specificationmyim

OpenAccessAvailableonlinehttp://arthritis-research.
com/content/11/5/R145Page1of13(pagenumbernotforcitationpurposes)Vol11No5ResearcharticleAnti-inflammatoryandarthriticeffectsofthiacremonone,anovelsulfurcompoundisolatedfromgarlicviainhibitionofNF-κBJungOkBan1,JuHoonOh1,TaeMyoungKim2,DaeJoongKim2,Heon-SangJeong3,SangBaeHan1andJinTaeHong11CollegeofPharmacyandMedicalResearchCenter,ChungbukNationalUniversity,12,Gaeshin-dong,Heungduk-gu,Cheongju,Chungbuk,361-763,Korea2CollegeofVeterinaryMedicine,ChungbukNationalUniversity,12,Gaeshin-dong,Heungduk-gu,Cheongju,Chungbuk,361-763,Korea3CollegeofAgriculture,LifeandEnvironmentsSciences,ChungbukNationalUniversity,12,Gaeshin-dong,Heungduk-gu,Cheongju,Chungbuk,361-763,KoreaCorrespondingauthor:JinTaeHong,jinthong@chungbuk.
ac.
krReceived:26Dec2008Revisionsrequested:18Feb2009Revisionsreceived:17Jul2009Accepted:30Sep2009Published:30Sep2009ArthritisResearch&Therapy2009,11:R145(doi:10.
1186/ar2819)Thisarticleisonlineat:http://arthritis-research.
com/content/11/5/R1452009Banetal.
;licenseeBioMedCentralLtd.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
AbstractIntroductionSulfurcompoundsisolatedfromgarlicexertanti-inflammatoryproperties.
Werecentlyisolatedthiacremonone,anovelsulfurcompoundfromgarlic.
Here,weinvestigatedtheanti-inflammatoryandarthritispropertiesofthiacremononethroughinhibitionofNF-κBsinceNF-κBisknowntobeatargetmoleculeofsulfurcompoundsandanimplicatedtranscriptionfactorregulatinginflammatoryresponsegenes.
MethodsTheanti-inflammatoryandarthritiseffectsofthiacremoneininvivowereinvestigatedin12-O-tetradecanoylphorbol-13-acetate-inducedearedema,carrageenanandmycobacteriumbutyricum-inducedinflammatoryandarthritismodels.
Lipopolysaccharide-inducednitricoxide(NO)productionwasdeterminedbyGriessmethod.
TheDNAbindingactivityofNF-κBwasinvestigatedbyelectrophoreticmobilityshiftassay.
NF-κBandinduciblenitricoxidesynthetase(iNOS)transcriptionalactivitywasdeterminedbyluciferaseassay.
ExpressionofiNOSandcyclooxygenase-2(COX-2)wasdeterminedbywesternblot.
ResultsTheresultsshowedthattopicalapplicationofthiacremonone(1or2μg/ear)suppressedthe12-O-tetradecanoylphorbol-13-acetate-induced(1μg/ear)earedema.
Thiacremonone(1-10mg/kg)administereddirectlyintotheplantarsurfaceofhindpawalsosuppressedthecarrageenan(1.
5mg/paw)andmycobacteriumbutyricum(2mg/paw)-inducedinflammatoryandarthriticresponsesaswellasexpressionofiNOSandCOX-2,inadditiontoNF-κBDNA-bindingactivity.
Infurtherinvitrostudy,thiacremonone(2.
5-10μg/ml)inhibitedlipopolysaccharide(LPS,1μg/ml)-inducednitricoxide(NO)production,andNF-κBtranscriptionalandDNAbindingactivityinadosedependentmanner.
TheinhibitionofNObythiacremononewasconsistentwiththeinhibitoryeffectonLPS-inducedinduciblenitricoxidesynthase(iNOS)andCOX-2expression,aswellasiNOStranscriptionalactivity.
Moreover,thiacremononeinhibitedLPS-inducedp50andp65nucleartranslocation,resultinginaninhibitionoftheDNAbindingactivityoftheNF-κB.
TheseinhibitoryeffectsonNF-κBactivityandNOgenerationweresuppressedbyreducingagentsdithiothreitol(DTT)andglutathione,andwereabrogatedinp50(C62S)-mutantcells,suggestingthatthesulfhydrylgroupofNF-κBmoleculesmaybeatargetofthiacremonone.
ConclusionsThepresentresultssuggestedthatthiacremononeexerteditsanti-inflammatoryandanti-arthriticpropertiesthroughtheinhibitionofNF-κBactivationviainteractionwiththesulfhydrylgroupofNF-κBmolecules,andthuscouldbeausefulagentforthetreatmentofinflammatoryandarthriticdiseases.
AIA:adjuvant-inducedarthritis;CCK-8:cellcountingkit-8;CO2:carbondioxide;COX-2:cyclooxygenase-2;DMEM:Dulbecco'smodifiedeaglemedium;DTT:dithiothreitol;ECL:enhancedchemiluminescence;EMSA:electromobilityshiftassay;GFP:greenfluorescentprotein;ICR:InstituteofCancerResearch;IκB:inhibitoryκB;IFN:interferon;IKK:inhibitoryκBkinase;IL:interleukin;iNOS:induciblenitricoxidesynthetase;LPS:lipopoly-saccharide;MMP:matrixmetalloproteinases;NF:nuclearfactor;NO:nitricoxide;PBS:phosphate-bufferedsaline;SD:Sprague-Dawley;TNF:tumornecrosisfactor;TPA:12-O-tetradecanoylphorbol-13-acetate.
ArthritisResearch&TherapyVol11No5Banetal.
Page2of13(pagenumbernotforcitationpurposes)IntroductionGarlichasbeenusedintraditionalmedicineasafoodcompo-nenttopreventthedevelopmentofcancerandcardiovasculardiseases,bymodifyingriskfactorssuchashypertension,highbloodcholesterolandthrombosis,andpreventingotherchronicdiseasesassociatedwithaging[1-4].
Thesepharma-cologicaleffectsofgarlicareattributedtothepresenceofpharmacologicallyactivesulfurcompoundsincludingdiallylsulfide,diallyldisulfide,allicin,anddipropylsulfide.
Thesecompoundshavebeenknowntoincreasetheactivityofenzymesinvolvedinthemetabolismofcarcinogens[5],andhaveanti-oxidativeactivities[6]aswellasanti-inflammatoryeffectsinvitroandinvivo[7-13].
Despitetheirwidespreadmedicinaluseandanti-inflammatoryeffects,littleisknownaboutthecellularandmolecularmechanismsofthecompo-nentsofgarlic.
Nuclearfactor(NF)-κBisafamilyoftranscriptionfactorsthatincludesRelA(p65),NF-κB1(p50andp105),NF-κB2(p52andp100),c-Rel,andRelB.
Thesetranscriptionfactorsaresequesteredinthecytoplasmbyinhibitory(I)κBs,whichpre-ventNF-κBactivation,andinhibitnuclearaccumulation.
