Zinc Prevents Abdominal Aortic Aneurysm Formation by Induction of A20-Mediated Suppression of ...

17 Pages·2016·2.43 MB·English

RESEARCHARTICLE
Zinc Prevents Abdominal Aortic Aneurysm
Formation by Induction of A20-Mediated
Suppression of NF-κB Pathway
Ya-WeiYan1☯,JunFan1☯,Shu-LingBai1*,Wei-JianHou1,XiangLi2,HaoTong1
1 DepartmentofTissueEngineering,SchoolofFundamentalScience,ChinaMedicalUniversity,Shenyang,
China,2 DepartmentofCellBiology,CollegeofBasicMedicine,ChinaMedicalUniversity,Shenyang,China
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
Chronicinflammationanddegradationofelastinarethemainprocessesinthedevelopment
ofabdominalaorticaneurysm(AAA).Recentstudiesshowthatzinchasananti-inflamma-
toryeffect.Basedonthese,zincmayrendereffectivetherapyforthetreatmentoftheAAA.
Currently,wewanttoinvestigatetheeffectsofzinconAAAprogressionanditsrelated
OPENACCESS molecularmechanism.RatAAAmodelswereinducedbyperiaorticapplicationofCaCl .
2
AAAratsweretreatedbydailyintraperitonealinjectionofZnSO orvehiclealone.Theaorta
Citation:YanY-W,FanJ,BaiS-L,HouW-J,LiX, 4
TongH(2016)ZincPreventsAbdominalAortic segmentswerecollectedat4weeksaftersurgery.Theprimaryrataorticvascularsmooth
AneurysmFormationbyInductionofA20-Mediated musclecells(VSMCs)werestimulatedwithTNF-αaloneorwithZnSO for3weeks.The
4
SuppressionofNF-κBPathway.PLoSONE11(2):
resultsshowedthatzincsupplementationsignificantlysuppressedtheCaCl -induced
e0148536.doi:10.1371/journal.pone.0148536 2
expansionoftheabdominalaorticdiameter,aswellasapreservationofmedialelastin
Editor:MasukoUshio-Fukai,UniversityofIllinoisat
fibersintheaortas.Zincsupplementationalsoobviouslyattenuatedinfiltrationofthemacro-
Chicago,UNITEDSTATES
phagesandlymphocytesintheaortas.Inaddition,zincreducedMMP-2andMMP-9pro-
Received:October21,2015
ductionintheaortas.Mostimportantly,zinctreatmentsignificantlyinducedA20expression,
Accepted:January19,2016 alongwithinhibitionoftheNF-κBcanonicalsignalingpathwayinvitroinVSMCsandinvivo
Published:February26,2016 inratAAA.Thisstudydemonstrated,forthefirsttime,thatzincsupplementationcouldpre-
ventthedevelopmentofratexperimentalAAAbyinductionofA20-mediatedinhibitionof
Copyright:©2016Yanetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative theNF-κBcanonicalsignalingpathway.
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
Introduction
credited.
Abdominalaorticaneurysm(AAA)isakindofseriousvasculardiseasewithhighincidence
DataAvailabilityStatement:Allrelevantdataare
andhighmortality.Furthermore,withthechangeofthelifestyleandanagingpopulation,the
withinthepaperanditsSupportingInformationfiles.
incidenceisarisingtrend[1].Itstypicalpathologicalchangesincludechronicinflammatory
Funding:ThisworkwassupportedbytheNational
cellsinfiltration,aorticelastinproteolyticdegradationandpathologicalremodeling.These
NaturalScienceFoundationofChinaunderContract
changesresultinthedestructionofelasticlamellarstructureintheaorticmediaandtherefore
(No.81571919)forFJ.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto gradualaneurysmaldilatationandevenfinallyrupture[2].
