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BackgroundOxaliplatinisathirdgenerationplatinumbasedchemotherapydru


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Document on Subject : "BackgroundOxaliplatinisathirdgenerationplatinumbasedchemotherapydru"— Transcript:

1 BackgroundOxaliplatinisathird-generation
BackgroundOxaliplatinisathird-generationplatinum-basedchemo-therapydrug,whichiswidelyusedasthefirst-linetreat-mentofmetastaticcolorectalcancer[1–5].Despiteitsefficacyagainstthetumor,ithasseriousneurotoxicity,adose-limitingsideeffect.Thisneurotoxicityischaracter-izedbyparesthesiaanddysesthesiainthehandsandfeet[6],andabout85to95%ofpatientsrapidlydevelopsignificantacuteneuropathicpainwithoutmotordys-functionshortlyafteranoxaliplatininfusion[7,8].Severaldrugs(e.g.gabapentinandduloxetine)arerecommendedtomitigatethissideeffect[9–11].Unfor-tunately,theseanalgesicscauseanothersideeffects,suchassomnolenceandnausea[12].Activationofglialcells,suchasastrocytesandmicro-glia,hasbeenobservedinthelumbarspinalcordinani-malmodelsofperipheralneuropathicpain[13–17].Uponactivation,astrocytesandmicrogliareleaseavar-ietyofsubstancesthatenhancethetransmissionofpain,suchaspro-inflammatorycytokines[18,19].Bothinter-leukin(IL)-1andtumornecrosisfactor-(TNF-)en-hancethespontaneousexcitatorypost-synapticcurrentsfrequencyofspinaldorsalhornneurons[20].Inanimalmodelsofchemotherapy-inducedperipheralneuropathy(CIPN),thecausalrelationshipbetweenglialactivationandneuropathicpainhasalsobeenreported[21–24].Oxaliplatintreatmentloweredthepainthresholdcom-binedwithasignificantincreaseinthenumberofGFAP(astrocyte)andIba-1(microglia)immunoreactivecellsinthespinaldorsalhorn[21,22].Inaddition,asinglein-jectionofoxaliplatininducesspinalglialactivationcoin-cidentwithpainbehaviorslikecoldandmechanicalallodynia[25].Buja,aprocessedAconitituber,isoneofthefre-quentlyusedherbalmedicineinseveraldiseases[25–27].Previousarticleshavereporteditsanalgesiceffectondifferentkindsofneuropathicpain,suchasdiabeticneuropathyandpostherpeticneuralgia[28,29].Bujainhibitedneuropathicmechanicalallodyniabysuppressingtheactivationofspinalastrocytesinanerveinjurymodel[26].Also,arecentclinicalstudyhasreportedthatBujareducedneuropathicpaininoxaliplatin-treatedcolorectalcancerpatients[30].Gyejigachulbu-tang(GBT),whichiscomposedofCinnamomiCortex(Yukgye;inKorean),PeoniaeRadix(Jakyak),AtractylodisLanceaeRhizoma(Bokryeng),ZiziphiFructus(Saenggang),GlycyrrhizaeRadix(Gamcho),ZingiberisRhizoma(Gungang)andAconitiTuber(Buja),showedapotentanalgesiceffectagainstoxaliplatin-inducedperipheralneuropathyinrats.SucheffectofGBTisassociatedwithdeactivationofspinalastrocytesandmicroglia[25].Inthepresentstudy,weinvestigatedwhetherBujare-lievesoxaliplatin-inducedcoldandmechanicalallodyniainrats,andifso,whethersuchanti-allodyniceffectofBujaisrelatedtothemodulationofglialactivationandpro-inflammatorycytokinesinthespinalcord.MethodsAnimalsYoungadultmaleSprague-Dawleyrats(DaehanBiolink,Chungbuk,Korea),weighingapproximately200–220g,atthebeginningoftheexperimentalprocedurewereused.Animalswerehousedincages(3–4ratspercage),andfedwithwaterandfoodadlibitum.Theroomwasmaintainedwitha12h-light/darkcycle(alightcycle;08:00–20:00,adarkcycle;20:00–08:00)andkeptat23±2°C.Allanimalswereacclimatedintheircagesfor1weekpriortoanyexperiments.AllproceduresinvolvinganimalswereapprovedbytheInstitutionalAnimalCareandUseCommitteeofKyungHeeUniversity(KHUASP(SE)-15-088)andwereconductedinaccordancewiththeguidelinesoftheInternationalAssociationfortheStudyofPain[31].DrugadministrationOxaliplatin(Sigma-Aldrich,StLouis,MO,USA)wasdeliveredatanamountof6mg/kg[32,33],dissolvedin5%glucose(Sigma-Aldrich)solutionataconcentrationof2mg/ml.Oxaliplatinwasadministeredintraperitone-ally(i.p.).Controlanimalsreceivedanequivalentvolumeof5%glucosesolutioni.p.asavehicle.Buja(BushiinJapanese;TJ-3023,TsumuraCo.Ltd.,Ibaraki,Japan)wasobtainedbyagenerousgiftfromProf.SchuichiKoizumi(DepartmentofNeuropharma-cology,Facultyofmedicine,UniversityofYamanashi).ThequalityofBujaisstrictlycontrolledbythemanufac-turer.Bujawassuspendedanddilutedwithdistilledwater(30mg/ml,300mg/kg,approximately2–2.2ml/rat)[26].OurpreliminarystudyusingseveraldosesofBujaconfirmedthat300mg/kgwastheoptimalconcen-tration.Bujawastreatedorallyfor5consecutivedaysafteranoxaliplatininjection.Equivalentvolumeofdis-tilledwater(DW)wasadministeredtocontrolanimals.Animalswerearbitrarilydividedinto4groups:Vehicle+DW,group1;Vehicle+Buja,group2;Oxaliplatin+DW,group3;Oxaliplatin+Buja;group4.BehavioraltestsForassessmentofoxaliplatin-inducedneuropathicpainbeforeandafterBujaadministraion,coldandmechan-icaltestwereperformed.Coldallodyniaweredeter-minedbycoldimmersiontestaspreviouslydescribed[34,35].Inbr

2 ief,ratswereplacedintoanacryliccylin-der
ief,ratswereplacedintoanacryliccylin-derholderwiththetailprotrudingandwereadaptedtothetestingenvironmentatleast30minpriortotesting.Afterimmersingthetailin4°Cwater,thetailwith-drawallatency(TWL)wasmeasuredwitha15scut-offtime.Thistestwasrepeated5timesat5minintervalsJungetal.BMCComplementaryandAlternativeMedicine (2017) 17:48 Page2of8 topreventtissuedamage.Theaverageofeachlatencywasusedtorepresentcoldallodynia(i.e.theshorterlatencywasconsideredthemoreseverecoldallodynia).Mechanicalallodyniawasevaluatedthewithdrawalre-sponseoftailusingaseriesofvonFreyfilaments(bend-ingforcesto0.4,0.6,1.0,2.0,4.0,6.0,8.0and15.0g;equivalentinlogunits:3.61,3.84,4.08,4.31,4.56,4.74,4.93and5.18;Stoelting,IL,USA),aspreviouslydescribed[25].Usingtheup-downmethod,the50%withdrawalthresholdwasdetermined[36].Ratswereimmobilizedinanacryliccylinderholderasmentionedabove.Testwasinitiatedwithafilamenthaving2.0gbendingforce.Whenwithdrawalofthetailwasobservedduringorrightafterstimulation,thiswasconsideredasapositiveresponse.Thefilamentwiththenextlowerbendingforcewasappliedafterapositiveresponsewasobserved,whereasthefilamentwiththenexthigherbendingforcewasappliedwhentherewasnoresponse(i.e.negativeresponse).ThisprocedurecontinueduntilthesixthvonFreyfilamentstimulationfromthefirststimulation(2.0g)oruntilthethirdchangeofresponse(positiveornegative)wasobserved.TheextremevaluesofseriesofvonFreyfilamentsweresetasacut-offvalue.Theseresponseswereconvertedinto50%thresholdvalueusingthefollowingformula:50%threshold=Xf+(XfisthevalueofthefinalvonFreyfilament[logunits],isthecorrectionfactorfromcalibrationtable,andisthemeandifferenceoflogunitsbetweenstimuli)[37].ImmunohistochemistryAttheendoftheexperiment(day5),theL4/L5seg-mentsofthespinalcordwereexposedfromthelumbarvertebralcolumnvialaminectomyandidentifiedbytra-cingthedorsalrootsfromtheirrespectivedorsalrootganglia(DRG).Animalswereperfusedusing0.1Mphosphatebufferedsaline(PBS),followedby4%parafor-maldehyde(BBCBiochemical,WA,USA).Sampledtissueswerepost-fixedin4%paraformaldehyde(BBCBiochemical)for24hat4°C,andthenpermeatedwith30%sucrose(Sigma-Aldrich)in0.1MPBSfor48hat4°C.Lumbarspinalcordsegmentswereembeddedinoptimalcuttingtemperature(OCT)compound(SakuraFinetek,Tokyo,Japan)ondryice.Usingcryostat(MicromHM505N;ThermoScientific,MA,USA),frozenspinalcordsegmentswerecutata20mthick-ness.Sectionswerecollectedin0.1MPBSat4°C.Thesesectionsweremountedonslideglass(Matsunami,Osaka,Japan)andincubatedfor1hin0.2%TritonX-100in0.5%bovineserumalbumin(BSA;BOVOGENbiologics,EastKeilor,Australia)solutionatroomtemperature(RT).Afterrinsingtheslideglasswith0.5%BSAsolution,doubleimmunostainingusingprimaryantibodiesraisedindifferentspecieswascarriedout.Thesectionswereincubatedovernightat4°Cwithpri-maryantibodies:mouseanti-glialfibrillaryacidicprotein(GFAP1:500;Millipore,CA,USA),rabbitanti-Iba-1(1:500;Wako,Osaka,Japan).Afterrinsingin0.5%BSAsolution,sectionswereincubatedatRTindarkfor1hwithsecondaryantibodies:anti-mouseandanti-rabbit-immunoglobulinG(IgG)labeledwithAlexaFluor488andAlexaFluor546(1:200;Invitrogen,USA).Confocallasermicroscope(LSM5Pascal,Zeiss,Oberkochen,Germany)wasusedtoobtainimmunofluorescentim-ages.QuantitativeanalysisofGFAPandIba-1positivecellswereperformedonthespinaldorsalhornimagestakenthrougha20X0.5NAobjective.ImageJ(https://imagej.nih.gov/ij/,NationalInstitutesofHealth,USA)wasusedforquantifyingtheGFAPpositivecellsandIba-1positivecells[22].ToquantifyGFAPorIba-1positivecells,numberofGFAPorIba-1positivecellsfromsixlumbarspinalcordsectionimagesofeachanimalwereaveraged.Sixanimalswereallocatedineachgroup.ELISAToinvestigatewhetherBujaadministrationdecreasesthequantityofTNF-orIL-1inthespinalcord,eachcytokinewasmeasuredbyenzymelinkedimmunosorb-entassay(ELISA).Theanimalsweresacrificedattheendoftheexperiment.Afterperfusionwith0.1MPBS,thelumbarspinalcordsegmentswereobtainedasde-scribedabove.Everycollectedtissuewasstoredin1mlRIPAbuffer(ThermoScientific)withproteaseinhibitorcocktail(Roche,Basel,Switzerland).SampleswereassayedusingacommercialratTNF(BDOptEIASetRatTNF,BDbiosciences,CA,USA)andmouseIL-1(BDOptEIASetmouseIL-1,BDbiosciences)ELISAkitfollowingthemanufacturer’sprotocol.Inbrief,1:10dilutionofserumwasusedforthequantificationofbothcytokines.Microtiterplateswerecoatedovernightat4°Cwithanti-ratTNForanti-mouseIL-1monoclonalanti-bodies(mAbs).Eachwellwasblockedwith10%fetalbo-vineserum(FBS;Gibco,Therm

3 oScientific)for1hatRT.Samplesandstandard
oScientific)for1hatRT.SamplesandstandardswereloadedafterwashingoutFBSandincubatedfor2hatRT.Biotinylatedanti-ratTNFandanti-mouseIL-1mAbswereaddedfor1hatRT.Streptavidin-horseradishperoxidaseconjugatewastreatedandincubatedfor30min.TMBsubstratesolution(BDBioscience)wastreatedfor30mins,andthenStopsolu-tionwasadded.WashingeachwellwithPBST(PBSwithTween-20;Sigma-Aldrich)wasperformedbetweeneverystep.Opticaldensity(O.D.)wasmeasuredat450nmwithcorrection570nm.O.D.wasmeasuredinamicroplatereader(Tecan).TotalamountofproteininsamplesweremeasuredusingBio-Radproteinassaykit(Bio-Rad,CA,USA).Allresultswerenormalizedtothetotalamountofproteinineachsample.Jungetal.BMCComplementaryandAlternativeMedicine (2017) 17:48 Page3of8 