20-induced
Hui
a,
a
University,Nanchang,Jiangxi330006,People''sRepublicofChina
Nanchang
articleinfo
detectableperipheralneuropathy.Distalsymmetricalpoly-
neuropathy(DSP)isamajorneurologicaldisorderinHIV/AIDS
patients(SchützandRobinson-Papp,2013;Maratouetal.,2009).
SymptomsofDSPincludeneuropathicpain,suchasallodynia,
14;Vermaetal.,
rootganglion
andperipheral
theperipheryto
2009).Thermal
areenhancedby
3;Maratouetal.,
al.,2012;Milligan
etal.,2000;Wallaceetal.,2007).Atpresent,clinicalin-
terventionsprovideonlysymptomaticreliefratherthanacure.
BecausethepathogenicmechanismofHIV-associatedchronicpain
isnotyetclear,theunderstandingofhowHIV-1infectionleadsto
chronicpainisveryimportantforthedevelopmentofeffective
therapies.
PurinergicP2receptorsareactivatedbyextracellularpurine
(ATP,ADP)and/orpyrimidine(UTP,UDP)nucleotides(Burnstock,
2013,2014;Idzkoetal.,2014;MagniandCeruti,2013).P2
Correspondingauthor.DepartmentofPhysiology,MedicalSchoolofNanchang
University,Nanchang,Jiangxi330006,People''sRepublicofChina.
E-mailaddress:liangsd@hotmail.com(S.Liang).
1
ContentslistsavailableatScienceDirect
Neurochemistry
NeurochemistryInternationalxxx(2017)1e8
Theseauthorscontributedequallytothiswork.
1.Introduction
Chronicneuropathicpainisacommonsymptominpatients
withhumanimmunode?ciencyvirus(HIV)-1infection.Glycopro-
tein120(gp120)isanHIV-1proteinthatcancausepainbehaviors
inanimalmodels(Hao,2013;Nasirinezhadetal.,2015;Yuanetal.,
2014;Zhengetal.,2011).Concomitantwithchronicpainmanifes-
tation,approximately30%ofHIV-1/AIDSpatientsdevelopclinically
hyperalgesia,anddysesthesia(Freemanetal.,20
2005;SchützandRobinson-Papp,2013).Dorsal
(DRG)afferent?bersaredistributedtobothcentral
terminalsaswellastransmitnoxiousstimulifrom
thecentralnervoussystem(Basbaumetal.,
hyperalgesiaandmechanicalallodyniainrats
peripheraladministrationofgp120(Hao,201
2009;HerzbergandSagen,2001;Kamermanet
?2017ElsevierLtd.Allrightsreserved.
Articlehistory:
Received2June2017
Receivedinrevisedform
2August2017
Accepted11August2017
Availableonlinexxx
Keywords:
HIVgp120-associatedneuropathicpain
P2Y
12
receptor
Dorsalrootganglia
Satellitegliacells
http://dx.doi.org/10.1016/j.neuint.2017.08.006
0197-0186/?2017ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleinpressas:Shi,L.,et
International(2017),http://dx.doi.org/10.101
abstract
Humanimmunode?ciencyvirus(HIV)envelopeglycoprotein(glycoprotein120,gp120)caninduce
chronicneuropathicpainbydirectlystimulatingprimarysensoryafferentneurons.Activationofsatellite
glialcells(SGCs)indorsalrootganglia(DRG)playsanimportantroleinthetransmissionofneuropathic
pain.TheP2Y
12
receptorisexpressedinSGCsofDRG.Inthisstudy,weinvestigatedtheroleoftheP2Y
12
receptorinHIVgp120-inducedneuropathicpain.Theresultsshowedthatperipheralnerveexposureto
HIVgp120increasedmechanicalandthermalhyperalgesiaingp120-treatedmodelrats.Thegp120
treatmentincreasedtheexpressionofP2Y
12
mRNAandproteininDRGSGCs.TreatmentwithP2Y
12
short
hairpinRNA(shRNA)inDRGSGCsdecreasedtheupregulatedexpressionofP2Y
12
mRNAandproteinin
DRGSGCsaswellasrelievedmechanicalandthermalhyperalgesiaingp120-treatedrats.Reductionof
P2Y
12
receptordecreasedco-expressionofP2Y
12
andglial?brillaryacidicprotein(GFAP),expressionof
GFAP,interleukin(IL)-1b,tumornecrosisfactor(TNF)-receptor1(TNF-R1),andphosphorylationofAkt
(p-Akt)proteinsinDRGofgp120-treatedrats.UpregulationofGFAPisamarkerofSGCactivation.