publish,orpreparationofthemanuscript. InflammationplayssignificantroleintheprogressionofAAA[3,4],whichmaybethe
potentialtreatmenttargetforAAA.RecentstudieshaveshownthatzincfingerproteinA20
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. couldpreventinflammatoryresponseinaorticallograftsanddevelopmentoftransplant
PLOSONE|DOI:10.1371/journal.pone.0148536 February26,2016 1/17
ZincPreventsAbdominalAorticAneurysmFormation
arteriosclerosis[5].ZincfingerproteinA20,azinc-fingertransactivatingfactor,wasidentified
asaprimaryresponsegenefollowinginflammatorystimulation(TNF,IL-1orLPS)ofhuman
umbilicalveinendothelialcells[6].A20canalsobeinducedinsmoothmusclecellsandexhibit
ananti-inflammatoryimpactbyblockadeofnuclearfactorκB(NF-κB)signaling[7,8].NF-κB
canpromotechronicinflammationintheaorticwall[9],andregulateMMPstranscription
[10].Inhumanandanimalsexperiment,inhibitionofNF-κBactivationcanpreventthedevel-
opmentofAAA[11,12].
Zincisoneofthemostcommontraceelementsinthehumanbody,andneededforDNA
synthesis,RNAtranscription,celldivisionandactivation.Zincplaysacriticalroleinwound
healing,biosynthesis,andhomeostasisofvariousconnectivetissues[13].Zincalsohasanti-
inflammatoryactionobviouslyregulatingpathogenesisoftheinflammation-relateddiseases
[14].Reportshaveshownthatdecreasedplasmazincandincreasedinflammatorycytokinesin
theelderlysubjectswerecorrectedbyzincsupplementation[15].Atsametime,zincdeficiency
willinducevascularinflammationassociatedwithNF-κBsignaling[16].ZinccaninduceA20
expressionandinhibitNF-κBactivation,decreaseincidenceofinfectionandgenerationof
inflammatorycytokinesinpatients[17].Basedonthese,wespeculatethatzincmayrender
effectivetherapyforthetreatmentoftheAAA.
Inthepresentstudy,thepurposeistodetectwhetherzincsupplementationcanpreventthe
developmentofexperimentalAAA,withspecialconcentrationontheregulationofA20-NF-
κBpathwayinvivoandvitrostudies.
MaterialsandMethods
Experimentagentsandinstruments
Anti-A20,anti-elastinantibodywasobtainedfromSantaCruzTechnology(SantaCruzBio-
tech,SantaCruz,CA,USA).Primarypolyclonalantibodiesagainstphospho-IKKβ(Ser180/
181),phospho-IκBα(Ser32/36),IKKβ,IκBα,andNF-κBp65arefromCellSignalingTechnol-
ogy.Anti-CD45,anti-CD68andanti-CD20werefromBoster,China.Anti-β-actin,anti-MMP-
2,anti-MMP-9werepurchasedfromBioss,China.Calciumchlorideanhydrous(CaCl )and
2
zincsulfateheptahydrate(ZnSO (cid:1)7H O)werepurchasedfromSigma.Diaminobenzidine
4 2
(DAB)andstrept-avidinbiotincomplex(SABC)immunohistochemicalkitwerepurchased
fromBoster(Wuhan,China).FetalbovineserumandDulbecco'smodifiedEagle'smedium
(DMEM)werepurchasedfromHyclone(Logan,Utah,USA).
Animalexperiments
30adultmale8-wk-oldWistarrats(obtainedfromtheexperimentalanimalcenterofChina
MedicalUniversity)weighing250to300gwererandomlydividedintothreegroups:control
group(withoutanytreatment),AAAgroup(treatedbyCaCl alone),zinc-administrated
2
group(treatedbyCaCl togetherwithzincintraperitonealinjection),with10ratspergroup.