ThedegradationofIκBsfacilitatesthemigrationofNF-κBintothenucleus,wheretheytypicallyformhomodimersorheterodim-ersthatbindtothepromotersofmanyinflammatoryresponsegenesandactivatetranscription[14,15].
Targeteddisruptionofthep50subunitofNF-κBreducesventricularruptureaswellasimprovingcardiacfunctionandsurvivalaftermyocar-dialinfarction,aproinflammatorydisease[16,17].
Itisalsowellappreciatedthatp50homodimersareimportantintheinflam-matorycytokinegenes,andthattheratioofp50relativetotheotherRel(p65)familymembersinthenucleusislikelytobeadeterminingfactorforgeneexpressionofinflammation.
NF-κBregulateshostinflammatoryandimmuneresponsepropertiesbyincreasingtheexpressionofspecificcellulargenes[18].
Theseincludethetranscriptionofvariousinflammatorycytokines,suchasIL-1,IL-2,IL-6,IL-8andTNF-α[19],aswellasgenesencodingcyclooxygenase-2(COX-2)andiNOS.
Asaresult,inhibitionofsignalpathwaysleadingtoinactivationofNF-κBisnowwidelyrecognizedasavalidstrategycombatingautoimmune,inflammatory,andosteolyticdiseases[20].
SeveralstudieshaveshownthatinhibitorsofNF-κBmaybeusefulinthetreatmentofinflammatorydiseasesincludingarthritis[21-23].
Anti-inflammatorydrugshavealsobeendem-onstratedtoinhibittheNF-κBpathway[24-26].
WerecentlyalsofoundthatinhibitionofNF-κBcanameliorateinflamma-toryresponses,andarthritis[27-30].
Severalrecentinvestiga-tionshaveshownthatsulfurcompoundscaneffectivelyinterferewiththeNF-κBpathway[31-33].
Inaseriesofphar-macologicalstudiesofsulfurcompoundingarlic,wefoundthattheantioxidantpropertiesofgarlic-waterextractisincreasedbyaraiseintheheatingtemperatureoftheextract.
Weisolatedandidentifiedthiacremonone,anovelandmajorsulfurcompund(0.
3%)ingarlic,andfoundthatithashigheranti-oxidantpropertiescomparedwithothersulfurcompounds[34,35].
Wealsoreportedaninhibitoryeffectofthiacre-mononeonNF-κBactivityincoloncarcinomacelllines,inpar-allelwiththeinhibitoryeffectofcoloncellgrowthandinductionofapoptosis[15].
Inthisstudy,weinvestigatedwhetherthiacremononeexertedanti-inflammatoryandarthritiseffectsthroughtheinhibitionofNF-κBactivity.
MaterialsandmethodsChemicalsCharacterizationofanovelsulfurcompoundisolatedfromgar-lic(namedthiacremonone)hasbeendescribedelsewhere[15,34].
ItsstructureisshowninFigure1.
Thiacremononewasresolvedin0.
01%dimethylsulfoxide,andtreatedatsamplesizesof2.
5,5and10μg/mlinculturecells.
CellcultureRAW264.
7,amousemacrophage-likecelllineandTHP-1,ahumanmonocyticcellline,wereobtainedfromtheAmericanTypeCultureCollection(Cryosite,LaneCove,NSW,Aus-tralia).
DMEM,RPMI,penicillin,streptomycin,andfetalbovineserumwerepurchasedfromGibcoLifeTechnologies(Rock-ville,MD,USA).
RAW264.
7cellsweregrowninDMEMwith10%fetalbovineserum,100U/mlpenicillin,and100μg/mlstreptomycinat37°Cin5%carbondioxide(CO2)humidifiedair.
THP-1cellsweregrowninRPMIwith10%fetalbovineserum,0.
05mM2-mercaptoethanol,100U/mlpenicillin,and100μg/mlstreptomycinat37°Cin5%CO2humidifiedair.
CellviabilityassayRAW264.
7cellswereplatedatadensityof104cells/wellin96-wellplates.
Todeterminetheappropriatedosethatisnotcytotoxictothecells,thecytotoxiceffectwasevaluatedinthecellsculturedfor24hoursusingthecellcountingkit-8assayFigure1ChemicalstructureofthiacremononeChemicalstructureofthiacremonone.
Availableonlinehttp://arthritis-research.
com/content/11/5/R145Page3of13(pagenumbernotforcitationpurposes)accordingtothemanufacturer'sinstructions(Dojindo,Gaith-ersburg,MD,USA).
Briefly,10μlofthecellcountingkit-8(CCK-8)solutionwasaddedtocellculture,andincubatedforafurther24hours.
Theresultingcolorwasassayedat450nMusingamicroplateabsorbancereader(Sunrise,Tecan,Swit-zerland).
Eachassaywascarriedoutintriplicate.
NitriteassayRAW264.
7cellswereplatedat2*104cells/wellin96-wellplateandthenincubatedwithorwithoutlipopolysaccharide(LPS;1μg/ml)intheabsenceorpresenceofvariousconcen-trationsofthiacremononefor24hours.
Thenitriteaccumula-tioninthesupernatantwasassessedbyGriessreaction[36].
Each50μlofculturesupernatantwasmixedwithanequalvol-umeofGriessreagent(0.
1%N-(1-naphthyl)-ethylenediamine,1%sulfanilamidein5%phophoricacid)andincubatedatroomtemperaturefor10minutes.
Theabsorbanceat550nmwasmeasuredinanautomatedmicroplatereader,andaseriesofknownconcentrationsofsodiumnitritewasusedasastandard.
ElectromobilityshiftassayElectromobilityshiftassay(EMSA)wasperformedasdescribedpreviously[15].
Briefly,1*106cells/mlwaswashedtwicewith1*PBS,followedbytheadditionof1mlofPBS,andthecellswerescrapedintoacoldEppendorftube.
Cellswerespundownat15,000gforoneminutes,andtheresultingsupernatantwasremoved.
SolutionA(50mMHEPES,pH7.
4,10mMKCl,1mMEDTA,1mMEGTA,1mMdithiothreitol,0.
1μg/mlphenylmethylsulfonylfluoride,1μg/mlpepstatinA,1μg/mlleupeptin,10μg/mlsoybeantrypsininhibitor,10μg/mlaprotinin,and0.
5%NonidetP-40)wasaddedtothepelletina2:1ratio(v/v)andincubatedonicefor10minutes.
SolutionC(solutionA+10%glyceroland400mMKCl)wasaddedtothepelletina2:1ratio(v/v)andvor-texedonicefor20minutes.
Thecellswerecentrifugedat15,000gforsevenminutes,andtheresultingnuclearextractsupernatantwascollectedinachilledEppendorftube.
Con-sensusoligonucleotideswereend-labeledusingT4polynucle-otidekinaseand(γ-32P)ATPfor10minutesat37°C.