StatisticalanalysisAllthedataarepresentedasmean±SEM(standarderrorofthemean).StatisticalanalysisandgraphicworkswereperformedwithPrism5.0(GraphPadsoftware,USA).One-wayanalysisofvariance(ANOVA)orTwo-wayANOVAfollowedbyBonferroni’smultiplecomparisontestwasusedforstatisticalanalysis.Inallcases,p0.05wasconsideredsignificant.ResultsAnti-allodyniceffectsofBujainoxaliplatin-injectedratsToinvestigatewhetherBujaalleviatesoxaliplatin-inducedneuropathicpain,coldandmechanicalallodyniawereassessedusingtailimmersiontestandvonFreyhairtest,respectively,inrandomlydivided4groupsofanimals(seeMethods).Significantcoldallodyniasign(i.e.decreasedTWLinresponsetocoldstimuli)wasobservedsinceday3afteranoxaliplatin(6mg/kg,i.p.)injection(p0.05atD+3,p0.001atD+5,group1vs.group3,Fig.1a).DailyoraldministrationofBuja(300mg/kg)for5consecutivedaysfollowinganoxali-platininjectionreversedsuchdecreaseinTWLtonor-mallevel(p0.001atD+5,group3vs.group4,Fig.1a).Formechanicalallodynia,anoxaliplatininjectioninducedasignificantdecreasein50%thresholdsinceday3(p0.001atD+3andD+5,group1vs.group3,Fig.1b).Bujaadministrationalsoreversedthismech-anicalallodyniasigntonormallevel(p.001atD+3andD+5,group3vs.group4,Fig.1b).Bujaadmin-istrationinvehicle-injectedrats(group2)showednoeffectonbehavioralresponsestocoldandmechanicalstimuli(pȃ&#x.500;0.05,vsgroup1,Fig.1aandb).Thesere-sultssuggestthatoraladministrationofBujapotentlyinhibitsoxaliplatin-inducedcoldandmechanicalallodyniainrats.SuppressiveeffectofBujaonactivationofspinalastrocytesinoxaliplatin-injectedratsTodeterminewhetherBujasuppressesglialactivationinthedorsalhornofspinalcordafteranoxaliplatininjection,activationofspinalglialcells(i.e.astrocytesandmicroglia)inlaminaeI-IIofthedorsalhornwasquantifiedusingimmunohistochemicalanalysis.AsshowninFig.2,anoxaliplatininjectionsignificantlyincreasedGFAP-positivecells(astrocytes)inthespinaldorsalhorn(p.001,group1vs.group3)andthesecellsinthegroup3exhibitedsomatichyper-trophywiththickprocesses(Fig.2a),whichisalsorepresentedbyenhancedintensityofimmunoreactivity(Additionalfile1:FigureS2),indicatingoxaliplatin-inducedactivationofspinalastrocytes.AdministrationofBujasignificantlyreducedsuchactivationofspinalastrocytesfollowinganoxaliplatininjection(p0.001,group3vs.group4,Fig.2).Co-immunolabelingsofGFAP(astrocytes)andIba-1(microglia)positivecellsinthesamespinalcordsampleswereperformedandthesecellswerenotco-localized(Additionalfile2:FigureS3).ThenumberofIba-Ipositivecells(micro-glia)inthespinaldorsalhornisalsoincreasedfol-lowinganoxaliplatininjection(p0.001,group1vs.group3,Additionalfile3:FigureS1)andthesecellsinthegroup3showedamoeboidshapeswiththickprocesses(Additionalfile3:FigureS1A),whichis Fig.1InhibitoryeffectofBujaoncoldandmechanicalallodyniainoxaliplatin-injectedrats.aTimecourseoftailwithdrawallatency(TWL)inresponsetocoldwater(4°C)stimuli.Anoxaliplatininjection(group3:Oxaliplatin+DW)inducedasignificantdecreaseinTWLsinceday3(D+3)comparedtoavehicleinjection(group1:Vehicle+DW).DailyoraladministrationofBuja(300mg/kg)for5daysfollowinganoxaliplatininjection(group4:Oxaliplatin+Buja)significantlyinhibitedcoldallodynia,whereasBujahadnoeffectonTWLinvehicle-injectedrats(group2:Vehicle+Buja).bTimecourseofmechanicalthreshold.Anoxaliplatininjection(group3)significantlydecreased50%thresholdsinceday3comparedtoavehicleinjection(group1).Bujaadministrationfollowinganoxaliplatininjection(group4)significantlyinhibitedmechanicalallodynia,whereasBujahadnoeffectonmechanicalthresholdinvehicle-injectedrats(group2).N=6rats/group.Dataarepresentedasmean±SEM.*p0.05,***p.001,vs.group1;###p0.001,vs.group3,bytwo-wayANOVAfollowedbyBonferroni’spost-testJungetal.BMCComplementaryandAlternativeMedicine (20