2
Allanimalexperimentswerecarriedoutinstrictaccordancewiththerecommendationsinthe
GuidefortheCareandUseofLaboratoryAnimalsoftheNationalInstitutesofHealth.The
protocolwasapprovedbytheInstitutionalAnimalCareandUseCommittee(IACUC)of
ChinaMedicalUniversity.TheapprovalreferencenumberisSCXK(Liao)2013–0001.Allrats
werehousedundera12-hourlight/darkcyclesandhadfreeaccesstoanormaldietandwater
adlibitum.RatAAAwasestablishedbyperivascularapplicationof0.5MCaCl aspreviously
2
described[18],whereassalinewasusedinthecontrolgroup.Inshort,theratswereanesthe-
tizedwithsodiumpentobarbital(40mg/kg,intraperitoneally),shearedanddisinfected,then
fixedontheoperatingtableinthesupineposition.Anincisionwasmadeontheabdominal
mediantoexposetheabdominalaorta.ThenspongecontainingCaCl wasdirectlyplacedon
2
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ZincPreventsAbdominalAorticAneurysmFormation
theabdominalaortaatapproximatelythecenterofthesectionbetweentherenalarteryandthe
iliacbifurcation.Thespongewasremovedafter15minfollowedbyclosingtheabdominalcav-
ityinlayers.
Afterthesurgery,zinc-administratedgroupanimalswereimmediatelyprovidedwithdaily
intraperitonealinjectionofZnSO atadoseof3mg/kg/day(physiologicaldose)[19,20]for4
4
weeks.AAAgroupratswereinjectedwiththeequivoluminalsaline.
Fourweeksaftertheoperation,theseratswereanesthetizedwithsodiumpentobarbital
(60mg/kg,absolutelethaldose,intraperitoneally)andtreatedaorticsegmentswereexcised,
fixedin4%phosphate-bufferedparaformaldehyde,embeddedinparaffinforthefollowing
assays.ChangesoftheaorticexternaldiameterwereanalyzedbyapplicatingSZH
stereomicroscope.
Histologicalexamination(Orceinstaining,HEstaining)
SerialsectionsoftheabdominalaorticsegmentswithOrceinstainingandHEstainingaccord-
ingtothestandardtechniqueswereusedtoobservethechangesoftheelasticlamellaeandthe
morphologyofaorticwalls,respectively.Theexperimentalresultswereanalyzedbyusing
MetaMorph/DP10/BX41microscopeimageanalysissystem.
Immunohistochemicalstaining
Anti-CD45(1:200,leucocytes,Boisynthesis,China),anti-CD68(1:100,macrophages,Boster
Biotechnology,China)andanti-CD20(1:100,Blymphocytes,BosterBiotechnology,China)
wereusedtolocateandspecifyinflammatorycellsinfiltrationintheabdominalaorticwalls.
Immunohistochemicalstainingwasperformedon5μmsections.Theyweredewaxed,rehy-
dratedingradedalcohols,blockedforinhibitingendogenousperoxidaseactivitybyusing3%
hydrogenperoxidase,andpreincubatedwith5%normalbovineserumalbumin(BSA).Appro-
priatedilutedrabbitpolyclonalprimaryantibodieswereappliedtothesections.Subsequently,
theywereseriallyincubatedwithbiotinylatedanti-mouseIgGandthesupersensitivestreptavi-
din-biotincomplex(SABC)accordingtothemanufacturer’sdirections.Immunecomplexes
werevisualizedbyusing3,3'-diaminobenzidine(DAB),andcellnucleuswerecounterstained
withhematoxylin.Allsectionsweredehydratedandcoveredbycoverglasseswithneutralbal-
sam.Forthenegativecontrolexperiments,wesubstitutedphosphatebuffersolution(PBS)for
theprimaryantibody.Theinfiltrationofinflammatorycellswasdeterminedbycountingthe
meannumberofnucleisurroundedbypositiveimmunostainingwithin400×highpowerfields
(HPF).Non-specificstainingwasnotobserved.
