ cage tilt soiledcage and pair-housing (Table 1) All of the stressors whichwere applied individually and continuously were randomlyscheduled over a 1-week period and repeated throughoutthe 5-week procedure The control group was housed in aseparate room and had no contact with the stressed animalsThese rats were deprived of food and water for the 18 hpreceding each sucrose test but otherwise food and waterwere freely available in the home cage

24 Sucrose Preference Test Before the beginning of CUMSprocedure all rats were given 1 sucrose solution for 24hours Then both sucrose solution and fresh water weremade accessible to the rats for another 24 hours Afterdeprived of drinking for 23 hours the rats were given both1 sucrose solution and fresh water for 1 hour again Afterthis sucrose consumption training phase the animals wererandomly divided into 5 groups (8 rats per group) control-vehicle (saline) CUMS-vehicle (saline) CUMS-fluoxetine(positive control 10mgkg) and CUMS-nobiletin at a doseof 20mgkg and 40mgkg Nobiletin was suspended insaline with 10 (vv) Tween-80 (polyoxyethylene sorbitanmonooleate) and fluoxetine hydrochloride was dissolvedin 09 physiological saline Four groups except control-vehicle were exposed to the CUMS procedure for 5 weeksand treated All drugs were administered by gavage in avolume of 10mLkg body once daily at 1100 AM for 5 weeksThrough the period of CUMS and treatment sucrose prefer-ence test were conducted following an 18-h food and waterdeprivation at 1100 AM every Tuesday Sucrose preferencewas calculated as sucrose preference () = sucrose intake

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Time and length of stressors used in the CMS procedureThese stressors which were applied continuously were randomly scheduledover a 1-week period and repeated throughout the 5-week period At the same time animals were treated with nobiletin (20 and 40mgkgPO) fluoxetine (10mgkg PO) or saline

Monday Tuesday Wednesday Thursday Friday Saturday SundayFood and water deprivation 1600997888rarr1000 0800ndash2000Stroboscopic illumination 1200ndash2000 2000997888rarr0800White noise 2000997888rarr0800 0800ndash2000Lightdark succession every 2 h 0800ndash2000 0800ndash2000Overnight illumination 2000997888rarr080045∘ cage tilt 997888rarr1600 2000997888rarr0800 2000997888rarrSoiled cage 0800ndash2000Pair-housing 2000997888rarr0800Sucrose preference test 1000ndash1200

(mL)[sucrose intake (mL) +water intake (mL)]times 100 Thetreatment protocol of dose and administration route used fornobiletin and fluoxetine was adopted according to our andother previous studies and was calculated based on the bodysurface area of the rat [11 21]

25 Blood Sampling and Tissue Extraction After the lastsucrose preference all rats were decapitated between 1200PM (midday) and 200 PM to avoid fluctuation of hormonelevels Blood samples were collected immediately The brainregion of hippocampus was isolated immediately and thenstored at minus80∘C for later analysis of mRNA and protein levels

26 Serum Corticosterone Assay Blood was collected on iceand separated in a refrigerated centrifuge at 4∘C (4000timesgfor 10min) Serum was stored at ndash20∘C until assays wereperformed Serum corticosterone levels were measured usinga commercial kit (Enzo Life Sciences Plymouth MeetingUSA) based on enzyme immunoassay

The concentrations of corticosterone in each sample weredetermined in duplicate and the average of the two valueswas used as the value for that rat

27 Real-Time PCR Total RNA was extracted from hip-pocampus using Trizol reagent following the manufacturerrsquosinstructions Reverse transcription was performed usingM-MLV reverse transcriptase for cDNA synthesis Real-time PCR reactions were performed using a SYBR PremixEx Taq Kit in ABI-7500 system The BDNF (NM 012513sense primer 51015840-TGTGACAGTATTAGCGAGTGGGT-31015840and antisense primer 51015840-CGATTGGGTAGTTCGGCATT-31015840) TrkB (NM 012731 sense primer 51015840-CTTATGCTT-GCTGGTCTTGG-31015840 and antisense primer 51015840-GGGTAT-TCTTGCTGCTCTCA-31015840) synapsin I (NM 019133 senseprimer 51015840-CCCTTCATTGATGCTAAATACG-31015840 and anti-sense primer 51015840-GTTGACCACAAGTTCCACGAT-31015840) andthe internal control GAPDH (NM 017008 sense primer 51015840-ACCACAGTCCATGCCATCAC-31015840 and antisense primer51015840-TCCACCACCCTGTTGCTGTA-31015840) primers were usedThe fluores-cence signal was detected at the end of each cycleMelting curve analysis was used to confirm the specificity

of the products The results were analyzed by the 2minusΔΔCTmethod

28 Western Blot Brain samples were homogenized in alysis buffer containing 50mMTris-HCl (pH74) 1mMEDTA150mM NaCl 1 Triton X-100 1 sodium deoxycholate01 SDS 1mM trichostatin A and phosphatase inhibitorcocktail The homogenates were centrifuged at 14000timesg for20min at 4∘C and the supernatants were collected The pro-tein concentrationwas determined by a BCA assay Total pro-teinswere separated by SDS-PAGEand transferred to a PVDFmembrane Following blocking in 3 BSATBST at roomtemperature for 1 h the membranes were incubated withthe appropriate primary antibodies at 4∘C overnight (anti-BDNF 1 500 anti-TrkB 1 1000 antisynapsin I 1 1000 andanti-GAPDH 1 5000) After being washed with TBST forthree times the membranes were incubated with an HRP-labeled secondary antibody (1 4000) The blots were washedagain for three times byTBSTbuffer and the immunoreactivebands were detected by using the enhanced chemilumi-nescence method Western blot bands were scanned byHewlett-Packard Scanjet 5590 and subsequently analyzeddensitometrically with Bio-Rad Quantity One software Theresults were normalized using GAPDH expression as theinternal standard

29 Statistical Analyses All data were expressed as mean plusmnSEM SPSS software (version 130) was used for statisticalanalyses Data of sucrose preference and body weight fromthe CUMS were analyzed using a repeated ANOVA withtreatment as between factor and time (weeks) aswithin factorFor biochemical analysis a one-way ANOVA followed bypost-hoc Dunnettrsquos test was performed A value of 119875 lt005 was considered statistically significant for analysis Thefigures were obtained by GraphPad Prism (version 5)

3 Results

31 Effects of Nobiletin on the Sucrose Preference and BodyWeight Gain in the CUMS Figure 1(a) presented the sucrosepreference in rats at baseline (week 0) and during a 5-week CUMS period Separated one-way ANOVA revealed no


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Figure 1 Effects of nobiletin on the sucrose preference (a) and body weight (b) in CUMS-induced rats The data represented the values ofmeanplusmn SEM (119899 = 8) 119875 lt 005 and

