Inhibition of inducible nitric oxide synthase by Acanthopana

JournalofEthnopharmacology118(2008)231–236

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Ethnopharmacology

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InhibitionofinduciblenitricoxidesynthasebyAcanthopanaxsenticosusextractinRAW264.7macrophages

Qiu-YeLina,b,Li-JiJina,Zhen-HuiCaob,Yong-PingXua,

ab

DepartmentofBioscienceandBiotechnology,DalianUniversityofTechnology,2LinggongRoad,GanjingziDistrict,Dalian,Liaoning116024,People’sRepublicofChinaFacultyofAnimalScienceandTechnology,YunnanAgriculturalUniversity,Kunming650201,People’sRepublicofChina

articleinfoabstract

Aimofthestudy:TheherbAcanthopanaxsenticosus(Siberianginseng)haslongbeenusedasatradi-tionalmedicine.However,littleisknownaboutanti-in ammatoryeffectsanditsmechanismsofaction.Excessproductionofnitricoxide(NO)isoneofthecharacteristicsofin ammation.InthisstudyweexaminedtheeffectsofA.senticosusextract(ASE)onNOproductionandinduciblenitricoxidesynthase(iNOS)geneexpressioninlipopolysaccharide(LPS)plusinterferon-gamma(IFN- )-stimulatedRAW264.7macrophagesandinvestigateditsmechanismsofanti-in ammatoryactivity.

Materialsandmethods:RAW264.7macrophagesweretreatedwith10 g/mlLPSplus20U/mlIFN- inthepresenceorabsenceofASE.NOproductionandiNOSgeneexpressionwereinvestigated.WefurtherevaluatedtheeffectofASEonoxidativestress-sensitivetranscriptionnuclearfactor-kappaB(NF- B)activation.

Results:ASEsigni cantlysuppressedNOproductionandiNOSgeneexpressioninadose-dependentman-ner.ASEalsoreducedDNA-bindingactivityofNF- BinLPSplusIFN- stimulatedRAW264.7macrophages.FurtherstudiesindicatedthatLPSplusIFN- -inducedinhibitoryfactor-kappaBalpha(I- B )degradationandp65nucleartranslocationwereinhibitedinRAW264.7macrophagesexposedtoASE.Moreover,ASEinhibitedtheLPSplusIFN- mediatedincreaseinintracellularperoxidesproduction.

Conclusions:TheseresultssuggestASEsuppressesiNOSgeneexpressionthroughtheinhibitionofintra-cellularperoxidesproduction,whichhasbeenimplicatedintheactivationofNF- B.

©2008ElsevierIrelandLtd.Allrightsreserved.

Articlehistory:

Received19December2007

Receivedinrevisedform1April2008Accepted4April2008

Availableonline11April2008Keywords:

AcanthopanaxsenticosusAraliaceae

Anti-in ammatoryeffectNitricoxide

InduciblenitricoxidesynthaseNuclearfactor-kappaB

1.Introduction

Nitricoxide(NO)isashort-livedbiomoleculethatmediatesmanybiologicalactivitiesincludingvasoregulation,plateletaggre-gationandneurotransmission(Moncadaetal.,1992;Nathan,1992).IthasalsobeenassociatedwithhostdefensemechanismswithNOproductionbyactivatedmacrophagesmediatingimmunefunctionsincludingantimicrobialandantitumoractivities(MacMickingetal.,1997).However,excessNOproductionhasbeenimplicatedinsepticshockandautoimmunediseases(MacMickingetal.,1995;Guziketal.,2003).Forthisreason,inhibitionofNOproductionhasbeenidenti edasapromisingtherapyforthetreatmentforin ammatorydiseases(Sautebin,2000;Tsaoetal.,2005).

