J. Coloproctol. (Rio J.) vol.36 número2

w w w . j c o l . o r g . b r

Journal

of

Coloproctology

Original

Article

Effect

of

lecithin

on

oxidative

stress

in

an

experimental

model

of

rats

colitis

induced

by

acetic

acid

Josieli

Raskopf

Colares

,

Elizângela

Gonc¸alves

Schemitt

,

Renata

Minuzzo

Hartmann

,

Rosa

Maria

Moura

,

Maria

Isabel

Morgan-Martins

,

Henrique

Sarubbi

Fillmann

,

Lúcio

Fillmann

,

Norma

Possa

Marroni

a_{BioHealth,UniversidadeLuteranadoBrasil(ULBRA),Canoas,RS,Brazil}

b_{LaboratoryofOxidativeStressandAntioxidants,UniversidadeLuteranadoBrasil(ULBRA),Canoas,RS,Brazil}

c_{LaboratoryofHepatologyandExperimentalGastroenterology,HospitaldeClínicasdePortoAlegre(HCPA),UniversidadeFederaldoRio}

GrandedoSul(UFRGS),PortoAlegre,RS,Brazil

d_{MedicalSciences,UniversidadeFederaldoRioGrandedoSul(UFRGS),PortoAlegre,RS,Brazil}

e_{AppliedToxicology,UniversidadeLuteranadoBrasil(ULBRA),Canoas,RS,Brazil}

f_{PontifíciaUniversidadeCatólicadoRioGrandedoSul(PUCRS),PortoAlegre,RS,Brazil}

g_{Physiology,UniversidadeFederaldoRioGrandedoSul(UFRGS),PortoAlegre,RS,Brazil}

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t

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o

Articlehistory:

Received12November2015 Accepted26March2016 Availableonline13April2016

Keywords: Ulcerativecolitis

Inflammatoryboweldisease Lecithin

Oxidativestress

a

b

s

t

r

a

c

t

Ulcerativecolitis(UC)isaninflammatorydiseasethataffectsthebowels.Reactiveoxygen species(ROS)areinvolvedintheprogressofUC.

Objective:EvaluatetheantioxidanteffectoflecithininanexperimentalmodelofacuteUC

inducedbyadministrationofaceticacid(AA)inrats.

Methods:Lecithin(0.5mL/kg/day)administeredorally2daysbeforeandafterinductionof

colitiswith4%AAinavolumeof4mL.Twenty-fivemaleWistarratsweredividedin5groups: control(CO);control+lecithin(CO+LE);colitis(CL);colitis+lecithin(CL+LE);lecithin+colitis (LE+CL).Analsphincterpressure,LPO(TBARS),andantioxidantactivityofenzymes super-oxidedismutase(SOD)andcatalase(CAT)weremeasured,andahistologicalanalysiswith H&Ewasperformed.

Resultsanddiscussion:AnalsphincterpressurewassignificantlysmallerintheCOgroup,

lecithintreatmentincreaseditinpre-andpost-treatedgroups.LPOandSODactivitywere increasedintheCOgroupanddecreasedinthelecithin-treatedgroups.CATactivitywas increasedinCOgroupanddecreasedinlecithingroups.Thehistologicalanalysisshowed damagetothebowelswithdestructionofcrypts,edema,andinflammatoryinfiltrate.Use oflecithinpreservedthecryptsanddecreasedtheedema.

∗ _{Correspondingauthor.}

E-mail:henrique@fillmann.com.br(H.S.Fillmann). http://dx.doi.org/10.1016/j.jcol.2016.03.002

Conclusion: Ulcerativecolitisincreasedlipidperoxidation,andtheuseoflecithinwas effec-tivereducingdamagetothebowelsinthemodelofexperimentalcolitis.

