Braz. j. . vol.83 número6

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www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

The

potential

role

of

amlodipine

on

experimentally

induced

bacterial

rhinosinusitis

夽

Arzu

Tatar

a,∗

,

Mukadder

Korkmaz

b

,

Muhammed

Yayla

c

,

Elif

Polat

d

,

Hakan

Uslu

e

,

Zekai

Halici

f

_,

_Secil

_N.

_Parlak

d

a_Ataturk_University,_Medical_Faculty,_Department_of_{Otorhinolaryngology,}_Head_and_Neck_Surgery,_Erzurum,_Turkey b_Ordu_University,_Medical_Faculty,_Department_of_{Otorhinolaryngology,}_Head_and_Neck_Surgery,_Ordu,_Turkey c_Kafkas_University,_Medical_Faculty,_Department_of_{Pharmacology,}_Kars,_Turkey

d_Ataturk_University,_Medical_Faculty,_Department_of_Embryology_and_Histology,_Erzurum,_Turkey e_Ataturk_University,_Medical_Faculty,_Department_of_Medical_{Microbiology,}_Erzurum,_Turkey f_Ataturk_University,_Medical_Faculty,_Department_of_{Pharmacology,}_Erzurum,_Turkey

Received16January2016;accepted9August2016 Availableonline28September2016

KEYWORDS

Rhinosinusitis; Non-antibiotic; Amlodipine; Antioxidants; Guineapig

Abstract

Introduction:Antibioticsarefrequentlyusedforthetreatmentofrhinosinusitis.Concernshave beenraised regardingtheadverseeffectsofantibioticsandgrowingresistance. Thelackof developmentofnewantibioticcompoundshasincreasedthenecessityforexplorationof non-antibioticcompoundsthathaveantibacterialactivity.Amlodipineisanon-antibioticcompound withanti-inflammatoryactivity.

Objective: Inthisstudyweaimedtoinvestigatethepotentialroleofamlodipineinthe treat-mentofrhinosinusitisbyevaluatingitseffectsontissueoxidativestatus,mucosalhistologyand inflammation.

Methods:Fifteen adult albino guinea pigs were inoculatedwith Staphylococcus aureus and treatedwithsaline,cefazolinsodium,oramlodipinefor7days.Thecontrolgroupwascomposed by five healthy guinea pigs. Animals were sacrificed after the treatment. Histopathologi-calchanges wereidentifiedusingHematoxylin-Eosin staining.Inflammationwas assessedby PolymorphonuclearLeukocyteinfiltrationdensity.Tissuelevelsofantioxidants(superoxide dis-mutase,glutathione)andanoxidativeproduct(malondialdehyde)weredetermined.

Results:In rhinosinusitis induced animals, amlodipine reduced loss ofcilia, lamina propria edema and collagendeposition compared to placebo (saline) andalthough notsuperior to cefazolin,amlodipinedecreasedpolymorphonuclearleukocyteinfiltration.Thesuperoxide dis-mutaseactivityandglutathionelevelswerereduced,whereasthemalondialdehydelevelswere

夽 _Please_cite_this_article_as:_Tatar_A,_Korkmaz_M,_Yayla_M,_Polat_E,_Uslu_H,_Halici_Z,_et_al._The_potential_role_of_amlodipine_on_{experimentally} inducedbacterialrhinosinusitis.BrazJOtorhinolaryngol.2017;83:619---26.

∗_{Corresponding}_author.

E-mail:arzutatar@atauni.edu.tr(A.Tatar).

PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.

http://dx.doi.org/10.1016/j.bjorl.2016.08.006

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increasedsignificantlyinallthree-treatmentgroupscomparedtothecontrolgroup.Amlodipine treatedgroupshowedsignificantlyincreasedsuperoxidedismutaseandglutathionelevelsand decreasedmalondialdehydelevelscomparedtoalltreatmentgroups.

Conclusion:Thenon-antibioticcompoundamlodipinemayhavearole inacuterhinosinusitis treatmentthroughtissueprotective,antioxidantandanti-inflammatorymechanisms.