Therefore,P2Y
12
shRNAtreatmentdecreasedHIVgp120-inducedmechanicalandthermalhyperalgesia
ingp120-treatedrats.
c
NursingCollege,MedicalSchoolofNanchang
d
DepartmentofCellBiology,MedicalSchoolofUniversity,Nanchang,Jiangxi330006,People''sRepublicofChina
DepartmentofPhysiology,MedicalSchoolofNanchangUniversity,Nanchang,Jiangxi330006,People''sRepublicofChina
b
JiangxiProvincialKeyLaboratoryofAutonomicNervousFunctionandDisease,Nanchang,Jiangxi330006,People''sRepublicofChina
P2Y
12
shRNAtreatmentrelievedHIVgp1
inrats
LiranShi
a,b,1
,BingWu
a,b,1
,ZhihuaYi
a,b,c
,Shanhong
HuilongYuan
a,b
,TianyuJia
a,b
,ShuangmeiLiu
a,b
,
HongXu
a,b
,ChunpingZhang
b,d
,ShangdongLiang
journalhomepage:www.elsevier.com/locate/n
al.,P2Y
12
shRNAtreatmentreliev
6/j.neuint.2017.08.006
neuropathicpain
Zhao
a,b
,LifangZou
a,b
,LinLi
a,b
,
Liu
a,b
,YunGao
a,b
,GuilinLi
a,b
,
b,
International
ci
edHIVgp120-inducedneuropathicpaininrats,Neurochemistry
International
receptorsconsistofmetabotropic(i.e.,G-protein-coupled)P2Yre-
ceptorsandionotropicP2Xreceptors(i.e.,nucleotide-gatedion
channels)(Burnstock,2013,2014;Idzkoetal.,2014;Magniand
Ceruti,2013).ATPisreleasedfrombothneuronsandglialcells
(FieldsandBurnstock,2006;VerderioandMatteoli,2011;Sperl
C19
agh
etal.,1995,1998;Sperlaghetal.,1997).Satelliteglialcells(SGCs)are
themostabundantcelltypeinDRG(CostaandMoreira,2015;
Hanani,2005).Afterperipheralnerveinjury,SGCsundergostruc-
turalchangesandproliferate(Hanani,2005;Jasminetal.,2010).
Neuronegliainteractionsinvolvepurinergicsignaling(Fieldsand
Burnstock,2006;Rajasekharetal.,2015;VerderioandMatteoli,
2011).SGCsofDRGexpresstheP2Y
12
receptor(Katagirietal.,
2012;Kobayashietal.,2008,2013).TheP2Y
12
receptorpartici-
patesinthetransmissionofnociceptivesignals(Burnstock,2013;
HorvC19athetal.,2014;Katagirietal.,2012;MagniandCeruti,
2013).However,itisuncertainwhethertheP2Y
12
receptoris
involvedinthepathogenicmechanismsunderlyingHIVneuro-
pathicpain.Inthisexperiment,ouraimwastoinvestigatetherole
oftheP2Y
12
receptorinHIVgp120-inducedneuropathicpain.
2.Materialsandmethods
2.1.Animals
MaleSprague-Dawleyrats(200e250g)werehousedforatleast
oneweekbeforethestartoftheexperimentandmaintainedunder
temperature-controlledconditionsat22
C14
Cand60%humiditywith
freelyavailablefoodandwater.Allexperimentsconformedtothe
ethicalregulationsoftheInternationalAssociationfortheStudyof
PainandwereapprovedbytheNanchangUniversityAnimalCare
andUseCommittee.
2.2.Reagents
Recombinantenvelopeglycoprotein120(gp120)Fragment
421e438,whichwasderivedfromtheCD4attachmentregionof
HIVgp120,wasobtainedfromSigma-Aldrich.Gp120waskeptin
stocksolutionataconcentrationof50mMandtemperature
ofC080
C14
C.Fifteenminutesbeforetesting,thestocksolutionwas
dilutedtothedesiredconcentrationswith0.1%ratserumalbumin
(RSA,Sigma-Aldrich)onice.
2.3.Neuropathicpainmodelofperineuralgp120application
Ratswererandomlydividedintofourgroups:shamoperation
group(shamgroup),HIV-gp120group(gp120group),gp120group
treatedwithP2Y
12
shorthairpinRNA(shRNA)(gp120tP2Y
12
shRNA)andgp120grouptreatedwithscrambledshRNA
(gp120tNCshRNA).Eachgroupcontained8rats.Forperineural
HIV-gp120application,previouslydescribedmethodswereused
(HerzbergandSagen,2001;Yietal.,2017).Brie?y,ratswere
anesthetizedbyinjectingthemintraperitoneallywith10%chloral
hydrate,andtheleftsciaticnervewasexposedinthepoplitealfossa
withoutperineuriumdamageunderasepticsurgicalconditions.A
2C26mmstripofoxidizedregeneratedcellulosethathadprevi-
ouslybeensoakedin250mlof0.1%RSAinsalinecontaining400ng
ofgp120(orin0.1%RSAinsalinefortheshamgroup)wasloosely
wrappedaroundthesciaticnerve.Then,thenervewasgentlyput
backintoplaceandtheincisionswereclosedwith4/0sutures.Per
themanufacturer''sinstructionsfortheEntranster?-invivo
transfectionreagent(EngreenBiosystemCompanyofBeijing),20ml
ofatransfectioncomplexconsistingofshRNA(P2Y
12
orNCshRNA)
andtransfectionreagentataratioof1:2wasintrathecallyinjected
intoratsofthegp120tP2YshRNAandgp120tNCshRNA
L.Shietal./Neurochemistry2
12
groupsonday7.Ratsintheshamandgp120groupsreceivedan
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreli
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
equalamountofsaline.ThreedifferentshRNAstargetingsequences
ofP2Y
12
(NM_022800.1)andanegativecontrolscrambledshRNA
(nothomologoustoanygene)weresynthesizedbyRiboBioCo.Ltd.
