w w w . j c o l . o r g . b r
Journal
of
Coloproctology
Original
Article
Antioxidant
effect
of
mesalazine
in
the
experimental
colitis
model
induced
by
acetic
acid
Rosa
Maria
Moura
a,
Renata
Minuzzo
Hartmann
b,c,
Francielli
Licks
a,c,
Elizângela
Gonc¸alves
Schemitt
a,b,c,
Josieli
Raskopf
Colares
a,c,
Mariana
do
Couto
Soares
a,c,
Lucio
Sarubbi
Fillmann
d,
Henrique
Sarubbi
Fillmann
c,d,
Norma
Possa
Marroni
a,b,c,∗aUniversidadeLuteranadoBrasil(ULBRA),LaboratóriodeEstresseOxidativoeAntioxidantes,Canoas,RS,Brazil bUniversidadeFederaldoRioGrandedoSul(UFRGS),Pós-graduac¸ãoemCiênciasMédicas,PortoAlegre,RS,Brazil
cUniversidadeFederaldoRioGrandedoSul(UFRGS),HospitaldeClínicasdePortoAlegre(HCPA),LaboratórioExperimentalde
HepatologiaeGastroenterologia,PortoAlegre,RS,Brazil
dPontifíciaUniversidadeCatólicadoRioGrandedoSul(PUCRS),PortoAlegre,RS,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12November2015 Accepted21March2016 Availableonline14April2016
Keywords:
Oxidativestress Inflammation Nitricoxide
a
b
s
t
r
a
c
t
Introduction:Inflammatoryboweldisease(IBD)ischaracterizedbyachronicinflammation ofthegastrointestinaltract,withoutspecificcauseorpathogen.
Objective:Theeffectofmesalazineinacolitismodelinducedbyaceticacid(AA)was evalu-ated.
Methods:We used 40 Wistar rats, ±350g, divided into 4 groups: control (CO); con-trol+mesalazine(CO+M);colitis(CL)andcolitis+M(CL+M)at24and48hoftreatment. TheanimalsreceivedthesubstancesbyanintracolonicenemaofAA4%andtreatment withmesalazinePO20mg/kgaftercolitisinduction.
Results:Mesalazinereducedtissuedamageinthegut,normalizedsphincteranalpressure levelsanddecreasedlipidperoxidation,metabolitesofnitricoxideandiNOSandNF-kB expressioninthetreatedgroupsinbothtreatmenttimepoints(24and48h),aswellasthe activityofantioxidantenzymes.
Conclusion: Mesalazinewaseffectiveinreducingtissuedamageandoxidativeand inflam-matorydamage,restoredantioxidantcapacityandincreasedanalsphincterpressurelevels, possiblyduetoitsantioxidanteffect.
©2016SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.This isanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:nmarroni@terra.com.br(N.P.Marroni).
http://dx.doi.org/10.1016/j.jcol.2016.03.003
Efeito
antioxidante
da
mesalazina
no
modelo
de
colite
experimental
induzida
por
ácido
acético
Palavras-chave:
Estresseoxidativo Inflamac¸ão Óxidonítrico
r
e
s
u
m
o
Introduc¸ão: Adoenc¸ainflamatóriaintestinal(DII)caracteriza-seporumainflamac¸ãocrônica dotratogastrointestinalsemumacausaoupatógenoespecífico.
Objetivo: Foiavaliadooefeitodamesalazinanomodelodecoliteinduzidaporácidoacético (AA).
Materialemétodos: Foramutilizados40ratoswistar,±350gramas,divididosem4grupos: Controle(CO);Controle+Mesalasina(CO+M);Colite(CL)eColite+M(CL+M)nostemposde 24e48horasdetratamento.Osanimaisforamsubmetidosàadministrac¸ãointracolônica porenemacomsoluc¸ãodeAAa4%etratamentocommesalazinanadoseoralde20mg/kg apósainduc¸ãodacolite.
Resultados: Amesalazinareduziuaslesõesteciduaisnointestino,normalizouosníveis depressãoanalesfincteriana,reduziualipoperoxidac¸ão,metabólitosdoóxidonítricoe expressãodaiNOSedoNF-kBnosgrupostratadosemambosostemposdetratamento(24 e48horas),bemcomoaatividadedasenzimasantioxidantes.
