Electroacupuncture at Guanyuan (CV 4), Zusanli

18 Pages·2017·2.75 MB·English

RESEARCHARTICLE
Electroacupuncture at Guanyuan (CV 4),
Zusanli (ST 36) and Baihui (DU 20) regulate the
aging-related changes in gene expression
profile of the hippocampus in sub-acutely
aging rats
JianminLiu1,2☯,JingLiu1,2☯,Guang’anWang3,GuangyaLiu1,2,HuanjiaoZhou1,YunFan1,
FengxiaLiang1,2,HuaWang1,2*
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a1111111111 1 Acupuncture-moxibustionandOrthopedicCollege,HubeiUniversityofChineseMedicine,Wuhan,China,
2 HubeiProvincialCollaborativeInnovationCenterofPreventiveTreatmentbyAcupuncture&Moxibustion,
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Wuhan,China,3 TheThirdClinicalMedicineCollege,HenanUniversityofChineseMedicine,Zhengzhou,
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China
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☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS Abstract
Citation:LiuJ,LiuJ,WangG,LiuG,ZhouH,Fan
Y,etal.(2018)ElectroacupunctureatGuanyuan Toinvestigatethemolecularmechanismsofsub-acutelyaginganddemonstratetheeffect
(CV4),Zusanli(ST36)andBaihui(DU20)regulate ofelectroacupuncture(EA)attheGuanyuan(CV4),Zusanli(ST36)andBaihui(DU20)
theaging-relatedchangesingeneexpression
acupointonthesub-acutelyagingbrain,cDNAmicroarraysandbioinformaticsanalyses
profileofthehippocampusinsub-acutelyaging
werecarriedout.ThirtySprague-Dawley(SD)maleratswereselectedandrandomly
rats.PLoSONE13(1):e0191623.https://doi.org/
10.1371/journal.pone.0191623 dividedintothreegroups:thecontrolgroup(C),thesub-acutelyagingmodelgroup(M)and
theelectroacupuncturegroup(M+EA).Sub-acutelyagingmodelratswereobtainedbyD-
Editor:CristoforoScavone,UniversidadedeSao
Paulo,BRAZIL galactoses.c.injectioncontinuouslyfor40days.TotalRNAwasextractedfromthehippo-
campusareaofbrainsinthreegroupsforcDNAmicroarrays.Thedataofdifferentgroups
Received:November8,2016
werecomparedandanalyzedbydifferentialexpressionanalysis,Geneontology(GO)term
Accepted:December14,2017
enrichment,KyotoEncyclopediaofGenesGenomes(KEGG)pathwayenrichmentand
Published:January19,2018
quantitativereal-timePCR.Accordingtotheresults,4052DEgeneswereidentifiedinour
Copyright:©2018Liuetal.Thisisanopenaccess study.Amongthem,therewere3079differentiallyexpressed(DE)genesbetweengroupM
articledistributedunderthetermsoftheCreative
andgroupC,andthesegenesareassociatedwiththeagingofrats.Moreover,983genes
CommonsAttributionLicense,whichpermits
wereexpresseddifferentlyingroupM+EAcomparedwithgroupM,revealingthatpointssti-
unrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalauthorand mulicouldregulategeneexpressioninbrainwithaging.Geneontology(GO)termenrich-
sourcearecredited. mentandKEGGenrichmentwereperformedtofurtherclassifythedifferentialexpression
DataAvailabilityStatement:Allrelevantdataare genes.ImportantGOtermsandKEGGpathwaysconnectedwithsub-acutelyagingEA
withinthepaperanditsSupportingInformation effectswereidentified.Atlast,3significantdifferentiallyexpressedgeneswereselectedfor
files.
real-timequantitativePCRtoclarifythecDNAmicroarrayresults.Inconclusion,thecDNA
Funding:Thisworkwassupportedby2016 microarraydatafirstcomparedandanalyzedthedifferencesofgeneexpressionprofilein
scientificresearchguidanceprojectsofHubei
thehippocampusofratsindifferentgroups,whichcontributetoourknowledgeonthe
province(No.B2016111).Thefundershadnorole
molecularmechanismsofEAtowardssub-acutelyaging.
instudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthe
manuscript.
