Xiaoyaosan Improves Depressive-Like Behaviors in Mice through Regulating Apelin-APJ System

14 Pages·2017·7.99 MB·English

molecules
Article
Xiaoyaosan Improves Depressive-Like Behaviors in
Mice through Regulating Apelin-APJ System
in Hypothalamus
ZhiyiYan1 ID,HaiyanJiao1,XiufangDing1,QingyuMa2,XiaojuanLi1,QiuxiaPan1,
TingyeWang1,YajingHou1,YoumingJiang1,YueyunLiu1andJiaxuChen1,2,*
1 SchoolofTraditionalChineseMedicine,BeijingUniversityofChineseMedicine,Beijing100029,China;
[email protected](Z.Y.);[email protected](H.J.);[email protected](X.D.);
[email protected](X.L.);[email protected](Q.P.);[email protected](T.W.);
[email protected](Y.H.);[email protected](Y.J.);[email protected](Y.L.)
2 Formula-patternResearchCenter,SchoolofTraditionalChineseMedicine,JinanUniversity,
Guangzhou510632,Guangdong,China;[email protected]
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:28March2018;Accepted:2May2018;Published:3May2018
Abstract: Background: The apelin-APJ system has been considered to play a crucial role in HPA
axis function, and how the traditional Chinese compound prescription Xiaoyaosan regulates the
apelin-APJ system as a supplement to treat depressive disorders. Objective: To investigate the
depression-likebehaviorsandexpressionofapelinandAPJinhypothalamusofchronicunpredictable
mildstress(CUMS)miceandstudywhetherthesechangesrelatedtotheregulationofXiaoyaosan.
Methods: 60adultC57BL/6Jmicewererandomlydividedintofourgroups,includingcontrolgroup,
CUMSgroup,Xiaoyaosantreatmentgroupandfluoxetinetreatmentgroup. Miceinthecontrolgroup
andCUMSgroupreceived0.5mLphysiologicalsalineonceadaybyintragastricadministration.
Mice intwo treatment groupsreceivedXiaoyaosan (0.25 g/kg/d) andfluoxetine (2.6 mg/kg/d),
respectively.After21daysofmodelingwithCUMS,theexpressionofapelinandAPJinhypothalamus
weremeasuredbyreal-timefluorescencequantitativePCR,westernblotandimmunohistochemical
staining. Thephysicalcondition,bodyweight,foodintakeandbehaviortestssuchasopenfieldtest,
sucrosepreferencetestandforceswimmingtestweremeasuredtoevaluatedepressive-likebehaviors.
Results: Inthisstudy,significantbehavioralchangeswerefoundinCUMS-inducedmice,meanwhile
the expressions of apelin and APJ in the hypothalamus were changed after modeling. The body
weight,food-intakeanddepressive-likebehaviorsinCUMS-inducedmicecouldbeimprovedby
Xiaoyaosan treatment which is similar with the efficacy of fluoxetine, while the expressions of
apelinandAPJinhypothalamusweremodifiedbyXiaoyaosan. Conclusions: Thedatasuggestthat
apelin-APJsystemchangesinthehypothalamusmaybeatargetofdepressivedisorders,andthe
beneficialeffectsofChinesecompoundprescriptionXiaoyaosanondepressive-likebehaviorsmaybe
mediatedbytheapelin-APJsystem.
Keywords: Xiaoyaosan;depression;apelin;APJ;chronicunpredictablemildstress;hypothalamus
1. Introduction
Depression is a common mental health problem in modern society, and one of its important
inducing factors is the stress response, which is defined as an uncoordinated state of organism or
the instability of the internal environment [1]. The main characteristic of stress response is the
hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and autonomic nervous system
afterstimulationbytheinternalandexternalenvironment[2]. TheHPAaxis,asaneuroendocrine
Molecules2018,23,1073;doi:10.3390/molecules23051073 www.mdpi.com/journal/molecules
Molecules2018,23,1073 2of14
immunenetworkhub,hastheresponsibilitytomaintainastableinternalenvironmentandprovide
physiologicalandpsychologicalresponsestotheexternalenvironmentandemotionalstimulation[3].
Therefore, hyperactivity of HPA axis is one of the most common neurological manifestations of
depression. Corticotropin-releasingfactor(CRF)isakeyfactorinregulatingthestressresponse,and
thehypersecretionofCRFinthehypothalamusisthebasisofHPAaxisdysfunction[4]. Thesecretion
of CRF in the hypothalamus increased when a subject is exposed to stressors, then the secretion
of adrenocorticotropic hormone (ACTH) in the pituitary increases, which leads to the increase of
glucocorticoid (GC) secreted by the adrenal gland [5]. The main sites of action of GC in the brain
are the hippocampus and cerebral cortex, and excess GC can damage the brain by affecting the
glucocorticoidreceptor(GR)[6],whiletheexcessofGCcausesanegativefeedbackregulationinthe
dysfunctionoftheHPAaxis,whichleadstohyperactivityoftheHPAaxis[7].
Putative apelin receptor (APJ) related to the angiotensin receptor AT1, is isolated from the
human genome in 1993 for the first time [8]. The APJ endogenous ligand (apelin), as a kind of
bioactive peptide, has extensive physiological functions through the interaction with its specific
receptorAPJ[9]. Theapelin-APJsystemisinvolvedinthefunctionalactivitiesofmanysystemssuch
asthecardiovascularsystem,centralnervoussystem,endocrinesystem,immunesystem,digestive
systemandcirculatorysystem[10–13]. APJandapelinaredistributedinthehypothalamicnuclei[14],
especiallyinthesupraopticnucleus(SON)andparaventricularnucleus(PVN)[15]. InO’Carrollet
al.’sstudy,stresscoulddecreasetheexpressionofapelininthePVNandthecontentofcortisolin
plasma, and increase the expression of APJ which is associated with the expression of apelin and
cortisol(CORT),whichmeansthattheapelin-APJsystemplaysanimportantroleinthegeneration
ofGCandnegativefeedbackoftheHPAaxis[16]. Apelin-13canpromotethereleaseofsecretionof
ACTHandCORTbystimulatingthereleaseofCRFandAVPinthehypothalamus,whichsuggests
thattheregulationofapelindependsonCRFandAVP[17]. Furthermore,thecentralinjectionof(Pyr1)
apelin-13canincreasetheexpressionofc-fosgeneinPVN[18],andapelincouldsignificantlystimulate
thereleaseofCRFandAVPinhypothalamicexplantsinvitro[19]. Thesestudiesindicatethatthe
apelin-APJsystemplaysaroleinregulatingtheactivityoftheHPAaxis.
