Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes

24 Pages·2016·8.56 MB·English

RESEARCHARTICLE
Flavonoid Compound Icariin Activates
α
Hypoxia Inducible Factor-1 in Chondrocytes
and Promotes Articular Cartilage Repair
PengzhenWang1,2☯,FengjieZhang2,3☯,QilingHe4,JianqiWang5,HoiTingShiu3,
YinglanShu2,3,WingPuiTsang2,3,ShuangLiang2,3,KaiZhao2,3,ChaoWan1,2,3*
1 MinistryofEducationKeyLaboratoryofRegenerativeMedicine(JinanUniversity-TheChineseUniversity
ofHongKong),Guangzhou510000,China,2 SchoolofBiomedicalSciencesCoreLaboratory,Instituteof
StemCell,GenomicsandTranslationalResearch,ShenzhenResearchInstitute,TheChineseUniversityof
HongKong,Shenzhen518057,China,3 MinistryofEducationKeyLaboratoryofRegenerativeMedicine
(TheChineseUniversityofHongKong-JinanUniversity),SchoolofBiomedicalSciences,Facultyof
Medicine,TheChineseUniversityofHongKong,Shatin,HongKongSAR,China,4 Departmentof
Microbiology,UniversityofAlabamaatBirmingham,Birmingham,AL35294,UnitedStatesofAmerica,
5 DepartmentofChemistry,FacultyofScience,TheChineseUniversityofHongKong,Shatin,HongKong
SAR,China
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS
Abstract
Citation:WangP,ZhangF,HeQ,WangJ,ShiuHT,
ShuY,etal.(2016)FlavonoidCompoundIcariin
ActivatesHypoxiaInducibleFactor-1αin Articularcartilagehaspoorcapabilityforrepairfollowingtraumaordegenerativepathology
ChondrocytesandPromotesArticularCartilage
duetoavascularproperty,lowcelldensityandmigratoryability.Discoveryofnovelthera-
Repair.PLoSONE11(2):e0148372.doi:10.1371/
peuticapproachesforarticularcartilagerepairremainsasignificantclinicalneed.Hypoxia
journal.pone.0148372
isahallmarkforcartilagedevelopmentandpathology.Hypoxiainduciblefactor-1alpha
Editor:ValérieGeoffroy,INSERM-universityParis
(HIF-1α)hasbeenidentifiedasakeymediatorforchondrocytestoresponsetofluctuations
7,FRANCE
ofoxygenavailabilityduringcartilagedevelopmentorrepair.ThissuggeststhatHIF-1αmay
Received:January8,2015
serveasatargetformodulatingchondrocytefunctions.Inthisstudy,usingphenotypiccellu-
Accepted:January18,2016
larscreenassays,weidentifythatIcariin,anactiveflavonoidcomponentfromHerbaEpime-
Published:February3,2016 dii,activatesHIF-1αexpressioninchondrocytes.Weperformedsystemicinvitroandinvivo
Copyright:©2016Wangetal.Thisisanopen analysistodeterminetherolesofIcariininregulationofchondrogenesis.Ourresultsshow
accessarticledistributedunderthetermsofthe thatIcariinsignificantlyincreaseshypoxiaresponsiveelementluciferasereporteractivity,
CreativeCommonsAttributionLicense,whichpermits whichisaccompaniedbyincreasedaccumulationandnucleartranslocationofHIF-1αin
unrestricteduse,distribution,andreproductioninany
murinechondrocytes.ThephenotypeisassociatedwithinhibitingPHDactivitythrough
medium,providedtheoriginalauthorandsourceare
credited. interactionbetweenIcariinandironions.TheupregulationofHIF-1αmRNAlevelsinchon-
drocytespersistsduringchondrogenicdifferentiationfor7and14days.Icariin(10−6M)
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. increasestheproliferationofchondrocytesorchondroprogenitorsexaminedbyMTT,BrdU
incorporationorcolonyformationassays.Icariinenhanceschondrogenicmarkerexpres-
Funding:TheworkwaspartiallysupportedbyHong
KongRGCGRF(475910),NationalNaturalScience sioninamicromasscultureincludingSox9,collagentype2(Col2α1)andaggrecanas
FoundationofChina(NSFC)(81130034),Guangdong determinedbyreal-timePCRandpromotesextracellularmatrix(ECM)synthesisindicated
ScienceandTechnologyDepartment(20130501C),
byAlcianbluestaining.ELISAassaysshowdramaticallyincreasedproductionofaggrecan
andShenzhenSynergisticInnovationProgram
andhydroxyprolineinIcariin-treatedculturesatday14ofchondrogenicdifferentiationas
GJHS20120702105445379toCW.Thefundershad
noroleinstudydesign,datacollectionandanalysis, comparedwiththecontrols.Meanwhile,theexpressionofchondrocytecatabolicmarker
decisiontopublish,orpreparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0148372 February3,2016 1/24
IcariinActivatesHIF-1αtoPromoteCartilageRepair
CompetingInterests:Theauthorshavedeclared genesincludingMmp2,Mmp9,Mmp13,Adamts4andAdamts5wasdownregulatedfollow-
thatnocompetinginterestsexist. ingIcariintreatmentfor14days.Inadifferentiationassayusingbonemarrowmesenchymal
stemcells(MSCs)carryingHIF-1αfloxedallele,thepromotiveeffectofIcariinonchondro-
genicdifferentiationislargelydecreasedfollowingCrerecombinase-mediateddeletionof
HIF-1αinMSCsasindicatedbyAlcianbluestainingforproteoglycansynthesis.Inanalgi-
natehydrogel3Dculturesystem,IcariinincreasesSafraninOpositive(SO+)cartilagearea.
