Molecules Free Full

中安在线

2018-06-09

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Molecules   Free Full

(PF,(1)),amonoterpeneglycoside,isoneoftheprincipalbioactivecomponentsextractedfromtherootofpallthathasbeenusedinTraditionalChineseMedicineforthousandsofyears[,].Recently,,protectthecerebralischemiainjury,haveeffectsonthetreatmentofAlzheimer’sdisease,relievepainandimproveneuralsynapseplasticity[,,].PFisahighlywater-solublephenolicglucosidewithpoorliposolubility[,],anddoesnoteasilypassthroughtheblood–brainbarrier(BBB).Therefore,en(TCM),certainTCM,calledmessengerdrugs,areconsideredcapableofintroducingthemaineffectivedru(Hort.)isonesuchmessengerdrug,whichisfrequentlyusedinthetreatmentofencephalopathy[].Ourpreviousresearchandsomereportshaveshownthatitcouldenhancedrugabsorptionthroughgastrointestinaltract[],andincreasethedistributionofdrugsinbraintissue[,,].,whichcontainsPall.,Hort.,(Oliv.)Diels.,(Schw.)Wolf,(Sam.),hasbeenusedtotreatischemicstrokeandAlzheimer’sdiseases[,].,whichhasbeencommonlyusedforthetreatmentofstroke,alsocontainschuanxiongandPF[].BuyangHuanwudecoctioncontainingchuanxionghasabettertherapeuticeffectoncerebralischemiathanBuyangHuanwudecoctionwithoutchuanxiong,,t,lactones,alkaloids,volatileoilandotheringredients[].Volatileoilofchuanxiongcouldenhancedrugpermeationthroughskin[],promotethesurvivalofcerebralcortexneurons,andalleviatethedamagescausedbyischemiareperfusion[].Ligustilide((2),LIG),SenkyunolideI((3),SENI)andSenkyunolideA((4),SENA),phthalidecompoundsinvolatileoil,havevariouspharmacologicaleffectscloselyassociatedwithchuanxiong[,,]andcouldbeabsorbedintoratbraintissuesthroughBBB[].Inthisarticle,LIG,SENIandSENAwereregardeda(P-gp),encodedbytheABCB1gene,belongstotheATP-bindingcassettesuperfamilyandisconstitutivelyexpressedattheluminalmembraneofthebrainendothelialcells[].ThebiologicalfunctionofP-gpistoprotectb,itcausestheintracellul,compoundsthatinhibitP-gpactivitiesmayhavethepotentialtoimprovetheclinicaloutcomesofdrugs[].Untilnow,itwasunclearwhetherLIG,SENAandSENI(MDCK-MDR1)cell,whichishumanP-gp-overexpressing,ers,whicharealsofoundincerebralendothelialcells,andhavebeenreportedasasuitablemodelforBBB[,].MDCK-MDR1cellwasconsideredtobeoneofpromisingcellstodeterminethepotentialdistributionofdrugsinbrainandthetransportmechanismofdrugsthatpassedtheBBB[,].ThepresentstudywasundertakentoexplorethetransportmechanismofPFusingMDCK-MDR1cellsmodes,toevaluatetheinfluenceofLIG,SENIandSENAonthetransportofPF,doftheMTTassay,–800μg/,,SENAandSENIgroupshavenocellcytotoxicityintheconcentrationrangeof0–120μg/μg/mLPFcombinedwiththreeingredientsofchuanxiong(/)havenocellcytotoxicityintheconcentrationrangeof0–80μg/mLcalculatedwithLIG,SENI,().Ingeneral,drugswithhighPapp(1×105cm/s)canbewell-absorbed,whilethosewithlowPapp(1×106cm/s)arepoorlyabsorbed[].ThePapp(A→B)×106cm/→Bdirectiondidnotdiffersignificantly(),andthefluxesofPFwerelinearlyassociatedwiththeconcentration(),whichindicatedthatthetransportofPFwasconcentrationdependent,=(=,=3),andthelinearequationfrombasolateralsidetoapicalsidewasy=+(=,=3),→A/A→BpermeabilityofPFwasmorethan2intheMDCK-MDR1cells,whichindicatedthatthetransportmechanismofPFmightbepassivediffusionasthedomina,aP-gpinhibitor,verapamil(VER),wasaddedtomediumthatcontained400μg/mLPF().ThePapp(A→B)(×106cm/s).InthepresenceofP-gpinhibitor,theB→A/A→,whichsuggestedthatPFwasP-gpsubstance[].Althoughthesurfaceareaismuchlargerforthetranscellularroute,theparacellularrouteisthepreferredabsorptionpathwayformanywater-solubleandpoorlylipid-solubledrugs,ionizeddrugs,nctionoftheendotheliocyteandincreasetheparacellularpermeationofhydrophilicmacromolecules[,].Ifthetransportmechanismofadruginvolvesparacellulardiffusion,itstransp,inthisstudy,weinvestigatedthetransportofPFintheabsenceorpresenceofEDTA-Na2()./LEDTA-Na2wasadded().ThePapp(A→B)ofPFincreaseduptoaboutfive-foldincomparisonwiththenon-EDTA-Na2added(×106cm/s).–druginteractionexperimentofPFtransportwascarriedoutinMDCK-MDR1cellstoinvestig,causedbyvariousconcentrationsofLIG,SENAandSENI,→BdirectionwithrisingconcentrationofLIG,(40and80μg/mL),theA→BfluxesofPFinMDCK-MDR1weresignificantlyincreased(,respectively);inthepresenceofSENIorSENA(20,40,and80μg/mL),thePapp(A→B)ofPFinMDCK-MDR1weresignificantlyincreased(,,,respectively);and80μg/mLofSENAhasthestrongestpromotingeffect,foritsignificantlyincreasedbyapproximately109%(TEER)ofthecellmonolayerintheabsenc(20μg/mL)andSENA(80μg/mL)significantlydecreasedduringpermeationstudies().TEERofcellstreatedwithLIG(80and40μg/mL),SENA(40and20μg/mL)andSENI(80μg/mL)markedlydecreasedduringpermeationstudies().AdecreaseinTEERduringthepermeationexperimentcanbeexplainedbythemodulationandopeningofthetightjunctionscausedbythepermeationenhancerLIG,SENIandSENA,(Rho123),anincreasedcellularaccumulationofRho123isgenerallyconsideredamarkerofdiminishedP-gpeffluxfunction[].Theeffectsofdiffere,asP-gpinhibitor,significantlyenhancedtheintakeofRho123().LIG,SENAandSENIcouldincreasetheintakeofRho123inadose-dependentmanner().Comparedwithcontrolgroup,80μg/mLofSENAsignificantlyincreasedbyapproximately87%intakeofRho123(),while80μg/mLofLIGandSENImarkedlyincreasedbyapproximately35%().While40μg/mLofLIG,SENIandSENAincreasedbyapproximately15%intakeofRho123whencomparedwithcontrolgroup,therewasnosignificantdifference().ThesedatasuggestedthatLIG,SENIandSENAcoulddown-regulateP-gpeffluxfunctiontoenhancethetransportofP-gpsubstrates,,,correspondingtoP-gp(A).ItwasfoundthatP-gpexpressionsinMDCK-MDR1cellsweremarkedlydown-regulatedbyLIG,,P-gpexpressionsofcellstreatedwithLIG(20μg/mL)andSENA(20μg/mL)significantlydown-regulated().P-gpexpressionsofcellstreatedwithLIG(40,80μg/mL),SENA(40,80μg/mL)andSENI(20,40,80μg/mL)markedlydown-regulated().ThesedatasuggestedthattheenhancedtransportsofPFcanbeexplainedbythereducedexpressionofP-gpinMDCK-MDR1cellscausedbyLIG,()isc,,itminimizesalltheligandposesandbasedontheligandsscoretheyareranked[,,].Thedockinganalysisinrevealedtheinterpsubstrate,,,SENAandSENIwithlowerLibDockScorecanbeexplainedbythattheyenhancedthetransportofPFb(PET)cellcultureinsertsand12-wellplates(12mmdiameter,μmporesize)werepurchasedfromCorningCostarCorporation(Cambridge,MA,USA).PFandverapamilwereobtainedfromtheNationalInstitutefortheControlofPharmaceuticalandBiologicalProducts(Beijing,China).LIG,SENIandSENAwerepurchasedfromChengduHerbpurifyCo.,Ltd.(Chendu,China).Rhodamine123waspurchasedfromNanjingKeyGENBioTeCHCo.,Ltd.(Nanjing,China).Acetonitrile(MredaInc.,Merck,Darmstadt,Germany),Ltd.(Shanghai,China).CellswereculturedinDulbecco’smodifiedEagle’smedia(DMEM,containing-glutamineand25mMHEPES),supplementedwith10%heat-inactivatedfetalbovineserum(FBS)aswellas100U/mLpenicillinand100μg/°Cwith5%,000cells/cm2,andweregrowntocon,LIG,SENIandSENAtointerferewiththegrowthofMDCK-MDR1cellswadeterminedwiththeaidofthe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT),SENIandSENAweredissolvedanddilutedbyHBSScontaining1%alvolumeof200μLofculturemediumatadensityof1×105cells/°Cina5%,themediumwasremovedandreplacedw,20μLofa5mg/mLMTTsolutioninculturemediumwasaddedtoeachwellandthemixtureswereincubatedat37°ialdehydrogenaseandassumesthatcellviability(correspondingtothereductiveactivity)isproportionaltotheproductionofpurpleformazan,(BioTek,Winooski,VT,USA).Wellswithdrugsandcellswasusedassamplegroup,edasablankcontrol,trol,apercentageoftheabsornHank’sbufferedsaltsolution(HBSS,)for30minat37°(TEER)valuesweremeasuredacrossthemonolayersusingaMilliporeMillicellERSsystemequippedwithchopstickelectrodes(MilliporeCorporation,Billerica,MA,USA).ApproximateMDCK-MDR1valuesrangedfrom500to800Ω·(A)→basolateral(B)transporter,(B)→apical(A)[].ThefinalorganicsolventconcentrationinHBSSwasalwayskeptbelow1%,aconcentration,whichdidnotaltercellviabilityorpermeability[].PFtransportwasstudiedatfiveconcentrations:100,200,400,600and800μg/,SENAandSENIonPFtransportwerestudiedat13,20,40and80μg/mLconcentrationsinthepresenceof400μg/μmol/°(tostudyB→Atransport)and500μLofaliquotsweretakenfromthebasalside(tostudyA→Btransport)at30,60,90,120,μLofPFsampleswasmixedwith180μLmethanol,andthen20μμg/mLwasadded,byvortexagitationfor3minandthencentrifugedfor10minat16,/,anautosamplerSIL-30AC,acolumnovenCTO-30AC,asolventdegasserDGU-20A3andacontrollerCBM-20AfromABSciex(Framingham,MA,USA)(100×,μm,Torrance,CA,USA).Thecolumnovenwasmaintainedat30°s:5%B,0–;5%–20%B,–;20%–40%B,–;40%–90%B,–;90%B,–;90-5%B,;and5%B,–,/μs(ABSciex):TurboIonSpray(TIS)temperature,650°C;ionsprayvoltage,4500V;curtaingas,nitrogen,30;nebulizinggas,50;TISgas,50;declusteringpotential,130eVforPF,0eVforinternalstandard(IS,gastrodin);collisionenergy,30eVforPF,–,–;()aywascalculatedusingthefollowingEquation(1):。

Molecules Free Full ”秦怀对孙依依的立场转变显然有些不太顺应,想不明确本人那里说错话了,明显看着有门的工作,怎样忽然就这样了呢。 Molecules Free Full