Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via

15 Pages·2015·2.38 MB·English

RESEARCHARTICLE
Electroacupuncture Ameliorates Acute Renal
Injury in Lipopolysaccharide-Stimulated
Rabbits via Induction of HO-1 through the
PI3K/Akt/Nrf2 Pathways
Jian-boYu*☯,JiaShi☯,YuanZhang☯,Li-rongGong,Shu-anDong,Xin-shunCao,Li-liWu,
Li-naWu
DepartmentofAnesthesiology,TianjinNankaiHospital,TianjinMedicalUniversity,Tianjin,China
a11111
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
Electroacupunctureatselectacupointshavebeenverifiedtoprotectagainstorgandysfunc-
OPENACCESS
tionsduringendotoxicshock.And,hemeoxygenase(HO)-1asaphaseIIenzymeandanti-
Citation:YuJ-b,ShiJ,ZhangY,GongL-r,DongS-a,
oxidantcontributedtotheprotectionofkidneyinsepticshockrats.Thephosphatidylinositol
CaoX-s,etal.(2015)Electroacupuncture
AmelioratesAcuteRenalInjuryin 3-kinase(PI3K)-AktpathwaymediatedtheactivationofNF-E2relatedfactor-2(Nrf2),which
Lipopolysaccharide-StimulatedRabbitsviaInduction wasinvolvedinHO-1induction.Tounderstandtheefficacyofelectroacupuncturestimula-
ofHO-1throughthePI3K/Akt/Nrf2Pathways.PLoS
tioninamelioratingacutekidneyinjury(AKI)throughthePI3K/Akt/Nrf2pathwayandsubse-
ONE10(11):e0141622.doi:10.1371/journal.
quentHO-1upregulation,adoseofLPS5mg/kgwasadministeredintravenouslyto
pone.0141622
replicatetherabbitmodelofAKIinducedbyendotoxicshock.Electroacupuncturepretreat-
Editor:BenedettaBussolati,CenterforMolecular
mentwashandledbilaterallyatZusanliandNeiguanacupointsforfiveconsecutivedays
Biotechnology,ITALY
whileshamelectroacupunctureatnon-acupointsascontrol.Resultsdisplayedthatelectro-
Received:April26,2015
acupuncturestimulationsignificantlyalleviatedthemorphologicrenaldamage,attenuated
Accepted:October7,2015
renaltubularapoptosis,suppressedtheelevatedbiochemicalindicatorsofAKIcausedby
Published:November2,2015 LPS,enhancedtheexpressionsofphospho-Akt,HO-1protein,Nrf2totalandnucleoprotein,
Copyright:©2015Yuetal.Thisisanopenaccess andhighlightedtheproportionsofNrf2nucleoproteinasaparallel.Furthermore,partialpro-
articledistributedunderthetermsoftheCreative tectiveeffectsofelecroacupuncturewerecounteractedbypreconditioningwithwortmannin
CommonsAttributionLicense,whichpermits
(theselectivePI3Kinhibitor),indicatingadirectinvolvementofPI3K/Aktpathway.Inconsis-
unrestricteduse,distribution,andreproductioninany
tently,wortmanninpretreatmentmadelittledifferencetotheexpressionsofHO-1,Nrf2
medium,providedtheoriginalauthorandsourceare
credited. nucleoproteinandtotalprotein,whichindicatedthatPI3K/Aktmaybenottheonlypathway
responsibleforelectroacupuncture-affordedprotectionagainstLPS-inducedAKI.These
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. findingsprovidenewinsightsintothepotentialfutureclinicalapplicationsofelectroacupunc-
tureforAKIinducedbyendotoxicshockinsteadoftraditionalremedies.
