Rev. Bras. Anestesiol. vol.67 número6

REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

www.sba.com.br

SCIENTIFIC

ARTICLE

Dexmedetomidine

preconditioning

protects

against

lipopolysaccharides-induced

injury

in

the

human

alveolar

epithelial

cells

Lei

Zhang

,

Xian-Jin

Zhou

,

Li-Ying

Zhan

,

Xiao-Jing

Wu

,

Wen-Lan

Li

,

Bo

Zhao

,

Qing-Tao

Meng

,

Zhong-Yuan

Xia

a_{WuhanUniversity,RenminHospital,DepartmentofAnesthesiology,Wuhan,Hubei,China}

b_{WuhanUniversity,RenminHospital,LaboratoryofAnesthesiologyandCriticalCareMedicine,Wuhan,Hubei,China} c_{TongjiUniversity,FirstMaternityandInfantHospital,DepartmentofAnesthesiology,Shanghai,China}

Received17March2016;accepted27February2017 Availableonline26June2017

KEYWORDS Dexmedetomidine; Lipopolysaccharides; Preconditioning; Acutelunginjury; Alveolarepithelial cell

Abstract

Backgroundandobjectives: Dexmedetomidine (DEX) has demonstrated the preconditioning

effectandshownprotectiveeffectsagainstorganizeinjury.Inthisstudy,usingA549(human

alveolar epithelial cell) celllines, we investigated whether DEX preconditioning protected

againstacutelunginjury(ALI)invitro.

Methods:A549 were randomly divided into four groups (n=5): control group, DEX group,

lipopolysaccharides(LPS) group,andD-LPS (DEX+LPS)group. Phosphatebuffer saline(PBS)

orDEXwereadministered.After2hpreconditioning,themediumwasrefreshedandthecells

were challengedwith LPSfor 24honthe LPSand D-LPSgroup. Thenthe malondialdehyde

(MDA),superoxidedismutase(SOD), Bcl-2,Bax,caspase-3andthecytochromecintheA549

weretested.Theapoptosiswasalsoevaluatedinthecells.

Results:ComparewithLPSgroup,DEXpreconditioningreducedtheapoptosis(26.43%±1.05%

vs.33.58%±1.16%,p<0.05)intheA549,whichiscorrelatedwithdecreasedMDA(12.84±1.05

vs. 19.16±1.89nmoL.mg−1 _{protein, p<0.05) and increased SOD activity (30.28±2.38 vs.}

20.86±2.19U.mg−1 _{protein, p<0.05). DEX preconditioning also increased the Bcl-2 level}

(0.53±0.03vs.0.32±0.04,p<0.05)anddecreasedthelevelofBax(0.49±0.04vs.0.65±0.04,

p<0.05),caspase-3(0.54±0.04vs.0.76±0.04,p<0.05)andcytochromec.

Conclusion:DEX preconditioning hasa protectiveeffect against ALI in vitro. The potential

mechanismsinvolvedaretheinhibitionofcelldeathandimprovementofantioxidation.

©2017SociedadeBrasileiradeAnestesiologia.PublishedbyElsevierEditoraLtda.Thisisan

openaccessarticleundertheCCBY-NC-NDlicense(

http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗_{Correspondingauthor.}

E-mail:674612814@qq.com(Z.Y.Xia). http://dx.doi.org/10.1016/j.bjane.2017.02.002

PALAVRAS-CHAVE Dexmedetomidina; Lipopolissacarídeos;

Pré-condicionamento; Lesãopulmonar aguda;

Célulasepiteliais alveolares

Pré-condicionamentocomdexmedetomidinaprotegecontralesõesinduzidas porlipopolissacarídeosemcélulasepiteliaisalveolareshumanas

Resumo

Justificativaeobjetivos: Dexmedetomidina(DEX)demonstroupossuirefeitopré-condicionante

e também efeitos protetores contra lesão organizada. Neste estudo, usando células A549

(célulasepiteliaisalveolareshumanas),investigamosseopré-condicionamentocomDEX

pro-porcionariaprotec¸ãocontralesãopulmonaraguda(LPA)invitro.

