高浓度葡萄糖下铁的神经毒性作用.docx
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高浓度葡萄糖下铁的神经毒性作用.docx
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高浓度葡萄糖下铁的神经毒性作用
Neurotoxiceffectsofironoverloadunderhighglucoseconcentration*
ShiZhao1,LinZhang1,ZihuiXu1,WeiqunChen2
1DepartmentofEndocrinology,WuhanCentralHospital,Wuhan430014,HubeiProvince,China2CentralLaboratory,WuhanCentralHospital,Wuhan430014,HubeiProvince,China
ShiZhaoandLinZhangcontributedequallytothisstudy.
Correspondingauthor:
ShiZhao,Professor,Master’ssupervisor,Chiefphysician,DepartmentofEndocrinology,WuhanCentralHospital,Wuhan430014,HubeiProvince,China,zhaoshiwuhan@.
Received:
2013-08-29
Accepted:
2013-11-02
(N201304023)
Acknowledgments:
TheauthorswouldliketothankXuZHfromtheDepartmentofEndocrinology,WuhanCentralHospital,Wuhan,HubeiProvince,Chinaforassistingwithpapereditinganddatadiscussion.
Funding:
ThestudywassupportedbytheNaturalScienceFoundationofHubeiProvince,No.2010CDB09001.
GraphicalAbstract
Abstract
Ironoverloadcanleadtocytotoxicity,anditisariskfactorfordiabeticperipheralneuropathy.However,theunderlyingmechanismremainsunclear.Weconjecturedthatironoverload-inducedneurotoxicitymightbeassociatedwithoxidativestressandtheNF-E2-relatedfactor2(Nrf2)/AREsignalingpathway.Asaninvitrocellularmodelofdiabeticperipheralneuropathy,PC12cellsexposedtohighglucoseconcentrationwereusedinthisstudy.PC12cellswereculturedwithferricammoniumcitrateatdifferentconcentrationstocreateironoverload.PC12cellsculturedinferricammoniumcitrateunderhighglucoseconcentrationhadsignificantlylowcellviability,ahighrateofapoptosis,andelevatedreactiveoxygenspeciesandmalondialdehydelevels.Thesechangesweredependentonferricammoniumcitrateconcentration.Nrf2mRNAandproteinexpressionintheferricammoniumcitrategroupswereinhibitedmarkedlyinadose-dependentmanner.Allchangescouldbeinhibitedbyadditionofdeferoxamine.TheseresultsindicatethatironoverloadaggravatesoxidativestressinjuryinneuralcellsunderhighglucoseconcentrationandthattheNrf2/AREsignalingpathwaymightplayanimportantroleinthisprocess.
KeyWords
neuralregeneration;peripheralnerveinjury;ironoverload;oxidativestress;diabeticperipheralneuropathy;reactiveoxygenspecies;highglucose;PC12cells;Nrf2/ARE;grants-supportedpaper;neuroregeneration
Authorcontributions:
ZhaoSconceivedanddirectedthestudy,revisedthemanuscript.ZhangLdesignedthestudy,conductedtheexperiments,analyzedthedata,anddraftedthemanuscript.XuZHrevisedthepaper.ChenWQguidedthestudyandprovidedtechnicalsupport.Allauthorsapprovedthefinalversionofthepaper.
Conflictsofinterest:
Nonedeclared.
Authorstatements:
Themanuscriptisoriginal,hasnotbeensubmittedtoorisnotunderconsiderationbyanotherpublication,hasnotbeenpreviouslypublishedinanylanguageoranyform,includingelectronic,andcontainsnodisclosureofconfidentialinformationorauthorship/patentapplication/fundingsourcedisputations.
INTRODUCTION
Diabeticperipheralneuropathyisoneofmostcommonchroniccomplicationsinducedbydiabetichyperglycemia,andisassociatedwithaxonalatrophy,bluntedregenerativepotential,demyelination,andlossofperipheralnervefibers[1].Althoughnumerousfactorscontributetodiabeticperipheralneuropathy,includinginsulin-inducedresistancetoneuronaltrophicsupport[2],decreased(Na/K)-ATP-aseactivity[3]andSchwanncelldysfunction[4],increasedoxidativestressandmitochondrialdysfunctionseemintimatelyassociatedwithnervedysfunctionanddiminishedregenerativecapacity.Oxidativestressandapoptosishavebeenfoundtoplaycrucialrolesindiabeticperipheralneuropathy[5-6].Underhyperglycemia,largeamountsofreactiveoxygenspeciesareproducedbythemitochondrialrespiratorychain,andneuronalapoptosisisincreased[7].Despiteadvancesinunderstandingtheetiologyofdiabeticperipheralneuropathy,fewapprovedtherapiesexistforthepharmacologicalmanagementofthedisease.Therefore,identifyingnoveltherapeuticstrategiesremainsparamount.
Ironisubiquitousincellsandisessentialforbiologicalfunctioning.Normalironbalanceismaintainedbymeticulousregulationofitsabsorptionfromtheintestineandreleasefrommacrophages.Itismodulatedinresponsetorequirementfrombodyironstoresanddemandfromerythropoiesistopreventdeleteriousextremesofirondeficiencyorexcess[8].However,withoutadequatemanagement,excessamountsoffreeironmaycauseprogressivedamage.Inrecentyears,therehasbeenincreasinginterestinbrainironmetabolismduringnormalageing,particularlyasexcessiveirondepositionhasbeenfoundinneurologicaldisorders[9].Ironoverloadisalsoariskfactorfordiabetes.Thelinkbetweenironanddiabeteswasfirstrecognizedinpathologicconditions(hereditaryhemochromatosisandthalassemia),buthighlevelsofdietaryironalsoconferdiabetesrisk.Ironplaysadirectandcausalroleindiabetespathogenesis,whichinvolvesbothβcellfailureandinsulinresistance.Ironalsoregulatesmetabolisminmosttissuesinvolvedinenergyhomeostasis,withtheadipocyteinparticularhavinganiron-sensingrole.Themolecularmechanismsunderlyingtheseprocessesarenumerousandincompletelyunderstood,butincludeoxidativestressandthemodulationofadipokineandintracellularsignaltransductionpathways[10].