Conclusão:Amesalazinademonstroueficácianareduc¸ãodaslesõesteciduais,danos oxida-tivos einflamatórios, restabeleceua capacidadeantioxidantee aumentouos níveisde pressãoanalesfincteriana,possivelmentepeloseuefeitoantioxidante.
©2016SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Inflammatoryboweldisease(IBD)ischaracterizedbychronic inflammationofthegastrointestinaltractwithoutaspecific causeorpathogenandaffectsmillionsofpeopleworldwide. Inrecentyears,therehasbeenanincreaseinthenumberof diagnosedcasesofIBDinyoungpeople;thisincreasehasbeen accompaniedbysignificantprogressintheinvestigationofits pathophysiology.1–3
Unspecific ulcerative colitis (UUC) is included in this classification and is characterized by mucosal ulceration, asubmucosal inflammatoryinfiltrateand eventually extra-intestinalmanifestations.4–6
AnuncontrolledinflammationinUUCisprobablytheresult oftheinteractionbetweengeneticpredisposition,changesin theinnateimmunesystemandanexaggeratedresponseof adaptiveimmunity.Theinflammatoryinfiltratethatoccursin UUCpossiblyisduetoaruptureofthecolonicbarrierfollowed bybacterialinvasion;thus,pro-inflammatory cytokinesare released,withthegenerationoffreeradicalsandincreased productionofnitricoxide(NO).7–10 Thesefactorscancause
injuryandtissuedestruction.10–12
The enzyme nitric oxide synthase (NOS) is responsible forNOsynthesis,through theconversionofl-arginineand oxygen into l-citrulline and NO. The generation of nitric oxide (NO) is critical for vascular integrity, bowel motility andrelaxationoftheanalsphinctermuscle.7,8,13Theenzyme
induciblenitricoxidesynthase(iNOS)hasacytostaticeffect oninhibiting enzymes containingironandalsobycausing DNA fragmentation. The synthesis of iNOS is induced by lipopolysaccharides(LPS),freeradicalsoranyotherdetectable changeintheintra-orextracellularenvironment,activating thetranscriptionofpro-inflammatorygenes.14
ActivationofnuclearfactorkappaB(NF-kB)isrestricted to areas with inflammatory activity of mononuclear cells and epithelial cells in the lamina propria, an effect that wasdemonstratedinanimalmodelsofinflammatorybowel disease.Whenactivated,NF-kBinducestranscriptionof proin-flammatory genes, including interleukins (IL-1, 6, 8) and iNOS.7
Mesalazine or5-aminosalicylic acid (5-ASA)has chemo-preventiveeffectsagainstcolorectalcancerbyincreasingp53 proteingeneexpressionthroughepigeneticmechanisms.The anticancereffectsofmesalazinecanalsobemediatedbyits abilitytoremovemoleculesthatcauseoxidativedamageto mucosa.15StudiesofIkedaetal.suggestthatmesalazinecan
reducetumorcells,butnottheproliferationofnormal epithe-lialcells.16
Another mechanism ofactionof5-ASA is relatedto its abilitytointerferewiththeproductionofpro-inflammatory cytokines byinhibitionofNF-kBactivity,inhibiting natural killercellsoflymphocytesandmonocytesinthemucosaand reducingtheproductionoffreeradicals.17
ThepathophysiologyofUUCisnotyetfullyunderstood. Todate,fewstudieshaveevaluatedtheantioxidanteffectsof 5-ASAinexperimentalmodelsofcolitis.Inviewofthese con-siderations,thisstudyaimstoevaluatetheantioxidantaction ofmesalazineinreducingoxidativedamageinan experimen-talmodelofcolitisinducedbyaceticacid.
Material
and
methods
Animals
–ULBRA,Canoas,RS,undertheapprovalofthisCommittee (project2015-22P),andwiththeguidelines ofthe European UnionofAnimalExperimentation86/609EEC.18
FortymaleWistarratsweighingapproximately300gwere used;the animals were divided into four groupsaccording tothetimeoftreatment(24and48h)–control(CO)(n=5), control+mesalazine(CO+M)(n=5),colitis(CL)(n=5)and col-itis+mesalazine(M+CL)(n=5).Theanimals werekeptina vivariumofthe Universidade Luterana doBrasil,in plastic boxesof47cm×34cm×18cmlinedwithwoodshavingsina 12-hlight/darkcycle (illuminationfrom 7to19h)and tem-peraturebetween20 and25◦C.Water and foodwere given
adlibitum.Themodelchosenfortheinductionofcolitiswas adaptedfromthosedescribedbyYamadaetal.andTannahill etal.19,20AllanimalswerepreviouslyanesthetizedandCLand
CL+Mgroupsweresubjectedtointracolonicadministration ofa solution ofacetic acid diluted to 4% and with a vol-umeof4mLbyenema;andanimalsofCOandCO+Mgroups weresubmittedtotheintracolonicadministrationofsaline 0.9%withavolumeof4mL,alsobyenema.The administra-tionofmesalazineinCO+MandCL+Mgroupswascarried outPOatadoseof20mg/kgdilutedin1mLofsaline0.9%, andtheother groupsreceived1mLofsaline0,9%(adapted fromHirotanietal.21).Thedrugadministeredismarketedas
Mesacol®(NycomedPharma).Eachtabletcontains1200mgof
mesalazine.
Aftertheperiodoftreatmentofeachgroup(24and48h), theanimalswereanesthetizedwithxylazine2%atadoseof 8mg/kgand ketamine98mg/kgintraperitoneally.Following thisprocedure,themeasurementofanalsphincterpressure wascarriedoutandaportionofabout8cmoftheintestinewas removedforhistologicalandimmunohistochemicalanalysis andalsoforother biochemicalstudies.Finally,theanimals werekilledbyexsanguinationunderdeepanesthesia.18
Measurementofanalsphincterpressure
Afterinductionofdisease and treatment withmesalazine, animals were anesthetized with the aim of obtaining anal sphincter pressures. An anorectal manometry device (Proctossystem-Viotti–SP)withaballooncatheterand mea-surementsincm H2Owas used. Theballooncatheterwas
introducedinto theanalcanaland thenretracted,so asto record the sphincter pressure. Three sequential measure-ments were performed and then the average of the three valueswasobtained,inordertoobtaintheresultofpressure.22
Histologicalanalysis
Inhistologicalstudy,afragmentoftheintestinewasimmersed inbuffered formalin.Thenthe fragmentwas embeddedin paraffinblocksandsectionedonarotarymicrotome (thick-ness,3m).Thesectionswerestainedinhematoxylin–eosin (HE)forroutinehistologicevaluation.Theslideswere ana-lyzedunderaNIKONLabophotbinocularmicroscope(200×
magnification). Thehistological analysiswas based on the presenceofinflammatoryinfiltrate,edema,necrosis accord-ingtothedamageindexshowninTable1andadaptedfrom Millaretal.23
Table1–Indexofmacroscopicandmicroscopicchanges
inthecolonicmucosa.
Scoring Microscopicanalysis
0 Normal
1 Slightdamageandsomepointsofinflammatory infiltrateinthemucosa
2 Damageandmoderateinflammatoryinfiltratein twoormoreareasofthemucosa
3 Damageandsevereinflammatoryinfiltrateinthe mucosawithlossofepithelium
Lipidperoxidation
Samplesofboweltissuewereplacedintesttubeswitha mix-tureoftrichloroaceticacid(TCA)10%andthiobarbituricacid (TBA)0.67%.Subsequently,thesampleswereheatedinawater bathfor15minandcooled oniceforapproximately5min. TBAreactswithlipidperoxidationproductsformingaSchiff base;TCAisusedtodenatureproteinspresentinthemixture, aswellastoacidifythereaction.Aftercoolingthesamples, 1.5mLofn-butyl alcoholwas addedto extractthe formed pigment. Thesampleswereplacedonastirrerfor45sand centrifugedfor10minat3000rpm.Finally,thestainedproduct presentinthesupernatantwasreadonaspectrophotometer atawavelengthof535nm.TheTBARSconcentrationobtained wasexpressedinnmolpermilligramofprotein.24
Activityofantioxidantenzymessuperoxidedismutase (SOD)andglutathioneperoxidase(GPx)
The activity ofthe enzymesuperoxide dismutase (SOD)is defined by its ability to inhibit a detection system which reactswithsuperoxideradical.TheSODmeasurement tech-nique is based on the inhibition of this reaction. To that end,adrenalinewasused;wheninanalkalinemedium,this substancebecomesadrenochrome,yieldingO−whichisthe
enzymesubstrate;thus,itsdefinitionoccursbytheamount ofSOD, whichhasthe abilitytoinhibit by50%the rateof oxidationofthedetectoradrenaline.25
Thisenzymecatalyzesthereactionofhydroperoxidewith reducedglutathione(GSH)toformoxidizedglutathione(GSSG) andtheproductfromhydroperoxidereduction.
