Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro aWord文件下载.docx

Neferinea bisbenzylisoquinline alkaloid attenuates bleomycininduced pulmonary fibrosis in vitro aWord文件下载.docx

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Abstract

Inthisstudy,weevaluatedthepotentialantifibroticpropertyofneferine,abisbenzylisoquinlinealkaloidextractedfromtheseedembryoofNelumbomuciferaGaertn.Intratrachealbleomycinadministrationresultedinpulmonaryfibrosis14and21daysposttreatment,asevidencedbyincreasedhydroxyprolinecontentinbleomycingroup（255.77±

97.17µ

g/lungand269.74±

40.92µ

g/lung）comparedtoshamgroup（170.78±

76.46µ

g/lungand191.24±

60.45µ

g/lung）,andthehydroxyprolinewassignificantlysuppressed（193.07±

39.55µ

g/lungand201.08±

71.74µ

g/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.

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.Sequential4µ

msectionsofthelungswerestainedwithroutinehematoxylin-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