119875 lt 001 versus control-vehicle group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehicle group

statistical significance effect on sucrose preference (119865(4 35) =014 119875 gt 005) among the groups on the baseline test Arepeated ANOVA with treatment as independent factor andweek as repeated factor revealed that there was a statisticallysignificant effect of treatment (119865(3 28) = 1392 119875 lt 001)and treatmenttimesweek interaction (119865(15 140) = 227 119875 lt001) but not effect of week (119865(5 140) = 190 119875 gt 005) onthe sucrose preference

Furthermore the sucrose preference of CUMS-vehiclerats was significantly lower compared to control-vehicleanimals at weeks 2 3 4 and 5 (119865(1 14) = 860 1603 25311798 119875 lt 005 119875 lt 001 119875 lt 001 119875 lt 001 resp)Nobiletin treatment gradually reversed the CUMS-induceddeficit in sucrose intake the onset of amelioration that isa significant increase in sucrose preference as compared toCUMS-vehicle was seen at week 2 and remained in followingweeks treated with 40mgkg nobiletin (119875 lt 005 119875 lt 001119875 lt 001 119875 lt 001 resp) In addition sucrose preferencewas reversed by nobiletin (20mgkg 119875 lt 001 119875 lt 001resp) and fluoxetine (10mgkg 119875 lt 001 119875 lt 001 resp) atweeks 4 and 5

As illustrated in Figure 1(b) the body weight of animalsin each group does not have a significant difference at thebeginning A repeated ANOVA showed that during the 5-week experiment period the body weight in the wholeexperiment showed a continual increase (119865(5 140) = 5682119875 lt 001) However only fluoxetine increased the bodyweight compared with the CUMS-vehicle group at week 5by post hoc test (119875 lt 005) No significant differences wereobserved among the rest of the groups

0

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lowastlowast lowastlowast lowastlowast

Figure 2 Effects of nobiletin on the serum corticosterone levels inCUMS-induced ratsThedata represented the values ofmeanplusmn SEM(119899 = 8 in ELISA assay)

119875 lt 001 versus control-vehicle grouplowastlowast

119875 lt 001 versus CUMS-vehicle group

32 Effects of Nobiletin on the Serum Corticosterone LevelsThe effects of nobiletin on the serum corticosterone levels intheCUMS rats were shown in Figure 2TheCUMSprocedurecaused a significant increase in serum levels of corticosteronein rats (119865(1 14) = 2498 119875 lt 001) Compared with CUMS-vehicle animals chronic treatment with 20 and 40mgkg


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(c)

Figure 3 Effects of nobiletin on the hippocampal BDNF (a) TrkB (b) and synapsin I (c) mRNA expression in CUMS-induced ratsThe datarepresented the values of meanplusmn SEM (119899 = 6 in PCR assay) 119875 lt 005 and

119875 lt 001 versus control-vehicle group lowast119875 lt 005 and lowastlowast119875 lt 001versus CUMS-vehicle group

nobiletin significantly reversed CUMS-induced elevation incorticosterone (119875 lt 001 119875 lt 001 resp) In additionthe positive drug fluoxetine (10mgkg) also decreased thecorticosterone levels in serum (119875 lt 001)

33 Effects of Nobiletin on the BDNF Expression The mRNAand protein expression of BDNF in the hippocampus werepresented in Figures 3(a) 4(a) and 4(b) respectively Thelevels of BDNF mRNA (119865(1 10) = 1751 119875 lt 001) andprotein (119865(1 10) = 4212 119875 lt 001) in the hippocampuswere significantly decreased by CUMS Chronic nobiletintreatment at 20 (mRNA 119875 lt 005 protein 119875 lt 001 resp)and 40mgkg (mRNA 119875 lt 001 protein 119875 lt 001 resp)markedly elevated BDNF expression in the hippocampusFluoxetine also elevated BDNF mRNA and protein expres-sion in this region (119875 lt 001 119875 lt 001 resp)

34 Effects of Nobiletin on the TrkB Expression In thehippocampus TrkB mRNA and protein expression wasslightly decreased (not statistically significant) by CUMSprocedure compared with the control-vehicle group (Figures3(b) 4(a) and 4(c)) Compared with CUMS-vehicle rats

chronic nobiletin treatment markedly elevated TrkB protein(119875 lt 005 119875 lt 001 resp) but not mRNA expression Inaddition fluoxetine also increased hippocampal TrkB proteinexpression (119875 lt 001) in CUMS animals

35 Effects of Nobiletin on the Synapsin I Expression TheCUMS procedure significantly decreased the synapsin ImRNA (119865(1 10) = 703 119875 lt 005) and protein (119865(1 10) =550 119875 lt 005) expression in the hippocampus (Figures 3(c)4(a) and 4(d)) The synapsin I mRNA and protein levelswere significantly increased in this region after treatmentwithnobiletin (mRNA 119875 lt 001 119875 lt 001 protein119875 lt 005119875 lt 001 resp) or fluoxetine (119875 lt 001 119875 lt 001 resp) inrats exposed to the CUMS procedure

4 Discussion

As proposed by Willner CUMS appears more suitable forstudying the neurobiological basis of depression and themechanisms of antidepressant agents as compared to acutestress models such as FST or TST [17 18] Therefore inthe present study we investigated the effects of nobiletin on


BDNF

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iletin

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lowastlowastlowast

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Figure 4 Effects of nobiletin on the hippocampal BDNF TrkB and synapsin I protein expression in CUMS-induced rats (a) Representativewestern blot images of BDNF TrkB and synapsin I are shown and BDNF (b) TrkB (c) and synapsin I (d) results were quantified and are themeanplusmn SEM (119899 = 6 inWestern assay) 119875 lt 005 and

119875 lt 001 versus control-vehicle group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehiclegroup

the hippocampal BDNF-TrkB system in the CUMS proce-dure The CUMS animals exhibited a persistent reductionin responsiveness to pleasurable stimuli (a specific hedonicdeficit) which is measured by a decrease in their sucrosepreference an indicator of anhedonia-like behavioral changein the CUMS [17] CUMS-induced reduction in sucrosepreference was also confirmed in our present study Asreported with conventional antidepressants [11 25] chronictreatment with 20 and 40mgkg nobiletin caused a reversalof sucrose preference in rats exposed to CUMSThese resultsfurther confirmed the antidepressant-like actions of oralnobiletin administration in animal models of depressionMoreover the results obtained from our study also indi-cated that nobiletin reversed the emerging anhedonia atweek 2 suggesting that nobiletin produced rapidly actingantidepressant-like responses However there was a delayin the onset of the fluoxetine since 4 weeks of fluoxetinetreatment was needed for a complete recovery of anhedoniaTherefore it suggests that nobiletin displayed a faster onset

of action compared to the serotonin reuptake inhibitorfluoxetine The rapid onset of nobiletin is supported by itsrapid absorption in rodents [26 27] The concentration ofnobiletin in rat brain increased to a maximum of 420120583gmLwithin 10 h after oral administration of 50mgkg nobiletinand the mean area under curves (AUC