Funding:Financialsupportforthisresearchwas
providedbygrantNo.81372096fromtheNational
NaturalScienceFoundationofChina,Beijing,China,
andgrantNo.12ZCZDSY013300fromtheKey
ProjectsintheTianjinScience&TechnologyPillar
Program,Tianjin,China.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0141622 November2,2015 1/15
PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Septicshockisthemostcommoncontributingfactortoacutekidneyinjury(AKI)incritically
patients,andevenprogressintoacuterenalfailure[1].Thecombinationofacuterenalfailure
andsepsisaccountsformorethan70%mortality,despitemultifariouspotentialinterventions
[2].Lipopolysacchride(LPS)isaprimaryinitiatorofinflammatoryandhemodynamicpertur-
bationsofsepsisandinvolvedinexperimentalAKIstudies[3].Thereisexperimentalevidence
thatendotoxin-relatedincreaseintumornecrosisfactor-α(TNF-α)andoxygenradicalsare
majorcausesofAKIduringendotoxicshock[4,5].Hemeoxygenase(HO)-1,astheratelimit-
ingenzymeofhemedegradation,wasfirstrecognizedinpreservingrenalfunctionandreduc-
ingmortality[6].OurpriorstudiesdefinitelyelucidatedtheprotectiveroleofHO-1onthe
kidneyinendotoxicshockrats[7].ThebiologicaleffectofHO-1attributedtothedegradation
ofhemeandtheproductsofenzymaticreactionincludingbiliverdinandcarbonmonoxide,
whichexertedantioxidant,antiproliferativeandalsoanti-inflammatoryproperties[8].
AcupuncturerepresentsapotentiallyvaluableadjuncttopreventclinicaldisordersinChina
andotherAsiancountriesforthepast3000years[9].Itwasproventhatelectricalstimulation
ofthevagusnervecouldmodulatesystemicinflammatoryresponsestoendotoxin,inhibitthe
synthesisofTNFandpreventthedevelopmentofshock[10].Inaddition,electroacupuncture
wasfoundtoproduceneuro-protectiveeffectbyenhancingtheactivitiesofantioxidant
enzymesandreducingtheextentoflipidperoxidation[11].Traditionally,Zusanli(ST36)and
Neiguan(PC6),twospecificpoints,wereconsideredofchoiceforgoodhealthcare.Recent
studieshaveshownthatST36acupuncturepretreatmentexertedprotectiveeffectsagainstsep-
sis-inducedkidneyinjury[12].Besides,LiuetalindicatedthatelectroacupunctureatPC6
retrievedbloodpressureandimprovedendotoxicshockinrats[13].However,littlewasknown
aboutitsmechanismsofmodulation.
SeveraltranscriptionalfactorsincludingNF-E2relatedfactor-2(Nrf2),activatorprotein
(AP)-1andnuclearfactor-κβ(NF-κβ)havebeenimplicatedintheregulationofHO-1expres-
sion[14].Amongthese,Nrf2playedacrucialroleinmountingtheinnateimmuneresponse
andmaintainingredoxhomeostasis,whichdeterminedsurvivalduringendotoxicshock[15].
Moreover,onlyphosphatidylinositol3-kinase(PI3K)-relatedsignalpathwaycontrolledthe
activationofNrf2wasinvolvedintheinductionofHO-1[16,17].Meanwhile,PI3K/Aktpath-
waywasthoughttobepivotalinthemaintenanceofhomeostasisandtheintegrityofthe
immuneresponseduringsepsis[18].Ourpreliminarystudieshaveconfirmedthat
electroacupunctureexertedanti-inflammatoryorantioxidantcapacitytoreverselunginjury
inducedbyendotoxicshockthroughupregulationofHO-1andactivationofNrf2/AREpath-
way[19].Nevertheless,theroleofHO-1inprotectiveeffectofelectroacupunctureagainst
endotoxicshockinducedAKIandtheregulatorymechnismshavebeenunconfirmed
systematically.
Basedonthesepreviousdata,wehypothesizedthatelectroacupuncturestimulationatbilat-
eralST36andPC6acupointscouldattenuateAKIevokedbyendotoxicshockinrabbitsviathe
inductionofHO-1throughmodulatingPI3K/Akt/Nrf2pathway.
