J. Coloproctol. (Rio J.) vol.36 número4

(1)

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

Journal

of

Coloproctology

Original

Article

Inflammatory

bowel

and

oxidative

stress

changes

in

an

experimental

model

of

portal

hypertension:

action

of

N-acetylcysteine

Francielli

Licks

a,∗

,

Renata

Minuzzo

Hartmann

b

,

Elizângela

Schemitt

b

,

Josieli

Raskopf

Colares

d

_,

_Lúcio

_Sarubbi

_Fillmann

c

_,

_Henrique

_Fillmann

c

_,

Norma

Possa

Marroni

a,b,d

a_Universidade_Federal_do_Rio_Grande_do_Sul_(UFRGS),_Programa_de_Pós_Graduac¸ão_em_Ciências_Biológicas,_Porto_Alegre,_RS,_Brazil b_Universidade_Federal_do_Rio_Grande_do_Sul_(UFRGS),_Programa_de_Pós_Graduac¸ão_em_Medicina,_Porto_Alegre,_RS,_Brazil

c_Pontifícia_Universidade_Católica_do_Rio_Grande_do_Sul_(PUCRS),_Porto_Alegre,_RS,_Brazil

d_Universidade_Luterana_do_Brasil_(ULBRA),_Programa_de_Pós_Graduac¸ão_em_BioSaúde,_Canoas,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28April2016

Accepted7May2016

Availableonline6July2016

Keywords: N-acetylcysteine

Portalhypertension

Intestine

Oxidativestress

Inflammation

a

b

s

t

r

a

c

t

Introduction:Portalhypertension(PH)ischaracterizedbyvasodilatationintheportalsystem

andthebowelisoneoftheseverelyaffectedorgans.N-acetylcysteine(NAC)isamolecule

withimportantpropertiesandwidelyusedinclinicalpractice.

Objective:ToevaluateNACactioninthebowelofanimalssubmittedtotheanimalmodelof

partialportalveinligation(PPVL).

Methods:18maleWistarratsweredividedintothreeexperimentalgroups(n=6):

sham-operated (SO), PPVL, and PPVL+NAC. On the 8th day after surgery, N-acetylcysteine

(10mg/kg,ip)wasadministereddailyfor7days.Onthe15thdaytheanimals’bowelwas

collectedforoxidativestressanalysis,immunohistochemistryandWesternblot.We

evalu-atedtheexpressionofNF-KBandTNF-␣byimmunohistochemistryandofiNOSbyWestern

blot.LipidperoxidationwasassessedbyTBARStechnique,andtheactivitiesofantioxidant

enzymessuperoxidedismutase(SOD)andglutationperoxidase(GPx)werechecked.

Results:WeobservedanincreasedexpressionofNF-KBandTNF-␣inPPVLgroup,andan

increasediNOSexpressionassessedbyWesternblot.NACreducedtheexpressionofall

proteinsevaluated.Wealsoobservedanincreaseinoxidativestressinthebowelofmice

PPVLgroupcomparedtocontrols(SO),andNACwaseffectiveinreducingthesevaluesin

PPVL+NACgroup.Also,areductionintheactivityofSODandGPxenzymeswasobserved

inthediseasedgroup,andNACwasabletorestoretheactivityoftheenzymesassessed.

Conclusion: Wesuggesttheanti-inflammatoryandantioxidantactionofNACinthebowel

ofanimalssubmittedtoPPVLmodel.

isanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_.

E-mail:francielli.licks@gmail.com.br(F.Licks).

http://dx.doi.org/10.1016/j.jcol.2016.05.005

(2)

Alterac¸ões

intestinais

inflamatórias

e

de

estresse

oxidativo

em

modelo

experimental

de

hipertensão

portal:

ac¸ão

da

N-acetilcisteína

Palavras-chave: N-Acetilcisteína

HipertensãoPortal

Intestino

EstresseOxidativo

Inflamac¸ão

r

e

s

u

m

o

Introduc¸ão: AHipertensãoPortal(HP)écaracterizadaporumavasodilatac¸ãonosistema

portal,eointestinoéumdosórgãosgravementeacometidos.AN-acetilcisteína(NAC)é

umamoléculacomimportantespropriedades,amplamenteutilizadanaclínica.