XiaoyaosanisaclassicChinesecompoundprescriptionthathastheeffectofinvigoratingthe
spleen,dispersingthestagnatedliver-energyandnourishingblood[20]. Ithasbeenwidelyusedto
treatmentaldisorders,particularlydepression[21,22]. ModernstudieshaveindicatedthatXiaoyaosan
has an anti-depressant effect through regulation of the HPA axis [23]. The apelin-APJ system is
involvedinthedisordersofHPAaxiscausedbydepression,therefore,thepurposeofthethisstudyis
tocharacterizetheexpressionofapelinandAPJinhypothalamusofmicewithchronicunpredictable
mildstress(CUMS),andobservetheeffectofXiaoyaosaninterventionontheapelin-APJsystemto
determinetheanti-depressantmechanismofactionofXiaoyaosan.
2. Results
2.1. EffectofXiaoyaosanonPhysicalCondition,BodyWeightandFoodIntakeofCUMS-InducedMice
TheobservationstocharacterizetheeffectsofXiaoyaosanincludedthephysicalcondition,food
intake and body weight, and were carried out in mice with the aim of evaluating the efficacy of
XiaoyaosaninCUMS-inducedmice. Aftermodelingfor21days,comparedwiththecontrolgroup
themiceintheCUMSgroupexhibitedsignsoffatigue,disheveledhair,lessactivity,flockingtogether
behaviorandloosestools,whilethephysicalconditionofthetwotreatmentgroupswasbetterthan
thatoftheCUMSgroup(Figure1a). AsshowninFigure1b,c,bodyweightandfoodintakeineach
groupshowednosignificantdifferencesatday0,andthebodyweightofmiceinallgroupsalsohad
nosignificantdifferencesatday0andday7(p>0.05,respectively),thedataofday14showedthat
themiceintheCUMSgrouphadasignificantdecreaseinbodyweightcomparedwithcontrolgroup
mice(p<0.05),thenthedifferenceofbodyweightbetweenCUMSgroupandcontrolgroupbecame
moreobviousbyday21(p<0.01),furthermore,thebodyweightsofmiceintheXiaoyaosantreatment
Molecules2018,23,1073 3of14
Mgorloecuulpes w20e18r,e 23h, ixg h er than those of the CUMS group mice (p < 0.05), while the body weight of3 mof i1c4e
inthe fluoxetinetreatment grouphad nosignificantdifferencecompared withCUMSgroupmice
0.05). The data of food intake showed that the food intake of mice in CUMS group decreased at day14
(p>0.05). ThedataoffoodintakeshowedthatthefoodintakeofmiceinCUMSgroupdecreasedat
and day 21 compared with control group (p < 0.01), meanwhile, the food intake of Xiaoyaosan and
day14andday21comparedwithcontrolgroup(p<0.01),meanwhile,thefoodintakeofXiaoyaosan
fluoxetine treatment groups at day 14 and day 21 were higher than those of CUMS group mice (p <
andfluoxetinetreatmentgroupsatday14andday21werehigherthanthoseofCUMSgroupmice
0.01).
(p<0.01).

FFigiguurree 11. .(a(a) )PPhhyyssicicaal lccoonndditiitoionn oof fmmicicee inin aalll lggrroouuppss wwaass oobbsseerrvveedd aat tththee eenndd oof fmmooddeelilningg; ;(b(b) )BBooddyy
wweeigighht twwaass rreeccoorrddeedd oonnccee aa wweeeekk dduurriningg ththee mmooddeelilningg ppeerrioiodd; ;(c(c) )FFoooodd inintatakkee wwaass rreeccoorrddeedd oonnccee aa
wweeeekk dduurriningg tthhee mmooddeelliinngg ppeerriioodd. .DDaattaa wweerree eexxpprreesssseedd aass mmeeaannss ±± SSEEMM (n(n == 1155),) ,*** *pp << 00.0.011 vveerrssuuss
ccoonntrtrool lggrroouupp; ;Δ∆Δ ∆p p< <0.001.0 v1evresrussu CsUCMUMS Sgrgoruopu.p .
2.2. Effect of Xiaoyaosan on Depressive-Like Behaviors of CUMS-Induced Mice
2.2. EffectofXiaoyaosanonDepressive-LikeBehaviorsofCUMS-InducedMice
For the purpose of further evaluating the depressive-like behaviors, several behavioral tests
Forthepurposeoffurtherevaluatingthedepressive-likebehaviors,severalbehavioraltestswere
were carried out, including sucrose preference test (SPT), force swimming test (FST) and open field
carriedout, includingsucrosepreferencetest(SPT),forceswimmingtest(FST)andopenfieldtest
test (OFT). The sucrose preference test is a classic method for detecting loss of pleasure due to
(OFT).Thesucrosepreferencetestisaclassicmethodfordetectinglossofpleasureduetodepression,
depression, and the reduced preference for sucrose in the test is a key indicator of depression in
and the reduced preference for sucrose in the test is a key indicator of depression in rodents [24].
rodents [24]. The SPT results of each group were basically the same at day 0 (both p > 0.05), but the
TheSPTresultsofeachgroupwerebasicallythesameatday0(bothp>0.05),butthesucrosepreference
sucrose preference of mice in the CUMS group was significantly lower compared with mice in the
ofmiceintheCUMSgroupwassignificantlylowercomparedwithmiceinthecontrolgroupatday
control group at day 21 (p < 0.01), meanwhile the sucrose preference of mice in the two treatment
21(p<0.01),meanwhilethesucrosepreferenceofmiceinthetwotreatmentgroupwassignificantly
group was significantly higher than those of the CUMS group mice (p < 0.01). The forced swimming
higherthanthoseoftheCUMSgroupmice(p<0.01). Theforcedswimmingtestisabehavioraldespair
test is a behavioral despair test to evaluate the efficacy of antidepressant drugs, where the immobility
testtoevaluatetheefficacyofantidepressantdrugs,wheretheimmobilitytimeofmiceintheCUMS
time of mice in the CUMS group was significantly longer compared with control group mice at day
group was significantly longer compared with control group mice at day 21 (p < 0.01, Figure 2c).