ThisphenotypeisaccompaniedbyupregulationofHIF-1α,increasedproliferatingcell
nuclearantigenpositive(PCNA+)cellnumbers,SOX9+chondrogeniccellnumbers,and
Col2expressioninthenewlyformedcartilage.Coincidewiththemicromassculture,Icariin
treatmentupregulatesmRNAlevelsofSox9,Col2α1,aggrecanandCol10α1inthe3Dcul-
tures.Wethengeneratedalginatehydrogel3DcomplexesincorporatedwithIcariin.The3D
complexesweretransplantedinamouseosteochondraldefectmodel.ICRSIIhistological
scoringat6and12weekspost-transplantationshowsthat3Dcomplexesincorporatedwith
Icariinsignificantlyenhancearticularcartilagerepairwithhigherscoresparticularlyin
selectedparametersincludingSO+cartilagearea,subchondralboneandoverallassess-
mentthanthatofthecontrols.TheresultssuggestthatIcariinmayinhibitPHDactivitylikely
throughcompetitionforcellularironionsandthereforeitmayserveasanHIF-1αactivator
topromotearticularcartilagerepairthroughregulatingchondrocyteproliferation,differentia-
tionandintegrationwithsubchondralboneformation.
Introduction
Articularcartilage,ahighlyorganizedavascularconnectivetissuewithsubstantialdurability,
haslimitedregenerativecapacityfollowingtraumaordegenerativepathology.Thedeficitis
likelyduetoinsufficiencyofresidentmesenchymalstemcells(MSCs)andlowmetabolicactiv-
ityofchondrocytesinarticularcartilage.Currenttherapeuticstrategiesforarticularcartilage
repairhavetwomainfocuses:marrowstimulationandcell/tissue-basedtransplantation[1].
ThemarrowstimulatingsurgeriessuchastranscorticalPridiedrilling,abrasionarthroplasty
andmicrofracture[2,3]inducebloodsupplyandrecruitlocalstem/progenitorcellsintothe
affectedlesionfrombonemarrowthroughthesubchondralbone.Thecell/tissue-basedtrans-
plantationsfillthecartilagedefectsandpromoteregenerationwithautologouschondrocytes,
osteochondralallografts,cartilageallografts,orMSCs[4,5].Bothstrategiesoftenincludebio-
materialsasscaffoldscombinedwithbiomechanicalorbiochemicalsignalstobetterfillthe
defectareas,enhancemarrowstimulation,maintainchondrogenicphenotype,orpromote
chondrogenesisinvivoorinexvivocultures[1].Thebiochemicalsignalswidelystudiedare
growthfactorssuchasTGFbetafamilymembers(e.g.TGF-β1,2,3,andBMPs),IGF-1and
FGF-2thatareidentifiedasfunctionalstimulitopromotechondrogenicdifferentiationand
cartilagegrowth[6–11].However,theexogenousgrowthfactorsarecostlyandsubjecttoquick
degradation,andtheirclinicalefficacyandsafetyremaintobeestablished,raisingthedemand
fornovel,effective,safe,bio-stableandlow-costalternatives.
Chondrogenesisisregulatedbymultiplemechanismsincludingtheaforementionedgrowth
factorsemanatingfromthesurroundingmatrix,cytokines,oxygensupplyandmechanical
force,amongwhichhypoxiaisahallmarkforarticularcartilagedevelopmentandregeneration
andfunctionsasastimulusforinitiationofgeneprogramsregulatingchondrogeniccellprolif-
eration,differentiationandmetabolism.TheMSCsorchondrocytesarereadilylocatedina
PLOSONE|DOI:10.1371/journal.pone.0148372 February3,2016 2/24
IcariinActivatesHIF-1αtoPromoteCartilageRepair
hypoxicmicroenvironment,andrespondtotheoxygenfluctuationsthroughtranscriptionfac-
torhypoxiainduciblefactor-alpha(HIF-α)duringcartilagedevelopmentandrepair.HIF-αis
identifiedasakeymediatorforoxygensensingbymammaliancells.HIFisanαβheterodimer,
inwhichtheαsubunitisinduciblebyhypoxia,whereastheβsubunitisconstitutively
expressedinthenucleus[12].HIF-αexistsasthreeisoformsHIF-1α,HIF-2α,andHIF-3α.