MaterialsandMethods
Animalpreparation
Forthecurrentstudy,two-month-oldmaleNewEnglandwhiterabbitsweighing1.5–2.0kg
wereobtainedfromLaboratoryAnimalCenterofNankaiClinicalInstitutionofTianjinMedi-
calUniversity.Allanimalexperimentswerecarriedoutinstrictaccordancewiththerecom-
mendationsintheGuidefortheCareandUseofLaboratoryAnimalsoftheNationalInstitutes
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PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
ofHealth.TheprotocolswereapprovedbytheCommitteeontheEthicsofAnimalExperi-
mentsofTianjinNankaiHospital(NKH-20131107).Therabbitswerehousedat18~22°C,and
acclimatizedtoa12/12-hlight-darkregimen.Foodandwaterweresuppliedadlibitumfora
5-dayperiodbeforetheexperimentprocedures.Thenightbeforesurgeryrabbitsweredenied
accesstofoodbuthadaccesstowater.Alloperationswereperformedundertheanesthesiaof
urethane,andalleffortsweremadetominimizethesufferingoftheanimals.
Electroacupunctureprotocols
Twopairsofstainlesssteelneedles(diameter0.3mm)wereinsertedperpendicularlytoadepth
of5mmandthenheldinplace.TheselectedacupointswereZusanli(ST36),locatedbetween
thetibiaandfibularapproximately5mmlateraltotheanteriortubercleofthetibia,andNei-
guan(PC6),locatedbetweentheradiusandulnaprobablyone-sixththelengthofthelinefrom
thewristtoelbow[12,20].Aseriesofnonacupointslocated5mmlateraltoST36orPC6origi-
nallocationservedascontrols.Asdescribedindetailpreviously[19,21],stimulation(alternat-
ingdisperse-densewaveof2Hzand15Hz,onceaday)weredeliveredusinganelectrical
stimulationdevice(HANSG6805-1A,HuayiCo,Shanghai,China)for15minutes.Current
intensity((cid:1)1.0mA)wasadjustedtoelicitslighttwitchesofthelimbs.Electroacupuncturewas
initiatedforafive-consecutive-daybeforetheexperimentandthroughouttheoperatingsteps
for6hduringtheexperimentalday[22].Foracupuncturesessions,therabbitswerelightly
immobilizedusinghandstominimizestress.Acupuncturepointswereidentifiedbyanexperi-
encedacupuncturist(ZY),whowasnotinvolvedindataanalysis.
Experimentaldesign
Rabbitsweresedatedwith5mg/kgof20%urethaneviathemarginalearvein.Anesthesiawas
maintainedwithcontinuousinfusionofketamineat3mg/kg/hthroughouttheexperiment.
Briefly,tracheotomywasperformedasepticallysoastofacilitatespontaneousrespiration.The
animalswereplacedonaheatingpadunderaradiantheatlamptokeepthebodytemperature
at37.7–40.3°C.APE-50catheterwasinsertedthroughtherightinternalcarotidarterytomon-
itorarterialpressurecontinuouslybyusingHelligemonitorinstrument(Germany).Theinter-
naljugularveinwascannulatedwitha24-gcatheterfordrugsadministration.Aretention
catheterwasinsertedintothebladderviaurethraforurinecollection.LactatedRinger’ssolu-
tion(8ml/kg/h)wasinfusedintravenouslyforthedurationofeachexperiment.
Sixtyrabbitswererandomlyassignedtosixgroups(n=10/group):groupELW,W,C,EL,
SEL,andL(Fig1).Rabbitssubjectedto0.5ml(5mg/kg)LPS(L2630,sigma,USA)ingroup
ELW,EL,SELandLtomimicendotoxicshock-inducedacutekidneyinjury[23].Toserveas
controls,therabbitsingroupCreceivedanequalamountofnormalsaline.Electroacupuncture
treatmentwasappliedbilaterallyatST36andPC6pointsingroupELandELW,whilegroup
SELacupuncturedatnon-acupointsasdescribedabove.ToblockPI3K/Aktsignalingpathway,
wortmannin(theselectivePI3Kinhibitor,0.6mg/kg;Selleck,USA)wasadministeredingroup
ELWintravenously30minbeforeLPSinjection[24].Meanwhile,itsdrugvehicle,dimethyl
sulfoxide(DMSO,0.08ml/kg)wasgiveninothergroupsinstead,andWgrouprabbitswere
acceptedwortmanninalone.Meanarterialpressure(MAP)wasmonitoredconsecutivelyafter
LPSadministration.MAPdidnotdecreasewithin2horrabbitsdiedwithin6hweredeemedas
theexclusioncriteriaofcurrentstudy[7].
