biopsyproven nonalcoholic fatty liver disease.docx

biopsyproven nonalcoholic fatty liver disease.docx

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biopsyprovennonalcoholicfattyliverdisease

Nameofjournal:

WorldJournalofGastroenterology

ESPSManuscriptNO:

11492

Columns:

CASECONTROLSTUDY

Increasedcirculatingzonulininchildrenwithbiopsy-provennonalcoholicfattyliverdisease

PacificoLetal.ZonulinandpediatricNAFLD

LuciaPacifico,EneaBonci,LidiaMarandola,SaraRomaggioli,StefanoBascetta,ClaudioChiesa

LuciaPacifico,SaraRomaggioli,StefanoBascetta,DepartmentofPediatricsandChildNeuropsychiatry,SapienzaUniversityofRome,Rome,00161Rome,Italy

EneaBonci,LidiaMarandola,DepartmentofExperimentalMedicine,SapienzaUniversityofRome,00161Rome,Italy

ClaudioChiesa,InstituteofTranslationalPharmacology,NationalResearchCouncil,00133Rome,Italy

Authorcontributions:

PacificoL,BonciE,andChiesaCdesignedthestudy,analyzedthedataandwrotethemanuscript;RomaggioliS,andBascettaScollectedthedata;MarandolaLperformedthemeasurementsandanalyses.AlltheAuthorsparticipatedinthecriticalreviewandinthefinalapprovalofthemanuscript.

Correspondenceto:

ClaudioChiesa,MD,InstituteofTranslationalPharmacology,NationalResearchCouncil,ViaFossodelCavaliere100,00133Rome,Italy.claudio.chiesa@r.it

Telephone:

+39-6-49979215Fax:

+39-6-49979216

Received:

May23,2014Revised:

June22,2014

Accepted:

July16,2014

Publishedonline:

Abstract

AIM:

Toinvestigatethepotentialassociationofcirculatingzonulinwiththestageofliverdiseaseinobesechildrenwithbiopsy-confirmednonalcoholicfattyliverdisease（NAFLD）.

METHODS:

Acase-controlstudywasperformed.Caseswere40obesechildrenwithNAFLD.ThediagnosisofNAFLDwasbasedonmagneticresonanceimaging（MRI）withhighhepaticfatfraction（HFF≥5%）,andconfirmedbyliverbiopsywith≥5%ofhepatocytescontainingmacrovesicularfat.Controlswereselectedfromobesechildrenwithnormallevelsofaminotransferases,andwithoutMRIevidenceoffattyliveraswellasofothercausesofchronicliverdiseases.Controlswerematched（1-to1）withthecasesonage,gender,pubertalstageandascloselyaspossibleonbodymassindex-standarddeviationscore.Allparticipantsunderwentclinicalexamination,laboratorytestsincludingzonulin,inflammatoryandmetabolicparameters,andMRIformeasurementofHFFandvisceraladiposetissue.

RESULTS:

ZonulinvaluesweresignificantlygreaterinobesesubjectswithNAFLDthaninthosewithoutNAFLD[median（interquartilerange）,4.23（3.18-5.89）vs3.31（2.05-4.63）,P<0.01].InpatientswithNAFLD,zonulinconcentrationsincreasedsignificantlywiththeseverityofsteatosisandtheSpearman’scoefficientrevealedapositivecorrelationbetweenzonulinvaluesandsteatosis（r=0.372,P<0.05）;however,wedidnotfindasignificantcorrelationbetweenzonulinandlobularinflammation（P=0.23）,ballooning（P=0.10）,fibrosisscore（P=0.18）,orpresenceofnonalcoholicsteatohepatitis（P=0.17）.Withintheentirestudypopulation,zonulinlevelswerepositivelyassociatedwithgamma-glutamyltransferase,2-hinsulin,HFF,andnegativelyassociatedwithwhole-bodyinsulinsensitivityindexWBISI,afteradjustmentforage,genderandpubertalstatus.WhentheassociationswererestrictedtothegroupofNAFLDpatients,2-hinsulin,hepaticfat,andWBISIretainedstatisticalsignificance.

CONCLUSION:

CirculatingzonulinisincreasedinchildrenandadolescentswithNAFLDandcorrelateswiththeseverityofsteatosis.

©2014BaishidengPublishingGroupInc.Allrightsreserved.

Keywords:

Zonulin;Intestinalpermeability;Nonalcoholicfattyliverdisease;Nonalcoholicsteatohepatitis;Children

Coretip:

Alterationingutmicrobiotafollowedbyimpairmentofintestinalwallintegritymayplayanimportantroleinthepathogenesisofnonalcoholicfattyliverdisease（NAFLD）.Zonulinisamediatorknowntoregulateintestinalpermeabilitybymodulatingintracellulartightjunctions.Weshowedthatzonulinconcentrationsareincreasedinobesechildrenwithbiopsy-provenNAFLDandcorrelatewiththeseverityofsteatosis,butnotwiththepresenceofnonalcoholicsteatohepatitis（NASH）,lobularinflammationorfibrosisscore.ThesefindingsmaywellfitwiththerecenttheorysuggestingthatsimplesteatosisandNASHaredifferentandnotnecessarilyinter-relateddiseases.

