Doxycycline(hyclate)isatetracyclineantibioticandbroad-spectrummetalloproteinase(MMP)inhibitor.

Doxycyclineshowsexcellenteffectivenessandtime-dependentcharacteristicsagainstM.gallisepticumstrainS6invitro^[2].Osteoblastsexposedtothecompositecontaining25μg/mLdoxycycline(DOX)/β-cyclodextrin(βCD)hasincreasedcellproliferation(p<0.05)=""compared=""to=""control=""osteoblast=""cultures=""at=""all=""experimental=""time=""points,=""reaching=""a=""maximum=""in=""the=""second=""week.=""alkaline=""phosphatase=""(ap)=""activity=""and=""collagen=""secretion=""levels=""are=""also=""elevated=""in=""osteoblasts=""exposed=""to=""the=""dox/βcd=""composite=""(p=""><0.05=""vs.=""controls)=""and=""reach=""a=""maximum=""after=""14="">^[3].Doxycycline(20nM)inhibitsECM(extracellularmatrix)productionandremodelinginbothSMC(smoothmusclecell)typesofcultures,andsynthesisofcollagensandisoprenylatedproteinsinSMC-Ch(acholesterol-richdiet)isahigherthaninSMC-C(astandarddiet)^[4].

Inheterozygous(HT)Col3a1knockoutmice,after3monthsoftreatmentwithdoxycyclineorplacebo,9-month-oldHTorwild-type(WT)micearesubjectedtosurgicalstressingoftheaorta.A3-foldincreaseinstress-inducedaorticlesionsfoundinuntreatedHTmice1weekafterintervention(cumulativescore4.5±0.87versus1.3±0.34inWT,p<0.001) is="" fully="" prevented="" in="" the="" doxycycline="" (25="" or="" 100="" mg/kg,="" p.o.)-treated="" group="">^[1].

• [1].WilfriedBriest,etal.DoxycyclineamelioratesthesusceptibilitytoaorticlesionsinamousemodelforthevasculartypeofEhlers-Danlossyndrome.JPharmacolExpTher.2011Jun;337(3):621-7.

  [2].ZhangN,etal.ThePK/PDInteractionsofDoxycyclineagainstMycoplasmagallisepticum.FrontMicrobiol.2016May4;7:653.

  [3].TrajanoVC,etal.Osteogenicactivityofcyclodextrin-encapsulateddoxycyclineinacalciumphosphatePCLandPLGAcomposite.MaterSciEngCMaterBiolAppl.2016Jul1;64:370-5.

  [4].Palomino-MoralesR,etal.Inhibitionofextracellularmatrixproductionandremodelingbydoxycyclineinsmoothmusclecells.JPharmacolSci.2016Mar25.Epubaheadofprint

  [5].WangX,etal.FTOisrequiredformyogenesisbypositivelyregulatingmTOR-PGC-1αpathway-mediatedmitochondriabiogenesis.CellDeathDis.2017Mar23;8(3):e2702.

Gelatin(0.1%(w/v)isaddedtostandardLaemmLiacrylamidepolymerizationmixture.Tissueextractismixed1:2withsamplebuffer[250mMTris-ClpH6.8,10%(w/v)SDS,20%(v/v)glycerol,0.005%(w/v)bromphenolblue].Serumisdiluted1:10withelectrophoresisbuffer(2.5mMTris,20mMglycine,0.005%SDS)andmixed1:2withsamplebuffer.TwentyμLsareloadedafter10-minincubationatroomtemperaturewithoutboiling.Afterelectrophoresisat90V,thegelsaresoakedin2.5%(w/v)TritonX-100,incubated2to3daysat37°Cingelatindigestionbuffer[50mMTris-Cl,pH8.0,8mMCaCl₂,10mMZnSO₂,0.02%(w/v)NaN₃],stainedin0.05%CoomassieblueR-250inaceticacid/methanol/water(1:4.5:4.5byvolume),destainedin10%aceticacidand5%methanol,andscannedforlysisbandintensity.ThelysisbandintensityisproportionaltogelatinaseactivityandisquantifieddensitometricallybyusingOne-DimensionalScansoftware.Theresult,anumberbetween0.07and3.75,isnormalizedtotheproteincontentbydividingthedensitometryresultwiththerelativeopticaldensityfromtheBCAproteinassaykitresult.Theresultisusedfortheanalysisasthearbitraryunit.ForthetotalMMPactivityresultsoflysisbandsofpro-MMP-9,activeMMP-9,pro-MMP-2,andactiveMMP-2areadded.AproteinsizeMarkerisusedtodeterminethecorrectsize.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

Doxycyclineisdilutedinculturemediumataconcentrationof10μg/mL.