Gelshiftreactionswereassembledandallowedtoincubateatroomtemperaturefor10minutesfollowedbytheadditionof1μl(50,000to200,000cpm)of32P-labeledoligonucleotideandanother20minutesofincubationatroomtemperature.
Subse-quently1μlofgelloadingbufferwasaddedtoeachreactionandloadedontoa4%nondenaturinggelandelectrophoreseduntilthedyewas75%ofthewaydownthegel.
Thegelwasdriedat80°Cforonehourandexposedtofilmovernightat70°C.
TherelativedensityoftheproteinbandswasscannedbydensitometryusingMyImage(SLB,Seoul,Korea),andquantifiedbyLabworks4.
0software(UVPInc.
,Upland,CA,USA).
TherelativedensityoftheDNA-proteinbindingbandswasscannedbydensitometryusingMyImage(SLB,Seoul,Korea),andquantifiedbyLabworks4.
0software(UVPInc,Upland,CA,USA).
TransfectionandassayofluciferaseactivityRAW264.
7cells(5*106cells)wereplatedin24-wellplatesandtransientlytransfectedwithpNF-κB-Lucplasmid(5*NF-κB;Stratagene,LaJolla,CA,USA)oriNOS-luciferasereporterplasmid[37]orp50(C62S)mutantplasmidsusingamixtureofplasmidandlipofectAMINEPLUSinOPTI-MENaccordingtomanufacturer'sspecification(Invitrogen,Carlsbad,CA,USA).
Cellsweretransientlyco-transfectedwithpEGFP-C1vector(Clontech,PaloAlto,CA,USA)withWelFect-EXPLUStransfectionreagent(WelGENEInc.
,Daegu,Korea)accordingtothemanufacturer'sinstructions.
After24hourstransfection,expressionofgreenfluorescentprotein(GFP)wasdetectedbyfluorescencemicroscopy(DASmicroscope:LeicaMicrosystems,Inc.
,Deefield,IL,USA).
ThetransfectionefficiencywasdeterminedasthenumberofGFP-expressingcellsdividedbythetotalcellnumbercounted*100.
ThetransfectedcellsweretreatedwithLPS(1μg/ml)anddif-ferentconcentrations(2.
5,5and10μg/ml)ofthiacremononeforeighthours.
Luciferaseactivitywasmeasuredbyusingtheluciferaseassaykit(Promega,Madison,WI,USA),andread-ingtheresultsonaluminometerasdescribedbythemanufac-turer'sspecifications(WinGlow,BadWildbad,Germany).
WesternblotanalysisWesternblotanalysiswasperformedasdescribedpreviously[15].
Themembranewasincubatedforfivehoursatroomtem-peraturewithspecificantibodies:mousepolyclonalantibodiesagainstp50andp-IκB(1:500dilution,SantaCruzBiotechnol-ogyInc.
SantaCruz,CA,USA),rabbitpolyclonalforp65andIκB(1:500dilution,SantaCruzBiotechnologyInc.
,SantaCruz,CA,USA)andiNOSandCOX-2(1:1000dilution,Cay-manChemical,AnnArbor,MI,USA).
Theblotwasthenincu-batedwiththecorrespondingconjugatedanti-mouseimmunoglobulinG-horseradishperoxidase(1:4,000dilution,SantaCruzBiotechnologyInc.
,SantaCruz,CA,USA).
Immu-noreactiveproteinsweredetectedwiththeenhancedchemi-luminescence(ECL)westernblottingdetectionsystem(GEHealthcareBiosciences(formerlyAmershamBiosciences),LittleChalfont,Buckinghamshire,UK).
TherelativedensityoftheproteinbandswasscannedbydensitometryusingMyIm-age(SLB,Seoul,Korea),andquantifiedbyLabworks4.
0soft-ware(UVPInc.
,Upland,CA,USA).
Assayof12-O-tetradecanoylphorbol-13-acetate-inducedearedemainmiceThemaleInstituteofCancerResearch(ICR)miceandmaleSprague-Dawley(SD)ratsusedhereweremaintainedinaccordancewiththeNationalInstituteofToxicologicalResearchoftheKoreaFoodandDrugAdministrationguide-linesforthecareanduseoflaboratoryanimals.
TheprotocolwasapprovedbytheInstitutionalAnimalCareandUseCom-mitteeatChungbukNationalUniversity.
12-O-tetradecanoyl-phorbol-13-acetate(TPA;1μg/ear)aloneorincombinationArthritisResearch&TherapyVol11No5Banetal.
Page4of13(pagenumbernotforcitationpurposes)withthiacremonone(1or2μg/ear)inacetone(10μl)wasappliedtotherightearofICRmice.
Controlmicereceivedacetonealone.
Avolume(10μL)ofthiacremonone(1or2μg/ear)containingacetonewasdeliveredtoboththeinnerandoutersurfacesoftheear30minutesafterTPAapplication.
After24hours,thetipoftheearthicknesswasmeasuredusingverniercalipers(MitutoyoCorporation,Kawasaki,Japan),andearpunchbiopsies6mmindiameterweretakenandweighed.
Followingthis,themiceweresacrificedbycer-vicaldislocation.
Theincreaseinthicknessorweightoftheearpuncheswasdirectlyproportionaltothedegreeofinflamma-tion[38].
WefurtherinvestigatedtheexpressionofiNOSandCOX-2bywesternblotanalysis,andtheactivationofNF-κBbyEMSAineachearpunchbiopsies.
Carrageenan-inducedpawedemainflammatorymodelandMycobacteriumbutyricum-inducedarthritismodelTheanti-inflammatoryandanti-arthriticpropertyofthiacre-mononewastestedinmaleSDratsusingthecarrageenanpawedematestaccordingtothemethodofSugishitaandcolleagues[39]andaMycobacteriumbutyricum-inducedarthriticmodelasdescribedelsewhere[27].
Thiacremonone(1or2mg/kg),indomethacin(positivecontrol,10mg/kg)orvehicle(saline)wasadministereddirectlyintotheplantarsur-faceoftherighthindpaw30minutesafterinjectionofcarra-geenan(0.
05ml;3%,w/vinsaline)intothesubplantarareaoftherighthindpaw.
Thevolumesoftheinjectedandcontralat-eralpawsweremeasuredatone,two,three,andfourhoursafterinductionofedemausingaplethysmometer(Letica,Comella,Spain).
Wenextinvestigatedtheantiarthriticeffectofthiacremononeinachronicadjuvant-inducedarthritis(AIA)animalmodel.
AIAwaselicitedinSDratsbytheinjectionof0.
1mlofM.
butyricum(10mg/ml)insaline,intothesubplantarareaoftherighthindpaw.
Pawvolumesweremeasuredatthebeginningoftheexperimentusingawater-displacementplethysmometer.
Animalswithedemavaluesof1.
1mllargerthannormalpawswerethenrandomizedintotreatmentgroups.
