The Protective Effects of Areca catechu Extract on Cognition and Social Interaction Deficits in a

12 Pages·2015·4.47 MB·English

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2015, Article ID 426092, 11 pages
http://dx.doi.org/10.1155/2015/426092
Research Article
Areca catechu
The Protective Effects of
Extract on Cognition and Social Interaction Deficits in
a Cuprizone-Induced Demyelination Model
AbulimitiAdilijiang,1,2TengGuan,3JueHe,2KellyHartle,2
WenqiangWang,1andXinMinLi2
1XiaMenXianYueHospital,XianYueRoad387-399,Xiamen361012,China
2DepartmentofPsychiatry,FacultyofMedicineandDentistry,UniversityofAlberta,
1E7.31WalterC.MackenzieHealthSciencesCentre,Edmonton,AB,CanadaT6G2B7
3DepartmentofHumanAnatomyandCellScience,FacultyofMedicine,UniversityofManitoba,745BannatyneAvenue,
Winnipeg,MB,CanadaR3E0J9
CorrespondenceshouldbeaddressedtoWenqiangWang;[email protected];[email protected]
Received19December2014;Revised9February2015;Accepted10February2015
AcademicEditor:DidierStien
Copyright©2015AbulimitiAdilijiangetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Schizophreniaisaseriouspsychiatricillnesswithanunclearcause.Onetheoryisthatdemyelinationofwhitematterisoneofthe
mainpathologicalfactorsinvolvedinthedevelopmentofschizophrenia.ThecurrentstudyevaluatedtheprotectiveeffectsofAreca
catechunutextract(ANE)onacuprizone-induceddemyelinationmousemodel.TwodosesofANE(1%and2%)wereadministered
orallyinthedietfor8weeks.Animalssubjectedtodemyelinationshowedimpairedspatialmemoryandlesssocialactivity.In
addition,micesubjectedtodemyelinationdisplayedsignificantmyelindamageincortexanddemonstratedahigherexpressionof
NG2andPDGFR𝛼andAMPKactivation.ANEtreatmentnotonlysignificantlyenhancedcognitiveabilityandsocialactivity,but
alsoprotectedmyelinagainstcuprizonetoxicitybypromotingoligodendrocyteprecursorcell(OPC)differentiation.Inaddition,
ANEtreatmentdemonstratedsignificantdephosphorylationofAMPK𝛼,indicatingaregulatoryroleforANEinschizophrenia.
ThisstudyshowedthatANEtreatmentmayenhancecognitiveabilityandsocialactivitybyfacilitatingOPCdifferentiationand
protectingagainstmyelindamageincortex.ResultsalsosuggesttheAMPKsignalingpathwaymaybeinvolvedinthisprocess.
1.Introduction been limited due to the relatively poor understanding of
theneuropathologyofthisdisorder[2].Recently,increasing
Schizophreniaisaseriouspsychiatricillnessthataffectsabout evidencesuggeststhatoligodendrocytes(OL)playanimpor-
1% of the population [1]. The symptoms of schizophrenia tantroleinthepathogenesisofschizophrenia[3].Therefore,
include“positivesymptoms”(hallucinations,delusions,and neuroprotectiveagentsthatdirectlytargetwhitematterinjury
disorganized thinking), “negative symptoms” (diminished mightbeatherapeutictargetforschizophrenia.
emotionalresponses,socialwithdrawal,bluntedaffect,scar- Inthesearchfornewtherapeuticagentstotreatneurolog-
cityofspeech,andlethargy),andcognitivesymptoms(atten- icaldisorders,therehasbeensignificantprogressinthearea
tion deficits, impaired executive function, and memory). of medicinal plants. Areca nuts (from Areca catechu, L.),
Schizophrenia is responsible for enormous healthcare and popularlyknownas“betelnuts,”arewidelyusedintraditional
nonhealthcare related expenditures worldwide. Although Chinesemedicinefortreatmentofconstipation,oedema,ber-
antipsychotic drugs acting through multiple receptors have iberi,anddyspepsia.Arecanutsproduceavarietyofpharma-
beenrevolutionaryforcontrollingpositivesymptoms,nega- cological effects such as heightened alertness and euphoria,
tivesymptomsandcognitivedeficitsremainrelativelyunaf- [4].ScientificstudiesconductedontheArecanuthaveshown
fected. The development of novel antipsychotic drugs has that it possesses antioxidant [5], antischizophrenic [6–10],
2 Evidence-BasedComplementaryandAlternativeMedicine
memory-protective [11], analgesic, and anti-inflammatory (CCAC). The experimental protocol was approved by the
[5] effects. Phytochemical analysis of the crude extract and UniversityCommitteeonAnimalCareandSupply(UCACS)
the aqueous fraction demonstrated the presence of alka- andtheUniversityofManitobaAnimalCareCommittee.