0ndash119905) in brain was2066120583gsdothmL which indicated that significant amount ofnobiletin reached in brain rapidly As a result the pharma-cokinetic profile of nobiletin supports that fast and significantbrain permeability of nobiletin is enough to trigger rapidsignal transduction cascades to produce therapeutic effects

Since controversy exists regarding the influence of bodyweight change on sucrose consumption in the CUMS [28] itis important to measure this variable Present study demon-strated that CUMS did not affect the body weight in the 5-week paradigm Although some studies showed that CUMScaused a body weight reduction [11 29 30] several previousreports also demonstrated that CUMS exerted only slightlyor no effect on body weight of rats [24 31] These results


suggest some dissociation between body weight change andanhedonia in the CUMS [32] although body weight lossis also a diagnostic criterion for a clinical depression [33]In addition the effect of fluoxetine on body weight is stillcontroversial Our data displayed that fluoxetine but notnobiletin increased the body weight compared with CUMS-vehicle group However no significant effect of fluoxetineon body weight was shown in another preclinical study [11]These discrepancies may be produced by many parametersin the experimental design mainly including strains ofanimals experimental conditions and stressed time [34]On the other hand according to some preclinical reportsnobiletin can induce fatty acid oxidation suppress adipocytedifferentiation and enhance adiponectin secretion and lipol-ysis which resulted in reduction of body weight gain [35ndash37] Therefore the inability of nobiletin to produce bodyweight gain could be explained by that the restoration ofeating behavior (anhedonia improvement) is counteracted byincreased lipolysis

Multiple mechanisms are responsible for the devel-opment of depression The hypothalamic-pituitary-adrenal(HPA) axis is involved in the pathogenesis of depressionand plays a key role in mediating the responses to variousstressful stimuli and the effects of the antidepressant [38]Theexposure to an acute stressor activates the HPA axis resultingin a cascade of endocrine events This cascade finally stimu-lates the production and release of glucocorticoids [39] Thehippocampus has very high levels of glucocorticoid receptor(GR) and provides negative feedback to the hypothalamusto prevent further release of glucocorticoids [40] How-ever once the stress sustains glucocorticoid hypersecretionwill reduce GR expression and impair negative feedbackinhibition Subsequently hippocampus will be damagedreduced in size or functionally suppressed [41] As a resultfurther harmful processes will be done resulting in cognitivedeficits decreased appetite and behavioral anhedonia [42]Corroborated by observations from our previous and otherstudies [2 25 43] the present study exhibited that the HPAhyperactivity induced by CUMS as indicated by an increasedserum corticosterone level was ameliorated by nobiletin andfluoxetine administration for 5 weeks The finding suggestedthat the antidepressant-like actions of oral nobiletin treat-ment might be partly related to the modulation of the HPAaxis activity Studies of both serotonergic and noradrenergicantidepressants suggested that successful treatment normal-ized the measures of the HPA axis function in depressedpatients and stressed animals [44 45] These antidepressantsimproved hippocampal function stimulated GR expressionand then restored the negative corticosteroid feedback [46]As a result the normalized negative feedback reducedserum corticosterone levels mediated through corticotropin-releasing hormoneadrenocorticotropic hormone inhibition[47] Based on our previous report that nobiletin producedan antidepressant-like effect through serotonergic noradren-ergic and dopaminergic systems [21] we speculate that theaction of nobiletin on the HPA axis is thus likely to beexerted via the monoaminergic systems However it shouldbe noted that the mechanism of nobiletin to inhibit serumcorticosterone secretion is complicated since glucocorticoid

is affected by many factors and there is no consistent evi-dence of a simple relationship between the monoaminergicsystem and HPA axis [48] Therefore there would be adual system for the HPA restoration directly via unknownmechanisms (such as cytokines) and via monoaminergicsystems [49 50]

In recent years the biological research of depressionhas moved beyond the monoamine hypothesis [23] Neu-rotrophins and their receptors are also involved in thefunction of the hippocampus where some of them in par-ticular BDNF and TrkB receptor are widely expressed [51]Increasing clinical and experimental evidence indicated thatBDNF played a role in the pathophysiology of depression andthat antidepressants may in part exert their effects throughregulation of BDNF [7] For example downregulation ofBDNF expression was found in hippocampal region fromdepressed suicide victims relative to matched controls ina clinical study [52] Many of the chronic stressors suchas CUMS or social defeat stress have also been foundto robustly downregulate hippocampal BDNF mRNA andprotein expression in rodents [11 53] Consistently datadisplayed in our study also indicated that 5-week CUMSsignificantly decreased the hippocampal BDNF mRNA andprotein expression In contrast to the effects of CUMSnobiletin and fluoxetine treatments restored the reduction ofBDNF expression in the model which was partly consistentwith a previous study that nobiletin possessed neurotrophicaction in PC12 cells [54]

In parallel with BDNF increased mRNA expression ofhippocampal TrkB receptor is involved in the therapeuticaction of antidepressant treatment in rats [55] TrkB receptoras well as BDNF in hippocampus is decreased in depressedpatients [56] Chronic antidepressant treatment has beenreported to enhance hippocampal neurogenesis as well as toincrease the expression of BDNF and TrkB receptor in animalmodels [7 57] BDNF initiates TrkB receptor-dependent dif-ferent intracellular signaling pathway and exhibits beneficialeffect for the treatment of depression in experimental studies[58] Although BDNF-TrkB signaling is downregulated inhippocampus according to animal models of stress andordepressive-like behavior [59 60] some studies failed to detectevidence for depression with reduced BDNF levels in meta-analyses [61] or heterozygous mutant 119887119889119899119891+minus mice [62]However in contrast to the unclear association with thepathophysiology of depression the neurotrophin signalingappears to be required for antidepressant activity In aprevious study drugs that block TrkB signaling pathway canbreak the antidepressant-like effect of BDNF in rats [59]In contrast to our data on BDNF mRNA expression hip-pocampal TrkB mRNA expression did not modify after the5-week CUMS procedure or drug treatments However TrkBprotein expression increased following nobiletin or fluoxetinetreatment although the inhibitory effect of CUMS did notreach a significant level This finding suggests that nobiletinmay affect the rate of TrkB protein synthesis or degradationbut not TrkB mRNA Although the mechanism of nobiletin-induced TrkB protein upregulation is unknown the roleof ubiquitin-proteasome pathway has been suggested for


ligand-induced TrkB degradation [51 60] Further studies arenecessary to determine whether ubiquitin system is involvedin nobiletin-induced TrkB regulation In addition it shouldbe noted that a recent study found that 5mgkg fluoxetinetreatment administration for 5 weeks did not alter expressionof hippocampal TrkB protein in rats [24] The reason for thisdiscrepancymay be due to different time points atwhichTrkBexpression was assessed after the last exposure to the stressor[63] In their study after 5-week procedure of CUMS theanimals were submitted to behavioral tests for two weeks andsacrificed at week 7 [24] Another reason could possibly beattributed to the different doses and administration routes offluoxetine used

