Rev. bras. farmacogn. vol.25 número6

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Anti-inflammatory

effects

of

methanolic

extract

of

green

algae

Caulerpa

mexicana

in

a

murine

model

of

ulcerative

colitis

Mariana

A.O.

Bitencourt

a

_,

_Hylarina

_M.D.

_Silva

a

_,

_Gisely

_M.F.

_Abílio

b

_,

_George

_E.C.

_Miranda

c

_,

Adolpho

M.A.

Moura

d

_,

_João

_X.

_de

_{Araújo-Júnior}

e

_,

_Ericka

_J.D.

_Silveira

a

_,

_Barbara

_V.O.

_Santos

b

_,

Janeusa

T. Souto

a,∗

a_Departamento_de_{Microbiologia}_e_{Parasitologia}_e_Departamento_de_Odontologia,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brasil b_Programa_de_{Pós-graduac¸}_ão_em_Produtos_Naturais_e_Bioativos_Sintéticos_Universidade_Federal_da_Paraíba,_João_Pessoa,_PB,_Brasil

c_Laboratorio_de_Algas_Marinhas,_Departamento_de_Ecologia_{Sistemática,}_Universidade_Federal_da_Paraíba,_João_Pessoa,_PB,_Brasil d_Centro_de_Pesquisa_Ageu_Magalhaes,_Fundac¸_ão_Oswaldo_Cruz,_Cidade_{Universitária,}_Recife,_PE,_Brasil

e_Laboratorio_de_Química_Medicinal,_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_de_Alagoas,_Maceió,_AL,_Brasil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28April2015 Accepted1October2015 Availableonline26October2015

Keywords: Caulerpamexicana

Greenalgae Inflammation Ulcerativecolitis Cytokines Tissuedamage

a

b

s

t

r

a

c

t

Inflammatoryboweldiseases,whichincludeCrohn’sdiseaseandulcerativecolitis,arecharacterizedby chronicandrelapsedgutinflammation.Caulerpamexicanaisatypeofgreenmarinealgaethatcanbe foundintropicalareas,suchastheBrazilianCoastland.Thesemacrophytesexhibitinvitroandinvivo anti-inflammatorypropertiessuchastheabilitytoreducebothcellmigrationtodifferentsitesandedema formationinducedbychemicalirritants.TheaimofthisstudywastoexaminetheeffectoftheC.mexicana methanolicextractonthetreatmentofcolitisinducedbydextransodiumsulfate.Acuteexperimental colitiswasinducedinBALB/cmicebytreatmentwith3%dextransodiumsulfateorallyfor14days.During this14-dayperiod,C.mexicanamethanolicextract(2mg/kg/day)wasgivenintravenouslyonalternate days.Treatmentwiththemethanolicextractsignificantlyattenuatedbodyweightlossandsevereclinical symptoms.Thiswasassociatedwitharemarkableameliorationofcolonicarchitecturedisruptionand asignificantreductioninpro-inflammatorycytokineproduction.Theseresultssuggestthatthe anti-inflammatoryactionofC.mexicanamethanolicextractoncolorectalsitesmaybeausefultherapeutic approachforinflammatoryboweldiseases.

Introduction

Inflammatorybowel diseases(IBD)includingCrohn’sdisease (CD)andulcerativecolitis(UC),arecharacterizedbychronicand relapsedgutinflammation.UCisassociatedwithintestinal inflam-mationandoftenresultsinweightloss,diarrheaaccompaniedby blood and mucus,fever, gastric dysmotilityand colon shorten-ing(Hendricksonetal.,2002;ByrneandViney,2006;Cho,2008). Traditionaltherapeuticagents,suchas5-aminosalicylates(5-ASA) andcorticosteroids,arestillusedtotreatIBD.Some immunomod-ulators,such asazathioprine and 6-mercaptopurine,as wellas antibioticsarebecomingimportantinsteroidresistantand steroid-dependent patients (Cho et al., 2011). Considering the serious sideeffects associatedwiththeconventionaltreatment, natural

∗ Correspondingauthor.