loids (i.e., arecaidine, arecoline, guvacine, and guvacoline), C57BL/6 mice were divided into the following four
anthraquinones, coumarins, flavonoids, saponins, sterols, groups (𝑛 = 10/group): control (regular chow and distilled
hydroxychavicol,procyanidins,tannins,terpenes,andgallic waterfor9weeks);CPZ(distilledwaterandregularchowfor
acid [12]. Several studies have shown that schizophrenic oneweekand0.2%CPZchowfor8weeks);andCPZ+ANE1
patientswhochewedbetelnutsscoredsignificantlyloweron andCPZ+ANE2(distilledwaterandregularchowforfirst
bothpositiveandnegativesymptomscalesthannonchewers week and 0.2%CPZ + ANE (1% or2%) chow for8 weeks).
onthePositiveandNegativeSyndromeScale(PANSS)[6–10]. Eachgroupwasseparatelycaged.
ThehypothesisisthatArecacatechunutsmayexerttheirben-
eficialeffectsonschizophreniathroughthemainactiveingre-
2.3.BehavioralTesting
dient, arecoline. However, the antischizophrenic effect of
Arecacatechunutsandtheirmechanismremainsunclear. 2.3.1.SpatialWorkingMemoryTest. Spatialworkingmemory
Based on pharmacological properties of Areca catechu wasevaluatedbymeasuringspontaneousalternationbehav-
nuts, it was proposed that Areca catechu nut extract (ANE) iorina𝑌-maze.Thistestisbasedonthenaturaltendencyof
might ameliorate schizophrenic symptoms by targeting of rodents to explore novel environments and to recall which
oligodendrocytes (OLs) to prevent demyelination of white areashavebeenexplored.The𝑌-mazehasthreearms(each
matter.Totestthishypothesis,weusedacuprizone-(CPZ-) 30cmlong),markedA,B,andC.Micewereplacedindivid-
induceddemyelinationanimalmodel.Withthegoalofather- uallyattheendofonemazearmandallowedtoroamfreely
apeuticinterventionforschizophrenia,thecurrentstudywas for8min.Thetotalnumberofarmentriesandtheseriesin
designedtoevaluatetheeffectsofANEoncognition,social which they occurred were recorded. Overlapping entrance
interaction, OL restoration, and myelin repair in CPZ- sequences(e.g.,ABCandBCA)weredefinedasthenumber
induceddemyelination. ofalternations.Alternationwascalculatedasapercentage:
Percent alternation
2.MaterialsandMethods
(number of alternations) (1)
= ×100;
2.1. Areca catechu Nut Extraction. The Areca catechu nut
(total number of arm entries−2)
extractwasprovidedbytheTCMdepartmentoftheXianyue
HospitalinXiamen(China).FreshArecacatechunuts(2kg)
see[21].
werepurchasedfromXiamenChineseTraditionalMedicine
Hospital and authenticated by a pharmacist (Feng Tang) in
2.3.2. Sociability and Social Preference Tests. The sociability
TCM Department of Xiamen Xianyue Hospital, where a
test was used to examine whether the test mouse preferred
voucher specimen (20120910) was deposited. Briefly, Areca
socialinteractionwithunfamiliarconspecificanimalstoatest
catechunutswerecleanedofadulterants,crushedtoopenup
areawithoutanimals.Thistestwasadaptedfromthethree-
thecrestoftheseedandsoakedin4Lof50%aqueous-ethanol
chamber paradigm test known as Crawley’s sociability [14]
forcoldmacerationforaperiodof7daysatroomtempera-
withseveralpreviouslyreportedmodifications[15].Thetest
ture.ThesolutionwasfilteredthroughWhatmanqualitative
wasperformedinanovelenvironmentunknowntothetest
filter paper (Grade 1) and the filtrate was collected. The
mouse.Thisnovelenvironmentwasacagewiththreeinter-
crushed nuts were resoaked and refiltered twice. The com-
connectedcompartments.Thedimensionsofeachcompart-
bined filtrates were concentrated in a rotary evaporator at
∘ mentwere23.3-cmlong,40-cmwide,and22-cmhigh.The
40 Cunderreducedpressuretoyieldaviscous,darkbrown
walls dividing the compartments had rectangular openings
Arecacatechucrudeextract(Ac.Cr)weighing200g(yield10%
∘ (10-cm wide and 10-cm high) allowing movement between
w/w). This extract was dried in an oven at 90 C and then
stored at −4∘C until use. It was mixed into powered mouse thecompartments.
