Antiinflammatory effect of a fatty acid mixture with high v 9:v 6 ratio and low v 6: v 3 ratio on rats submitted to dental extraction

Anti-in

fl

ammatory

effect

of

a

fatty

acid

mixture

with

high

v

-9:

v

-6

ratio

and

low

v

-6:

v

-3

ratio

on

rats

submitted

to

dental

extraction

Radamés

Bezerra

Melo

a

,

Paulo

Goberlânio

de

Barros

Silva

b,

*

,

Reinaldo

Barreto

Oriá

c

,

José

Ulisses

de

Souza

Melo

d

,

Conceição

da

Silva

Martins

c

,

Aline

Matos

Cunha

a

,

Paulo

Roberto

Leitão

Vasconcelos

a

a_{DepartmentofSurgery,FacultyofMedicine,FederalUniversityofCeara,Fortaleza,Ceara,Brazil} b_{FacultyofDentistry,UniversityCenterChristus,Fortaleza,Ceara,Brazil}

c_{DepartmentofMorphology,FacultyofMedicine,FederalUniversityofCeara,Fortaleza,Ceara,Brazil} d_{FacultyofMedicine,StateUniversityofCeara,Fortaleza,Ceara,Brazil}

ARTICLE INFO

Articlehistory:

Received17January2016

Receivedinrevisedform30October2016 Accepted11November2016

Keywords:

Surgery Oral

Woundhealing Antioxidants

Anti-inflammatoryagents

ABSTRACT

Objective:Toevaluatetheanti-inflammatoryeffectofpretreatmentforthreedays withafattyacid mixturewithhighv-9:v-6ratioandlowv-6:v-3ratioonratssubmittedtodentalextraction.

Materialandmethods:Thirty-twomaleWistarrats(270–310g)wererandomlydistributedinfourgroups (n=8/group):theshamcontrolgroupandthenegativecontrolgroupreceivedsaline;thehighomega-6/ lowomega-9groupreceivedisolipidfattyacidwithhighv-6:v-3ratioandlowv-9:v-6ratio;thehigh omega-3/lowomega-6groupreceivedfattyacidwithlowv-6:v-3ratioandhighv-9:v-6ratio.Saline andoilswereadministeredbygavagefor4daysbeforeexodontiaand3daysaftersurgery,followedby euthanasia.Masseteredemawasevaluatedclinicallyandtissuesamplesweresubmittedtoosteoclast count(H&E),myeloperoxidaseassay,andwesternblotting(tumornecrosisfactor-alphaand interleukin-1beta).

Results:Inthehighomega-3/lowomega-6group,asignificantdecreasewasobservedinmasseteredema (p<0.0001),myeloperoxidase(p<0.0001),osteoclasts(p=0.0001)andTNF-aexpression(p<0.0001), butnotinIL-1bexpression.

Conclusion:Theingestionoffattyacidwithhighv-9:v-6ratioandlowv-6:v-3ratiosignificantly reducedinflammatoryresponseinratssubmittedtodentalextraction.

ã2016ElsevierLtd.Allrightsreserved.

1.Introduction

Historically, nutrients have been observed as a source of caloriesforcellhomeostasis,andaminoacidshavebeenobserved as a source for protein synthesis. However, current evidence supportsthenotionthatatargetedsupplyofnutrientscanimprove

healthoutcomesbymodulatingimmuneandmetabolicresponses (Alexander,1998;Lietal.,2012;Stables&Gilroy,2011).

Nutraceuticals, which arenutrientsat pharmaceuticaldoses, canmodifybiologicalresponsesinhumansaloneorinconjunction withothersupplements.Themostwidelystudiednutraceuticals are the

v

-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), glutamine, arginine and nucleotides (Stableforth,Thomas,&Lewis,2009).

Marinefishoils,especiallyEPAandDHA,areparticularlyrichin

v

-3fattyacids(Alexander,1998).Someauthorshaveshownthat oilswithahigh

v

-6:

v

-3ratiocanpromotediseasesbystimulating theproductionofinflammatorymediators(Allayee,Roth,&Hodis, 2009).The opposite effect may beachieved by ingestinglarge amountsofoilwithalow

v

-6:

v

-3ratio.

