Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downreg.docx

Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downreg.docx

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PropofolexertsitsantiinflammatoryeffectinAlveolartypeIIepithelialcellsthroughdownreg

Propofolexertsitsanti-inflammatoryeffectinAlveolartype

epithelialcellsthroughdownregulationofCD14andTLR4expression

LingMa,WeiminChen,XiuyingWu,LingxinMeng,YujiFujino

Authors:

LingMa,M.D.1,XiuyingWu,M.D,Ph.D2，WeiminChen,M.D,Ph.D3,YujiFujino,M.D,Ph.D4

1.Lecturer,DepartmentofAnesthesiology,ShengjingHospitalofChinaMedicalUniversity,Shenyang,China

2.AssociateProfessor,DepartmentofAnesthesiology,ShengjingHospitalofChinaMedicalUniversity,Shenyang,China

3.Professor,DepartmentofAnesthesiology,ShengjingHospitalofChinaMedicalUniversity,Shenyang,China

4.AssociateProfessor,DepartmentofAnesthesiologyandIntensiveCareMedicine,OsakaUniversityMedicalSchool,Osaka,Japan

Correspondingauthor:

WeiminChen.

Mailingaddress:

DepartmentofAnesthesiology,No.36SanhaoStreet,HepingDistrict,Shenyang,110004,China.

Phone:

+86-13386861177Email:

chenwm@sj-hospital.org

Runningtitle:

propofolandalveolartype

epithelialcell

Summary

Background:

WeinvestigatedwhetherLPSinducedinflammationinATIIisthroughCD14andTLR4andtheeffectofdifferentdosageofpropofolontheinflammationinprimaryculturedratalveolarepithelialtype

（AT

）cells.

Methods:

TheculturedAT

cellswererandomlyassignedtooneofthefollowingfivegroups:

GroupC:

AT

cellsinuntreatedgroup（control）wasculturedintheabsenceofpropofolandLPS;GroupLPS:

treatedwith1μg.-1mlLPS;GroupP1:

treatedwith1μ.ml-1LPSand25μMpropofol;GroupP2:

treatedwith1μg.ml-1LPSand50μMpropofol;GroupP3:

treatedwith1μg.ml-1LPSand100μMpropofol.AT

cellsinuntreatedandpropofolcontrolgroupswereculturedat37°Cfor3h.CD14andTLR4mRNAweredetectedusingreal-timePCR.WesternblotwereusedtodetectCD14andTLR4proteinexpression.CD14andTLR4expressionontheAT

cellswereimagedusingimmunofluorescence.TNF-αproductionweredeterminedusingELISAkit.

Results:

LPSstimulationresultedinanincreasedCD14andTLR4expressionandincreasedTNF-αproductioninAT

cells.Propofol,atconcentrations

50µM,significantly（P<0.05）anddose-dependentlydecreasedCD14andTLR4mRNAexpressionandproteinexpressioninAT

cells.ThiswasaccompaniedbydecreasesinTNF-αproduction（P<0.05）.

Conclusion:

Theseresultssuggestthatpropofol,atclinicalrelevantconcentrations,canreduceinflammatoryresponseinLPS-inducedAT

cellsinjurythroughdownregulationofCD14andTLR4expression.

Keywords:

propofol;alveolartype

epithelialcell;CD14;Tolllikereceptor-4

Introduction:

Thelungrepresentsasitefortheinvasionofvariousbacteriaorbacterialproducts.Alongwithalveolarmacrophages,pulmonaryepithelialcellsarethefirstcellstobechallengedbypathogenicmicroorganisms.ratalveolarepithelialtype

（AT

）cellssynthesizeandsecretesurfactant,controlthevolumeandcompositionoffluidinthealveolarspace,andproliferateanddifferentiateintoalveolartypeIepithelialcellsafterlunginjurytomaintaintheintegrityofthealveolarlining1,2.Recently,therehavebeenpublicationsreportingtheinvolvementofAT

inmodulatingthedevelopmentofinflammatoryreactionswithinthealveolus3.AT

secretechemokinesinresponsetoinflammatorystimuliinvitro,suggestingapotentialphysiologicroleinacuteinflammatorylunginjury4,5.