21 (p < 0.01, Figure 2c). Similar to the SPT results, the immobility time of the two treatment group
Similar to the SPT results, the immobility time of the two treatment group mice was significantly
mice was significantly lower than those of CUMS group mice (both p < 0.01) indicating that both
lowerthanthoseofCUMSgroupmice(bothp<0.01)indicatingthatbothXiaoyaosanandfluoxetine
Xiaoyaosan and fluoxetine had antidepressant effects in the CUMS model mice. The open field test
hadantidepressanteffectsintheCUMSmodelmice. Theopenfieldtestreflectstheindependentand
reflects the independent and exploratory behaviors of mice in a strange environment [25]. Among all
exploratorybehaviorsofmiceinastrangeenvironment[25]. Amongallthegroups, therewasno
the groups, there was no significant difference in the results of OFT at day 0 (both p > 0.05, Figure
significantdifferenceintheresultsofOFTatday0(bothp>0.05,Figure3a,c,e). Aftermodelingfor
3a,c,e). After modeling for 21days, the total movement distance and the number of entries into the
21days,thetotalmovementdistanceandthenumberofentriesintothecentralareaofmiceinthe
central area of mice in the CUMS group were significantly less than those of control group mice (both
CUMSgroupweresignificantlylessthanthoseofcontrolgroupmice(bothp<0.01,Figure3b,d),while
p < 0.01, Figure 3b,d), while the Xiaoyaosan and fluoxetine treatments remarkably reversed the
theXiaoyaosanandfluoxetinetreatmentsremarkablyreversedtheCUMS-induceddecreaseinthe
CUMS-induced decrease in the total movement distance and number of entries into the central area
totalmovementdistanceandnumberofentriesintothecentralarea(bothp<0.01).
(both p < 0.01).
The residence time of mice in the CUMS group was significantly longer than in control group
mice (p < 0.05, Figure 3f), while Xiaoyaosan and fluoxetine could also reverse this CUMS-induced
Molecules 2018, 23, x 4 of 14
Molecules 2018, 23, x 4 of 14
behavior change (p < 0.05). Representative movement trails of mice in each group in the OPT at day
Molbeceuhleasv2i0o1r8 ,c2h3a,1n0g7e3 (p < 0.05). Representative movement trails of mice in each group in the OPT at d4aoyf 14
21 are shown in Figure 4.
21 are shown in Figure 4.

FigFFuiirggeuu2rre.e (22a.. ) ((aSa))P SSTPPoTTf oomff mimceiicceien iinne aeecaahcchhg grgorrououupppa aatttd ddaaayyy 0 00 ( ((bbbaaassseeellliiinnneee)));;; (((bbb))) SSSPPPTTT oooff f mmmiicciecee iinnin eeaeacachhc h ggrrgooruuoppu paatt a ddtaadyya 22y11;2; 1 ;
(c)(F(ccS)) TFFSSoTTf mooff i cmmeiiccinee ieinna ceehaaccghhr oggruroopuupapt aadtt addyaayy2 1 22.11.D. DDaataattaaw wweeerrereee eexxxppprrreeesssssseeeddd aaasss mmmeeeaaannnsss ±±± SSSEEEMMM ((nn( n == = 11551))5,, )**,*** p*p p<< <00..00011.0 1
vervvsueerrsssucuoss n cctoornnottlrrgoolrl oggurroopuu;pp∆;;∆ ΔΔΔpΔ pp< <<0 00.0..00111 v vveeerrrsssuuusss CCCUUUMMMSSS gggrrrooouuuppp...

Figure 3. (a) The total moving distance of mice in each group at day 0 (baseline); (b) The total moving
FigFuirgeu3re. (3a. )(aT)h Tehteo ttoatlaml movoivnigngd disitsatannceceo offm miiccee iinn eeaacchh ggrroouupp aatt ddaayy 00 ((bbaasseelilninee);) (;b(b) T)hTeh etottoatla mlmovoinvgin g
distance of mice in each group at day 21; (c) The number of entries into central area of mice in each
disdtaisntcaencoef omf imceicien iena ecahchg rgoruopupa tatd dayay2 211;;( c(c))T Thhee nnuummbbeerr ooff eennttrriieess iinnttoo cceenntrtaral laraerae aofo mfmiciec eini neaecahc h
group at day 0 (baseline); (d) The number of entries into central area of mice in each group at day 21;
group at day 0 (baseline); (d) The number of entries into central area of mice in each group at day 21;
groupatday0(baseline);(d)Thenumberofentriesintocentralareaofmiceineachgroupatday21;
(e) The residence time of mice in each group at day 0 (baseline); (f) The residence time of mice in each
(e) The residence time of mice in each group at day 0 (baseline); (f) The residence time of mice in each
(e)Theresidencetimeofmiceineachgroupatday0(baseline);(f)Theresidencetimeofmiceineach
group at day 21. Data were expressed as means ± SEM (n = 15), * p < 0.05, ** p < 0.01 versus control
group at day 21. Data were expressed as means ± SEM (n = 15), * p < 0.05, ** p < 0.01 versus control
groupatday21.Datawereexpressedasmeans±SEM(n=15),*p<0.05,**p<0.01versuscontrol
group; Δ p < 0.05, ΔΔ p < 0.01 versus CUMS group.
grou∆p; Δ p < 0.05∆, ∆ΔΔ p < 0.01 versus CUMS group.
group; p<0.05, p<0.01versusCUMSgroup.