Changesinmetabolicresponsesanddietarylipidlevelshave been shown to impactoxidative stress(Melo et al., 2014) and inflammatoryresponsesinaccordancewiththeratiobetweenthe ingested polyunsaturatedfattyacids (Calder,2003).In addition, Abbreviations:EPA,eicosapentaenoicacid;DHA,docosahexaenoicacid;v-3,

omega-3;v-6,omega-6;G1,shamcontrolgroup;G2,negativecontrolgroup;G3, positivecontrolgroup;G4,treatmentgroup;POD3,thirdpostoperativeday;MPO, myeloperoxidase;CTAB, cetrimoniumbromide; TNF-a, tumorenecrosisfactor alpha;IL-1b, interleukin 1beta; EDTA,ethylenediaminetetraacetic acid;ECL, electrogeneratedchemiluminescence;COX,cyclooxygenase;LOX,lipoxygenase; AA,arachidonicacid;PG,prostaglandin;LT,leucotrien;TXA,tromboxan.

* Corresponding authorat: Facultyof Dentistry,University CenterChristus, CampusParqueEcológico,RuaJoãoAdolfoGurgel,133,Papicu,CEP:60192 345, Fortaleza,Ceará,Brazil.

E-mailaddresses:paulo_goberlanio@yahoo.com.br,

paulo.goberlanio@gmail.com(P.G.deBarrosSilva).

http://dx.doi.org/10.1016/j.archoralbio.2016.11.009

0003-9969/ã2016ElsevierLtd.Allrightsreserved.

–

ContentslistsavailableatScienceDirect

Archives

of

Oral

Biology

fattyacids affecteicosanoid synthesis and actas growthfactor mediators,therebycontributingtocellgrowthandepithelialcell differentiation(Capone,Bagga,&Glaspy,1997).

The

v

-3and

v

-6fattyacidfamilymembersaremetabolizedby thesame enzyme system and, therefore,compete forenzymes (Dyeberg, 1986). This competition impacts the production of eicosanoids,includingprostaglandins,thromboxanesand leuko-trienes(Fischer,1989).Eicosanoidsmetabolizedfrom polyunsatu-rated

v

-6 fattyacidsact aspro-inflammatory mediators,while thosemetabolizedfrom

v

-3fattyacidshaveanti-inflammatory properties.

Oneofthegreatestconcernsinthepostoperativecareoforal surgerypatients is inflammation,which causes discomfort and increasestheriskoflockjaw,especiallyinthefirstthreedays.To determineiffattyacidsupplementationcouldreduceinfl amma-tionandlock-jawafteratoothextraction,weevaluatedtheeffects ofafattyacidmixturewithahigh

v

-9:

v

-6ratiooralow

v

-6:

v

-3 ratio on the inflammatory response in rats following dental extraction.

2.Materialsandmethods

2.1.Studydesign

This controlledexperimental studyincluded32 male Wistar rats(weighing270–310g) supplied bytheexperimental animal facility of the Federal University of Ceará. The animals were acclimatedforoneweekundercontrolledlaboratoryconditions (24_{C,relativehumidity40–60%,12-hcircadiancycle,foodand}

wateradlibitum)andthenrandomlyassignedtofourexperimental groups (n=8/group). The sham group received saline without exodontia(0.9%NaCl).Thenegativecontrolgroupreceivedsaline andexodontia(0.9%NaCl).Thehighomega-6/lowomega-9group receivedexodontiaandanisolipidfattyacid(

a

-linolenicacid)with ahigh

v

-6:

v

-3ratio(8:1)andalow

v

-9:

v

-6ratio(0.4:1).Thehigh omega-3/lowomega-6groupreceivedexodontiaandamixtureof fattyacids (docosahexaenoic acid,

a

-linolenicacid and eicosa-pentaenoic acid) with a low

v

-6:

v

-3 ratio (1.4:1) and a high

v

-9:

v

-6ratio(3.4:1).