4 17) 17:48 Page4of8 representedbyenhanced
17) 17:48 Page4of8 representedbyenhancedintensityofimmunoreactiv-ity(Additionalfile1:FigureS2).However,Bujaadministrationdidnotchangesuchmicroglialactiva-tion,i.e.increasedIba-1positivecellsandalteredmorphology(p�0.05,group3vs.group4,Additionalfile3:FigureS1).TheseresultssuggestthatBujasup-pressesactivationofastrocytesinthespinaldorsalhornfollowinganoxaliplatininjectionwithoutaffect-ingmicroglialactivation.Bujadown-regulatesthelevelsofspinalpro-inflammatorycytokinesinoxaliplatin-injectedratsPro-inflammatorycytokines,IL-1andTNF-,weremea-suredwithELISAcarriedoutattheendoftheexperiment(day5).AsshowninFig.3,thelevelsofIL-1andTNF-inthespinalcordweresignificantlyincreasedafteranoxaliplatininjection(p0.001forIL-1,p.05forTNF-,group1vs.group3).TreatmentofBujareversedthisup-regulationofIL-1andTNF-inthespinalcordtonormallevel(p0.001forIL-1,p.01forTNF-,group3vs.group4).TheseresultssuggestthatBujacansuppresstheoxaliplatin-inducedincreaseinthelevelsofspinalpro-inflammatorycytokines.DiscussionOxaliplatin,awidelyusedchemotherapeuticagent,isknowntoevokeperipheralneuropathyevenafterasin-gleinjection[1–3,25].Variouskindsofexperimentsare Fig.2Bujaattenuatestheactivationofspinalastrocytesinoxaliplatin-injectedrats.ARepresentativeimagesofGFAPpositivecellsinthespinaldorsalhornofgroup1:Vehicle+DW(a),group2:Vehicle+Buja(b),group3:Oxaliplatin+DW(c)andgroup4:Oxaliplatin+Buja(d).NotetheincreasednumberofGFAPpositivecellsandthealteredmorphology(somatichypertrophywiththickprocesses)inthegroup3(c),indicatingactivationofastrocytes.BQuantificationresultofGFAPpositivecells.Sixlumbarspinalcordsectionimagesfromsingleanimalwereaveraged.N=6rats/group.Dataarepresentedasmean±SEM.***p0.001,vs.group1;###p0.001,vs.group3,byone-wayANOVAfollowedbyBonferroni’spost-test Fig.3Bujasuppressestheoxaliplatin-inducedup-regulationofspinalpro-inflammatorycytokines.a,bQuantificationofpro-inflammatorycytokines,IL-1(a)andTNF-(b),inthespinalcord.N=6rats/group.Dataarepresentedasmean±SEM.*p0.05,***p0.001,vs.group1;##p.01,###p0.001,vs.group3,byone-wayANOVAfollowedbyBonferroni’spost-test.group1:Vehicle+DW,group2:Vehicle+Buja,group3:Oxaliplatin+DWandgroup4:Oxaliplatin+BujaJungetal.BMCComplementaryandAlternativeMedicine (2017) 17:48 Page5of8 stillunderwaytodivulgetheexactmechanismofoxaliplatin-inducedperipheralneuropathyanditsoptimaltreatmentmethodhasnotbeendevelopedyet[23,38].Althoughitsexactpathophysiologyisnotclearlyunderstood[23],accumulatingevidencesimplythatactivationofspinalglia,suchasastrocytesandmicroglia,playanimportantroleinoxaliplatin-inducedneuropathicpain[21,22,25,39].Activatedspinalgliawereobservedafteroxaliplatininjection,andintrathecalinjectionofminocyclineandfluorocitrate,whichde-creasetheactivationofastrocytesandmicrogliarespect-ively,effectivelyattenuatedneuropathicpain[22].Activatedgliaareknowntocontributetoneuropathicpainbyreleasingpro-inflammatorycytokinessuchasIL-1andTNF-[13–15],andsuppressingtheactiva-tionofastrocytesandmicrogliadown-regulatedtheexpressionofpro-inflammatorycytokines,whichledtothealleviationofnerveinjury-inducedneuropathicpain[40,41].Intrathecalinjectionofpro-inflammatorycyto-kinessuchasIL-1andTNF-