高浓度葡萄糖下铁的神经毒性作用.docx

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