Objetivo: Avaliaraac¸ãodaNACnointestinodeanimaissubmetidosaomodeloanimalde

ligaduraparcialdaveiaporta(LPVP).

Métodos: Foramutilizados18ratosmachosWistardivididosemtrêsgruposexperimentais

(n=6):Sham-operated(SO),LPVP,LPVP+NAC.No8◦_dia_após_a_cirurgia,_a_{N-acetilcisteína}

(10mg/kg,ip)foiadministradadiariamentedurante7dias.No15◦_dia_foi_coletado_o_intestino

dosanimaisparaanálisesdeestresseoxidativo,imunohistoquímicaeWesternblot.Nós

avaliamosaexpressãodoNF-kbeTNF-␣porimunohistoquímicaedaiNOSporWestern

blot.Alipoperoxidac¸ãofoiavaliadapelatécnicadeTBARS,easatividadesdasenzimas

antioxidantesSuperóxidoDismutase(SOD)eGlutationaPeroxidase(GPx)foramverificadas.

Resultados: Observamosumaumentoda expressãodoNF-kbeTNF-␣nogrupoLPVP,e

aumentonaexpressãodaiNOSavaliadaporWesternblot.ANACreduziuaexpressãode

todasasproteínasavaliadas.Observamosumaumentodoestresseoxidativonointestino

dosratosdogrupoLPVPcomrelac¸ãoaoscontroles(SO),sendoaNACeficaznareduc¸ão

dessesvaloresnogrupoLPVP+NAC.Ainda,umareduc¸ãonaatividadedasenzimasSODe

GPxnogrupodoente,sendoaNACcapazderestauraraatividadedasenzimasavaliadas.

Conclusão: Sugerimosaac¸ãoanti-inflamatóriaeantioxidantedaNACnointestinode

ani-maissubmetidosaomodeloLPVP.

´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Portalhypertension(PH)isasyndromewhoseclinicalpicture

