Netrin1 Protects against LArginineInduced Acute Pancreatitis in Mice.docx
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Netrin1 Protects against LArginineInduced Acute Pancreatitis in Mice.docx
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Netrin1ProtectsagainstLArginineInducedAcutePancreatitisinMice
Netrin-1ProtectsagainstL-Arginine-InducedAcutePancreatitisinMice
∙Abstract
∙Introduction
∙MaterialsandMethods
∙Results
∙Discussion
∙AuthorContributions
∙References
∙ReaderComments(0)
∙Figures
Abstract
Acutepancreatitis(AP)isacommoninflammatorydiseasemediatedbydamagetoacinarcellsandsubsequentpancreaticinflammationwithinfiltrationofleukocytes.Theneuronalguidanceprotein,netrin-1,hasbeenshowntocontrolleukocytetraffickingandmodulateinflammatoryresponsesinseveralinflammation-baseddiseases.Thepresentstudywasaimedtowardinvestigatingtheeffectsofnetrin-1inaninvivomodelofAPinmice.APwasinducedinC57BL/6micebyadministrationoftwointraperitonealinjectionsofL-Arginine(4g/kg).Miceweretreatedwithrecombinantmousenetrin-1atadoseof1µg/mouseorvehicle(0.1%BSA)intravenouslythroughthetailveinimmediatelyafterthesecondinjectionofL-Arginine,andevery24hthereafter.Miceweresacrificedatseveraltimeintervalsfrom0to96haftertheinductionofpancreatitis.Bloodandtissuesamplesofpancreasandlungwerecollectedandprocessedtodeterminetheseverityofpancreatitisbiochemicallyandhistologically.Immunohistochemicalstainingdemonstratedthatnetrin-1wasmainlyexpressedintheisletcellsofthenormalpancreasandtheAPmodelpancreas,andthepancreaticexpressionofnetrin-1wasdown-regulatedatboththemRNAandproteinlevelsduringthecourseofAP.Exogenousnetrin-1administrationsignificantlyreducedplasmaamylaselevels,myeloperoxidaseactivity,pro-inflammatorycytokineproduction,andpancreasandlungtissuedamages.Furthermore,netrin-1administrationdidnotcausesignificantinhibitionofnuclearfactor-kappaBactivationinthepancreasofL-Arginine-inducedAP.Inconclusion,ournoveldatasuggestthatnetrin-1iscapableofimprovingdamageofpancreasandlung,andexertinganti-inflammatoryeffectsinmicewithsevereacutepancreatitis.Thus,ourresultsindicatethatnetrin-1mayconstituteanoveltargetinthemanagementofAP.
Citation:
ChenJ,CaiQ-p,ShenP-j,YanR-l,WangC-m,etal.(2012)Netrin-1ProtectsagainstL-Arginine-InducedAcutePancreatitisinMice.PLoSONE7(9):
e46201.doi:
10.1371/journal.pone.0046201
Editor:
JuanSastre,UniversityofValencia,Spain
Received:
April28,2012;Accepted:
August28,2012;Published:
September27,2012
Copyright:
©Chenetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:
ThestudywasfundedbyShanghaiMunicipalScienceandTechnologycommission(no.10JC1412700)andtheNaturalScienceFoundationofChina(no.30901770andno.81150110493).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
Competinginterests:
Theauthorshavedeclaredthatnocompetinginterestsexist.
Introduction
Acutepancreatitis(AP)isaninflammatoryconditionwithaclinicalcoursethatvariesfrommildtosevere[1].SevereAPremainsalife-threateningdiseasewithahighmortalityrateamongadefinedproportionofthoseaffected[2].ThecurrentmanagementofAPislimitedtosupportivecareandtreatmentofcomplicationswhentheydevelop;thus,aneffectivetreatmentisurgentlyneeded[3],[4].Inpastdecades,intenseeffortshavebeendevotedtoelucidatingtheeventsresponsiblefortheinitiationandseverityofthediseasesothatnoveltherapeutictargetsmaybeidentified.Althoughtheexactmechanismsareforthemostpartunknown,thereissomeevidencethattheseverityandoutcomeofAPmightbedeterminedbytheacinarcellresponsetoactivationoftrypsinogen,aswellastheeventsthatoccursubsequenttoacinarcellinjury,includingactivationoftranscriptionfactorssuchasnuclearfactor-kappaB(NF-κB),recruitmentofinflammatorycells,andgenerationofinflammatorymediators[1],[5].Inlightofthisknowledge,inhibitionoftheinflammatorypathwayseemstobethemostpromisingapproachforpreventingthedevelopmentofthedisease.
Overthepastfewyears,theneuronalguidanceprotein,netrin-1,hasreceivedconsiderableattentionforitspotentialroleininflammation-baseddiseases.Netrin-1wasoriginallyidentifiedasadiffusiblefactorreleasedbyneuralfloorplatecellsinthedevelopingspinalcordthatregulatesaxonaloutgrowthandgrowthconemigration[6],[7].Subsequentstudiesfoundthatnetrin-1isalsoexpressedoutsidethecentralnervoussystemandcontrolsleukocytetraffickingfromthevascularspaceintositesofacuteinflammation[8],[9].Recentstudiesinvariousinflammation-baseddiseases,suchaskidneyischemiareperfusioninjury,hypoxia-inducedinflammation,acutelunginjury,peritonitis,andinflammatoryboweldisease,showedthatnetrin-1holdsanti-inflammatorypotentialandcanreducelocalinflammatorytissueinjury[10]–[14].However,theroleofnetrin-1inacutepancreatitis,tothebestofourknowledge,hasnotbeeninvestigatedtodate.