E-mail:jtsouto@cb.ufrn.br(J.T.Souto).

products,includingthosefrommarineorigin,havebeenstudiedto aidintheimprovementofIBDclinicalsymptoms(D’Orazioetal., 2012).

Caulerpamexicana isa green marinealgae, fromthe Bryop-sidalesOrderandtheCauleparceaeFamily, whichcanbefound especiallyintropicalareassuchastheBrazilianCoastland(Neto etal.,2008).Thesemacrophyteshavemanybioactivecompounds, suchaspolysaccharides,terpenesandflavonoids,whichhave dif-ferent pharmacological activitieswith antitumor,antiprotozoal, antiviral,antioxidant,antinoceptive,anti-inflammatoryand anti-coagulanteffects(Rochaetal.,2007;Souzaetal.,2009a,b;Matta etal.,2011;Torresetal.,2014).Recently,weshowedthataqueous andmethanolicextractsofC.mexicanahaveinvitroandinvivo

anti-inflammatory propertiesthat reduce thepro-inflammatory cytokine levels in macrophage culture supernatants stimulated withLPSand decreasethecell migration todifferentsitesthat occurs following stimulation with zymosan. The extracts also reduceedemaformationinducedbychemicalirritants(Bitencourt etal.,2011).

http://dx.doi.org/10.1016/j.bjp.2015.10.001

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Theoral administrationofdextransulfate sodiumsalt (DSS) iswidelyusedasamodelforUC.Thismodelischaracterizedby mucosalinfiltration ofinflammatory cells, epithelialinjury and ulceration(Kimetal.,2010).Anotherstudyreportedthatmicewith DSS-inducedcolitisexhibitedphenotypiccharacteristicssimilarto humanacuteandchronicUC(Okayasuetal.,1990).Wepreviously showedthatC.mexicanaextractsexhibitanti-inflammatory activ-ity(Bitencourtetal.,2011).Therefore,theaimofthisstudywasto examinethetreatmenteffectsoftheC.mexicanamethanolicextract onDSS-inducedcolitis.

Materialsandmethods

ExtractionandisolationofthemethanolicextractofC.mexicana

ThegreenalgaeCaulerpamexicanawascollectedinthecoastal regionofBessabeach(7◦₀₃′₅₂′′_S/34◦₄₉′₅₁′′_W),_Joao_Pessoa,_in_the ParaibaStateofBrazilinApril2008.Thespecimenwasidentified byDr.GeorgeEmmanuelCavalcantideMiranda.Voucher speci-mensofC.mexicana(JPB13985)havebeendepositedintheLauro PiresXavierHerbariumattheFederalUniversityofParaiba,Brazil. Freshalgaesampleswerewashed,dried,lyophilizedand exhaus-tivelyextractedwithmethanolinaSoxhletapparatus,toobtainthe methanolicextract.

Animals

MaleBALB/cmice(6–8weeksold)wereusedin the experi-ment.Allmicewerehousedfivepercageataroomtemperature of22±2◦_C_and_under_a₁₂_h:12_h_light/dark_cycle._They_had_free accesstofood andwater. Groups of fiveanimals wereused in eachtestgroupandcontrolanimalsreceivedsalineonly.Allinvivo

experimentswereapprovedby“EthicsCommitteeonAnimalUse, CEUA/UFRN,” under protocol number 044/2009, which was in accordancewiththeguidelinesoftheBrazilianCommitteefor Ani-malExperimentation(COBEA).