isestablishedbytheemergenceofananatomicalobstaclein

theportalsystem.Thisobstacle,whichblocksthebloodflow,

causesblooddammingatthesiteofobstruction.The

compen-satorymechanismofdecompressionintheportalsystemis

thedevelopmentofanimportantvasodilationinthe

splanch-nicterritory;inturn,this eventisresponsibleforthemain

complicationsoftheportalhypertensionsyndrome.1

Wecancorrelatethedevelopmentofahyperdynamic

col-lateralcirculationwithoneofthemajorcomplicationsfrom

PH:thebleedingfromgastrointestinalvarices,aneventthat

is triggeredwhen the portal pressure gradient rises above

12mmHg.2_The_progressive_vasodilation_in_the_splanchnic

ter-ritoryisresponsiblefortheappearanceofthesevaricoseveins,

themostimportantbeingthoselocatedinthestomachand

bowel.These conditionsare known asPortal Hypertension

Gastropathy(PHG)andPortalHypertensiveColopathy(PHC),

respectively,andthefirstoftheseconditionsisalreadywell

establishedintheliterature.3

IntestinalchangespresentinPHarestillbeingelucidated

andwere graduallyidentifiedover thelastdecade asbeing

mainlyoneofthecausesoffatalgastrointestinalbleedingin

patientswithPH.4_The_pattern_of_lesions_in_cases_of_PH_can

befoundinotherpartsofthegastrointestinaltract,including

theintestine,5_due_to_mucosal_edema,_inflammatory_diseases,

andectopicandanorectalvarices.6

The experimental model of Partial Portal Vein Ligation

(PPVL)hasbeenusedbymanyauthorstostudythe

molecu-larchangesinpre-hepaticportalhypertension.7–9_In_rats,_the

hemodynamicchangespresentinPHshowuparoundtheday

14aftersurgery,andhyperdynamiccirculationand

splanch-nicvasodilationareprevailingconditionsinanimalssubjected

toaPPVLmodel.10_In_addition,_PHC_and_{encephalopathy}_are

among the most important manifestations resulting from

thisexperimentalmodel,andinflammatorymechanismsare

aggravatingfactorsinbothmanifestations.11

Inflammationisaneventoftenassociatedwithinjuriesof

different origins.In the caseofPH, systemic and

splanch-nic vascularresponsesappeartoplayanimportantrolein

the pathogenesisofhyperdynamiccirculationand are very

similar tothose producedinthe post-traumatic

inflamma-toryresponse.Themechanicalstresscausedbytheincreased

bloodflowinthesplanchnicterritorystimulatesthe

endothe-liumtosecretevasoactivesubstances,cytokinesandgrowth

factors,andthisisatriggeringfactorforlocalorgeneralized

inflammation.12

With respect to local inflammation, it is important to

mention that the mucosa ofthe gastrointestinal tract isa

major reservoir ofmacrophagesand mast cells,and these

cellslocatedintheintestineareconsideredaseffectorcells

that participate in the first line of defense of our body.13

Inthecaseofinflammation,theintestinalmucosaacquires

a phenotypic pro-inflammatory profile, secreting cytokines

that can amplify the systemic inflammatory vascular

(3)

Amongthecytokinessecretedbytheintestineafterthe

ini-tialstimulation,onemustmentionthetumornecrosisfactor

(TNF-␣),releasedbymastcells oftheintestineand

mesen-tericlymphnodes.Thisproinflammatorycytokineisprobably

relatednotonlytotheinflammatoryprocesspresentinPHbut

alsotothedevelopmentofhyperdynamiccirculation,sinceit

stimulatestheprimaryrouteofsplanchnicvasodilation,nitric

oxide.14 _In _addition, _TNF-␣ _is _an_{extracellular} _stimulus _for

releaseofanotherpro-inflammatorycytokine,NF-kB.This

fac-torinducesphosphorylationinIkB,thecytoplasmicinhibitory

proteinthatpreventsthenucleartranslocationofNF-kB.15

The activation of nitric oxide (NO) is the main event

proposedasatriggering factorforthe developmentof

col-lateralcirculationincasesofPH.NOissynthesizedbynitric

oxidesynthase(NOS),including,amongitsmajorisoforms,

endothelialnitricoxidesynthase(eNOS),neuronalnitricoxide

synthase(nNOS),andinduciblenitricoxidesynthase(iNOS).

This latter isoform is associated with increased levels of

NOproduction.16_iNOS _is_mainly_expressed _in_macrophages

and smooth muscle cells, especially after the stimulation

by lipopolysaccharides, or inflammatory cytokines.17 _This

enzymeismodulatedbytranscriptionfactors,amongwhich

NF-kBisconsidered theprimary mediatorofits activation,

which,inturn,canbeactivatedbytheoxidativestress.18

Inflammatory cells are important sources for the

gen-eration ofreactive oxygen species; thus the oxidative and

inflammatory damage acts synergistically in the

develop-mentandworseningofconditionsinwhichtheseeventsare

present.19

With HP, the role of oxidative stress is associated with

the overproduction of nitric oxide, which determines the

productionofhighlyreactive species,forexample,

peroxy-nitrite(ONOO ).20 _Using _the _PPVL _model, _previous _studies

publishedbyourstudygrouphavealreadyshownthis

signif-icantinvolvementinthepathogenesisofpre-hepaticportal

hypertension9,21,22_as_well_as_the_{participation}_of

inflamma-tioningastricinjuryinthisexperimentalmodel.23

Thisstudy aimedtoevaluatetheinvolvementof

oxida-tiveandinflammatorystressintheintestinalmucosaofrats

withpre-hepaticportalhypertension.Inaddition,weaimed

toevaluatetheantioxidantandanti-inflammatoryactionof

N-acetylcysteineinintestinalinjuriespresentinPPVLmodel.

Material

and

methods

Ethics

Theexperimentalprocedureswithanimalswerecarriedout

inaccordance withthe current Brazilian legislationin the

practiceofscientificresearch(Law11,794,OfficialGazette–

October8,2008),EuthanasiaPracticeGuidelinesofCONCEA

(2013)andBrazilianGuidelinesfortheCareandUseofAnimals

forScientificandDidacticPurposes–DBCA(2013).

Animals

TheanimalswereacquiredinthevivariumofHCPAaccording

tothespecificationsoftheAnimalExperimentationUnit(UEA)

andwere maintainedthroughoutthe experimentinplastic

cagesmeasuring47cm×34cm×18cm,linedwithwood

shav-ings, in a 12-h light/dark cycle and at a temperature of

22±4◦_C._All_animals_were_fed_a_commercially_available

ani-malfood(Purina®_–_Nutripal,_Porto_Alegre,_RS,_Brazil)_and_had

accesstowateradlibitum.