Samplingandstorage
Theoverallhealthstatusofallrabbitsenrolledincurrentstudyweremonitoredevery30
min~1hforindicatorsofdistressfollowingtheinjectionofLPS.Inparticular,aswiththe
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PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
Fig1.Schematicdiagramofdepictingtheexperimentalprotocols.“EA”presentedelectroacupunctureatbilaterallyZusanliandNeiguanacupoints;
“SEA”presentedshamelectroacupunctureatnon-acupoints;“none”meansnoelectroacupuncturepretreatmentforfivedays.“surgery”meansthe
experimentaloperationofrabbits.“.“Brace”meanselectroacupunctureorshamelectroacupuncturethroughouttheoperationofrabbits.
doi:10.1371/journal.pone.0141622.g001
suggestionsofNemzeketal,rabbitswereeuthanizedbyCO2asphyxiationeitherattheendof
theexperiment,namelyat6hafterLPSadministration,oruponsignsofimminentdeath
includinginabilitytoambulate,laboredbreathingandcyanosis,prolongedhypothermia,and
unconsciousnesstoexternalstimuli[25,26].Thebloodsamplewasdrawnfromtherightinter-
nalcarotidarteryintoEDTAtubesimmediately.Then,thebloodspecimenwascentrifugedat
3000rpmfor15minat4°Candthesupernatantwasfrozenat-80°Cforsubsequentanalysis.In
addition,urinesamplesfromthebladderwereextracted.Aftertherabbitswereeuthanized,the
kidneyswereharvestedimmediatelyandquicklyperfusedwithphosphate-bufferedsaline
(PBS)toremoveblood.Theleftkidneytissuesweresnapfrozeninliquidnitrogenandstored
at-80°Cforlateranalyses.Portionsoftheremnantkidneytissueswerefixedin4%formalde-
hydeforhistopathologicalanalysis.
Biochemicalmeasurements
Superoxidedismutase(SOD)activitiesandmalondialdehyde(MDA)contentsintherenaltis-
sueswererespectivelyquantifiedbyspectrophotometry[27]andbymeansofLoewenberg
[28],bothofwhichwereexpressedasperunitofproteindeterminedbytheLowrymethod
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PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
[29].Besides,Plasmalevelsofbloodureanitrogen(BUN)andcreatinine(Cr)wereevaluated
byaHitachi7060FullyAutomatedBiochemistryAnalyzer(Tokyo,Japan).Urineconcentra-
tionsofN-acetyl-glucosaminidase(NAG)weredeterminedbyparanitrophenolcolorimetry
(KitsuppliedbyShanghaiDeboBiotechnologylimitedcompany,Shanghai,China).Inaddi-
tion,tumornecrosisfactor-alpha(TNF-a)andinterleukin-10(IL-10)levelsinplasmawere
measuredwithcommerciallyavailableenzyme-linkedimmunosorbentassaykits(R&Dsys-
tems,Minneapolis,MN,USA).Alltheprocedureswereperformedaccordingtomanufacturer
protocols.