Bothsalineandoil(1.2g/kg/day)wereadministeredbygavage 4daysbeforeand3daysaftertheextractionofthefirstlowerleft molar.Thecompositionofthefattyacidsadministeredinthehigh omega-6/lowomega-9groupandthehighomega-3/lowomega-6 groupsare shown in Table1. Thetest oil mix consistedof the followingediblenutritionaloils:sunflowerhigholeic(48%),canola (25%),fish(15%)andolive(12%).Theoilproportionsweredesigned toprovide a combination of antioxidant (

v

-9:

v

-6=3.7:1) and anti-inflammatoryproperties (

v

-6:

v

-3=1.4:1). The isolipid oil mixconsisted of the following edible nutritional oils: soybean (97%)andcorn(3%).Theoilproportionsweredesignedtoexclude antioxidant(

v

-9:

v

-6=0.4:1) and anti-inflammatory properties (

v

-6:

v

-3=8:1).

After4daysofgavage,theanimalswereanesthetizedwithan intramuscularinjectionofafreshlypreparedmixtureofketamine

(90mgkg 1_{)andxylazine(10mgkg} 1_{)andsubmittedtoatooth}

extractionor shamsurgery (Silvaet al.,2015).Threedaysafter surgery,theanimalswereeuthanizedbycervicaldislocation,and alveolarmucosaandalveolarbonesampleswerecollected.

2.2.Determinationofmyeloperoxidaseinalveolarmucosa

Myeloperoxidase (MPO), an enzyme expressed mainly in azurophilicneutrophilgranulocytes,isawidelyusedquantitative markerofneutrophilinfiltrationininflamedtissues.MPOactivity canbedeterminedbycolorimetryandELISA(Bradley,Christensen, &Rothstein,1982).

Followingeuthanasia,aspecimenofthealveolarmucosawas collected,weighedandfreeze-storedat 80_{Cuntilrequiredfor}

theassay.Thetissuesampleswerehomogenizedina0.5%CTAB (cetrimoniumbromide)solution(50mgtissue/1mLCTAB),frozen, thawed and homogenized again. Subsequently, the samples (1500g) were centrifuged for 12min at 4_{C. The supernatant}

wastransferredtoanEppendorftubeandcentrifugedfor10minto remove contaminants. Ninety-six-well plates were filled with supernatant(7

m

L)andtestsolution(200

m

L)preparedwith5mg o-dianisidine,15

m

L1% H2O2,3mLphosphate bufferand 27mL

H2O.Theabsorbancewasreadinaspectrophotometerat450nm

(t0=0min;t1=1min).TissueMPOlevelsweredeterminedusing

H2O2asa substrate.Aunit ofMPOwasdefined astheamount

required to convert 1

m

mol H2O2 into water in 1min at 22C

(Barretoetal.,2008).WhenH2O2isdegraded,asuperoxideradical

is released, which produces o-dianisidine as a function of the reaction time. Absorbance was plotted on standard neutrophil curvesandexpressedasMPO/mgtissue(MPOactivity).

2.3.Determinationofmasseteredema

Themassetermuscleextendsfromthezygomaticarchtothe ramusofthemandible.Priortosurgery,afteranesthetizing the animal (t0), a caliper was introduced into the mouth and the

middleportionofthemasseterwasmeasured(mm).Threedays after surgery, prior toeuthanasia, the masseter was measured again. Measurements were made in duplicate by a blinded investigator.

2.4.Histologicalprocessingandhistomorphometricanalysis (osteoclastcount)

Following euthanasia, the hemi-mandibles, from which the molarswereextracted,wereexcised,fixedin10%formaldehydefor 24h,decalcifiedina7%formicacidsolutionfor24handprocessed forhistology(hematoxylinandeosin).