Cellcultureandexperiments
PrimaryaorticVSMCswereobtainedbyexplantculturesaspreviousmethod[21].Adult
healthyWistarratswereanesthetizedwithsodiumpentobarbital(60mg/kg,absolutelethal
dose,intraperitoneal)andthentheiraortaswereexcisedfromthesectionbetweentherenal
arteryandtheiliacbifurcation,separatedlooseconnectivetissueandcollateralvessels,scraped
offintimallayer,thencutinto1–2mm2smallsegmentsandtransferredinto25cm2tissuecul-
tureflaskssupplementedwithhighglucoseDMEM(H-DMEM)plus20%foetalbovineserum
(FBS),100U/mLpenicillin,and100U/mLstreptomycin,After4days,cellsfreedoutaround
thetissueandsubculturewasdonewhencellsreached80%confluence.Afterthethirdpassage,
theVSMCswereidentifiedwithImmunofluorescenceassayforα-SMA(α-smoothmuscle
actin).
ThefourthtosixthpassageVSMCsweretreatedwithno-additives,TNF-αaloneandTNF-
αtogetherwithzinc.TNF-αandzincsulfateheptahydrateweredissolvedinDMEMand
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ZincPreventsAbdominalAorticAneurysmFormation
addedat10ng/mLand15μMrespectively,thenculturedcontinuouslyfor3weeks,medium
wasreplacedevery3days.Theallcellswereserum-starvedinDMEMcontainingnoFBSfor24
htosynchronizetheminG phaseofthecellcyclepriortotreatments.
0
WesternBlotting
TotalproteinswereextractedfromtheabdominalaortictissuesandvariouslytreatedVSMCs.
TheproteinconcentrationsweredeterminedusingaBCAproteinassaykit(Beyotime).After
denaturingtheproteinswithloadingbuffer,thesameamountofprotein(40mg)and2μlpre-
stainedproteinmolecularweightmarkerwereusedforWesternblotanalysiswithprimary
antibodiesagainstA20(1:500,SantaCruz),phospho-IKKβ(1:800,Ser180/181;CellSignaling),
phospho-IκBα(1:800,Ser32/36;CellSignaling),IκBα(1:1000,CellSignaling),andNF-κBp65
(1:1000,CellSignaling),MMP-2,9(1:300,Boster,China)withβ-actin(1:1000,Bioss,China)as
positivecontrol,andwiththerespectivelyappropriateHRP-conjugatedsecondaryantibody.
Specificantibodybindingwassubsequentlyvisualizedusinganenhancedchemiluminescence
(ECL)kit(BeyotimeBiotechnology,Shanghai,China)followingthemanufacturer’sinstruc-
tionsusingChemDocXRSwithQuantityOnesoftware(BioRad,Hercules,CA).Theexperi-
mentswererepeatedthreetimes.Theintensityofeachbanddeterminedbydensitometryusing
imageanalyzingsoftwareindicatedtheexpressionlevelofthespecific-protein.
Statisticalanalysis
Allvalueswereexpressedasthemean±SEM.Analysisofvariancewasusedtodeterminethe
significanceofdifferencesinmultiplecomparisons.TheseresultswereprocessedusingtheSta-
tisticalPackagefortheSocialSciences,version16.0(SPSSInc.,Chicago,IL,USA).Compari-
sonsbetweengroupswereconductedusingtheTtest.P<0.05wasconsideredstatistically
significant.
Results
ZincsupplementationpreventedtheprogressionofCaCl -induced
2
AAAs
FourweeksafterCaCl -inducedAAAsurgery,theratsweresacrificedandtheaortatissues
2
werecollected.First,theinhibitoryeffectsofzincontheAAAdevelopmentweredetermined
byobservingtheaneurysmalincidence,aorticexternaldiameterandthemorphological
changes.TheaneurysmalincidenceintheAAAgroupwas70%,whilethatinthezinc-adminis-
tratedgroupwasonly20%.TheaorticexternaldiameterintheAAAgroupincreasedobviously
comparedwiththatinthecontrolgroup(controlgroup,1.38±0.02mm;AAAgroup,2.43
±0.17mm).However,theadministrationofzincsignificantlydecreasedtheaorticdiameter
comparedwiththeAAAgroup(1.57±0.09mm)(Fig1Aand1D).
