Enhancement of immune cytokines and splenic CD4+ T cells by electroacupuncture at ST36 ...

13 Pages·2017·2.25 MB·English

RESEARCHARTICLE
Enhancement of immune cytokines and
splenic CD4+ T cells by electroacupuncture at
ST36 acupoint of SD rats
LongyunChen1☯,AnliXu1☯,NinaYin2,MinZhao1,ZhigangWang3,TaoChen2,
YishengGao2,ZebinChen2*
1 DepartmentofBiochemistry,BasicMedicalCollege,HubeiUniversityofChineseMedicine,Wuhan,China,
2 DepartmentofAnatomy,BasicMedicalCollege,HubeiUniversityofChineseMedicine,Wuhan,China,
3 DepartmentofPathogenicBiology,BasicMedicalCollege,HubeiUniversityofChineseMedicine,Wuhan,
China
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a1111111111 ☯Theseauthorscontributedequallytothiswork.
a1111111111 *[email protected]
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Abstract
ElectroacupunctureattheST36acupointcanenhancethebody’simmunefunction.How-
ever,themechanismforthisenhancementhasnotbeenfullydescribed.Ourstudywas
OPENACCESS
designedtoinvestigatetheeffectofelectroacupunctureontheimmunefunctionofSprague-
Citation:ChenL,XuA,YinN,ZhaoM,WangZ,
Dawley(SD)rats.Theratswererandomlydividedintothreegroups:acontrolgroup,anon-
ChenT,etal.(2017)Enhancementofimmune
cytokinesandsplenicCD4+Tcellsby acupointgroup(abdominalmuscleacupuntured)andaST36acupointgroup.Ourresults
electroacupunctureatST36acupointofSDrats. showedthatsuccessiveelectroacupunctureattheST36acupointfor3dsignificantly
PLoSONE12(4):e0175568.https://doi.org/
enhancedtheinterferon-γ(IFN-γ)levelintheserumofSDrats.Theresultsalsoshowedthat
10.1371/journal.pone.0175568
theserumandextractsfromspleencellsoftheST36acupointgroupcontainedhigherlevels
Editor:BrijSingh,UniversityofNorthDakota
ofinterleukin(IL)-2andIL-17comparedtothoseoftheothertwogroups.Immunohisto-
SchoolofMedicineandHealthSciences,UNITED
chemicalanalysisshowedthatelectroacupunctureappliedtotheST36acupointenhanced
STATES
theexpressionlevelofCD4inspleencells.Furthermore,itwasobservedthatCD4co-local-
Received:October2,2016
izedwithtransientreceptorpotentialvanilloid(TRPV)channelsatthemembraneofsplenic
Accepted:March28,2017
CD4+TcellsandtheexpressionlevelofCD4wasrelatedtoTRPVchannelsintheelectroa-
Published:April13,2017 cupuncturetreatment.Theseobservationsindicatedthatelectroacupuncturestimulationat
Copyright:©2017Chenetal.Thisisanopen theST36acupointenhancedthelevelofimmunecytokinesandsplenicCD4+Tcells
accessarticledistributedunderthetermsofthe throughTRPVchannelsinthissystem.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation Introduction
files.
Electroacupuncture(EA)treatmentisoneofthemostpopularapproachesincomplementary
Funding:Thisstudywassupportedbygrantsfrom medicineandhealthmaintenance.Ithasbeenshowntohavesomeanalgesiceffectsinreliev-
theHealthandFamilyPlanningCommissionof
ingpain[1–5].Recently,someexperimentalandclinicalstudieshaveshownthatsequential
HubeiProvince(no.2013Z-Z01)andHubei
electroacupuncturestimulationappliedtotheST36acupoint(Zusanliacupuncturepoint)per-
ProvincialDepartmentofEducation(no.
formedwellinthetreatmentofstress-inducedimmunodeficiency[5,6].Apreviousstudy
B2016112).Thefunders’websitesarehttp://www.