Afterthehemi-mandibleswereembeddedinparaffinblocks, 4

m

m-thicksectionswerecutusingasemi-automaticmicrotome. The sections were stained with hematoxylin and eosin for examinationunder aconventional light microscope(magnifi ca-tion:400x).Fivemicro-fieldswereselectedformicrographsusing aLeicaDFC295microscopecoupledtoaLeicaDM2000microscope with LAS software (Leica Heerbrugg, Switzerland). The images wereexportedandanalyzedwithImageJsoftware.Thecellcounter command was used to count the osteoclasts. To minimize observationbias,theslideswererandomized,andtheinvestigator wasblindedtothegroupassignments.Thesumoffivemicrographs wasconsideredasampleunitforthestatisticalanalysis(Kimetal., 2012).

2.5.WesternblotforTNF-

a

andIL-1

b

ToevaluatetheproteinexpressionofTNF-

a

andIL-1

b

,alveolar ridgesegmentswere homogenizedin RIPA lysisbuffer(50mM Table1

Composition of fatty acid mixes used in Group 3 (isolipid) and Group 4 (inflammatoryandantioxidant).

Composition Sourceofv-3 Ratio Highomega-6

lowomega-9

v 6+v-3 Cornoil Soybeanoil

ALA v 6:v-3=8:1

v 9:v-6=0.3:1

Highomega-3 lowomega-6

v 9+v-6+v-3 Oliveoil Canolaoil Fishoil ALA(35%) EPA(39%) DHA(26%)

v 6:v-3=1.4:1

Tris–HCl[pH8.5],50mMNaCl,0.1mMEDTA,1%Tween20,1mM dithiothreitol)andproteaseinhibitor(1

m

Linhibitor:100

m

LRIPA). For the protein extraction, the alveolar ridge samples were centrifuged(17min,4_{C,13,000rpm),andthesupernatant was}

collected. Protein concentrations were determined using the bicinchoninicacidassayaccordingtothemanufacturer’sprotocol. Then, equal amounts of protein (50

m

g) were separated by electrophoresisina 10%sodiumdodecylsulfate-polyacrylamide geland transferredtoa nitrocellulosemembrane (Hybond-ECL, AmershamPharmaciaBiotech,Amersham,UK). Themembranes wereblockedwith 5%non-fat dry milk for onehour and then incubatedfor1hatroomtemperaturewithanti-

b

-actin(1:1000), anti-TNF-

a

(1:400)andanti-Il-1

b

(1:400)(SantaCruz Biotechnol-ogy,SantaCruz,CA).Subsequently,themembraneswerewashed and incubated for 1h at room temperature with horseradish peroxidase-conjugated secondary antibody (monkey anti-rabbit immunoglobulinG;1:1000;AmershamPharmaciaBioTech,UK). Afterwashing,themembraneswereincubatedwitha chemilumi-nescence reagent (ECL; Amersham Pharmacia Biotech) and exposed to HyperWlm ECL (Amersham Pharmacia Biotech) to develop the western blot. The densitometry analysis was performedwithImageJsoftwareversion1.4(NationalInstitutes ofHealth,USA).Theresultswereexpressedastherelativedensity oftheTNF-

a

orIL-1

b

/

b

-actinbands(Limaetal.,2012).

2.6.Statisticalanalysis

TheShapiro-Wilktestwasusedtoassessthenormalityofthe data distribution. All data were presented as the meanSEM. GraphPad Prismsoftware version 5.0 (GraphPad Software,Inc., California Corporation, San Diego,California) was used for the statisticalanalysis.Aone-wayANOVAwas usedtocomparethe groups.Thelevelofstatisticalsignificancewassetat5%(p<0.05).

2.7.Ethicalconsiderations

Approvalfortheexperimentaluseoflaboratoryanimalswas obtained by the local ethics committee on animal use (CEUA, formerlyCEPA)and filedunder protocol#73/2011(29February 2012)incompliancewithFederalLaw#11.794(8October2008) andDecree#6.689(15July2009)regulatingLaw#11794(http:

www.planalto.gov.brccivil03Ato2007-20102008LeiL11794.htm).