Undernormoxia,HIF-1αishydroxylatedbyprolylhydroxylaseatspecificprolineresidues,
ubiquitinatedthroughinteractionwiththevonHippel-Lindautumorsuppressorprotein
(pVHL),andsubsequentlydegradedbytheproteasome.Underhypoxia,theactivityofthe
HIF-targetingprolylhydroxylaseenzymes(PHD1,PHD2,andPHD3)isinactivated,therefore
HIF-1αisnothydroxylatedandaccumulatedinthecytoplasm,andthentranslocatesintothe
nucleusanddimerizeswithHIF-1βtotransactivatedownstreamtargetgenes[13].
Usinggeneticapproach,itisshownthatHIF-1αisrequiredforchondrocytesurvivaland
growtharrestduringdevelopment[14].HIF-1αdeletioninmouselimbbudmesenchyme
leadstosignificantlyreducedexpressionofSox9,akeytranscriptionfactorofchondrocytedif-
ferentiation,inhypoxicprechondrogeniccondensationresultingindifferentiationarrestand
thussevereskeletalmalformations[15].HIF-1α-nullhypoxicchondrocytesexhibitssignifi-
cantlydecreasedaggrecanandCol2expression,implyingtheimportantroleofHIF-1αincon-
trollinghypoxia-inducedECMsynthesisinchondrocytes[16].Itisalsodemonstratedthatthe
HIF-1αpathwayisactivatedduringboneregeneration[17–20].Prolylhydroxylaseinhibitor,
Deferoxamine(DFO),asanHIF-1αactivator,improvesskeletalrepairthroughpromoting
angiogenesisandcartilaginouscallusformation[20].TheHIF-1αdownstreamtargeterythro-
poietin(EPO)promotesbonehealingthroughenhancingcartilaginouscallusformationand
angiogenesis[21].HypoxicconditionsenhancecartilageformationofhumanMSCsandsup-
presstheirhypertrophicdifferentiation[20,21].Theaboveinformationindicatesthatthehyp-
oxia/HIF-1αpathwaymayserveasamoleculartargettofacilitatechondrogenesis.
Inthisstudy,basedoncellularscreenandfunctionalassays,weidentifyIcariin,aflavonoid
compoundderivedfromHerbaEpimedii,asanactivatorofHIF-1αinchondrocytes.Chondro-
cytestreatedwithIcariinhaveincreasedmRNAandproteinlevelsandnucleartranslocationof
HIF-1α.IcariinincreasestheproliferationandenhancesdifferentiationandECMproduction
ofchondrocytesinmicromassor3Dalginatehydrogelcultures,whichisassociatedwithupre-
gulationofHIF-1α.Inanosteochondraldefectmodelinmice,Icariinpromotesarticularcarti-
lagerepairwithimprovedICRSIIhistologicalscorecomparedwiththecontrols.Theseresults
suggestthatIcariinmayserveasanHIF-1αactivatortopromotearticularcartilagerepair
throughregulatingchondrocyteproliferation,differentiationandECMsynthesis.
MaterialsandMethods
Chemicalandreagents
IcariinwasobtainedfromBeijingAokeBiotechnologyCO.LTD(A0145).Stocksolutionsof
icariinwerepreparedindimethylsulfoxide(DMSO)(SigmaCo,StLouis,Mo)andstoredat
−20°C.Thefinalconcentrationofdimethylsulfoxideusedintheculturewas0.01%(v/v).Iron
(II)sulfateheptahydrate(FeSO )waspurchasedfromSigma(SigmaCo,StLouis,Mo).
4
Luciferasereporterassay
7000C2C12cellswereseededin96-wellplatesfor24handtransfectedwith100ngofHRE-Luc
reporterconstructusingLipofectamineLTXreagent.0.05μgofpRL-CMVplasmidexpressing
Renillialuciferasewasco-transfectedineachwelltomonitorthetransfectionefficiency(Pro-
mega).C2C12cells,amousemyoblastcellline,hadbeenusedinHRE-Lucreporterassays
[22].At24hpost-transfection,cellsweremaintainedinnormalmediumwithorwithout
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
Icariinforfurther24h.Luciferaseactivitywasmeasuredbyperformingdual-luciferase
reporterassayasdescribedbythemanufacturer(Promega).Relativeluciferaseactivitywasnor-
malizedwithRenillaluciferaseactivityineachwellandthencomparedtheluciferaseintensity
ofIcariintreatedgroupwiththecontrolgroup.