hbwsjs.gov.cn/andhttp://www.hbe.gov.cn/.Zebin demonstratedthatelectricalstimulationattheST36acupointscanactivatetheimmunesystem
PLOSONE|https://doi.org/10.1371/journal.pone.0175568 April13,2017 1/13
ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Chenreceivedthefunding.Hehadaroleinthe andhelpsupportanti-cancertreatments[7–9].However,thecurativemechanismsofelectroa-
studydesign. cupunctureremainpoorlyunderstood,whichlimitstheapplicationofelectroacupunctureon
Competinginterests:Theauthorshavedeclared alargerscale.
thatnocompetinginterestsexist. CD4+Tcellsareoneoftheimportantcellsinthehumanimmunesystem.Tcellsplaya
veryimportantroleintheimmunesystem.CD4+Tcells,whichareknownasthe"helper"of
theimmunesystem,protectthebodyagainstmicrobessuchasviruses.TheCD4+Tcellisan
importantcomponentoftheadaptiveimmuneresponse.Afterdifferentstimuli,CD4+Tcells
candifferentiateintodifferentsubsetsofcells,includingTh1andTh2cells,follicularhelper
(Tfh)cells,Th17cells,andregulatoryTcells(T ).ThefunctionsoftheCD4+Tcellare
REG
diverse,suchastheactivationofimmunecells,directcytotoxiceffects,andimmunoregulation
[10].
FreeCa2+ionsareusedassecondmessengersformostcells,includingimmunecells.Rest-
ingTcellsandBcellsareknowntomaintainlowintracellularCa2+concentrations[11].Some
receptorslocatedontheimmunecellsurfaceareknowntoincreasetheconcentrationofintra-
cellularCa2+,suchastheTcellreceptor,theBcellreceptorandco-stimulatoryreceptors[12].
ApreviousstudyalsonotedthatCa2+signalinginimmunecellsisimportantforthedifferenti-
ationofimmunecellsandgenetranscription[11–13].Ithasbeenshownthattheactivationof
TcellscancauseanincreaseintheIFN-γandIL-2levels[14].Further,electroacupuncturehas
beendemonstratedtoenhancethelevelsofsplenicIFN-γandIL-2releasedbyhelperTcells
[15,16].HowtheincreasesintheIFN-γandIL-2levelswereinducedbyelectroacupuncture
requiresfurtherstudy,andtheeffectofelectroacupunctureonsplenicTcellsneedstobe
explored.Hence,theaimsofthisstudyweretoinvestigatetheeffectandmechanismofelectro-
acupunctureonthereleaseofimmunecytokinesandtheactivationofsplenicTcells.
Materialsandmethods
Animalsandelectroacupuncturetreatment
AdultmaleSprague-Dawley(SD)ratsweighting200±25gwerepurchasedfromtheExperi-
mentalAnimalCenterofHubeiUniversityofTraditionalChineseMedicine(medicallabora-
toryanimalcertificatenumber:SCXK(Hubei)2015–0018).TRPV1knockout(TRPV1-/-)
micewerepurchasedfromNanjingBiomedicalResearchInstituteofNanjingUniversity
(medicallaboratoryanimalcertificatenumber:SCXK(Jiangsu)2015–0001).Alltheanimals
werekeptintheExperimentalAnimalCenterofHubeiUniversityofChineseMedicine.The
ExperimentalAnimalCenterofHubeiUniversityofChineseMedicineobtainedalaboratory
animaladministrativelicensefromtheScienceandTechnologyDepartmentofHubeiProv-
ince(number:SYXK(Hubei)2012–0067).Theanimalswererandomlydividedinto3groups:
(a)acontrolgroupinwhichtheanimalsreceivednotreatmentandwerehousedattheanimal
facilities(20–23˚C,50%±5%humidity,12-hourlight/darkcyclewithlightsonat8:00am);
(b)anon-acupointgroupinwhichelectroacupuncturestimulationwasappliedtotheabdomi-
nalmuscleasdescribedinapreviousstudy[16];(c)andaST36acupointgroupinwhichelec-
troacupuncturestimulationwasappliedtotheST36acupoint.ThelocationofST36acupoint
wasshowninFig1Aand1B.Twosterilizedacupunctureneedles(diameter0.16mm,length
25mm,Zhongyantaihe,Tianjin,China)wereinsertedattheST36acupointortheexternal
obliqueabdominalmuscletoadepthof4–5mm,andtheneedleswereconnectedbytheirhan-
dlestoaHANS(Han’sAcupointNerveStimulator)electro-stimulator.Thedisperse-dense
wavesatthealternatingfrequencies2Hzand15Hzandanintensityof1mAwereusedas
electricalstimulationfor30min.Thestimulationprotocolwasrepeatedeachdayfor1,3,7or
14days.Femaleanimalswereexcludedfromourstudy.Allmanipulationswereperformed
between8:00a.m.and12:00a.m.everydaytominimizetheinfluenceofcircadianrhythms.