Toinitiatethesociabilitytest,thetestmousewasplacedin
chow at concentrations of 1% and 2% on the day of the
thecentralcompartment(empty).Intheleftcompartment,a
experiment.
conspecific stranger mouse (CSM) to the test mouse was
placedunderasmallwirecage(diameter11.0cm).CSMwere
2.2.AnimalsandExperimentalManipulations. MaleC57BL/6 randomlyselectedfrommiceofthesameagemaintainedin
mice (7 weeks old) were purchased from Charles River separatecages.Intheemptyrightcompartment(emptycage,
Canada (Montreal, Canada). After 1 week of acclimation, EC), an empty wire cage of the same size and shape was
the mice were fed a standard rodent chow (LabDiet, PMI placed.Aftera5minhabituationperiod,the10minsociabil-
Nutrition International, LLC, Brentwood, MO, USA). The itytestwasperformed.Sociabilityindex(SI)wasdefinedas
milledchowcontained0.2%CPZ(w/w)(Sigma-Aldrich,St. theratioofeitherfrequencyordurationbetweenCSMside
Louis, MO, USA) for eight weeks to induce demyelination andECside,aspreviouslydescribed[15].
[13].Thecontrolmicereceivedstandardrodentchowwithout Followingthesociabilitytest,asocialpreferencetestwas
CPZ.Theanimalstudiesinthisstudywereperformedwithin performed in the same cage. In this test, the CSM in the
theguidelinessetbytheCanadianCouncilonAnimalCare left compartment contained a familiar mouse (FM) and a
Evidence-BasedComplementaryandAlternativeMedicine 3
stranger mouse (novel mouse, NM) was placed in a wire polyvinylidene fluoride membranes. The membranes were
cageintherightcompartment.Asbefore,thetestmousewas incubatedinablockingsolution(5%skimmilkinPBS)for
∘
placed in the empty central compartment, and the 10min 1hat22 C,followedbyincubationwiththeprimaryantibody
social preference test was conducted. The social preference inablockingsolution.
test evaluates the duration of social interaction (i.e., time Anti-MBP (1:5000) (Santa Cruz Biotechnology, CA,
spent) with either FMNM mice by the test mouse. Social USA) was used to detect MBP. Anti-PDGFR𝛼 (1:2500)
preference index (SPI) was defined as the ratio of either (Santa Cruz Biotechnology, CA, USA) was used to detect
frequencyordurationbetweennovelsideandfamiliarside,as PDGFR𝛼asmarkerofOPCs.
ThemembraneswerethenwashedwithPBSthreetimes,
previously described [15]. The duration and direction of
followed by incubation in the blocking solution containing
mousemovementswererecordedandanalyzed.TheNMwas
therabbitanti-goat,goatanti-mouse,orgoatanti-rabbitsec-
also randomly selected from separate group of mice of the
ondaryantibodies(1:10,000).AfterthreerinsesinPBS,the
same age. The NM had not been used in any previous
immunoreactivebandsweredevelopedusinganECLdetec-
experiments.
tion kit (Amersham Biosciences, Baie d’Urfe, Quebec). To
confirmtheequalamountsofloadingsamples,𝛽-actinwas
2.4. Tissue Preparation and Immunohistochemical Staining. alsolabeled(1:5000;Sigma,St.Louis,MO,USA)throughthe
󸀠
At the end of the behavior tests, mice were anesthetized same procedures as described above. Phospho-5 AMP-
withsodiumpentobarbital(50mg/kg)andperfusedintracar- activated protein kinase (AMPK𝛼) (Thr172) antibodies
diallywithphosphate-bufferedsaline(PBS)followedby4% (1:1000) and AMPK𝛼 antibodies (1:1000) (Cell Signaling
Technology, Inc., Danvers, MA, USA) were incubated with
paraformaldehyde in PBS. The brains were postfixed over-
frontal cortex samples in 5% w/v BSA, 1X Tris-buffered
nightin4%paraformaldehyde.Thefixedbrainswererinsed
∘
∘ saline,and0.1%Tween-20at4 Cwithgentleshaking,over-
threetimeswith0.01MPBS,chilledin30%sucroseat4 Cfor
oneday,andfrozenat−80∘C.Serialcoronalsections(30𝜇m) night. After incubation in a secondary anti-rabbit antibody
(1:5000) diluted in 5% w/v BSA, 1X Tris-buffered saline,
of brains were cut using a sliding microtome (Thermo;
and0.1%Tween-20,theimmunoreactivebandwasvisualized
Kalamazoo,MI,USA).
usinganECLdetectionkit(AmershamBiosciences,Bucking-
Frozensections(30𝜇m)wereincubatedwith0.3%H2O2 hamshire,UK).