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Effectofelectroacupunctureonsub-acutelyagingbrain
Competinginterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Inrecentyears,thenumberofolderpersonsisincreasingrapidly.Underthiscircumstance,
theproblemofagingisdrawingmoreandmoresocialattention.Agingisaccompaniedby
cognitivedeclineinamajorsegmentofthepopulationandistheprimaryriskfactorforAlz-
heimer’sdiseaseandotherprevalentneurodegenerativedisorders.Aspeoplebecomeolder,
theage-relatedchangeswilloccur,suchaschangesinthefunctionsandcompositionofthe
humanbody[1,2].Coupledwithphysiologicchanges,therearealsomedicalconditionsthat
risemorecommonlywithadvancingage.Thesechangesandconditionsincreaseanolder
adult’svulnerabilitytoandinjuriesfromabuseorneglect[3].Accordingtoformalresearch,
agingisassociatedwithdeteriorationoftheimmunesystem(immunosenescence),an
increasedsusceptibilitytoinfection,autoimmunediseaseandcancerandreducedresponsive-
nesstovaccination[4,5].Akeyfeatureoftheagedhumanimmunesystemistheaccumulation
ofhighlydifferentiatedCD8(+)CD28(-)Tcells,aphenomenonthatnegativelyinfluences
immunefunctionintheelderly.Asaresult,themechanismsthatregulatesurvivalordeathof
CD8(+)CD28(-)Tcellshasbecomethefocusofmanyresearchers[6].Tocombatimmunose-
nescence,alotofstrategiesareemerging,focusingoncellularandgenetictherapies,which
includebonemarrowtransplantationandgeneticreprogramming[7].Exceptforimmunose-
nescence,brainagingprocessesarealsoenormouslycomplexphenomenathatcaninclude
cognitivedeclineandincreasetheriskofAlzheimer’sdisease(AD)[8].Therearedifferent
kindsofmethodstocombatage-relatedbraindiseasesordelaybrainaging,includingdrug
therapyandnon-drugtherapy[9].Forinstance,Mannosylatedliposomalflavonoidisusefulin
combatingage-ralatedischemia-reperfusioninducedoxidativedamageinratbrain[10].
Moreover,Caloricrestriction(CR)andtransgenicoverexpressionofendogenousantioxidants
therapywereproventodelayorinhibitanumberofage-associatedpathologicandbiologic
changesinthebrain,thustohavelife-extendingfunction[11,12].
Acupunctureisatraditionaltherapyappliedforthousandsofyears,anditwasalsoapower-
fulnon-drugtherapywhichisusedextensivelyinOrientalMedicineandhasemergedasan
importantmodalityofcomplementaryandalternativetherapytoWesternMedicine[13].In
thetheoryoftraditionalChinesemedicine,itisproposedthatacupuncturecanstrengthenthe
humanbodytoresistdiseasesbypuncturingneedlesatcertainpoints[14].Ithasalsobeen
provedthatacupuncturetheorycouldapplytoratsandthattheresultsofacupunctureexperi-
mentsbasedonratsagreewiththeresultsbasedonhuman.Untilnow,rats’modelshavebeen
appliedtotheacupunctureresearchofvariouskindsofdiseases,suchashypertension,Alzhei-
merdisease,diabetes,aging,andhaveachievedgreatsuccess[15].Forexample,previous
researchdemonstratedthatacupunctureat“Taichong”(LR3)hasimmediateeffectonpatients
withhypertensionof1or2degree,andtheloweringextentispositivelyrelatedwiththeblood
pressurebeforeacupuncture[16].Thenotherresearchgroupsprovedthatmoderate-stimula-
tionof“Taichong”(LR3)canlowerbloodpressureandplasmaEA-1levelinhypertension
rats.Sothespontaneouslyhypertensiverats(SHR)modelwasusedbymanyresearchersto
seekabetteracupunctureparameterforclinicaltreatmentofhypertension[17,18].Double
reinforcing-oneunblockingacupuncturewasprovedtohaveasignificantlyeffectonsenile
immunologicfunction[19].Otherresearchesdemonstratedthatdoublereinforcing-one
unblockingacupuncturecouldalsoimprovethespatiallearningandmemoryinagingmodel
rats[20,21].