HEstainingshowedthattherewereorderlyandclearlayersintheabdominalaorticwallsin
thecontrolgroup,whiletheabdominalaorticwallsintheAAAgroupbecamedisorganized
andthinner.However,theabdominalaorticwallsinthezinc-administratedgrouphadalmost
thesamemorphologyasthenormalabdominalaorta(Fig1B).
Aorticextracellularmatrixproteolyticdegradationisoneofthemaincharacteristicchanges
oftheaneurysm.Orceinstainingwasusedtodetecttheelastinchangesintheaorta.Orcein
stainingshowedthattheelasticlamellaeofthecontrolgroupwerecorrugatedappearanceand
integratedstructurewithoutanydamage.TheelasticlamellaeintheAAAgroupwereflator
evendisruptedanddispersed.Butzinc-supplementationpreservedthewaveelasticlamellae
andalleviatedtheelastinfragmentation(Fig1C).
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ZincPreventsAbdominalAorticAneurysmFormation
Fig1.EffectofzincontheAAA.(A)Representativeimagesofthediametersoftheaortas.(B)Representativemacroscopicmorphologicalchangesbylow-
powermicrographsofhematoxylinandeosinstaining.(C)RepresentativeorceinstainingshowingelasticlamellaechangesofAAAsample.(D)Thediameter
oftheaortas.(E)Thenumberoftheelasticlamellae.AL:aorticlumen.Scalebars:200μm.Resultswerepresentedasmean±SEM.n=10foreachgroup.*
p<0.05versuscontrolgroup,#p<0.05versusAAAgroup.
doi:10.1371/journal.pone.0148536.g001
Inaddition,zinc-supplementationreversedthedecreasedelasticlamellaenumberinthe
wallofAAA.Orceinstainingdemonstratedthattheelasticlamellaenumberwas11±2inthe
controlgroup,6±1intheAAAgroupand9±2inthezinc-administratedgroup(Fig1E).At
sametime,theintervalofelasticlamellaeincreasedintheAAAgroupcomparedwiththatin
thecontrolgroup,whiletightelasticlamellaearesimilartothenormalaorticwallinthezinc-
administratedgroup.
Zincpreservedtheelastin
PrimaryaorticVSMCswerecultured.At3dayofculture,somecellsappearedaroundthetis-
sueaortasegments.Thecellsexhibitedtypicalspindle-likemorphologyunderopticalmicro-
scope(S1Fig).Inaddition,thecellswerestainedwithα-SMAantibody,nonon-stainingcells
wereobserved(S1Fig).TheresultsconfirmedthattheisolatedcellswereVSMCs.VSMCswere
treatedwithTNF-αtomimictheinflammationenvironmentoftheAAAinvivo.Westernblot
exhibitedTNF-αtreatmentdecreasedtheelastinlevelintheVSMCs,whiletheVSMCstreated
withTNF-αtogetherwithzincshowedsignificantlyhigherexpressionlevelofelastinthanthat
intreatedwithTNF-αalone(Fig2A).Westernblotfurtherexhibitedthattherewasmoreelas-
tinexpressionintheaortasegmentsofthezinc-administratedgroupthanthatoftheAAA
group(Fig2B).Thereforeweconcludedthatzinccouldpreventtheelastindegradationin
VSMCsinvitroandinAAAinvivo.
