Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

Tillu, Melemedjian, Asiedu, Qu Ning, De, Dussor Gregory, Price

Description

Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors. Results To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE 2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment. Conclusions These results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 2
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Tillu
etal
.
MolecularPain
2012,
8
:5
http://www.molecularpain.com/content/8/1/5

RESEARCH

MOLECULAR PAINOpenAccess

ResveratrolengagesAMPKtoattenuateERKand
mTORsignalinginsensoryneuronsandinhibits
incision-inducedacuteandchronicpain
DiptiVTillu
1
†
,OhannesKMelemedjian
1
†
,MarinaNAsiedu
1
,NingQu
1
,MilenaDeFelice
1
,GregoryDussor
1,2
and
TheodoreJPrice
1,2,3*

Abstract
Background:
Despiteadvancesinourunderstandingofbasicmechanismsdrivingpost-surgicalpain,treating
incision-inducedpainremainsamajorclinicalchallenge.Moreover,surgeryhasbeenimplicatedasamajorcause
ofchronicpainconditions.Hence,moreefficacioustreatmentsareneededtoinhibitincision-inducedpainand
preventthetransitiontochronicpainfollowingsurgery.WereasonedthatactivatorsofAMP-activatedprotein
kinase(AMPK)mayrepresentanoveltreatmentavenueforthelocaltreatmentofincision-inducedpainbecause
AMPKactivatorsinhibitERKandmTORsignaling,twoimportantpathwaysinvolvedinthesensitizationof
peripheralnociceptors.
Results:
TotestthishypothesisweusedapotentandefficaciousactivatorofAMPK,resveratrol.Ourresults
demonstratethatresveratrolprofoundlyinhibitsERKandmTORsignalinginsensoryneuronsinatime-and
concentration-dependentfashionandthattheseeffectsaremediatedbyAMPKactivationandindependentof
sirtuinactivity.Interleukin-6(IL-6)isthoughttoplayanimportantroleinincision-inducedpainandresveratrol
potentlyinhibitedIL-6-mediatedsignalingtoERKinsensoryneuronsandblockedIL-6-mediatedallodyniainvivo
throughalocalmechanismofaction.Usingamodelofincision-inducedallodyniainmice,wefurtherdemonstrate
thatlocalinjectionofresveratrolaroundthesurgicalwoundstronglyattenuatesincision-inducedallodynia.
IntraplantarIL-6injectionandplantarincisioninducespersistentnociceptivesensitizationtoPGE
2
injectionintothe
affectedpawaftertheresolutionofallodyniatotheinitialstimulus.Wefurthershowthatresveratroltreatmentat
thetimeofIL-6injectionorplantarincisioncompletelyblocksthedevelopmentofpersistentnociceptive
sensitizationconsistentwiththeblockadeofatransitiontoachronicpainstatebyresveratroltreatment.
Conclusions:
Theseresultshighlighttheimportanceofsignalingtotranslationcontrolinperipheralsensitizationof
nociceptorsandprovidefurtherevidenceforactivationofAMPKasanoveltreatmentavenueforacuteand
chronicpainstates.

Background
surgery[2].Despiteimprovementsinpost-surgicalpain
Incisionassociatedwithsurgerycausesacutepainandtreatmentstrategies,theincidenceofmoderatetosevere
surgeryhasbeenidentifiedasapotentialmajorcauseofpainaftersurgeryisstillhighinseveralpatientpopula-
chronicpainconditions[1-3].Between10and50%oftions[4,5].Moreover,theexactmechanismsinvolvedin
patientsdevelopchronicpainfollowingsurgicalproce-thedevelopmentofpersistentpainfollowingsurgery
duressuchasgroinherniarepair,breastandthoracichavenotbeenelucidated.Interleukin6(IL-6),apro-
surgery,legamputation,orcoronaryarterybypassinflammatorycytokine,isasignificantmediatorofnoci-
ceptiveplasticityinpre-clinicalpainmodelsandis
*Correspondence:[e-mail]
implicatedinseveralhumanpainconditions.SerumIL-
†
Contributedequally
6levelsincreasesignificantlyinpatientsimmediately
1DepartmentofPharmacology,UniversityofArizona,1501NCampbellAve,
aftersurgery[6-8]andcirculatingIL-6levelsarepro-
POBOX245050,Tucson,AZ85724,USA
Fulllistofauthorinformationisavailableattheendofthearticle
portionaltotheextentoftissueinjuryduringan
©2012Tilluetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Tillu
etal
.
MolecularPain
2012,
8
:5
http://www.molecularpain.com/content/8/1/5

