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Curcumin restores Nrf2 levels and prevents quinolinic acid-induced neurotoxicityby Julio Tobn

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CurcuminrestoresNrf2levelsandpreventsquinolinicacid-inducedneurotoxicity

IvnCarmona-Ramreza,AbelSantamarab,JulioC.Tobn-Velascob, c,MarisolOrozco-Ibarra d,IrmaG.Gonzlez-Herrerab,JosPedraza-Chaverr c,PerlaD.Maldonadoa,

aPatologaVascularCerebral,InstitutoNacionaldeNeurologayNeurociruga ManuelVelascoSurez,MxicoD.F.,14269,MxicobAminocidosExcitadores,InstitutoNacionald eNeurologay Neurociruga ManuelVelascoSurez,MxicoD.F.,14269,Mxico

cDepartamentodeBiologa,FacultaddeQumica,UniversidadNacionalAutnomadeMxico,MxicoD.F.,04510,MxicodNeurobiologaMolecularyCelular,INNN-UNAM,InstitutoNacionaldeNeurologayNeurociruga ManuelVelascoSurez,MxicoD.F.,14269,Mxico

Received5July2011;receivedinrevisedform13December2011;accepted22December2011

Abstract

Neurologicaldiseasescompriseagroupofheterogeneousdisorderscharacterizedbyprogressivebraindysfunctionandcelldeath.Inthenextyears,these

diseasesareexpectedtoconstituteaworld-widehealthproblem.Becauseexcitotoxicityandoxidativestressareinvolvedinneurodegenerativediseases,it

becomesrelevantto describepharmacological therapies designed to activate endogenouscytoprotective systems. Activationof transcription factor Nrf2

stimulatescytoprotectivevitagenesinvolvedinantioxidantdefense.Inthiswork,weinvestigatedtheabilityoftheantioxidantcurcumintoinducetranscription

factorNrf2inaneurodegenerativemodelinducedbyquinolinicacidinrats.Animalswereadministeredwithcurcumin(400mg/kg, p.o.)for10days,andthen

intrastriatallyinfusedwithquinolinicacid(240nmol)onday10oftreatment.Curcuminpreventedrotationbehavior(6dayspost-lesion),striatalmorphological

alterations(7 dayspost-lesion)and neurodegeneration(1 and3 dayspost-lesion)i nducedbyquinolinicacid.Curcuminalso reducedquinolinicacid-inducedoxidativestress(measuredasproteincarbonylcontent)at6h post-lesion.Theprotectiveeffectsofcurcuminwereassociatedtoitsabilitytopreventthe

quinolinicacid-induceddecreaseofstriatalintra-nuclearNrf2levels(30and120minpost-lesion),andtotalsuperoxidedismutaseandglutathioneperoxidase

activities(1daypost-lesion).Therefore,resultsofthisstudysupporttheconceptthatneuroprotectioninducedbycurcuminisassociatedwithitsabilityto

activatetheNrf2 cytoprotectivepathwayandto increasethetotal superoxidedismutaseandgluta thioneperoxidaseactivities.

2013ElsevierInc.Allrightsreserved.

Keywords: Nrf2;Curcumin;Quinolinicacid;Oxidativestress

1.Introduction

Neurodegenerationistheresultofpathologicalprocessesproduc-

ingsevereandspecificpatternsofbraincelldamageinaconcertedmanner [1,2].Neurodegenerativeeventsconstituteamajorcausefor

development of neurological disorders. Humandiseases coursingwithneurodegenerationinvolveexcitotoxicityasatriggeringevent

fortheinitiationofdeadlycascades [3-5].Excitotoxicityiscurrentlydefinedasatoxicprocesscharacterizedbyasustainedstimulationof

excitatoryaminoacidreceptors [6-8],mainlyinvolvingN-methyl-D-aspartatereceptors(NMDAr).Differenttoxiceventsderivedfrom

excitotoxicityhavebeencharacterizedinexperimentalmodels,and

they include upregulation of detrimental signalingpathways,dis-

ruptedCa2+ homeostasis,andrecruitmentofreactiveoxygenand

nitrogenspecies(ROS/RNS),withfurtheroxidative/nitrosativestress

[2,8-11],ultimatelyleadingtocelldeath.Quinolinic acid (QUIN) is a glutamatergic agonist acting on

NMDAr, preferentially in discrete populations of these receptorscontaining the NR2A and NR2B subunits. This metabolite is

synthesized in the kynurenine pathway, and is normallypresentat nanomolar concentrations in human and rat brains [12].