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig1.(AandB)ThelocationofST36acupointonthebodyofSDrats.
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Aftertheelectroacupuncturetreatment,theanimalswereanesthetizedbyanintraperitoneal
injectionwith50mg/kgpentobarbitalsodiumforthefollowingexperiments.Allexperiments
wereperformedatroomtemperature.Alltheanimalswereallowedtocalmdowninthenew
environmentforatleast30min.
PreparationofextractfromST36acupointarea
Topreparetheextract,theratswereanesthetizedbyanintraperitonealinjectionwith50mg/
kgpentobarbitalsodium.Then,thetissuesaroundtheST36acupoint(10mmlength,10mm
widthand5mmdepth)werecutoff,weighed,mincedintosmallpiecesandhomogenizedin
coldPBS(10mgtissueper100μlPBS).Theresultingsuspensionwassubjectedtofurther
breakdownviaultrasonication,andthehomogenateswerethencentrifugedat1500gfor15
min.Thesupernatantswerecollectedandstoredat-80˚C.
ELISAassay
AnELISAassaywasusedtoassesstheIFN-γ,IL-2andIL-17levelsintheserumandextracts
fromtheST36acupointarea.Aftertheelectroacupuncturetreatment,oneeyeoftheanesthe-
tizedratswaspulledoutusingtweezers,andthefreshbloodwascollectedina10mlEPtube.
Thetubesweremaintainedat4˚Cfor24h,andtheserumwasharvestedandstoredat-80˚C.
Theserumandextractsweresubjectedtoanenzyme-linkedimmunosorbentassay(ELISA)to
detectIFN-γ,IL-2andIL-17expressionaccordingtothemanufacturer’sprotocol(Thermo
Fischer).Allassaysweredoneatleastthreetimesinthreeseparateexperiments.
Immunohistochemicalanalysis
Thetissuesweredehydratedusingsequentialincubationswith75%,85%,90%,95%and100%
ethanol.Thetissuesweretreatedwithatransparentagentafterthedehydration.Transparent
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
tissuesweresoakedandembeddedinparaffinwax.Theywereslicedandplacedonslides.The
slideswereimmersedinxylenefor10minutes.Then,thesectionsweredehydratedbysequen-
tialincubationswith100%,95%,80%and60%ethanolfor5minuteseach.Thesectionswere
rinsedwithdistilledwaterthreetimesfor3minuteseach.Theslidesweretransferredtoa
microwave-proofcontainerandcoveredwithcitratebufferforantigenretrieval.Sectionswere
blockedusing5%normalblockingserumfor1hour.Then,theywereincubatedwiththe
respectiveprimaryantibodyfor1houratroomtemperature.Followingprimaryantibody
incubation,theslideswereincubatedinasufficientamountofperoxidase-labeledpolymerfor
30minutesandthesubstratefor5–10minutes,successively,untilabrowncolordeveloped.
Then,theslidesweretreatedbyhematoxylincounterstaining.Afterdehydrationandmount-
ing,theslideswereobservedunderamicroscope.
SDS-PAGEandWB
Thetissuesofthespleenwerecutout,weighedandmincedintosmallpiecesasbefore.Then,
thetissueswerehomogenizedinlysisbuffer(20mMTris-HCl[pH8.0],100mMNaCl,1.9%
[wt/vol]TritonX-100,1mM1,4-dithiothreitol,and5%[vol/vol]glycerol)onicefor30min.