GPxactivitycanbestudiedbymeasuringtherateofNADPH consumptioninasystemcontainingGSHinthepresenceof glutathionereductase(GR).Thetechniqueconsistsof deter-mining the spectrophotometric activity of the enzyme, by measuring therate ofNADPH oxidation inareactive mix-ture. GPxactivity wasmeasured inaspectrophotometerat 340nmandexpressedasnmolperminutepermilligram pro-tein(nmol/min/mgprot).26
Levelsofnitricoxidemetabolites–nitritesandnitrates
ofsulfanilamide and naphthylethylenediamine specific for NO2−).Thereadingwasperformedinamicroplatereaderat
540nmandtheresultswereexpressedinmmolofNO2/NO3.27
Immunohistochemistryoftheenzymeinduciblenitric oxidesynthase(iNOS)andnuclearfactorkappaB(NF-kB)
For immunohistochemistry, the bowel samples were incu-bated with 10% of rabbit serum at room temperature for blockingpossibleundesiredreactionsofthesecondary anti-body.Slideswereincubatedwithrabbitpolyclonalantibodies againstiNOSandNF-kB(iNOSandNF-kB:SantaCruz Biotech-nology,UnitedStates)inadilutionof1:200overnightat4◦C.
After this period, the solution with the primary antibody wasremoved,theslideswerewashedinbuffer andfurther incubated with the secondary antibody (biotinylated anti-rabbitIgG, Santa Cruz Biotechnology,Santa Cruz,CA, USA) for30min atroom temperature. Next, the secondary anti-bodywasremovedandafterwardthesampleswere treated with EnVision reagent and then washed three times with buffer.Nucleiwerecounterstainedwithhematoxylin.Finally, theslideswerewashedtwotimeswithethanolandxylenefor 10s.
The images were photographed through a microscope equippedwitha digitalcamera inorderto captureimages usingthesoftwareImage-Plus(MediaCybernetics,Bethesda, USA);thisdone,theexpressionofiNOSandNF-kBwas car-ried out bydigital analysisby Adobe Photoshop® software
CS3 extended, version 10.0, by counting all pixels stained bytheimmunohistochemistryreaction.Thelevelofprotein expression(iNOS and NF-kB) was determined by multiply-ing the averagedensity ofthe imageby the percentageof thoseareaspositivelystainedbytheantibodies(brown stain-ingareas).
Statisticalanalysis
From the collected data, the means and standard devia-tions of each group were calculated using the statistical softwareGrapPadINSTAT,version3.0.Thetestusedfor anal-ysisofvarianceoftheresultswasANOVA,followedbythe Student–Newman–Keulstestforparametricdata.Theresults wereconsideredstatisticallysignificantwhenasignificance levelofatleast5%(p<0.05)wasobtained.
Results
Histologicalanalysis
Throughthehistologicalanalysis,wefoundthattheanimals inCLgroupshowedthedestructionofcrypts(CP)andedema ofthesubmucosa(SB)intheanimals’intestine.No histologi-calchangeswerefoundinCOandCO+Mgroups.Thegroups treatedwithmesalazine–M+CL(24and48h)showed preser-vationofCPandadecreaseofsubmucosaledema.Theresults ofmicroscopicdamageindexesshowedlessdamageinthe treatedgroups–CL+Minbothtimes(24and48h)(Fig.1Aand B)whencomparedtocolitisgroup.
CO
A
B
CL
CL
CP
SB
CO
CP
SB E SB
CO 0+0
0+0. 2.4+0.2a 1.2+0.4b Groups Microscopic
CO+M
CO+M CP
CP SB
SB SB
SB CP
CP CP
CL
E SB
CP
E
E CL+M
CL+M
CL+M CO+M
CL
CL+M
CO 0+0
0+0. 2.7+0.2a 1.6+0.2b Groups Microscopic
CO+M
Fig.1–Effectofmesalazineinbowelmacroscopicchanges inanexperimentalmodelofcolitis.
CO,control;CO+M,control+mesalazine,CL,colitis;CL+M, colitis+mesalazine;CP,crypts;E,edema;SB,submucosa. aSignificantdifferencebetweenCLandCO/CO+M.
bSignificantdifferencebetweenCL+MandCL.