©2016SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Efeito

da

lecitina

sobre

o

estresse

oxidativo

no

modelo

experimental

de

colite

induzida

por

ácido

acético

em

ratos

Palavras-chave: Retocoliteulcerativa

Doenc¸ainflamatóriaintestinal Lecitina

Estresseoxidativo

r

e

s

u

m

o

Aretocoliteulcerativa(RCUI)éumadoenc¸aintestinalinflamatória.Espéciesreativasde oxigênio(ERO)estãoenvolvidasnoprogressodaRCUI.

Objetivo:AvaliaroefeitoantioxidantedelecitinaemmodeloexperimentaldeRCUIinduzida

pelaadministrac¸ãodeácidoacético(AA)emratos.

Métodos:ALecitina(0,5mL/kg/dia)foiadministradaporviaoral2diasanteseapósainduc¸ão

decolitecomAA.VinteecincoratosWistarmachosforamdivididosem5grupos: con-trole(CO);controle+lecitina(CO+LE);colite(CL);colite+lecitina(CL+LE);lecitina+colite (LE+CL).Foramavaliadas:pressãodoesfíncteranal,lipoperoxidac¸ão(LPO),atividade antiox-idantedasenzimassuperóxidodismutase(SOD)ecatalase(CAT),efoirealizadaumaanálise histológicacomH&E.

Resultadosediscussão:ApressãodoesfíncteranalfoisignificativamentemenornogrupoCL,

otratamentocomlecitinaaumentouapressãonosgrupospréepóstratados.ALPOe ativi-dadedaSODaumentaramnogrupoCLediminuiramnosgrupostratadoscomlecitina. A atividadeda CATfoiaumentadanogrupoCLediminuiunosgruposcomlecitina.A análisehistológicamostroudanosaointestinocomdestruic¸ãodascriptas,edemae infil-tradoinflamatório.Ousodelecitinaproporcionouumapreservac¸ãodascriptasediminuic¸ão doedema.

Conclusão: ARCUIaumentaaLPO,autilizac¸ãodelecitinafoieficaznareduc¸ãodosdanos

aointestinoinduzidoporAAnomodelodecoliteexperimental.

©2016SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda.Este éumartigoOpenAccesssobalicençadeCCBY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Inflammatoryboweldisease(IBD)ischaracterizedbychronic inflammation of the gastrointestinal tract, and numerous physiopathogenic mechanisms may be associated with its etiology, such as those of genetic, dietary, immunological, infectious,parasitical, post-radioactive, ischemic and envi-ronmentalorder.1_{Idiopathiculcerativerectocolitis(IURC)and} Crohn’sdiseasearethemostcommonformsofincidenceof IBDanditsetiologyisnotfullyclarified.2,3

IBDposes a serious global healthproblem, as it affects primarilyyoungpeople andhassevereandchronicclinical presentations,occurringallovertheworld.3

Research suggests that oxidative stress may be impor-tant inthe activity and developmentofIBD. Other studies showedthatreactiveoxygenspecies(ROS)aregeneratedin excess in individuals with colitis as compared to normal individuals.2,4

TheexperimentalmodelofcolitisperformedbyFillmann etal.1_{suggeststhatbesidesROS,nitricoxideisinvolvedinthis} situation,triggeringinhibitoryactiononsmoothmuscles, pro-motingrelaxationoftheanalsphincterandtherebyadecrease inanalsphincterpressurelevels.

Theincreaseinthe generationofROSinulcerative coli-tistriggersanimbalanceinthecellredoxstatusandthereby anincreaseinfreeradicals(FR).Suchincreaseoverwhelms the antioxidant defense capabilitiesof the cell, thus char-acterizing oxidativestress(OS),whichinturn triggerslipid peroxidation(LPO),leadingtodisruptionofdisulfidebridgesof lipidsbybreakingthemandlossofcellintegrity,destabilizing itandleadingtocelldeath.5