In addition to increasing BDNF levels and TrkB signal-ing antidepressants also increase its downstream target ofsynaptic protein in rodents [64 65] For example fluoxetine-induced changes in synaptic protein expression were medi-ated by TrkB signaling in rats [12] Synapsins are the mostabundant synaptic proteins and play multiple roles in synap-tic transmission and plasticity [66] Reduction of synapsinin the hippocampus was found in patients with mentaldisorder [67] CUMS procedure has been demonstrated toalter brain structure and function in rat causing suppressionof synaptic plasticity in the hippocampus [68] Collectivelysince BDNF-TrkB signaling pathway canmediate behaviouralresponses to antidepressants and induce neuronal plasticitywe investigated the contribution of the signaling to nobiletin-induced changes in synapsin I expression one of the majorsynaptic vesicle-associated proteins Consistentwith previousstudies [69 70] CUMS suppressed the expression of synapsinI at both themRNA and protein levels Chronic nobiletin andfluoxetine completely abolished this CUMS-induced deficitThis finding suggests that nobiletin exerts downstream effectson synapsin I similar to those of classical antidepressantfluoxetine

In addition glucocorticoids play a critical role in medi-ating stress-induced downregulation of BDNF in the hip-pocampus in animals [63] Repeated corticosterone admin-istration like stress decreased the BDNF levels in thehippocampus of normal or adrenalectomized rats [71 72]A significant hypersecretion of corticosterone with down-regulation of hippocampal BDNF was also found in theGR-deficient mice [73] In contrast there was a significantinduction in BDNF expression in rats after removal of theadrenal glands [74] Moreover BDNF-enriched synaptic pro-tein of synapsin I was suppressed by glucocorticoid in vitro[54] Together with the literatures we speculated that BDNF-TrkB pathway restoration by nobiletin may be mediated bycorticosterone

Taken together the results of the present work showedthat anhedonic behavior as well as downregulation ofhippocampal BDNF TrkB and synapsin I expression wasobserved in rats subjected to CUMS The results also sug-gested that nobiletin counteracted the ability of CUMS toinduce depression-like behavior in rats and might involvemaintenance of hippocampal BDNF-TrkB pathwayHowevernonstressed rats treated with nobiletin did not adopt in ourpresent study data concerning behavioral and biochemicalchanges in nonstressed rats would certainly provide some

additional explanations on the antidepressant-like mecha-nism of nobiletin so this is one of the limitations of thepresent study Finally since enhanced induction expression ofBDNF in response to chronic antidepressant agent treatmentscould promote neuronal survival and protect neurons fromthe damaging effects of stress in rat brain [36] BDNFTrkB-dependent neurogenesis involved in the antidepressant-likemechanism of nobiletin will be further investigated by usinginhibitorsactivators or gene block-outsilence technique

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The project was supported by Grants from the Natural Sci-ence Foundation of Fujian Province of China (no 2011J05081)and the National Natural Science Foundation of China (no81202940)

References

[1] World Health Organisation Depression 2007 httpwwwwhointmental healthmanagementdepressiondefinitionen

[2] Y Liu N Yang W Hao et al ldquoDynamic proteomic analysisof protein expression profiles in whole brain of Balbc micesubjected to unpredictable chronic mild stress implicationsfor depressive disorders and future therapiesrdquo NeurochemistryInternational vol 58 no 8 pp 904ndash913 2011

[3] E J Huang and L F Reichardt ldquoNeurotrophins roles inneuronal development and functionrdquo Annual Review of Neuro-science vol 24 pp 677ndash736 2001

[4] S D Skaper ldquoThe neurotrophin family of neurotrophic factorsan overviewrdquo in Neurotrophic Factors vol 846 of Methods inMolecular Biology pp 1ndash12 2012

[5] S A Heldt L Stanek J P Chhatwal and K J ResslerldquoHippocampus-specific deletion of BDNF in adultmice impairsspatial memory and extinction of aversive memoriesrdquo Molecu-lar Psychiatry vol 12 no 7 pp 656ndash670 2007

[6] F Angelucci S Brene and A A Mathe ldquoBDNF in schizophre-nia depression and corresponding animal modelsrdquo MolecularPsychiatry vol 10 no 4 pp 345ndash352 2005

[7] E Castren and T Rantamaki ldquoThe role of BDNF and its recep-tors in depression and antidepressant drug action reactivationof developmental plasticityrdquo Developmental Neurobiology vol70 no 5 pp 289ndash297 2010

[8] W Li and J Keifer ldquoRapid enrichment of presynaptic proteinin boutons undergoing classical conditioning is mediated bybrain-derived neurotrophic factorrdquo Neuroscience vol 203 pp50ndash58 2012

[9] M Fahnestock M Marchese E Head et al ldquoBDNF increaseswith behavioral enrichment and an antioxidant diet in the ageddogrdquo Neurobiology of Aging vol 33 no 3 pp 546ndash554 2012

[10] T Rantamaki P Hendolin A Kankaanpaa et al ldquoPharmaco-logically diverse antidepressants rapidly activate brain-derivedneurotrophic factor receptor TrkB and induce phospholipase-C120574 signaling pathways in mouse brainrdquo Neuropsychopharma-cology vol 32 no 10 pp 2152ndash2162 2007

[11] Y Zhang F Gu J Chen and W Dong ldquoChronic antidepres-sant administration alleviates frontal and hippocampal BDNF


deficits in CUMS ratrdquo Brain Research vol 1366 pp 141ndash1482010

[12] O F OrsquoLeary X Wu and E Castren ldquoChronic fluoxetinetreatment increases expression of synaptic proteins in the hip-pocampus of the ovariectomized rat role of BDNF signallingrdquoPsychoneuroendocrinology vol 34 no 3 pp 367ndash381 2009

[13] D Taliaz N Stall D E Dar and A Zangen ldquoKnockdownof brain-derived neurotrophic factor in specific brain sitesprecipitates behaviors associated with depression and reducesneurogenesisrdquo Molecular Psychiatry vol 15 no 1 pp 80ndash922010

[14] T Saarelainen P Hendolin G Lucas et al ldquoActivation of theTrkB neurotrophin receptor is induced by antidepressant drugsand is required for antidepressant-induced behavioral effectsrdquoJournal of Neuroscience vol 23 no 1 pp 349ndash357 2003