Analsphincterpressure
The sphincter anal pressure results showed a significant decreaseincolitisgroupandasignificantincreaseingroups treatedwithmesalazineinbothtreatmenttimes(24and48h)
versuscolitisgroup(p<0.001)(Fig.2AandB).
Lipidperoxidation
Theresultsoflipidperoxidation(LPO)usingthetechniqueof substancesthatreactwiththepresenceofthiobarbituricacid (TBARS)showedasignificantincreaseinLPOinLCgroupversus
Pressure - 24 hours A
B
CO CO+M CL CL+M
80
60
40
20
0
Pressure - 48 hours
CO CO+M CL CL+M
80
60
40
20
0
cm/H
2
O
cm/H
2
O
a
b
a
b
Fig.2–Effectofmesalazineinthelevelsofanalsphincter pressureinanexperimentalmodelofcolitis.Theresults correspondtomean±standarderror(SE).
CO,control;CO+M,control+mesalazine;CL,colitis;CL+M, colitis+mesalazine.
Analsphincterpressureat24and48h.
aSignificantdifferencebetweenCLandCO/CO+M.
bSignificantdifferencebetweenCL+MandCL.
Table2–Effectofmesalazineinthelevelsoflipid
peroxidation(LPO)andactivityofantioxidantenzymes
superoxidedismutase(SOD)andglutathioneperoxidase
(GPx)inanexperimentalmodelinducedbyaceticacid.
Theresultscorrespondtomean±standarderror(SE).
Groups 24h
LPO SOD GPx
CO 0.26±0.03 4.96±1.40 0.27±0.03 CO+M 0.22±0.03 3.66±0.76 0.25±0.02 CL 0.87±0.05a 14.35±2.68a 0.16±0.01a CL+M 0.44±0.05b 10.00±1.54b 0.23±0.01b
Groups 48h
LPO SOD GPx
CO 0.27±0.05 0.57±0.15 0.58±0.09 CO+M 0.30±0.01 0.72±0.17 0.51±0.06 CL 0.66±0.08a 2.83±1.18a 0.23±0.03a CL+M 0.49±0.05b 1.05±0.26b 0.46±0.04b
LPO24h(p<0.001);48h(p<0.05).SOD24and48h(p<0.05).GPx24 and48h(p<0.05).
CO,control;CO+M,control+mesalazine;CL,colitis;CL+M, coli-tis+mesalazine.
a SignificantdifferencebetweenCLandCO/CO+M.
b SignificantdifferencebetweenCL+MandCL.
NO2/NO3 - 24 hours A
CO CO+M CL CL+M
CO CO+M CL CL+M
mmol/L
B
mmol/L
50
40
30
20
10
0
50
40
30
20
10
0
NO2/NO3 - 48 hours
a
b
a
b
Fig.3–EffectofmesalazineinthelevelsofNOmetabolites inanexperimentalmodelofcolitis.Theresultscorrespond tomean±standarderror(SE).
CO,control;CO+M,control+mesalazine;CL,colitis;CL+M, colitis+mesalazine.
NOat24and48h(p<0.001).
aSignificantdifferencebetweenCLandCO/CO+M.
bSignificantdifferencebetweenCL+MandCL.
groupstreatedwithmesalazineinbothtreatmenttimes(24 and48h)versusCLgroup(Table2A–p<0.001;B–p<0.05).
Activityofantioxidantenzymessuperoxidedismutase (SOD)andglutathioneperoxidase(GPx)
Asignificantincreaseintheactivityofantioxidantenzyme SODinbothtreatmenttimes(24and 48h)wasobservedin the LC group versus CO and CO+M groups, and a signif-icant reduction inCL+M group versus SC group (Table 2A andB–p<0.05).AsignificantdecreaseintheactivityofGPx enzymeinbothtreatmenttimes(24and48h)wasobserved intheLCgroupversusCOandCO+Mgroups,anda signifi-cantincreaseinCL+MgroupversusCLgroup(Table2A;B–
p<0.05).
Levelsofnitricoxidemetabolites–nitritesandnitrates
ImmunohistochemistryandquantificationofiNOSand NF-kB
TheCLanimalsinbothtreatmenttimes(24and48h)showed astrongpositivestainingforiNOSandNF-kBwhichwasnoted bythebrownstaining.COandCO+Mgroupsshowedno pos-itivestaining.Thetreatmentwithmesalazinediminishedthe stainingforiNOSandNF-kB.Likewise,inthequantificationof theexpressionofiNOSandNF-kB,asignificantdecreaseinthe CL+Mgroupwasobserved,comparedtoCLgroup.Allimages arefeaturedin200×magnification(Fig.4AandBandFig.5A andB–p<0.001).