© 2016 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE

Rinossinusite; Nãoantibiótico; Amlodipina; Antioxidantes; Cobaia

Opapelpotencialdaamlodipinanarinossinusitebacterianainduzida experimentalmente

Resumo

Introduc¸ão:Antibióticos sãofrequentemente utilizadospara otratamento de rinossinusite. Questõestêmsidolevantadassobreosefeitosadversosdosantibióticosearesistência cres-cente.Afaltadedesenvolvimentodenovoscompostosantibióticosaumentouanecessidadeda explorac¸ãodecompostosnão-antibióticosquepossuematividadeantibacteriana.Aamlodipina éumcompostonão-antibióticocomatividadeanti-inflamatória.

Objetivo:Oobjetivodesseestudofoiinvestigaropapelpotencialdaamlodipinanotratamento darinossinusite,avaliandoseusefeitossobreoestadooxidativodotecido,histologiadamucosa einflamac¸ão.

Método: QuinzecobaiasalbinasadultasforaminoculadascomStaphylococcusaureusetratadas comsoluçãosalina,cefazolina,ouamlodipinadurante7dias.Ogrupocontroleincluiucinco cobaiassaudáveis.Osanimaisforamsacrificadosapósotratamento.Alteraçõeshistopatológicas foramidentificadasutilizando-seacoloraçãodeHematoxilina-Eosina.Ainflamaçãofoi avali-ada peladensidade deinfiltração deleucócitospolimorfonucleares.Foramdeterminadosos níveisteciduaisdeantioxidantes(superóxidodismutase,glutationa)eumprodutodeoxidação (malondialdeído).

Resultados: Emanimaiscomrinossinusiteinduzida,aamlodipinareduziu aperdadoscílios, edemadalâminaprópriaedeposiçãodecolágenoemcomparaçãocomogrupoplacebo(solução salina)eemboranãosejasuperioràcefazolina,aamlodipinadiminuiuainfiltraçãodeleucócitos polimorfonucleares.Osníveisdeatividadedasuperóxidodismutaseeglutationaforam reduzi-dos,enquantoosníveisdemalondialdeídoaumentaramsignificativamentenostrêsgruposde tratamentoemcomparaçãoao grupocontrole. Ogrupo tratadocomamlodipinaapresentou aumentosignificantedosníveisdesuperóxidodismutaseeglutationaediminuiçãodosníveis demalondialdeídoemcomparaçãocomtodososgruposdetratamento.

Conclusão:Ocompostonãoantibióticoamlodipinapodeterumpapelnotratamentoda rinoss-inusiteagudaatravésdemecanismosprotetoresdetecido,antioxidanteseanti-inflamatórios. © 2016 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Publicado por Elsevier Editora Ltda. Este é um artigo Open Access sob uma licença CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Rhinosinusitis is characterized by inflammation of the sinus and nasal mucosa. It is one of the most common healthproblems and accountsfor moreoutpatient antibi-otic prescriptions than any other diagnosis.1 _Both _host and environmental factors play a role in the develop-mentof rhinosinusitis. The pathophysiologycausing acute rhinosinusitis involves a series of changes that lead to obstruction of the sinus ostia, swelling and inflammation ofthemucosa,mucousstasis, impairedmucociliary clear-ance, and microbial infection. The goals of treatment areto reduce inflammation, eradicate infection,improve drainageandaerationofnasalandsinusmucosa,andrestore

mucociliaryfunction.2_Common_medical_therapies_for_acute rhinosinusitis include antibiotics, nasal saline irrigation, decongestants, antihistamines, mucolytics, topicalor sys-temic corticosteroids, and anti-inflammatorydrugs. Little evidence supports the use of decongestants and antihis-tamines,althoughtheymayhelptoreducerhinorrhoeaand nasalcongestion.Nasalirrigationwithsalineisusuallyused inacuterhinosinusitisandmayimprovemucociliary clear-ancebutevidenceislimitedtosupportitsuse.3,4

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trialsshowedthatuse ofantibiotics for acute rhinosinusi-tisoffersasmalltherapeutic benefitover placebo,witha correspondingriseintheriskforadverseevents.5