Groupsandexperimentalprotocols

Forthisstudy,weused18maleWistarrats(±250g)whichwere

divided randomly into three groups (n=6): sham-operated

(SO),PPVL,andPPVL+NAC.

On the first day of the experiment, the animals were

weighed and anesthetized with ketamine (100mg/kg) and

xylazine(10mg/kg)intraperitoneally(IP).Weperformedlocal

asepsisandthenmadeamid-ventrallaparotomywitha

care-ful exposure of bowel loops with a gauze pad soaked in

saline.Allanimalsunderwentthesame surgicalprocedure;

however, the animalsin group SO were submitted onlyto

the manipulation ofthe portal vein. The animals of PPVL

and PPVL+NACgroupswere submittedtoanexperimental

modelofPartialPortalVeinLigature,describedbySikuleretal.

1985.7

Weuseda20Gneedletopromoteapartialobstructionof

theportalvein;forthispurpose,boththevesselandthe

nee-dle were tiedwith silk3-0. Theimmediate vasodilationof

the splanchnicterritorywas observedand thenthe needle

wasgentlywithdrawn,leavingonlytheportalveinpartially

occluded.Next,intestinalloopswerereplacedintothe

abdom-inalcavityoftheanimals,andaninfusionof10mLofsaline

wasadministered,andtheperitoneumwassuturedwith

con-tinuouspoints.Theepitheliallayerwasclosedwithindividual

sutures. Theabove-described model features apre-hepatic

portalhypertension.7

Aftercompletionofthesurgicalprocedures,theanimals

wereplacedinindividualcagesfortheirrecoveryunder

anal-gesiawithdipyrone(200mg/kg);thefirstadministrationwas

performed byintramuscular route and the remaining was

orallyadministeredwithafrequencyof8/8hduringthe

sub-sequent72h.

Sevendays aftersurgery,thetreatment wasinitiatedin

the respectivegroups.Theanimals inOSand PPVLgroups

received only the vehicle (0.9% NaCl, 0.6mL IP). On the

other hand, the animals in PPVL+NAC group received

N-acetylcysteine(SigmaChemicalCo.,St.Louis,MO,USA;CAS

registrynumber616-91-1)atadoseof10mg/kgdissolvedin

0.6mLof0.9%NaCl.Thetreatmentwascarriedoutbeginning

onthe8thday,forsevendays.

Euthanasiaandtissuecollection

Attheendoftreatmentonday15,the animalswereagain

weighedandanesthetizedusingthesameprotocoldescribed

above.Afteraninspectionoftheanimal’sstateof

anesthe-sia,anewlaparotomywasmadeforremovaloftheintestines

forsubsequentanalysis.Aportionofthecollectedmaterial

wasstoredinafreezerat−80◦_C,_and_the_remainder_was_fixed

in 10% buffered formalin for24h. After this period, 3-mm

sections oftheparaffin blockwere obtainedusinga rotary

(4)