A10mg/kgdoseofthiacremonone,indomethacin(positivecontrol)orvehicle(saline)wassubcutaneouslyadministeredintotheplantarsurfaceoftherighthindpawfromday1today20postAIAinduction.
Themagnitudeoftheinflammatoryresponsewasevaluatedbymeasuringthevol-umesofbothhindpaws.
Onday21postAIAinduction,ratsunderanesthesiawereplacedonaradiographicboxatadis-tanceof90cmfromanx-raysource.
Radiographicanalysisofarthritichindpawswasperformedusinganx-raymachine(BLD-150RK,HradecKrálové,CzechRepublic)witha40KWexpositionfor0.
01seconds.
Pawswereorientedhorizontally,relativetothedetector.
Radiographswerescoredbyaninves-tigatorwhowasblindedtothetreatmentinformation,usingthefollowingscale:0=nobonedamage,1=tissueswellingandedema,2=jointerosion,and3=boneerosionandosteo-phyteformation.
DataanalysisDatawereanalyzedusingone-wayanalysisofvariancefol-lowedbyTuckeytestasaposthoctest.
Differenceswerecon-sideredsignificantatP<0.
05.
ResultsInhibitoryeffectofthiacremononeonTPA-inducedearedemainmiceThiacremononewasevaluatedforitsanti-inflammatoryactivityagainstTPA-inducededemaformationandinflammatorygeneexpressionaswellasNF-κBactivityinmice.
Topicalapplica-tionof1μgTPAinacetonetotheearofamouseincreasedtheaverageweightoftheearfrom4.
3mgto7.
2mgat24hourspostapplication(Figure2a).
Topicalapplicationof1or2μgthiacremononetogetherwith1μgTPAtotheearsofmiceinhibitedtheTPA-inducededemaofmouseearsby44or98%,respectively(Figure2a).
WefurtherinvestigatedtheeffectofthiacremononeoniNOSandCOX-2expressionandNF-κBactivityineachearpunchbiopsiesbywesternblotanalysisandEMSA.
Thiacremononedose-dependentlyinhib-itedTPA-inducedexpressionofiNOSandCOX-2(Figure2b).
ThiacremononealsoinhibitedTPA-inducedNF-κBDNA-bind-ingactivity(Figure2c)aswellasthenucleartranslocationofp50andp65andphosphorylationofIκBα(Figure2d).
Inhibitoryeffectofthiacremononeoncarrageenanandadjuvant-inducedarthritisTheanti-inflammatoryactivityofthiacremononewasalsodem-onstratedinthecarrageenanpawedematestinSDrats.
Directadministrationofthiacremonone(1or2mg/kg)intotheplantarsurfaceoftherighthindpaw30minutesbeforeinjec-tionofcarrageenan(0.
05ml;3%,w/vinsalineintothesub-plantarareaoftherighthindpaw,1.
5mg/paw)showedgreatlyreducedcarrageenan-inducedpawedema(40%reductioncomparedtocontralateralpaws;Figure3a).
Adose-depend-entinhibitionoftheexpressionofiNOSandCOX-2(Figure3b)aswellastheactivationofNF-κBDNA-bindingactivity(Figure3c)accompaniedbyaninhibitionofp50andp65nucleartranslocationandphosphorylationofIκBα(Figure3d)wasalsoreported.
InachronicratAIAmodel,oraladministra-tionofthiacremonone(5or10mg/kg)for20dayssignificantlyreducedadjuvant-inducedhindpawedemaformation(Figure4a).
Aradiographicexaminationofhindpawsrevealedtissueswellingatthepawofadjuvant-injectedrats.
However,theseeffectsweremarkedlyreducedbythiacremononetreatment,anditsinhibitoryeffectwascomparablewithindomethacin(10mg/kg;Figure4b).
Treatmentwiththiacremononedidnotaffectprogressionofbodyweight,anddidnotshowanybehavioralalternation(datanotshown),suggestingthatthia-cremononeitself(10mg/kg)didnotcauseanytoxicresponse.
Thiacremononedose-dependentlyinhibitedtheexpressionofiNOSandCOX-2(Figure4c).
Italsosuppressedtheactiva-tionofNF-κBDNA-bindingactivity(Figure4d)aswellasthenucleartranslocationofp50andp65andphosphorylationofIκBα(Figure4e).
Availableonlinehttp://arthritis-research.
com/content/11/5/R145Page5of13(pagenumbernotforcitationpurposes)EffectofthiacremononeonNF-κB-luciferaseactivityandNF-κBDNAbindingactivityTotestwhetherthiacremononewasabletoattenuateNF-κB-mediatedpromoteractivity,weusedaluciferasereportergeneexpressedunderthecontrolfiveκBcis-actingelements.
RAW264.
7cellsweretransientlytransfectedwithanNF-κB-dependentluciferasereporterconstructaccordingtotheman-ufacturef'sspecifications(Promega,Madison,WI,USA).
ThecellswerethentreatedwithLPS(1μg/ml)orco-treatedwithLPSandthiacremononeforsixhours.
Treatmentofcellswiththiacremononeresultedinaconcentration-dependentsup-pressionofluciferaseactivityinducedbyLPS(Figure5a).
TodeterminewhetherthiacremononewasalsoabletoinhibittheDNA-bindingactivityofNF-κBinRAW264.
7cells,nuclearextractsfromco-treatedcellswerepreparedandassayedforNF-κBDNA-bindingactivitybyEMSA.
LPSinducedastrongNF-κBDNA-bindingactivitythatwasattenuatedbyco-treat-mentofthecellswiththiacremononeinadose-dependentmanner(Figure5b).
TreatmentofcellswithLPS(1μg/ml)increasedthenucleartranslocationofNF-κBsubunitsp65andp50.
However,inthepresenceofthiacremonone,nucleartranslocationofp50andp65wasinhibitedinadose-dependentmanner(Figure4c).
ThiacremononealsoinhibitedLPS-induceddegradationofIκB-α(increasephosphorylation)inRAW264.
7cells(Figure5c).
WealsofoundthatexposureofRAW264.
7cellstothia-cremononeforonehourinhibitedtheDNA-bindingactivityofNF-κBthatwasinducedbyTNF-α(10ng/ml),IL-1α(10ng/ml)andinterferon-γ(IFN-γ;10ng/ml;Figure5d).
Thedose-Figure2EffectsofthiacremononeonTPA-inducedearedema,andexpressionofiNOSandCOX-2inmiceEffectsofthiacremononeonTPA-inducedearedema,andexpressionofiNOSandCOX-2inmice.
(a)12-O-tetradecanoylphorbol-13-acetate(TPA;1μg/ear)aloneortogetherwiththiacremonone(Thia;1or2μg/ear)in10μlacetonewastopicallyappliedtotherightearofInstituteofCan-cerResearch(ICR)mice(n=6).
ThethicknessorweightoftheearpunchesweredeterminedasdescribedinMaterialsandMethods.
