Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downregWord格式文档下载.docx
《Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downregWord格式文档下载.docx》由会员分享,可在线阅读,更多相关《Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downregWord格式文档下载.docx(16页珍藏版)》请在冰点文库上搜索。
![Propofol exerts its antiinflammatory effect in Alveolar type II epithelial cells through downregWord格式文档下载.docx](https://file1.bingdoc.com/fileroot1/2023-5/1/f4ffefa5-04a2-45ab-aca6-c4dd7f7d4a78/f4ffefa5-04a2-45ab-aca6-c4dd7f7d4a781.gif)
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
50µ
M,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
)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
cellsinuntreatedgroup(control)wasculturedfor3hintheabsenceofpropofol(AstraZeneca,Basiglio,Italy)andLPS(E.ColiO55:
B5,Sigma);
treatedwith1μg.-1mlLPSfor3h;
treatedwith1μ.ml-1LPSand25μMpropofolfor3h;
treatedwith1μg.ml-1LPSand50μMpropofolfor3h;
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