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ZincPreventsAbdominalAorticAneurysmFormation
Fig2.ZincpreservedtheelastinintheVSMCsinvitro.(A,n=3independentexperiments)andintheaortasinvivo(B,n=10foreachgroup).Theβ-actin
wasusedasaninternalcontrol.Resultswerepresentedasmean±SEM.**p<0.01versuscontrolgroup,#p<0.05versusAAAgroup(orTNF-αgroup).
doi:10.1371/journal.pone.0148536.g002
ZincreducedtheinflammationintheAAA
ToevaluatetheeffectofzincontheinflammationinAAA,theimmunostainingofleukocytes
(CD45),macrophages(CD68)andBcells(CD20)inaortaswasconducted.Thenumberof
CD45+,CD68+andCD20+cellswasincreasedinaortasofAAAgroupversuscontrolgroup,
indicatingthatinfiltrationofmacrophagesandBcellswereincreasedinAAA.However,zinc-
administrationabolishedleukocytes,macrophagesandBcellsinfiltrationinaortasofAAA
group,suggestingthatzinccanreducetheinflammationintheAAAasshowninFig3.
ZincinducedtheexpressionofA20inabdominalaortainvivoand
VSMCsinvitro
PreviousstudiesshowedthatzinccaninduceA20proteinexpressionbyinhibitinginflamma-
tion[22,23].WeexaminedexpressionofA20proteininabdominalaortasegmentsandpri-
maryculturesofVSMCstreatedasdescribedinMaterialandmethods.Invivoexperiment,
therewaslowerA20expressionintheAAAgroupaortasthanthatinthecontrolgroupaortas.
However,zincsupplementationsignificantlyreversedthelowA20expressioninabdominal
aortas(Fig4A).Invitroexperiment,theprimaryVSMCsweretreatedwithTNF-αinthepres-
enceorabsenceofzinc,TNF-αalonedecreasedA20expression,butcombinedwithzinccould
increaseA20expression(Fig4B).TheseresultsdemonstratedthatA20expressioncanbe
inducedbyzincadministrationinAAA.
ZincinhibitedtheNF-κBactivation
NF-κBactivationispivotaltotheinflammationprocess.Therefore,wewanttoknowwhether
zinccaninhibittheNF-κBactivationintheAAA.TheNF-κBp65subunitonlyslightly
expressedintheaortasinthecontrolgroup,butitsexpressionevidentlyincreasedintheAAA
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ZincPreventsAbdominalAorticAneurysmFormation
Fig3.Immunohistochemicalstainingshowedtheinfiltrationoftheinflammatorycells.CD45(A),CD68(B)orCD20(C)positivecellsinvesselwalls.
(D),thenumberoftheinflammatorycellsbystatisticalanalysis.Browndepositsindicatepositivestaining.Resultswerepresentedasmean±SEM.n=10for
eachgroup.*p<0.05versuscontrolgroup,#p<0.05versusAAAgroup.AL:aorticlumen.Scalebars:200μm.
doi:10.1371/journal.pone.0148536.g003
group.However,zinc-administrationapparentlyreducedNF-κBp65expressionintheaortas
(Fig5A).Invitroexperiment,TNF-αaloneobviouslyincreasedtheNF-κBp65subunitexpres-
sionintheVSMCscomparedwithcontrolgroup,whileNF-κBp65subunitexpressionwassig-
nificantlydown-regulatedintheVSMCsinTNF-αtogetherwithzincgroup(Fig5B).Theabove
resultsindicatedzincinhibitedtheNF-κBactivationintheAAA.
Zincdecreasedthephospho-IKKβ,phospho-IκBαandpreventedthe
degradationofIκBα
ThecanonicalNF-κBsignalingregulatesexpressionoflargenumberofmoleculesinvolvedin
inflammation.TheeffectofzinconthecanonicalNF-κBsignalingwasdetected.Comparedto
thecontrolgroup,thep-IKKβandp-IκBαincreasedsignificantly,whiletheIκBαdecreased
distinctlyintheabdominalaorticwallsintheAAAgroup.However,zincadministration
remarkablyreducedthep-IKKβandp-IκBαandpreventedtheIκBαdegradation(Fig6).In
vitroexperiment,TNF-αaloneobviouslyincreasedthegenerationofp-IKKβandp-IκBαand
degradedtheIκBαintheVSMCscomparedwiththatinthecontrolgroup.But,thegeneration
ofp-IKKβandp-IκBαclearlydecreasedandtheIκBαsignificantlyincreasedintheVSMCsin
thepresenceofzinc(Fig7).IKKβlevelwassimilaramongdifferentgroupsinvivoandinvitro.