Immunohistochemistry

The histological technique of immunohistochemistry was

usedtoevaluatetheexpressionofthenuclearfactorkappa

B(NF-kB)andtumornecrosisfactor(TNF-␣)intheanimal’s

intestine. Thereacquisitionofthe antigenwas carried out

usingabufferat100◦_C;_{subsequently,}_the_activity_of

endoge-nous peroxidase was blocked by incubation with absolute

methanol.Theslideswereincubatedwithrabbitpolyclonal

antibody (NF- [sc-9072], 1: 200, Santa Cruz Biotechnology,

SantaCruz,CA,USA)andgoatmonoclonalantibody(TNF-␣

[sc-1351],1:200, SantaCruz Biotechnology,Santa Cruz,CA,

USA)overnightat4◦_C._Next,_the _material_was_washed_and

incubatedwiththesecondaryantibodygoatanti-rabbit

IgG-HRP(sc-2004)for30minatroomtemperature.Theslideswere

analyzedusingamicroscopeequippedwithadigitalcamera,

andtheimageswerecapturedusingtheImage-Plussoftware

(MediaCybernetics,Bethesda,MD,USA).Quantificationofthe

markingofbothanalyteswascarriedoutbydigitalanalysis

withAdobePhotoshop®_CS3_extended_10.0,_using_the

count-ingofbrowncolorstainedpixels.Theexpressionlevelwas

determinedbymultiplyingtheaveragedensityoftheimage

bythepercentageofthestainedareas.24

Westernblot

The cytosolic extracts prepared on the basis of intestinal

homogenateswereusedinWesternblot,andproteinvalues

were determined bythe Bradford method.25 _Then, _protein

lysateswerefractionatedbypolyacrylamidegel

electrophore-sisat9–12% inan electrophoresisbuffer(25mM Tris,0.2M

glycine,3.5mM SDS,pH8.8)and thentransferredto

mem-branes of polyvinylidene fluoride (PVDF). Blocking of the

membranes was done using a 5% solution of skimmed

milk powder in PBS-Tween. Then, the PVDF membrane

was incubated overnight at 4◦_C _with _the _specific

mono-clonalprimary antibody,mouse polyclonalantibody(NOS2

[sc-7271], Santa Cruz Biotechnology, Santa Cruz,CA, USA).

Aftertheovernightincubation,themembraneswerewashed

withTTBSandincubated for1hatroom temperaturewith

the secondary antibody IgG-HRP sc-2005, anti-goat donkey

(sc-2020, Santa Cruz Biotechnology, Santa Cruz, CA, USA,

1:4000).Afterrevealed,thebandswerequantifiedusingthe

Scion Imageprogram, v. 4.02 for Windows(Scion

Corpora-tion,Frederick,USA).Theresultswereexpressedinarbitrary

units.26

Biochemicalanalyses

Homogenate

Theintestinesofthe animalswere homogenizedfor1min

withUltra-Turrax(IKA-WERK)inthepresenceofpotassium

chloride (KCl) 1.15% (5mL/g of tissue) and

phenylmethyl-sulfonyl fluoride (PMSF) at a concentration of 100mM in

isopropanol(10␮L/mL KCl added). Then, the homogenates

werecentrifugedfor10minat3000rpm(1110×g)ina

refrig-erated centrifuge (SORVALLRC-5B Refrigerated Superspeed

Centrifuge)andthesupernatantwasremovedandfrozenina

freezerat−80◦_C_for_subsequent_{measurements.}27

Proteincontent

The protein concentration in the homogenates was

deter-minedbytheBradfordmethod,withbovinealbumin(SIGMA)

usedasastandard.Thesamplesweremeasured

spectropho-tometrically at 595nm, and the values were expressed in

mg/mL. These values were used to calculate subsequently

TBA-RS and the values of antioxidant enzymes SOD and

GPx.25

Determinationofoxidativestress

For the determination of lipid peroxidation, we used the

method ofreactive substances to thiobarbituricacid

(TBA-RS).Thetechniqueconsistsinheatingthehomogenateinthe

presenceofthiobarbituricacid,withconsequent formation

ofapinkcolorproduct,measuredinaspectrophotometerat

535mm.Theappearanceofcoloroccursduetothepresence

ofmalondialdehydeandothersubstancesfromlipid

peroxida-tioninbiologicalmaterial.0.5mLofthiobarbituricacid(TBA)

0.67%, 0.25mL of distilled water, 0.75mL oftrichloroacetic

acid(TCA)10%,and0.25mLofthehomogenatewereplaced

in a test tube, in this order.TBA reacts with lipid

peroxi-dation productsforminga Schiffbase,and TCAexerts the

function ofdenaturation of the proteins present and also

acidifies thereactionmedium.Then,each tubewasstirred

and heated to100◦_C_for₁₅_min._After_that, _the_tubes _were

cooled and 1.5mLof n-butylalcohol was added toextract

the pigment formed. The tubes were placed on a shaker

(Biomatic) for 45s and centrifuged for 10min at 3000rpm

(1110×g). Finally, thestained productwas takenaway and

thereadingwastakenusingaspectrophotometer (CARY3E

–UV–VisibleVarianSpectrophotometer)atawavelengthof

535nm.TheconcentrationofTBA-RSisexpressedasnmol/mg

protein.28

Theactivityofsuperoxidedismutase(SOD)isdefinedbyits

abilitytoinhibitadetectionsystemwhichreactswithO2•−.