Theorganismhasadefensesystemagainstoxidantagents composed of enzymatic activity, i.e., enzymes superoxide dismutase(SOD),catalase(CAT)andglutathioneperoxidase (GPx);andnon-enzymaticagents,suchasglutathione(GSH), vitamins(A,C,E),flavonoids,andothercompoundspresentin food,suchaslecithin.Thefunctionofantioxidants(AOX)isto maintaintheredoxbalancebykeepingROSlevelslow,thereby preventingtheformationoffreeradicalssuchassuperoxide anion,hydrogenperoxide,andthemostharmfulone,hydroxyl radical.1,2,5

apolarportion,therebyinsertingitselfinthecellmembrane, maintainingits integrity, asit can react withfreeradicals, sweepingthemandpreventingLPO.7

Thelecithinmoleculeiscomposedofcholine,phosphate and fatty acids, and its importance lies in the fact that it isthelargestlipid componentofthebody.Cholinepresent inlecithinisthedietary component requiredforthe func-tioning of all cells. Lecithin or its metabolites, including phospholipids,ensurethe structuralintegrityandsignaling functions of cell membranes.8 _{Choline acts as a} precur-sorforthe biosynthesisofphosphatidylcholine (PC),which in turn plays an important role in the intestinal absorp-tionoflipids,asit increasesthemicellarsolubilityforming chylomicrons.9

Inthisstudyweevaluatedtheabilityoflecithintoreduce tissueoxidativestressintheexperimentalmodelofcolitisby aceticacidinWistarrats.

Materials

and

methods

Twenty-fivemaleWistarratsweighing300gwereused.They were divided infive groupsas follows:control(CO), colitis (CL),control+lecithin(CO+LE),colitis+lecithin(CL+LE)and lecithin+colitis(LE+CL).

Theanimalswere keptinthe vivariumofthe Universi-dadeLuteranadoBrasil(ULBRA)throughouttheexperiment, in12hlight/darkcycleandtemperaturebetween20and25◦_C.

Waterandfoodweregivenadlibitum.Themodelchosenfor colitisinductionwasadaptedfromYamada10_andTannahill etal.11 _{Theanimalsreceivedintracolonicadministrationof} 4%acetic acid ina volume of4mL byenema. Thegroups receivedlecithin0.5mL/animal48hbeforeandimmediately after induction of colitis once a day until the end of the experiment.Thedrugusedinthisexperimentwasfromthe SunflowerIndustryandlaboratoryFitotherapicLtdaand con-tained0.5mgofeggoil.

After pressure measurements, animals were anaes-thetizedwithxylazinehydrochloride50mg/kgandketamine hydrochloride 100mg/kg body weight ip for removal of the distal colon (8cm). Subsequently, euthanasia was per-formed by exsanguination under anesthesia. Experiments followed a protocol approved by the Animal Ethics Com-mittee of the Lutheran University of Brazil (ULBRA) with therecommendationsoftheEuropeanUnionregarding ani-mal experimentation: Directive of the European Counsel 86/609/EEC.12

Analsphincterpressuremeasurements

Before euthanasia the animals were lightly anaesthetized withIsoflurane®_{tomeasureanalsphincterpressure.} Anorec-talmanometrywasperformedincmofH2O(Proctossystem, Viotti,SP)withaballooncatheter.13

Histologicalanalysis

Forhistologicalexamination,aportionoftheintestinewas placed in buffered formalin and subsequently included in paraffin blocks to obtain 3-␮m thick cuts using a rotary

microtome. We performed standard histological examina-tion stainingwithhematoxylin–eosin(HE). Theslideswere analyzedwithabinocularmicroscopeLABOPHOTNIKONat magnificationof200×.