However, under pathological conditions, kynurenine pathway isstimulatedto increasethe levels of QUIN, thereforeaugmenting

therisk forexcitotoxic events.Then, thistoxicmetabolite exertsexcessiveexcitationofNMDArandrecruitsenhancedcytoplasmic

Ca2+ concentrations, mitochondrial dysfunction, decreased ATP

levels,cytochromecreleaseandoxidativestress,furtherleadingto

selectiveloss of GABAergic and cholinergic neurons [13]. Indeed,wheninjectedintothebrain,QUINreproducesneurodegenerative

events in rodents, resembling those observed in the brains of

patients with Huntington's disease [14]. In addition, the intras-

triatalinfusionofQUINtorodentsstimulateslipidperoxidationin

thisregionat shorttimes [15],andthese findingsarerelatedto

increased extracellular levels of hydroxyl radical (OH) in the

samebrainregion [16].Furthermore,QUINisknowntogeneratea

dysregulation in the oxidant/antioxidant ratio by affecting the

Available online at www.sciencedirect.com

JournalofNutritionalBiochemistry24(2013)14 24

ThisworkwassupportedbyCONACYT(GrantNo.103527toPDMand

129838toJPCH)andbyPAPITIN121910. Correspondingauthor. PatologaVascular Cerebral,InstitutoNacional

deNeurologayNeurocirugaManuelVelascoSurez,InsurgentesSur3877,

MxicoD.F.,14269,Mxico.Tel.:+525556063822x2009;fax:+52555424

0808.

E-mailaddress: [email protected](P.D.Maldonado).

0955-2863/$-seefrontmatter2013ElsevierInc.Allrightsreserved.

http://dx.doi.org/10.1016/j.jnutbio.2011.12.010

reducedglutathione:oxidizedglutathione(GSH:GSSG)ratio,aswell

a s depleting the activity of Cu,Zn-SOD at different post-lesion

times [17], alsorecruitingthe early andtime-dependent formation

of peroxynitrite (ONOO-) as a key RNS contr ibuting to this

game,CA,USA).Allotherreagentswerepurchasedfromotherknowncommercial

sources.DeionizedwaterfromaMilli-QSystemofMillipore(Bedford,MA,USA)was

usedforthepreparationofallsolutions.

2.2.Animals

15I.Carmona-Ramrezetal./JournalofNutritionalBiochemistry24(2013)14 24

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para gm . otewort y, t ese an o t er a t erat ons n uce

by QUIN can be prevented by different antioxidants such as

melatonin, sodium selenite, L-carnitine, epigallocatechin gallate,

etc. [15,19-21].Thisevidencehighlightsasubstantialcontributionof oxidative stress to the toxic pattern elici ted by QUIN.

Consequently,itcanbeassumedthatagentsexhibitingantioxidantproperties may have potential therapeutic valueat experimental

levelinthisandothermodels.Curcumin(CUR),thepolyphenolicnon-flavonoidyellowbioac-

tivecomponent of turmeric thepoweredrhizome of Curcuma

longa Linn ,hasbeenshowntoproduceawiderangeofpositive

biological effects through its antioxidant and anti-inflammatory

properties [22,23].CURisnon-toxicandhasbeenshowntobeapotent free radicalscavenger [24,25]. Inaddition,CUR, like other

polyphenols commonly found in plants, fruits and vegetables,

competes in efficacywith vitamins C and E [26], although recentfindingshaveattributedpartofitsanti-in flammatoryefficacytoits

morestablemetabolitetetrahydrocurcumin [27].Nevertheless,CUR

itself has been shown to cross the blood-brain barrier to exert

neuroprotective effects,suchas those reportedagainst homocyste-

ine-inducedcognitiveimpairment andoxidative stressin rats [25]. CUR

alsoinhibits amyloid toxicity in vivo [28] and improves memory

functionsinastreptozotocin-induceddementiamodelinratsthroughinsulin receptors [29]. One of the most relevant findingson CUR

researchis thedescriptionofits capacitytoupregulatethemaster

antioxidantcoordinator,transcriptionfactornuclear factorerythroid2-

related factor2 (Nrf2), whichin turn is responsible forphase 2

antioxidant and detoxification genes expression, such as heme

oxygenase-1,andthiseffectofCURisabletoprotecttheratbrain

fromfocalischemia [23].