ReactiveOxygenSpecies(ROS)areproducedunder phys-iological conditions and their production and antioxidant activityarebalanced.ExcessiveaccumulationofROScauses lipid and protein peroxidation and can lead to cell dam-age and death. Cells overcome this oxidative stress via

anti-oxidantdefence mechanisms thatinclude Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), Catalase (CAT),Glutathione(GSH),andperoxiredoxins. Malondialde-hyde(MDA),aproductoflipidperoxidation,isthereforea goodindicator ofcellulardamage.Recent studies showed thatROSactsassecond messengersfor inflammation acti-vationandplayaroleininflammation.6,7

Amlodipine (AML) is a dihydropyridine L-type cal-ciumchannelblockerwithsignificantantibacterialactivity against several gram-positive and gram-negative strains. It is reportedly the most powerful of the cardiovascu-lar drugs with antibacterialactivity.8,9 _AML _also_has _been reportedtohaveanti-inflammatoryactivity.AMLhasbeen showntodecreaseischemia-reperfusioninjurybyimproving theoxidativestatusinileumischemia-reperfusioninduced rabbits.10

The growthofantibioticresistanceandthelack of dis-covery of new antibiotic compounds have increased the necessityfor explorationofnon-antibioticcompounds like AML that have antibacterial activity. In both acute and chronicsinusitis,inflammationandedemaleadsto obstruc-tionofsinusostiathatfurtherexacerbatesthedisease.We supposethatanti-inflammatoryandantibacterialeffectsof AMLcanbebeneficialinthetreatmentofrhinosinusitis.

Theaimofthepresentstudywastoinvestigatepossible effectsof AML onoxidative status,inflammation, and tis-sueintegrityinananimalmodelofexperimentallyinduced acuterhinosinusitis.

Methods

Animals

Twentyadultalbinoguineapigswithnoevidenceofupper respiratorytractinfectionswereused.Eachanimalweighed 330---370g, and all were obtained from Ataturk Univer-sity’sExperimentalAnimalLaboratoryattheMedicinaland ExperimentalApplicationandResearchCentre.Theanimal experimentsandprocedureswereperformedinaccordance withnationalguidelinesfortheuseandcareoflaboratory animals, and the study was approved by Ataturk Univer-sity’slocalanimalcarecommittee(approvaln◦_2014-1/12). The guineapigswere housedin standard plasticcages on sawdust bedding in an air-conditioned room at 22◦_±₁◦_C and a 12:12h dark:light cycle. Standard guinea pig food andtapwaterweregivenadlibitum.Theadaptationtime beforetheexperimentwas2weeks.Theanimalswere ran-domly divided into four groups (five animals per group): one group served asa negative control (Group C; no rhi-nosinusitis induced, only treatedwith Intraperitoneal [IP] salineinjections),onegrouphadrhinosinusitisbutwasonly treated with IP saline injections (Group S; positive con-trol),onegrouphadrhinosinusitisandwastreatedwithoral

amlodipine(GroupSA),andonegrouphadrhinosinusitiswas treatedwithcefazolin(GroupSC).Animalsweresacrificed at7days.

Developmentoftheanimalmodel

Merocel(MedtronicXomed,Jacksonville,FL,USA)wascut withmicroforceps. Microscissors were used to shape and insertaspongeintothenostrils.Allfiveanimalsin theS, SC,andSAgroups,butnottheCgroup,wereadministered ketamine(i.m.,50mg/kg;Ketalar Pfizer,Istanbul,Turkey) and diazepam (i.m., 2mg/kg; Diazem, Deva, Istanbul, Turkey). The nasal dorsum was sterilized with povidone-iodine and gelatin sponges were inserted into the right nasal cavities. The nostrils were then inoculated with

Staphylococcusaureus(0.5mL)usingahypodermicsyringe

(Staphylococcus aureus strain ATCC25923 wassuspended

ata concentration of 900×106_CFU _{[Colony-forming} _units] permL).At24hafterthebacterialinoculation,theMerocel wasremovedfromthenasalcavities.Purulentnasal secret-ionswereobservedinthenasalcavitiesofanimals.