PacificoL,BonciE,MarandolaL,RomaggioliS,BascettaS,ChiesaC.Increasedcirculatingzonulininchildrenwithbiopsy-provennonalcoholicfattyliverdisease.WorldJGastroenterol2014;Inpress

INTRODUCTION

Parallelingtheworldwideepidemicofchildhoodobesity,nonalcoholicfattyliverdisease（NAFLD）hasbecomethemostcommoncauseofchronicliverdiseaseinchildrenandadolescents[1].NAFLDisaspectrumoffat-associatedliverconditionsthatcanresultinend-stageliverdiseaseandtheneedforlivertransplantation[2].Simplesteatosis,orfattyliver,occursearlyinNAFLDandmayprogresstononalcoholicsteatohepatitis（NASH）,fibrosisandcirrhosiswithincreasedriskofhepatocellularcarcinoma[2].GrowingevidencesupportstheconceptthatNAFLDisahighlyheritablediseaseinwhichgeneticvariationsandenvironmentalfactorscloselyinteracttodeterminethediseasephenotypeandprogressiontothemoreadvancedformsofthedisease[3].Recently,ithasbeensuggestedthatalterationingutmicrobiotafollowedbyimpairmentofintestinalwallintegritymayplayanimportantroleinthepathogenesisofNAFLD[4,5].Mieleetal[6]providedthefirstevidencethatNAFLDinadultsubjectsisassociatedwithincreasedgutpermeabilityandsmallintestinalbacterialovergrowth（SIBO）andthatthesefactorsareassociatedwiththeseverityofhepaticsteatosis[6].Specifically,theincreasedpermeabilityappearedtobecausedbydisruptionofintercellulartightjunctionsintheintestine.

Zonulinisamediatorknowntoregulateintestinalpermeabilitybymodulatingintracellulartightjunctions（TJ）[7-9].Humanzonulinisa47-kDaproteinthatincreasesintestinalpermeabilityinnonhumanprimateintestinalepithelia[8],participatesinthedevelopmentofintestinalinnateimmunity[10],andisoverexpressedinautoimmunedisordersinwhichTJdysfunctioniscentral,includingceliacdisease[11,12]andtype1diabetes[13].Circulatingzonulinisconsideredasausefulmarkerofintestinalpermeability[8,14].Inhumans,elevatedserumzonulinlevelshavebeendemonstratedtocorrelatewithincreasedintestinalpermeabilityassociatedwithalteredgeneticexpressionofintestinalTJproteins[13].Recentlypublishedstudieshaveshownhighercirculatingzonulinlevelsinobesethaninnonobeseadultsandinadultswithglucoseintolerancecomparedwithagroupwithnormalglucosetolerance[15,16].Thecurrentstudywasthereforedesignedtoinvestigatethepotentialassociationofcirculatingzonulinwiththestageofliverdiseaseinobesechildrenwithbiopsy-confirmedNAFLD.

MATERIALSANDMETHODS

Patientsandcontrols

Thestudypopulationconsistedofobesechildren[bodymassindex（BMI）abovethe95thpercentileforageandgender]seenintheHepatologyoutpatientClinicoftheDepartmentofPediatrics,SapienzaUniversityofRome,Italy,forchronically（atleast6mo）elevatedaminotransferaselevels.ThediagnosisofNAFLDwasbasedonmagneticresonanceimaging（MRI）withhighhepaticfatfraction（HFF≥5%）[17],andconfirmedbyliverbiopsy.Othercausesofchronicliverdisease,includinghepaticvirusinfections（hepatitisA-EandG,cytomegalovirus,andEpstein-Barrvirus）,autoimmunehepatitis,metabolicliverdisease,α-1-antitrypsindeficiency,cysticfibrosis,Wilson’sdisease,hemochromatosis,andceliacdiseasewereexcludedwithappropriatetests.Otherexclusioncriteriawerehistoryoftype1ortype2diabetes,renaldisease,foodallergy,totalparenteralnutrition,smokingoralcoholconsumption,anduseofhepatotoxicmedications.

Forcomparativepurposes,weenrolledagroupofcontrolsubjects,matchedforage,gender,pubertalstageandascloselyaspossibleforBMI-standarddeviationscore（SDS）.Thegroupwascomposedofobesechildrenwithnormallevelsofaminotransferases,andwithoutMRIevidenceoffattyliver（HFF<5%）aswellasofothercausesofchronicliverdisease（seeabove）.Controlswerealsoexcludediftheyhadhistoryoftype1ortype2diabetes,renaldisease,foodallergy,smokingoralcoholconsumption.Finally,theuseofanti-inflammatorydrugs,antibioticsorprobioticswasconsideredamongtheexclusioncriteriaforbothcasesandcontrols.

TheresearchprotocolwasapprovedbythelocalEthicsCommittee（PoliclinicoUmbertoIHospital,Rome,Italy）andwritteninformedconsentwasobtainedfromthenextofkin,caretakers,orguardiansonbehalfofthechildrenenrolledinthisstudy,inaccordancewithprinciplesofHelsinkiDeclaration.

Clinicalandlaboratorydata

Allparticipantsunderwentphysicalexaminationincludingmeasurementsofweight,standingheight,BMI,waistcircumference（WC）,anddeterminationofthestageofpuberty.Thepubertalstagewascategorizedintotwogroups（prepubertal:

boyswithpubichairandgonadalstageI,andgirlswithpubichairstageandbreaststageI;pubertal:

boyswithpubichairandgonadalstage≥IIandgirlswithpubichairstageandbreaststage≥II）.ThedegreeofobesitywasquantifiedusingCole’sleastmean-squaremethod,whichnormalizestheskeweddistributionofBMIandexpressesBMIasSDS[18].

Bloodsamplesweretakenfromeachsubject,afteranovernightfast,forestimationofglucose,insulin,Cpeptide,totalcholesterol,high-densitylipoproteincholesterol（HDL-C）,triglycerides,alanineaminotransferase（ALT）,aspartateaminotransferase,gamma-glutamyltransferase（GGT）,high-sensitiveCreactivepr