AssessmentofDSS-inducedcolitis

Previously we evaluate the dose-response of C. mexicana

methanolicextractinperitonitis,earswellingandairpouch mod-els,andthedosethatshowedbetteranti-inflammatoryeffectwas 2mg/kg,andthisischosentobeusedincolitismodel(Bitencourt et al., 2011). Our colitis model was developed following the methodologydescribedbyKimetal.(2010)andMelgaretal.(2006) withtheaimofevaluatecolitisduringitsacutephase.Therefore, experimentalcolitiswasinducedbygivingmicedrinkingwaterad libitumcontainingDSS3%(w/v)for14days.Miceweremonitored carefullyeverydaytoconfirmthattheyconsumedapproximately equalvolumesofDSS-containingwater.Fortheexperiment,the miceweredividedintothreeexperimentalgroups.Thefirstgroup waskeptasvehicle-treatedcontrolandtreatedwithsalineinthe samerouteasthemethanolicextract,andthesecondgroupwas givendrinkingwater withDSSonlythroughoutthe experimen-talperiod.The othergroupconsisted ofmice receiving3%DSS andadministeredmethanolicextract(2mg/kg/day)byintravenous routeonalternatedaysfor14days.Themicetreatmentwiththe methanolicextractbeganonthesamedaythatDSSadministration indrinkingwater.

Evaluationofdiseaseactivityindex(DAI)

Themicewerecheckeddailyforcolitisdevelopmentby mon-itoringbodyweight,grossrectalbleedingandstoolconsistency. Theoveralldiseaseseveritywasassessedbyaclinicalscoring sys-temonascaleof0–4. Briefly,thescoringwasasfollows:0,no

weightloss,nooccultbloodinthestoolsandnormalstool con-sistency;1,weightlossof1–5%,nooccultbloodandnormalstool consistency;2,5–10%weightloss,positiveforfecaloccultblood andloosestools;3,10–20%weightloss,positiveforfecaloccult bloodandloosestools;and4,greaterthan20%weightloss,gross rectalbleedinganddiarrhea.

Colontissueculture

Intestinetissuesamplescorrespondingtothetransversecolon wereexhaustivelywashedwithRPMI-1640mediumcontaining2% fetalbovineserumandgentamicinbeforebeingcutintosmaller pieces.Then,approximately0.5cmoftissuewasplacedin1mlof 10%FBScontainingRPMI-1460mediumin24-welltissueculture platesandincubatedfor24hat37◦_C_in_5%_CO₂_._Thereafter,_the supernatantswerecollectedfordeterminationofIL-6,IL-12,TNF-␣, IFN-␥andIL-17byELISA.

ELISAforcytokines

TheIFN-␥,IL-17,IL-12,TNF-␣andIL-6concentrationsinthe cell-freeculturesupernatantsofthecolontissuesweremeasured intriplicate,utilizingasetenzyme-linkedimmunosorbentassay kit(eBioscience,SanDiego,CA,USA)accordingtomanufacturer’s instructions.

Histopathology

Aftertheperiodof inductionofcolitisbyDSS,animalswere euthanized;fragmentsof0.5cmofascendantcolonwereobtained, fixedinasolutionof4%formalinandusedforhistologicalanalysis byconventionaltissuepreparationmethods.Tissuesampleswere viewedunderthelightmicroscope(40×)afterhematoxylinand eosin(H&E)staining.

Results

TreatmentofmicewithC.mexicanamethanolicextract decreasedtheDAIaftercolitisinduction

Theparameterusedtoevaluatethedevelopmentofthedisease wastheDAI,whichisassociatedwithobservedclinicalsymptoms suchasweightloss,stoolconsistency,fecalbloodanddiarrhea.In ourmodeltheDAIwasevaluatedindividuallyduringthe14daysof thestudy.ThehighestDAIscorewasobservedinDSSgroup(Fig.1D, closedsquares),whichexhibitedintenseweightlossfromthe11th day,asshowninFig.1C(closedsquares),aswellaspersistentand severediarrhea(Fig.1A)andfecalblood(Fig.1B).Ontheotherhand, thegrouptreatedwiththemethanolicextracthadweight devel-opmentsimilartothegroupthatreceivednormalwater(Fig.1C, closedcycles),lowdiseasedevelopmentrate(Fig.1D,closed tri-angles),withnobloodinthestool(Fig.1B,closedtriangles)and littlediarrhea(Fig.1A,closedtriangles)comparedwiththegroup receivingDSS.Overall,theDAIscoreoftheC.mexicana’s methano-licextract-treatedgroupwassignificantlylowerthanthatofthe DSScontrolgroupfromdayfourfollowingadministrationofDSS.