(b)Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andtheexpressionofinduciblenitricoxidesynthetase(iNOS)andcyclooxygenase-2(COX-2)inmiceearedematissues(2lanes/eachgroup)wasdetectedbywesternblottingusingspecificantibodies.
β-actinpro-teinwasusedasaninternalcontrol.
(c)DNA-bindingactivityofnuclearfactor(NF)-κBwasdeterminedbyelectromobilityshiftassay(EMSA)innuclearextractsfrommiceearedematissues(2lanes/eachgroup)asdescribedinMaterialsandMethods.
(d)Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andnucleartranslocationofp50andp65,anddegradationofinhibitory(I)κBinmiceearedematis-sues(2lanes/eachgroup)wasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
Valuesaremean±standarddeviation(n=6).
#indicatessignificantlydifferentfromcontrolgroup(P<0.
05).
*P<0.
05indicatestatisticallysignificantdiffer-encesfromtheTPA-treatedgroup.
ArthritisResearch&TherapyVol11No5Banetal.
Page6of13(pagenumbernotforcitationpurposes)dependentinhibitoryeffectofthiacremononeonLPS-inducedDNAbindingactivityofNF-κBwasalsoseeninTHP-1cells(Figure5e).
ThisDNA-bindingactivityofNF-κBwasconfirmedbycompetitionassaysaswellasbysupershiftassays.
Inthepresenceofap50antibody,theDNA-bindingactivitiesofNF-κBshowedasupershift.
However,inthepresenceofap65antibody,theDNA-bindingactivityofNF-κBwasdecreasedwithoutasupershift,suggestingthatp50mightbeatargetofthiacremonone,interferingwiththeDNA-bindingactivityofNF-κB(Figure5c).
EffectofthiacremononeonLPS-inducedNOproductionaswellasexpressionofiNOSandCOX-2inRAW264.
7cellsTheeffectofthiacremonone(2.
5,5,10μg/ml)onLPS-inducedNOproductioninRAW264.
7cellswasinvestigatedbymeasuringtheaccumulatednitrite,asestimatedbyGriessreaction,intheculturemedium.
Afterco-treatmentwithLPSandthiacremononefor24hours,LPS-inducednitriteconcen-trationinthemediumwasdecreasedremarkablyinaconcen-tration-dependentmanner.
TheIC50valueofthiacremononeininhibitingLPS-inducedNOproductionwas8μM(Figure6a).
ToinvestigatewhethertheinhibitoryeffectofthiacremononeaffectedNOproductionviainhibitionofcorrespondinggeneFigure3Effectofthiacremononeoncarrageenan-inducedarthritisinratsEffectofthiacremononeoncarrageenan-inducedarthritisinrats.
(a)Thiacremonone(Thia;1and2mg/kg)orindomethacin(Indo;10mg/kg)orvehicle(saline)wasorallyadministered30minutesbeforecarrageenan(0.
05ml;3%,w/vinsaline)intotheplanterareaoftherighthindpawofrat(n=10).
Thevolumesoftheinjectedpawsweremonitoredforfourhoursin10ratspereachgroupasdescribedinMaterialsandMethods.
(b)Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andtheexpressionofinduciblenitricoxidesynthetase(iNOS)andcyclooxygenase-2(COX-2)inratpawarthritistissueswasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
(c)DNA-bindingactivityofnuclearfactor(NF)-κBwasdeterminedbyelectromobilityshiftassay(EMSA)innuclearextractsfrommicepawarthritistissues(3lanes/eachgroup)asdescribedinMaterialsandMethods.
(d)Equalamountsoftotalproteins(40μg/lane)weresub-jectedto10%SDS-PAGE,andnucleartranslocationofp50andp65,anddegradationofinhibitory(I)κBinratpawarthritistissueswasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
Valuesaremean±standarddeviation(n=10).
#indi-catessignificantlydifferentfromcontrolgroup(P<0.
05).
*P<0.
05indicatestatisticallysignificantdifferencesfromthecarrageenan-treatedgroup.
Availableonlinehttp://arthritis-research.
com/content/11/5/R145Page7of13(pagenumbernotforcitationpurposes)expression,iNOSluciferaseactivityandexpressionofiNOSandCOX-2wasdetermined.
TranscriptionalregulationofiNOSexpressionbythiacremononewasdeterminedinRAW264.
7transfectedwithiNOS-luciferaseconstructcontainingmurineiNOSpromoter(-1592/+183)fusedtoluciferasegeneasareporter[39].
ThiacremononeinhibitedLPS-inducediNOSluciferaseactivityinaconcentration-dependentmanner(Figure6b).
UponLPStreatmentfor24hours,iNOSexpres-sionwasalsosignificantlyincreasedinRAW264.
7cells,andco-treatmentofcellswithLPSanddifferentconcentrationofthiacremononedecreasedLPS-inducediNOSexpressioninaconcentration-dependentmanner(Figure6c).
InagreementwiththeinhibitoryeffectonNOgeneration,thedensitometrydatashowedthattheiNOSexpressionwasinhibitedbythia-cremononeinaconcentration-dependentmanner.
AsNOcaninduceCOX-2expression,andCOX-2isalsoanenzymetoregulateinflammation,theexpressionofCOX-2wasinvesti-gated.
ConsistentwiththeinhibitoryeffectoniNOSexpres-sion,thiacremononeinhibitedLPS-inducedCOX-2expression,buttheextentwasmuchlessthanoniNOS(Fig-ure6c).
Figure4Effectofthiacremononeonadjuvant-inducedarthritisinratsEffectofthiacremononeonadjuvant-inducedarthritisinrats.
(a)Thiacremonone(Thia;10mg/kg)andindomethacin(Indo;10mg/kg)wereorallyadministeredfor20daysafterinjectionofadjuvantintotheplantarsurfaceofrighthindpawof10ratspergroup.
Hindpawvolumeandclinicalscoreweredeterminedfor20daysasdescribedinMaterialsandMethods.
(b)Aradiographicexaminationofhindpawsrevealedtissueswellingatthepawafter20days.
Theclinicalvaluewasdeterminedin10ratsasdescribedinMaterialsandMethods.
(c)Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andtheexpressionofinduciblenitricoxidesynthetase(iNOS)andcyclooxygenase-2(COX-2)inratpawarthritistissues(3lanes/eachgroup)wasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcon-trol.
(d)DNA-bindingactivityofnuclearfactor(NF)-κBwasdeterminedbyelectromobilityshiftassay(EMSA)innucleusextractfromratpawarthritistissues(3lanes/eachgroup)asdescribedinMaterialsandMethods.
(e)Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andnucleartranslocationofp50andp65,anddegradationofinhibitory(I)κBinratpawarthritistissueswasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
Valuesaremean±standarddeviation(n=10).
#indicatessignificantlydif-ferentfromcontrolgroup(P<0.
05).
*indicatessignificantlydifferentfromtheMycobacteriumbutyricum-treatedgroup(P<0.