Sofar,thefunctionofacupunctureinregulatingtheimmunesystemhasbeenrevealedin
manyresearches.Forinstance,acupuncturecanenhanceanticancerandantistressimmune
functionandexertantiinflammationeffects.TheacupuncturepointST36(Zusanli)isan
importantacupointwhichiswidelyappliedinimmune-relateddiseases.M.Zhuetal.reported
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Effectofelectroacupunctureonsub-acutelyagingbrain
thatEApreconditioningatST36obviouslyamelioratedCLP-inducedintestinalinjuryand
highpermeabilityandreducedthemortalityofCLP-inducedsepsisratsthroughincreasing
theconcentrationofsIgAandthepercentageofCD3+,γ/δ,andCD4+Tcellsandtheratioof
CD4+/CD8+Tcells[22].Anotherresearchgroupdiscoveredanovelcholinergicanti-inflam-
matorypathwayactivatedbyacupunctureaswellasachemokine-mediatedproliferationof
opioid-containingmacrophagesininflamedtissuesinresponsetoacupuncture[23].Acu-
puncturewasprovedtoplayaroleindelayingbrainagingandtreatingage-relatedbraindis-
eases.“YiQi-TiaoXue, FuBenPeiYuan” acupuncturemethodhasbeenprovedtoimprove
cognitionabilityofdementiarat[24].Moreover,acupuncturecaninducedifferentcellprolif-
erationindifferentbrainregionsofSAMP8,whichbringsforththeneedtoexplorefurtherfor
themechanismofcognitivedeficitsandacupunctureinterventioninthisfield[25].
BasedonalongclinicexperienceandstudyingofthetraditionalChinesemedicinetheory,
theelectroacupuncturetreatmentwhichinsertpointsatGuanyuan(CV4),Zusanli(ST36)
andBaihui(DU20)withneedle,alsonamed“ShuangguYitong”(double-reinforcingandone-
unblocking)acupuncturetherapyhasbeenfoundthespecialeffecttoregulatetheimmunesys-
temforrats[26–28].Somemechanismsofhowthe“ShuangguYitong”electroacupuncture
therapyregulatestheimmunosenescencehavebeenstudiedinourpreviousresearch.Itturns
outthatEAatGuanyuan(CV4),Zusanli(ST36)andBaihui(DU20)coulddelayagingbyreg-
ulatingTcellsinproliferation,secretionofIL-2anditsreceptorandimprovetheexpressionof
CD8+,CD28+[22,23].FurtherstudyrevealsthatEAatGuanyuan(CV4),Zusanli(ST36)and
Baihui(DU20)coulddecreasethecontentofIL-1βandIL-6ofserum[28].Basedontheprevi-
ousresearchesandinordertogainabetterunderstandingofaging-relatedchangesandhow
“ShuangguYitong”EAaffectsthebrainsofsub-acutelyagingrats,DNAmicroarrayanalysis
wasexplored.Themicroarraydatademonstratedifferencesbetweencontrolgroup(C),the
electroacupuncturegroup(M+EA)andthesub-acutelyagingmodelgroup(M).Thediffer-
encesexistedingeneexpressionlevel,GOcategoriesandpathwaycategories,whichcontribute
toourknowledgeontheeffectofEAtothebrainofsub-acutelyagingratsinthemolecular
level.Importantgenesrelatedwithsub-acutelyagingandEAeffectswereselectedforfurther
study.
Materialsandmethods
Animaltreatment
ThirtySDmaleratsin3-month-oldwereobtainedfromtheAnimalExperimentalCenterof
HebeiMedicalUniversity,China[Experimentalanimalproductionlicensenumber:SCXK
(Hebei)2008-1-003].Alltheratswereraisedintheindividuallyventilatedcageswiththetem-
peraturebetween20˚Cto25˚C.Thehumiditywasbetween45%and55%.Lightwasprovided
from8amto8pmtosimulatethecircadianrhythmswhilefoodandwaterwasofferedsuffi-
ciently.AllanimaltreatmentswereapprovedbytheAnimalEthicsCommitteeoftheHubei
UniversityofChineseMedicine,No.[2016]IEC(010).