IsolationandcultureofmurinechondrocytesandbonemarrowMSCs
PrimarychondrocyteswereisolatedfromthegrowthplatesofnewbornC57BL/6miceusing
themethodadaptedfrompreviouslyestablishedprotocol[23].Briefly,thegrowthplateswere
digestedbycollagenaseIandcollagenaseD,andchondrocyteswereplatedontoa100mm
petridishwithDMEM(GIBCO,Invitrogen)supplementedwith1%(v/v)penicillin/streptomy-
cinsulfate(GIBCO,Invitrogen),1%(v/v)L-glutamine(GIBCO,Invitrogen)and10%(v/v)
fetalbovineserum(FBS,GIBCO,Invitrogen)at37°Cina5%CO humidifiedincubatorunder
2
normoxicconditions(21%oxygen).Onthesecondday,non-adherentcellswereremovedand
theculturesweregentlyrinsedwithPBStwiceandculturedinthefreshmedium.Adherent
cellswereculturedandmediumwaschangedevery2daysuntilthecellsbecameconfluent.
Thechondrocyteswerealsoculturedunderhypoxicconditions(2%oxygen).Cellsatearlypas-
sages(nomorethan3passages)wereusedtoperformtheexperimentssoastominimizethe
possibilityofphenotypicchangesoverprolongedculture.Cellsatthesamepassagewereused
withineachexperimenttoensurethecomparabilityamonggroups.Bonemarrowmesenchy-
malstemcells(MSCs)wereisolatedfrom4-weekoldmicecarryingHIF-1αfloxedallele(HIF-
1αflox/flox)byflushingoutthebonemarrowfromthelongbonesusingphosphate-buffered
saline(PBS),followedbyreplatingandadhesioncultureofthebonemarrowmononuclearcell
population.MSCscolonieswereexpandedfor14daysandthenpassagedforsub-culture.
MSCsweremaintainedinalpha-MinimumEssentialMedium(α-MEM,GIBCO,Invitrogen)
supplementedwithpenicillin(100U/ml),streptomycinsulfate(100g/ml),1%(v/v)L-gluta-
mineand15%(v/v)fetalbovineserum(FBS)(GIBCO,Invitrogen)at37°Cina5%CO
2
humidifiedincubator.
UV-Visspectrumassay
StocksolutionsofFeSO andIcariinwerepreparedindeionizedwater(DIwater)andDMSO,
4
respectively.ThestocksolutionswerefurtherdilutedwithDIwatertothedesiredconcentra-
tion.AlltheUV-VisspectrawererecordedonaCary5GUV-VIS-NIRspectrophotometerat
roomtemperature.
MTTassay
TheMTTmethodwasusedtoevaluatetheeffectoficariinonchondrocytesproliferation.
After3daysofculture,thesupernatantwasremoved,and10μlofMTTsolution(5mg/ml)
wasaddedtoeverywellandincubatedfor4hbeforetermination.Afterremovalofthe
medium,theformedformazanpigmentwasdissolvedwithDMSO.The150μlofpigment
solutionwasaddedtoeachwellofa96-wellmulti-plateandtheabsorbancewasmeasuredby
usingamicroplatereaderat570nm.Allexperimentswerecarriedoutinquadruplicate.
Bromodeoxyuridine(BrdU)incorporationassay
CellproliferationwasexaminedbyusingthecellproliferationELISABrdUassay(Roche)
accordingtothemanufacturer’sinstructions.Briefly,chondrocyteswerecultureduntilcon-
fluency,thenre-platedin96-wellplatesatadensityof5×103cellsinafinalvolumeof100μl
perwell.ThechondrocytesweretreatedwithdifferentconcentrationsofIcariinfor1dayor3
PLOSONE|DOI:10.1371/journal.pone.0148372 February3,2016 4/24
IcariinActivatesHIF-1αtoPromoteCartilageRepair
daysfollowedbyBrdU(10μM)treatmentfor4h.Then,thecellswerefixedandincubated
withperoxidase-conjugatedanti-BrdUantibodyfor90min.BrdUincorporationwasdetected
byincubatingthecellswithtetramethyl-benzidine(TMB)asasubstrate.Colordevelopment,
whichwasdirectlyproportionaltotheamountofDNAsynthesisandherebytothenumberof
proliferatingcells,wasquantifiedbymeasuringtheabsorbanceat370nmbyamicroplate
reader.Theexperimentswerecarriedoutintriplicate.
Colonyformationassay
Theprimarychondrogeniccellsisolatedfromgrowthplatesofnewbornmiceweretreated
withdifferentconcentrationsofIcariinfor24h,thentrypsinizedandreplated.500cellswere
platedin6-wellcultureplatesinaCO incubatorsuppliedwith5%CO for2weeks.Cellswere
2 2
fixedin10%(v/v)formalinfor10minandstainedwith0.1%(w/v)crystalviolet(Sigma)for10
min.Thecolonies(>50cells)werecountedusingadissectingmicroscope.Thesinglecellsthat
formcoloniescontainingmorethan50cellswerereferredtochondroprogenitors.Theexperi-
mentswereperformedintriplicate.