ThetreatmentofmicewithC.mexicanamethanolicextract amelioratedthetissuedamageinthecolonaftercolitisinduction

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5

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Vehicle DSS 3% DSS 3% + ME

Stool consistency score

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Bleending score

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Disease activity inde

x (D

AI)

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Fig.1.Thediseaseactivityindex(DAI)inmicetreatedwithCaulerpamexicanamethanolicextractinthecolitismodel.(A)Stoolconsistencyscore,(B)bleedingscore,(C) weightlossscoreand(D)totalDAIscore.Themicewereadministered3%DSSintheirdrinkingwater(adlibitum)for14dayswithorwithoutmethanolicextract(ME) treatment(2mg/kg/dayi.v.upto14days);salinewasusedasanegativecontrol.ChangesinDAIlevelwereevaluateddailythroughoutthe15-dayexperimentalperiod. *p<0.05inthevehicle-treatedcontrolgroupvs.theDSS-inducedcolitisgroup;significancesbetweentreatedgroupsweredeterminedbyANOVA.

waterwithoutDSSpresentednormalcolonstructureswithcrypts and other morphological structures preserved (Fig. 2A and B). On theotherhand, colon samplesfromthe micethat received DSSshowedtypicalinflammatorychangesinthecolon architec-tureand a diffuseinflammatory infiltrate,composedmainlyby mononuclearcells.Atcertainpointstheinflammationwasmore intense,promotingsmallfociofglandulardestructionand mod-erateulceration,cryptdilationandgobletcelldepletionasshown inFig.2CandD.Althoughtheyhadaslightinflammatory reac-tiondecrease,thegrouptreatedwiththemethanolicextracthad an acute inflammatory infiltrate observed on theentire length ofthemucosathat wasintenseinsomeareas,leading to glan-dulardestructionandulcerationpoints.Insomeareas,abscesses withexudatewerepresent,althoughthesewerelessintenseand morefocalwhencomparedwiththegroupthatreceivedonlyDSS (Fig.2EandF).

ThetreatmentofmicewithmethanolicextractofC.mexicana

decreasedTh1andTh17cytokineproductioninthecolonculture supernatant

Becausecytokinesareimportantmediatorsofinflammationin colitismodels,thenextstepwastoevaluatetheTh1andTh17 cytokinelevelsintheinvitrocolontissueculturesupernatant.The resultspresented inFig.3 showanintenseproduction ofIFN-␥ (Fig.3A),IL-6(Fig.3B),IL-12(Fig.3C),IL-17A(Fig.3D)andTNF-␣ (Fig.3E)inthecolonculturesupernatantsofmicethatreceived DSSintheirdrinkingwater.However,themicetreatedwiththe methanolicextractofC.mexicanaexhibitedasignificantdecreasein thelevelsofthesecytokinescomparedwiththegroupthatreceived DSSintheirdrinkingwater.

Discussion

UC,whichismajorformofIBD,isanonspecificinflammatory diseaseofthelargeintestine. Inaddition,it is alifelong illness with profound emotional and social impacts, it causes serious

intestinaltractdamageanditsprogressiontochronicUCcanleadto coloncancer(XavierandPodolsky,2007;ShanahanandBernstein, 2009).TheUCinducedinmicebydrinkingwatercontainingDSS leadstoclinicalsymptomssuchasweightloss,diarrhea,fecalblood, mucosalulcerationandcolonicshortening(Kimetal.,2010).Inthis study,weevaluatedthedevelopmentofthediseasefor14days (Melgaretal.,2006).ThehighestDAIscorewasobservedintheDSS groupthatdevelopedintenseweightloss,fecalbleedingand per-sistentandseverediarrhea.Ontheotherhand,theanimalstreated withthemethanolicextractdidnotshowtheclinicalsymptomsof colitisorweightloss.