Intestinehomogenates

The intestines were weighed and homogenized for 40s in an Ultra-Turrax (IKA-WERK) centrifuge at4◦_{C in the}

pres-enceof1.15%KCl(5mLpergramoftissue)andmethylphenyl sulfonylfluoride(MPSF)ataconcentrationof100mMin iso-propanol(10␮LpermLofKCladded).Thenthehomogenates

werecentrifugedfor10minat3000rpminarefrigerated cen-trifuge(SORVALLSuperT21 –CondensedOperatingKendro Laboratory Products –USA). Thesupernatant was pipetted into Eppendorf flasks and the precipitate was discarded. The samples were stored again at −80◦_{C for posterior}

analyses.14

Protein

ProteinswerequantifiedaccordingtoLowryandcolleagues, usingastandardsolutionofbovinealbuminataconcentration of1mg/mL.Samplesweremeasuredspectrophotometrically at 625nm, and values expressed in mg/mL. The values wereusedtocalculatethiobarbituricacidreactivesubstance (TBARS)andantioxidantenzymelevels.15

Lipidperoxidation

The amount of aldehydes generated by lipid peroxidation is measured by the TBARS method, which determines the amountofsubstancesreactingwiththiobarbituricacid. Sam-ples were incubated at 100◦_{C for 15min after addition of}

500␮L of 0.37% thiobarbituric acid in 15% trichloroacetic

acid and centrifuged at3000rpm (1612.8×g) for10minat 4◦_{C.Absorbancewasdetermined spectrophotometricallyat}

535nm.16

Antioxidantsenzymeanalyses

The analysis of superoxide dismutase (SOD) is based on theinhibitionofthereactionofthesuperoxideradicalwith adrenaline, detected spectrophotometricallyat 480nm and valuesexpressedinU/mgprot.17_{Theanalysisofcatalase(CAT)} activityisbasedonmeasuringthedecreaseinhydrogen per-oxide,detectedspectrophotometricallyat240nmandvalues expressedinpmol/mgprot.18

Statisticanalysis

70

A

B

Pressure

TBARS

a

a

b

b b

b

60

50

40

30

cm/H

2

O

nmoles/mgprot

20

10

0

5

4

3

2

1

0

CO CO+LE CL LE+CL CL+LE

CO CO+LE CL LE+CL CL+LE

Fig.1–(A)Meanvaluesofanalsphincterpressureinthe differentgroupsstudied(cm/H2O):*significantincreaseof analsphincterpressureintheCLgroupascomparedtothe othergroups(p<0.05),#significantdecreaseofanal

sphincterpressureintheLE+CLandCL+LEgroupsas comparedtotheCLgroup(p<0.05).

(B)Evaluationoflipoperoxidationthroughthetechniqueof thiobarbituricacidreactivesubstances(TBARS)(nmol/mg prot):*significantincreaseoflipoperoxidationintheCL groupascomparedtotheothergroups(p<0.05),

#_{significantincreaseoflipoperoxidationintheCLgroupas}

comparedtotheothergroups(p<0.05),#significant

decreaseoflipoperoxidationintheLE+CLandCL+LECL groupsascomparedtotheCLgroup(p<0.05).

Results

Analsphincterpressureandlipidperoxidation

Theadministrationoflecithinincreasedsphincterpressure by114%intheLE+CLgroupandby86%intheCL+LEgroup (Fig.1A).LPOevaluationbyTBARSshowedthatgroups receiv-inglecithintreatment(CL+LEandLE+CL)had significantly decreasedLPOascomparedtothegroupwithcolitis(p<0.05) (Fig.1B),whileintheCLgrouptherewasanincreaseof119% inrelationtoCOandCO+LEgroups.

Superoxidedismutase(SOD)andcatalase(CAT)activity

Fig.2showsthevaluesofSODandCATactivitiesacrossthe differentgroups.NotethatbothSODandCATdecreased sig-nificantlyinthegroupstreatedwithLE(CL+LEandLE+CL)as comparedtothecolitisgroup(p<0.05)(Fig.2AandB).