After1weekofadaptation,theratswererandomlydividedintothreegroupsasfollowing:
thecontrolgroup(C),thesub-acutelyagingmodelgroup(M)andtheelectroacupuncture
group(M+EA),10ratsforeachgroup.RatsingroupCwereraisednormallywithoutanytreat-
ments.Whilethesub-acutelyagingmodelratswereobtainedbyD-galactoses.c.injectioncon-
tinuouslyfor40dayssincethesecondweek.TheconcentrationofD-galactoses.c.was350mg.
kg-1.d-1,onetimeforeachday[29].RatsingroupMwereraisedwithoutanytreatmentsafter
modeling.WhiletheRatsofM+EAgroupstartedEAtreatmentatGuanyuan(CV4),Zusanli
(ST36)andBaihui(DU20)onceadayaftermodelingfor27days(theproceduresareshownas
following).TheprocessoftheexperimentwasshowninS1Fig.Thebodytemperature,tongue
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Effectofelectroacupunctureonsub-acutelyagingbrain
temperatureandtheweightofratsindifferentgroupsweremeasuredeverythreedaysduring
themodelmakingandelectroacupuntureprocedurestomakesurethatthephysiologicalindi-
catorsofratsarenormal.Alleffortsweremadetominimizesuffering.
Electroacupuncture(EA)treatment
Sterileacupunctureneedles[size:φ0.30(cid:3)25mm(diameter:0.30millimeter,length:25millime-
ter);madebySuzhouAcupuncture&MoxibustionApplianceCo.,Lid,Suzhou,P.R.China]
andthepulsegenerator(6805-II,ShanghaiTaichengtechnologydevelopmentco.,LTD)were
usedduringtheEAtreatment.Needleswereinsertedperpendicularlyintothemusclelayerat
Guanyuan(CV4),Zusanli(ST36)andinsertedhorizontallyintosubgalealtissueatthepoint
ofBaihui(DU20)toadepthof2mm.TheGuanyuan(CV4)acupointwasconnectedtothe
negativechargeofthepulsegeneratorandtheZusanli(thebilateralZusanliwereusedalter-
natelyintheEAtreatment)wasconnectedtothepositivecharge(continuouswave:2Hz,1
mA,lasted15min).Baihui(DU20)wasstimulatedwithneedlesatthesametime:Afterthe
needlehasreacheditsdesiredlocation,totwirlandrotatetheneedlebackwardandforward
continuouslywiththefrequency2–3timespersecondfor30seconds.Thesamemanipulation
wasdoneafterevery5minutesforthreetimesuntilwithdrawingtheneedle.TheEAtreatment
wasperformedeverydayexceptSundayandlastedfor27days.Thiselectroacupuncture(EA)
treatmentprotocalhasbeendepositedintheprotocols.io.Thedigitalobjectidentifier(DOI)
linkis:http://dx.doi.org/10.17504/protocols.io.ky8cxzw.
Tissuesampling
AttheendoftheEAtreatment,ratsfromthreegroupswerefastedovernight.Thenalltherats
werekilledbydislocationofcervicalvertebraandthebrainswerequicklyexcisedafterintra-
peritonealanesthesiausingpentobarbitalnatrium.Thecollectedsampleswerewashedbythe
coldnormalsaline.Subsequently,thehippocampusareawasdivided,frozenintheliquid
nitrogenandthenkeptunder-80˚CforRNAextraction.Thehippocampusareaoftenratsin
eachgroupmakesuponesample,andthenthethreesamplesfromgroupC,groupMand
groupM+EAwerepreparedfortheRNAextraction.
RNApreparationandquantitativereal-timePCR(q-PCR)
RNAisoPlus(TaKaRaBiotech.Co.,Dalian,China)wasusedtoextractthetotalRNAaccording
tothemanufacturer’sprotocol.AllRNAsamplesweretreatedwithDNaseI(TaKaRaBiotech.
Co.,Dalian,China)andfrozenat−80˚CbeforeDNAmicroarrayexperiment.RNAquantityand
puritywasassessedusingNanoDropND-1000(Passcriteriaforabsorbanceratiosareestablished
atA260/A280(cid:21)1.8andA260/A230(cid:21)1.6).RINvaluesareascertainedusingAgilentRNA6000
NanoassaytodetermineRNAintegrity(PasscriteriaforRINvalueisestablishedat(cid:21)7indicating
acceptableRNAintegrity).gDNAcontaminationwasevaluatedbygelelectrophoresis.