(Guangzhou,China).TheoptimalP2Y
12
shRNA(datanotshown)
wasselectedbyreal-timeRT-PCR.Thesequenceswereasfollows:
sense5
0
-CACCGCTTCGTTCCCTTCCACTTTGCGAACAAAGTGGAAGGG
AACGAAGC-3
0
,antisense3
0
-CGAAGCAAGGGAAGGTGAAACGCTTGT
TTCACCTTCCCTTGCTTCGAAAA-5''.Rats''behaviorwasassessedby
thesameinvestigatorat0,1,4,7,10,12and14daysafterthestartof
theexperiment.Thetesterwasblindtothegroupdesignation.At
day14,theanimalsweresacri?cedwithCO
2
,andtheleftL4e6
DRGswereremoved.
2.4.Measurementofthemechanicalwithdrawalthreshold(MWT)
ABME-404electronicmechanicalstimulator,whichwaspro-
videdbyInstituteofBiomedicalEngineeringofChineseAcademyof
MedicalSciences,wasusedtoevaluatetheMWTbetween
10:00e12:00.Theendfacediameterofthetestneedle,pressure
measurementrange,andpressuremeasurementresolutionofthis
equipmentwere0.6mm,0.1e50g,and0.05g,respectively.Ani-
malswereplacedinacleanglassboxthatwaspositionedonthe
sieveofthemetalframeforanacclimationperiodofatleast1h.
Thetestneedletouchedthelefthindpawsuntiltheanimalwith-
drewitspaws.Thecomputerrecordedpressurevaluesautomati-
cally.Thisprocedurewasperformed5timesforeachratatintervals
C215min,andtheMWTwascalculatedasthemeanvalueofthese
measurements(Linetal.,2010;Nasirinezhadetal.,2015).The
baselinewascalculatedfromanaverageof5consecutivewith-
drawalresponsesofthelefthindpaw.
2.5.Measurementofthethermalwithdrawallatency(TWL)
DeterminationoftheTWLwasperformedusingaBME-410C
ThermalPawStimulationSystem.Theanimalswereleftina
transparent,bottomlessboxonaglassplateforanacclimation
periodofatleast1h.Abeamofradiantheatorientedtheplantar
surfaceoftheirpaws.TWLwastakenasanindexofthenociceptive
threshold.Thelightbeamwasswitchedoffwhentheanimallifted
itspaw.Thetimeonthescreenwasdesignatedasthepawwith-
drawallatency.Thehindpawsweretestedalternatelyat5-min
intervals.Thecutofftimeforheatstimulationwas30s.
2.6.Quantitativereal-timePCR
DRGswereisolatedimmediatelyand?ushedwithice-coldPBS.
TotalRNAsampleswerepreparedusingtheTRIzolTotalRNARe-
agent(BeijingTiangenBiotechCo.).cDNAsynthesiswasperformed
with2mgoftotalRNAusingtheRevertAid?HMinusFirstStrand
cDNASynthesisKit(Fermentas,Burlington,Ontario,Canada).The
primersweredesignedwithPrimerExpress3.0software(Applied
Biosystems),andthesequenceswereasfollows:forP2Y
12
,forward
5
0
-CTTCGTTCCCTTCCACTTTG-3
0
andreverse5
0
-AGGGTGCTCTCCTT-
CACGTA-3’;forb-actin,forward5
0
-TAAAGACCTCTATGCCAACA-
CAGT-3
0
andreverse5
0
-CACGATGGAGGGGCCGGACTCATC-3’.
QuantitativePCRwasperformedusingSYBR
?
GreenMasterMixin
anABIPRISM
?
7500SequenceDetectionSystem(AppliedBio-
systems,Inc.:FosterCity,CA).Geneexpressionwasquanti?edusing
theDDCTmethodwithCTasthethresholdcycle.Therelativelevels
oftargetgenes,normalizedtothesamplewiththelowestCT,were
reportedas2
C0DDCT
.
2.7.Double-labeledimmuno?uorescence
xxx(2017)1e8
Double-labeledimmuno?uorescencewasperformedas
evedHIVgp120-inducedneuropathicpaininrats,Neurochemistry
0.3%TritonX-100for30minatroomtemperature.Primaryanti-
bodiesagainstGFAP(mouseanti-GFAP,Millipore)andP2Y
12
(rabbit
(1:5000concentration,AlomoneLabs,Jerusalem,Israel),and
mousemonoclonalanti-b-actinantibodies(1:800concentration,
International
BeijingZhongshanBiotechCo.,China)at4
C14
Covernight.The
membraneswerewashedthreetimeswithTBSTandincubatedfor
1hatroomtemperaturewithahorseradishperoxidase-conjugated
secondaryantibody(goatanti-rabbitIgGandgoatanti-mouseIgG,
1:2000concentration,BeijingZhongshanBiotechCo.)inblocking
buffer.Afteranotherwashcycle,thelabeledproteinswerevisual-
izedbyenhancedchemiluminescence(ECL)withaBio-Radsystem.