Guineapigsweretreated twicedailyfor a7 dayswith injection of saline IP in Group S and Group C and cefa-zolinsodium (i.m., 50mg/kg/day) in group SC. Group SA was treated with per oral amlodipine 5mg/kg/day (dis-solvedinsaline,twicedaily)for7days.The animalswere thensacrificedwithalethaldoseofthiopentalsodiumand decapitated.Theexternalnasaldorsumwassterilizedwith povidone-iodineswab,andafterskinelevation,thelateral nasalwalls andmaxillary sinuses were removed fromthe right nasalcavity. The mucosae of thelateral nasal walls werestoredat−80◦_C_for_biochemical_analysis.

Histopathologicalanalysis

Thespecimens of allguineapig wererapidlyfixed in 10% bufferedformalinfor24hforhistologicalexamination.After thefixation, tissue sampleswere routinely processedand embedded in paraffin wax from that 5␮m-thick sections werecut.Then,sixsectionswereobtainedforeachguinea pigtissueandplacedontopositivelychargedslides.The sec-tionswerestainedwithMayer’sHematoxylin&Eosinafter the deparaffinization and rehydration of them. Later, all sections were examined and photographed under a light photomicroscope(NikonEclipseE600,Japan)for histopatho-logicalexamination.

Semi-quantitativeanalysis

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Group C

Group S

Group SC

Group SA

V

E

L.P

Figure1 Micrographoftissuesforallgroups.E,Epithelium;LP,LaminaPropria;V,Vasodilatationsinveins,yellowstar;goblet cell,redstar;laminapropriaedema,greenarrow;neutrophiles,redarrow;macrophages,orangearrow;degenerativeepithelium cell,yellowarrow;cilias,H&Estaining.

Stereologicalanalysis

Stereologicalanalysesweremadeusingastereology work-stationconsistingofstereologysoftware(StereoInvestigator version9.0,Microbrightfield,Colchester,VT,USA),a mod-ified light microscope (Leica DM4000 B, Germany) and a motorizedsystemwhichcanmovethesectioninbothxandy

direction.Theopticfractionatorframemethodwasusedto countPolymorphonuclearLeukocytes(PMNLs)inthelamina propriaofsixtissuesectionsforeachguineapig.The sec-tionswereexaminedundera40×LeicaPlanApoobjective (NA=1.40).ThedensityofPMNLswasestimatedaccording todefinedbyKaraandcolleagues.13

Biochemicalanalysis

Lateral nasal wall mucosa samples sized 0.5cm2 _were usedforbiochemicalanalysis.SuperoxideDismutase(SOD) activity,14 _Glutathione_(GSH)15 _levels,_and_{Malondialdehyde} (MDA)16 _levels_were_determined_in_duplicate_for_each sam-plesupernatantand standardsat roomtemperature using a modified method and an ELISA reader. The average absorbanceofeach sampleandstandard werecalculated. A standard curve was plotted and the linear relationship equation was obtained from the absorbance of the stan-dards. Linear SOD, GSH, and MDA concentrations were calculatedaccordingtothisequation.TheresultsforSOD, GSH,andMDAinthetissueswereexpressedasU/mgprotein, mmoL/mLprotein,andnmoL/mgprotein,respectively.All dataarepresentedasmean±standarddeviation(SD)based onpermgprotein.Proteinconcentrationsweredetermined bythe Lowry methodusing commercial proteinstandards

(Totalproteinkit-TP0300-1KT;SigmaChemicalCo.,Munich, Germany).

Statisticalanalysis

All data are expressed as group mean±SD and analyzed using SPSS (IBM SPSS Statistics 20.0, IBM Corporation, Somers, NY, USA). Kolmogorov---Smirnov test was applied for analysis of data distribution. The stereological and semi-quantitativehistopathologicaldatawereanalyzed by the nonparametric Kruskal---Wallis test followed by the Mann---Whitney U-test. The parametric test of analysis of variance (ANOVA) and LSDtest for mean separation were usedtoanalyzethebiochemicaldata. Acriticalvaluefor significanceofp<0.05wasusedthroughoutthestudy.

Results

Histopathologicalresults

Group C(Control) ---epithelial structurewasseen normal. Nocilialosswasobserved.Littleinflammatorycell infiltra-tionwasevidentin thelamina propriaandnoedemawas observed(Fig.1;Table1).

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Table1 Thestatisticalresultsofsemi-quantitativeassessmentofhistopathologicalchangesofallgroups.