DespiteafewgroupshavealreadydescribedprotectiveeffectsofCURon QUIN-inducedtoxicity includingantiperoxidativeactionsin rat brain homogenates [30], as well as antiexcitotoxic and

antioxidant effectsin primaryculturesof humanneurons [21] ,

to our knowledge there are no reports on the effects of thispolyphenoliccompoundinthetoxicmodelinducedbyQUINunder

invivoconditions,noronapossibleinvolvementofNrf2inCUR-induced neuroprotection. Therefore, theaim of thiswork wasto

evaluateifCURcanattenuateorpreventtheQUIN-inducedinvivotoxicity in rats, and whether this effect is dependenton Nrf2

upregulation.OurresultssupportaroleoftheNrf2pathwayintheneuroprotectiveactionsofCURinthisparadigm.

2.Methodsandmaterials

2.1.Reagentsandchemicals

CUR,QUIN,apomorphine,2,4-dinytrophenilhydrazine(DNPH),guanidinehydro-

chloride,4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES),sodium fluo-

ride(NaF), phenylmethylsulfonylfluoride,sodium pyrophosphate,sodium vanadate

(Na3VO4), ethylenediaminetetraacetic acid(EDTA), ethylene glycoltetraacetic acid

(EGTA),Nonidet P-40,glycerol, sodiumdodecylsulfate (SDS),leupeptin, glutathione

(GSH),glutathionereductase,NADPH,xanthine,xanthineoxidase,nitrobluetetrazo-

lium(NBT),andbovineserumalbumin(BSA),wereobtainedfromSigma-Aldrich(St.

Louis,MO,USA).Fluoro-jadeB(F-JB)wasobtainedfromMillipore(Bedford,MA,USA).

Anti-Nrf2antibodyusedforimmunofluorescence(C-20,sc-722)andWesternblot(sc-13032),anti-Lamin B1 antibody (sc-20682), and 4,6-diamidino-2-phenylindole

dihydrochloride (DAPI, sc-3598)were obtained from Santa CruzBiotechnology

(SantaCruz,CA,USA).Anti- -tubulinantibody(ab-11307)wasfromAbcam(San

Francisco,CA,USA). AlexaFluor488 goatanti-rabbitIgG secondaryantibodywas

obtained from Invitrogen (Carlsbad, CA,USA). Goat-antirabbit IgG horseradish

peroxidase-conjugate(62-6120)and goat-antimouseIgG horseradishperoxidase-

conjugate(sc-2005)secondaryantibodiesusedforWesternblotwerefromZymed

(San Francisco, CA, USA) and Santa Cruz Biotechnology(SantaCruz,CA, USA),

respectively.Vectashield mountingmedium wasobtained fromVector Labs(Burlin-

MaleWistarratsinitiallyweighing250 300g,wereusedthroughoutthestudy.

Forallexperimentalpurposes,animalswerehoused fivepercageinacrylicboxesand

providedwithstandardcommercialratchow(LabRodentDiet5001;PMIFeedsInc.,

Richmond,IN,USA)andwateradlibitum.Thehousingroomwasmaintainedunder

constantconditionsofhumidity(50%10%),temperature(25C3C)andlighting

(12 h dark-lightcycles). All procedures with animals were carried outstrictly

accordingto theNationalInstitutes ofHealth Guidelinesfor thecareand useof

laboratoryanimals andthelocalguidelinesontheethicaluseofanimalsfromthe

Health Ministry of Mexico. During the experiments, all efforts were made to

minimizeanimal suffering.

2.3.Experimentaldesign

All experiments were performedduringthe morning (starting every dayat

8:00AM).Theanimalswererandomlydividedintofourgroups,asmentionedbelow:

1)controlgroup(CT),treatedwithvehicleplussaline;2)CURgroup,treatedwithCUR

plussaline;3)QUINgroup,treatedwithvehicleplusQUIN;and4)CUR+QUINgroup,

treatedwithCURplusQUIN.AnimalsfromCURandCUR+QUINgroupsreceivedCUR(100,200or400mg/kg/day,p.o.)for10consecutivedays.CTandQUINgroupsreceived