Histopathologicalanalysis

Theslideswerestainedwithhematoxylin–eosin(HE)and ana-lyzedat200×magnification.Fig.3Ashowsaphotomicrograph

400

USOD/mgprot

nmoles/mgprot

SOD

A

B

a

b _b

a

b b

300

200

100

0

1

0.8

0.6

0.4

0.2

0

CO CO+LE CL LE+CL CL+LE

CO CO+LE CL LE+CL CL+LE

Fig.2–(A)MeanvaluesofSODactivityinthebowelacross thedifferentgroupsstudied(USOD/mg/prot):*significant increaseofSODactivityintheCLgroupascomparedtothe othergroups(p<0.05),#significantdecreaseofSODactivity

inLE+CLandCL+LEgroupsascomparedtotheCLgroup (p<0.05).

(B)MeanvaluesofCATactivityinthebowelsofthe differentgroupsstudied:*significantincreaseinCAT activityintheCLgroupsascomparedtotheothergroups (p<0.05),#significantdecreaseofCATactivityintheLE+CL

andCL+LEascomparedtotheCLgroup(p<0.05).

ofananimalinthecontrolgroup(CO).Notetheintegrityof crypts(CP)withsimpleglandularepitheliumandnormal sub-mucosa(SM).Fig.3Bshowsaphotomicrographofananimalin thelecithincontrolgroup(CO+LE)withsimilararchitectureto thecontrolgroup.Fig.3Cshowsaphotomicrographfromthe colitisgroup(CL).Notethechangesinthearchitectureofthe colon,destructionofCP,extensivesubmucosaledema(E)and inflammatoryinfiltrate(IF).Fig.3Dshowsprophylactic treat-mentwithlecithin(LE+CL),withlesspreservationofCPand nodecreaseinE.Fig.3Eisfromananimalinthecolitisgroup treatedwithlecithin(CL+LE).NotethepreservationofCPwith glandularepithelium,andlessinflammatoryinfiltrate.

Discussion

Theetiologyofulcerativecolitisisnotwellunderstand,and there are several experimental models with similar phys-iopathogeny that are used toinvestigate its toxic or acute presentation. Acetic acid causes intestinal injury, and the developmentofinflammationisconsidered oneofthe fea-turesofcolitis.19

A

CP

CP

SM

CP

CP

CP

SM

SM

CO

CL

CO+LE 200x

200x

200x

LE+CL

CL+LE

200x 200x

B

C

D

E

Fig.3–Photomicrographofthedistalportionoftheintestineinthedifferentgroups.(A)Control(CO);(B)control+lecithin (CO+LE);(C)colitis(CL);(D)lecithin+colitis(LE+CL);(E)colitis+lecithin(CL+LE).Magnification200×.

animalsinthecolitis(CL)andinthelecithin-treated(LE+CL and CL+LE) groups, where the inflammatory process was foundtobereduced.

Amicroscopicanalysiswithhematoxylin-eosin(HE) con-firmsinjuryresultingfrominflammatoryprocessinthecolitis group(Fig.3C),wherethereisdisruptionofcryptsandedema inthesubmucosa.Inanimalsofthecontrolgroups,onenotes thatthe architectureofthe intestinaltissueshows normal cryptsandsubmucosa.

Preservationandrestorationofthecryptsand reduction ofedemawere observedintheanimalsreceiving LEeither beforeoraftercolitisinduction,whichdemonstratesthatLEis abletoreduceinflammation,corroboratingotherstudiesthat usedotherantioxidantsandwhichdemonstratedreduction ofinjuriesbroughtaboutbycolitis.4,20,21

Oxidativedamageisassociatedwithcolitisbytheincrease inROSandNOS,withgenerationoffreeradicalswhichcause celldestructionleadingtoleukocyteinfiltrationandreleaseof inflammatorymediatorsaswellascytokines,triggering oxida-tivestress.1,19,22

By evaluatingtheLPOtriggeredbytheoxidativeprocess (Fig.1B),weseethatLEwaseffectiveinreducingLPOinthe CLgroups,bothinanimalspre-treatedwithLEandinthose post-treatedwiththedrug.Thisisevidenceofasignificant reductionintheoxidativedamageinducedbycolitis.