MicromasscultureandAlcianbluestaining
Chondrocyteswereseededina4-wellplateatadensityof1.0×105cellsperwellinafinalvol-
umeof10μlandincubatedfor4hat37°Ctoallowadherence.0.5mloffreshmediawasadded
toeachwell.Onthesecondday,mediawerechangedtochondrogenicmediumcontainingvari-
ousconcentrationsofIcariin.ThechondrogenicmediumcontainedDulbecco’smodifiedEagle’s
medium(DMEM)(highglucose)with10%(v/v)fetalbovineserum(FBS),ITS+Premix1:100
dilution,1%(v/v)penicillin/streptomycin,100mML-glutamine,50μg/mlascorbicacid(pH
7.1)and10mM-glycerophosphate(Sigma).After7or14days,wellswerewashedoncewith
PBSandthenfixedfor20minwith0.5mlof10%(v/v)neutralbufferedformalin.Thepellets
werethenwashedthreetimeswithsteriledistilled,deionizedwater,withthelastwashbeingleft
onthecellsfor15min.Subsequently,0.5mlof1%(w/v)Alcianblue(Sigma)inglacialacetic
acid(3%)(v/v)wasaddedtoeachwellfor30minor1hatroomtemperature.Aftertwowashes
with70%(v/v)ethanolandthreewasheswithsteriledistilled,deionizedwater,photomicro-
graphsofthestainedcellmasswereobtained.TheECMproteoglycanwasstainedblue.
ELISAassay
HydroxyprolineandaggrecaninchondrocyteswereassayedusingELISAkitsaccordingto
manufacturer’srecommendations.ELISAkitsusedweremousehydroxyprolineELISAkit
(CUSABIO)andmouseAGCELISAkit(HXBIO).Opticaldensitywasdeterminedusinga
microplatereader.Allexperimentswerecarriedoutintriplicate.
Three-dimensional(3D)alginate-chondrocytesbeadsculture
Chondrocytesweretrypsinizedandresuspendedin2%(w/v)alginatesolutionin0.9%(w/v)
sodiumchlorideataconcentrationof4×106cells/mlandmixedwellbypipettingupand
down.Thealginate/cellmixture(20μl)wasdroppedintosterile102mMCalciumChloride
solution.The3Dalginate-chondrocytesbeadswereformedintheCalciumChloridesolution
forthreeminutesandthenwashedtwicewith0.9%(w/v)SodiumChloride.The3Dalginate-
chondrocytesbeadswerethentransferredtoaculturedishcontainingchondrogenicmedium
withorwithoutIcariin(10−6M)andculturedintheincubatorat37°C.Themediumwas
changedevery3daysupto21days.Thealginate/cellcomplexeswereprocessedformRNA
expression,histologicalcharacterizationandimmunohistochemistryanalysis.
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
Osteochondraldefectmodelinmice
ExperimentalprocedureswerecarriedoutaccordingtoprotocolsapprovedbyAnimalExperi-
mentationEthicsCommittee,TheChineseUniversityofHongKongandAnimal(Controlof
Experiments)OrdinancefromDepartmentofHealth,HongKongSAR.Theosteochondral
defectmodelinmicewasadaptedfrompreviouslyestablishedprocedure[24].Briefly,the
C57BL/6malemice(20–25g)wereanesthetizedusingxylazine(5mg/kg)andKetamine(40
mg/kg)cocktail,understerileconditions.Theinter-chondylenotchofthedistalfemurwas
exposedandanosteochondraldefectwith1mmindiameterand2mminheightwascreated
witha21Gneedle.Thedefectswereimplantedwiththe3Dalginate-Gelfoamcomplexesincor-
poratedwithorwithoutIcariin.Beforethesurgery,the3Dalginate-Gelfoamcomplexeswere
generatedasfollows.Icariinwasdissolvedin4%(w/v)alginateacidsodium(Sigma)solution
tomakeaworkingsolutionat1x10−6M.Allthesolutionswerefreshlyprepared.TheGel-
1
foam wasthenpre-humidifiedwithsteriledeionized,distilledH Oanddriedinsterilized
2
gauze,andwasshapedindesignedcubicstructure(2X2X2mm3).10μlofalginatesolution
containingIcariinornoIcariinwascarefullyaddedintothe3DGelfoamscaffoldfollowedby
immersingwith102mMcalciumchloridesolution.Theimplantswerethenincubatedin37°C
for5minutes.The3Dcomplexesweretrimmedto1mmindiameterand2mminheightfor
implantation.5micewereusedineachgroup.Aftersurgery,theanimalswereadministered
withanalgesics(BuprenorphineHCl,0.02mg/Kg/day)onceperdayforthreedays.Theani-
malsweremonitoredeverydaypost-surgeryinthefirstweek,thentwotimeseachweekupto
12weeks.Thereisnounintendeddeathofanimalduringthestudy.Sampleharvestingand
autopsyexaminationwereperformedat2weeks,6weeksand12weekspost-surgery,theani-
malsweresacrificedbycervicaldislocationunderanaesthesiawith2%(v/v)Isoflurane.The
cartilagesamplesofthedistalfemurwereprocessedforhistologicalanalysis.