Thehomogenateswerecentrifugedat12,000rpmfor5minat4˚C.Aftercentrifugation,the
supernatantswerecollected.Theconcentrationoftheproteinineachsamplewasdetermined
usingtheBradfordmethod.Equalamountsofproteinwereseparatedby12%SDS-PAGEand
transferredontoanitrocellulosemembrane(GEHealthcare).Themembranewasblocked
with5%nonfatmilkandincubatedwiththeprimaryantibodiesforIL-2andIL-17,followed
byincubationwiththegoatanti-rabbitsecondaryantibodies.
DetectionofCa2+concentration
TheserumwassubjectedtoacolorimetricmethodforthedetectionoftheCa2+concentration
accordingtothemanufacturer’sprotocol(CalciumAssayKit,BioVision).
TodetecttheCa2+concentrationinspleencells,thefreshspleentissuewasmadeintoasin-
glecellsuspension(2x106cellsperEPtube).Thecellswereresuspendedinserum-free
mediumatafinalconcentrationof2.5μmol/LofFluo3-Am.Thesuspensionwasplacedat
37˚Cat5%CO for30min.Thesuspensionwascentrifugedat1500rpmfor5min,andthe
2
supernatantwasdiscarded.Thecellswerewashedwithcalcium-freePBStwiceandsuspended
in500μlcalcium-freePBS.TheCa2+concentrationofthespleencellswasdetectedbyflow
cytometry.
Immunofluorescenceanalysis
Thespleenwastreatedusingthemethoddescribedintheimmunohistochemicalanalysiswith
differentprimaryandsecondaryantibodies.ThespecificprimaryAbsforCD4,TRPV1or
TRPV4wereused.Followingtheprimaryantibodyincubation,thegoatanti-rabbitIgG(Cy3)
orgoatanti-mouseIgG(FITC)fluorescencesecondaryantibodieswereadded.DAPIwasused
tostainthenucleusfor5min.
Results
EffectofelectroacupunctureontheexpressionofIFN-γ
ApreviousstudyreportedthatelectroacupunctureappliedtotheST36acupointforthreecon-
secutivedaysenhancedthelevelofIFN-γinthespleenofrats[15,17].Thus,wewantedto
determinewhetherelectroacupuncturecausesthelevelofIFN-γintheserumtoincrease.Male
SDratsweredividedintothreegroups:acontrolgroup,anon-acupointgroupandaST36
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig2.EffectofelectroacupunctureatST36acupointonIFN-γlevelofSDrats.(A)Electroacupuncturewasapplied
totheleftandrightST36acupointinthesameratfor30min.Theelectroacupunturestimulationwasrepeatedeachday
for1,3,7or14consecutivedaysondifferentrats.TheIFN-γlevelinserumofSDratswasdetected.(D1=1day)(B)
ElectroacupunctureattheST36acupointcausedtheIFN-γlevelinserumincreased.*P<0.05forST36acupointgroup
versuscontrolgroupandnonacupointgroup.(C)AftertheratsweretreatedbyelectroacupunctureattheST36
acupoint,theIFN-γlevelinextractsfromST36acupointareaincreased.*P<0.05forST36acupointgroupversuscontrol
groupandnonacupointgroup.(D)TheIFN-γlevelinspleenwasdetectedviaimmunohistochemicalanalysis.The
positivesignalisbrownordarkbrown.Mock=Control;Nonacupoint=electroacupunctureatabdominalmuscle;
ST36=electroacupunctureattheST36acupoint.
https://doi.org/10.1371/journal.pone.0175568.g002
acupointgroup.Forthenon-acupointgroup,electroacupuncturewasappliedtotheabdomi-
nalmuscle.ElectroacupunctureattheST36acupointwasappliedtotheSDratsfor1,3,7or
14consecutivedays.ThelevelofIFN-γintheserumwasdetectedviatheIFN-γELISAassay.