[15] L M Monteggia B Luikart M Barrot et al ldquoBrain-derivedneurotrophic factor conditional knockouts show gender dif-ferences in depression-related behaviorsrdquo Biological Psychiatryvol 61 no 2 pp 187ndash197 2007

[16] B E Leonard ldquoStress norepinephrine and depressionrdquo Journalof Psychiatry and Neuroscience vol 26 pp S11ndashS16 2001

[17] P Willner A Towell D Sampson S Sophokleous and R Mus-cat ldquoReduction of sucrose preference by chronic unpredictablemild stress and its restoration by a tricyclic antidepressantrdquoPsychopharmacology vol 93 no 3 pp 358ndash364 1987

[18] P Willner ldquoValidity reliability and utility of the chronic mildstress model of depression a 10-year review and evaluationrdquoPsychopharmacology vol 134 no 4 pp 319ndash329 1997

[19] H Onozuka A Nakajima K Matsuzaki et al ldquoNobiletina citrus flavonoid improves memory impairment and Abetapathology in a transgenic mouse model of Alzheimerrsquos diseaserdquoJournal of Pharmacology and Experimental Therapeutics vol326 no 3 pp 739ndash744 2008

[20] Y Yamamoto N Shioda F Han et al ldquoNobiletin improvesbrain ischemia-induced learning and memory deficits throughstimulation of CaMKII and CREB phosphorylationrdquo BrainResearch vol 1295 pp 218ndash229 2009

[21] L T Yi H L Xu J Feng X Zhan L P Zhou and C C CuildquoInvolvement of monoaminergic systems in the antidepressant-like effect of nobiletinrdquo Physiology and Behavior vol 102 no 1pp 1ndash6 2011

[22] S Campbell and GMacqueen ldquoThe role of the hippocampus inthe pathophysiology of major depressionrdquo Journal of Psychiatryand Neuroscience vol 29 no 6 pp 417ndash426 2004

[23] O Berton and E J Nestler ldquoNew approaches to antidepressantdrug discovery beyond monoaminesrdquo Nature Reviews Neuro-science vol 7 no 2 pp 137ndash151 2006

[24] M First I Gil-Ad M Taler I Tarasenko N Novak and AWeizman ldquoThe effects of fluoxetine treatment in a chronicmild stress rat model on depression-related behavior brainneurotrophins and ERK expressionrdquo Journal of Molecular Neu-roscience vol 45 no 2 pp 246ndash255 2011

[25] L T Yi J M Li Y C Li Y Pan Q Xu and L D KongldquoAntidepressant-like behavioral and neurochemical effects ofthe citrus-associated chemical apigeninrdquo Life Sciences vol 82no 13-14 pp 741ndash751 2008

[26] S P Singh Wahajuddin D Tewari K Patel and G KJain ldquoPermeability determination and pharmacokinetic studyof nobiletin in rat plasma and brain by validated high-performance liquid chromatography methodrdquo Fitoterapia vol82 no 8 pp 1206ndash1214 2011

[27] D Saigusa M Shibuya D Jinno et al ldquoHigh-performanceliquid chromatography with photodiode array detection fordetermination of nobiletin content in the brain and serumof mice administrated the natural compoundrdquo Analytical andBioanalytical Chemistry vol 400 no 10 pp 3635ndash3641 2011

[28] P Willner J L Moreau C K Nielsen M Papp andA Sluzewska ldquoDecreased hedonic responsiveness followingchronic mild stress is not secondary to loss of body weightrdquoPhysiology and Behavior vol 60 no 1 pp 129ndash134 1996

[29] V A Edgar G A Cremaschi L Sterin-Borda and A MGenaro ldquoAltered expression of autonomic neurotransmitterreceptors and proliferative responses in lymphocytes from achronic mild stress model of depression effects of fluoxetinerdquoBrain Behavior and Immunity vol 16 no 4 pp 333ndash350 2002

[30] W Liu and C Zhou ldquoCorticosterone reduces brain mito-chondrial function and expression of mitofusin BDNF indepression-like rodents regardless of exercise preconditioningrdquoPsychoneuroendocrinology vol 37 no 7 pp 1057ndash1070 2012

[31] G D Gamaro M E Prediger J Lopes M G Bassani and CDalmaz ldquoFluoxetine alters feeding behavior and leptin levelsin chronically-stressed ratsrdquo Pharmacology Biochemistry andBehavior vol 90 no 3 pp 312ndash317 2008

[32] P C Casarotto and R Andreatini ldquoRepeated paroxetine treat-ment reverses anhedonia induced in rats by chronic mild stressor dexamethasonerdquo European Neuropsychopharmacology vol17 no 11 pp 735ndash742 2007

[33] American Psychiatric Association Diagnostic and StatisticalManual ofMental Disorders APA PressWashington DC USA4th edition 2000

[34] S Pothion J C Bizot F Trovero and C Belzung ldquoStraindifferences in sucrose preference and in the consequences ofunpredictable chronic mild stressrdquo Behavioural Brain Researchvol 155 no 1 pp 135ndash146 2004

[35] K Kunimasa S Kuranuki N Matsuura et al ldquoIdentifica-tion of nobiletin a polymethoxyflavonoid as an enhancer ofadiponectin secretionrdquo Bioorganic and Medicinal ChemistryLetters vol 19 no 7 pp 2062ndash2064 2009

[36] K Kanda K Nishi A Kadota S Nishimoto M C Liu andT Sugahara ldquoNobiletin suppresses adipocyte differentiationof 3T3-L1 cells by an insulin and IBMX mixture inductionrdquoBiochimica et BiophysicaActa vol 1820 no 4 pp 461ndash468 2012

[37] Y S Lee B Y Cha S S Choi et al ldquoNobiletin improves obesityand insulin resistance in high-fat diet-induced obese micerdquoTheJournal of Nutritional Biochemistry vol 24 no 1 pp 156ndash1622013

[38] M Aihara I Ida N Yuuki et al ldquoHPA axis dysfunction inunmedicated major depressive disorder and its normalizationby pharmacotherapy correlates with alteration of neural activityin prefrontal cortex and limbicparalimbic regionsrdquo PsychiatryResearch vol 155 no 3 pp 245ndash256 2007

[39] L Manthey C Leeds E J Giltay et al ldquoAntidepressant use andsalivary cortisol in depressive and anxiety disordersrdquo EuropeanNeuropsychopharmacology vol 21 no 9 pp 691ndash699 2011

[40] N Rohleder J M Wolf and O T Wolf ldquoGlucocorticoid sen-sitivity of cognitive and inflammatory processes in depressionand posttraumatic stress disorderrdquo Neuroscience and Biobehav-ioral Reviews vol 35 no 1 pp 104ndash114 2010

