Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro a.docx

Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro a.docx

《Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro a.docx》由会员分享，可在线阅读，更多相关《Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro a.docx（21页珍藏版）》请在冰豆网上搜索。

Neferineabisbenzylisoquinlinealkaloidattenuatesbleomycininducedpulmonaryfibrosisinvitroa

Neferine，abisbenzylisoquinlinealkaloidattenuatesbleomycin-inducedpulmonaryfibrosisinvitroandinvivo

LiboZhao1,2,LinHuang1,JialingWang2,YuzhenLi1

1.PekingUniversityPeople’sHospital,Beijing,China

2.DepartmentofPharmacology,TongjiMedicalCollege,Wuhan,China

Abstract

Inthisstudy,weevaluatedthepotentialantifibroticpropertyofneferine,abisbenzylisoquinlinealkaloidextractedfromtheseedembryoofNelumbomuciferaGaertn.Intratrachealbleomycinadministrationresultedinpulmonaryfibrosis14and21daysposttreatment,asevidencedbyincreasedhydroxyprolinecontentinbleomycingroup（255.77±97.17µg/lungand269.74±40.92µg/lung）comparedtoshamgroup（170.78±76.46µg/lungand191.24±60.45µg/lung）,andthehydroxyprolinewassignificantlysuppressed（193.07±39.55µg/lungand201.08±71.74µg/lung）byneferineadministration（20mg/kg,b.i.d）.Theattenuated-fibrosisconditionwasalsovalidatedbyhistologicalobservations.Biochemicalmeasurementsrevealedthatbleomycincausedasignificantdecreaseinlungsuperoxidaedismutase（SOD）activity,whichwasaccompaniedwithsignificantincreaseinmalondialdehyde（MDA）levelsandmyeloperoxiase（MPO）activityonthe7thand14thday.However,neferinereversedthedecreaseinSODactivityaswellastheincreaseofMDAandMPOactivity.Enzyme-linkedimmunosorbentassayandradio-immunityassayshowedthattreatmentwithneferinealleviatedbleomycininducedincreaseofproinflammatorycytokinessuchastumornecrosisfactor（TNF）-,interleukin（IL）-6andendothelin（ET）1inplasmaorintissue.Additionally,neferineblockedbleomycininducedincreasesofNF-κBinnuclearextractsandTGF-β1intotalproteinextractsofmurineRAW264.7macrophages.Inconclusion,neferineattenuatesbleomycin-inducedpulmonaryfibrosisinvitroandinvivo.Thebeneficialeffectofneferinemightbeassociatedwithitsactivitiesofanti-inflammation,antioxidation,cytokineandNF-κBinhibition.

Keywords:

Bleomycin;Pulmonaryfibrosis;Neferine;Mouse;Macrophage

Introduction

Pulmonaryfibrosisisachronicandprogressivelungdiseasewithanaveragesurvivalof3yearsfromtheonsetofdyspnoea[1,2].Thediseasecanbeidiopathicordevelopedasacomplicationofmanyrespiratoryandsystemicdiseasesandbecharacterizedbyexcessivedepositionofextracellularmatrixproteinswithinthepulmonaryinterstitium,leadingtoimpairedgastransferandrespiratoryfailure.Althoughtheetiologyofpulmonaryfibrosishasnotbeenclearlyclarifiedyet,inflammation,oxidativestressandinjuriesfromcytokinesaredefinitelyinvolvedinpathogenesis[3-9].Inflammationresponseistheinitialresponsefollowinginjurieschallenge.Activatedinflammatorycellssuchasneutrophilsandmacrophagesaccumulateinthelowerairways,releasingharmfulamountsofreactiveoxygenspecies（ROS）andavarietyofharmfulcytokinesandgrowthfactorsthatregulatetheproliferation,chemotactism,andsecretaryactivityofalveolarfibroblastsinalveolarwall.Theactivatedfibroblaststhenproduceincreasingamountsofmatrixproteins,distortingthenormallungarchitecture.Onthebasisoftheseconcepts,targetedinhibitionofinflammation,oxidativestressandcytokinereleaserepresentpossiblestrategicpointsfortherapeuticintervention.

Uptonowtherehasbeennosatisfactorytreatmentforpulmonaryfibrosis.Corticosteroidscontinuetobetheprimarymodeoftreatmentrecommendationsforthisdisorder[10-13].Despitethepotentiallyfavorableeffectsofthesedrugsonseveralinflammatoryprocesses,only15~30%ofpatientswithpulmonaryfibrosiscouldbenefitfromthem.Thusthedevelopmentofeffectiveagentstoamelioratepulmonaryfibrosisisurgentlyneeded[14].Intratrachealadministrationofbleomycinisthemostwidelyusedexperimentalmodeloflungfibrosis,sincethepathologyinrodentsisverysimilartohuman.Recently,manyagents,forexample,pirfenidone[15],PG490-88[16],LLDT-8[17],taurineandniacin[18]wereinvestigatedwiththismodel.