TheresultsshowedthatelectroacupunctureattheST36acupointforthreeconsecutivedays
significantlyincreasedtheIFN-γlevelinserum(Fig2Aand2B).Basedontheseresults,the
electroacupuncturetreatmentatST36acupointwasonlyappliedforthreeconsecutivedaysin
ourresidualresearch.WefurtherstudiedtheeffectofelectroacupunctureontheIFN-γlevelin
theextractsfromtheST36acupointarea.TheIFN-γELISAassaydemonstratedthattheIFN-γ
levelintheextractsoftheST36acupointgroupincreasedbecauseoftheelectroacupuncture
(Fig2C).Consistentwithapreviousstudy[15],ourresultsdemonstratedthatelectroacupunc-
tureattheST36acupointenhancedthelevelofIFN-γinthespleen,asshownbyimmunohis-
tochemicalanalysis(Fig2D).
TheeffectofelectroacupunctureappliedtotheST36acupointontheIL-
2andIL-17levels
BecauseIFN-γ,IL-2andIL-17arethecytokinesreleasedbyhelperTcells,weexamined
whetherelectroacupunctureattheST36acupointenhancedT-cellcytokineproduction.First,
theserumandthetissueextractwereprepared,andtheexpressionleveloftheendogenous
cytokineswasexamined.AsshowninFig3Aand3B,theIL-2andIL-17levelsintheserumof
theST36acupointgroupweresignificantlyhigherthanthoseofthenon-acupointandcontrol
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig3.EffectofelectroacupunctureatST36acupointonIL-2andIL-17levelsofSDrats.(AandB)IL-2
andIL-17levelofserumincreasedinratstreatedbyelectroacupunctureattheST36acupoint.*P<0.05for
ST36acupointgroupversuscontrolgroupandnonacupointgroup.(CandD)TheexpressionlevelsofIL-2
andIL-17inextractsfromspleencellswereanalyzedviawesternblot.GAPDHwasusedascontrol.
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groups.However,therewasnodifferencebetweentheacupuncturedabdominalmuscleand
thoseofthecontrolrats.
TodeterminewhethertheIL-2andIL-17levelswerealsoincreasedintheextractsfromthe
spleen,westernblotanalysiswasused.AsshowninFig3Cand3D,theincreasesintheIL-2
andIL-17levelswereaccompaniedbyelectroacupunctureattheST36acupoint.Thesefind-
ingsindicatedthattheelectroacupunctureappliedtotheST36acupointactivatedhelperT
cellsandenhancedthereleaseofT-cellcytokines.
ElectroacupunctureappliedtotheST36acupointenhancedsplenic
CD4+cells
ItisknownthatCD4moleculesmainlydistributeonthesurfaceofThelpercellsubsets.To
investigatetheeffectofelectroacupunctureonhelperTcells,immunohistochemicalanalysis
wasused.Fig4Aand4BshowthattheelectroacupunctureappliedtotheST36acupointsignif-
icantlyincreasedtheproportionofTcellswiththeexpressionofCD4.Thisresultmaysuggest
thattheenhancementofthedifferentiationandproliferationofsplenicCD4+Tcellswas
inducedbyelectroacupuncturestimulationattheST36acupoint.
EffectofelectroacupunctureonthedistributionofCa2+
IthasbeenreportedthatahigherlevelofintracellularCa2+isimportantforIL-2receptor
expression,IL-2productionandTcellactivation[18].Weassessedwhetherthelevelofintra-
cellularCa2+waschangedbyelectroacupuncture.TheconcentrationofCa2+inthespleencells
wasdetectedviaflowcytometry.AsshowninFig5A,theintracellularCa2+leveloftheST36
acupointgroupwashigherthanthatofthenon-acupointandcontrolgroups.Becausethe
levelofintracellularCa2+increased,wehypothesizedthatthelevelofextracellularCa2+may
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig4.TheexpressionlevelofCD4inspleenwasassayedviaimmunohistochemicalanalysis.Thepositivesignalisbrownordarkbrown.
(A)TheexpressionlevelofCD4increasedinspleencellsoftheratstreatedbyelectroacupunctureattheST36acupoint.(B)Proportionsof
immunopositiveCD4+Tcells.