Thebandintensitywasquanti?edusingImage-ProPlussoftware.
Therelativebandintensitiesofthetargetproteinswerenormalized
againsttheintensityoftherespectiveb-actininternalcontrols
(Amadi-Obietal.,2007).
2.9.Statisticalanalysis
SPSS20softwarewasusedtocalculatestatisticalsigni?cance
(p<0.05)throughoutthestudy.Statisticalcomparisonswere
performedwithone-wayanalysesofvariance(ANOVA)followedby
Fisher''sposthoctestsformultiplecomparisons.Theresultswere
anti-P2Y
12
,AlomoneLabs,Jerusalem,Israel)weredilutedtoaratio
of1:100.ThesectionswerewashedagaininPBSandincubated
withasecondaryantibody(goatanti-rabbitTRITC,JacksonImmu-
noResearch,Inc.,WestGrove,PA,USA)andgoatanti-mouseFITC
(BeijingZhongshanBiotechCo.)dilutedtoa1:200concentrationin
PBScontaining1%BSAfor1hat37
C14
C.Finally,thesectionswere
washedinPBS,andtheresultswereassessedusinga?uorescence
microscope(Olympus,Tokyo,Japan).Datawerecollectedfromsix
animalsineachgroup.Five?eldscontainingapproximately50
neuronseachwererandomlyselected,anddatafromeachanimal
wereaveraged.
2.8.Westernblotanalysis
DRGswereremovedandstoredatC080
C14
C.Thegangliawere
homogenizedinRIPAlysisbuffer(50mMTris-Cl,pH8.0,150mM
NaCl,0.1%sodiumdodecylsulfate(SDS),1%NonidetP-40,0.02%
sodiumdeoxycholate,100mg/mLphenylmethylsulfonyl?uoride,
and1mg/mLAprotinin)containingaproteaseinhibitor.Ganglia
wereincubatedonicefor30minandsubsequentlycentrifuged
(12,000g;10min).Thesupernatantswerecollectedandassayed
fortheproteinconcentrationwiththeBCAProteinAssayKit.These
supernatantsweredilutedwith6C2loadingbufferandheatedto
95
C14
Cfor5min;aliquotscontaining20mgofproteinfromeach
groupwerethenseparatedusingaBio-Rad10%SDSepolyacryla-
midegelelectrophoresissystemandtransferredtopolyvinylidene
?uoride(PVDF)membranes.Themembranewasblockedwith5%
non-fatdrymilkin1C2TBSTfor2hatroomtemperature,followed
byincubationwithrabbitanti-P2Y
12
(1:800concentration,Abcam,
USA),rabbitanti-interleukin(IL)-1b,rabbitanti-tumornecrosis
factor(TNF)-receptor1(TNF-R1)(1:5000concentration,Abcam,
USA),rabbitanti-phosphorylated-Akt(p-Akt)andrabbitanti-Akt
previouslydescribed.Inbrief,rats’DRGswereremovedand?xedin
4%paraformaldehyde(PFA)for2hatroomtemperature.DRGs
weredehydratedin30%sucroseand4%PFAovernight.Then,10-
mm-thicksectionswerecutusingacryostatandmountedonthe
slides.ThesectionswerewashedinPBSandincubatedinblocking
solutioncontaining3%bovineserumalbumin(BSA)inPBSwith
L.Shietal./Neurochemistry
reportedasthemeans±SEM.
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreliev
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
3.Results
3.1.EnhancedP2Y
12
mRNAandproteinexpressioninDRGofgp120-
treatedrats
TheP2Y
12
receptorisinvolvedinneuropathicpain(HorvC19ath
etal.,2014;Katagirietal.,2012).TheexpressionlevelsofP2Y
12
mRNAweremeasuredbyreal-timePCR.Theexpressionvaluesfor
P2Y
12
mRNAexpressioninthegp120groupwerehigherthanthose
intheshamgroup(p<0.01)(Fig.1A).Theexpressionlevelsfor
P2Y
12
mRNAinthegp120tP2Y
12
shRNAgroupwerelowerthan
thoseinthegp120group(p<0.01).Nosigni?cantdifferencewas
foundbetweenthegp120groupandgp120tP2Y
12
NCshRNA
group(p>0.05).
TheexpressionlevelsforP2Y
12
proteinweremeasuredby
Westernblotting.Theopticaldensityvalues(ODV)forP2Y
12
pro-
teinexpressionwereincreasedinthegp120groupcomparedwith
theshamgroup(p<0.01)(Fig.1B).Theexpressionlevelsofthe
P2Y
12
proteininthegp120tP2Y
12
shRNAgroupwerelowerthan
thoseinthegp120group(p<0.01).Nosigni?cantdifferencewas
foundbetweenthegp120groupandgp120tP2Y
12
NCshRNA
group(p>0.05).Therefore,P2Y
12
receptorisinvolvedinneuro-
pathicpaininHIVgp120-treatedrats.