TheresidencetimeofmiceintheCUMSgroupwassignificantlylongerthanincontrolgroup
mice(p<0.05,Figure3f),whileXiaoyaosanandfluoxetinecouldalsoreversethisCUMS-induced
Molecules2018,23,1073 5of14
behaviorchange(p<0.05). RepresentativemovementtrailsofmiceineachgroupintheOPTatday21
areshowninFigure4.
Molecules 2018, 23, x 5 of 14
Molecules 2018, 23, x 5 of 14

Figure 4. The movement trails of mice in each group at day 21 assessed by video tracking software in
Figure4.Themovementtrailsofmiceineachgroupatday21assessedbyvideotrackingsoftwarein
the OFT that displayed the locomotor function.
theFiOguFTre t4h.a Tthdei smpolavyeemdetnht etrlaoiclso mof omtoicref uinn ecaticohn g.roup at day 21 assessed by video tracking software in
the OFT that displayed the locomotor function.
2.3. Effect of Xiaoyaosan on the Expression of Apelin and APJ in Hypothalamus of CUMS-Induced Mice
2.3. EffectofXiaoyaosanontheExpressionofApelinandAPJinHypothalamusofCUMS-InducedMice
2.3. EffeIcnt oofr dXeira otoya doestaenr monin teh ew Ehxepthreesrs iXoina ooyf aAopsealnin r eangud lAatPedJ itnh eH ayppeoltihna-lAamPJu ssy osft eCmU iMn Sth-Ien mduocuesde Mmiocde el
Ionf odredpererstsoiodne, ttehrem eixnperewsshioenths eorfX aipaeolyina oasnadn ArePgJ uwlaetreed mtheaesaupreedli.n F-AirsPt Josfy asltle, mthein qtRhTe-mPCoRu saendm odel
In order to determine whether Xiaoyaosan regulated the apelin-APJ system in the mouse model
of dewpersetsesrino nbl,ott hreesuelxtsp rreevsesaiolends thoaft athpee l2i1n-daanyd CUAMPJS wcoeurled smigenaisfiucarnedt d.ecFrierassteo thfea allp,etlhine leqvReTl i-nP CthRe and
of depression, the expressions of apelin and APJ were measured. First of all, the qRT-PCR and
westehrynpbotlhoatlraemsuuslt osfr CevUeMalSe dmtichea (tbtohteh 2p1 <- d0a.0y1,C FUigMurSe 5cao,uc)l,d ansidg tnhiefi cmaincet dine cthree aXsieaothyaeoaspane ltirnealtemveenlti nthe
western blot results revealed that the 21-day CUMS could significant decrease the apelin level in the
hypogthroaulapm shuoswoefdC aU siMgnSifmicaincet i(nbcorethaspe i<n 0a.p0e1l,inF liegvuerle co5ma,pc)a,readn dwtithhe thmei CceUiMnSt hgeroXuipa omyiaceo s(pa n< 0tr.0e1a)t.m ent
hypothalamus of CUMS mice (both p < 0.01, Figure 5a,c), and the mice in the Xiaoyaosan treatment
The 21-day CUMS could also significantly increase the APJ level in the hypothalamus of CUMS mice
groupshowedasignificantincreaseinapelinlevelcomparedwiththeCUMSgroupmice(p<0.01).
group showed a significant increase in apelin level compared with the CUMS group mice (p < 0.01).
(both p < 0.01, Figure 5b,d), and the mice in the Xiaoyaosan treatment group showed a significant
The21-dayCUMScouldalsosignificantlyincreasetheAPJlevelinthehypothalamusofCUMSmice
Thed e2c1r-edaasye iCnU AMPJS l ceovuell dco amlspoa sriegdn wifiictha nthtley CinUcMreSa sger tohuep A mPiJc ele (vpe <l i0n.0 t1h)e, ahnydp ao tshimalialmaru rse soufl tC wUaMs Sa lmsoi ce
(bothp<0.01, Figure5b,d), andthemiceintheXiaoyaosantreatmentgroupshowedasignificant
(bootbhs epr v< e0d. 0in1, thFeig fuluroe x5ebti,nde) ,t raenadtm thenet mgriocue pin (p t h< e0 .X05ia, op y<a 0o.s0a1n, r etrsepaetcmtiveenlty g). ro up showed a significant
dedcreecaresaesien inA APJPlJe lveevlecl ocommppaarreeddw wiitthh tthhee CCUUMMSS ggrroouupp mmiiccee ((pp << 00.0.10)1,) a,nadn da asismimilairla rresruesltu wltaws aaslsaol so
obosebrsverevdeidn inth tehefl uflouxoexteitnineet rtereaatmtmeennttg grroouupp ((pp << 00..0055,, pp << 00.0.011, ,rerespspecetcitvievleyl)y. ).

Figure 5. (a) The mRNA expressions of apelin in each group; (b) The mRNA expressions of APJ in
each group; (c) The protein level of apelin in each group; (d) The protein level of APJ in each group.
Data were expressed as means ± SEM (n = 5), ** p < 0.01 versus control group; Δ p < 0.05, Δ Δ p < 0.01
versus CUMS group.
FigFuigruer5e. 5(.a ()aT) hTehem mRRNNAAe exxpprreessssiioonnss ooff aappeelliinn iinn eeaacchh ggrroouupp; ;(b(b) )TThhe emmRRNNAA exepxrpersesisosnios nosf oAfPAJ PinJ in
eacehacghr goruopu;p(;c ()cT) hTehep proroteteininl elevveell ooff aappeelliinn iinn eeaacchh ggrroouupp; ;(d(d) )TThhe epprortoetieni nlelveevl eolf oAfPAJ PinJ ienaceha cghrogurpo.u p.