in0.01MPBSfor30minatroomtemperature(RT)toquench
endogenous peroxidase activity. The sections were then
blockedwith10%goatseruminPBSor10%rabbitserum(for 2.6.ImageAnalysis. Toquantifythenumberofcellsexpress-
MBPstaining)for1hatRT.Afterblocking,thesectionswere ing NG2 and PDGFR𝛼, three digital images of coronal
incubatedat4∘Covernightwiththeprimaryantibodiesinthe sectionsfromeachanimal(includingthemidlineandthetwo
blockingsolution.Afterrinsing,thesectionswereincubated edges of the corpus callosum (CC) in each section) were
examined. The positive cell counts are expressed as the
for1hatRTwiththeselectivebiotin-conjugatedsecondary
averageofpositivecellsintwoareasofthreecoronalsections,
antibody (1:1000; Vector Laboratories, Burlingame, CA,
500𝜇m apart, between 1 and −1mm from the bregma. The
USA).Stainingwasperformedwithanavidin-biotincomplex
averageswerecalculatedfromatleasteightmicepergroup.
kit(VectorLaboratories,Burlingame,CA,USA)andvisual-
Three digital images from coronal sections (including
ized with using 0.025% 3,3-diaminobenzidine as the chro-
cerebral cortex) of each animal were analyzed for MBP
mogen(DAB,Sigma-Aldrich,St.Louis,MO,USA).
staining.Aminimumofeightanimalswereanalyzedineach
Goatpolyclonalantibodiesdirectedagainstmyelinbasic
group.ThepercentareaofMBP-positivestainingwascalcu-
protein(MBP)(1:500;SantaCruzBiotechnology,CA,USA)
latedinaselectedareaofthecortex.Resultsareexpressedas
were used to detect myelin components. Rabbit polyclonal
percentMBP-positiveareacomparedtocontrol.Imageswere
NG2antibodies(1:250;Chemicon,Temecula,CA,USA)and
obtainedusinganOlympusBX-51lightmicroscope(Olym-
PDGFR𝛼antibodies(1:250;SantaCruzBiotechnology,CA)
pus,RichmondHill,ON,Canada).Theimageswereanalyzed
were used as a marker for oligodendrocyte progenitor cells usingImage-ProPlussoftware(Version6.1,MediaCybernet-
(OPCs)[16,17]. ics,Inc.,SilverSpring,MD,USA).
2.5. Western Blot Analysis. The frontal cortex samples were 2.7. Statistical Analyses. All data were expressed as the
homogenizedinradioimmunoprecipitationassaylysisbuffer mean±thestandarderrorofthemean(SEM).Tocompare
(50mMTris,150mMNaCl,1%NP40,0.5%sodiumdeoxy- experimental and control groups, a one-way ANOVA was
cholate,and0.1%sodiumdodecylsulfatewithfreshlyadded performedfollowedbyDunnett’stest.Whenstatisticallysig-
proteaseinhibitorcocktail(Sigma,St.Louis,MO,USA).The nificantdifferenceswerefound,Newman-Keulstestwasused
supernatantwascollectedaftercentrifugationat12,000rpm asposthoctesttodeterminethestatisticaldifferencebetween
∘
for10minat4 C.ABCAproteinkit(Pierce,Nepean,Ontario, groups.AllstatisticalanalyseswereperformedwithGraph-
Canada) was used to quantify total protein concentration. Pad Prism, Version 5 (GraphPad Software, Inc., San Diego,
Samplesoftheproteinswereloadedonto10%sodiumdode- CA, USA). A 𝑃 value of <0.05 was considered statistically
cylsulfate-polyacrylamidegelandsubjectedtoelectrophore- significant. The figures were created using the GraphPad
sis at 70V and 110V. The proteins were then transferred to Prism5StatisticalAnalysisSystem.
4 Evidence-BasedComplementaryandAlternativeMedicine
40 80
n) ### ###
30 mi 60
min) 8e ( ∗∗∗
z
8 a
Arms entries ( 2100 Ynation in-m 4200
er
Alt
0 0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
Figure1:TheeffectofArecanutextract(ANE)onspatialworkingmemoryincuprizone-(CPZ-)inducedmousedemyelinationmodel.After
1weekofacclimation,maleC57BL/6micewerefedrodentchowcontaining0.2%CPZ(w/w)for8weekstoinducedemyelination.Micein
theANEtreatmentgroupswerefedrodentchowcontaining0.2%CPZplus1%ANE(ANE1)or2%ANE(ANE2).Thecontrolmicereceived
normalrodentchow.Datawereexpressedasthemean±SEM.∗∗∗,###𝑃<0.001.∗∗,##𝑃<0.01.∗,#𝑃<0.05.∗indicatescomparisonwiththe
controlgroupwhile#indicatescomparisonwiththeCPZgroup.
3.Results diminished the duration of time spent in the novel side of
the apparatus compared with control mice (𝑃 < 0.01).