Groups N◦_of_subjects _LPE _LC _V _GCN _CD

C 5 0.14±0.07d _0.09_±_0.11d _0.12_±_0.04d _1.11_±_0.10d _0.14_±_0.05d

S 5 1.12±0.20c _2.90_±_0.10a _2.85_±_0.19b _2.94_±_0.16a _2.91_±_0.10a

SC 5 2.06±0.16a _0.40_±_0.09b _1.70_±_0.27c _1.18_±_0.18c _1.07_±_0.06b

SA 5 1.08±0.21b _0.16_±_0.09c _2.92_±_0.08a _1.98_±_0.47b _1.01_±_0.17c

C,controlgroup;S,rhinosinusitis+salinegroup;SC,rhinosinusitis+cefazolingroup;SA,rhinosinusitis+amlodipinegroup;LPE,lamina propriaedema;LC,lossofcilia;V,vasodilatation;GCN,gobletcellnumber;CD,collagendeposition.

Thedifferentsuperscriptletters(a,b,candd)showthatthevaluesinthesamecolonare statisticallydifferentfromeachother (p<0.05).Valuesgiveninthetablearemean±standarddeviation.UsedtheKruskal---Wallisrangetestoptionandwereconsideredto besignificantatp<0.05(allgroups).

Table2 Thestatisticalresultsofthenumericaldensities ofPolymorphonuclearLeukocyte(PMNL) infiltrationinthe treatmentandcontrolgroups.

Groups PMNL(mean±SD)

C 1.405±0.034d

S 4.105±0.062a

SC 1.796±0.008c

SA 2.514±0.064b

Thedifferentsuperscriptletters(a,b,candd)showthatthe valuesarestatisticallydifferentfromeachother(p<0.05). Val-uesgiveninthetablearemean±standarddeviation.Usedthe Kruskal---Wallisrangetestoptionandwereconsideredtobe sig-nificantatp<0.05(allgroups).

C, control group; S, rhinosinusitis group; SC, rhinosinusi-tis+cefazolingroup;SA,rhinosinusitis+amlodipinegroup.

lamina propria weredistinguished. Differently fromother groups macrophage infiltrations were seen and collagen depositionwasincreasedinthisgroup(Fig.1;Table1).

Group SC(Rhinosinusitis+cefazolinsodium)---nociliary loss wasdetected. The number of goblet cells and colla-gendepositionwerelessthanGroupS.Dilationsofvessels weresimilartoGroupC.Themostlaminapropriaedemawas seeninthisgroup.Degenerativeepitheliumcellswereless thanGroupS. Differentlyfromtheothergroups increased connectivetissuecellswereprominent(Fig.1;Table1).

Group SA (Rhinosinusitis+amlodipine)--- this groupwas similar to Group C than the other experimental groups. Nociliary loss wasdetected. The numbers of gobletcells werelessthanGroupS,collagendepositionandlamina pro-pria edema were similar to Group C. Vasodilatations was increased than theother experimental groups. Degenera-tiveepitheliumcellswerelessthantheotherexperimental groups(Fig.1;Table1).

Stereologicalresults

Statisticalanalysisrevealedthatthereweresignificant dif-ferences between all groups (p<0.05). When the mean numerical densities of PMNLs for all groups compared,it wasseenGroupShadthehighestvalue.TheGroupSAvalue followedGroupS,while theGroup SChadsimilarvalueto GroupC(Table2).

60

50

40

30

20

10

0 C d

a

b

c

S SC

Groups

SOD activity U/ml

SA

Figure2 Superoxidedismutase(SOD)activitiesofthestudy groups.All groups showed a statistical difference fromeach other. Group S presented the lowest SOD activity. C, con-trol group; S, sinusitis group; SC, sinusitis+cefazolin group; SA,sinusitis+amlodipinegroup.Used theanalysisofvariance (ANOVA)andLSDtestsandwereconsideredtobesignificantat

p<0.05(allgroups).Thebarsinthedifferentserialshownby thedifferentletters(a,b,c,andd)arestatisticallydifferent fromeachother.