The immunohistochemical staining result further indicated that the mice in the CUMS group
DaDtaatwa ewreereex epxrpersessesdeda sams meaenasns± ± SSEEMM ((nn == 55)),, **** pp << 00..0011 vveerrssuuss ccoonnttrrooll ggrroouupp; ;Δ∆ p p< <0.00.50,5 Δ,Δ∆ p∆ <p 0<.001. 01
had a lower expression of apelin and a higher expression of APJ in the PVN and SON compared with
vervseurssuCs UCMUMSgS rgoruopu.p.
the control group (both p < 0.01, Figures 6 and 7), while Xiaoyaosan reversed the CUMS-induced
changes of apelin and APJ in the PVN and SON (both p < 0.01), and the expression of APJ in the PVN
The immunohistochemical staining result further indicated that the mice in the CUMS group
Tanhde SimONm uofn mohicies tionc fhluemoxiectainles ttareinatimngenret sguroltufpu wrtahse arlisnod loicwateerd thtahna tthteh eCUmMicSe ginrotuhpe mCiUceM (bSogthro pu <p had
had a lower expression of apelin and a higher expression of APJ in the PVN and SON compared with
alow0e.0r1e)x. pressionofapelinandahigherexpressionofAPJinthePVNandSONcomparedwiththe
the control group (both p < 0.01, Figures 6 and 7), while Xiaoyaosan reversed the CUMS-induced

changes of apelin and APJ in the PVN and SON (both p < 0.01), and the expression of APJ in the PVN
and SON of mice in fluoxetine treatment group was also lower than the CUMS group mice (both p <
0.01).
Molecules2018,23,1073 6of14
controlgroup(bothp<0.01,Figures6and7),whileXiaoyaosanreversedtheCUMS-inducedchanges
ofapelinandAPJinthePVNandSON(bothp<0.01),andtheexpressionofAPJinthePVNandSON
ofmMicoeleciunlefls 2u0o18x, e23t,i nx e treatmentgroupwasalsolowerthantheCUMSgroupmice(bothp<60 o.f0 114) .
Molecules 2018, 23, x 6 of 14

FiguFreig6u.re(a 6).R (ae)p Rreespernesteantitvaetivmei mcriocgroragprahpshos foifm immmununoohhisistotocchheemmiiccaall ssttaaiinniinngg ffoorr aappeelilnin (s(scacalel ebbara r= =505 0µm,
Figure 6. (a) Representative micrographs of immunohistochemical staining for apelin (scale bar = 50
400×μmm,a 4g0n0i×fi mcaatginoinfi)caintiothn)e iPn VthNe PaVndN SaOndN S;O(bN); T(bh)e Trhees urelstuoltf oimf iamgaegae naanlaylysissiso off aappeelliinn iinn PPVVNN; ;(c()c )The
μm, 400× magnification) in the PVN and SON; (b) The result of image analysis of apelin in PVN; (c)
The result of image analysis of apelin in SON. MOD, mean optical density. Data were expressed as
resulTthoef riemsualgt eof ainmaalgyes iasnaolfysaips eolfi napienlinS iOn NSO.NM. OMDO,Dm, meaenano oppttiiccaall ddeennssitiyty. .DaDtaa twaewre eerxepreexspserde sasse d as
meanmmsee±aannsSs E± ± MS SEEM(Mn ( =(nn 5= = )5 ,5)*), *,* **p* p <p < <0 0 .0.00.0111 vv veeerrrsssuuusss c ccooonnnttrtrorooll gl grgrorououpup;p ;Δ Δ;ΔΔ ∆p p ∆< < p0 0.0.<011 0v v.e0er1rssuvuses rC CsUuUMsMCSS Ug grMroouuSppg. . roup.

FiguFreig7u.re(a 7). (Rae) pRreepsreensetanttiavtievme micircoroggrraapphhss ooff iimmmmuunnoohhisitsotochcehmemicaicl astlasitnaiinngi nfogr fAoPrJA (sPcaJl(es bcaalre = b5a0r μ=m5, 0µm,
Figure 7. (a) Representative micrographs of immunohistochemical staining for APJ (scale bar = 50 μm,
400×40m0×a gmnaifigncaiftiicoanti)oinn) tihn ethPeV PNVNan adnSdO SNO;N(b; ()bT) hTehree rseuslutlot foifm imagaegea naanlaylysissiso offA APPJJi ninP PVVNN;; ((cc)) TThhee result
400× magnification) in the PVN and SON; (b) The result of image analysis of APJ in PVN; (c) The
ofimrreaesgsueultlat o noffa i limmyasagigsee oa afnnAaalylPysJsiisis no ofSf A OAPNPJJ .i inMn S SOOONDN., .M mMOeOaDDn, ,m ompeeataincn ao olppdttieiccanalsl d idteyen.nssDiittyay.t .aD Dawattaea rw weeeerrxee pe exrxeppsrrseeesssdseedad sa asms m meeaeanansnss± ± ± SEM
SEM (n = 5), ** p < 0.01 versus control gr∆o∆up; ΔΔ p < 0.01 versus CUMS group.
(n=5S)E,M** (pn< = 05.)0, 1**v pe <r s0u.0s1c voenrstruosl cgornotruopl ;groupp;< ΔΔ0 p.0 <1 0v.0e1r svuesrsCusU CMUSMgSr ogruopu.p.
Molecules2018,23,1073 7of14
3. Discussion
CUMSisacommonmethodtoestablishastressdiathesismodelofdepressioninmodernresearch,
andthefactthatthreeweeksofmodelingcandevelopsignificantdepressive-likechangeshasbeen
widelycertified[26,27]. Meanwhile,theC57BL/6Jmousehasahomogeneousresponsetostress,soit
isoftenusedtostudystress-inducedchangesinthebody[28,29]. Therefore,thisstudyestablisheda
micedepressionmodelbythreeweeksofCUMS,andobservedtheeffectofXiaoyaosanintervention
onthedepressive-likebehaviorsandthechangesoftheapelin-systeminthehypothalamus.