For this purpose, adrenalineis used, which, inanalkaline

medium,turnstoadrenochrome,producingO2•−,whichitis

thesubstrateoftheenzyme.Beforeperformingthe

determi-nation withthehomogenate,measurement ofthe reaction

medium(50mMglycine-NaOH,pH9.6)wascarriedoutwith

50␮Lofadrenaline(60mM,pH2.0),correspondingto100%of

the reaction. Thismixturewas stirredand readat480nm.

Subsequently, different volumesof the homogenate(50␮L,

25␮L,and10␮L)wereadded,andtheinhibitionofthe

reac-tionwasmeasured.Theenzymaticactivitywasexpressedas

SODunits/goftissue(quantityofSODabletoinhibitin50%

theadrenalinereductionrate).29

Thedeterminationofglutathioneperoxidase(GPx)using

the Flohé–Guntzler method30 _consists _in _measuring _the

NADPHconsumptionrateinasystemcontainingGSH;the

oxi-dationisrecordedspectrophotometricallyatawavelengthof

340nm.Tothisend,2.7mLofaregulatingsolutionofNa+and

K+phosphates(100mM,pH7.0)with50␮LofNADPH(10mM),

150␮LofBOOH(10mM)and50␮Lofglutathionereductase

(12U/mL)wereplacedinaquartzcuvette.Themixturewas

readfor1min;atthispoint,abaselinewasestablished,and

then50␮LofGSH(100mM)and50␮Lofthehomogenatewere

(5)

A

B

C

TNF-α

120 000

SO

Expression of the positiv

e pix

els

,%

PPVL ∗

#

PPVL+NAC 100 000

80 000

60 000

40 000

20 000

0

Fig.1–ImmunohistochemistryofTNF-␣.Effectsofpartialportalveinligation(PPVL)andN-acetylcysteine(NAC)

administrationonTNF-␣expression.SO,Sham-operatedgroup;PPVL,partialportalveinligation;PPVL+NAC,partialportal veinligationtreatedwithNAC.*p<0.001,#_p_<_0.001_(n₌_6).

atanabsorbanceof340nm. Theactivity wasexpressed in

nmol/min/mgprotein.

Statisticalanalysis

Alldatawerepresentedasamean±standarderror.Statistical

analyseswerecalculatedusingGraphpadInstatsoftware,

ver-sion3.0forWindows.Analysisofvariance(ANOVA)andthe

Student–Newman–Keulstestwasusedformultipleanalyses,

andthesignificancelevelwassetatp<0.05(5%).

Results

Immunohistochemistry

InassessingtheexpressionoftheproteinTNF-␣inthe

differ-entexperimentalgroups,weobservedasignificantincreasein

itsmarkingsinPPVLversusSOgroup(p<0.001).The

adminis-trationofN-acetylcysteineintheproposeddoseinthisstudy

was ableto reducethe expression ofTNF-␣ inPPVL+NAC

group(p<0.001)(Fig.1).

The expression of NF-kB was increased in animals

subjected to the experimental model of PPVL versus

con-trols (p<0.001), and the values of PPVL+NAC group were

significantlyreducedversusvaluesforanimalsofthediseased

group(p<0.001)(Fig.2).

Westernblot

Using Western blot, we observed a significant increase in

iNOSexpressionintheintestineofanimalsofthediseased

group(p<0.01).TheanimalstreatedwithNAChadtheirvalues

reduced,asobservedinPPVL+NACgroup(p<0.01)(Fig.3).

Biochemicalanalyses

Anincreasewasobservedinlipidperoxidation,evaluatedby

TBA-RSinanimalsfromPPVLversusOSgroup(p<0.05),and

NACwas abletoreducetheselevels whenadministeredto

PPVL+NACgroup(p<0.01)(Fig.4).

SODactivitywasalsoevaluatedintheintestineofanimals

ofdifferentexperimentalgroups;theactivitywasreducedin

diseasedversuscontrolanimals(p<0.01),andthetreatment

hasproveneffectiveinincreasingtheactivityofthisenzyme

(p<0.05)(Fig.5).

Another antioxidant enzyme, GPx, was also evaluated

in this study; We observed a similar behavior in PPVL

(p<0.05),andinPPVL+NACgroupsversusOSgroup(p<0.01)

(6)

Discussion

Theportalhypertensionsyndromeshowsascharacteristics

the occurrence of ascites, hepatic encephalopathy, and a

hyperdynamiccollateralcirculation31_;_this_latter_occurrence

isconsideredthemaincauseofworseningofthegeneral

con-dition.