Discussion
Thepathophysiologyofulcerativecolitisisnotyetfully under-stood;thus,thestudywithexperimentalmodelscanhelpto determinethefactorsinvolvedinthisdisease,alsoallowing thestudyoftherapeuticagentsinordertoachievemore effec-tivetreatments. Colitisinduced byacetic acid enema have similaritieswithUUCinhumanswithrespecttohistological andmetabolicfeatures,promotingthedevelopmentof sub-mucosal edema,inflammatoryinfiltrate,colonic ulceration, destructionofcryptsanddepletionofgobletcells.Considering thatthemodelevaluatesacutecharacteristics,onecan inves-tigatethecomponentsinvolvedininflammationandevaluate theeffectivenessofnewtherapiesintheacutephaseofthe disease.7,28
Thehistologicalanalysisdemonstratedthetrophiceffectof mesalazineinthecolon,improvingthecolonicstructureand decreasingthesizeandseverityoftheinjury.Inthetreated groups(24and48h)aninitialinjurywasobservedwherethe antioxidantactionofmesalazineled tothe preservationof themucosasinceitsapicalpartstillhadsomemucosallesions anditsbasalpartwasintact.Intheirstudy,Songetal. demon-stratedthattheconcomitantadministrationofbutyrateand mesalazine in a model of experimental colitis induced by dextran sulfate sodium (DSS) improved colonic injury, the inflammatoryprofileofthe mucosaandcecallymphnode. Normalizationofneutrophil,eosinophilandactivatedBandT lymphocytesinfiltratewasobserved,highlightingthe poten-tialuseofbutyrateasanadjunctinthetreatmentofUUC.29,30
Inexperimentaltrials,5-ASAwasadministeredin combi-nationwithNAC(N-acetylcysteine)inratswith TNBS-(2,4,6-trinitrobenzenesulfonicacid)inducedcolitis,anditwasfound thatthecombinedtreatmentshowedsynergismversus single-agenttreatment,promotingrepairofinflamedmucosa.4
Amongthe factorsrelatedtoUUC,onecandescribethe infiltrationofneutrophils dueto their abilitytorelease RL andproinflammatorycytokineswiththeincreaseofLPO.13On
theotherhand,oxidativestresshasitsdamageminimizedby antioxidantdefensesofendogenousorexogenousnature.In theirstudy,Harrisetal.demonstratedthatRLplaysan impor-tantroleinthepathogenesisassignedtoLPOpresentincell membranes.BiopsiesperformedinpatientswithUUCShowed highLPOindexversusnormalsubjects.31
Inourstudy,weobservedthatmesalazinedecreasedLPO intreatedanimals(24and48h),similartothecontrolgroup findings, possiblyby its antioxidant action.Similar results
were foundin contemporary studies that used substances withantioxidantactionasglutamine,melatonin,Boswellia ser-rataandquinceextractinexperimentalmodelsofulcerative colitis.7,13,28
Oxidative stress has its damage minimized by non-enzymatic antioxidant defense systems, such as vitamins and/or enzymes.Inourstudy,weobservedincolitisgroup anincrease ofSOD inresponsetotheincrease ofLPOdue tooxidative stress,and areductionintheseparametersin bothgroupstreatedwithmesalamine(24and48h).GPx activ-ity was decreased in colitis group and increased in those groupstreated(24and48h).Ourresultsshowthatmesalazine increasedenzymeactivity intreated groups,confirming its actiononoxidativestress.Ourresultscorroboratethestudyof Arabetal.thatevaluatedtheactivityofantioxidantenzymes SODandGPxinanexperimentalmodelofTNBS-induced col-itis. Aftertreatmentwithtelmisartan,asignificantincrease wasdemonstratedintheactivityofSODandGPx;this out-comehighlightstheantioxidanteffectsofthesetreatments.12
TheimplicationofoxidativestressinthepathogenesisofUUC has been emphasized byseveral clinical and experimental studies,wherethegenerationofRLandNOisrelatedto intesti-nallesions.10,11,13inthis study,the levelsofanalsphincter