BecauseUCischaracterizedbyanintenseinflammatory pro-cess, the ability of themethanolic extractto reduce thecolitis clinicalsymptomsdemonstratestheextract’santi-inflammatory activity.Inaddition,thecolonhistologicalanalysisshowsthatthe animalsthatreceivedwaterwithDSSpresentedtypical inflamma-torychangesintheircolonarchitecturethatwasslightlyattenuated whentheanimalsweretreatedwiththemethanolicextract.These effectsonclinicalsymptomsandonhistologicalparameterscould be due to the presence of antioxidant compounds, suchas ␤ -caroteneand␣-tocopherolthathavebeenisolatedfromC.mexicana

(Sousaetal.,2008).Theinhibitionoffreeradicalsbythese com-poundscouldbedecisiveindecreasingtheoxidativestressthat causesdamagetotheintestinalepithelialbarrier(ZhuandLi,2012; Zhengetal.,2012).Furthermore,wereportedpreviouslythatthe

C.mexicanamethanolicextractinhibitscellmigrationinamodel ofperitonitis(Bitencourtetal.,2011)andwebelievethatthis inhi-bitioncouldalsobehappeninginthecolitismodelpresentedhere, contributingtotheattenuationoftheclinicalsymptomsaswell asleadingtotheslightimprovementonthehistologicalanalysis observedbytheinflammatoryinfiltratereductioninthecolonsof

C.mexicanatreatedanimals.

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Fig.2. TissuedamageinthecolonofmicewithDSS-inducedcolitistreatedwithmethanolicextractofCaulerpamexicana.Salinewasusedasanegativecontrol(AandB). Representativesectionsofcolontissuefrommicethatreceivedintheirdrinkingwater3%DSSwhichweretreatedwithmethanolicextractofC.mexicana(2mg/kg,i.v.,up to14days),showinganimprovementininflammatoryprocessesandrestorationofmucosaltissue(EandF)andcolonsectionsfromanimalsthatwerenottreatedwiththe extractunderstudy(CandD),showingthedestructionofthemucosaltissuewhichhasbeenreplacedbyinflammatorygranulationtissue(arrow).Histologicalchangeswere determinedbyH&Estaining.OriginalmagnificationinAandFwas100×,inBandDwas200×andinCandEwas40×.

modelisreliableforstudyingthepathogenesisofUCandfor test-ingdrugs orphytochemicals for treatment(Cunha etal., 2013; Luceroetal.,1996).Theliteratureshowsthatthereisanimbalance betweenpro-inflammatoryandanti-inflammatorycytokinesinUC (Papadakis,2004).Pro-inflammatorycytokinesareresponsiblefor determiningthenatureof theimmuneresponse inUC andcan stimulatetherapidsynthesisandsecretionofinflammatory medi-ators,suchasreactiveoxygenandnitrogenspecies,leukotrienes, plateletactivatingfactorandprostaglandins(Neuman,2007).The hyperactivationofimmunecellsisanotherimportantfactorinIBD progressionandisassociatedwithhighlevelsofpro-inflammatory cytokinesfromTh1andTh17patternofimmuneresponse,such as TNF-␣, IL-6 and IFN-␥, which are known to be involved in colondamage(Gálvez,2014).Cytokinesareimportantmediators

ofinflammationandelevatedlevelsofpro-inflammatorycytokines areobservednotonlyintheinflamedgutofIBDpatientsbutalsoin animalswithDSS-inducedcolitis(Neuman,2007).Becauseofthis itisinterestingtoevaluateifthepatternofimmuneresponsein colitismodelinducedbyDSScouldbechangedbytreatmentwith themethanolicextractofC.mexicanaandthispatterncanbe eval-uatedbythetypeofTh1andTh17cytokinesproducedatthesite ofinjury.