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decrease.Tocheckthis,wedetectedtheCa2+concentrationintheserumbyusingacolorimet-
ricmethod.TheresultsshowedthattheCa2+concentrationintheserumdecreasedbecauseof
theelectroacupunctureattheST36acupoint(Fig5B).Thesefindingssupporttheideathat
electroacupunctureappliedtotheST36acupointledtoCa2+influxinspleencells.
TRPV1andTRPV4locatedatthesurfaceofsplenicCD4+Tcells
TRPVchannelsareagroupofnon-selectivecationchannelsinTcells.Apreviousstudydem-
onstratedthatTRPV1andTRPV4belongtotheTRPVsubfamilyandareexpressedendoge-
nouslyinhumanandmiceTcells.TheactivationofTRPV1andTRPV4leadstoaCa2+influx
inpurifiedmurineTcells[14].IthasalsobeenreportedthatelectroacupunctureattheST36
acupointhasaneffectonTRPV1andTRPV4[19,20].Toprobethecorrelationbetweenthe
CD4andTRPVchannels,wedetectedtheexpressionofTRPV1andTRPV4inCD4+Tcells
viaimmunofluorescence.WefoundthatTRPV1andTRPV4co-localizedwithCD4atthe
membraneofsplenicCD4+Tcells(Fig6Aand6B).Thisfindingsuggestedthatelectroacu-
punctureattheST36acupointledtoaCa2+influxinsplenicTcellsviaTRPVchannels.
Fig5.TheconcentrationofCa2+inspleencellsandserum.(A)Thevaluesmentionedintheuppercornerofeachflowcytometricdot-plot
indicatetheconcentrationofCa2+inspleencells.Representativedot-plotsofthreeindependentexperimentsareshown.Theconcentrationof
Ca2+increasedinspleencellsoftheratstreatedbyelectroacupunctureattheST36acupoint.(B)TheconcentrationofCa2+inserumoftheratsin
eachgroupwasdetected.ElectroacupunctureattheST36acupointcausedthedecreasingofCa2+contentinserum.*P<0.05forST36acupoint
groupversuscontrolgroupandnonacupointgroup.
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig6.CD4andTRPVchannelsco-localizedatTcellmembrane.(A)Confocalimagesdemonstratedthat
CD4andTRPV1co-localizedinTcellmembrane.CellswereimmunostainedwithspecificantibodiesforCD4
andTRPV1.(B)CD4andTRPV4co-localizedatTcellmembrane.
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EffctofelectroacupunctureatST36acupointonTRPV1-/-mice
InordertocheckiftheTRPVisnecessaryforelectroacupunctureenhancedimmunefunction,
electroacupunctureatST36acupointwasappliedtoTRPV1-/-mice.Theresultsshowedthat
theexpressionlevelofIFN-γhadnoobviouschangeinserumandspleenofTRPV1-/-mice
withtheelectroacupunctureatST36acupoint(Fig7A,7Band7C).WithoutTRPV1,thereis
nosignificantincreaseintheproportionofTcellswiththeexpressionofCD4underelectroa-
cupuncturetreatment(Fig7D).WealsofoundthattheconcentrationofCa2+inthespleen
cellsoftheST36acupointgroupwithTRPV1knockoutdidn’tincreasesignificantly(Fig7E).
TheseresultssuggestedthatelectroacupunctureatST36acupointhadlittleeffectonTRPV1
-/-mice.