05).
ArthritisResearch&TherapyVol11No5Banetal.
Page8of13(pagenumbernotforcitationpurposes)TodisprovetheinhibitoryeffectofthiacremononeonNOpro-ductionviainhibitionofcellgrowth,thecytotoxiceffectofthi-acremononewasevaluatedintheabsenceorpresenceofLPSintheRAW264.
7cellsbyCCK-8assay.
Thiacremonone(upto10μg/ml)didnotaffectthecellviabilityintheabsenceofLPS(datanotshown)orthepresenceofLPSinRAW264.
7cells(Figure6d).
Therefore,thiacremononeinhibitedLPS-inducedNOproductioninRAW264.
7cellswithoutanytoxiceffect.
Suppressionofthiacremonone-inducedinhibitionofDNAbindingactivityofNF-κBandcellgrowthbythiolreducingagents,andinthecellstransfectedwithmutantp50WefurthertestedwhethertheinhibitionofNF-κBwasduetoaninteractionbetweenthesulfhydrylgroupofthep50subunitofNF-κBandthiacremonone,aspreviouslyseenincoloncan-cercells[15].
Cellswereco-treatedwiththiacremononeandreducingagents,dithiothreitol(DTT)orglutathioneforoneFigure5EffectofthiacremononeonLPS-inducedNF-κBactivationinRAW264.
7andTHP-1cellsEffectofthiacremononeonLPS-inducedNF-κBactivationinRAW264.
7andTHP-1cells.
(a)RAW264.
7cellsweretransfectedwithp-NF-κB-Lucplasmid(5*nuclearfactor(NF)-κB),andthentreatedwithlipopolysaccharide(LPS;1μg/ml)aloneorincombinationwiththiacremonone(Thia;2.
5,5,10μg/ml)at37°Cforsixhours.
LuciferaseactivitywasthendeterminedasdescribedinMaterialsandMethods.
(b)TheDNA-bindingactivityofNF-κBwasinvestigatedusingelectromobilityshiftassay(EMSA)asdescribedinMaterialsandMethods.
NuclearextractsfromRAW264.
7cellswithLPSalone(1μg/mL)orincombinationwiththiacremonone(2.
5,5,10μg/ml)weresubjectedtoDNA-bindingreactionswith32Pend-labeledoligonucleotidespecifictoNF-κB.
ThespecificDNA-bindingactivityofNF-κBcomplexisindicatedbyanarrow.
(c)Forcompetitionassays,nuclearextractsfromRAW264.
7cellstreatedwithLPS(1μg/ml)wereincubatedforonehourbeforeEMSAwithunlabeledNF-κBoligonucleotideorlabeledNF-κBoligonucleotide.
Forsupershiftassays,nuclearextractsfromRAW264.
7cellstreatedwithLPS(1μg/ml)wereincubatedforonehourbeforeEMSAwithspecificantibodiesagainstthep50andp65NF-κBisoforms.
SSindicatessupershiftband.
(d)Cellstreatedwith1μg/mLofLPSonlyorLPSplusdifferentconcentrations(2.
5,5,10μg/ml)ofthiacremononeat37°Cforonehour.
Equalamountsoftotalprotein(40μg)weresubjectedto10%SDS-PAGE.
Nucleartranslocationofp50andp65,anddegradationofinhibitory(I)κBweredetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
(e)NuclearextractsfromRAW264.
7cellswithanotherinduceralone(TNF-α(10ng/ml),IL-1α(10ng/ml),IFN-γ(10ng/ml))orincombinationwiththiacremonone(10μg/ml)weresubjectedtoDNA-bindingreactionswith32Pend-labeledoligonucleotidespecifictoNF-κB.
ThespecificDNA-bindingofNF-κBcomplexisindicatedbyanarrow.
(f)NuclearextractsfromTHP-1cellswithLPSalone(1μg/mL)orincombinationwiththiacremonone(2.
5,5,10μg/ml)weresubjectedtoDNA-bindingreactionswith32Pend-labeledoligonucleotidespecifictoNF-κB.
ThespecificDNA-bindingofNF-κBcomplexisindicatedbyanarrow.
Values(A,BandC)aremean±standarddeviationofthreeindependentexperimentsperformedintriplicate.
#indicatessignificantlydifferentfromcontrolgroup(P<0.
05).
*indicatessignificantlydifferentfromtheLPS-treatedgroup(P<0.
05).
Availableonlinehttp://arthritis-research.
com/content/11/5/R145Page9of13(pagenumbernotforcitationpurposes)hour,andthentheDNA-bindingactivityofNF-κBwasexam-ined.
Wefoundthatthesereducingagentssignificantlysup-pressedtheinhibitoryeffectsofthiacremononeontheDNA-bindingandtranscriptionalactivityofNF-κB(Figures7a,b).
Furthermore,DTTandglutathionesuppressedtheinhibitoryeffectsofthiacremononeonNOgeneration(Figure7c)andiNOSluciferaseactivity(Figure7d).
TakingintoconsiderationthesupershiftoftheDNA-bindingactivitiesofNF-κBuponadditionofanti-p50antibody,andthesuppressiveeffectofDTTandglutathioneonthiacremonone-inducedinhibitionofDNA-bindingactivityofNF-κBandNOgeneration,wepostulatedthatthesulfhydrylresidueinp50mightbeatargetofthiacremonone.
Totestthispostulation,wefurtherstudiedtheinhibitoryeffectsofthiacremononeontheDNA-bindingactivityofNF-κBandNOgenerationinp50mutantcells(C62S),wherethecysteineresidueat62ofp50wasreplacedbyserine.
Asexpected,therewasareductionintheinhibitoryeffectofthiacremononeontheDNA-bindingactivityofNF-κB(Figure7e)andonNOgeneration(Figure7f)Figure6EffectofthiacremononeonLPS-inducedNOgeneration,expressionofiNOSandCOX-2andcellviabilityinRAW264.
7cellsEffectofthiacremononeonLPS-inducedNOgeneration,expressionofiNOSandCOX-2andcellviabilityinRAW264.
7cells.
(a)Thecellsweretreatedwith1μg/mLoflipopolysaccharide(LPS)onlyorLPScombinedwithdifferentconcentrations(2.
5,5,10μg/ml)ofthiacremonone(Thia)at37°Cfor24hours.
Nitricoxide(NO)generationwasdeterminedinculturemediumasdescribedinMaterialsandMethods.
(b)Thecellsweretran-sientlytransfectedwithaninduciblenitricoxidesynthetase(iNOS)-luciferaseconstruct,andactivatedwithLPS(1μg/ml)aloneorLPScombinedwiththeindicatedconcentrationsofthiacremononeforeighthours.
Luciferaseactivitywasthendetermined.
Quantificationofbandintensitiesfromthreeindependentexperimentalresultswasdeterminedbyadensitometry,andthevalueunderthebandindicatefolddifference(average)fromuntreatedcontrolgroup.