NOissynthesizedfroml-argininebynitricoxidesynthase(NOS)withNAPDHandoxygenascosubstrates(GrovesandWang,2000).InducibleNOSisthekeyenzymeinvolvedinNOpro-ductionbymacrophagesstimulatedbybacterialendotoxinof

Correspondingauthor.Tel.:+8641184706345;fax:+8641184706359.E-mailaddress:autumndlut@http://(Y.-P.Xu).0378-8741/$–seefrontmatter©2008ElsevierIrelandLtd.Allrightsreserved.doi:10.1016/j.jep.2008.04.003

lipopolysaccharide(LPS)andproin ammatorycytokinessuchasinterferon-gamma(interferon- )andtumornecrosisfactor-alpha(TNF- )(Moncadaetal.,1992;NathanandXie,1994).Activityoftheinduciblenitricoxidesynthase(iNOS)isregulatedatdiffer-entlevels,fromtranscriptional,posttranscriptional,translational,throughtoposttranslationalsteps(Xieetal.,1994;Vodovotzetal.,1996;Rao,2000).ExpressionoftheiNOSgeneinmacrophagesisregulatedmainlyatthetranscriptionallevel,particularlybynuclearfactor-kappaB(NF- B)(Xieetal.,1994).Inthecytosol,NF- Bisexpressedconstitutivelyashomo-orhetero-dimerslinkedtoinhibitoryI Bproteins(Baldwin,1996).WhencellswerestimulatedbyLPS,IFN- orotherin ammatorycytokines,NF- Bwasactivatedthroughphosphorylation,ubiquitination,andproteasome-mediateddegradationoftheI Bproteins,followedbytranslocationofNF- Btothenucleusandinductionofgenetranscriptionthroughbindingtothecis-actingNF- Belement(Baldwin,1996;Karin,1999).

Acanthopanaxsenticosus(Rupr.&Maxim.)Harms.(Syn.Eleuthe-rococcussenticosus(Rupr.andMaxim.)Maxim.),alsotermedSiberianginseng,belongstothefamilyAraliaceaeandisa1.5–2.6mhighshrubfoundinChina,Korea,JapanandRussia.InChina,

232Q.-Y.Linetal./JournalofEthnopharmacology118(2008)231–236

JapanandRussia(BrekhmanandDardymov,1969;Farnsworthetal.,1985),theextractoftherootbarkofAcanthopanaxsenticosushasbeenusedforthecontrolofbloodpressure,formentalandemotionalproblems,asanalepticoragentstocopewithstress.Inaddition,therootsandstemsofAcanthopanaxsenticosushavebeenreportedtohavepharmacologicalactioninthetreatmentofvariousdiseases,suchaschronicrenalfailure,rheumatics,diabetesmelli-tus,chronicbronchitis,hypertension,gastriculcersandischemicheartdiseases(Nishibeetal.,1990;Fujikawaetal.,1996;DavydovandKrikorian,2000).InKorea,thestemshavealsobeenusedclin-icallyforthetreatmentofallergy(Yietal.,2002).Acanthopanaxsenticosusistheof ciallylistedintheChinesePharmacopoeiaandisamongthe10mostpopularsupplementsusedintheUnitedStates(Li,2001).However,littleisknownaboutanti-in ammatoryeffectsanditsmechanismsofaction.

OurpreviousstudieshaveshownthatAcanthopanaxsenticosusextract(ASE)inhibitsNOproductionbymurinemacrophagesinvitroandinvivo(Linetal.,2007).Inthisstudy,wereporttheeffectsofASEoniNOSexpressioninRAW264.7macrophagesanddescribepossiblemechanismsofanti-in ammatoryactivity.2.Materialsandmethods2.1.Chemicalsandreagents

Dulbecco’smodi edEagle’smedium(DMEM)cellcul-turemediumwasobtainedfromInvitrogenCo.(Carlsbad,CA).LPS(Escherichiacoliserotype055:B5),2 ,7 -dichloro uoresceindiacetate(DCFH-DA),mousemonoclonalantibodyagainst -act …… 此处隐藏：25037字，全部文档内容请下载后查看。喜欢就下载吧 ……