pressure,asaphysiologicalparameterofnitricoxideactivity, weremeasured.Theresultsconfirmasignificantdecreasein analsphincterpressuremeasurementsinanimalsofthe col-itisgroup,andasignificantincreaseinNOlevels.Thegroups treatedwithmesalamine(24and48h)showedanincreasein analsphincterpressureandasignificantreductioninNO lev-els, comparedtowhatwas foundincolitis group animals. Experimental animalstudies indicate that nitric oxide and vasoactiveintestinalpolypeptidehaveaninhibitoryactionon smoothmuscle,promotingrelaxationoftheanal sphincter, thusdecreasinganalsphincterpressurelevels.7,8,13
Our results corroborate those of Hartmann et al. who demonstratedasignificantincreaseinnitricoxidelevelsinthe intestineofanimalswithcolitisandaconsequentdecreasein analsphincterpressurelevelsincolitisgroupanimals, sug-gesting the involvement of relaxationof sphincter muscle withincreasingnitric oxidelevels.Fillmann etal.observed anincreaseinnitricoxidelevelsand adecreasein sphinc-teranalpressurelevelsinanimalswithinducedcolitis;and whentheseanimalsweretreatedwiththeantioxidant glu-tamine,theseauthorsobservedasignificantdecreaseofnitric oxide levels and a consequent increase in sphincter anal pressure.7,13
Carrieretal.foundthatmicesubjectedtocolitisdueto DSS showedincreasesinoxidativestressandin inflamma-torymarkersasfollows:.TNF-alpha,IL-1andNF-kB.32Other
studieshavealsoassociatedincreasesofNOandincreasesin iNOSexpressionincolitismodels.8,9Inourstudy,weobserved
increasedexpressionofiNOSandNF-kBincolitisgroup ani-mals.Thegrouptreatedwithmesalamineshoweddecreased expressionoftheseproteins. Similarresultswere foundin thestudybyArabetal.whousedtelmisartanastherapyfor experimentalcolitisandobserveddecreasedlevelsofNF-kB evaluated by immunohistochemistry, besidesa diminished iNOSgeneexpressionevaluatedbyPCR.12Davaatserenetal.
CO
CL CL+M
CO+M
iNOS 24 hours
iNOS 48 hours
iNOS 24 hours iNOS 48 hours CO
CL CL+M
CO+M
CO CO+M CL CL+M CO CO+M CL CL+M
50
Expression of the positiv
e pix
els
, %
Expression of the positiv
e pix
els
, %
50
40 45
40
30 35 30
25 20 20
15 10 5 10
0 0
a
b a
b
Fig.4–Effectofmesalazineintheexpressionoftheenzymeinduciblenitricoxidesynthase(iNOS)inanexperimental modelofcolitis.Theresultscorrespondtomean±standarderror(SE).
CO,control;CO+M,control+mesalazine;CL,colitis;CL+M,colitis+mesalazine. iNOSat24and48h(p<0.001).
aSignificantdifferencebetweenCLandCO/CO+M.
NF-kB 24 hours
NF-kB - 24 hours
CO CO+M CL CL+M CO CO+M CL CL+M
50 a
b
a
b
70 80
60
50
40
30
20
10
0
Expression of the positiv
e pix
els
, %
Expression of the positiv
e pix
els
, %
45 40 35 30 25 20 15 10 5 0
NF-kB 48 hours
NF-kB - 48 hours CO
CL CL+M
CO+M CO+M
CO
CL CL+M
Fig.5–EffectofmesalazineintheexpressionofnucleartranscriptionfactorkappaB(NF-kB)inanexperimentalmodelof colitis.Theresultscorrespondtomean±standarderror(SE).
NF-kBat24and48h(p<0.001).
aSignificantdifferencebetweenCLandCO/CO+M.
bSignificantdifferencebetweenCL+MandCL.
diminished iNOS expression in groups treated with vari-ousdosesofthis compound.3 Kretzmann etal.observed a
decreaseintheexpressionofp65andp50subunitsofNF-kB inanimalstreatedwithglutamineintheexperimentalmodel ofTNBS-inducedcolitis.8
Althoughthepathogenesisisnotyetfullyunderstood,the treatmentsavailablehavebeenabletointervene,improving thequalityoflifeofpatients.Therefore,itissuggestedthat mesalazinedeterminesanimprovementintheinflammatory bowelpicture,alsothankstoitsantioxidantaction.
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