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0 10 20 30 40

IFN-γ

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IL-12 (pg/ml)

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Fig.3. LevelsofTh1andTh17cytokinesobservedinculturedcolonictissueofmicewithDSS-inducedcolitistreatedwithCaulerpamexicanamethanolicextract.Micewere administered3%DSSintheirdrinkingwater(adlibitum)for14dayswithorwithoutC.mexicanamethanolicextract(2mg/kg/dayi.v.upto14days);salinewasusedasa negativecontrol.Theproductionof(A)IFN-␥,(B)IL-6,(C)IL-12,(D)IL-17Aand(E)TNF-␣weredeterminedasdescribedinMaterialandMethodssection.Valuesrepresent mean±SD(n=5)*p<0.05inthevehicle-treatedcontrolgroupvs.theDSS-inducedcolitisgroup;significancebetweenthetreatedgroupswasdeterminedbyANOVA.

methanolicextractexertsananti-inflammatoryeffectinUCby neg-ativelyregulatingthepro-inflammatorycytokinelevels.Reduction ofthelevelsoftheseinflammatorymediatorsmayhaveoccurredby twopossiblemechanisms.Thefirstisassociatedwiththecapacity oftheC.mexicanamethanolicextracttodirectlyinhibitcytokine secretionfrominnateimmunecells,aspreviouslydemonstrated (Bitencourtetal.,2011),andthelatterisrelatedtothereductionof cellmigrationtothecolonicmucosaasanindirectmechanismto decreasecytokinelevels.

Currently some studies have shown the efficacy of nat-ural products in the treatment of experimental IBD. These studies demonstrated that curcumin–piperine mixtures in self-microemulsifying(CUR-PIP-SMEDDS),ellargicacid,mangiferinand olipeinameliorate theDSS-induced colitis in mice throughthe reductioninDAIandhistopathologicallesion,and downregulat-inginflammatorymediatorssuchasMPOactivity,IFN-␥,TNF-␣ andIL-6levels,COX-2andiNOSexpressionandinterferingwith the signaling pathways of p38 MAPK, NF-␬B, and STAT3 (Dou et al., 2014; Li et al., 2015; Giner et al., 2011; Marín et al., 2013).

The green seaweed C. mexicana is composed of numerous compoundsendowedwithpotentialbiologicalactivities( Carrillo-Dominguezet al.,2002).Someof them couldcontribute tothe negativeeffectsmediatedbythemethanolicextractoncytokine secretionatthecolonicmucosaandoninflammatorycell migra-tion to the colon. In fact, compounds like caulerpin and other purifiedindolicalkaloidsfromCaulerpaspeciessuchasCaulerpa racemosa, have proven anti-inflammatoryactivity reducing cell migration in peritonitis models (Bamias and Cominelli, 2007) and sulphated polysaccharides isolated from C. mexicana has anti-inflammatory and antinociception activity (Carneiro et al., 2014).

Therefore,newstudiesareneededtoisolatethecompounds fromtheextract andevaluatetheirmechanismsof action. Fur-thermore,consideringthegreatreductionintheclinicalsymptoms andcytokinesecretion,butonlyslightameliorationof histologi-caldamage,itispossiblethattheassociationoftheC.mexicana

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Conclusions

Insummary,ourdatashowthatthetreatmentof micewith methanolicextractofC.mexicanasignificantlyamelioratethe clin-icalsignsobservedinUCanddecreasethelevelsofcytokinefrom Th1(IFN-g,IL-12andTNF-a)andTh17(IL-6andIL-17)immune responsepatternandthisdecreasecouldbeassociatedwith reduc-tionoftissue damageobservedinthecolonoftheanimalsthat receivedDSS.Thereforethisextractappearspromisingforresearch onmetabolitesthatmaybeeffectiveinthetreatmentofUC.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Authors’contributions

MAOB and HMDS (MSc students) contributed to biological studies, runningthe laboratory work, analysisof the data and draftedthepaper.GMFA(MScstudent)contributetoC.mexicana

extractproduction.GECMcontributedincollectingplantsample and identificationand herbarium confection. AMAMcontribute withthemiceproduction.JXAJcontributedtocriticalreadingof themanuscript.EJDScontributetohistopathologyanalysis.JTSand BVOSdesignedthestudy,supervisedthelaboratoryworkand con-tributedtocriticalreadingofthemanuscript.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Acknowledgments

The authors are grateful to the BNB (482012), CNPq (478655/2007-0)forthejointfundingofthisresearchprojectand byfellowshipsfromCAPES.

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