Histopathologicalexamination
Briefly,formalin-fixedparaffin-embeddedkidneyweresectionedat4μmandstainedwith
hematoxylinandeosinforgeneralhistologicalassessment.Thesemiquantitativeanalysisofthe
kidneyslideswereconductedbyatrainedhistopathologistwithnoknowledgeoftreatment
allocation.Eachsuccessivefieldwasassessedindividuallyforseverityoftubulointerstitial
lesionsandgradedasfollows[30,31]:0,normal;1,areasoftubularepithelialcellswelling,vac-
uolardegeneration,necrosisanddesquamationinvolving<25%ofcorticaltubules;2,similar
changesinvolving>25%but<50%ofcorticaltubules;3,similarchangesinvolving>50%
but<75%ofcorticaltubules;4,similarchangesinvolving>75%ofcorticaltubules.Atleast10
differentfieldsofeachslicewerevisualizedbymicroscopy(×400).Themeanscoresofallfields
weretakenasrenalinjuryscoresofabovekidneysections
TUNELstaining
Paraffin-embeddedrenalsections(4μm)werestainedwiththeterminaldeoxynucleotidyl
transferasemediateddeoxyuridintriphosphatenick-endlabeling(TUNEL)assayusinganin
situapoptosisdetectionkit(Roche,Germany)accordingtothemanufacturer’sinstructions.
BrownlabeledTUNELpositivecellswerecountedintenrandomlyselectedfieldsfromeach
slidesatamagnificationof×200,andthreedifferentkidneysectionswereobservedforperani-
mals(n=10/group)bylightmicroscopy.TheTUNEL-positivecellsforeachfieldwerequanti-
fiedasthepercentageoftotalrenalcellscountedbyablindobserver[21].
Westernblotanalysis
Tissuesampleswerehomogenizedinlysisbuffersolution(13.2mmol/LTris-HCl,5.5%glyc-
erol,0.44%SDSand10%β-mercaptoethanol).Afterthesupernatantswerecollected,thepro-
teinextractswereobtainedbyusingthetotalandnuclearproteinisolationkit(Thermo,USA).
ProteinlevelsweredetectedbytheBicichoninicacid(BCA)assaykit(Thermo,USA).Equal
amountsofseparatedprotein(50μg)werefractionatedby12%SDS-PAGEandtransferredtoa
PVDFmembrane(Bio-Rad,USA).Blotswereincubatedovernightwithat4°Cwithpolyclonal
rabbitantibodiesagainstphosphor(Ser473)-specificAkt(1:300,BiossInc,USA),HO-1(1:800,
Abcam,UK)andNrf2(1:300,Biorbyt,UK).Then,blotswerewashedtriplefor10minwith
TBS-0.05%Tween20,andincubatedwith1:3000dilutionofthehorseradishperoxidase
(HRP)-conjugatedgoatanti-rabbitIgG(Biorbyt,UK)for1hat37°C.Antigenicdetectionwas
visualizedbyenhancedchemiluminesence(Bio-Rad,USA)asdescribedbyWangetal[5],and
therelativedensityofbandswasquantifiedbydensitometry(MolecularAnalystImage-
analysisSoftware,Bio-Rad,USA).Thedensitometervaluesofmeasuredproteinswerenormal-
izedtoβ-actinusedasloadingcontrols.
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PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
Immunoflurescenceassays
ToestimatetheintracellulardistributionofNrf2nucleoprotein,immunoflurescenceanalysis
wasperfomedaspreviouslydescribed[19].Kidneytissueswereembeddedinparaffin,sec-
tionedat4μm,andthendewaxedinxyleneandrehydratedwithgradedethanolsolutions.
Afterproteinblockade,thesectionswereincubatedovernightat4°CwiththepolyclonalNrf2
primaryantibodyconjugatedtoFITC(dilution1:150,Biorbyt,UK).Followingwashingtriple
inphosphate-bufferedsaline(PBS),slideswerecounterstainedwith4’,6-diamidino-2-pheny-
lindole(DAPI)(Roche,Shanghai,China)tovisualizethenuclei.Theimageswerecapturedby
afluorescentmicroscope(OlympusU-25ND25,Tokyo,Japan)coupledtoadigitialcamera.