First-strandcDNAwaspreparedbyAll-in-oneFirststrandcDNASynthesisKit(Geneco-
poeia,Guangzhou,China)followingthemanufacturer’sprotocol.ThentheBIO-RADCFX96
q-PCRsystem(SYBRGreenIfluorescentdyedetection)wasusedtoperformtheqRT-PCR.
ThemRNAabundancewasnormalizedwiththehousekeepinggeneβ-actin,andtherelative
expressionlevelswerecalculatedusingthe2-ΔΔCtmethod[30].
Microarray
Microarrayanalysiswascarriedouttoinvestigategeneexpressionpatternsinthehippocam-
pusofratsindifferentgroups.6-OHDA-targetedtranscriptswereanalyzedwithWholeRat
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Effectofelectroacupunctureonsub-acutelyagingbrain
Genome4×44Kmicroarrays(Geneexpressionhybridizationkit,Agilent)accordingtothe
manufacturer’sinstruction.RosettaResolverSystemR(RosettaBiosoftware)wasusedtopro-
cessdataanalysis:1.Rosettaprofileerrormodelcalculation:theerrorduetorandomfactors
andsystematicbiasesareestimatedbyRosettaerrormodelwhichcancapturethepredictable
behaviorofthevarianceinmicroarraymeasurement[31];2.Squeezereplicatedprobes:the
repeatedprobeswithinonechipareaveraged;3.Normalizeintensities:Medianscalingper-
formedondatasetwithoutflaggedandcontroldata;4.Pearson’scorrelationcoefficient:statis-
ticalanalysiscalculatedonthreetechnicalreplicatestoassessreproducibility;Thereplicated
analysisoftheRNAfromthesamesamplecandecreasethetechnicalvariationandthefalse
positive,makingtheresultsmoreaccurately.5.Mergetechnicalreplicatedata:Averageinten-
sityvaluescalculatedontechnicalreplicates;6.Pairwiseratiocalculation:Probefiltering,nor-
malization,pair-wisecomparisonanderror-weightedmodelingareperformedbasedon
customers’designatedsamplegroups;7.Differentiallyexpressedgenelists:Standardselection
criteriatoidentifydifferentiallyexpressedgenesareestablishedat|Foldchange|(cid:21)1and
P<0.05.Student’st-test(two-tailed)wasusedfordataanalysisthisstudy.8.Theheatmap
wasobtainedusingthesoftwareHemI[32],whiletheVenndiagramwasobtainedusing
VENNY[33].WeappliedKOBASsoftwaretotestthestatisticalenrichmentofdifferential
expressiongenesinKEGGpathways.
Results
RNApreparation
TotalRNAwasextractedfromthehippocampusofratsindifferentgroupsandtreatedwith
DNaseI.TheabsorbanceratiosofA260/A280ingroupC,MandM+EAwere1.99,1.99and
1.88,respectively.WhiletheabsorbanceratiosofA260/A230were1.98,2.16and1.96.RINval-
uesinthreegroupswereallabovethepasscriteria(8.5,9.0and8.3respectively)(Table1).The
resultsindicatingthattheRNApurityandintegrityaresuitableforcDNAmicroarray
experiment.
Identificationofdifferentiallyexpressedgenesbetweendifferentgroups
Todemonstratethemolecularmechanismsofthesub-acutelyagingandEAeffects,thediffer-
entiallyexpressed(DE)genesbetweendifferentgroupswereidentifiedandanalyzed.Firstly,
theheatmapwasmadetocomparetheexpressionpatternsofDEgenesinthreegroups.There
are4052DEgenesidentifiedinourstudy(S1Table).AscanbeseeninFig1,thegeneexpres-
sionpatternofgroupMwasquitedifferentfromthatofgroupC,revealingthatthesub-acutely
agingwasassociatedwiththeexpressionchangingofalargesumofgenesinthehippocampus.
Meanwhile,theexpressionlevelofDEgenesingroupM+EAwasdifferentfromthatingroup
M,whichsuggestedthatEAhaschangedthegeneexpressionpatternofbraininsub-acutely
agingrats.