3.1.TheEffectofArecaNutExtract(ANE)onSpatialWorking These findings indicated significant lower social preference
Memory. Spatialworkingmemorywasanalyzedinmicesub- forCPZ-treatedmicecomparedtocontrolmice(𝑃 < 0.01).
jected to demyelination by CPZ using the 𝑌-maze test. The ANE treatment reversed the social preference impairment
percentage of alternation behaviors significantly decreased and increased the duration of time spent in the novel side
in CPZ-treated mice compared with the control group anddecreasedthetimespentonthefamiliaranimalside.A
(𝑃 < 0.001) (Figure1). This finding indicated significantly statisticallysignificantimprovementwasobservedinthe2%
impairedspatialworkingmemory.Alternationinthe𝑌-Maze ANE treatment group (𝑃 < 0.05) compared with the CPZ
significantly increased after treatment with both doses of group(Figure2).TheseresultssuggestedthatANEtreatment
ANE. These results suggest that ANE treatment improved improved deficits in social interaction in mice subjected to
spatialworkingmemory. CPZ-induceddemyelination.
3.2.TheEffectofArecaNutExtract(ANE)onSocialInteraction
Behaviors. It has been reported that animals subjected to 3.3. The Effect of Areca Nut Extract (ANE) on Matured
CPZ-induced demyelination animals displayed less social Oligodendrocytes and Progenitor Cells. MBP immunostain-
interaction [13]. Stay durations on each side of a social test ingresultsshowedthatCPZ-treatedanimalshadsignificant
boxweremeasuredaftera5-minhabituationperiod.Inthe myelin sheath disruption in the frontal cortex. The optical
sociability test, CPZ-treated mice spent less time in the density of MBP was significantly decreased in the CPZ
compartment with a CSM compared with control group group compared to control animals (𝑃 < 0.01). The 2%
(Figure2). ANE-treated mice showed a trend toward an concentration of ANE produced a statistically significant
increaseddurationwithCSM.However,thesechangeswere increaseinMBPopticaldensitycomparedtothatoftheCPZ-
notstatisticallysignificant. treated group (𝑃 < 0.05) (Figure3). Western blot analysis
CPZ-treatedmicespentmoretime(𝑃 < 0.01)intheEC showedthatMBPproteinexpressioninfrontalcortextissue
compartment than control mice, and the mice treated with decreased significantly (𝑃 < 0.05) after chronic CPZ
2%ANEspendsignificantlylesstimeintheECcompartment exposurecomparedtocontrolmice.ANEtreatmenttended
(𝑃<0.01),comparedwithCPZ-treatedmice(Figure2).The toupregulateMBPexpression,andcomparedtoCPZ-treated
change in social interaction was expressed as a sociability mice, 2% ANE treatment showed a statistically significance
index,definedastheratioofdurationofstaybetweenCSM increaseinMBPexpression(𝑃<0.05)(Figure4).Treatment
side and EC side, which showed ANE treatment inhibited with2%ANEproducedobviousprotectiveeffectsfromCPZ-
the decrease in sociability induced by CPZ. However, these inducedmyelinsheathdamage.
resultswerenotstatisticallysignificant. NG2- and PDGFR𝛼-positive cells are a group of OPCs
The social preference for novelty, which determines thathavethepotentialtodevelopintomatureOLsintheadult
whetherthetestanimalpreferssociallynovelversusfamiliar brain [16]. After demyelination with CPZ, the numbers of
animals, was also investigated. CPZ exposure significantly NG2-andPDGFR𝛼-positivecellsinthecortexweredramat-
increased the latency for engagement with socially familiar icallyhigher(Figures5and6)thanthoseinthecontrols.The
animals(𝑃 < 0.01).Inaddition,CPZtreatmentsignificantly numbersofNG2-andPDGFR𝛼-positivecellsinthecortexof
Evidence-BasedComplementaryandAlternativeMedicine 5
80 100
e (s)
ger sid 60 e (s) 80 ∗∗
n d
c stra pty si 60
cifi 40 em ##
nspe n in 40
o o
on in c 20 Durati 20
ati
ur
D 0 0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
1.5 80
∗∗
e (s) 60
Sociability index 10..05 ation in familiar sid 4200 ##
ur
D
0.0 0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
100 3
#
80
el side (s) 60 # ce index 2
v ∗∗ n
o e
n in n 40 prefer ∗∗
Duratio 20 Social 1
0 0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
Figure2:TheeffectofArecanutextract(ANE)onsociabilityandsocialpreferencebehaviortestedinsocialinteractionincuprizone-(CPZ-)
inducedmousedemyelinationmodel.After1weekofacclimation,maleC57BL/6micewerefedrodentchowcontaining0.2%CPZ(w/w)for
8weekstoinducedemyelination.MiceintheANEtreatmentgroupswerefedrodentchowcontaining0.2%CPZplus1%ANE(ANE1)or2%
ANE(ANE2).Thecontrolmicereceivednormalrodentchow.Datawereexpressedasthemean±SEM.∗∗,##𝑃<0.01.∗,#𝑃<0.05.∗indicates
comparisonwiththecontrolgroupwhile#indicatescomparisonwiththeCPZgroup.