(c)Thecellsweretreatedwith1μg/mLofLPSonlyorLPScombinedwithdifferentconcentrations(2.
5,5,10μg/ml)ofthi-acremononeat37°Cfor24hours.
Equalamountsoftotalproteins(40μg/lane)weresubjectedto10%SDS-PAGE,andtheexpressionofiNOSandCOX-2wasdetectedbywesternblottingusingspecificantibodies.
β-actinproteinwasusedasaninternalcontrol.
(d)RAW264.
7cellsweretreatedwithvariousdoses(2.
5,5,10μg/ml)ofthiacremononefor24hours.
Morphologicalchangeswereobservedundermicroscope(magnifica-tion,*200).
CellviabilitywasdeterminedbytheCCK-8assaydescribedinMaterialsandMethods.
CellswereincubatedwiththiacremononeintheabsenceorpresenceofLPS.
Resultsweregiveninpercentagerelatedtountreatedcontrols.
Allvalues(A,B,CandD)representthemeans±standarddeviationofthreeindependentexperimentsperformedintriplicate.
#indicatessignificantlydifferentfromcontrolgroup(P<0.
05).
*indi-catessignificantlydifferentfromtheLPS-treatedgroup(P<0.
05).
ArthritisResearch&TherapyVol11No5Banetal.
Page10of13(pagenumbernotforcitationpurposes)inthesep50mutantcells.
Theseresultsclearlysuggestedthatthiacremononemediateditseffectsthroughmodulationofcysteineresiduesofthep50subunitofNF-κB.
DiscussionTheactivationofiNOScatalyzestheformationofalargeamountofNO,whichplaysakeyroleinthepathogenesisofavarietyofinflammatorydiseases[40-43].
ActivationofNF-κBiscriticalintheinductionofiNOS[44-46].
Therefore,agentsthatinhibitNF-κB,resultingindecreasediNOSexpressionandNOgeneration,mayhavebeneficialtherapeuticeffectsinthetreatmentofinflammatorydiseases.
Thiacremononeinhib-itedLPS-inducediNOSandCOX-2expressionaccompaniedbyareductioninNOgeneration.
ConsistentwithitsinhibitoryactivityonNOproduction,thiacremononealsodecreasedNF-κBactivity.
TheinhibitoryeffectsofthiacremononeontheNF-κBDNA-bindingactivitieswerealsodemonstratedinmacro-phagesstimulatedbyTNF-α,IFN-γ,andIL-1α.
ThepromoteroftheiNOSgenecontainstwomajordiscreteregionssyner-gisticallyfunctioningtowardthebindingoftranscriptionfactorNF-κB,whichismainlyactivatedbyLPSandIFN-γ,andIL-1α[47,48].
Therefore,thesedataindicatedthatthiacremononecouldinterferewithNF-κB-mediatedsignalsinvolvingthepro-ductionofpro-inflammatorymoleculeNO,andthusgiveanti-inflammatoryresponses.
InvivoanimalstudiesshowedthatthiacremononeinhibitedTPA,carrageenanandM.
butyricum-inducedpawedema.
Treatmentofthiacremononealsoresultedinagreatreductionoftissueswellingandosteophyteformationinachronicarthri-tisratmodel.
Paralleledwiththeseinhibitoryeffects,thiacre-mononealsoinhibitedTPA,carrageenanandM.
butyricum-Figure7AbolitionoftheinhibitoryeffectofthiacremononebyDTTandglutathioneGSH,andinthecellsharboringmutantp50onNOgenerationandDNAbindingactivationofNF-κBAbolitionoftheinhibitoryeffectofthiacremononebyDTTandglutathioneGSH,andinthecellsharboringmutantp50onNOgenerationandDNAbindingactivationofNF-κB.
(a)RAW264.
7cellsgrowninsix-wellplateswerecotreatedwithindicatedconcentrationsofdithiothreitol(DTT)(100nM)orglutathione(GSH;100μM)withthiacremonone(Thia;10μg/ml)foronehour.
Nuclearextractswerethenpreparedandexaminedbyelectro-mobilityshiftassay(EMSA)asdescribedinMaterialsandMethods.
(b)Thecellsweretransientlytransfectedwithnuclearfactor(NF)-κB-luciferaseconstruct,andwereco-treatedwithindicatedconcentrationsofDTT(1to100nM)orGSH(1to100μM)withthiacremonone(10μg/ml)foreighthours,andthentheluciferaseactivitywasdetermined.
(c)Thecellswereco-treatedwithindicatedconcentrationsofDTT(1to100nM)orGSH(1to100μM)with1μg/mLoflipopolysaccharide(LPS)onlyorLPSplusthiacremonone(10μg/ml)at37°Cfor24hours.
Nitricoxide(NO)generationwasdeterminedinculturemediumasdescribedinMaterialsandMethods.
(d)Thecellsweretransientlytransfectedwithinduciblenitricoxidesyn-thetase(iNOS)-luciferaseconstruct,andwereco-treatedwithindicatedconcentrationsofDTT(1to100nM)orGHS(1to100μM)withthiacre-monone(10μg/ml)foreighthours,andthentheluciferaseactivitywasdetermined.
(e)RAW264.
7cellsweretransfectedwithp50mutant(C62S)plasmidat37°Cforsixhours,andthenNF-κBDNA-bindingactivitywasdeterminedafteronehouroftreatmentwiththiacremononebyelectromobil-ityshiftassay(EMSA)asdescribedinMaterialsandMethods.
(f)NOgenerationwasdeterminedinculturemediumasdescribedinMaterialsandMethods.
RAW264.
7cellsweretransfectedwithp50mutant(C62S)plasmidat37°Cforsixhours,andthenNOgenerationwasdeterminedafter24hourstreatmentwiththiacremononeasdescribedinMaterialsandMethods.
Allvaluesrepresentthemeans±standarddeviationofthreeinde-pendentexperimentsperformedintriplicate.
#indicatessignificantlydifferentfromcontrolgroup(P<0.
05).
*P<0.
05indicatestatisticallysignifi-cantdifferencesfromtheLPS-treatedgroup.
Availableonlinehttp://arthritis-research.
com/content/11/5/R145Page11of13(pagenumbernotforcitationpurposes)inducediNOSandCOX-2expression,aswellasNF-κBactiv-ityinvivo.
ThiacremononeinhibitedtheproductionofTNF-αaswellastheexpressionofmatrixmetalloproteinases(MMP-3and9)andchemokinesinthesetissues(datanotshown).
ActivationoftheNF-κBpathwayresultsinthetransactivationofamultitudeofresponsivegenesthatcontributetowardtheinflammatoryphenotype,includingTNF-αfrommacrophages,MMPsfromsynovialfibroblastsandchemokinesthatrecruitimmunecellstotheinflamedpannus.
Thisislargelyaconse-quenceoftheactivationoftheNF-κBpathwaythatinvolveshomodimersandheterodimersofp50/p65[49].