Histologyandimmunohistochemistry
Forhistologicevaluation,thesampleswerefixedin4%(w/v)paraformaldehyde(PFA)inPBS,
decalcified,andembeddedwithparaffin.Thesectionswerecutwithamicrotomeatthethick-
nessof5μm.ThesectionswerestainedwithHematoxylin&EosinorSO/fastgreenstaining.
Forthe3Dculturesystem,alginate/chondrocytescomplexeswererinsedinPBSat4°C,fixed
for24hwith4%(w/v)PFA,soakedinaseriesofgradientsucrose,embeddedinOCT,and
solidifiedbyliquidnitrogen.FrozensectionswerecutforstandardHematoxylin&Eosin,
Alcianblue,andSO/fastgreenstaining.Forimmunohistochemistryanalysis,endogenousper-
oxidaseactivitywasquenchedby3%(v/v)hydrogenperoxide.Thesectionswereblockedwith
3%(v/v)bovineserumalbumin(BSA)andincubatedwiththeprimaryantibodiesofrabbit
polyclonalanti-Sox9(Abcam),rabbitpolyclonalanti-CollagentypeII(Col2),rabbitpolyclonal
anti-HIF-1α(NovusBiologicals)andmousemonoclonalanti-PCNA(proliferatingcellnuclear
antigen)(CellSignaling)respectively.AdjacentsectionswereincubatedwithIgGasnegative
controls.HistostainPlusKitwasusedforcolordevelopmentwithdiaminobenzidine(DAB)
accordingtothemanufacturer’sinstructions(Invitrogen).
Histologicalassessment
TheInternationalCartilageRepairSociety(ICRS)IIscorewasusedtoassesstheinvivocarti-
lagerepair[25].Thecentralportionoftheosteochondraldefectwasdefinedasregionofinter-
estandserialadjacentsectionswereemployedforanalysis.Threeblindedreadersgradedthe
cartilagesectionsaccordingtoICRSIIparametersandcriteria.Theadaptedhistological
parametersinclude:(1)matrixstaining(metachromasia):0%,nostaining;100%,full
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
metachromasia;(2)subchondralbone:0%,abnormal;100%,normalsubcondralbone;(3)
overallassessment:0%,bad(fibroustissue);100%,good(hyalinecartilage).
Immunoblotting
Westernblottinganalysiswasperformedusingstandardprotocol.Inbrief,whole-celllysate
wasobtainedbyusingcelllysisbufferinthepresenceofaproteaseinhibitorcocktail(Sigma).
EqualamountofproteinwasloadedontoanSDSmini-PAGEsystemafterconcentrations
weredetermined,andtransferredontoaPVDFmembraneusingaBio-Radsemi-drytransfer
system.Proteintransferefficiencyandsizedeterminationwereverifiedusingprestainedpro-
teinmarkers.Membraneswereblockedwith5%(w/v)drymilkinTris-bufferedsalinewith
Tween-20for1hatroomtemperatureandsubsequentlyincubatedovernightwithprimary
antibodiesat4°C.Theprimaryantibodiesusedincludedanti-HIF-1α,anti-EGLN2/PHD1,
anti-EGLN3/PHD3(NovusBiologicals,Littleton,CO)andanti-PHD2(H-40)(SantaCruz,
CA).SignalsweredetectedusinganHRP-conjugatedsecondaryantibodyandtheSuperSignal
WestFemtoMaximumSensitivitySubstrate(Pierce,ThermoFisherScientific,Inc.,Rockford,
IL,USA).ThequantitationofimmunoblottingbandswasperformedusingNIHImageJ1.48
software.Thevalueswereobtainedafternormalizationusingthebeta-actinsignalintensity
andcomparedwiththatofthenon-treatmentcontrol.
Immunofluorescenceandconfocalmicroscopy
Chondrocyteswereplatedonlysine-treatedglasscoverslipsplacedin6-wellplates.Aftercul-
turingfor24hundernormalcondition,cellswerestarvedfor12handtreatedwithIcariin
(10−6M)for8h.Thecellswerethenfixedwith4%(w/v)PFAfor10minatroomtempera-
ture,permeabilizedwith0.1%(v/v)TritonX-100for3minutes,andincubatedwiththepri-
maryantibodyofrabbitanti-HIF-1α(Novus,1:100)overnightat4°C.Afterrinsing,the
coverslipswereincubatedwithfluorescenceconjugatedsecondaryantibody(Molecular
Probes;Invitrogen)andprotectedfromlightfor30minatroomtemperature,andfinally
mountedinVECTASHIELDmountingmediawithDAPI(VectorLaboratories).Fluores-
cencelocalizationwasdetectedbyconfocalmicroscopewithalaser-scanningmicroscope
(FV10-ASW1.7;Olympus).
Quantitativereal-timepolymerasechainreaction(qPCR)
TotalRNAwasextractedfromchondrocytesusingTRIzolreagent(Invitrogen).Firststrand
cDNAwassynthesizedfrom1μgoftotalRNAinthepresenceofoligo-dT12-18primer(Invi-
trogen)andMMLVreversetranscriptaseaccordingtomanufacturer’sinstructions(Promega).