Theoverlaycolorofbluestaininginnucleus,accompaniedwithgreenstainingbothincyto-
plasmandnucleuswasconsideredtobepositive.Asemiquantitativenumericscoreswere
determinedforkidneysamplesbasedonthepercentageofpositiveproteininfivefieldsofeach
slicesata400multiplesignalmagnificationusingImage-proplussoftware(Imageproplus6.0,
mediaCybernetics,USA)[32,33].
Statisticalanalysis
Dataareexpressedasmeans±SD.Forsignalcomparisons,renalinjuryscoreswereanalyzed
bythenon-parametricunpairedMann-Whitneytest.Besides,parametricdatawereanalyzed
forstatisticalsignificancebymeansofone-wayANOVA,followedbytheBonferroni-Dunn
methodformultiplecomparisons.TheanalyseswerecarriedoutwithGraphPadPrism5.0
(GraphPadsoftware,LaJolla,CA,USA).Pvalues<0.05wereconsideredtoindicatestatistical
significance.
Results
RenalSODactivitiesandMDAcontents
AsshowninFig2,LPSpotentlydecreasedtheactivitiesofSOD,whileincreasedthecontents
ofMDAcomparedtogroupC(allP<0.01).Bycontrast,electroacupuncturetreatmentsignifi-
cantlycounteractedthealterationsinducedbyLPS(P<0.01)(augment25.4%forSOD,attenu-
ation20.2%forMDA).Nosignificantinfluencesinaboveparameterswerediscoveredwhen
comparedgroupSELwithgroupL(P>0.05).Additionally,administrationofwortmanninin
groupELWcouldreversetheprotectiveeffectsofelectroacupunctureatST36andPC6
(P<0.05).
PlasmaTNF-aandIL-10
PlasmalevelsofTNF-αandIL-10at6hafterLPSinjectionwereshowninFig2.Electroacu-
puncturestimulationobviouslyattenuatedTNF-aby29.9%andenhancedIL-10by32.6%in
groupELcomparedwithgroupLorSEL(P<0.01).However,aboveprotectiveeffectswere
neutralizedtosomeextentbytheselectivePI3Kinhibitor,wortmannin(P<0.05).Therewere
nosignificantdifferencesbetweengroupLandgroupSEL(P>0.05).
PlasmaBUN,CrandurineNAG
DatainTable1revealedthatrabbitsfromgroupL,EL,SELandELWpossessedhigherlevels
ofBUN,CrandNAGthangroupCorW(P<0.01).Besides,asmarkersofrenalfunction,both
BUNandCr,aswellasNAGweredistinctlyreducedinelectroacupuncturetreatmentgroup
comparedwithgroupL(P<0.01).Wortmanninpretreatmentnotablyrestrainedthelevelsof
theseindicatorsingroupELW(P<0.05).Wedidnotfindasignificantdifferenceinthelevels
ofBUN,CrandNAGbetweengroupSELandgroupL(P>0.05).
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Fig2.AnalysisofSODactivities(A),MDAcontents(B)intherenaltissue,aswellasplasmaIL-10(C)
andTNF-αlevels(D)ineachgroups.ElectroacupunctureatbilaterallyZusanliandNeiguanacupoints
significantlyincreasedSODactivities,decreasedMDAcontentsaccompaniedwithincreasedlevelsofIL-10
anddecreasedTNF-αlevels.Pretreatmentwithwortmanninsignificantlyrestrainedtheaboveeffectsof
electroacupunctureingroupELW.Valueswerepresentedasmean±SD(n=10).Thedatawereanalyzed
usingone-wayANOVAandtheBonferronitestformultiplecomparisons.Significantdifferenceswere
indicatedwithanasterisk:*P<0.05,**P<0.01.
doi:10.1371/journal.pone.0141622.g002
Table1. ComparisonofplasmaBUNandCr,aswellasurineNAGlevelsinthestudygroups.