Fig.1

OurlaboratoryhasconsistentlyinvestigatedtheprotectiveeffectsofsometraditionalChineseMedicineonpulmonaryfibrosisanddemonstratedthatisoliensinine,abisbenzylisoquinlinealkaloidisolatedfromtheseedembryoofNelumbomuciferaGaertn.,abatestheaccumulationofcollagenofthelunginbleomycin-orparaquat-inducedpulmonaryfibrosismodels[19].Neferineisanotherbisbenzylisoquinlinealkaloid（Fig.1）extractedfromthesameseedembryo,NelumbomuciferaGaertn,andtheamountofneferineisthreetimesofisoliensinineintheseed.Previousstudieshavedocumentedthatneferinehasextensivepharmacologicaleffectsinthecardiovascularsystem[20-24].Inrecentyears,ithasbeenreportedthatneferineinhibitstheproliferationofvascularsmoothmusclecells（VSMCs）[25]andhypertrophicscarfibroblasts[26].Moreover,whenneferineislocallyinjectedintothenudemicewithscars,itcouldhelpreducethevolumeofthescars,decreasethecontentsofcollagenandacidicmucopolysaccharid[27].Apharmacokineticstudyshowedthattheconcentrationofneferineisveryhighinthelungsofrats[28].Thusthepresentinvestigationwastotestwhetherneferinehasaneffectonbleomycin-inducedfibrosis,whichisassociatedwiththeattenuationofinflammation,oxidativestressandinjuriesfromcytokines.

Fig.1

Methods

Animalsanddrugs

WithapprovalbytheInstitutionalCareInvestigationCommittee,120Kunmingmiceweighing20to25gprovidedbytheDepartmentofExperimentAnimalsofTongjiMedicalCollegeofHuazhongUniversityofScienceandTechnologywerehousedinanimallaboratoryofDepartmentofPharmacology.A12hlight/darkcyclewasmaintainedandthetemperaturewascontrolled23±2°C.Allmicewereacclimatizedtotheirnewsurroundingsfor1weekpriortoexperimentalprocedures.

BleomycinHydrochloridepurchasedfromNipponKayakuCo.,Ltdwasdissolvedinsterile0.9%salineonthedayofintratrachealinstillationatadoseof0.1mgin0.05mLsolutionpermouse.NeferinewasextractedfromtheseedembryoofNelumbomuciferaGaertnbythePhytochemistryLaboratoryoftheDepartmentofPharmacology,TongjiMedicalCollegeofHuazhongUniversityofScienceandTechnology.ThequalityofneferinewastalliedwithmonomerstandardstipulatedbythenationalpharmacopoeiaofChina（FW=625,purity>98%byHPLC）.

Animalmodelofbleomycin-inducedpulmonaryfibrosis

Asingle-dosebleomycin-mousemodelofacutelunginjurythateventuatesintofibrosishasbeenpreviouslyestablishedinourlaboratoryandthesamemodelwasusedinthepresentstudy.Briefly,afterbodyweightswererecorded,micewereanesthetizedviaintraperitonealinjectionof40mg/kgpentobarbitalsodiumsolution.Theskinandsubcutaneoustissueoverlyingtheproximalportionofthetracheawereexposedbya5mmtransversalincisiontoallowinsertionintothetracheaofaneedlecontainingtheinstilledsolution.Themicewereshakentofacilitatedistributionofthebleomycinsolutionorsalinethroughoutthelung.Theincisionwasclosedbyasinglesuture.Thenthe120micewererandomizedinto4groupsassham（treatedwithsaline）,bleomycin,neferine（20mg/kg,b.i.d）,andpirfenidone（100mg/kg,b.i.d）.Saline,neferineorpirfenidonewasadministeredwithintragastricinjectiononthesameday.Here,pirfenidone，agenerallyacceptedantifibrosticaagentwasusedasapositivecontrol.Wechosethesedosesofneferineandpirfenidoneaccordingtotheresultsofpreliminaryexperimentsortherapeuticdoseinclinicaltrial[29-32].Foreachoftheforgoingthreegroups（36miceineachgroup）,animalsweredividedintothreesubgroups,whichweresacrificedonthe7th,14thor21stdayafterintratrachealtreatment,individually.Forthepirfenidonegroup（12mice）,animalsweresacrificedonthe21stdayposttreatment.Plasmaandlungswereisolatedandpreparedforvariousmeasurements.

Pathologicalexaminations

Thelungswerefixedin4%paraformaldehydefor24handthenprocessedforparaffinembedding.Sequential4µmsectionsofthelungswerestainedwithroutinehematoxylin-eosinforgeneralmorphologyandstainedwithMasson’strichromeforevaluationoffibrosisunderaphotomicroscope（OlympusBX51Tokyo,Japan）.EachsuccessivefieldwasindividuallyassessedfortheseverityofinterstitialfibrosisusingthesemiquantitativegradingsystemdescribedbyAshcroftandcoworkers[33].Thegradeofpulmonaryfibrosiswasscoredinablindedfashionbyexamining30randomlychosenregionspersampleatamagnificationof100×.Ascorerangingfrom0（normallung）to8（totalfibrosis）wasassigned.Themajorcriteriaforgradingpulmonaryfibrosisincludedinflammatorycellinfiltration,edema,interstitialthickeningofalveolarorbronchiolarwalls,andcollagendeposition,asfollows.Grade0=normallung;Grade1=minimalfibrousthickeningofalveolarorbronchialwalls;Grades2–3=moderatethickeningofwallswithoutobviousdamagetolungarchitecture;Grades4–5=increasedfibrosiswithdefinitedamagetolungarchitectureandformationoffibrousbandsorsmallfibrousmass;Grades6–7=severedistortionofstructureandlargefibrousareas;‘honeycomblung’wasplacedinthiscategory;Grade8=totalfibrousobliterationofthefield.Themeanscoreofallfieldswastakenasthefibrosisscoreofthatlungsection.

HydroxyprolineAssay

Toestimatethetotalamountofcollagenasanindicatorofpulmonaryfibrosis,thehydroxyprolinecontentofthelungswasmeasuredbyaspectrophotometricassayaccordingtotheproceduredescribedbyWoessner[34].Briefly,lungswerehomogenizedinsalinew