LEisamoleculewithamphotericcharacteristic,thereby adheringtotheplasmamembrane,whichismadeupof phos-pholipids. Therebyit subtracts the anions offree radicals, sweepingthemandpreventingthemfromtriggeringLPO,as seenintheTBARSanalysis.

Tahan et al.23 _{administered melatonin astreatment for} experimentalcolitisandshowedsignificantreductioninthe treatedversusuntreatedgroupwithcolitis.Al-rejaieetal.22 used naringenin (a flavonoid present in citrus fruits) as treatment and observedreduced LPOin thetreated group, suggestingROSreduction.

importantroleinprovisionfortheprocessesofmotility regu-lationandcytoprotectionofthelargeintestine.6,24

Inthisstudy,themeasurementofanalsphincterpressure byanorectalmanometryshowedthatthe colitisgrouphad significantdecreaseinsphincterpressureascomparedtothe othergroups.Inapreviouswork,Hartmannetal.2_foundthat animalswithcolitisinducedbyaceticacidshowedsignificant increaseinNOintheintestinewithconsequentdecreaseof sphincterpressure.

ResearchbyFillmannetal.25_{;Kretzmannetal.}20_;Hartmann etal.,2_{showedthatadministrationofsuchantioxidant} sub-stances as glutamineand Boswellia serrata were associated withincreaseofanalsphincterpressureintreatedanimals. Thesefindingsareinagreementwithourdata,asthe admin-istrationofLEinthedifferenttreatedgroupswaseffectivein increasinganalsphincterpressure,possiblybydecreasingNO, inflammationandLPO,aspreviouslyreported.

Theenzymesystem,suchassuperoxidedismutase(SOD) and catalase (CAT), prevents accumulation of superoxide anion(O2•)andhydrogenperoxide(H2O2),preventingthe for-mationofthemostreactivefreeradical,thehydroxylradical (HO•_{), thus being considered the maindefenseline. These}

enzymesarefoundnotonlyinmitochondriabutalsointhe cytosol(CAT),wheremostfreeradicalsaregenerated.5 How-ever,SODincreasemayresultinincreaseintheformationof H2O2,whichmaycauseitsaccumulationintissuesand con-sequentincreaseinHO•_{throughtheFenton&Haber–Weiss}

reaction,leadingtooxidativestress.24

Wefounda significant increasein SODand CAT activi-tiesinthe colitisgroup.Wesuggest thatthis increasewas duetotheactivationofamechanismtocompensateforthe damagecausedbythe actionofacetic acidintheintestine oftheanimals.SODiscrucialfortheredoxbalance, dismu-tatingthe superoxideanioninto hydrogen peroxide(H2O2), andCATdegradesintowaterandoxygen,therebyavoiding the formationofHO• _radical26 _{protectingagainst oxidative} damage.Lecithin-treated animals (LE+CLand CL+LE) pre-sented reducedSOD and CATactivities, becausetreatment significantlydecreaseslipoperoxidationandrestoresthe oxi-dant/antioxidant balance in the organism, thus protecting againsttheoxidativedamageinducedbyaceticacid.

Wecanconcludethataceticacidiseffectiveasan exper-imentalmodelin inducingcolitis,leading toinflammation andoxidativedamageinthelargebowel.Lecithin administra-tionwaseffectiveinreducingtheinflammatoryprocessinthe bowels,decreasingoxidativestressbysignificantlydecreasing LPOandrestoringantioxidant defenses.Thesefindings are supportive of the use of antioxidants in the treatment of inflammatoryboweldisease,butfurtherstudiesarerequired toclarifytheprotectiveeffectinhumans.

Funding

CNPq,CAPES,FAPERGS.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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