3.Results

3.1.MPOactivityinalveolarmucosa

The exodontia procedure raised MPO levelsin the negative controlgroup(vs.sham:5.111.08)and significantlydecreased MPOlevelsinthealveolarmucosaofanimalsinthehighomega-3/

lowomega-6group(6.380.48)comparedtonegativecontrols (10.680.81)(p<0.001).Nodifferenceswereobservedbetween the high omega-6/low omega-9 group (9.830.52) and the negativecontrolgrouporthehighomega-3/lowomega-6group (Fig.1A).

3.2.Masseteredema

The exodontia procedure increased masseter edema in the negativecontrolgroup(vs.sham:0.120.11).Theincreaseinthe massetermusclethicknesswassignificantlysmallerin thehigh omega-3/low omega-6group (0.490.04) than in thenegative control group (2.150.06) or the high omega-6/low omega-9 group(1.860.07)(Fig.1B).

3.3.Histopathologicalandhistomorphometricfindings(osteoclast count)

On the third postoperative day, visible, although negligible, microscopicchangeswereobservedintheshamgroup(Fig.2A).In contrast,thetissuefromthenegativecontrolgroupandthehigh omega-6/lowomega-9groupexhibitedextractionsitesfilledwith granulationtissueandnumerous,mostlybinucleated,osteoclasts intheproximityofthebonetissuewithevidentHowship’slacunae (amicroscopicsignofosteoclastactivity)(Fig.2BandC).Inthe high omega-3/low omega-6 group, a small number of bi- or multinucleated osteoclasts were observed adhering to the mineralized bone matrix through Howship’s lacunae (Fig. 2D). Nosignsoflocalinfectionwereobserved.

Osteoclastsweresignificantlymorenumerousinthenegative controlgroup(65.713.3)thanintheshamgroup(0.80.6),but no significant difference was observed between the negative control group and the high omega-6/low omega-9 group (50.66.5).Thenumberofosteoclastswassignificantlyreduced in thehighomega-3/lowomega-6group(31.12.6)(p=0.0001, ANOVA/Tukey)(Figs.1Cand2).

3.4.WesternblotforTNF-

a

andIL-1

b

TheTNF-

a

/

b

-actinratiowassignificantlyhigherinthenegative controlgroup(35.58.8)thanintheshamgroup(2.10.5),butno significantdifferencewasobservedbetweenthenegativecontrol groupandthehighomega-6/lowomega-9group(44.95.8).The TNF-

a

/

b

-actinratiowassignificantlylowerinthehighomega-3/ lowomega-6group(8.83.3)thaninthenegativecontrolgroup or the high omega-6/low omega-9 group (p<0.0001; ANOVA/ Tukey)(Fig.3A).

TheIL-1

b

/

b

-actinratiowassignificantlyhigherinthenegative control group (44.95.2) than in the sham group (17.54.1) (p=0.0113, ANOVA/Tukey), but no significant difference was

Fig.1.Dosageofmyeloperoxidase(MPO)(A),masseteredema(B)andosteoclastcount(C)intissuefromthesiteofdentalextractioninfourgroupsofrats:Sham(n=8), negativecontrol(saline,n=8),highomega-6/lowomega-9(isolipidfattyacidmixwithhighv-6:v-3ratioandlowv-9:v-6ratio)andhighomega-3/lowomega-6 (anti-inflammatoryandantioxidantfattyacidmixwithlowv-6:v-3ratioandhighv-9:v-6ratio).Resultsexpressedasmeanvaluesstandarderrorofthemean.*p<0.05;**

observedbetweenthenegativecontrolgroupandthehigh omega-6/lowomega-9group(38.57.3)orthehighomega-3/low omega-6group(38.64.1)(Fig.3B).