Discussion
InChina,acupuncture,especiallyattheST36acupoint,hasalonghistoryofuseasasupple-
mentarytherapyforthetreatmentofacuteandchronicdiseases.Althoughthedetailedmecha-
nismsunderlyingelectroacupunctureneedfurtherstudy,severalstudieshaveindicatedthat
localmolecularandcellularchangesoccuratandaroundthelocationoftheacupoint.Some
mechanismshavebeenproposedtoexplaintheeffectsofelectroacupuncture,including1)the
extracellularsignal-regulatedkinase(ERK)pathway[20,21],2)theregulationofthenervous
controlsystem(neurotransmitterregulation)[22,23],and3)anincreaseinfunctionofthe
immunesystem[8,15,16].Inaddition,ithasbeensuggestedthatthemorphologicalchanges
inducedbyacupunctureinfibroblastsandconnectivetissueareinvolvedinthetherapeutic
effectofacupuncture[24,25].However,theexactmechanismsbywhichacupuncturefavor-
ablyincreasesthefunctionofimmunesystemarenotwellknown.Ourpresentstudyclearly
showedthatelectroacupuncturestimulationatthebilateralST36acupointonceaday(30min)
for3dayssignificantlyenhancedthelevelsofIFN-γ,IL-2andIL-17(Figs2and3).Italso
showedthatelectroacupunctureappliedtotheST36acupointincreasedthenumberofCD4+
Tcellsinthespleencomparedtothoseobservedinthenon-acupointandcontrolgroups
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
Fig7.ElectroacupunctureatST36acupointhadlittleeffectonTRPV1-/-mice.(A)TheIFN-γlevelsinserumofTRPV1-/-miceweredetected.
(B)TheexpressionofTRPV1inspleenwasdetected.(C)TheexpressionlevelsofIFN-γinspleenweredetectedviawesternblot.GAPDHwasused
ascontrol.(D)ProportionsofimmunopositiveCD4+TcellsinspleenofTRPV1-/-mice.(E)TheconcentrationofCa2+inspleencellsofTRPV1-/-
mice.Thevaluesmentionedintheuppercornerofeachflowcytometricdot-plotindicatetheconcentrationofCa2+inspleencells.Representativedot-
plotsofthreeindependentexperimentsareshown.TheconcentrationofCa2+hadnosignificantincreaseinspleencellsoftheTRPV1-/-micetreated
byelectroacupunctureattheST36acupoint.
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(Fig4Aand4B).Theseresultsindicatedasignificantlypositivecorrelationbetweenthelevels
ofimmunecytokinesandCD4+Tcells.
CD4isamarkerforhelperTcells.IL-2isprimarilyreleasedbyactivatedhelperTcells[26].
Intheimmunesystem,IL-2servesasanimportantfactorintheup-regulationofnaturalkiller
(NK)activity.IFN-γcanbereleasedbybothhelperTcellsandIL-2-activatedNKcells[27–
29].IL-17isreleasedbyIL-17-producingCD4+Tcells(Th17cells).Asagrowthfactorfor
botheffectorandregulatoryTcells,IL-2hasbeenshowntoenhancetheindirectrelationship
thatexistsbetweenT andTh17cells.ApreviousstudydemonstratedthatT canbecon-
REG REG
vertedintoIL-17-expressingCD4+TcellsinthepresenceofexogenousIL-2[30].Considering
thesereportsandthepresentresults,itispossiblethattheenhancementofIFN-γ,IL-2andIL-
17byelectroacupunctureattheST36acupointisdue,inpart,tothedifferentiationandactiva-
tionofCD4+Tcells.
ItisgenerallyacceptedthatCa2+signalingisimportantinthecontextofTcellactivation
anddifferentiation.TheinvolvementofCa2+intheregulationoftheimmunesystemhasbeen
demonstratedviathemodulationofextracellularandintracellularCa2+concentrationsusing
severalCa2+chelatingagents.IthasbeenreportedthatEGTA,asachelatorofextracellular
Ca2+,inhibitstheriseincytoplasmicCa2+,IL-2productionandthefurtherproliferationof
naiveTcellsinresponsetoactivationstimuli[31].Additionally,Tcellactivationmediatedby
concanavalinA(ConA)wassustainedforalongertimebecauseoftheimmediateriseinintra-
cellularCa2+.Similarly,theexpressionofTcellantigenreceptorβ(TCR-β)isregulatedbythe
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ElectroacupuctureatST36acupointcouldenhanceimmunefunction
depletionofintracellularCa2+[18].Consideringthesereports,weassessedthelevelofintracel-
lularCa2+inspleencells.TheresultsshowedthatthelevelofintracellularCa2+increased
becauseofelectroacupunctureattheST36acupoint(Fig5A).Atthesametime,adecreasein
theCa2+concentrationintheserumwasobserved(Fig5B).Theseresultsprovedthattherise
intheintracellularCa2+inducedbyelectroacupunctureattheST36acupointwasresponsible
forthedifferentiation,proliferationandactivationofsplenicCD4+Tcells.