[41] S Thakker-Varia and J Alder ldquoNeuropeptides in depressionrole of VGFrdquoBehavioural Brain Research vol 197 no 2 pp 262ndash278 2009

[42] E J Doczy K Seroogy C R Harrison and J P HermanldquoHypothalamo-pituitary-adrenocortical axis glucocorticoids


and neurologic diseaserdquo Immunology and Allergy Clinics ofNorth America vol 29 no 2 pp 265ndash284 2009

[43] Y C Li J D Shen J Li R Wang S Jiao and L T YildquoChronic treatment with baicalin prevents the chronic mildstress-induced depressive-like behavior involving the inhi-bition of cyclooxygenase-2 in rat brainrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 40 pp 138ndash143 2013

[44] G Nikisch ldquoInvolvement and role of antidepressant drugsof the hypothalamic-pituitary-adrenal axis and glucocorticoidreceptor functionrdquoNeuroendocrinology Letters vol 30 no 1 pp11ndash16 2009

[45] I Kitayama A M Janson A Cintra et al ldquoEffects of chronicimipramine treatment on glucocorticoid receptor immunore-activity in various regions of the rat brain Evidence forselective increases of glucocorticoid receptor immunoreactivityin the locus coeruleus and in 5-hydroxytryptamine nerve cellgroups of the rostral ventromedial medullardquo Journal of NeuralTransmission vol 73 no 3 pp 191ndash203 1988

[46] N Barden ldquoModulation of glucocorticoid receptor gene expres-sion by antidepressant drugsrdquo Pharmacopsychiatry vol 29 no1 pp 12ndash22 1996

[47] L S Brady H J Whitfield Jr R J Fox P W Gold and MHerkenham ldquoLong-term antidepressant administration alterscorticotropin-releasing hormone tyrosine hydroxylase andmineralocorticoid receptor gene expression in rat brain Ther-apeutic implicationsrdquo The Journal of Clinical Investigation vol87 no 3 pp 831ndash837 1991

[48] L Lanfumey R Mongeau C Cohen-Salmon and M HamonldquoCorticosteroid-serotonin interactions in the neurobiologi-cal mechanisms of stress-related disordersrdquo Neuroscience andBiobehavioral Reviews vol 32 no 6 pp 1174ndash1184 2008

[49] P A Zunszain C Anacker A Cattaneo L A Carvalho and CM Pariante ldquoGlucocorticoids cytokines and brain abnormali-ties in depressionrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 35 no 3 pp 722ndash729 2011

[50] N Barden ldquoImplication of the hypothalamic-pituitary-adrenalaxis in the physiopathology of depressionrdquo Journal of Psychiatryand Neuroscience vol 29 no 3 pp 185ndash193 2004

[51] M T Sommerfeld R Schweigreiter Y A Barde and E HoppeldquoDown-regulation of the neurotrophin receptor TrkB followingligand binding Evidence for an involvement of the proteasomeand differential regulation of TrkA and TrkBrdquo The Journal ofBiological Chemistry vol 275 no 12 pp 8982ndash8990 2000

[52] F Karege G VaudanM Schwald N Perroud and R LaHarpeldquoNeurotrophin levels in postmortem brains of suicide victimsand the effects of antemortem diagnosis and psychotropicdrugsrdquo Molecular Brain Research vol 136 no 1-2 pp 29ndash372005

[53] B Haenisch A Bilkei-Gorzo M G Caron and H BonischldquoKnockout of the norepinephrine transporter and pharmaco-logically diverse antidepressants prevent behavioral and brainneurotrophin alterations in two chronic stressmodels of depres-sionrdquo Journal of Neurochemistry vol 111 no 2 pp 403ndash4162009

[54] H C Lai M J Wu P Y Chen et al ldquoNeurotrophic effect of cit-rus 5-hydroxy-3 6 7 8 31015840 41015840-hexamethoxyflavone promotionof neurite outgrowth via cAMPPKACREB pathway in PC12cellsrdquo PLoS ONE vol 6 no 11 Article ID e28280 2011

[55] M Nibuya S Morinobu and R S Duman ldquoRegulation ofBDNFand trkBmRNA in rat brain by chronic electroconvulsive

seizure and antidepressant drug treatmentsrdquo Journal of Neuro-science vol 15 no 11 pp 7539ndash7547 1995

[56] Y Dwivedi H S Rizavi R R Conley R C Roberts C ATamminga andGN Pandey ldquoAltered gene expression of brain-derived neurotrophic factor and receptor tyrosine kinase Bin postmortem brain of suicide subjectsrdquo Archives of GeneralPsychiatry vol 60 no 8 pp 804ndash815 2003

[57] D T Balu and I Lucki ldquoAdult hippocampal neurogenesisregulation functional implications and contribution to diseasepathologyrdquoNeuroscience and Biobehavioral Reviews vol 33 no3 pp 232ndash252 2009

[58] TNumakawa S Suzuki E KumamaruNAdachiMRichardsand H Kunugi ldquoBDNF function and intracellular signaling inneuronsrdquo Histology and Histopathology vol 25 no 2 pp 237ndash258 2010

[59] Y Shirayama A C H Chen S Nakagawa D S Russell andR S Duman ldquoBrain-derived neurotrophic factor producesantidepressant effects in behavioral models of depressionrdquoJournal of Neuroscience vol 22 no 8 pp 3251ndash3261 2002

[60] A Kutiyanawalla A V Terry Jr and A Pillai ldquoCysteamineattenuates the decreases in TrkB protein levels and theanxietydepression-like behaviors in mice induced by corticos-terone treatmentrdquo PLoS ONE vol 6 no 10 Article ID e261532011

[61] M Gratacos J R Gonzalez J M Mercader R de Cid MUrretavizcaya and X Estivill ldquoBrain-derived neurotrophicfactor Val66Met and psychiatric disorders meta-analysis ofcase-control studies confirm association to substance-relateddisorders eating disorders and schizophreniardquo Biological Psy-chiatry vol 61 no 7 pp 911ndash922 2007

[62] Y Ibarguen-Vargas A Surget P Vourcrsquoh et al ldquoDeficit inBDNF does not increase vulnerability to stress but dampensantidepressant-like effects in the unpredictable chronic mildstressrdquo Behavioural Brain Research vol 202 no 2 pp 245ndash2512009

[63] H D Schmidt and R S Duman ldquoThe role of neurotrophicfactors in adult hippocampal neurogenesis antidepressanttreatments and animal models of depressive-like behaviorrdquoBehavioural Pharmacology vol 18 no 5-6 pp 391ndash418 2007