Métodos: CélulasA549foramaleatoriamentedistribuídasemquatrogrupos(n=5):grupo

con-trole, grupo DEX, grupo lipopolissacarídeos (LPS) e grupo D-LPS (DEX+LPS). Administramos

soluc¸ãodePBS(tampãofosfato-alcalino)ouDEX.Após2hdepré-condicionamento,omeiofoi

renovadoeascélulas desafiadascomLPSpor24hnosgruposLPSeD-LPS.Emseguida,

mal-ondialdeído(MDA),superóxidodismutase(SOD),Bcl-2,Bax,caspase-3ecitocromocemA549

foramtestados.Apoptosetambémfoiavaliadanascélulas.

Resultados: Emcomparac¸ãocomogrupoLPS,opré-condicionamentocomDEXreduziua

apop-tose(26,43%±1,05%vs.33,58%±1,16%,p<0,05)emcélulasA549,oqueestácorrelacionado

comadiminuic¸ãodeMDA(12,84±1,05vs.19,16±1,89nmoL.mg−1_{deproteína,p<0,05)e}

aumentodaatividadedeSOD(30,28±2,38vs.20,86±2,19U.mg−1_{deproteína,p<0,05).O}

pré-condicionamentocomDEXtambémaumentouoníveldeBcl-2(0,53±0,03vs.0,32±0,04,

p<0,05)ediminuiuoníveldeBax(0,49±0,04vs.0,65±0,04,p<0,05),caspase-3(0,54±0,04 vs.0,76±0,04,p<0,05)ecitocromoc.

Conclusão:Opré-condicionamentocomDEXtemefeitoprotetorcontraLPAinvitro.Os

poten-ciaismecanismosenvolvidossãoinibic¸ãodamortecelularemelhoradaantioxidac¸ão.

©2017SociedadeBrasileiradeAnestesiologia.PublicadoporElsevierEditoraLtda.Este ´eum

artigo OpenAccess sobumalicenc¸aCCBY-NC-ND(

http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Acute Lung Injury (ALI) and its most severe form, Acute RespiratoryDistress Syndrome(ARDS),is arelatively com-mon syndrome in critically ill patients associated with high morbidity and mortality. Whichare characterizedby high-proteinpulmonaryedemaandseverehypoxemic respi-ratory failure and may result from many clinical insults, includingsepsisandpneumonia.1---3_{Despiteintenseresearch}

andan improved understanding of the pathophysiologyof ALI/ARDS,therearenospecificpharmacologicaltreatments ofprovenbenefitforthem.Sothemorbidityandmortality remainssignificantat35%---40%.3---5

Preconditioninghasbeen reportedtoofferaneffective protection against ALI by a previous stimulus.6,7 _{To date,}

it has been extensively studied for its potential to offer a unique opportunity to exert protective effects in clini-cal practice. Today, we know that the protection can be elicitedmoresafely bymanydrugs suchas dexmedetomi-dine(DEX).8,9

DEXhasbeencommonlyusedasasedativeinclinical sett-ings.Recently,investigatorsfoundthatDEXwascapableof mimickingthepreconditioningeffectandshownprotective effects against organize injury caused by lipopolysaccha-rides(LPS).10,11_{ThatevidenceindicatesthatDEXmayhave}

protectiveeffectagainstALI.Here,weusedtheLPS-induced A549 injury model to simulate ALI in vitro, investigat-ingwhetherDEXpreconditioningwouldproduceprotection againstALI. Wehave alsoinvestigatedthe effects of DEX preconditioningoncelldeathandantioxidationfunctionto gainabetterinsightintothemechanism(s).

Materials

and

methods

Cellcultureandtreatments

Human alveolar epithelial cell line A549 was obtained fromtheChinaCenterforTypeCultureCollection(CCTCC, Wuhan University, Wuhan, China). A549 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2mmol.L−1 _{glutamine, 100U.mL}−1 _{penicillin and}

100mg.mL−1_{streptomycin,andmaintainedinahumid}

envi-ronmentat37◦_Cand5%CO2.