vehiclefor10days.CURwasdissolvedincarboxymethylcellulose5%(vehicle).Onday

10,animalswereintrastriatallyinfusedwith1 lofQUINorsaline.QUIN-treatedrats

receivedasingleinfusionofQUIN(240nmol/L)intherightstriatum,accordingtothe

followingstereotaxiccoordinates:+0.5mmanteriortobregma,-2.6lateraltobregma,

and+4.5mmventraltothedura [31]. TheeffectsofincreaseddosesofCUR(100,200

and400mg/kg)oncirclingbehavior(6dayspost-lesion)andhistologicalalterations

(7days post-lesion)inducedby QUINwere firstevaluatedto select an optimum

protective doseof CUR.The mostprominent protective effect wasobservedwith

400mg/kg,sothisdosewasusedfromthispointonforfurtherassays.Neurodegenera-

tion(1and3dayspost-lesion),proteincarbonyllevels(6hpost-lesion),Nrf2activation

(30and120minpost-lesion),andactivitiesoftotalsuperoxidedismutase(SOD)and

glutathioneperoxidase(GPx)(1daypost-lesion)weredetermined.

2.4.Circling behavior

Motoralterations,assessedasrotationbehavior,wereevaluatedinanimalsfrom

allexperimentalgroups,accordingtopreviousreports [20,32].SixdaysafterQUIN

infusion,animalswereadministeredwithapomorphine(1mg/kg, s.c.)andseparatedintoindividualacrylicboxcages.Fiveminuteslater,thenumberofipsilateralrotations

tothelesionedsidewasrecordedfor1h.Eachrotationwasdefinedasacompleteturn

(360).Dataareexpressedasthetotalnumberofipsilateralturnsin1h.

2.5.Histologicalexamination

Braintissueswerecollectedandhistologicallyprocessedaccordingtoprevious

descriptions [20,32].Sevendaysafterintrastriatallylesioned,animalsfromallgroups

wereanesthetized i.p.with0.5mLofsodiumpentobarbitalandperfusedtranscardially

with 0.9%salinesolution containing heparin(200/1 v/v), followedby 4%p-

formaldehydeat4C.Brainswereremoved,post-fixedin4%p-formaldehydefor7

days andembedded in paraffin. Fixedtissues were serially sectionedin an 820

HistoSTAT microtome(AmericanInstrument Exchange, Inc.,Haverhill,MA, USA).

Striatalsections(5 m-thick)wereobtainedevery100 m,coveringatotaldistanceof

300 m(100 manteriorand100 mposteriortotheneedletract).Allsectionswere

stainedwith hematoxylin-eosin(H&E)to visualizecell bodies,usingan imageanalyzer

IM100(LeicaCambridge,UK).

2.6.Quantitativeassessmentof lesions

Cellcountingin histologically preparedsectionswas performedas previously

described [32].Thegeneralcriteriatoscoredamagedcellsincludedpyknoticnuclei,

cytoplasmic vacuolation, neuronal atrophy, interstitial edema and necrosis. The

numberof damagedand preservedneuronswas obtainedfrom 5randomly selected

fieldsinthreesectionslidesperbrain.Dataareexpressedaspercentageofdamaged

neuronsper field.

2.7.Detectionof neurodegenerationusingF-JBstaini ng

Braintissuewascollectedandprocessedaccordingtopreviousreports [33,34].

Oneand three days after intrastriatallylesioned,animals from all groups were

anesthetized i.p. with0.5 mLof sodium pentobarbital andtranscardiallyperfused

with 0.9%saline solution containingheparin (200/1v/v), followed by 4% p-

formaldehyde at4C. Brains wereremovedand post-fixedin 2%p-formaldehyde

and30%sucrosefor7daysat4C.Tissueswerethensubmergedinoptimalcutting

temperaturecompoundandfrozenoverliquidnitrogen,followedbycryosectionat

-25Cina cryostatmicromHM520(ThermoScientific,Pittsburg,PA,USA).Sections

(22 m-thick) were mounted onto glass slides coated with 0.5% gelatin and

immersedin 1%NaOHand80% ethanol for5 min, followedfor2 minin70%

ethanol.Slidesweretransferredto0.06%KMnO4 for10minonashakingtableto

assess consistent backgroundsuppression betweensections,and thenrinsed in

distilledwaterfor2min.Sectionswerestainedina0.0004%F-JBplus0.0002%DAPI

solutionfor20min.Slideswererinsedthricewithdistilledwaterfor1mineachstep.

Excesswaterwasremoved,placingthesectionsonaslidewarmerat50Cuntilfully

dry(510min),andthenclearedbyfurtherimmersioninxyleneforatleast1min

beforecoverslippingwithanon-aqueous,nonfluorescentplasticmountingmedia.