Thecollateral circulationdevelopsinordertodivertthe

blood flow from the obstructed territory; and the

obstruc-tion may be situatedbefore (pre-hepatic), into (hepatic) or

after(post-hepatic)theliver.32_Regardless_of_the_place_of_this

obstruction,the unblocking compensatory mechanism will

eventuallyresultintheformationofvaricoseveinsdistributed

alongthedigestivetractofthepatient.

Ingeneral,varicoseveinslocatedinthecolonareprevalent

inthececumandrectosigmoidregion33_and_characterize

por-talhypertensioncolopathy(PHC).Inthisscenario,theriskof

bleedingthroughcolonicvaricesis1–8%34_;_on_the_other_hand,

forrectalvaricoseveins,theriskisfrom44to89%incirrhotic

patients.6 _In_addition,_vascular_ectasia_and_{microcirculatory}

changesalongtheintestinalmucosadamageitsintegrityand

promotethedevelopmentofalocalinflammatoryprocess.35

Theintestinalinflammatoryconditionpresentincasesof

portalhypertensionisconsideredanaggravatingfactorinthe

pathogenesis ofthe disease.Among the pro-inflammatory

cytokines involved in the process, TNF-␣ is considered as

an importantmediator, being produced byintestinal mast

cells.36 _In _this _study,_we _observed_a _significant _increase_of

thiscytokineintheintestineofanimalsofPPVLgroup(Fig.1).

ThesamebehaviorwasobservedintheevaluationofNF-kB,

whichwasincreasedinrelationtotheanimalsofSOgroup

(Fig.2).Thesedataareconsistentwithstudiespublished,that

reportleukocyteinfiltrationandaninflammatoryprocessin

thissameexperimentalmodel.37

N-acetylcysteinewasabletoreducetheexpressionofboth

cytokinesevaluatedintheintestineofanimalsofPPVL+NAC

group. Theanti-inflammatory propertiesofNAC havebeen

previously described in inflammatory bowel diseases with

encouragingresults,38_and_these_results_may_be_related_to_its

thiol group, which isimportantto combatoxidative stress

and inflammation.39 _In _addition, _the _treatment _with _NAC

suppressestheactivationofNF-kBandthesubsequent

pro-duction of its cytokines and also blocks TNF-␣ activation,

causingstructuralchangesinitsreceptor.40

Theinvolvementofnitricoxideinthisexperimentalmodel

iswellestablishedintheliterature.Withtheprogressionof

thedisease,theimmunesystemisactivatedandtheinducible

formofNOS(iNOS)undergoesup-regulation.40_In_this_study,

theanimalsofPPVLgroupdemonstratedasignificantincrease

intheexpressionofiNOSversusanimalsofOSgroupinthe

evaluation by Westernblot (Fig. 3). A previous study,

pub-lishedbyourresearchgroup,observedthesamebehaviorof

thisenzymeinthestomachofanimalssubjectedtothesame

experimentalmodelofpartialportalveinligation.23

A

NF-kB

160 000

140 000

120 000

100 000

80 000

60 000

40 000

20 000

0

SO

∗

#

Expression of the positiv

e pix

els

, %

PPVL PPVL+NAC

B

C

(7)

SO PPVL PPVL+NA C

120 kDa

42 kDa

iNOS

∗

# β–action

TBARS

4

3

2

1

0

SO PPVL PPVL+NAC

Relativ

e r

a

tio (iNOS/

β

-action)

Fig.3–WesternblotofiNOS.Effectsofpartialportalvein ligation(PPVL)andN-acetylcysteine(NAC)oniNOS expression.SO,Sham-operatedgroup;PPVL,partialportal veinligation;PPVL+NAC,partialportalveinligationtreated withNAC.*p<0.01,#_p_<_0.01_(n₌_6).

Inthisstudy,N-acetylcysteinewasabletoreducethe

lev-elsofiNOSintotheintestineofanimalsofPPVL+NACgroup.