[64] J Alfonso L R Frick D M Silberman M L Palumbo A MGenaro and A C Frasch ldquoRegulation of hippocampal geneexpression is conserved in two species subjected to differentstressors and antidepressant treatmentsrdquo Biological Psychiatryvol 59 no 3 pp 244ndash251 2006

[65] V S Barbiero R Giambelli L Musazzi et al ldquoChronicantidepressants induce redistribution and differential activationof alphaCaM kinase II between presynaptic compartmentsrdquoNeuropsychopharmacology vol 32 no 12 pp 2511ndash2519 2007

[66] F Cesca P Baldelli F Valtorta and F Benfenati ldquoThe synapsinskey actors of synapse function and plasticityrdquo Progress inNeurobiology vol 91 no 4 pp 313ndash348 2010

[67] M P Vawter L Thatcher N Usen T M Hyde J E Kleinmanand W J Freed ldquoReduction of synapsin in the hippocampusof patients with bipolar disorder and schizophreniardquoMolecularPsychiatry vol 7 no 6 pp 571ndash578 2002

[68] M Joels H Karst D Alfarez et al ldquoEffects of chronic stress onstructure and cell function in rat hippocampus and hypothala-musrdquo Stress vol 7 no 4 pp 221ndash231 2004

[69] R Silva A R Mesquita J Bessa et al ldquoLithium blocks stress-induced changes in depressive-like behavior and hippocampalcell fate the role of glycogen-synthase-kinase-3120573rdquoNeurosciencevol 152 no 3 pp 656ndash669 2008


[70] N Li R J Liu J M Dwyer et al ldquoGlutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral andsynaptic deficits caused by chronic stress exposurerdquo BiologicalPsychiatry vol 69 no 8 pp 754ndash761 2011

[71] M J M Schaaf J de Jong E R de Kloet and E VreugdenhilldquoDownregulation of BDNF mRNA and protein in the rathippocampus by corticosteronerdquo Brain Research vol 813 no 1pp 112ndash120 1998

[72] Q Q Mao Z Huang S P Ip Y F Xian and C T Che ldquoPeonyglycosides reverse the effects of corticosterone on behavior andbrain BDNF expression in ratsrdquoBehavioural Brain Research vol227 no 1 pp 305ndash309 2012

[73] S Ridder S Chourbaji R Hellweg et al ldquoMice with genet-ically altered glucocorticoid receptor expression show alteredsensitivity for stress-induced depressive reactionsrdquo Journal ofNeuroscience vol 25 no 26 pp 6243ndash6250 2005

[74] H M Chao R R Sakai L Y Ma and B S McEwen ldquoAdrenalsteroid regulation of neurotrophic factor expression in the rathippocampusrdquo Endocrinology vol 139 no 7 pp 3112ndash3118 1998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


reviewed earlier strongly suggest that the behavioral effectsof antidepressants require functional BDNF signaling in thebrain

Stress is an important precipitant factor in depressionand the changes in various body systems that occurred indepression are similar to those observed in response to stress[16] Chronic unpredictable mild stress (CUMS) the mostpromising rodent model for depression is widely used forpreclinical testing and screening of antidepressants [17] Inthe CUMS paradigm animals are subjected to a variety ofmild stressors presented intermittently for prolonged periodsof time which mimic chronic stressful life events and resultin a behavioral deficit anhedonia a core symptom of humandepression [18] Anhedonia is monitored by a reduction insucrose preference which can be restored by therapeuticallyeffective drugs for the treatment of depression [11]

Nobiletin a dietary constituent of flavonoids isolatedfrom citrus fruits has been reported to upregulate synaptictransmission and improve memory impairment in rodents[19 20] Our previous study found that nobiletin adminis-tration significantly reduced the immobility time in both thetail suspension test (TST) and forced swimming test (FST)without accompanying changes in locomotor activity in theopen-field test (OFT) in mice [21] Furthermore we alsodemonstrated that the antidepressant-like effect of nobiletinwas mediated by monoaminergic systems in this studyHowever until now the neurotrophic molecular expressionunderlying the antidepressant-like effect of nobiletin remainsunknown

Therefore considering that hippocampus is critical forstress learning and memory processes in depression and inthe antidepressant response to pharmacotherapy [22ndash24] theaim of the present study was to evaluate the effects of dailynobiletin treatment on sucrose preference and correspondingchanges in BDNF TrkB receptor and the downstream targetsynapsin I in the hippocampus after CUMS

2 Materials and Methods

21 Animals Male Sprague-Dawley rats (260ndash300 g) werepurchased from Laboratory Animal Centre Fujian MedicalUniversity Fujian Province China Animals were single-housed under a normal 12-h12-h lightdark cycle with thelights on at 0700 AM Ambient temperature and relativehumidity were maintained at 22 plusmn 2∘C and at 55 plusmn 5 andanimals were given a standard chow and water ad libitum forthe duration of the study except when the CUMS procedureor sucrose preference test was required The animals wereallowed to acclimatize to these conditions for 1 week beforeany experimental procedure was initiated At the beginningof the experiments the average body weight of rats wasapproximately 310 g All procedures were performed in accor-dance with the published guidelines of the China Council onAnimal Care (regulations for the Administration of Affairsconcerning experimental animals approved by the StateCouncil on October 31 1988 and promulgated by Decreeno 2 of the State Science and Technology Commission onNovember 14 1988)

22 Chemicals and Reagents Nobiletin (purity gt98 byHPLC) was obtained from Shanxi Huike Botanical Devel-opment Co Ltd (Xirsquoan China) Fluoxetine hydrochloridewas purchased from Changzhou Siyao Pharmaceuticals CoLtd (Changzhou China) All primers used in this studywere designed and synthesized by Sangon Biotech Co Ltd(Shanghai China)The anti-BDNF (Catalog number sc-546detecting mature BDNF) and anti-TrkB (Catalog numbersc-12 detecting full-length TrkB) antibody and the respec-tive secondary antibodies were purchased from Santa CruzBiotechnology Inc (Santa Cruz USA) The antisynapsin I(Catalog numberAB1543 detecting synapsin I) antibodywaspurchased from Millipore Corporation (Billerica USA) Theanti-GAPDH (Catalog number KC-5G5 detecting GAPDH)antibody was purchased from Kangcheng Biotech (Shang-hai China) Trizol reagent was purchased from Invitrogen(Carlsbad USA) Reverse transcriptase moloney murineleukemia Virus (M-MLV) used for cDNA synthesis wasfrom Promega Corporation (Madison USA) All otherreagents used in polymerase chain reaction (PCR) andwestern blot were purchased from Sangon Biotech Co Ltd(Shanghai China)