Quantitativereal-timePCRwasperformedwithSYBRPremixExTaq(Takara)inABIFast
Real-timePCR7900HTSystem(AppliedBiosystems).Allsampleswereperformedintripli-
cate.β-actinwasusedasaninternalcontrol.PrimersequenceswereshowninTable1.
Statisticalanalysis
AlldatawereexpressedasMean±SDandanalyzedusingSPSSversion17.0software(SPSS
Inc.,Chicago,IL).Thenormalityofthedataandtheequalvariancesacrossthegroupswere
validatedbytheShapiro-Wilk’stestandtheLevene’stestrespectively.Thedifferencesbetween
groupswereanalyzedbyusingStudent’st-testorANOVA.Allexperimentswereperformedin
triplicateunlessotherwisestated.P<0.05wasdefinedassignificancelevel.
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
Table1. SequencesofPrimersUsedforQuantitativeReal-timePCR.
Gene Forwardprimer Reverseprimer
Sox9 AGGAAGCTGGCAGACCAGTA TCCACGAAGGGTCTCTTCTC
Col2α1 CTACGGTGTCAGGGCCAG GCAAGATGAGGGCTTCCATA
Aggrecan CTGAAGTTCTTGGAGGAGCG CGCTCAGTGAGTTGTCATGG
HIF-1α TGGCTCCCTATATCCCAATG GGTCTGCTGGAACCCAGTAA
Clo10α1 CCTGGTTCATGGGATGTTTT ACCAGGAATGCCTTGTTCTC
Mmp2 CCAGCAAGTAGATGCTGCCT GATGGCATTCCAGGAGTCTG
Mmp9 TAGCTACCTCGAGGGCTTCC GTGGGACACATAGTGGGAGG
Mmp13 CTGGACCAAACTATGGTGGG GGTCCTTGGAGTGATCCAGA
Adamts4 CAGTGCCCGATTCATCACT GAGTCAGGACCGAAGGTCAG
Adamts5 CGAAGAGCACTACGATGCAG TGGAGGCCATCATCTTCAAT
β-actin GTTGTCGACGACGAGCG GCACAGAGCCTCGCCTT
doi:10.1371/journal.pone.0148372.t001
Results
IcariinactivatesHIF-1αexpressioninchondrocytesbyinhibitingPHD
throughcompetitionforironions
IcariinhasachemicalformulaC H O (Fig1A)withmolecularmass676.662g/mol.We
33 40 15
screenedIcariinforitsabilitytoactivateahypoxiaresponsiveelement(HRE)constructdriving
aluciferasereporter(HRE-Luc)inC2C12cells.Undernormoxia(21%oxygen),Icariin
increasedluciferasereporteractivityinC2C12cellsinadose-dependentmanneratconcentra-
tionsrangingfrom10−8Mto10−6M,whileshowedaninhibitoryeffect(Fig1B)athigher
doses(10−5Mand10−4M).ThechangesinHREreporteractivityinC2C12cellsuponIcariin
treatmentwereinlinewiththeupregulationofHIF-1αproteinlevel(Fig1C)andincreased
HIF-1αnuclearlocalization(Fig1D)inprimaryculture-derivedchondrocytesfollowingtreat-
mentwithIcariinfor8hours.IcariinstimulatedanincreaseinHIF-1αproteinlevelinthe
chondrocytesundernormoxiaby1.62fold,whichwascomparabletothe1.60-foldaccumula-
tionofHIF-1αwhenthechondrocyteswereexposedtohypoxia(2%oxygen),whilenoadditive
orsynergisticeffectwasobservedwhenthecellsweretreatedwithIcariinunderhypoxia(Fig
1C).Whenthechondrocytesweresubjectedtochondrogenicdifferentiation,Icariinsignifi-
cantlyelevatedHIF-1αmRNAlevelsfollowing7or14daysoftreatment(Fig1Eand1F).It
wasdocumentedthatsomeflavonoids(e.g.quercetin,galangin)chelatedcellularironions,a
requiredcofactorforPHDactivity,leadingtotheinhibitionofPHDs-catalyzedHIFprolyl
hydroxylationandsubsequentlyHIF-1α/2αaccumulation[26].Wethusdeterminedwhether
Icariin,asaflavonoidcomponent,inducedtheaccumulationofHIF-1αinchondrocytes
throughthesimilarmechanismasotherflavonoids[27].WeemployedUV-Visspectrum
assaystocomparethedifferenceintheabsorptionspectraofIcariin,FeSO andtheirmixture
4
solutions.AfteraddingFeSO toIcariinsolutionandbeingincubatedat37°Cfor12h,there
4
wasaclearredshiftoftheabsorptionpeaksoriginallylocatedatλ=268nmandλ=319nmfor
Icarrinsolution.Moreover,anewabsorptiondomaininthevisiblelightregionattheλ=
425nmwasobservedinthemixturesolution,whichdidnotexisteitherinIcariinorFeSO
4
solution(Fig1G).ThisabsorptiondomainhasbeenpredictedbytheDensityfunctionaltheory
(DFT)computationusingGaussian03inasystemcomposedofferrousionandquercetin,as
whichIcariinhasthesamecorestructure[27].Thenewabsorptiondomainendowsthelight