4.Discussion

Arachidonicacid(

v

-6) andEPA(

v

-3)arebothsubstrates of cyclooxygenase(COX)and lipoxygenase(LOX). Whenincreased, EPAactsasa competitiveinhibitor,preventing arachidonicacid (AA)fromenteringthecascadeandgeneratingpro-inflammatory endproducts.Highconsumptionoflinoleicacidfavorstheincrease of AA in cell membrane phospholipids, thereby increasing the productionofprostaglandin(PG)E2andleukotriene(LT)B4viathe

COXand5-LOX pathways,respectively.Theingestionof fish oil introduces EPA into membrane phospholipids, inhibiting AA

metabolismdue tocompetitionforthesameenzyme pathways (COXand5-LOX)andthereby promotingtheformationofPGE3

(instead ofPGE2)and LTB5 (instead ofLTB4), whichare weaker

inflammatory mediators (James, Gibson, & Cleland, 2000). In general, linoleic acid is a precursor for the synthesis of pro-inflammatoryeicosanoids,suchasthromboxaneA2(TXA2),PGI2,

PGE2andLTB4(Jamesetal.,2000;Kelley,2001).

Amongthemediators,PGE2 andLTB4 havethegreatest

pro-inflammatorypotential. PGE2 induces fever, promotes

vasodila-tion, increases vascular permeability and enhances pain and edemacausedbyotheragents,suchasbradykininandhistamine. However, PGE2 actsas an anti-inflammatorymediator when it

suppressesTNF-

a

andIL-1

b

productionandasan immunosup-pressantwhenitinhibitslymphocyteproliferation,naturalkiller cell activityand IL-2 and interferonproduction. LTB4 increases

vascular permeability, blood flow and leukocyte chemotaxis, which triggersthe release of lysosomalenzymes and increases theproductionofreactiveoxygenspecies,TNF

a

,IL-1andIL-6.In allofthesescenarios,LTB4actsasapro-inflammatorymediator

(Grimble,2002).Inthepresentstudy,TNF-

a

productionwaslikely suppressedduetothesubstitutionofLTB5forLTB4.Aspreviously

mentioned, thromboxanes are also a product of eicosanoid metabolism.TXA2(themainbyproductofAA)promotesplatelet

aggregation,leukocyteadhesionandsmoothmusclecontraction (Calder,2003).

Alternatively,theincreasedavailabilityof

v

-3fattyacids(such as linolenic acid, EPA and DHA) favors the synthesis of anti-inflammatoryeicosanoids, suchas PGE3,TXA3 and LTB5.Sucha

balanceis lessfavorableforthegenerationofpro-inflammatory mediators, and some immunosuppressive effects may be lost (Robert,2005).

Excessive

v

-6fattyacidmetabolismresultsintheproductionof pro-inflammatory mediators,vasoconstriction,increasededema, increasedchemotaxisofpolymorphonuclearleukocytes,increased pro-inflammatory cytokine signaltransduction and local tissue toxicity,potentiallyleading toinflammatory conditions,suchas systemic inflammatoryresponse syndromeand multiple organ failuresyndrome.Loweringthe

v

-6:

v

-3ratiointhedietmakesit possibletoprovidethepro-inflammatoryproductsrequiredfor immuneresponsesandattenuationofexcessiveinflammatoryand oxidative conditions, as demonstrated in experimental animal studiesandclinicaltrials(Basetal.,2007;Meloetal.,2014;Sijben &Calder,2007).

Inourstudy,theingestionofoilswithareduced

v

-6:

v

-3ratio wasassociatedwithanti-inflammatoryeffects,reducedmasseter edemaandlowerMPOlevelsinalveolarbonetissue.Asthefirst cells to migrate from the blood vessels to the site of injury, neutrophilsplayaprotectiverolethroughphagocytosis.Inthefirst daysfollowinginjury,neutrophilsaccountforapproximately50% ofall cellsat thewoundsite. Eventually,thelocalstimulusfor recruitment (pro-inflammatory cytokine synthesis) wanes and infiltration clears in a few days as the lysed neutrophils are phagocytosed by macrophages (Alves, Godoy, Figueiredo, & Menezesetal.,2007).ThereductioninTNF-

a

expressionobserved

inourstudymaybeexplainedbyareductioninthesynthesisof pro-inflammatorymediators(suchasLTB4)inducedbyantioxidant

and anti-inflammatory agents. Thedecrease in MPOlevels and masseteredema duetoreducedleukocytechemotaxis,vascular permeabilityandbloodflowmayalsobeattributedtoantioxidant andanti-inflammatorymediators(Grimble,2002).