theANEtreatmentgroupweresignificantlylowerthaninthe ANE might target OPCs and facilitate OPC differentiation
CPZgroup.Westernblotanalysisofcortextissuealsoshowed intomatureOLstopreventmyelinsheathdamage.
upregulation of PDGFR𝛼 protein expression, although the
quantitative analysis did not show statistical significance. 3.4.TheEffectofArecaNutExtract(ANE)onAMP-Activated
A significant decrease in PDGFR𝛼 protein expression was ProteinKinase(AMPK)Expression. Ithasbeendemonstrated
observedforbothconcentrationsofANE(𝑃<0.05,forboth thatArecanutconstituentsmodulatemetabolicsignalsreg-
groups versus CPZ) (Figure4). These results suggested that ulatingacrucialstress-sensingenzyme,AMPK[18].Protein
6 Evidence-BasedComplementaryandAlternativeMedicine
Control×4 CPZ×4
CPZ+ANE1×4 CPZ+ANE2×4
150
ol)
ntr
o
c
of 100
% #
P ( ∗∗
B
M
y
nsit 50
e
d
al
c
pti
O
0
Control CPZ CPZ+ANE1 CPZ+ANE2
Group
Figure3:Arecanutextract(ANE)treatmentmaypreventthedemyelinationprocessinthefrontalcortex.PhotomicrographsshowedMBP
immunostaininginthefrontalcortexinthecontrolgroup,CPZ,CPZ+1%ANE(ANE1),CPZ+2%ANE(ANE2)groups,respectively.The
graphshowsthemeanMBP-immunostainingopticaldensityinthefrontalcortexforeachtreatmentgroup.Dataarerepresentedasthemean
±SEM.∗∗,##𝑃<0.01.∗,#𝑃<0.05.∗indicatescomparisonwiththecontrolgroupwhile#indicatescomparisonwiththeCPZgroup.
expressionincorticalspecimenswasanalyzedtodetermine 4.Discussion
theeffectofANEonAMPKkinase.PhosphorylatedAMPK𝛼
wassignificantlyincreasedinmicesubjectedtoCPZ-induced The focus of this study was the neuropathological features
demyelinationcomparedwithcontrolmice(𝑃<0.05).After of white matter in schizophrenic brains and animal models
treatment with 1% and 2% ANE, phosphorylated AMPK𝛼 designedtomimicthisneuropathology.Withlow-doseCPZ
ratios were significantly reduced compared with the CPZ administration, rodents showed selective demyelination
group(𝑃 < 0.001;𝑃 < 0.001,resp.).TotalAMPKexpression lesions in the prefrontal cortex, which made this model
did not show significant differences between the groups especially attractive for schizophrenia studies [17, 19, 20].
(Figure7). The data suggested that ANE treatment might PreviousstudieshaveshownCPZ-induceddemyelinationin
suppressAMPKactivation. mice resulted in deficits spatial working memory, social
Evidence-BasedComplementaryandAlternativeMedicine 7
MBP PDGFR𝛼
𝛽-actin 𝛽-actin
1.5 1.5
1.0 # n 1.0
𝛽-actin 𝛼𝛽/-acti # #
MBP/ 0.5 ∗ DGFR 0.5
P
0.0 0.0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
Figure4:TheeffectofArecanutextract(ANE)treatmentonoligodendrocytelineages.FiguresshowrepresentativeWesternblotimages
ofMBPandPDGFR𝛼-immunoreactivebandsfromcortexsamplesofthetreatmentgroups.Densitometricquantificationshowstheratioof
MBPandPDGFR𝛼normalizedto𝛽-actin.Resultsshownarepresentedasthemean±SD.∗∗,##𝑃<0.01.∗,#𝑃<0.05.∗indicatescomparison
withthecontrolgroupwhile#indicatescomparisonwiththeCPZgroup.
interaction, and executive function. The abnormal behav- ANE showed protective effects on myelin degradation and
iors,demonstrabledemyelination,andoligodendrocyteloss increasedMBPexpressioninthecortex.
observed in CPZ-treated mice suggested a correlation DuringdosingwithCPZ,matureOLsundergoapoptosis.