TRPV1andTRPV4arenon-selectiveCa2+ionchannelsbelongingtotheTRPVsubfamily.
TheyareendogenouslyexpressedandfunctioninthesplenicTcellsofmice[14].Thefunc-
tionalroleofTRPV1andTRPV4inthecontextofTcellactivationandeffectorfunctionshas
beendescribedinapreviousstudy.Inthisstudy,itwasfoundthatCa2+influxwasinduced
byTRPV1andTRPV4activationinpurifiedmurineTcells.IfTRPV1andTRPV4were
inhibitedbyspecificinhibitors,theConA-drivenmitogenicactivationofTcellsdecreasedto
significantlevels.TcellactivationwasalmostabolishedbythecombinationofTRPV1and
TRPV4inhibitors.ThisfindingindicatesthatTRPV1andTRPV4playanimportantrolein
Tcellactivation.Italsohasbeenreportedthattheinhibitionofthesechannelshadastrong
inhibitoryeffectonthereleaseofcytokines,suchasTNF,IL-2andIFN-γ[14].FromFig6A
and6B,wecanseeTRPV1andTRPV4wereexpressedandco-localizedwithCD4atthe
membraneofsplenicTcells.Inpreviousstudy,ithasbeenreportedthatelectroacupunture
atST36acupointhadeffectonTRPV1[32].Sowecheckedtheeffectofelectroacupuncture
treatmentonTRPV1-/-mice.OurresultsshowedthatelectroacupunctureatST36acupoint
hadlittleeffectonTRPV1-/-mice(Fig7).Accordingtotheseresults,weproposethatthe
activationofTRPVchannelsbyelectroacupunctureattheST36acupointareresponsiblefor
theCa2+influxinspleencells.
Asacriticalcomponentoftheadaptiveimmunesystem,Tlymphocytesmediateprotection
againstinfectionandmalignancybutarealsoinvolvedinmanyimmunepathologies.T-cell
developmentplaysakeyroleinregulatingadaptiveimmuneresponses.Itisalsousedasa
modelsystemforstudyingcelldifferentiation.T-celldevelopmenthasbeenofinterestto
immunologistsforalongtime.Theeffectofacupunctureonimmunefunctionandthetreat-
mentofacuteandchronicdiseaseshasbeendemonstratedbyclinicalandexperiment
research.OurfindingsindicatedthatelectroacupunctureattheST36acupointenhance
immunefunctionthroughtheactivationsplenicTcells.
Inconclusion,thepresentstudyhasdemonstratedthatelectroacupunctureattheST36acu-
pointwasabletoregulatetheproductionofimmunecytokines(IFN-γ,IL-2andIL-17)and
thedifferentiationandactivationofsplenicTcells,whichwasmediatedbytheregulationof
extracellularandintracellularCa2+concentrations.OurresultsalsosuggestedthatCa2+influx
inspleencellsinducedbyelectroacupunctureattheST36acupointmightbemediatedby
TRPVchannels.Furtherresearchonthemechanismofhowelectroacupunctureenhances
immunefunctioniscurrentlybeingcarriedout.
Supportinginformation
S1Table.SpecificcriteriaformonitoringSDrats.Wetooknotesonfoodintake,water
intakeandbodyweightofSDratsinaweektomonitoranimalhealth.Electroacupuncture
treatmentwasappliedfromday5today7.
(XLSX)
S2Table.SpecificcriteriaformonitoringTRPV1knockoutmice.Wetooknotesonfood
intake,waterintakeandbodyweightofTRPV1knockoutmiceinaweektomonitoranimal
health.Electroacupuncturetreatmentwasappliedfromday5today7.
(XLSX)
PLOSONE|https://doi.org/10.1371/journal.pone.0175568 April13,2017 10/13