Therealwaysexistsweightlossandappetitedecreaseduringthedepressionprocess[30]. After
modelingforthreeweeks,themiceinthetwotreatmentgroupshadabetterfoodintakecondition
thantheCUMSgroup,whichsuggestedthatXiaoyaosanandfluoxetinehadsimilareffectsonappetite
regulation. Apreviousstudyshowedthattheuseoffluoxetinemightresultinweightloss,although
it also has also been reported to have the opposite effects of weight gain and hyperphagia [31,32],
sotheeffectsoffluoxetineonweightmaybemediatedbypartiallydistinctmechanismsandinvolve
somemetaboliceffects[33,34]. Meanwhile,Xiaoyaosanhadagoodeffectintheregulationofbody
weight[35],thesameresultswererevealedinthisstudy. TheSPT,FSTandOFTwereusedtoevaluate
thedepressive-likebehaviorsofCUMS-inducedmice. TheSPTcouldreflectthestress-inducedmental
stateandestimatetheanti-depressanteffectofdrugs[36]. TheSPTresultsindicatedthatthreeweeksof
CUMShadanobviousinfluenceonthetastechangesofmice,theXiaoyaosanandfluoxetinetreatments
couldsignificantlyreversetheCUMS-inducedanhedonia. TheFSTintheanimalmodelsofdepression
had good sensitivity and specificity for antidepressant treatments [37]. It had a similar results as
SPTinthisstudythatfurtherconfirmedtheefficacyofXiaoyaosanandfluoxetine. TheOFT,asan
animal psychological test, is commonly used to assay the locomotor function and emotionality of
rodents[38]. Ithasthecharacteristicsofsimpleoperation,goodfeasibilityandaccuratedatarecords.
Thetotalmovementdistance,numberofentriesintothecentralareaandresidencetimewereselected
to assess the different behaviors of mice in each group. When mice were in a state of fatigue and
depression,theirlocomotoractivitywoulddecreaseaccordingly,sothetotalmovementdistanceof
miceintheCUMSgroupwassignificantlylowerthanthatinthecontrolgroup. Thenumberofentries
intothecentralareareflecttheanxietyandadaptabilityofmicetothenewenvironment,andanxious
ordepressedmiceprefertomovearoundtheboxratherthanmovingintothecentralarea,thusthe
numberofentriesintocentralareaofmiceinCUMSgroupwassignificantlylowerthanthatinthe
controlgroup. Inaddition, theresidencetimerepresentstheretentiontimeofmicetoexplorethe
surroundingenvironment,thecuriosityofmicetothestrangeenvironmentdecreasedwiththeincrease
ofresidencetime,henceanobviousincreasecouldbeseenintheresidencetimeofmiceintheCUMS
groupfromthedata.Accordingtotheresultsdiscussedinpreviousstudies[21],ourstudyalsoshowed
theobviousantidepressanteffectofXiaoyaosanorfluoxetineaccordingtotheresultsofOFT,SPTand
FST,furthermore,anxiety-likebehaviors,suchassignificantdecreaseinnumberofentriesintocentral
areaoftheOFTcouldalsobeimprovedbythetreatment.
The HPA axis is a vital stress response site, and is also an important regulatory pathway of
endocrineactivity,HPAaxishyperactivityhappenswhendamageoccurstothenegativefeedback
systemoftheHPAaxis,andthentheneuroendocrineimmunenetworkbecomesimbalancedwhich
isoneofthemainmanifestationsofdepression[39,40],sothehypothalamus,astheessentialcenter
oftheHPAaxis,isthekeyregiontostudythecentralmechanismofdepressive-likebehaviors. Itis
reportedthatthestructuresofapelinandAPJaresimilarinmanyspeciessuchashumans,rats,mice
andcows,andthedistributionofapelinandAPJhasahighdegreeofoverlapinthebody[41–43]. Asa
pairofbiologicalactivepeptides,theroleofapelinandAPJinthefunctionofhypothalamushasbeen
exploredinmanystudies[44–46],andthechangesoftheapelin-APJsysteminthestress-inducedHPA
axisdysfunctionarealsoastudypointinthementaldisorders[47]. Tofigureoutwhetherapelin-APJ
systemisinvolvedinthefunctionalmechanismofXiaoyaosan,thelevelsofapelinandAPJinthe
hypothalamuswereobserved. ItwasfoundthatCUMScouldinhibittheapelinlevelandincrease
theexpressionofAPJinthehypothalamus,whichmeantthatstressmightleadtothereductionof
Molecules2018,23,1073 8of14
apelin,thenaffectitsinteractionwithAPJ,thenweakenthefunctionoftheapelin-APJsystem,andthe
reasonforthesechangesmayberelatedtocompensatedagonistlossbyincreasingthereceptor. The
sameresultswerereflectedinbothPVNandSON,whichwasasupplementtopreviousresearch[16].
TheresultsindicatedthatXiaoyaosancouldpromotetheapelin-APJsystemwhichwasmanifested
in the significant up-regulation of apelin levels and the normal range of APJ expression. Besides,
someresearchshowedthatXiaoyaosancouldregulatethecontentofCRF[48],andthefunctionof
the apelin-APJ system was associated with CRF [49], which suggests that the regulating effect of
Xiaoyaosan to the apelin-APJ system might be mediated by CRF, but certainly further research is
necessary. Fluoxetineisakindofselectiveserotoninreuptakeinhibitor(SSRI)thatiswidelyusedin
thetreatmentofmentaldiseasessuchasdepression,panicdisorders,obsessive-compulsivedisorders
and bulimia nervosa [50]. The 5-HT system is associated with the HPA axis, and brain 5-HT can
enhancetheactivityofHPA,thusactivatingthesecretionofCRFinthehypothalamus,thenpromote
the secretion of ACTH [51], so fluoxetine was used to set up a reference drug group in this study
to evaluate the influence of Xiaoyaosan on CUMS-induced disorder of the hypothalamus. In our
study,theexpressionofapelininthefluoxetinetreatmentgrouphadnoobviouschangecompared
withtheCUMSgroup,whichillustratedthatdifferentfromXiaoyaosan,thetreatmentoffluoxetine
mightnotregulatetheapelinlevelinthehypothalamusofCUMS-inducedmice. Furtherstudiesare
neededtoexaminetheroleofXiaoyosanintheregulationoftheapelin-APJsystem,andmorerelative
brainregionsincludingcortex,hippocampusandamygdalastillneededtobeexamined,inorderto
comprehensivelyanalysetheperformanceoftheapelin-APJsysteminmentaldisorders.