betweenwhitematterdamageandcertainbehavioralabnor- OPCsproliferateduringrecoveryinthesubventricularzone,
malities[13,21–23]. migrating to the demyelinated areas of the CC and cortex,
Impairment of working memory has been widely and develop into mature OLs [21]. In animals subjected to
reported in patients with schizophrenia [24]. Some studies demyelination,adramaticincreaseinNG2-positivecellswas
haveindicatedthatimpairedspatialworkingmemoryhasa observed in the frontal cortex and CC, but not in the sub-
strongcorrelationwithnegativesymptomsofschizophrenia ventricularzone.Theseresultsindicatethatthemigrationof
andcouldbeconsideredanendophenotypeofschizophrenia NG2-positivecellstothedemyelinatedareasofthecortexand
[21].Inthe𝑌-mazetest,ANEsignificantlyenhancedspatial CCoccurredatalatestageofdemyelination[16].Duringthe
working memory in CPZ-treated mice. This was consistent recovery period, the number of NG2-positive cells was
with the findings of Joshi et al. [25], which suggested that reduced with increasing recovery time [21]. PDGFR𝛼 and
Areca catechu extract produced a greater increase in spatial NG2 antibodies were used to detect OPCs. In our study,
memoryandlearningowingtoahighercontentofarecoline. PDGFR𝛼-positiveandNG2-positiveOPCsincreasedinthe
In addition, the phenolic fraction such as hydroxychavicol cortex of mice subjected to CPZ-induced demyelination.
wasalsofoundtoimprovecognitivedeficits[26]. ANE treatment reduced the numbers of PDGFR𝛼-positive
ApreviousstudyshowedthatCPZ-treatedmicedemon- and NG2-positive OPCs in the frontal cortex. It has been
strateddeficitsinsocialinteractionstartingat4weeksafter reportedthattheArecanutpossessesantioxidativeeffects[5].
CPZ exposure when demonstrable loss of myelin sheath This antioxidative capability of ANE might help to prevent
andMBPwereobserved[13].Thesefindingssuggestedthat myelin damage, subsequently decreasing the compensatory
normalbehaviorinthesocialinteractiontestreliedonintact increaseofNG2andPDGFR-𝛼.Myelin-formingcellsinthe
whitematter.Ourresultsshowedlesssocialabilityandsocial central nervous system can arise from OPCs under certain
preferenceinmicetreatedwithCPZfor8weeks.ANEtreat- conditions. Exposure of OPCs to PDGF led to continuous
mentimprovedsocialinteractiontimeinCPZ-treatedmice. self-renewal without differentiation. In contrast, OPCs not
MBP immunostaining showed obvious demyelination in exposedtomitogensdifferentiateintooligodendrocytesand
the cortex after 8 weeks of CPZ treatment. Western blot donotproliferate[27].TheWesternblotresultsindicatedthat
analysis also demonstrated decreased expression of MBP. ANE decreased platelet-derived growth factor receptor
8 Evidence-BasedComplementaryandAlternativeMedicine
Control CPZ
CPZ+ANE1 CPZ+ANE2
150
ol) ∗∗∗
ntr
o
c
% of 100 ###
cells ( ###
+
2
NG 50
of
y
nsit
e
D
0
Control CPZ CPZ+ANE1 CPZ+ANE2
Group
Figure5:Arecanutextract(ANE)decreasedNG2-positivecellsincortex.PhotomicrographsshowNG2-immunostaininginthefrontal
cortexofmicefromthetreatmentgroups.ThebargraphshowsthemeannumberofNG2-positivecellsinthefrontalcortexasapercentage
ofcontrol.Dataarerepresentedasthemean±SEM.∗∗∗,###𝑃 < 0.001.∗∗,##𝑃 < 0.01.∗,#𝑃 < 0.05.∗indicatescomparisonwiththecontrol
groupwhile#indicatescomparisonwiththeversusCPZgroup.
(PDGFR𝛼) expression and thus might arrest OPCs prolif- of neurodegenerative diseases [28]. In our study, chronic
eration and stimulate differentiation into oligodendrocytes. administrationofCPZcausedactivationofAMPKshownas
Although a search of the literature did not produce any increasedphosphorylationlevelofAMPK𝛼(p-AMPK𝛼),and
publications describing a direct effect of ANE on NG2 and differentconcentrationofANEsuppressedAMPKactivation.