Lipopolysaccharide（LPS）triggersaphysicalassociationbetweenclusterofdifferentiation14（CD14）andTolllikereceptor4（TLR4）6,7.InflammatoryresponsetoendotoxinislargelymediatedthroughCD14andTLR48,9.TheactivationofCD14andTLR4leadstoproinflammatorycascadeincludingtheexpressionofproinflammatorymediators,suchasTNF-α.CD14andTLR4havebeenfoundonAT

10,11andcouldthusplayanimportantroleintheinnateimmuneresponseatthealveolarsurfacearea.

Littleisknownabouttheinteractionofgenerallyusedintravenousanestheticpropofol（2,6-diisopropylphenol）withATII.OurgoalistoinvestigatewhetherLPSinducedinflammationinATIIisthroughCD14andTLR4andtheeffectofdifferentdosageofpropofolontheinflammation.

MethodsandMaterials

Isolationofalveolarepithelialcellandprimaryculture

ATIIwereisolatedfrommaleSPFdegreeWistarrats（180-250g）,followingtheprotocolaccordingtothemethodofRichardetal12.Briefly,afterintraperitonealsodiumpentobarbital（60mg.kg-1）andheparin（400U.kg-1）,thethoraciccavitywasopenedandtheinferiorvenacavawascut.Thelungswereperfusedviatherightatriumwithsterilesalineandinflatedsimultaneouslywithair.Afterremovalfromthethoraciccavity,theywerewashed6timeswithnormalsalineanddigestedenzymaticallywithtypsin.Thelungswerethenmincedinfetalbovineserum（FBS）andDNase

（Roche）,andtheresultingsuspensionwasfilteredthrough150μmand30μmmeshonce.ThecellmixturewaspurifiedbycentrifugationonadiscontinuousPercollgradient（density1.089and1.04,400g,20min）.Theinterfacewascollectedandafterrewashingthecellswereseededonto6-wellculturedishesatadensityof1×106cells/dishinDulbecco'smodifiedEaglemedium（DMEM）with10%inactivatedFBS,penicillin（100U.ml-1）,streptomycin（100μg.ml-1）at37℃inthehumidifiedatmosphereof95%air/5%CO2andculturedfor18-20hours.

Characterizationoftype

pneumocyte

AlkalinePhosphataseHistochemicalStaining

AlkalinephosphatasestainingservedtoidentifyATIIcellsinprimaryculturedcellsgrownonglasscoverslips.Briefly,thecellswerefixedfor15minatroomtemperaturein4%paraformaldehydesolution,rinsedfor1minindistilledwater,andair-dried.FixedcellswereincubatedwithBCIP/NBTstain（SigmaChemical,StLouis,Mo）for10minatroomtemperature.Thesampleswerethenrinsedwithdistilledwaterfor1minandair-dried.Thecellswerecounterstainedwith0.1%neutralredsolution（Sigma）for1minatroomtemperature,rinsedfor1min,air-dried,andphotographedunderlightmicroscopy.Bluestainingidentifiedcellswithhighalkalinephosphataseactivity,whereasothercells,suchasmacrophages,werecounterstainedinred.

Transmissionelectronmicroscopy

Cellswerefixedin2.5%glutaraldehyde,washedthreetimesin0.1Mphosphatebufferedsaline（PBS）andseriallydehydratedinacetoneandembeddedinEpon812.Ultrathinsections（70nm）wereexaminedwithatransmissionelectronmicroscope（JEM-1200EX,JEOL,Tokyo,Japan）.