4. MaterialsandMethods
4.1. Animals
Specific-pathogenfree(SPF)maleC57BL/6Jmice(SYXK(Jing)2012-0001)aged12weekswere
purchasedfromBeijingVitalRiverLaboratoryAnimalTechnologyLimitedCompany(Beijing,China).
All mice were adaptive fed for 7 days separately in a standard animal feeding room before the
experimentscommenced(roomtemperature: 21±1◦C;relativehumidity: 30–40%;lightcondition:
a12h/12hdark/lightcycle). AllanimalexperimentswereapprovedbytheInstitutionalAnimal
CareandUseCommitteeatBeijingUniversityofChineseMedicineandconformedtotheexisting
currentanimalwelfareguidelines. Theexperimentalprotocolsappliedinthisstudywereperformed
inaccordancewithapprovedguidelines.
4.2. PreparationofDrugs
ThecompoundprescriptionusedintheexperimentisXiaoyaosanwhichcontainsthefollowing
eighttraditionalChinesedrugs: RadixAngelicaeSinensis,RadixPaeoniaeAlba,Poria,RadixBupleuri,
RadixGlycyrrhizae,RhizomaAtractylodisMacrocephalae,HerbaMenthaeandRhizomaZingiberis
Recens,inaratioof6:6:6:6:3:6:2:2. ThesemedicinalherbswerepurchasedfromtheMedicinalMaterials
CompanyofBeijingTongrentang(Beijing,China)andextractedintheChinesemedicinepreparation
roomofChina-JapanFriendshipHospitalasdescribedpreviously. Theextractionratewas18.8%[52].
4.3. CUMSProcedureandDrugAdministration
A total of 60 mice were randomly divided into four groups (n = 15) according to their body
weights: controlgroup(nostresswithphysiologicalsaline),CUMSgroup(CUMSplusphysiological
saline),Xiaoyaosantreatment(CUMSplusXiaoyaosan)groupandfluoxetinetreatmentgroup(CUMS
plusfluoxetine). Allmicewerehousedseparatelyincages,andthestressassaymethodsusedwere
restraintstress(3h),fooddeprivation(24h),waterdeprivation(24h),hightemperaturestress(50◦C,
5min),ice-coldswimming(5min),unpredictableshocks(50mV,oneshock/10s,30sduration,15times
intotal),tailclamp(1min),noiseenvironment(60Hz,1h)andwetandsoiledcage(24h),eachstress
methodwasusedfor2or3times,theCUMSmodelinglastedfor21days. Miceinthecontrolgroup
Molecules2018,23,1073 9of14
andCUMSgroupreceived0.5mLphysiologicalsalinebyintragastricadministration,miceinthetwo
treatmentgroupsreceivedXiaoyaosan(0.25g/kg/d)andfluoxetine(2.6mg/kg/d),respectively[53].
Furthermore,toobservethephysicalconditionofthemiceduringtheCUMSprocedure,thebody
weightandfoodintakeofeachmousewererecordedweekly(day0,day7,day14andday21)until
theendofmodeling,thedataofthedaybeforeexperiment(day0)wasrecordedasbaseline.
4.4. SucrosePreferenceTest
Thesucrosepreferencetestwasperformedatday0andday21aspreviouslydescribed[53]. After
waterandfooddeprivation24h,eachmousewasgiventwobottlescontainingdeionizedwaterand
1%sucrosesolution. Theratiooftheamountofsucrosesolutiontothatoftotalsolutionconsumed
within1hwascalculatedwhichrepresentedtheparameterofhedonicbehavior.
4.5. ForcedSwimmingTest
The forced swimming test was performed at day 21 as previously described [54], mice were
individuallyplacedinaclearglassaquarium(19cmindiameter×24cmhigh)containing6-cmdeep
water(24±1◦C)for6min. ThewholetimewasrecordedusingaHDcamera,andtheimmobility
timewasrecordedduringthefinal5min. Themousewasconsideredtobeimmobilewhenitstopped
swimmingandremainedfloatingonthewater. Thewaterintheaquariumwaschangedaftereach
mousetoavoidolfactorycuesleftbythepreviousmice.
4.6. OpenFieldTest
Theopenfieldtestwasperformedatday0andday21aspreviouslydescribed[55].Theactivityof
miceineachgroupweremeasuredina40cm×40cm×15cmwoodenboxwithoutceiling,theinside
wallandfloorofwhichwerecoveredbyblackpaint. Thefloorwasdividedinto25equalsquaresby
whitelines,eachmousewasplacedrightinthecenterandstartedtorecordthemovementcondition
for5minbyHDcamera. Theboxwascleanedaftereachmousetoavoidolfactorycuesleftbythe
previousmice. TheEthoVision3.0sofwarewasusedtoevaluatethetotalmovingdistance,residence
timeandnumberofentriesintocentralarea.
4.7. SampleCollectionandPreparation
Aftermodelingfor21days,themiceineachgroupwereanesthetizedbyintraperitonealinjection
of3%sodiumpentobarbital. Thehypothalamusoffivemiceineachgroupwerecollectedinfrozen
pipesandstoredat−80◦Cforwesternblottesting,thenthehypothalamusoffivemiceineachgroup
werecollectedinfrozenpipeswithRNApreservationsolutionforqRT-PCR,thewholebrainsofthe
remainingmiceafterfixationofarterialperfusionwerestoredin4%paraformaldehydesolutionat
4◦Cfortissueslicingandimmunohistochemistry.