ZincalleviatedtheMMP-2andMMP-9expressionintheAAA
MMP-2andMMP-9coulddegradeelasticfibersandwereconsideredasespeciallyimportant
moleculesinthepathogenesisofAAAs[24].Westernblotresultrevealedthattheexpressionof
theMMP-2andMMP-9wassignificantlyup-regulatedintheaortasoftheAAAgroupcom-
paredwiththatofthecontrolgroup,whilezinc-administrationsignificantlydecreasedthe
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ZincPreventsAbdominalAorticAneurysmFormation
Fig4.ZincinducedtheexpressionoftheA20intheaortasinvivo(A,n=10foreachgroup)andintheVSMCsinvitro(B,n=3independent
experiments).Theβ-actinwasusedasaninternalcontrol.Resultswerepresentedasmean±SEM.**p<0.01versuscontrolgroup,##p<0.01versusAAA
group(orTNF-αgroup).
doi:10.1371/journal.pone.0148536.g004
Fig5.ZincinhibitedtheactivationoftheNF-κBintheaortasinvivo(A,n=10foreachgroup)andintheVSMCsinvitro(B,n=3independent
experiments).Theβ-actinwasusedasaninternalcontrol.Resultswerepresentedasmean±SEM.**p<0.01versuscontrolgroup,##p<0.01versusAAA
group(orTNF-αgroup).
doi:10.1371/journal.pone.0148536.g005
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ZincPreventsAbdominalAorticAneurysmFormation
Fig6. Theexpressionofthep-IKKβ,p-IκBα,IKKβandIκBαintheabdominalaortasinvivo(A).Zincdecreasedthephospho-IKKβ(B),phospho-
IκBα(C)anddegradationofIκBα(E).TheleveloftheIKKβhadnosignificantdifference(D).Theβ-actinwasusedasaninternalcontrol.Resultswere
presentedasmean±SEM.n=3independentexperiments.*p<0.05versuscontrolgroup,#p<0.05versusAAAgroup.
doi:10.1371/journal.pone.0148536.g006
MMP-2andMMP-9expressionintheaortas(Fig8).Soweconfirmthatzincsupplementation
caninhibittheexpressionoftheMMP-2andMMP-9intheAAA.
Discussion
Ourresultsdemonstratedthatzincsupplementationsignificantlysuppressedtheabdominal
aortaexpansion,preservedelastinfibersandpreventedthedevelopmentofexperimentalAAA.
Furthermore,studiesrevealedthatzinccouldreducetheinfiltrationofinflammatorycellsand
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Fig7. Theexpressionofthep-IKKβ,p-IκBα,IKKβandIκBαintheVSMCsinvivo(A).Zincdecreasedthephospho-IKKβ(B),phospho-IκBα(C)and
degradationofIκBα(E).TheleveloftheIKKβhadnosignificantdifference(D).Theβ-actinwasusedasaninternalcontrol.Resultswerepresentedas
mean±SEM.n=3independentexperiments.*p<0.05versuscontrolgroup,#p<0.05versusAAAgroup.
doi:10.1371/journal.pone.0148536.g007
theexpressionofMMP-2andMMP-9inAAA.WealsofoundthatzincinducedA20expres-
sionandinhibitedthecanonicalNF-κBsignalingpathwayinvitroinVSMCsandinvivoin
AAA.ThesefindingspointtoanewstrategytotreatAAA.
InflammationisoneofthemainreasonsresponsibleforthedevelopmentofAAA.The
inflammatorycellsproduceMMPstodegradetheelastinintheaorticwall,especiallyMMP-2
andMMP-9.Inourexperiment,theAAAinratswasinducedbyperiaorticapplicationofcal-
ciumchloride(CaCl )[25].TheinflammatoryaspectofouranimalAAAresemblesthatfound
2
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