evokedhyperalgesiaandallodyniainnaiveanimals[42,43].Furthermore,block-ingtheactionofIL-1andTNF-usingIL-1receptorantagonistandanti-TNFserumalleviatednerveinjury-inducedneuropathicpain[44,45].Inananimalmodelofoxaliplatin-inducedneuropathicpain,releaseofIL-1andTNF-fromactivatedspinalgliawereobserved[39,46].IntrathecallyinjectedA3adenosinereceptoragonistspre-ventedtheactivationofastrocytesandtheincreaseofpro-inflammatorycytokinesinthespinalcordandsignifi-cantlyattenuatedneuropathicpain[46].Therefore,target-ingspinalglialactivationandpro-inflammatorycytokinescouldbeanidealstrategytoattenuateoxaliplatin-inducedneuropathicpain.BujaisagenerallyusedherbalmedicineinEast-Asia,suchasKorea,JapanandChina.Itisalsoakeycompo-nentinGBT,whichhasbeentraditionallyusedagainstcold-induceddisordersbasedontheSangHanLun[25].Inaddition,GBTshowedanti-allodyniceffectonoxaliplatin-inducedneuropathicpain[25].Recentexper-imentsconductedonbothhumanandanimalshaveshownthatBujasignificantlyimprovedcoldormechan-icalallodynia[26,28,30].Inthepresentstudy,fivecon-secutiveoraladministrationsofBuja(300mgkg1perday)markedlyalleviatedoxaliplatin-inducedcoldandmechanicalallodynia.Theseresult

5 sstronglysuggestthatBujahaspotentefficac
sstronglysuggestthatBujahaspotentefficacyonoxaliplatin-inducedneuro-pathicpain.OurexperimentshowedthatoraladministrationofBujadecreasedthecoldandmechanicalallodyniaviasuppressingtheactivationofspinalastrocytes.ThisresultissimilartothatofShibataetal.[26]where,Bujawassuggestedtocontrolneuropathicpainviainhibitionofactivatedastrocytesinnerveinjurymodel.Inneuro-pathicstate,extracellularsingle-regulatedkinase(ERK)pathwaysinastrocyteswereactivatedbyIL-1andIL-18frommicrogliaandpromotedthesynthesisofpro-inflammatorycytokines,IL-1andTNF-[47,48].BujadirectlyaffectedinhibitionofERK1/2-phosphorla-tion,whichresultedinsuppressionofthespinalastro-cytes[26].However,inourpreviousstudyconductedwithGBT(400mgkg1forfivedays),GBTattenuatedoxaliplatin-inducedcoldandmechanicalallodyniabyde-creasingtheactivationofbothspinalastrocytesandmicroglia[25].AlthoughbothBujaandGBTalleviatedoxaliplatin-inducedcoldandmechanicalallodynia,Bujaonlyattenuatedtheactivationofspinalastrocytes,whereasGBTsuppressedtheactivationofbothastro-cytesandmicroglia.Lorenzoetal.[22]mentionedthatalthoughtheactivationofbothspinalastrocytesandmicrogliaareimportantinoxaliplatin-inducedneuro-pathicpain,down-regulatingonlyastrocytesormicrogliacanleadtotheattenuationofpain.Intheactionofsup-pressingtheactivatedgliabyGBT,activatedastrocytesinhibitionisachievedbyBuja,andanothercomponentofGBTmayberesponsiblefortheattenuationofmicro-glia.Ifanysinglemedicinalherb,whichhaskeyeffect,isomitted,aspecificactivityfromcomplexformulasdisap-pears[49].Furthermore,studiesofanothercomponentinGBTareontheprogress.Pro-inflammatorycytokines,suchasIL-1andTNF-,releasedfromactivatedglia,actonthespinaldorsalhornneuronsandinfluenceexcitatoryneurotransmis-sions[14,15].IL-1,byinducingthephosphorylationofaspecificN-methyl-D-aspartate(NMDA)receptorsub-unit,increasestheinfluxofcalciumionthroughNMDAreceptorchannelandtheproductionofnitricoxide.IL-1alsoincreasesthegenerationofprostaglandinE2,whichamplifytheexcitabilityofpain-projectionneurons[14].TNF-,byenhancingtheactivationof-Amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid(AMPA)receptor,increasestheexcitatorypost-synapticcurrentsfrequencyinthespinaldorsalhorn[20].Theseinterac-tionsbetweencytokinesandneuronscontributetocen-tralsensitization[50]andfurtherenhanceneuropathicpain.Inourresult,thesuppressionofactivatedspinalastrocytesandthedecreaseofIL-1andTNF-levelsinthespinalcordwerecoincidedaftertreatmentofBuja.GBTalsodown-regulatedreleaseofspinalpro-inflammatorycytokines[51].Takenalltogether,ourfindingssuggestthatBujastronglyalleviateoxaliplatin-inducedcoldandmechanicalallodyniaviasuppressionofactivatedspinalastrocytesanddown-regulationofpro-inflammatorycytokines.ConclusionInconclusion,thisstudyclearlydemonstratedthereliev-ingeffectofBujaonoxaliplatin-inducedcoldandmech-anicalallodynia.Also,BujasignificantlysuppressedtheJungetal.BMCComplementaryandAlternativeMedicine (2017) 17:48 Page6of8 activatedspinalastrocytesanddown-regulatedpro-inflammatorycytokinesafteranoxaliplatininjection.TheseresultsaltogethersuggestthatBujamaybeaneffectivealternativetotreatoxaliplatin-inducedneuro-pathicpain.AdditionalfilesAdditionalfile1:FigureS2.Intensityofimmunoreactivity(IM)ofGFAPandIba-1positivecells.IntensityofIMofGFAPandIba-1positivecellsincreasedingroup3.Ingroup4,IMofGFAPpositivecellsweresignificantlydecreased,whereaslittlechangesofIba-1positivecellsIMwasobserved.Dataindicatethatrelativemeanimmunofluorescenceintensityofasinglecell(n=10).Dataarepresentedasmean±SEM.***p0.001,vs.group1;###p0.001,vs.group3,byone-wayANOVAfollowedbyBonferroni’spost-test.group1:Vehicle+DW,group2:Vehicle+Buja,group3:Oxaliplatin+DWandgroup4:Oxaliplatin+Buja.(PDF7kb)Additionalfile2:FigureS3.Representativeco-immunolabelingimageofGFAPandIba-1positivecellsinthespinaldorsalhorn.Co-immunolabelinginthesamesectionshowedthespatiallydifferentdistributionofastrocytes(GFAP-positivecells)andmicroglia(Iba-1positivecells)inthespinaldorsalhorn(a).Separatedimagesforastrocytes(b)andmicroglia(c)werealsopresented,respectively.(PDF236kb)Additionalfile3:FigureS1.SpinalmicrogliaactivationwasnotsuppressedbyBuja.(A)RepresentativeimagesofIba-1positivecellsinthespinaldorsalhornofgroup1:Vehicle+DW(a),group2:Vehicle+Buja(b),group3:Oxaliplatin+DW(c)andgroup4:Oxaliplatin+Buja(d).NotetheincreasednumberofIba-1positivecellsandthealteredmorphology(somatichypertrophywiththick

6 processes)inthegroup3(c),indicatingactiv
processes)inthegroup3(c),indicatingactivationofmicroglia.(B)QuantificationresultofIba-1positivecells.Sixlumbarspinalcordsectionimagesfromsingleanimalwereaveraged.N=6rats/group.Dataarepresentedasmean±SEM.***p0.001,vs.group1;###p0.001,vs.group3,byone-wayANOVAfollowedbyBonferroni’spost-test.(PDF260kb)AbbreviationsAMPA:-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid;ANOVA:Analysisofvariance;BSA:Bovineserumalbumin;CIPN:Chemotherapy-inducedneuropathicpain;DRG:Dorsalrootganglia;DW:Distilledwater;ELISA:Enzyme-linkedimmunosorbentassay;ERK:Extracellularsingle-regulatedkinase;FBS:Fetalbovineserum;GBT:Gyejigachulbu-Tang;GFAP:Glialfibrillaryacidicprotein;IgG:ImmunoglobulinG;IL-1:Interleukin-1;IM:Immunoreactivity;mAbs:Monoclonalantibodies;NMDA:N-methyl-D-aspartate;O.D.:Opticaldensity;OCT:Optimalcuttingtemperature;PBS:Phosphatebufferedsaline;PBST:PhosphatebufferedsalinewithTween20;RT:Roomtemperature;SDrat:Sprauge-Dawleyrat;SEM:Standarderrorandmean;TNF-:Tumornecrosisfactor-;TWL:TailwithdrawallatencyAcknowledgementsWewouldliketoexpresssincerethankstoProf.KoizumiS.forhisgenerousgiftofBujaandvaluablediscussiononourmanuscript.FundingThisworkwassupportedbyagrantoftheKoreaHealthTechnologyR&DProjectthroughtheKoreaHealthIndustryDevelopmentInstitute(KHIDI),fundedbytheMinistryofHealth&Welfare,RepublicofKorea(grantnumber:HI14C0738).Fundershavenoroleinthedesignofthestudyandcollection,analysis,andinterpretationofdataandinwritingthemanuscript.AvailabilityofdataandmaterialsAlldatageneratedoranalyzedduringthisstudyareincludedinthispublishedarticleanditsadditionalfiles.Authors’contributionsYJ,JHL,SHYandSKKconceivedanddesignedthestudy.YJ,JHLandWKperformedtheexperimentsandanalyzedthedata.YJ,JHL,WKandSKKwrotethemanuscript.Allauthorsreadandapprovedthefinalmanuscript.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.ConsentforpublicationNotapplicable.EthicsapprovalAllproceduresinvolvinganimalswereapprovedbytheInstitutionalAnimalCareandUseCommitteeofKyungHeeUniversity(KHUASP(SE)-15-088)andwereconductedinaccordancewiththeguidelinesoftheInternationalAssociationfortheStudyofPain.Authordetails1DepartmentofClinicalKoreanMedicine,GraduateSchool,KyungHeeUniversity,Seoul02447,RepublicofKorea.2DepartmentofScienceinKoreanMedicine,GraduateSchool,KyungHeeUniversity,Seoul02447,RepublicofKorea.3DepartmentofPhysiology,CollegeofKoreanMedicine,KyungHeeUniversity,Seoul02447,RepublicofKorea.4DepartmentofDigestivesystemofInternalMedicine,CollegeofKoreanMedicine,KyungHeeUniversity,Seoul02447,RepublicofKorea.Received:19July2016Accepted:5January2017 References1.WindebankAJ,GrisoldW.Chemotherapy-inducedneuropathy.JPeripherNervSyst.2008;13(1):27–46.2.McWhinneySR,GoldbergRM,McLeodHL.PlatinumNeurotoxicityPharmacogenetics.MolCancerTher.2009;8(1):10–6.3.RaymondE,FaivreS,WoynarowskiJM,ChaneySG.Oxaliplatin:mechanismofactionandantineoplasticactivity.SeminOncol.1998;1998:4–12.4.FormigaMN,FanelliMF,DettinoALA,NicolauUR,CavicchioliM,LimaENP,deMelloCAL.Isearlyresponseby18F-2-fluoro-2-deoxy-D-glucosepositronemissiontomography-computedtomographyapredictoroflong-termoutcomeinpatientswithmetastaticcolorectalcancer?JGastrointestOncol.2016;7:365–72.5.YoshinoT,UetakeH,TsuchiharaK,ShitaraK,YamazakiK,OkiE,SatoT,NaitohT,KomatsuY,KatoT.PARADIGMstudy:Amulticenter,randomized,phaseIIIstudyof5-fluorouracil,leucovorin,andoxaliplatin(mFOLFOX6)pluspanitumumaborbevacizumabasfirst-linetreatmentinpatientswithRAS(KRAS/NRAS)wild-typemetastaticcolorectalcancer.ASCOAnnuMeetProc.2016;2016:TPS776.6.ExtraJM,EspieM,CalvoF,FermeC,MignotL,MartyM.PhaseIstudyofoxaliplatininpatientswithadvancedcancer.CancerChemotherPharmacol.1990;25(4):299–303.7.LehkyTJ,LeonardGD,WilsonRH,GremJL,FloeterMK.Oxaliplatin-inducedneurotoxicity:Acutehyperexcitabilityandchronicneuropathy.MuscleNerve.2004;29(3):387–92.8.PasettoLM,D’AndreaMR,RossiE,MonfardiniS.Oxaliplatin-relatedneurotoxicity:Howandwhy?CritRevOncolHematol.2006;59(2):159–68.9.WolfS,BartonD,KottschadeL,GrotheyA,LoprinziC.Chemotherapy-inducedperipheralneuropathy:preventionandtreatmentstrategies.EurJCancer.2008;44(11):1507–15.10.GamelinL,Boisdron-CelleM,DelvaR,Guérin-MeyerV,IfrahN,MorelA,GamelinE.PreventionofOxaliplatin-RelatedNeurotoxicitybyCalciumandMagnesiumInfusionsARetrospectiveStudyof161PatientsReceivingOxaliplatinCombinedwith5-FluorouracilandLeucovorinforAdvancedColorectalCancer.ClinCancerRes.2004;10(12):4055–61.11.MarianiG,Garr

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