4.8. QuantitativeReal-TimePolymeraseChainReaction(qRT-PCR)Analysis
Total RNA of hypothalamus from each mouse was extracted using Trizol reagent (Applied
Biosystems,Waltham,MA,USA),andthentheRevertAidFirstStrandcDNASynthesisKit(Termo
FisherScientifc,Waltham,MA,USA)wasusedtosynthesizecDNA.Thesequencesforprimersshowed
inTable1weredesignedbasedonpublishedmRNAsequencesinNCBI,andthensynthesizedbya
professionalbiotechnologycompany(SangonBiotechCo.,Ltd.,Shanghai,China). GAPDH(BBILife
Science,Amherst,MA,USA)wasusedashouse-keepinggeneinthestudy. TheqRT-PCRreaction
systemwaspreparedbySYBR® GreenPCRMasterMix(AppliedBiosystems)inafinalvolumeof
50µLandperformedonMulticolorReal-timePCRDetectionSystem(Bio-Rad,Hercules,CA,USA).
The thermal cycling conditions were as follows: 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C
for 15 s and 58 ◦C for 1min. The relative expression of mRNA in each sample was calculated by
2−∆∆Ctmethod.
Molecules2018,23,1073 10of14
Table1.PrimersequencesusedintheqRT-PCRanalysis.
Gene Sequences
forward 5(cid:48)-CTGCTCTGGCTCTCCTTGAC-3(cid:48)
Apelin
reverse 5(cid:48)-CTCGAAGTTCTGGGCTTCAC-3(cid:48)
forward 5(cid:48)-CGCTCATTCCTGCCATCTAC-3(cid:48)
APJ reverse 5(cid:48)-AAGGTCAAGTCAGCCACTGC-3(cid:48)
4.9. WesternBlotAnalysis
The total protein of hypothalamus from each mouse was extracted using RIPA Lysis buffer
(Biomiga,Santiago,CA,USA)andtheBCAproteinassaykit(TermoFisherScientific)wasusedto
determinetheproteinconcentration. TheproteinexpressionsofapelinandAPJweremeasuredby
westernblotandtheprocedurewasperformedaspreviouslydescribed[56]. The12%SDS-PAGEwas
selectedtoconducttransmembraneaccordingtorelativemolecularweightofapelinandAPJ,andthen
proteinsweretransferredontopolyvinylidenefluoride(PVDF)membranes. PBSTwith5%nonfat
milkwasusedtoblockthemembranes,andtheprimaryantibodieswereapelin(M-77)(SC-33805,
1:100 dilution, Santa Cruz, Dallas, TX, USA), Apelin receptor Antibody (APJ) (PA5-21306, 1:500
dilution,ThermoScientific)andβ-TubulinMonoclonalAntibody(A01030,1:5000dilution,Abbkine,
Inc.,Redlands,CA,USA).TheSuperSignal®WestPicoChemiluminescentSubstrate(ThermoScientific)
wasusedtodevelopthemembranesandtheTanon-5200analysissystem(Tanon,Shanghai,China)
wasusedforexposure,thentheopticaldensityofproteinbandswerereadbyTanonGissoftware.
4.10. ImmunohistochemicalStainingAnalysis
Thewholebrainsin4%paraformaldehydesolutionwereusedtoprepareparaffinsliceswhich
includedPVNandSONbasedonthemousebrainstereotaxicatlas[57]. Theimmunohistochemical
stainingprocedure was performed as previously described [52]. In brief the steps were as follows:
(1) the slices were dewaxed in graded xylene thrice each for 15 min; (2) dehydrated using graded
ethanolthriceeachfor1min;(3)flushedwithtapwaterfor3min,thensoakedindeionizedwater
thriceeachfor1min;(4)heatinducedepitoperepair(HIER)wasusedforantigenretrieval;(5)washed
theslicesbyPBST(0.01MPBSwith0.1%Tween-20)thriceeachfor5min,andincubatedwith3%H O
2 2
for10min,thenwashedagain;(6)incubatedwithlowlenthalserumfor60min,thenincubatedwith
primaryantibody(apelin: 1:200dilution;APJ:1:1000dilution)at4◦Covernight;(7)washedtheslices
byPBSTthriceeachfor5min,thenincubatedwithpolymerhelperfor30minatroomtemperature
andwashedagain;(8)incubatedwiththeHRPconjugatedanti-rabbitIgGpolymerfor30minatroom
temperature;(9)washedtheslicesbyPBSTthriceeachfor5min,thentheDABsolutionwasusedfor
coloration;(10)thesliceswererinsedwithtapwatertostopthecolorreaction,thensealedwithneutral
gumafterthecounterstaining.
Aimageanalyzer(MIAS99,Fubo-TechCo,Beijing,China)withacolorvideocamera(TK-C1381,
JVC,Beijing,China)andBX50microscope(OlympusCo.,Tokyo,Japan)wasusedtocollectimagesof
SONandPVNathighpowermagnification(400×),andtheImageproPlus6.0softwarewasusedto
obtaintheaverageopticaldensity(AOD)whichiswidelyusedintheimageanalysis[58,59].
4.11. StatisticalAnalysis
DataoftheresultswereprovidedintheSupplementaryMaterialsfile,andthedatawereexpressed
asmeans ± standarderrorsofthemeans(SEM).SPSS21.0softwarewasusedtoanalyzethedata,
one-wayanalysisofvariance(ANOVA)ornon-parametrictestwasusedforgeneraldatabasedon
normalitytestandhomogeneitytestforvariance,andleastsignificantdifference(LSD)methodwas
adopted for the comparisons between groups. Besides, repeated measurement process of general
linearmodel(GLM)wasusedtoconductone-wayANOVAanalysisforrepeatedmeasureddata(body
weightandfoodintake),andmultivariateanalysisprocessofvariancewasusedtomakecomparisons