Experimentaldesign

TheculturedAT

cellswererandomlyassignedtooneofthefollowingfivegroups:

GroupC:

AT

cellsinuntreatedgroup（control）wasculturedfor3hintheabsenceofpropofol（AstraZeneca,Basiglio,Italy）andLPS（E.ColiO55:

B5,Sigma）;GroupLPS:

treatedwith1μg.-1mlLPSfor3h;GroupP1:

treatedwith1μ.ml-1LPSand25μMpropofolfor3h;GroupP2:

treatedwith1μg.ml-1LPSand50μMpropofolfor3h;GroupP3:

treatedwith1μg.ml-1LPSand100μMpropofolfor3h.

RealTimePCR

TotalRNAwasextractedbyTRIzolreagent（Invitrogen,Carlsbad,CA）.RealtimePCRswereconductedwithSYBRPrimeScriptTMRT-PCRkit（Cat.no.DRR063S,TaKaRaBiotechnology,Dalian,China）accordingtothemanufacturer’sinstructions.Briefly,totalRNA（500ng）wasreverse-transcribedintocDNAusing0.5μlPrimeScriptRTEnzymeMix

0.5μlRandom6mers.QuantitativerealtimePCRwasdoneusinganABIPRISM®7500Real-TimePCRSystem（PEAppliedBiosystems,FosterCity,CA）.Thereactionswerecarriedoutonmultiple8-wellstripes.GAPDHwasamplifiedonthesameplatesandusedtonormalizethedata.Thereactionvolumewas25μlcontaining12.5μloftheSYBRPremixEXTaq,0.5μlofROXreferenceDye

2.5μleachof2μMforwardandreverseprimers,5μlofRNasefreewater,and2μlofthecDNAsamples.Real-timePCRswereperformedintriplicateforeachsampleandatleast3differentsetsofcellpreparationswereused.Thethermalcyclingconditionsusedwere:

95℃for10sfollowedby40cyclesat95℃for5s,60℃for34s.PCRproductswereheatedto95℃for15s,annealedat60℃for1minandthenheatedfrom60℃to95℃for15stoobtainmeltingcurve.DissociationcurveanalysiswasperformedforeachgenetoensurethespecificityofPCRproducts.Serialdilutions（10-fold）ofoneofthesampleswereusedtogeneratethestandardcurveforeachPCR.AllsenseandantisenseprimersweresuppliedbySangon（Shanghai,China）.Theoligonucleotidesprimersusedforreal-timePCRwere:

ratCD14:

forwardATTCCCGACCCTCCAAGT;reverseCCAGCAGTATCCCGCAGT;ratTLR4:

forwardGAGGACTGGGTGAGAAACGA;reverseGAAACTGCCATGTCTGAGCA;ratGAPDH:

forwardATGTTCCAGTATGACTCCACTCACG;reverseGAAGACACCAGTAGACTCCACGACA.

Immunoblotting

Cellswerelysedusingacommerciallyavailablekit（KGP250;NanjingKeygenBiotechCo.Ltd.,Nanjing,China）.ProteinconcentrationwasdeterminedusingtheEasyQuantproteinassaykit（Cat.no.DQ101,TransGenBiotechCo.Ltd.,Beijing,China）andboiledfor5minat100℃.EachvesselhomogenatewasdilutedwithPBStoobtainthesameproteinconcentration.Then20μgofproteinperlanewasseparatedona10%SDS-polyacrylamidegelsandtransferredtoanitrocellulosemembrane（Bio-Rad,Hercules,CA）.Thetransferswereblockedovernightwith5%dryskimmilkpower.Themembraneswereincubatedwithanti-CD14（sc-9150）,anti-TLR4（sc-16240）oranti-β-actin（sc-47778;allSantaCruzBiotechnology,CA）antibodiesat1:

200dilutionsfor2hat37℃.AfterbeingwashedinPBS3times,themembraneswereincubatedwith1:

2000horseradishperoxidase-conjugatedanti-rabbit,-goat,or-mouseIgGs（Pierce,Rockford,IL）for1hatroomtemperature.Theblotswerewashedagain.Theindividualtargetproteinswere