PDGFR-𝛼,afuturestudyinvestigatingthepossibleeffectof A greater role for AMPK in regulating cell growth and
ANEonNG2andPDGFR-𝛼inthemicewithoutacuprizone- proliferationhasbeenproposedbasedonrecentstudies[29].
diet is necessary to evaluate the possible direct effect of AMPK activating agents cause dephosphorylation of glyco-
ANE on NG2 and PDGFR-𝛼. Moreover, further studies are gensynthasekinase-3𝛽(GSK-3𝛽)[30].GSK3𝛽wasfoundto
necessary to elucidate the effects of ANE on spontaneous suppressoligodendrocytedifferentiationandmyelinationin
remyelinationaftercuprizone-dietatdifferenttimepoints. vivo [31]. Alterations in GSK-3𝛽 and 𝛽-catenin have been
AMPKisaserine/threonineproteinkinase.Activationof reported in schizophrenic patients and have been targeted
AMPKthroughoxidationinthecerebellummaycontribute byantipsychoticdrugs[32,33].Invitrostudieshavedemon-
to neurodegeneration, and AMPK signaling pathways have strated that ANE treatment induced the phosphorylation
been proposed as therapeutic targets for the treatment of GSK-3𝛽 [34–36]. 𝛽-catenin expression was increased by
Evidence-BasedComplementaryandAlternativeMedicine 9
Control CPZ
CPZ+ANE1 CPZ+ANE2
150
ol)
ntr
o ∗
c
of
% ##
ells (100 ###
c
+
𝛼
R
F
G 50
D
P
of
y
nsit
De 0
Control CPZ CPZ+ANE1 CPZ+ANE2
Group
Figure6:Arecanutextract(ANE)decreasedPDGFR𝛼-positivecellsinthefrontalcortex.ThebargraphshowsthemeannumberofPDGFR𝛼-
positivecellsinthefrontalcortexasapercentageofcontrol.Dataarepresentedasthemean±SEM.∗∗∗,###𝑃<0.001.∗∗,##𝑃<0.01.∗,#𝑃<0.05.
∗indicatescomparisonwiththecontrolgroupwhile#indicatescomparisonwiththeCPZgroup.
arecoline in a dose-dependent manner [37]. The results of investigations may be required to (1) identify the profile
this study suggest that ANE treatment may facilitate OPC of ANE by using HPLC and determine the active compo-
differentiationandmyelinationprocessesandthattheAMPK nentsresponsibleforthepreventativeeffectonwhitematter
signalingpathwaymaybeinvolvedinthisprocess. demyelinationand(2)revealwhetherarecolineandphenolic
compounds in ANE attenuate demyelination and enhance
oligodendrocyte development synergistically and whether
5.Conclusion theyhavedualeffects:abiphasicdose-responserelationship.
ThisstudyshowedthatANEtreatmentmayenhancecogni-
tiveabilityandsocialactivitybyfacilitatingOPCdifferenti- ConflictofInterests
ation and protecting myelin damage in cortex. The AMPK
signalingpathwaymaybeinvolvedinthisprocess.Giventhe The authors declare that there is no conflict of interests
limitations of the current study, detailed pharmacological regardingthepublicationofthispaper.
10 Evidence-BasedComplementaryandAlternativeMedicine
1.5
∗
p-AMPK
n 1.0
cti ###
a
𝛽-
t-AMPK PK/
M
###
A 0.5
p-
𝛽-actin
0.0
Control CPZ CPZ+ANE1 CPZ+ANE2
Group
1.5 2.0
∗∗
1.5
n K
cti 1.0 MP
a
𝛽MPK/- K/total A 1.0 ###
A P
al 0.5 M ###
Tot p-A 0.5
0.0 0.0
Control CPZ CPZ+ANE1 CPZ+ANE2 Control CPZ CPZ+ANE1 CPZ+ANE2
Group Group
Figure7:TheeffectofArecanutextract(ANE)treatmentonAMP-activatedproteinkinase(AMPK)expression.Representativeimmunoblots
ofAMPKexpressionincortexsamplesareshown.Densitometricquantificationshowstheratioeachproteinexpressionnormalizedto𝛽-
actinexpression.Resultsarepresentedasthemean±SD.∗∗∗,###𝑃<0.001.∗∗,##𝑃<0.01.∗,#𝑃<0.05.∗indicatescomparisonwiththecontrol
groupwhile#indicatescomparisonwiththeCPZgroup.
Acknowledgments [5] A. M. Bhandare, A. D. Kshirsagar, N. S. Vyawahare, A. A.
Hadambar,andV.S.Thorve,“Potentialanalgesic,anti-inflam-
This work was supported by the Canadian Institutes of matoryandantioxidantactivitiesofhydroalcoholicextractof
HealthResearch(CIHR)funding.TheauthorsthankXiamen ArecacatechuL.nut,”FoodandChemicalToxicology,vol.48,no.
Xian Yue Hospital for providing Abulimiti Adilijiang with 12,pp.3412–3417,2010.
“professorship for international research” scholarships to [6] A.Bales,M.J.Peterson,S.Ojha,K.Upadhaya,B.Adhikari,and
completetheworkinUniversityofAlberta,Canada. B.Barrett,“Associationsbetweenbetelnut(Arecacatechu)and
symptomsofschizophreniaamongpatientsinNepal:alongi-
tudinalstudy,”PsychiatryResearch,vol.169,no.3,pp.203–211,
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