yellowcolortothemixture,asshownintheinsetofFig1G,whilebothIcariinandFeSO solu-
4
tionswerealmostcolorlessatthesameexperimentalcondition,indicatingthatsomenew
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
Fig1.IcariinupregulatesHIF-1αexpressioninchondrocytesbyinhibitingPHDsactivitythroughcompetitionforironions.(A)Thechemicalformula
ofIcariin.(B)HypoxiaresponseelementluciferasereporterassayinC2C12cellstreatedwithIcariinatindicatedconcentrations.(C)Westernblotanalysisfor
HIF-1αproteinexpressioninprimaryculture-derivedchondrocytesundernormoxiaorhypoxiaortreatedwithorwithoutIcariin(10−6M)for8h.β-actinused
astheloadingcontrol.(D)DetectionofHIF-1αnuclearlocalizationinIcariin(10−6M)-treatedchondrocytesbyimmunofluorescencestainingunderconfocal
microscope.(E,F)ChondrocyteswereculturedandinducedtodifferentiateinchondrogenicmediuminthepresenceorabsenceofIcariin(10−6M)for7or14
days.HIF-1αmRNAlevelsweredetectedbyreal-timePCRinIcariin-treatedchondrocytescomparedwiththatofthecontrolcells.*P<0.05,**P<0.01,
n=3.(G)UV-VisspectraoftheIcariin,FeSO andtheirmixture(n :n =3:1,C =0.5mM)inaqueoussolutionafterincubationat37°Cfor12h;
4 Icariin FeSO4 Icariin
C =0.5mM;C =1mM;Theinsetshowsthevisualappearanceofeachspecies.(H)WesternblotanalysisforHIF-1αproteinexpressionin
Icariin FeSO4
chondrocytestreatedwithorwithoutIcariin(10−6M)andFeSO (100μM)for12h.(I)WesternblotanalysisforPHDsandHIF-1αproteinexpressionin
4
chondrocytestreatedwithorwithoutIcariin(10−6M)for12h.InallFigs,ICA,Icariin.
doi:10.1371/journal.pone.0148372.g001
speciesformedafterthemixingandincubation,probablycausedbytheinteractionbetween
Fe2+andIcariin.Coincidewiththisobservation,wefoundthatthepositiveeffectofIcariinon
HIF-1αwaseliminatedbyFeSO (Fig1H),suggestingthatIcariinmightfunctionasaHIF-1α
4
activatorbyinhibitingPHDactivitythroughcompetitionforcellularironions.Interestingly,
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IcariinActivatesHIF-1αtoPromoteCartilageRepair
theproteinexpressionofPHD2andPHD3butnotPHD1wasalsoinhibitedbyIcariintreat-
ment,whichwasinlinewiththeupregulationofHIF-1α(Fig1I).
Icariinincreaseschondrocytesproliferation
WeassessedtheeffectofIcariinontheviabilityofchondrocytesbyMTTassay.Chondrocytes
weretreatedwithdifferentconcentrationsofIcariinfor3days.Theresultsshowedthatthe
cellsdidnotonlysurvivetheprolongedtreatmentofIcariinatawiderangeofdoses(from
10−7Mto10−5M)butalsosignificantlyincreasedtheirnumbersandachievedthehighestvia-
bilityupon10−6MofIcariintreatmentcomparedwiththecontrolgroup(Fig2A).Tofurther
confirmthatIcariinhasapositiveeffectonproliferationofchondrocytes,weperformedBrdU
incorporationassayandcolony-formationassay.AsshowninFig2B,10−6MofIcariin
increasedtheBrdUincorporationinchondrocytestothelevelthatwassignificantlyhigher
thanthatinthecontrolgroupafteronedayofcultureandthiseffectwasfurtherenhanced
afterthreedaysofculture.Colonyformationassayshowedthatthechondroprogenitorstreated
Fig2.Icariinincreaseschondrocytesproliferation.(A)MTTassayforcellviabilityofchondrocytestreatedwithorwithoutIcariin(0M,10−7M,10−6M,
10−5M)for3days.Treatedgroupscomparedwithcontrolgroup,*P<0.05,**P<0.01,n=3.(B)BrdUincorporationassayforchondrocytestreatedwithor
withoutIcariin(0M,10−7M,10−6M,10−5M)for1dayor3days.Treatedgroupscomparedwithcontrolgroup,*P<0.05;***P<0.001,n=3.(C)Colony
formationassayforchondroprogenitorcellstreatedwithIcariin(10−7M,10−6M,10−5M)for24hfollowedby14dayssub-culture.(D)Quantitationofthe
colonynumbersfrom(C),*P<0.05,n=3.
doi:10.1371/journal.pone.0148372.g002
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