Biochemicalresults

Theactivity of SODandthe levelof GSH, aswell aslipid peroxidation(MDA) levels,were evaluatedin allanimals. The resultsareshown in Figs. 2---4,respectively. The SOD (p<0.004)activityandGSH(p<0.003)levelswerereduced, whereastheMDA (p<0.003)levelswere increased signifi-cantlyinallthree-treatmentgroupscomparedtothecontrol group.Withinthetreatmentgroups,SOD(p<0.004)andGSH (p<0.003)levelswerehighestintheSAgroup,followedby theSCgroup,andwerelowestintheSgroup.MDA(p<0.003) levelswerelowestintheSAgroup,followedbytheSCgroup, andwerehighestintheSgroup.

Discussion

Themostcommonlyreportedpathogensinacutebacterial rhinosinusitis are streptococcus pneumonia, Haemophilus

influenzae,Moraxellacatarrhalis,Streptococcuspyogenes,

and Staphylococcus aureus. The Infectious Diseases

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9 8 7 6 5 4 3 2 1 0 C d a b c

S SC SA

Groups

GSH le

vel mmol/ml

Figure3 Glutathione (GSH) levels ofthe study groups.All groupsshowedastatisticaldifferencefromeachother.Group SpresentedthelowestGSHlevel.C,controlgroup;S,sinusitis group;SC,sinusitis+cefazolingroup;SA,sinusitis+amlodipine group. Used the Used the analysis of variance (ANOVA) and LSDtestsandwereconsideredtobesignificantatp<0.05(all groups).The barsinthedifferentserial shownby the differ-entletters(a,b,c,andd)arestatisticallydifferentfromeach other. 3.00 2.50 2.00 1.50 1.00 0.50 0.00

C S SC

Groups MD A le vel nmol/ml a d c b SA

Figure4 Malondialdehyde(MDA)levelsofthestudygroups. All groups showed a statistical difference from each other. Group S presented the highest MDA level. C, controlgroup; S, sinusitis group; SC, sinusitis+cefazolin group; SA, sinus-itis+amlodipinegroup.Usedtheanalysisofvariance(ANOVA) andLSDtestsandwereconsideredtobesignificantatp<0.05 (allgroups).Thebarsinthedifferentserialshownbythe differ-entletters(a,b,c,andd)arestatisticallydifferentfromeach other.

antibiotictherapiesisaworldwideclinicalthreat.Itis essen-tial todevelop novel antibacterialsto modulate thedrug stressresponseofbacteriaandhost defence mechanisms. Althoughourstudyisrepresentativeofacuterhinosinusitis, tissueprotectiveandanti-inflammatoryeffectsofAMLmay havearoleinpreventingtheprolongationofthediseaseand progressiontochronicstate.

Medicinal compounds used for the therapy of non-infectiouspathologyandthathaveantimicrobialproperties are called non-antibiotics. These compounds are divided into two classes: those with direct antibacterial activ-ity are called ‘‘antimicrobial non-antibiotics’’, while the second group consists of ‘‘helper compounds’’ and ‘‘macrophage modulators’’.18---20 _The _most _well-known non-antibiotic compounds are phenothiazines,21 chlor-promazine, thioridazine,22,23 _the _{anti-inflammatory} _drug diclofenac,24 _{antihistamines} _such _as _promethazine _and

diphenhydramine,andcardiovasculardrugssuchas amlodip-ine,dobutamine,lacidipine,nifedipine,andoxyfedrine.25---27 Theexactmechanismofnon-antibioticactionhasnotbeen determinedbuttheyhavebeenproposedtomodifycell per-meabilityandaffectpotassiumandcalciumeffluxpumpsin susceptiblebacteria.Drugresistancealsohasbeenreversed by additionof non-antibiotics suchasphenothiazines and chlorpromazine toan antibiotictowhichthe bacteriaare initiallyresistant.19

Amlodipine has been reported to have the most powerful antibacterialactivity amongcardiovascular non-antibiotics.11 _In _vitro _studies _also_suggested _that dihydro-pyridinecalciumantagonistsactasantioxidantsandreduce leukocyte-induced oxidationof low density lipoproteins.28 AMLalsohasbeensuggestedtoimproveendothelial dysfunc-tionindiabetesthroughantioxidantandanti-inflammatory mechanisms.29_It_has_been_shown_to_act_{synergistically}_with antibiotics andto have in vivoand in vitro antimicrobial activityagainstseveralbacteria.25