Treatment with antioxidants efficiently reduces the serum levels of nitric oxide and several pro-inflammatory mediators, includingTNF-

a

,IL-1

b

andIL-6.However,thereductionofTNF-

a

synthesis through the inhibition of oxidative stress is more efficientintheshorttermthanthereductionofIL-1

b

synthesis. LikeIL-6andnitricoxide,IL-1

b

seemstorequirelongerexposure beforeinhibitionoccurs(Ramirez-Ramirezetal.,2013).Thiswould explainwhythetreatmentpromptly inhibitedTNF-

a

synthesis, whilereducingIL-1

b

levelsonlyslightly.

The reduction of TNF-

a

synthesis not only helps minimize edemaafterdentalextractionbutalsoreducespain.Trismusisa majorindicatorofinflammationinclinicaltrialsandTNF-

a

acts swiftly by sensitizing nociceptors and limiting mouth opening (Bello,Adeyemo,Bamgbose,Obi,&Adeyinka,2011;Ribeiro,Vale, Ferreira,&Cunhaetal.,2000).Inhibitionbyspecificagents,suchas thalidomide,wasshowntoreducenociceptioninamurinemodel ofacutepawpain(Ribeiroetal.,2000).

Inaddition,TNF-

a

isintimatelyinvolvedinosteoclastogenesis. Duringthefirst3–7daysafterdentalextraction,TNF-

a

synthesisis usuallyupregulated andhyperinflammatoryconditions (suchas periodontaldisease)enhancelocalimmunostainingandprolong TNF-

a

superexpressionatthesiteofextraction,resultingingreater alveolarbonelossassociatedwithhyperactivationofosteoclasts (Kimetal.,2012).

The increased number of osteoclasts in situ is directly proportionaltotheincreaseintartrate-resistantacidphosphatase expression(Zenger,Ek-Rylander,&Andersson,2010)andoxidative stress,whichacceleratestheprocessofboneresorption(Halleen et al.,1999).Thus, drugs capableof reducing TNF-

a

levels and othermarkersinthesamefamily(such asreceptoractivatorof nuclearfactorkappa-Bligand)effectivelyinhibitboneresorption (Kimachi,Kajiya,Nakayama,Ikebe, &Okabe, 2011)by reducing metabolicactivityandosteoclasts attheextractionsite (Zenger

etal., 2010)and byreducing localoxidative stress(Lima etal., 2012),asobservedinthepresentstudy.

Inconclusion,thefattyacidmixturetestedinthisstudy(high

v

-9:

v

-6ratioandlow

v

-6:

v

-3ratio)effectivelyreducedTNF-

a

and MPO levels (but not IL-1

b

levels), masseter edema and osteoclast numbers at the site of dental extraction in rats. In additiontohavingaprotectiveeffectagainstatherosclerosisand plaquerupture, theanti-inflammatorypropertiesofmarinen-3 polyunsaturatedfattyacids(PUFAs)(Calder,2012)andthepresent antioxidanttherapyofferapromisingalternativeforthe preven-tionofbonelossafteratoothextraction.Thus,antioxidanttherapy isapotentiallyimportantadditiontodentalrehabilitationafter implant-supported mucous prostheses and dental implants. However,moreresearchis neededtoconfirmthefindings that reductionofinflammationintheearlyhealingstageaftertooth extractionresultsingreaterboneavailability.

Conflictofinterests

Theauthorsreportnoconflictsofinterestrelatedtothisstudy and agree with the decisions about it, approved the final manuscriptand referredtoit asoriginal manuscript,withtext, figures and photographs that have not appeared in any other publication,thismaterialhasbeensubmittedonlytoArchivesof OralBiology.

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