Alargebodyofevidenceshowsthatironoverloadiscloselyrelatedtodiabetesmellitusaswellasitschroniccomplications[11-15];oxidativestressandinflammatoryfactorsmayplayapivotalroleinthisrelationship[16].However,thereisnodirectevidenceonwhetherabnormalironmetabolismisrelatedtodiabeticneuropathy.
Inthisstudy,wemadeuseofacellularmodelofdiabeticperipheralneuropathyusingPC12cellsexposedtohighglucoseconcentration,andexaminedcellviabilityandapoptosisunderironoverload.Wemeasuredthelevelsofreactiveoxygenspeciesandmalondialdehyde,andtheexpressionofthetranscriptionalactivatorNF-E2-relatedfactor2(Nrf2).
RESULTS
Ironoverloadaggravatedhighglucoseconcentration-inducedneurotoxicityinPC12cells
Hyperglycemiawasrecentlyshowntoinduceoxidativestressandgeneratereactiveoxygenspeciesinneurons,resultinginneuronaldamageanddysfunction[17].Inaddition,highglucoseinducedoxidativedamageinPC12cells[18].Thus,wegeneratedacellculturemodelofdiabeticperipheralneuropathybyculturingPC12cellsinhighglucose(25mmol/L).Ironoverloadwascreatedbyexposuretoferricammoniumcitrate[19].Todeterminetheappropriateexperimentalconcentrationofferricammoniumcitrate,PC12cellsculturedunderhighglucose(25mmol/L)wereexposedto12differentconcentrationsofthecompound(0,12.5,25,50,100,200,300,400,500,600,700,800μmol/L)for24hours.
The3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide(MTT)assaywasusedtoassessPC12cellgrowthinhibition.FerricammoniumcitrateinhibitedthegrowthofPC12cellsinaconcentration-dependentmanner(Figure1A).Thedegreeofgrowthinhibitioncouldbedividedintothreephasesaccordingtoferricammoniumcitrateconcentration—theinitialphase(lessthan50μmol/L),therapidrisingphase(50–200μmol/L),andtheplateauphase(morethan200μmol/L).Thegrowthinhibitoryeffectwasstatisticallysignificantwhenthe25,100and400μmol/Ltreatmentdoseswerecomparedwitheachother(P<0.05orP<0.01).Hence,wechosethesethreeconcentrationsofferricammoniumcitrateforuseinthesubsequentexperiments.
Deferoxamineisachelatingagentusedtoremoveexcessfreeironfromthebody.Therefore,weexaminedtheeffectofdeferoxamineonourcellculturemodelofdiabeticperipheralneuropathy.AfterPC12cellswereexposedtohighglucose(25mmol/L)for24hours,ferricammoniumcitrateand/ordeferoxaminewereaddedandthecellswereculturedforanadditional24,48or72hours,andcellviabilitywasassessed(Figure1B–D).
B
A
C
D
E
Figure1PC12cellgrowthandviabilitywereinhibitedbyferricammoniumcitrate(FAC)andrescuedbydeferoxamine(DFO)(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromideassay).
(A)ThegrowthinhibitionratioofPC12cellsafterFACtreatment.Cellswereexposedto12differentconcentrationsofFAC(0,12.5,25,50,100,200,300,400,500,600,700and800μmol/L)underhighglucose(25μmol/L)for24hours.ThecellviabilityofPC12cellsafter24hours(B),48hours(C)and72hours(D)ofculture.CellmorphologyofPC12cellsat48hoursshowingthatcellssubjectedtohighglucoseand/orFACtreatmentfailedtoextendlongneuritescomparedwiththenormalglucoseconcentrationgroup.DeferoxamineprotectedPC12cellsbypromotingneuritegrowthandcellproliferation(E).
(A–D)AlldataarethepercentagetocontrolPC12cellswithoutsupplementationwithglucose,FACorDFO.Dataareshownasmean±SDfromtriplicateexperiments.One-wayanalysisofvariancewasadoptedformultiple-groupcomparison;two-tailedStudent’st-testwasusedforintergroupcomparison.aP<0.05,vs.HGG;bP<0.01,vs.25μmol/LFACgroup;cP<0.01,vs.100μmol/LFACgroup;dP<0.01,vs.400μmol/LFACgroup.
NGG:
Normalglucoseconcentrationgroup;HGG:
highglucoseconcentrationgroup;FAC25:
25μmol/LFACgroup;FAC100:
100μmol/LFACgroup;FAC400:
400μmol/LFACgroup;FAC+DFO:
400μmol/LFAC+200μmol/LDFOgroup.
Interestingly,ourdatashowedthatcellviabilityinthehighglucoseconcentrationgroupwassignificantlyhigherthaninthenormalglucoseconcentrationgroup24hoursafteraddingthedrug(P<0.05),buttherewasnosignifican
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- 关 键 词:
- 浓度 葡萄糖 神经 毒性 作用