Groups BUN(mmol/L) Cr(μmol/L) NAG(U/L)
ELW 9.91±1.35*# 108.9±16.83*# 39.8±9.55*#
W 5.64±0.95 36.1±3.48 13.2±5.16
C 5.68±0.87 38.4±3.56 13.7±5.23
EL 7.78±1.12*+ 82.6±13.85*+ 26.9±7.49*+
SEL 10.91±1.54*# 118.1±21.87*# 46.5±13.72*#
L 10.83±1.67*# 120.7±23.40*# 43.3±11.23*#
Abbreviations:BUN,bloodureanitrogen;Cr,creatinine;NAG,N-acetyl-glucosaminidase.Valueswere
expressedasmean±SD(n=10;one-wayANOVAfollowedbyBonferronipost-hoctestwasusedfor
statisticanalysisofthedata;
*P<0.01versusgroupC;
+P<0.01versusgroupL;
#P<0.05versusgroupEL).
doi:10.1371/journal.pone.0141622.t001
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Renalhistopathology
AsshowninFig3,noabnormalstructureswerevisibleingroupCandgroupW.Incompari-
son,kidneytissuesfromLPSgrouphadextensivecellularvacuolization,swellinganddesqua-
mationinproximaltubules,interstitialedemaandneutrophilinfiltration.Therewasadistinct
reductionintheextentofmorphologicaldamageinrabbitstreatedwithelectroacupunctureat
ST36andPC6.However,preprocessingwithwortmanninsuppressedtheefficacyofelectroa-
cupuncturetreatment.Theresults,presentedasthescoresofacutekidneyinjury,weresumma-
rizedinTable2.Consistently,thelevelsofkidneyinjuryscoresingroupELwerelowerthan
groupL,andhigherthangroupELW(P<0.05).Meanwhile,similarresultswereobservedin
groupSELandgroupL(P>0.05).
TUNELassay
Toassesswhetherelectroacupuncturestimulationdecreasedtheamountofrenaltubularcell
deathinLPS-inducedAKI,andtheproportionofapoptoticcellsinrenalslidesweredeter-
minedbyTUNELstaining(Fig4).AnelevatednumberofTUNEL-positivetubularcellswere
observedinrabbitssubjectedtoLPSthanthatofcontrols(P<0.05).IncontrasttogroupL,
electroacupuncturepreconditioningcausedasignificantdecreaseinthenumberofTUNEL-
positivecellsingroupEL,whilewortmanninpretreatmentingroupELWblockedtheprotec-
tiveeffectofelectroacupuncturestimulation(P<0.05).Additionally,thepercentageof
TUNEL-positivecellsingroupSELresembledthatofgroupL(P>0.05).
Fig3.Microphotographsofhistopathologicchangesofkidneysectionsstainedwithhematoxylinand
eosin.(originalmagnification×400):(W,C)Normalmorphologyofkidneys;(L,SEL)RabbitstreatedwithLPS
orshamelectroacupunctureshowinginfiltrationofinflammatorycells,tubularepithelialcellvacuolization,
swellinganddesquamation.(EL)Markedattenuationoftubulardamagesweredisplayedintreatmentwith
electroacupunctureatST36andPC6acupoints.(ELW)Preprocessingwithwortmanninsuppressedthe
protectiveefficacyofelectroacupuncturetosomeextent.Blackarrows:thedestructionofrenalcapsuleand
capsularspaceingroupELWwhileinfiltrationofinflammatorycellsingroupELorgroupSEL.
doi:10.1371/journal.pone.0141622.g003
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Table2. Semi-quantitativeanalysisofrenalinjuryscoresamongthefollowingsixgroups.
Groups RenalInjuryScores
ELW 2.5±1.08*#
W 0.4±0.52
C 0.5±0.53
EL 1.4±0.97*+
SEL 2.9±1.20*#
L 2.8±1.23*#
Valueswereanalyzedbythenon-parametricunpairedMann-Whitneytest.(n=10;
*P<0.05versusgroupC;
+P<0.05versusgroupL;
#P<0.05versusgroupEL).