Accordingtotheresults,therewere3079DEgenesbetweengroupMandgroupC,among
them1750geneswereup-regulatedand1329weredown-regulated.Thesegenesarerelated
withtheagingofbraininrats.WhencomparinggroupM+EAandgroupM,983DEgenes
Table1. QualitycontrolofRNAsampleinthreegroups.
Sample A260/A280 A260/A230 RIN Conc(ng/ul)
GroupC 1.99 1.98 8.5 1042.7
GroupM 1.99 2.16 9.0 734.0
GroupM+EA 1.88 1.96 8.3 705.8
https://doi.org/10.1371/journal.pone.0191623.t001
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Effectofelectroacupunctureonsub-acutelyagingbrain
Fig1.Heatmapofdifferentiallyexpressedgenesofthreesamples.
https://doi.org/10.1371/journal.pone.0191623.g001
wereidentified.Thenumberofup-regulatedgeneswas620whilethenumberofdown-regu-
latedgeneswas363,asshowninTable2.Furtheranalysiswasmadetocomparethenumber
andoverlappingrelationshipsofDEgenesbetweendifferentgroups.AccordingtoFig2A,the
comparinggroupsMvs.CandM+EAvsMshared445DEgenes.2634DEgeneswerepeculiar
tocomparinggroupMvs.Cand538DEgeneswerepeculiartoM+EAvsM.Thentheshared
445DEgeneswereanalyzedtofigureoutimportantgenesrelatedwithsub-acutelyagingand
EAeffects.Bycomparingtheup-regulatedgenesbetweenMvs.CandMvs.M+EA,weidenti-
fied348overlappingDEgenes,whichmeanEAcanreversethedown-regulateofthesegenes
inthesub-acutelyaginggroup.Besides,1404DEgeneswerepeculiartoMvs.Ccomparing
groupand274werepeculiartoMvs.M+EA(Fig2B).Likewise,bycomparingthedown-regu-
latedgenesofMvs.CandMvs.M+EA,78sharedDEgeneswereidentified.1251DEgenes
werepeculiartoMvs.Cdown-regulatedgenesand285werepeculiartocomparinggroupM
vs.M+EA(Fig2C).TheresultrevealsthatEAtreatmentcanup-regulatetheexpressionof78
geneswhichweredown-regulatedingroupM.Commonelementsin“MvsC-UP”and“Mvs
M+EA-UP”andin“MvsC-DOWN”and“MvsM+EA-DOWN”wereshowninS2Table.
Functionaldistributionofdifferentiallyexpressedgenes
TheidentifieddifferentiallyexpressedgeneswerefurtheranalyzedbyGeneontology(GO)
termenrichmentandKyotoEncyclopediaofGenesGenomes(KEGG)pathwayenrichment.
ComparinggroupsMvsCandM+EAvsMwerechosenforGOandKEGGanalysis.Accord-
ingtotheGOcategories,theidentifiedDEgeneswerecategorizedintothreemajorfunctional
groups:cellularcomponent,molecularfunction,andbiologicalprocess.Incomparinggroup
MvsC,theabundantgeneswerecategorizedinto20majorfunctionalgroupsbasedonthe
GOcategories,andproteinimportintonucleustranslocation,transgolginetworktransport
vesicleandkinaseactivoteracvitityarethetopthreefunctionalcategories,ascanbeenseenin
Table3.Likewise,incomparinggroupM+EAvsM,theabundantDEgeneswerecategorized
into20majorfunctionalgroups,andfunctionalmategoriesarepositiveregulationoftrans-
port,voltagegatedpotassiumchannelcomplexandvitaminbindingarethemostabundant
(Table4).