AllinvitrotreatmentsareperformedinSMCculturesat90%confluence,whenECMsynthesisinSMCstartstobeevident.Doxycycline(20nM)isdilutedinculturemediumataconcentrationof10μg/mL(20nM),atwhichnotoxicityorvariationinprimaryculturedSMCproliferationhasbeenreported,aswellasinothercelllinesandtheincubationtimeis48h.SMC-CandSMC-Chareseededatequalcelldensityin6-wellplatesand,whenconfluencereaches90%,1mLculturemediumisaddedtoeachwellcontaining3.7×10⁴ BqL-[5-³H]-proline(9.62×10¹¹Bq/mmol).After48 hincubation,cellsarelysedwith0.5mL0.5mol/LNaOHfor1h.Theresultingsolutionisneutralizedwithanequalamountof0.5mol/LHCl,and50μLareusedtomeasuretotalproteinswiththeBradfordmethod.Onevolumeof10%TCAisaddedtotheremaining250μLandcentrifugedat13,000gfor15 minat4°C.Theresultingprecipitateisdissolvedin100μL0.2mol/LNaOH,andthenneutralizedwith1mol/LHCl.ThesolutionisincubatedwithCollagenasebuffer(Tris-HCl,pH7.620mM,andCaCl₂250mMfinalconcentration)and10unitsofcollagenaseat37°Covernight.Then,150μL10%TCAareaddedandcentrifugedat13000gfor15 minat4°C.Theresultingsupernatantisaddedto4mLofscintillationfluidandtherADIoactivityismeasuredinaliquidscintillationcounterLS600TA.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

Doxycyclineispreparedinfooddiet.

Twogroupsof6-month-oldfemaleheterozygous(HT)Col3a1knockoutmicearetreatedfor3monthswithdoxycycline.Treatmentisprovidedwithfoodcontaining200or800mg/kgofdoxycycline.Becausepreliminarymeasuredfoodintakeofthesemiceisaveragedat3.5g/dayandtheaveragebodyweightofanimalsis25g,theaveragedrugdoseforlow-andhigh-dosegroupsis25(Doxy25)or100(Doxy100)mg/kgperday,respectively.Untreated(wildtype)WTandHTmicearemaintainedonaregulardiet(NIH-07mouse/ratdiet)andservedascontrols.After3months,undergeneralinhalationanesthesia(2%ofisofluraneinoxygen)andasepticconditions,theaBDominalaortasaresurgicallyexposedandstressedbythefollowingtechnique:thebloodflowisstoppedbyoccludingtheabdominalaortaagainstthespinalcolumnwithasterilecotton-tipapplicatorpressedattheleveloftherenalarteries.After30sasecondapplicatorispressedatthelevelofiliacbifurcationandthefirstapplicatorisabruptlyreleased,followedbyreleaseofthesecondapplicator.Theabdominalincisionissuturedclosed,andmicearereturnedtohomecages.Thetreatmentiscontinuedaftertheintervention.Oneweekafterinterventionmiceareeuthanizedbyanoverdoseofisoflurane.Bloodiscollected,andaortasandsegmentsofcolonandskinareharvested.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].WilfriedBriest,etal.DoxycyclineamelioratesthesusceptibilitytoaorticlesionsinamousemodelforthevasculartypeofEhlers-Danlossyndrome.JPharmacolExpTher.2011Jun;337(3):621-7.

  [2].ZhangN,etal.ThePK/PDInteractionsofDoxycyclineagainstMycoplasmagallisepticum.FrontMicrobiol.2016May4;7:653.

  [3].TrajanoVC,etal.Osteogenicactivityofcyclodextrin-encapsulateddoxycyclineinacalciumphosphatePCLandPLGAcomposite.MaterSciEngCMaterBiolAppl.2016Jul1;64:370-5.

  [4].Palomino-MoralesR,etal.Inhibitionofextracellularmatrixproductionandremodelingbydoxycyclineinsmoothmusclecells.JPharmacolSci.2016Mar25.Epubaheadofprint

  [5].WangX,etal.FTOisrequiredformyogenesisbypositivelyregulatingmTOR-PGC-1αpathway-mediatedmitochondriabiogenesis.CellDeathDis.2017Mar23;8(3):e2702.

480.9

C₂₂H₂₅ClN₂O₈

24390-14-5

RoomtemperatureincontinentalUS;mayvaryelsewhere

H₂O:26mg/mL

Doxycyclineisdissolvedin50 mLofdrinkingwatercontaining10%sucroseandthenmixedwith100 gofmousechow^[5].

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

• [1].WilfriedBriest,etal.DoxycyclineamelioratesthesusceptibilitytoaorticlesionsinamousemodelforthevasculartypeofEhlers-Danlossyndrome.JPharmacolExpTher.2011Jun;337(3):621-7.

  [2].ZhangN,etal.ThePK/PDInteractionsofDoxycyclineagainstMycoplasmagallisepticum.FrontMicrobiol.2016May4;7:653.

  [3].TrajanoVC,etal.Osteogenicactivityofcyclodextrin-encapsulateddoxycyclineinacalciumphosphatePCLandPLGAcomposite.MaterSciEngCMaterBiolAppl.2016Jul1;64:370-5.

  [4].Palomino-MoralesR,etal.Inhibitionofextracellularmatrixproductionandremodelingbydoxycyclineinsmoothmusclecells.JPharmacolSci.2016Mar25.Epubaheadofprint

  [5].WangX,etal.FTOisrequiredformyogenesisbypositivelyregulatingmTOR-PGC-1αpathway-mediatedmitochondriabiogenesis.CellDeathDis.2017Mar23;8(3):e2702.