operation,ratherthanbeingproportionaltothedura-
tionofthesurgicalprocedureitself[9].Furthermore,
IL-6levelshavebeenshowntobeelevatedinskin
aroundincisionsites[10,11]andithasbeenimplicated
inpreclinicalincision-inducedpainmodels[12-14].
Althoughthesereportsaresuggestiveofinvolvementof
IL-6inpost-surgicalpain,theprecisemechanismsby
whichIL-6drivespost-surgicalpainarepoorlyunder-
stood.However,IL-6hasbeenimplicatedasanimpor-
tantplayerinmanypreclinicalpainmodelsandelegant
geneticstudieshavedemonstratedthatIL-6
’
spainpro-
motingqualitiesaremediatedbyIL-6receptors
expressedbynociceptors[15,16].
RecentlywedemonstratedthatIL-6causesinduction
ofnascentproteinsynthesisinprimaryafferentneurons
andtheiraxonswhichcancontributetoincreasednoci-
ceptivesensitivity[17].WehavealsoshownthatAMP-
activatedproteinkinase(AMPK)activatorsreverse
mechanicalallodyniainneuropathicpainmodelsand
thatthesecompoundsnegativelyregulateproteinsynth-
esisinsensoryafferents[18].AMPK,theenergysensor
ofthecell,isaheterotrimericSer/Thrproteinkinase
activatedbyalterationsincellularAMP:ATPratio.
Onceactivated,AMPKinhibitsATPconsumingana-
bolicprocessessuchasproteintranslation[19].AMPK
activationachievestheseeffectslargelythroughinhibi-
tionofmammaliantargetofrapamycin(mTOR)signal-
ing[19]butAMPKactivationhasalsobeenlinkedto
inhibitionofmitogenactivatedproteinkinase(MAPK)
signaling[18,20].Wehypothesizedthatactivationof
AMPKsignalingpathwaymayrepresentanovelphar-
macologicalmechanismforthetreatmentofpost-surgi-
calpain.
Totestthishypothesis,wehaveutilizedresveratrol,a
naturalpolyphenolfoundinredgrapesandwine,which
haspreviouslybeenshowntoincreaseAMPKactivityin
neurons[21].Althoughseveralstudiesoriginally
describedresveratrolasanactivatorofsirtuinenzymes,
whichareNAD-dependentdeacetylases[22-25]these
resultshavebeenchallengedbasedonlackofspecificity
inscreeningassays[26,27].Moreover,severalrecentin
vivostudiesstronglysuggestthatresveratroleffectsare
independentofsirtuins.Ontheotherhand,resveratrol
isahighlypotentandefficaciousactivatorofAMPK
[28-30]anditsmetaboliceffectsaredependenton
a
subunitAMPKexpressionsuggestingthatAMPKisthe
majortargetforresveratrolinvivo[31]
Herein,wedemonstratethatresveratrolactivates
AMPKandsuppressestranslationregulationpathways
insensoryneuronsinadose-dependent,time-dependent
andreversiblemanner.Wealsoshowthatresveratrol
inhibitsbothacuteandpersistentsensitizationinanIL-
6-inducedhyperalgesicprimingmodelaswellasina
modelofpostsurgicalpain.Thesefindingssuggestthat

Page2of12

resveratrolmayhaveutilityinthetreatmentofpost-sur-
gicalpainandfurtherimplicateAMPKasanoveltarget
forthedevelopmentofanalgesics.
Results
Resveratrolsuppressessignalingtotranslationmachinery
insensoryneurons
WhileresveratrolhasbeenshowntostimulateAMPK
andinhibitmTORsignalingincell-linesandsome
neuraltissues,itseffectonsensoryneuronsisunknown.
Hence,wefirstaskedwhetherresveratroltreatment
influencedAMPKactivityorsignalingpathways
involvedinregulatingcap-dependentproteintranslation
inculturedtrigeminalganglion(TG)neuronsfrommice
growninthepresenceofNGF(50ng/ml)for5days.
TGculturesweretreatedwithvehicleorincreasingcon-
centrations(10,30or100
μ
M)ofresveratrol(Figure1)
for1h.ResveratrolactivatedAMPKinadosedepen-
dentmanner(Figure1A)andsuppressedactivityinsig-
nalingpathwaysthatpromotecap-dependent
translation.Specifically,thesechangesincludedsignifi-
cantlydecreasedphosphorylationofextracellularsignal
regulatedkinase(ERK,Figure1B)anditsdownstream
targetinvolvedintranslationcontroleukaryoticinitia-
tionfactor(eIF)4E(Figure1C).Resveratrolalso
decreasedAKT(Figure1D),mTOR(Figure1E)and
tuberinsclerosisprotein2(TSC2,Figure1F)phosphory-
lationindicatingnegativeregulationofthemTORpath-
wayinTGneurons.Consistentwiththisnotion,eIF4E
bindingprotein(4EBP,Figure1G)andribosomalS6
protein(rS6p,Figure1I),whicharedownstreammTOR
targets,demonstrateddecreasedphosphorylationupon
resveratroltreatment.Finally,resveratrolincreased
eIF4Gphosphorylation(Figure1H),aneffectthatcan
occurindependentlyofmTORsignaling[32]andthatis
uncoupledfromeIF4G-mediatedeIF3recruitment
[33,34].Wehaveobservedsimilareffectswithother
AMPKactivators(e.g.A769662)[18].Thus,incultured
TGneurons,resveratrolactivatesAMPKandsuppresses
signalingviatheERKandmTORpathwaystotransla-
tionmachinerysuggestingaconcentration-dependent
inhibitionofcap-dependenttranslationbyresveratrolin
sensoryneurons.
Havingestablishedaconcentration-dependenteffect
ofresveratrolonTGneuronsinculture,wenextasked
whethertheseeffectsweretimedependent.Resveratrol,
atamaximallyeffectivedose(100
μ
M),wasappliedto
TGneuronsfor10,30or100minandactivityinsignal-
ingpathwayswasassessedbyWesternblot(Figure2).
ResveratrolactivatedAMPKmaximallyat10and30
mintreatment(Figure1A).Similarly,resveratrolsup-
pressedactivityintheERK(Figure2Band2C)and
mTORpathways(Figure2D-I)overthetimecourseof
resveratrolexposure.

Tillu
etal
.
MolecularPain
2012,
8
:5
http://www.molecularpain.com/content/8/1/5

Page3of12

Figure1
ResveratrolsuppressesERKandmTORsignalinginsensoryneuronsinaconcentration-dependentmanner
.TreatmentofTG
neuronswithresveratrol(10,30,and100
μ
M)for1hinducesaconcentration-dependentincreaseinphosphorylat