3.2.ReductionoftheP2Y
12
receptordecreasedmechanical
hyperalgesiaandthermalhyperalgesiaingp120-treatedrats
Therewerenodifferencesinthemechanicalwithdrawal
threshold(MWT)betweengroupspriortotheexperiment.Onthe
4thand7thdaysoftheexperiment,MWTingp120andgp120tNC
shRNAratswaslowerthaninshamrats(p<0.05).Nosigni?cant
differenceswerefoundbetweenthegp120andgp120tNCshRNA
groups.OneweekafterinjectionwithP2Y
12
shRNA,theMWTof
gp120tP2Y
12
shRNAratswashigherthanthatofgp120rats
(p<0.01),butitremainedlowerthanthatofshamrats(p<0.05).
Therewasnosigni?cantdifferencebetweenthegp120and
gp120tNCshRNAgroups(Fig.2A).
TheeffectsofP2Y
12
shRNAonthermalhyperalgesiawereeval-
uatedwiththeThermalPawStimulationSystem.Onthe4thand7th
days,TWLingp120andgp120tNCshRNAratswasdecreased
comparedtoshamrats(p<0.05).TherewasnodifferenceinTWL
betweenthegp120andgp120tNCshRNAgroups.Onthe14thday
oftheexperiment,theTWLofgp120tP2Y
12
shRNAratswashigher
thanthatofgp120rats(p<0.01),butwasstilldecreasedrelativeto
shamrats(p<0.05).Nodifferencewasobservedbetweengp120and
gp120tNCshRNAanimals(Fig.2B).Theseresultsindicatedthat
P2Y
12
shRNAtreatmentincreasedtheMWTandTWLbydown-
regulatingP2Y
12
receptoringp120neuropathicpainmodelrats.
3.3.ReductionoftheP2Y
12
receptordecreasedco-expressionof
P2Y
12
andGFAPinDRGofgp120-treatedrats
Co-expressionofP2Y
12
andGFAPinDRGwasassayedbydouble
immuno?uorescence.Up-regulatedGFAP(amarkerofSGCs)in
DRGindicatesactivationofSGCsinnervousinjury.Fig.3Ashows
theco-expressionlevelsfortheP2Y
12
receptorandGFAPineach
group.P2Y
12
andGFAPwerecolocalizedinSGCsofDRG.After14
daysgp120application,theratioofGFAP/P2Y
12
-immunolabeled
cellswassigni?cantlyincreasedthanshamgroup(p<0.01)
(Fig.3B).TheP2Y
12
shRNAreducedtheratioofGFAP/P2Y
12
-
immunolabeledcellsincreasedbygp120(p<0.01).Thereisno
differencebetweengp120groupandthegp120tNCgroup.These
resultsdemonstratedthatgp120tP2Y
12
shRNAtreatment
decreasedup-regulationoftheP2YreceptorandGFAPco-
xxx(2017)1e83
12
expressionlevelsinDRG.
edHIVgp120-inducedneuropathicpaininrats,Neurochemistry
InternationalL.Shietal./Neurochemistry4
3.4.ReductionoftheP2Y
12
receptordecreasedexpressionofIL-1b
andTNF-R1inDRGofgp120-treatedrats
ActivationofDRGSGCscanincreasethereleaseofproin-
?ammatorycytokines,suchasinterleukin(IL)-1bandtumorne-
crosisfactora(TNF-a),andproin?ammatorycytokinesplayan
importantroleinthecreationandmaintenanceofpathological
pain(Sachsetal.,2002;Takedaetal.,2007,2009).Theexpression
levelsofIL-1bandTNF-R1proteininDRGweremeasuredby
Westernblotting(Fig.4).TheexpressionlevelsofIL-1bandTNF-R1
inthegp120groupwereincreasedcomparedtotheshamgroup
(p<0.01).TheexpressionlevelsofIL-1bandTNF-R1inthe
Fig.1.UpregulationofP2Y
12
mRNAandproteininDRGofgp120-treatedrats.
A.ExpressionofP2Y
12
mRNAinDRGwasmeasuredbyRT-qPCR.Expressioninthe
gp120groupwashigherthanintheshamgroup(p<0.01).Ingp120ratstreatedwith
P2Y
12
shRNA,P2Y
12
mRNAexpressionwassigni?cantlydecreasedcomparedwith
untreatedgp120rats(p<0.01).Theexperimentwasperformedthreetimes(n?8per
group).Dataarepresentedasthemeans±SEM.p<0.01comparedtothesham
group;##p<0.01comparedtothegp120-treatedgroup.
B.ExpressionofP2Y
12
proteininDRGwasassessedbyWesternblot.Proteinexpression
inthegp120-treatedgroupwasincreasedcomparedtotheshamgroup(p<0.01).In
gp120ratstreatedwithP2Y
12
shRNA,theexpressionofP2Y
12
proteinwassigni?cantly
lowerthaninuntreatedgp120rats(p<0.01).BargraphsshowtheratiooftheP2Y
12
proteinleveltob-actinlevelineachgroup.Dataaredisplayedasthemeans±SEM.
p<0.01comparedtotheshamgroup;##p<0.01comparedtothegp120-treated
group.