doi:10.1371/journal.pone.0141622.t002
Fig4.TUNELinsituassayoftubularcelldeathintherenaltissues.(originalmagnification×200):(A)RepresentativephotomicrographsofTUNEL
stainedrabbitkidneyscorrespondingtogroupELW,W,C,EL,SELandgroupL.Whitearrows:normalcells;Blackarrows:theTUNEL-positiverenaltubular
cells.(B)QuantificationofthenumberofTUNEL-positivecellsusingsemi-quantitativeanalysisofthepositivestainingfromtenrandomlyselectedfields.The
datawereexpressedasmean±SD(n=10;**P<0.01,usingone-wayANOVAandtheBonferronitestformultiplecomparisons).
doi:10.1371/journal.pone.0141622.g004
Renalphospho-Aktprotein,HO-1protein,Nrf2totalandnucleoprotein
expressions
ExposuretoLPSnotablyincreasedtheexpressionsofp-Aktprotein,HO-1protein,Nrf2total
andnucleoproteincomparedwithgroupC(P<0.05),illustratedinFig5.Besides,electroacu-
puncturetreatmentmarkedlyupregulatedthelevelsofabovementionedproteinsincontrastto
groupLorgroupSEL(P<0.05).Inwortmannin-treatedrabbits,phosphorylationofAktwas
substantiallydecreased(P<0.05),whileHO-1protein,Nrf2totalandnucleoproteinwere
slightlydecreasedcomparedwiththatingroupEL(P>0.05).However,therewereno
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PI3K/Akt/Nrf2PathwayinElectroacupunctureProtectionagainstAKI
Fig5.Westernblotanalysisofphospho-Aktprotein(A),HO-1protein(B),Nrf2total(C)andnucleoprotein(D)expressionsintherenaltissueofsix
groups.β-actinwasmonitoredastheinternalstandardtoensuresimilargelloadingofthestartingmaterialsineachsample.Blotimageswerecroppedfor
comparison(E).Electroacupuncturetreatmentsignificantlyincreasedthelevelsofphospho-Aktprotein,HO-1protein,Nrf2totalandnucleoproteincompared
withgroupLorgroupSEL.Inwortmannin-treatedrabbits,phosphorylationofAktwassignificantlydecreasedwhileHO-1protein,Nrf2totalandnucleoprotein
wasslightlydecreasedcomparedwithgroupEL.Allvalueswereexpressedasmean±SD(n=10;*P<0.05;**P<0.01,usingone-wayANOVAandthe
Bonferronitestformultiplecomparisons).Theblotswererepresentativeofthreeindependentexperiments.
doi:10.1371/journal.pone.0141622.g005
significantdifferencesbetweengroupSELandgroupLintermsofthemeasuredprotein
expressions(P>0.05).
RenaldistributionratioofNrf2nucleoproteinexpression
ThetranslocationofNrf2fromcytoplasmtonucleusbyelectroacupuncturecouldbevisualized
byImmunofluorescenceanalysis(Fig6).After6hofLPSadministration,nuclearlocalization
ofNrf2wasapparentcomparedtothenegligibledistributioninshamoperationgroup
(P<0.01).Inaddition,electroacupuncturetreatmentatST36andPC6acupointsdramatically
increasedthenuclearaccumulationofNrf2proteincontrastwithgroupLorgroupSEL
(P<0.01).Moreover,Wortmanninwasscarcelyeffectiveatinhibitingelectroacupuncture-
inducednucleartranslocationofNrf2,yetwortmanninalonehadnoeffect.Shamelectroacu-
puncturegroupexhibitedindistinguishableexpressionofNrf2nucleoproteincomparedwith
groupL(P>0.05).
Discussion
DatafromcurrentstudyrevealedthatelectroacupuncturestimulationatbilateralST36and
PC6acupointsdramaticallyalleviatedacutekidneyinjuryduringendotoxicshockinrabbits,
coincidentwiththeexpressionsofHO-1protein,Nrf2totalandnucleoproteinaswellasphos-
pho-Aktprotein.Furthermore,electroacupuncturetreatmentsignificantlyinhibitedLPS-
PLOSONE|DOI:10.1371/journal.pone.0141622 November2,2015 10/15