Inourstudy,histopathologicalexaminationshowedthat AML improvedlamina propriaedema, collagendeposition, decreasedciliarylosscomparedtosalinetreatedand cefa-zolin treated groups. Amlodipine group showed reduced PMNLinfiltrationandgobletcellnumbercomparedtosaline treated group. However in cefazolin treated group, gob-let cell number and PMNL infiltration was significantly lowerthanothertreatmentgroups.Amlodipinealsocaused increased vasodilatationcomparedtoall groups that may be attributed to the direct vasodilatatory effect of the compound. SOD and GSH are major components of the antioxidant mechanisms in tissue for combating ROS. In thepresentstudy,SODandGSHlevelswerehighestinthe healthycontrolgroupfollowedbytheSA,SC,andSgroups. LevelsofMDA,whichindicatetissuedamagebylipid perox-idation,werelowest inthecontrolgroupandfollowed by theSA, SC,andS groups,in increasingorder.In ourstudy AML better improved the oxidative status than cefazolin sodium. These findingssuggest thatAML improved antiox-idantdefencemechanismsandreducedMDAlevels,thereby preventingthetissuedamagecausedbyROS.

Although host defence mechanisms are basic ways to clearpathogenicmicroorganisms,theseverityofinfectious disease needs to be furthercontrolled by additional pro-tectivemechanismsthatlimittheextentoftissuedamage. Thisisreferredtoasdiseasetolerance,whichisabiologic phenomenonmainlybasedonprotectionofself-tissuesfrom immuneattack.Tissuedamagecontrolisaveryimportant componentofhostdefencemechanismsagainstinfectionas thisenforcesthebarrierfunctionofepithelialcellsto pre-ventpathogenaccesstohosttissuesandlimitsthedisease severitywithoutinterferingwithpathogenload.30

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severecasescomparedtocontrolsubjects,withagreater decreaseinseverecases.AstudyconductedbyWesterveld etal.reportedasignificantreductioninreducedglutathione levelsinmucosalsamplestakenfrompatientswithchronic sinusitis compared to healthy controls, but no informa-tionwasprovidedregardingtheseverityofdiseaseintheir study.32_Improvement_in_oxidative_status_of_sinonasal_mucosa anddecreaseininflammationandtissuedamagemay poten-tially help to treat acute rhinosinusitis and may prevent furtherprogressionofthediseasetochronicstate.

The exact mechanism of antimicrobial action of AML remains to be fully established. However, a reduction in theminimuminhibitoryconcentrationsofantibioticsbyits usecouldmakeitabeneficialauxiliarycompoundfor treat-ment of severe or recalcitrantbacterial infections of the upperrespiratorytract.AMLcanimproveantioxidantstatus andtissueintegrityand decreaseinflammation associated withbacterialrhinosinusitis.Thisdrugmaythereforehave apotential rolein thetreatment of rhinosinusitisby con-trollingtissuedamageandlimitingtheseverityofdisease.

The results of the present study indicated that AML improved SOD and GSH activities while decreasing MDA level,therebyenhancingtheoxidativestatusanddecreasing lipidperoxidationinthesinonasalmucosa.

Conclusion

This study evaluated the effect of AML on inflammation andoxidativestatusofsinonasalmucosaasasingleagent. ThecombinationofAMLandantibioticmayproduceamore dramaticdecreaseinoxidativestressandinflammationby acting synergistically withthe antibiotic.We believe that future studies with different microorganisms and studies thatinvestigatetheeffectofAMLco-administeredwithan antibiotic both in acuteand chronic rhinosinusitis models wouldbebeneficial.

Ethical

approval

Theanimalexperimentsandprocedureswereperformedin accordancewithnationalguidelinesfortheuseandcareof laboratoryanimals,andthestudywasapprovedbyAtaturk University’slocalanimalcarecommittee(approvalnumber: 2014-1/12).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

WethankProf.HasanTurkez,fromErzurumTechnical Uni-versityforstatisticalanalyses.

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