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreli
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
xxx(2017)1e8
gp120tP2Y
12
shRNAgroupweredecreasedrelativetothoseinthe
gp120group(p<0.01).Therewasnosigni?cantdifferencebe-
tweenthegp120andgp120tP2Y
12
NCshRNAgroups.P2Y
12
shRNAtreatmentmayhaveloweredup-regulationofIL-1band
TNF-R1inDRGandlessenedpainbehaviorsingp120neuropathic
painmodelrats.
3.5.ReductionoftheP2Y
12
receptordecreasedactivationofAktin
DRGofgp120-treatedrats
AktphosphorylationisamarkerofAktactivation.Theexpres-
sionlevelsofAktinDRGweredetectedbyWesternblotting(Fig.5).
Therewerenosigni?cantdifferencesintheexpressionlevelsofAkt
tob-actin(p>0.05,n?8foreachgroup).Theexpressionlevelsof
Fig.2.ReductionoftheP2Y
12
receptorintheDRGdecreasedmechanicalhyper-
algesiaandthermalhyperalgesiaingp120-treatedrats.
A.TheMWTinthegp120groupwaslowerthanintheshamgroupfrom4thdayto
14thday(p<0.05).Ingp120ratstreatedwithP2Y
12
shRNA,theMWTwashigherthan
intheuntreatedgp120group(p<0.01).Nodifferencewasfoundbetweenthe
gp120tNCgroupandgp120group(p>0.05).Eachgroupconsistedofeightrats.Data
aredisplayedasthemeans±SEM.p<0.05,p<0.01comparedtotheshamgroup;
#p<0.05,##p<0.01comparedtothegp120-treatedgroup.
B.TheTWLinthegp120groupwaslowerthanintheshamgroupfrom4thdayto14th
day(p<0.05).Ingp120ratstreatedwithP2Y
12
shRNA,theTWLwashigherthanthat
intheuntreatedgp120group(p<0.05).Nodifferencewasfoundbetweenthe
gp120tNCgroupandthegp120group(p>0.05).Eachgroupconsistedofeightrats.
Thedataaredisplayedasthemeans±SEM.p<0.05,p<0.01comparedtothesham
group;#p<0.05,##p<0.01comparedtothegp120-treatedgroup.
evedHIVgp120-inducedneuropathicpaininrats,Neurochemistry
L.Shietal./NeurochemistryInternationalxxx(2017)1e85
p-AktrelativetoAktinthegp120groupwereincreasedcompared
totheshamgroup(p<0.01,n?8foreachgroup),whichsuggested
thatAktwasactivatedinDRGofgp120-treatedrats.Theexpression
levelsofp-AktrelativetoAktinthegp120tP2Y
12
shRNAgroup
weredecreasedcomparedtothoseinthegp120group(p<0.01,
n?8foreachgroup).Nosigni?cantdifferencewasfoundbetween
thegp120andgp120tP2Y
12
NCshRNAgroups.Theseresults
indicatedthatP2Y
12
shRNAtreatmentmaysuppressthephos-
phorylationofAktinDRGofgp120neuropathicpainmodelrats.
Fig.3.ReductionoftheP2Y
12
receptordecreasedupregulationofP2Y
12
andGFAPco-e
A.Doubleimmuno?uorescencewasusedtomeasureP2Y
12
andGFAPco-expressionlevels
B.TheratioofGFAP/P2Y
12
-immunolabeledcellswassigni?cantlyincreasedthanshamgroup
increasedbygp120(p<0.01).Thereisnodifferencebetweengp120groupandthegp120t
group;##p<0.01comparedtothegp120-treatedgroup.
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreliev
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
4.Discussion
Chronicneuropathicpainisacommonpainsyndromeinpa-
tientswithHIV-1infection(Hao,2013;Nasirinezhadetal.,2015;
Yuanetal.,2014;Zhengetal.,2011).Althoughtheseverityand
frequencyofneuropathicpainarecorrelatedwiththeprogression
ofHIV-1infection,neuropathicpaincanappearatallstagesofHIV-
1infection.Thepresentstudydemonstratedthatperipheral
administrationofgp120enhancedthermalhyperalgesiaandme-
chanicalallodyniainrats.Theresultsindicatedthatgp120induced
pathologicalchangesinpainbehaviorsinanimalmodels.DRGhave
xpressionlevelsintheDRGofgp120-treatedrats.
intheDRGofgp120-treatedrats.
(p<0.01).TheP2Y
12
shRNAreducedtheratioofGFAP/P2Y
12
-immunolabeledcells
NCgroup.Dataaredisplayedasthemeans±SEM.p<0.01comparedtothesham
edHIVgp120-inducedneuropathicpaininrats,Neurochemistry
InternationalL.Shietal./Neurochemistry6
animportantroleinsensorysignalingunderphysiologicaland
pathologicalconditions.Thecurrentexperimentsdemonstrated
thattheP2Y
12
receptorwasincreasedinDRGofgp120-treatedrats,
leadingtoenhancedmechanicalandthermalhyperalgesia.The
Fig.4.ReductionoftheP2Y
12
receptordecreasedexpressionofIL-1bandTNF-R1
intheDRGofgp120-treatedrats.