TheA549atthelogphaseofgrowthwereseededintoa96 wellplate,2×103_{well.Afterovernightculture,thesecells}

wererandomlyallocatedtofourdifferenttreatmentgroups: theDEX(JiangsuHengruiMedicineCo.;Ltd.,Jiangsu,China) and D-LPS group received DEX (10␮g.mL−1_{); the control}

andLPS(Sigma---Aldrich,SanLuis,MO,USA)groupreceived the same volume of phosphate buffer saline (PBS). After 2hexposuretothepharmacologicalagenteachwellplate washedandthecellswerechallengedwithLPS(50␮g.mL−1₎

for24h ontheLPSand D-LPSgroup. Thenthecells were collectedforfurtheranalysis.

Measurementofmalondialdehyde(MDA)formation

andsuperoxidedismutase(SOD)activity

BCAproteinassaykit(BestBioCo.;Shanghai,China) accord-ingtothemanufacturer’sprotocol.

Westernblotting

Cellswerehomogenized and theprotein concentrationof itssupernatant wasdetermined by the BCA method. The supernatantcontaining 50␮gof protein wasseparatedby SDS---PAGE and transferred to a nitrocellulose membrane. Primary antibodies included caspase-3, Bcl-2, Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and ␤-actin (Abcam, Cambridge, MA, USA). The membrane was then incubated with an HRP-conjugated anti-rabbit secondary antibody(1:20,000;Pierce)for1handtheblotwas devel-opedwitha Supersignal chemiluminescence detectionkit (Pierce).The immunoblotting wasvisualized withaKodak X-ray Processor 102 (EastmanKodak, Rochester, NY, USA) and analyzed with Quantity One-4.2.3 software(Bio-Rad, Hercules,CA).

Cytochromecreleaseassay

Aspreviouslydescribed,12_{theA549cellswerefixedwith4%}

paraformaldehydefor15min,permeabilizedwith0.1% Tri-tonX-100for10min,andblockedusing3%serumdissolved inPBSfor30minatroomtemperature.Thecellswerethen probedwithanti-cytochromecantibody(1:100;SantaCruz Biotechnology)overnightat4◦_{C.Thecellswerewashedwith}

PBStwice and incubated withFITC conjugated secondary antibody(1:200;Biovision,China)for2hinthedarkat37◦_C.

Afterwashing,imagesofstainedcellswereobtainedusing afluorescencemicroscope.

Flowcytometricdetectionofapoptosis

Usingan AnnexinV-PE/7-AADApoptosisDetectionKit (Bio-Vision,MA,USA),theapoptoticcellswasevaluatedbyflow cytometryanalysis(FACScan,BDBiosciences,SanJose,CA, USA).13_{ThedatawereanalyzedusingtheCellQuestsoftware}

(BDBiosciences).

Statisticalanalysis

Thedataarereportedasthemean±SEMoffiveindependent experiments.ANOVAandStudent---Newman---Keuls(SNK)test wereperformedtodeterminestatisticalsignificance. Signif-icantdifferenceswereestablishedatp<0.05.

Results

EffectsofDEXonoxidativestressintheA549

Compared with the control group, LPS decreased SOD activity in the A549 (46.34±2.24U.mg−1 _{protein vs.}

20.86±2.19U.mg−1 _{protein, p<0.05) (Fig. 1A) and}

increased the formation of MDA (7.68±0.92nmoL.mg−1

protein vs. 19.16±1.89nmoL.mg−1 _{protein, p<0.05)}

(Fig. 1B). Such an effect was significantly attenuated

A

60

50

40

30

20

25

20

15

10

5

0 10

0

Control DEX

SOD (U.mg

-1

protein)

MDA (mmol.mg

-1 protein)

LPS D-LPS

Control DEX LPS D-LPS

∗ #

∗ #

∗

∗

B

Figure1 (A)SODactivityintheA549.(B)MDAlevelsinthe A549. Control, without injury; DEX, dexmedetomidine; LPS, lipopolysaccharide;D-LPS,DEX+LPS.Valueswerepresentedas mean±SEM,n=5foreachgroup.*p<0.05vs.Controlgroupand

#_{p<0.05vs.LPSgroup.}

by DEX preconditioning (30.28±2.38U.mg−1 _{protein and}

12.84±1.05nmoL.mg−1_{protein,p<0.05vs.LPSgroup).}

EffectsofDEXontheexpressionofBcl-2,Baxand

caspase-3

Compared with the control group, LPS decreased Bcl-2 level (0.79±0.03 vs. 0.32±0.04, p<0.05) and increased theexpressionofBax(0.29±0.03vs.0.65±0.04,p<0.05) in the A549 (Fig. 2A and B). Such an effect was signifi-cantlyattenuatedbyDEXpreconditioning(0.53±0.03and 0.49±0.04, p<0.05 vs. LPS group). Compared with the control group, LPS increased the expression of caspase-3 (0.36±0.03 vs.0.76±0.04, p<0.05) inthe A549 (Fig.2C andD).SuchaneffectwassignificantlyattenuatedbyDEX preconditioning(0.54±0.04,p<0.05vs.LPSgroup).