Inthedevelopingpancreas,netrin-1actsasaregulatorofcelladhesion,migration,anddifferentiation[15].Itwasthoughtthatnetrin-1wasabsentinthenormaladultpancreas,butnetrin-1hasbeenfoundinpancreaticadenocarcinomaandisimplicatedintumorigenesis[15],[16].Inaddition,arecentstudyrevealedthatnetrinproteinsarepresentinadultpancreaticislets,andnetrin-1modulatesbetacellapoptosissignalingviadependencereceptors[17].Thesefindingssuggestthatnetrin-1maybeinvolvedintheprogressionofpancreaticdiseases.APisacommoninflammatorydiseasemediatedbydamagetoacinarcellsandsubsequentpancreaticinflammationwithrecruitmentofleukocytes[1],[5].Wefirsthypothesizedthatnetrin-1couldprotectagainstacutepancreatitisbyinhibitingleukocyteinfiltrationandsuppressingtheinflammatoryresponse.Totestthishypothesis,weusedrecombinantmousenetrin-1toinvestigatetheeffectsofnetrin-1duringexperimentalpancreatitis.Wefoundthatexogenousnetrin-1administrationcouldreduceplasmaamylaselevels,myeloperoxidase(MPO)activity,pro-inflammatorycytokineproduction,andpancreaticandpulmonarydamageinthemousemodelofL-Arginine-inducedAP.
MaterialsandMethods
Experimentalprocedures
AllanimalexperimentswereapprovedbytheAnimalResearchCommitteeatShanghaiSecondMilitaryMedicalUniversityandwerecarriedoutinaccordancewithestablishedInternationalGuidingPrinciplesforAnimalResearch.C57BL/6mice(male,20–25g)wereusedandmaintainedintheAnimalHousingUnitinanenvironmentwithcontrolledtemperature(21–24°C)andlighting(12hlight-darkcycle).Standardlaboratorychowanddrinkingwaterwereprovidedadlibitum.Aperiodofatleast5dayswasallowedfortheanimalstoacclimatizebeforeanyexperimentalprocedureswereundertaken.
InductionofAP
L-ArgininehydrochloridewaspurchasedfromSigmaChemical(Sigma-Aldrich,St.Louis,MO,USA).Micewererandomlyassignedtothreegroups.Group1:
Animalsweregiventwointraperitonealinjectionsofnormalsaline,1hapart(Normalgroup).Group2:
AnimalswereinjectedintraperitoneallywithL-Arginineintwodoseof4g/kgbodyweighteach,1hapart(APgroup)[18].Group3:
Micewereadministeredrecombinantmousenetrin-1(R&DSystems,Minneapolis,MN)atadoseof1µg/mouseintravenouslythroughthetailveininavolumeof0.1mlimmediatelyafterthesecondinjectionofL-Arginine(0h)andevery24hthereafter(AP+Netrin-1group).ThenormalgroupandAPgroupofmicereceivedsimilarinjectionsofvehicle(0.1%BSA)atthesametimepoint.Animalsweresacrificedatseveraltimeintervalsfrom0to96hafterinductionofAP.Bloodsamplesweretakentodetermineserumamylaseandcytokinelevels.Thepancreasandrightlungwererapidlyremovedfromeachmouseformorphologicexaminationandscoring.Portionsoftheseorganswerealsostoredat−80°Cforfurtherinvestigation.
Morphologicalexamination
Samplesofpancreasandlungwerefixedin4%paraformaldehydebufferedwithPBS(pH7.4).Thetissueswerethenembeddedinparaffin,and5µmsectionswereprocessedforhematoxylinandeosin(H&E)stainingbystandardprocedures.Thepancreaticdamagewasevaluatedinablindedmannerusingapreexistingscoringsystemona0(absent)to4(extensive)scale,aspreviouslydescribed[19].Totalhistologicalscoresofpancreaswereobtained,representingthesumofthescoresforedema,inflammatorycellinfiltration,hemorrhage,andacinarcellnecrosis.Thelunginjurywasscoredaccordingtoinflammatorychanges,pulmonaryedema,andhemorrhageofalveoliandinterstitialtissue.Eachpathologicalchangewasscoredonascalefrom0–3(normal,0;minimalchange,1;mediumchange,2;andseverechange,3).
Amylaseestimation
Plasmaamylaseactivitywasmeasuredusingakineticspectrophotometricassay.Plasmasampleswereincubatedwithamylasereagent(Sigma)for2minat37°C,andabsorbancewasmeasuredeveryminuteforthesubsequent2minat405nmaccordingtothemanufacturer'sinstructions.Theresultingchangeinabsorbancewasusedtocalculateamylaseactivity.
MPOestimation
NeutrophilsequestrationinpancreasandlungwasquantifiedbymeasuringtissueMPOactivity.Forthemeasurements,tissuesampleswereimmediatelyhomogenizedonicein5volumesofnormalsaline.MPOactivitywasmeasuredusingtheMPOassaykit(NanjingJianchengCorp.,Nanjing,China),followingthemanufacturer'srecommendations.OneunitofMPOactivityisdefinedasdegrading1µmolo
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