23 CUMS Procedure The CUMS procedure was performedaccording to the traditional method described by Willner etal [17] with some modifications Briefly the weekly stressregime consisted of food and water deprivation stroboscopicillumination (150 flashesmin) white noise lightdark suc-cession every 2 h overnight illumination 45∘ cage tilt soiledcage and pair-housing (Table 1) All of the stressors whichwere applied individually and continuously were randomlyscheduled over a 1-week period and repeated throughoutthe 5-week procedure The control group was housed in aseparate room and had no contact with the stressed animalsThese rats were deprived of food and water for the 18 hpreceding each sucrose test but otherwise food and waterwere freely available in the home cage

24 Sucrose Preference Test Before the beginning of CUMSprocedure all rats were given 1 sucrose solution for 24hours Then both sucrose solution and fresh water weremade accessible to the rats for another 24 hours Afterdeprived of drinking for 23 hours the rats were given both1 sucrose solution and fresh water for 1 hour again Afterthis sucrose consumption training phase the animals wererandomly divided into 5 groups (8 rats per group) control-vehicle (saline) CUMS-vehicle (saline) CUMS-fluoxetine(positive control 10mgkg) and CUMS-nobiletin at a doseof 20mgkg and 40mgkg Nobiletin was suspended insaline with 10 (vv) Tween-80 (polyoxyethylene sorbitanmonooleate) and fluoxetine hydrochloride was dissolvedin 09 physiological saline Four groups except control-vehicle were exposed to the CUMS procedure for 5 weeksand treated All drugs were administered by gavage in avolume of 10mLkg body once daily at 1100 AM for 5 weeksThrough the period of CUMS and treatment sucrose prefer-ence test were conducted following an 18-h food and waterdeprivation at 1100 AM every Tuesday Sucrose preferencewas calculated as sucrose preference () = sucrose intake












3 Results



60

70

80

90

Week

Sucr

ose p

refe

renc

e (

)

0 1 2 3 4 5



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(a)

300

350

400

450

Body

wei

ght (

g)

Week0 1 2 3 4 5



lowast

(b)






0

50

100

150

(mgkg)

CUMS

Seru

m co

rtic

oste

rone

leve

ls (n

gm

L)








0

05

1

Tota

l BD

NF

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

(mgkg)

CUMS



lowastlowast

lowastlowast

lowast

(a)

Full-

leng

th T

rkB

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


(b)

Syna

psin

I m

RNA

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS




(c)








4 Discussion



BDNF

TrkB

Synapsin I

GAPDH

14kDa

145kDa

80kDa

36kDa

Vehi

cle

CUM

S-ve

hicle

Fluo

xetin

e

Nob

iletin

-20

Nob

iletin

-40

(a)

BDN

F le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

(mgkg)

CUMS



0

05

1 lowastlowast

lowastlowast

lowastlowast

(b)

TrkB

leve

lsfo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


lowastlowastlowast

lowastlowast

(c)

Syna

psin

I le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS



lowastlowastlowast

lowastlowast

(d)





















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



60

70

80

90

Week

Sucr

ose p

refe

renc

e (

)

0 1 2 3 4 5



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(a)

300

350

400

450

Body

wei

ght (

g)

Week0 1 2 3 4 5



lowast

(b)






0

50

100

150

(mgkg)

CUMS

Seru

m co

rtic

oste

rone

leve

ls (n

gm

L)








0

05

1

Tota

l BD

NF

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

(mgkg)

CUMS



lowastlowast

lowastlowast

lowast

(a)

Full-

leng

th T

rkB

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


(b)

Syna

psin

I m

RNA

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS




(c)








4 Discussion



BDNF

TrkB

Synapsin I

GAPDH

14kDa

145kDa

80kDa

36kDa

Vehi

cle

CUM

S-ve

hicle

Fluo

xetin

e

Nob

iletin

-20

Nob

iletin

-40

(a)

BDN

F le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

(mgkg)

CUMS



0

05

1 lowastlowast

lowastlowast

lowastlowast

(b)

TrkB

leve

lsfo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


lowastlowastlowast

lowastlowast

(c)

Syna

psin

I le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS



lowastlowastlowast

lowastlowast

(d)





















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















60

70

80

90

Week

Sucr

ose p

refe

renc

e (

)

0 1 2 3 4 5



lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

(a)

300

350

400

450

Body

wei

ght (

g)

Week0 1 2 3 4 5



lowast

(b)






0

50

100

150

(mgkg)

CUMS

Seru

m co

rtic

oste

rone

leve

ls (n

gm

L)








0

05

1

Tota

l BD

NF

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

(mgkg)

CUMS



lowastlowast

lowastlowast

lowast

(a)

Full-

leng

th T

rkB

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


(b)

Syna

psin

I m

RNA

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS




(c)








4 Discussion



BDNF

TrkB

Synapsin I

GAPDH

14kDa

145kDa

80kDa

36kDa

Vehi

cle

CUM

S-ve

hicle

Fluo

xetin

e

Nob

iletin

-20

Nob

iletin

-40

(a)

BDN

F le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

(mgkg)

CUMS



0

05

1 lowastlowast

lowastlowast

lowastlowast

(b)

TrkB

leve

lsfo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


lowastlowastlowast

lowastlowast

(c)

Syna

psin

I le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS



lowastlowastlowast

lowastlowast

(d)





















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

Tota

l BD

NF

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

(mgkg)

CUMS



lowastlowast

lowastlowast

lowast

(a)

Full-

leng

th T

rkB

mRN

Afo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


(b)

Syna

psin

I m

RNA

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS




(c)








4 Discussion



BDNF

TrkB

Synapsin I

GAPDH

14kDa

145kDa

80kDa

36kDa

Vehi

cle

CUM

S-ve

hicle

Fluo

xetin

e

Nob

iletin

-20

Nob

iletin

-40

(a)

BDN

F le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

(mgkg)

CUMS



0

05

1 lowastlowast

lowastlowast

lowastlowast

(b)

TrkB

leve

lsfo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


lowastlowastlowast

lowastlowast

(c)

Syna

psin

I le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS



lowastlowastlowast

lowastlowast

(d)





















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BDNF

TrkB

Synapsin I

GAPDH

14kDa

145kDa

80kDa

36kDa

Vehi

cle

CUM

S-ve

hicle

Fluo

xetin

e

Nob

iletin

-20

Nob

iletin

-40

(a)

BDN

F le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

(mgkg)

CUMS



0

05

1 lowastlowast

lowastlowast

lowastlowast

(b)

TrkB

leve

lsfo

ld o

f cha

nge

(nor

mal

ized

to G

APD

H)

0

05

1

(mgkg)

CUMS


lowastlowastlowast

lowastlowast

(c)

Syna

psin

I le

vels

fold

of c

hang

e(n

orm

aliz

ed to

GA

PDH

)

0

05

1

(mgkg)

CUMS



lowastlowastlowast

lowastlowast

(d)





















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article nobiletin ameliorates the deficits in...

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