TheKEGGpathwayenrichmentwerethenperformedtocategorytheDEgenesincompar-
inggroupMvsCandM+EAvsM.ComparedwithgroupC,5KEGGpathwaywereidentified
up-regulatedand9wereidentifieddown-regulatedingroupM.Theup-regulatedtermswere
asfollowing:rno04080:Neuroactiveligand-receptorinteraction,rno04916:Melanogenesis,
rno04270:Vascularsmoothmusclecontraction,rno04020:Calciumsignalingpathwayand
rno04912:GnRHsignalingpathway;thedown-regulatedtermswere:rno04070:Phosphatidy-
linositolsignalingsystem,rno04670:Leukocytetransendothelialmigration,rno04960:Aldo-
sterone-regulatedsodiumreabsorption,rno05223:Non-smallcelllungcancer,rno05214:
Glioma,rno05200:Pathwaysincancer,rno04662:Bcellreceptorsignalingpathwayand
Table2. ThenumberofDEgenesbetweendifferentsamples.
NO Comparison Up-regulated Down-regulated
1 Cvs.M+EA 1164 945
2 Mvs.M+EA 363 620
3 Mvs.C 1750 1329
https://doi.org/10.1371/journal.pone.0191623.t002
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Fig2.ComparisonsofthenumberandoverlappingrelationshipsofDEgenesbetweendifferentsamples.A.Purplecirclerepresentsnumber
ofDEgenesbetweengroupMandgroupC;yellowcirclestandfornumberofDEgenesbetweengroupM+EAandgroupM;B.Purplecircle
representsnumberofup-regulatedDEgenesbetweengroupMandgroupC;yellowcirclestandfornumberofup-regulatedDEgenesbetween
groupMandgroupM+EA;C.Purplecirclerepresentsnumberofdown-regulatedDEgenesbetweengroupMandgroupC;yellowcirclestandfor
numberofdown-regulatedDEgenesbetweengroupMandgroupM+EA.TheoverlappingregionrepresentssharedDEgenesoftwocomparable
groups.
https://doi.org/10.1371/journal.pone.0191623.g002
rno05222:Smallcelllungcancer(Fig3A).DEgenesinvolvedinthesepathwayswerelisted
inTable5.IncomparinggroupM+EAvsM,18KEGGpathwaytermswereup-regulated,
andthemostabundantfivetermswererno04070:Phosphatidylinositolsignalingsystem,
rno04720:Long-termpotentiation,rno04010:MAPKsignalingpathway,rno04310:Wntsig-
nalingpathwayandrno04020:Calciumsignalingpathway.ComparedwithgroupM,5KEGG
pathwaytermsweredown-regulatedingroupM+EA,includingrno04080:Neuroactive
ligand-receptorinteraction,rno04270:Vascularsmoothmusclecontraction,rno04916:Mela-
nogenesis,rno04020:Calciumsignalingpathwayandrno00350:Tyrosinemetabolism,as
showninFig3BandTable5.Notably,whilethegeneexpressioninphosphatidylinositolsig-
nalingsystempathwaywasdown-regulatedingroupMcomparedwithgroupC,itwasup-reg-
ulatedaftertheEAtreatment.Besides,whileKEGGpathwaysneuroactiveligand-receptor
interaction,vascularsmoothmusclecontraction,melanogenesisandcalciumsignalingpath-
waywereup-regulatedinthesub-acutelymodelgroup,theywerealldown-regulatedafterthe
EAtreatment.TheresultsrevealthatEAtreatmentcanaffecttheexpressionofgenesinthese
KEGGpathways,andreversingthegeneexpressionchangesinthesepathwayscanberegarded
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Effectofelectroacupunctureonsub-acutelyagingbrain