A.ExpressionoftheIL-1bprotein(normalizedtoeachb-actininternalsham)inthe
gp120groupwashigherthanintheshamgroup(p<0.01).Ingp120ratstreatedwith
P2Y
12
shRNA,expressionlevelsoftheIL-1bproteinwerelowerthaninthegp120
groupthatdidnotreceiveP2Y
12
shRNA(p<0.01).Nodifferencewasfoundbetween
thegp120tNCgroupandthegp120group(p>0.05).BargraphsshowtheratioofIL-
1bproteintob-actinineachgroup.Thedataaredisplayedasthemeans±SEM(n?8
pergroup).p<0.01comparedtotheshamgroup;##p<0.01comparedtothe
gp120-treatedgroup.
B.ExpressionoftheTNF-R1protein(normalizedtoeachb-actininternalsham)inthe
gp120groupwashigherthanintheshamgroup(p<0.01).Ingp120ratstreatedwith
P2Y
12
shRNA,TNFa-Rproteinexpressionwaslowerthaninthegp120group(p<0.01).
Nodifferencewasfoundbetweenthegp120tNCgroupandthegp120group
(p>0.05).BargraphsshowtheratioofTNFa-Rproteinleveltob-actinlevelineach
group.Dataaredisplayedasthemeans±SEM(n?8pergroup).p<0.01compared
totheshamgroup;##p<0.01comparedtothegp120-treatedgroup.
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreli
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
xxx(2017)1e8
P2Y
12
receptorparticipatesinthepathologicalprocessesofin-
?ammatoryandneuropathicpain(Burnstock,2013;Katagirietal.,
2012;MagniandCeruti,2013).Thus,theP2Y
12
receptorinDRGmay
beinvolvedinnociceptivetransmissionofneuropathicpaininHIV
gp120-treatedrats.Ourdataalsoshowedthatdown-regulationof
P2Y
12
reducedtheexpressionlevelsofP2Y
12
mRNAandprotein,
consequentlyrescuingMWTandTWLingp120-treatedrats.The
resultsfurtherindicatedthattheP2Y
12
receptorhasanimportant
roleinratDRGinthetransmissionofnociceptivesignalsinduced
byHIVgp120.
SGCstightlyensheaththesomaofDRGneurons(Costaand
Moreira,2015;Hanani,2005;Takedaetal.,2009).Intimateasso-
ciationsbetweenSGCsandneuronsfacilitatebidirectionalregula-
tionofSGCfunctionandneuronalexcitability.Afternerveinjury,
neuronalhyperexcitabilitycanresultinSGCactivationandthus
induceneuropathicpain(CostaandMoreira,2015;Hanani,2005;
Takedaetal.,2009;Yingetal.,2017).Elevationoftheactivation
markerGFAPindicatesactivationofSGCsinDRG.Thepresentstudy
showedthatGFAPexpressionwasincreasedinDRGSCGsafter
Fig.5.ReductionoftheP2Y
12
receptordecreasedactivationofAktintheDRGof
gp120-treatedrats.
Integratedopticaldensity(IOD)ratiosofAkttob-actinwerenotsigni?cantlydifferent
betweenthegp120andshamgroups(p>0.05).TheIODratioofp-AkttoAktwas
higherinthegp120groupthantheshamgroup(p<0.01,n?8foreachgroup).The
IODratioofp-AkttoAktinratstreatedwithgp120tP2Y
12
shRNAwassigni?cantly
lowerthaninthegp120group(p<0.01,n?8foreachgroup).Thedataaredisplayed
asthemeans±SEM,n?8.p<0.01comparedtotheshamgroup;##p<0.01
comparedtothegp120group.
evedHIVgp120-inducedneuropathicpaininrats,Neurochemistry
International
gp120treatment,whichsuggestsactivationofSGCsingp120rats.
P2Y
12
isexpressedinDRGSGCs(Katagirietal.,2012;Kobayashi
etal.,2013).Ourresultsalsoindicatedtheco-expressionvaluesof
P2Y
12
andGFAPinthegp120groupwereincreasedcomparedwith
thoseintheshamgroup.Thus,theP2Y
12
receptorisinvolvedinthe
transmissionofsignalsbyDRGSGCsresultingfromnerveinjuryin
gp120rats.P2Y
12
shRNAtreatmentinDRGSGCsdecreased
expressionofGFAPproteinandco-expressionofP2Y
12
andGFAPin
gp120-treatedrats.Simultaneously,theMWTandTWLwere
increasedingp120ratsafterP2Y
12
shRNAtreatmentcompared
withratstreatedwithgp120alone.Therefore,down-regulationof
theP2Y
12
receptordecreasedactivationofDRGSGCsandrelieved
painbehaviorsingp120-treatedrats.