EffectsofDEXoncytochromecrelease

A

Bcl _Caspase-3

Control

Dex LPS

D-LPS Control

Dex LPS

D-LPS

Bax

Bcl-2 ∗

∗ ∗

∗# ∗#

∗#

Bax

Relativ

e le

vel

Caspase-3

1

0.8 0.6

0.4

0.2 0

1

0.8 0.6

0.4

0.2 0

Control DEX Control DEX

LPS LPS

D-LPS D-LPS

β-action β-action

B

D

C

Figure2 (A)RepresentativepictureofBcl-2andBax.(B)ThelevelofBcl-2andBax.(C)Representativepictureofcaspase-3. (D)Thelevelofcaspase-3.Control,withoutinjury;DEX,dexmedetomidine;LPS,lipopolysaccharide;D-LPS,DEX+LPS.Valueswere presentedasmean±SEM,n=5foreachgroup.*p<0.05vs.Controlgroupand#_{p<0.05vs.LPSgroup.}

EffectsofDEXonapoptosis

Compared withthe controlgroup, LPSincreased the per-centage of apoptotic cells in the A549 (1.57%±0.52% vs. 33.58%±1.16%, p<0.05) (Fig. 4A and B). Such an effectwassignificantlyattenuatedbyDEXpreconditioning (26.43%±1.05%,p<0.05vs.LPSgroup).

Discussion

This study demonstrates that DEX preconditioning signifi-cantlydecreasedLPS-inducedinjuryintheA549.Themain findingsareasfollows:(1)DEXpreconditioningreducedMDA and increased SOD activity in the A549; (2) DEX precon-ditioningincreasedthe Bcl-2/Baxratioanddecreased the caspase-3andcytochromeclevel;(3)DEXpreconditioning attenuated apoptosisin the A549. This data provides the firstevidencethatDEXpreconditioningattenuatestheA549 injurycausedbyLPS.

Studieshavebeenreportedthatoxidativestressplaysan important roleon LPS-induced injury.14,15 _{Consistent with}

theprevious study,we also found the LPS-induced oxida-tivestressbyincreasedMDAbutdecreasedSODintheA549. MDAisthedegradationproductsoftheoxygen-derivedfree radicalsandlipidperoxidation.Therefore,theincreaseof MDAimpliesimpairmentofthenormalmembranestructure andoxidative tissuedamage.16 _{Incontrast,SODis thought}

tobe an important intracellular antioxidant enzyme with multiplebiologicalfunctions.Withanincreaseinthe antiox-idantenzymeSOD,itcanindicatethecellularcapabilityof scavengingandquenchingfreeradicals.17,18 _Previous

stud-iesalsoreportedthatDEXpreventedperoxidationreactions byincreasedSODbut decreasedMDAintissues.19,20 _Inthe

experiment, our results obviously demonstrated that DEX preconditioning resulted in an increase of the SOD activ-ityandadecreaseof theMDAin theA549.Thesefindings bothsuggestthatDEXpreconditioningcouldprotectagainst oxidativedamagebyLPS-inducedintheA549,thereby mod-ulatingthecellularinjuryanddysfunction.