ImmunofluorescencewasvisualizedwithanimageanalyzerIM100(LeicaCambridge,

UK)usingagreen filterforF-JBandbluefi lterforDAPI.

2.8.Protein carbonylcontent

Asan indexofproteinoxidation,proteincarbonylcontentinthestriataltissue(6h

post-lesion)wasdeterminedaspreviouslydescribed [32].Assessmentofcarbonyl

formationwasdoneonthebasisofformationofproteinhydrazonebyreactionwith

DNPH.Striatalhomogenateswereincubatedwith10%streptomycinsulfatetoremove

nucleicacidsovernightandcentrifugedat21,000gat4Cfor20min.Further,striatal

homogenates were treated with 10mM DNPH (in2.5M HCl) for1 h atroom

temperature,and10%trichloraceticacidwasaddedandcentrifugedat2,500gat4Cfor10min.Pelletwaswashedthreetimeswithethanol/ethylacetate(1:1),dissolved

with 6 M guanidine hydrochloride (inphosphatebuffer 20 mM,pH 7.4), andcentrifugedat5,000gat4Cfor3mintoremoveinsolublematerial.Absorbancewas

measuredat370nm.ProteincarbonylcontentisexpressedasnmolDNPH/mgprotein,

usingthe molarabsorptioncoefficient ofDNPH(22,000 M-1 cm-1).Totalprotein

concentration wasobtainedbyreadingopticaldensityat 280nm inblanktubes

preparedin parallel(treatedonlywith2.5M HCl),usinga standardcurveofBSA(0.25

2mg/ml)preparedin6Mguanidinehydrochloride.

2.9.Detectiono Nr2 b immuno luorescence

KCl,1mMphenylmethylsulfonyl fluoride,20mMNaF,1mMsodiumpyrophosphate,

1mMNa3VO4,1%NonidetP-40,and1 g/mLleupeptin).Homogenateswerethen

centrifuged at 500g for5 min, thesupernatants were recovered as cytosolic

fractions,and thenuclear pellets were washedin cold bufferB (10mM HEPES

pH=8.0,0.1mMEDTA,1mMphenylmethylsulfonyl fluoride,20mMNaF,1mM

sodiumpyrophosphate, 1 mM Na3VO4, 25% glycerol,0.1 M NaCl, and 1 g/mL

leupeptin).Afterasecondsteptofcentrifugationat500gfor5min,thenucleiwere

resuspendedin2%SDShypertoniccoldbufferC(10mMHEPESpH=8.0,0.1mM

EDTA,1mMphenylmethylsulfonyl fluoride,20mMNaF,1mMsodiumpyrophos-

phate, 1 mMNa3VO4, 25% glycerol, 0.4 M NaCl, and 1 g/mL leupeptin),and

sonicated.Cytosolicandnuclearfractionswereresolved inSDS-PAGE(10%), using60 g

(cytoplasmicfraction) or 80 g (nuclear fraction) protein/lane,transferred into

Immobilon-P membranes (PVDF, Millipore), and immunoblotted with primary

antibodies:anti-Nrf2,anti--tubulinandanti-LaminB1(1:1,000).Peroxidase-conju-

gatedsecondaryantibodies(1:10,000)wereusedtodetecttheproteinsofinterestby

enhancedchemiluminescence,accordingtoapreviousreport [35]. Thecorresponding

bandswerequantifiedbydensitometry.

2.11.SOD activity

TotalSODactivity(MnSODandCuZnSOD)inhomogenateswasdeterminedusing

xanthine-xanthineoxidasesystemtoreduceNBT.Mixturereactioncontainedinafinalconcentration:0.122mMEDTA,30.6 MNBT,0.122mMxanthine,0.006%BSA,and

49mMsodiumcarbonate.Fivehundred lofhomogenates(1:20)wereaddedto2.45 mlof themixture describedabove, then 50 l xanthine oxidase, in a final

concentrationof2.8U/l,wereaddedandincubatedinawaterbathat27Cfor30min.

Thereactionwasstoppedwith1mlof0.8mMcupricchlorideandtheopticaldensity

wasreadat560nm.OnehundredpercentofNBTreductionwasobtainedinatubein

whichthesamplewasreplacedby distilledwater.T heamountof proteinthatinhibited

NBTreductionto50%ofmaximumwasdefinedasoneunitofSODactivity.Results

wereexpressedasU/mgprotein.

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@@@@@_____Curcumin restores Nrf2 levels and prevents quinolinic acid-induced neurotoxicity☆ _ Julio Tobón - Academia