ActivationofDRGSGCscanincreasethereleaseofproin-
?ammatorycytokines.Enhancedlevelsofproin?ammatorycyto-
kinesaugmentabnormalneuronalexcitabilityandcontributeto
neuropathicpain(Kajanderetal.,1992;Sachsetal.,2002;Takeda
etal.,2007,2009).HIVgp120alsostimulatesthenervoussystem
toreleasepro-in?ammatorycytokines(Hao,2013;Yuanetal.,
2014;Zhengetal.,2011).Ourresultsindicatedthatup-regulation
oftheP2Y
12
receptoringp120-treatedratswasaccompaniedby
enhancedIL-1bproteinexpression.TNF-R1isactivatedbyTNF-ain
DRGneurons,increasingneuronalexcitability(Illesetal.,2012;
Kajanderetal.,1992).Ourresultsalsorevealedthattheexpres-
sionlevelsofTNF-R1inDRGofgp120-treatedratswereenhanced
comparedwithshamrats.Up-regulationoftheIL-1bproteinand
TNF-R1inDRGcanleadtoabnormalneuronalexcitability.Proin-
?ammatorycytokinesinDRGenhancedneuronalexcitabilityand
promotedhyperalgesiaingp120-treatedrats.P2Y
12
shRNAtreat-
mentdecreasedtheexpressionlevelsoftheIL-1bproteinandTNF-
R1inDRGandreducedhyperalgesiaingp120rats.P2Y
12
receptor
activationaugmentedIL-1breleaseinneuropathicpain(Horv
C19
ath
etal.,2014).Thus,down-regulationoftheP2Y
12
receptor
decreasedactivationofDRGSGCs,andthenreducedexpressionof
IL-1bandTNF-R1.Down-regulatedexpressionofIL-1bandTNF-R1
lessenedabnormalsignalbetweenneuronandSGCinDRGaswell
asrelievedneuropathicpainbehaviorsinducedbygp120
treatment.
ActivationoftheproteinkinaseB/Aktsignalingpathwayis
involvedinneuropathicpainmechanisms(Guedesetal.,2008;Xu
etal.,2007;Sunetal.,2006).Accordingly,inhibitionofprotein
kinaseB/Aktactivationreducedpainbehaviorsassociatedwith
neuropathicpain(Sunetal.,2006;Xuetal.,2007).TheP2Y
12
re-
ceptorisinvolvedinAktactivation(Irinoetal.,2008).Ourdata
indicatedthattheintegratedopticaldensity(IOD)ratioofp-Aktto
Aktingp120ratswashigherthaninshamrats.Itispossiblethat
AktphosphorylationinDRGisinvolvedinhyperalgesiaingp120-
treatedrats.AfterP2Y
12
shRNAtreatmentingp120rats,theIOD
ratiosofp-AkttoAktweresigni?cantlydecreased.Ourstudies
revealedthatP2Y
12
shRNAtreatmentsilencedup-regulationofthe
P2Y
12
receptoranddecreasedp-AktinDRGofgp120-treatedrats.
Consequently,thedegreeofp-Aktreductioninthegp120-treated
ratswascloselyassociatedwithreductioninpainbehaviorsafter
P2Y
12
shRNAtreatment.Therefore,weconcludethatsilencingthe
P2Y
12
receptorreducedAktactivationinDRGingp120-treatedrats.
Inconclusion,thepresentstudydemonstratedthatperipheral
nerveexposuretoHIVgp120increasedtheexpressionlevelsofthe
P2Y
12
receptorinDRGSGCsandenhancedmechanicalandthermal
hyperalgesiainratmodels.Up-regulationoftheP2Y
12
receptorin
DRGSGCsfurtherpromotedthereleaseofpro-in?ammatorycy-
tokines.UpregulationofIL-1b,TNF-R1andGFAPmayfacilitatethe
abnormalsignalbetweenneuronandSGCinDRGandleadtopain
behaviorsinducedbygp120.ReductionoftheP2Y
12
receptorin
DRGSGCsdecreasedupregulationofIL-1b,TNF-R1andGFAP,
L.Shietal./Neurochemistry
reducedp-Akt,andinhibitedabnormalsignalbetweenneuronand
Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreliev
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
SGCinDRGofgp120-treatedrats.Therefore,reductionoftheP2Y
12
receptorinDRGSGCsdecreasedhyperalgesiaingp120-treatedrats.
Con?ictofinterests
Theauthorsdeclaretheyhavenocon?ictsofinterestrelatedto
thismanuscript.
Acknowledgments:
ThesestudiesweresupportedbygrantsfromtheNational
NaturalScienceFoundationofChina(31560276,81570735,
81460200,81701114,81760152and81200853),theTechnology
PedestalandSocietyDevelopmentProjectofJiangxiProvince
(20151BBG70250and20151BBG70253),theNaturalScienceFoun-
dationofJiangxiProvince(20142BAB205028,20142BAB215027,
20171BAB205025and20121512040234),thegrant(2014-47)from
MajorDisciplinesofAcademicandTechnicalLeadersProjectof
JiangxiProvince,andtheEducationalDepartmentofJiangxiProv-
ince(GJJ13155andGJJ14319).
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Pleasecitethisarticleinpressas:Shi,L.,etal.,P2Y
12
shRNAtreatmentreli
International(2017),http://dx.doi.org/10.1016/j.neuint.2017.08.006
evedHIVgp120-inducedneuropathicpaininrats,Neurochemistry
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