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Man NSD AL_P
A M
Table3.ThemostenrichedGOtermsbetweengroup BiologicalProcess PROTEIN_IMPORT_INTO_NUCLEUS_TRANSLOCATION INTERACTION_WITH_HOST HEMOSTASIS ACUTE_INFLAMMATORY_RESPONSE REGULATION_OF_RAS_PROTEIN_SIGNAL_TRANSDUCTION CDC42_PROTEIN_SIGNAL_TRANSDUCTION NLS_BEARING_SUBSTRATE_IMPORT_INTO_NUCLEUS SMOOTH_MUSCLE_CONTRACTION_GO_0006939 BLOOD_COAGULATION COAGULATION WOUND_HEALING PROTEIN_IMPORT_INTO_NUCLEUS REGULATION_OF_SMALL_GTPASE_MEDIATED_SIGNAL_TR NUCLEAR_IMPORT REGULATION_OF_BODY_FLUID_LEVELS POSITIVE_REGULATION_OF_TRANSPORT NEGATIVE_REGULATION_OF_MULTICELLULAR_ORGANIS NEGATIVE_REGULATION_OF_CELL_CYCLE FEEDING_BEHAVIOR MITOTIC_SISTER_CHROMATID_SEGREGATION https://doi.org/10.1371/journal.pone.0191623.t003
PLOSONE|https://doi.org/10.1371/journal.pone.0191623 January19,2018 9/18
Effectofelectroacupunctureonsub-acutelyagingbrain
Y
T
VI
TI
C
A
MolecularFunction VITAMIN_BINDING AUXILIARY_TRANSPORT_PROTEIN_ACTIVITY VOLTAGE_GATED_POTASSIUM_CHANNEL_ACTIVITY METABOTROPIC_GLUTAMATEGABA_B_LIKE_RECEPTOR_ POTASSIUM_CHANNEL_ACTIVITY GUANYL_NUCLEOTIDE_EXCHANGE_FACTOR_ACTIVITY INWARD_RECTIFIER_POTASSIUM_CHANNEL_ACTIVITY KINASE_ACTIVATOR_ACTIVITY LOW_DENSITY_LIPOPROTEIN_BINDING NEUROPEPTIDE_HORMONE_ACTIVITY SMAD_BINDING PHOSPHOLIPASE_A2_ACTIVITY POTASSIUM_CHANNEL_REGULATOR_ACTIVITY SOLUTE_SODIUM_SYMPORTER_ACTIVITY RAS_GTPASE_BINDING COPPER_ION_BINDING PEPTIDE_RECEPTOR_ACTIVITY LIPID_TRANSPORTER_ACTIVITY SERINE_TYPE_ENDOPEPTIDASE_ACTIVITY SODIUM_CHANNEL_ACTIVITY
X
E
L
MP LE
O C
CellularComponent VOLTAGE_GATED_POTASSIUM_CHANNEL_C TRANS_GOLGI_NETWORK_TRANSPORT_VESI ENDOCYTIC_VESICLE ENDOSOME EARLY_ENDOSOME VESICULAR_FRACTION COLLAGEN MICROSOME CENTROSOME GOLGI_ASSOCIATED_VESICLE TRANSPORT_VESICLE TIGHT_JUNCTION CELL_SURFACE BASOLATERAL_PLASMA_MEMBRANE MICROTUBULE_ORGANIZING_CENTER APICAL_JUNCTION_COMPLEX APICOLATERAL_PLASMA_MEMBRANE CLATHRIN_COATED_VESICLE EXTRACELLULAR_SPACE COATED_VESICLE
Y
A
W
H
T
A
P
G_
N
C. ALI
p N
u G
upMandgro AL_PROCESS A_RECEPTOR_SI NSDUCTION
Table4.ThemostenrichedGOtermsbetweengro BiologicalProcess POSITIVE_REGULATION_OF_TRANSPORT INTERACTION_WITH_HOST ACUTE_INFLAMMATORY_RESPONSE POTASSIUM_ION_TRANSPORT CELL_FATE_COMMITMENT NEGATIVE_REGULATION_OF_MULTICELLULAR_ORGANISM REGULATION_OF_TRANSFORMING_GROWTH_FACTOR_BET DIGESTION FEEDING_BEHAVIOR ORGANELLE_LOCALIZATION MONOVALENT_INORGANIC_CATION_TRANSPORT ESTABLISHMENT_OF_ORGANELLE_LOCALIZATION REGULATION_OF_RAS_PROTEIN_SIGNAL_TRANSDUCTION CELLULAR_RESPONSE_TO_NUTRIENT_LEVELS CELLULAR_RESPONSE_TO_STRESS TRIACYLGLYCEROL_METABOLIC_PROCESS COFACTOR_TRANSPORT RESPONSE_TO_EXTRACELLULAR_STIMULUS REGULATION_OF_SMALL_GTPASE_MEDIATED_SIGNAL_TRA CDC42_PROTEIN_SIGNAL_TRANSDUCTION https://doi.org/10.1371/journal.pone.0191623.t004
PLOSONE|https://doi.org/10.1371/journal.pone.0191623 January19,2018 10/18