Control

Cytochrome c

DAPI

DEX LPS D-LPS

A

B

Control

LPS

D-LPS

DEX

100 ₁₀1 100

101

7-AAD

7-AAD

7-AAD

7-AAD

102 103 104

100 101 102 103 104

100 101 102 103 104

102 ₁₀3 ₁₀4

100 ₁₀1 ₁₀2 ₁₀3 ₁₀4 100 ₁₀1

40

∗

∗# 35

30

25

20

15

10

5

0

Control DEX

Apoptotic cells

,

(%)

LPS D-LPS

102 ₁₀3 ₁₀4 100

100 101 102 103 104

101 ₁₀2 ₁₀3 ₁₀4

Figure4 (A)Representativepictureofflowcytometer.(B)Thegradeofcellapoptosis.Control,withoutinjury;DEX, dexmedeto-midine;LPS,lipopolysaccharide;D-LPS,DEX+LPS.Valueswerepresentedasmean±SEM,n=5foreachgroup.*p<0.05vs.Control groupand#_{p<0.05vs.LPSgroup.}

Several evidences have indicated that oxidative stress wasinvolvedinunderlyingpathologicalmechanismsof LPS-inducedapoptosisinA549.21,22_{Assuggestedfromourresults,}

we also found that LPS had significantly reducedratio of Bcl-2/Bax associated with the increase of MDA and the decreaseofSODactivityinA549.BothBcl-2andBaxbelong to Bcl-2 family. However, Bcl-2 may be regarded as an importantcellularcomponentthatnotonlyguardsagainst apoptotic cell death but also influences multiple cellular events.Inrecentstudies,Bcl-2wasfoundtoprotectagainst LPS-induced injury in some organs including A549.23,24 _In

contrast,Baxexhibitsproapoptoticactions.WhenBaxwas

over expressed, it may form channels or pores allowing for the release of factorssuch ascytochrome cfrom the mitochondriatopropagateapoptosis.25 _{ThustheBcl-2/Bax}

proteinratioislikelycriticalforcellsurvivalafterinjury.24

Inthepresentstudy,ourresultsindicatedthatDEXincreased theexpressionofBcl-2anddecreasedtheexpressionofBax, leadingtoanincreaseoftheBcl-2/Baxratio.

Cytochrome c release and caspase-3 activation is piv-otalpointintheapoptoticcascadeandcanberegulatedby theBcl-2/Baxratio.26 _{Previousstudiesshowedthat}

depressed by Bcl-2. The released cytochrome c and acti-vatedcaspase-3cleavesdownstreamcriticalcellulartargets involved in chromatin condensation, DNA fragmentation, and cytoskeletal destruction, thereby expressing the dra-maticmorphologicalchangesofapoptosis.25---27_LPShasbeen

demonstratedtobeoneofthewaysofinducingcytochrome creleaseandcaspase-3activationinA549.28_Inthepresent

study,ourdata alsoshows thatcytochromec releaseand caspase-3activationsignificantlyincreasedafterLPS admin-istrationintheA549,consistentwiththepreviousstudies. Furthermore,we observedthat the cytochrome crelease and caspase-3 activation wasdepressedby DEX precondi-tioning.

Apoptosis is a fundamental process of cell death that occursviaactivation ofdistinctsignaling pathways involv-ing down-regulation of Bcl-2/Bax ratio and release of cytochromecandactivationofcaspase-3invitro.28,29

Ulti-mately,cellsundergodestructionandformationofapoptotic bodies.30 _{LPS have been shown to initiate this apoptotic}

cascade in A549.24 _{Using an in vitro model of ALI, our}

data demonstrate significant reduction of apoptosis after DEXpreconditioning,whichcorrelatedwithup-regulationof Bcl-2/Baxratioanddepressionofcaspase-3activationand cytochromecrelease.

However, the descriptive study has some obvious lim-itations that need to be addressed. Which included one sampling timepoint, thebrief periodof observation, and lackofcorrelationwithclinicalmeasurementsofALI. More-over, we haveonly investigatedsingleDEX concentration, whichisverylargetotheclinicalpractice.Comparedwith clinical,alargedoseisusuallyusedinlaboratory.31,32_Some

studies evenreporteda heavy dose in experiment.These may be related with different species.31,32 _{The relation}

betweendoseandeffectremainsunclear.Therefore,studies relatedtodose---effectrelationshipneedfurtherexploration inthemodel.

Summary

Our data show that DEX preconditioning can effectively attenuate the LPS-induced injury in A549. The protective effectsmayinvolveareductioninoxidativestressandcell deathinducedbyLPS.Theseexperimentalresultssuggested thatDEXmaybeefficaciousinthetreatmentofLPS-induced ALI.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

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