Wethusspeculatedthattheinvivoeffectsofthiacremononeonarthriticmodelsweremediatedbyitscombinedinhibitoryactionsonmultipleresponsesofsynovialcellsandinflamma-torycellsthroughtheinactivationofNF-κB.
Interestingly;wealsofoundthatthiacremononeinhibitedNF-κBandiNOSexpressioninculturedTHP-1monocytes.
Inlightofthesedata,theresultsofourstudyindicatethatinhibitionofNF-κBbythi-acremononecouldbebeneficialforthetreatmentofinflamma-torydiseasessuchasarthritis.
TheinhibitionofNF-κBactivationbythiacremononewasfoundtobesuppressedbytreatmentofcellswithreducingagentssuchasDTTandglutathione.
ThiswasaccompaniedbyasuppressionoftheinhibitoryeffectofthiacremononeonNOgeneration.
Thus,itispossiblethattheinhibitoryeffectsofthiacremononeonNF-κBactivitymaybemediatedbyoxidiz-ingthecriticalcysteineresiduepresentinNF-κBsubunits.
Wealsofoundthatinthepresenceofanantibodyagainstp50butnotp65,theNF-κBDNA-bindingactivitywassupershifted.
FurtherevidenceshowedthattheinhibitoryeffectofNOgen-erationandNF-κBactivitybythiacremononeinp50mutant(cysteinewasreplacedwithalanine)cellswassuppressed.
Itisnoteworthythatp50/p50homodimerismoreimportantthanp50/p65heterodimersintheregulationofinflammatorycytokinegenerationandinflammatorydiseases.
Itwasfoundthatincreasedcytokinelevelsinp50knockoutmicemayberelatedtothedifferenttranscriptionalactivityofp50/p50homodimerratherthanp65/p50heterodimerorp65/p65homodimer[50].
Targeteddisruptionofthep50subunitofNF-κBreducesatheroscleroticlesionswithaninflammatoryphe-notypeaswellasventricularruptureaftermyocardialinfarc-tion,aproinflammatorydisease[51,52].
Theseresultssuggestthatp50/p50maybemoreimportanttorelayinflammatorygeneexpressionthanthatofp65/p50orp65/65intheinflam-matoryresponses.
Therefore,therestudiessupportthepossi-bilitythatthesulfhydrylresidueofp50maybeatargetofthiacremononeinthepresentstudy.
Previousourstudydem-onstratedthatthiacremononeinhibitedcancercellgrowththroughinhibitionofNF-κB,andmaybep65isthetargetofthiacremonone[15].
Contrasttotheinflammatoryresponse,inthecancercells,p65maybeimportantintheactivationofNF-κB,andmanyofanti-cancerdrugstargetp65ofNF-κB.
Ourdatainthecancercellstudyisconsistentwiththosepreviouslyreportedfromotherlaboratorywithcaffeicacidphenethylester[53]andsesquiterpenelactoneparthenolide[54].
Sev-eralotherinvestigatorsdemonstratedthatsulfurcompoundsreactwithcysteineresiduesoftargetmoleculesinintracellularsignaltransductionproteinsincludingNF-κBthroughcysteine-cysteineinteractionorotherbindingways,andthusinhibitinflammatoryresponsesanddevelopmentofarthriticrheumatism[14,31,32].
Werecentlyalsodemonstratedthat2-hydroxycinnamaldehyde,asnakevenomtoxinandmelittininhibitinflammatoryresponsesandcancercellgrowththroughmodificationofsulfhydrylresiduesofNF-κBandregulatoryproteins(p50andp65aswellasIκBkinases(IKKs))[27,28,55].
Therefore,theinhibitionofNF-κBactivationbythi-acremononethroughdirectmodificationofp50maybeanimportantmolecularmechanismofthesuppressiveeffectofthiacremononeoninflammatoryresponsesandarthriticreac-tions.
However,inourpresentstudy,thiacremononeinhibitedboththeexpressionofIκBaswellasitsphosphorylation,buttheextentoftheinhibitionofphosphorylationwasmuchgreaterthantheinhibitionofIκBexpression.
Thus,theseresultscouldgivepossibilitiesthatthiacremononecansup-presstheexpressionofIκBandp-IκBaswellasinhibitphos-phorylation.
AsthesulfhydrylgroupofIKKsarealsoimportantintheactivityofIKKsaswellasNF-κB,thiacremononecouldbeeffectiveintheregulationofIKKs.
Wearecurrentlyinvesti-gatingtheseissues.
Theeffectivedoseofthiacremonone(10mg/kg)usedinthischronicAIAstudywascomparablewiththatoftheclassicanti-inflammatorydrugindomethacin.
Wedidnotdetectanysideeffectsofthiacremonone(lossofweightgainandanyobservedtoxicsigns)duringtreatmentfor20days.
Takentogether,thiacremonone,anovelsulfurcompoundisolatedfromgarlicinhibitediNOSexpressionandNOgenerationthroughpreventionofNF-κBactivityinvitro,andamelioratedinflammatoryresponsesandarthriticreactionsinacuteandchronicedemaandarthriticanimalmodels.
Thesedatasuggestthatthiacremononemaybepotentiallybeneficialforthepreventionofinflammatorydiseasessuchasarthriticrheu-matismwithcomparativelylowtoxiceffects.
ConclusionsOurresultsindicatethatthiacremononesuppressedtheTPA-inducedearedema,andcarrageenanandM.
butyricum-inducedarthritisthroughinhibitionofNF-κBDNA-bindingactivityandexpressionofiNOSandCOX-2.
IninvitrostudiesusingRaw264.
7andTHP-1cells,thiacremononealsoinhib-itedLPS-inducedNOproduction,NF-κBactivityandexpres-sionofiNOSandCOX-2,whichareclassicalmarkersofinflammation.
TheseinhibitoryeffectsweresuppressedbyreducingagentssuchasDTTandglutathione,andwereabro-gatedinthecellsexpressingp50(C62S)mutant.
Therefore,weconcludethatthiacremononeexerteditsanti-inflammatoryandanti-arthriticpropertiesthroughtheinhibitionofNF-κBactivationviainteractionwiththesulfhydrylgroupofNF-κBmolecules.
ArthritisResearch&TherapyVol11No5Banetal.
Page12of13(pagenumbernotforcitationpurposes)CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsJTHconceivedthedesignofthisstudyandcoordinatedallphasesofthepreparationofthemanuscript.
JOB,JHOandTMKperformedtheexperiments,andJOBparticipatedinthestatisticalanalysis.
DJKperformedradiographicanalysisofarthritichindpawsandHJisolatedthiacremononefromgarlicandprovided.
SBHparticipatedindataanalysisandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisworkwassupportedbytheKoreaScienceandEngineeringFoun-dation(KOSEF)grantfundedbytheKoreaGovernment(MOST)(R13-2008-00000-00).
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