Thisfindingisinagreementwithapreviouslypublishedstudy,

inwhichitwasreportedthatNACinhibitsnitricoxide

pro-ductionbycellsoftheimmunesystemandbytheinducible

isoformofNOS(iNOS).41

Theinvolvementofoxidativestressinthedevelopmentof

hyperdynamiccirculationwasinitiallyproposedbyFernand

etal.1998.42_Since_then,_several_studies_have_demonstrated_a

positivecorrelationoftheexperimentalmodelofpartialportal

veinligationwiththesameoxidativeimpairments.8,9,21–23

In the present study, we observed increased levels of

thiobarbituricacid reactivesubstances (TBA-RS)inanimals

subjectedtothisexperimentalmodel(Fig.4).Furthermore,the

increaseoflipidperoxidationwasaccomplishedbyreducing

TBARS 0.8

SO PPVL PPVL+NAC

∗

#

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

nmoles/mgprot

Fig.4–TBA-RS.Effectsofpartialportalveinligation(PPVL) andN-acetylcysteine(NAC)onTBA-RSvalues.SO,

Sham-operatedgroup;PPVL,partialportalveinligation; PPVL+NAC,partialportalveinligationtreatedwithNAC. *p<0.05,#_p_<_0.01_(n₌_6).

SOD

SO PPVL PPVL+NAC

∗

#

80 70 60 50 40 30 20 10 0

USOD/mgprot

Fig.5–SOD.Effectsofpartialportalveinligation(PPVL) andN-acetylcysteine(NAC)onSODactivity.SO,

Sham-operatedgroup;PPVL,partialportalveinligation; PPVL+NAC,partialportalveinligationtreatedwithNAC. *p<0.01,#_p_<_0.05_(n₌_6).

the activity ofantioxidant enzymes superoxide dismutase

(SOD)(Fig.5)andglutathioneperoxidase(GPx).Thisfinding

demonstratesapotentialconditionofoxidativestressinthe

bowelofexperimentalanimals.Thisisinagreementwith

pre-viousstudiesinwhichPPVLtriggeredaconditionofoxidative

stressinotherbodyorgansstudied:stomach9_and_liver.8_The

antioxidant action ofN-acetylcysteine revolves around the

fact thatthis moleculeisacysteine precursor forthe

syn-thesis ofGSH, and alsobyacting directly asa freeradical

scavenger.Thankstoitsantioxidantandanti-inflammatory

properties,NAChasbeenwidelystudiedinthetreatmentof

liverdiseases.40

Theauthorsofthisstudy havealreadyusedNACinthe

treatmentofexperimentalpre-hepatic portalhypertension,

withverypromisingresults.9,21_This_study_demonstrated_the

role of this moleculeinthe bowel of animalssubjected to

thesamemodel,whichpointstoasystemicpotentialofthis

drug.NACwasabletoreducelipidperoxidationlevels,as

mea-suredbyTBA-RS,andtorestoretheactivityofbothantioxidant

enzymesstudied(SODandGPx)intheanimals’bowel.Thus,

onecan say thatN-acetylcysteine actedasanantioxidant,

reducingoxidativestressintheintestinalmucosainanimals

withPPVL.

In conclusion, we point out an anti-inflammatory and

antioxidantsynergisticeffectofN-acetylcysteineinthebowel

ofanimalswithportalhypertension.Judgingbytheresults,

NACwasabletoreducetheintestinaldamageinanimalsby

GPx

SO PPVL PPVL+NAC

∗

#

0.7 0.6 0.5 0.4 0.4 0.2 0.1 0.0

nmolmin/mgprot

Fig.6–GPx.Effectsofpartialportalveinligation(PPVL)and N-acetylcysteine(NAC)onGPxactivity.SO,Sham-operated group;PPVL,partialportalveinligation;PPVL+NAC,partial portalveinligationtreatedwithNAC.*p<0.05,#_p_<_0.01

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reducingoxidativestressandinflammation-bothbeing

con-ditionsextremelyharmfulanddeterminantintheevolution

ofthediseaseherestudied.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This study received financial support from the Fundo de

IncentivoàPesquisaeEventos(FIPE ProjectNo.11-0293)of

Hospital de Clínicas de Porto Alegre (HCPA), Coordenac¸ão

de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES)

andConselhoNacionaldeDesenvolvimentoCientíficoe

Tec-nológico(CNPq).Wewantalsotothankthecontributionof

the ExperimentalHepatology andGastroenterology

Labora-tory(HCPA/UFRGS)andtheOxidativeStressandAntioxidants

Laboratory(ULBRA),placesofsupportanddevelopmentofthis

study.

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