模版Word格式文档下载.docx
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模版Word格式文档下载.docx
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participateinthehepatoprotectivemechanism,andalsosuggestedthatpharmacokineticsmightbechangedby
drug–herbinteraction.
KeywordsGanodermalucidumpolysaccharide(GLPS);
cytochromeP450;
immunehepaticinjury;
BacillusCalmetteGué
rin
(BCG);
HPLC;
rat
Ganodermalucidum(LEYSS,exFR.,G.lucidum)KARST
hasbeenprescribedtoimprovehealthandlongevityinthe
traditionalChinesemedicine,anditwasusedforthetreatment
ofneurasthenia,hypertension,hepatopathyandcarcinoma
forthousandsofyears.1)Morethanonehundred
speciesofbioactivecomponentshavebeenisolatedfromG.
lucidum,2)suchaspolysaccharides,triterpenoidsandalkaloids,
inwhichthatGanodermalucidumpolysaccharide
(GLPS)isoneofthemajoractivecomponents.Itsmultiple
pharmacologicaleffects,suchasantitumor,3)antioxidation,4)
immunomodulation5,6)andespeciallyhepatoprotection
againstchemicalorimmunehepaticdamage,4,7)havebeen
demonstratedinmanyanimalmodelsinvivoandinvitro.
However,theaccurateprotectivemechanismofGLPSonthe
liverdamageisstillunknown.
CytochromeP450(P450)monooxygenasesuperfamilyis
themostimportantphaseImetabolicenzymesysteminliver.
P450isnotonlyresponsiblefortheoxidativemetabolismof
numerousexogenouscompoundsandendogenoushormones,
butalsoplaysacriticalroleinvolvingintheactivationof
variouschemicaltoxicantandprocarcinogen.8)Thereare
researcheswhichhaveconfirmedthattheexpressionsand
activitiesofsomeP450isoenzymesmaybedown-regulated
undertheinflammatoryorinfectingcondition,andthen
resultinginchangeofthemetaboliccapabilityofthe
enzymes.9)However,whetherP450down-regulationisa
homeostaticmechanismorapathophysiologicalphenomenon
hasnotbeenelucidated.Ontheotherhand,becauseG.
lucidumiscurrentlypopularlyusedasself-medicationin
EastAsia,1)thus,itisneededtobeinvestigatedthatwhether
GLPSinfluencesP450metabolicactivity.
BacillusCalmetteGué
rin(BCG),aliveattenuatedMycobacterium
bovis,isclinicallyusedasapreventingvaccinefor
tuberculosisortherapeuticregimensforbladdercancer.10)On
theotherhand,somecasesofdisseminatedBCGinfection
havebeenproventoinducegranulomatoushepatitis,sepsis
andmultipleorganfailureinthesepatients.11)Severalanimal
experimentshaveconfirmedthatBCGinfectioncouldinduce
theimmunehepaticinjuryandproductionofinflammatory
cytokines,activefreeradicalsandnitricoxide(NO).12)Moreover,
itwasreportedthatmicrosomalP450contentandactivity
weresuppressedbyNOfrominduciblenitricoxidesynthase
(iNOS)duringBCGinfectioninrodentliver.13)Therefore,
thepharmacokineticsofP450metabolizedsubstrates
maybechangedbyBCGinclinicalpatients,leadingtoenhancement
ofthetherapeuticoradverseeffectsofdrugs.
Inourpreviousexperiment,ithasbeenobservedthat
GLPSinhibitsiNOSexpressionorNOproduction,andmitigates
immuneliverinjuryinducedbyBCGinmiceinvivo
andinvitro.7)Thepurposeofthepresentstudywastoinvestigate
theeffectofGLPSonthemetabolicactivitiesofthree
P450isoenzymesincludingCYP2E1,CYP1A2andCYP3A,
whichareabundantlyexpressedinliver,andfurthertoevaluate
whetherGLPSmodulatingP450activityinvolvedinthe
hepatoprotectivemechanismunderthesimilardamagedcondition
withourpreviousstudyinrats.
MATERIALSANDMETHODS
ChemicalsandReagentsNifedipine,oxidizednifedipine,
6-hydroxychlorzoxazone,b-nicotinamide-adeninedinucleotide
phosphate,reducedform(b-NADPH),and8-
chlortheophyllinewerepurchasedfromSigma-AldrichCo.
St.Louis,MO,U.S.A.),andphenacetinfromFlukaChemie
GmbH(Buchs,Switzerland).MycobacteriumbovisBCG
vaccine,acetaminophenandchloramphenicolwereordered
fromNationalInstitutefortheControlofPharmaceuticaland
BiologicalProducts(Beijing,China).Chlorzoxazonewas
kindlyprovidedbyProfessorXiumeiZhang,SchoolofMedicine,
ShandongUniversity(Shandong,China).Allother
chemicalsandsolventsusedwereofanalyticalgrade.
PreparationofGLPSGLPSwasprovidedbyFuzhou
InstituteofGreenValleyBio-PharmTechnology(Fuzhou,
China).Thepreparation,purificationandidentificationof
GLPSwereperformedaspreviouslydescribed.14,15)Briefly,
GLPSwasextractedbyhotwaterfromtheG.lucidumfruit-
ingbody(No.ofstrainGa0801),followedbyethanolprecipitation,
dialysis,andproteindepletionusingtheSevag
method.ThetotalyieldofGLPSwas0.82%(w/w)interms
oftheG.lucidumfruitingbody.Thepurityandmolecular
weightdistributionofGLPSweredeterminedbygelpermeation
chromatography(GPC)andHPLC.Monosaccharide
compositionwasdeterminedbygaschromatography(GC).
ThestructureoftheglycopeptideswasdetectedbyIR,1Hand
13C-NMR.TheGLPSwasapeptide-boundpolysaccharide
consistingofapproximately93.61%polysaccharideand
6.49%peptides.GLPShadamolecularweightof584900,
consistingD-rhamnose,D-xylose,D-fructose,D-galactose,Dmannose,
D-glucose,anduronicacidwithamolarratioof
0.793:
0.964:
2.944:
0.167:
0.389:
7.94:
0.33.Theglycoside
linkagewasmajorb-withminora-bonding.Thepeptides
consistedof16aminoacids(Asp,Thr,Ser,Glu,Gly,Ala,
Cys,Val,Met,Ile,Leu,Phe,Lys,His,Arg,Pro).Asahazelcolored
water-solublepowder,GLPSwasdissolvedindistilled
waterandstoredat4°
Cbeforeuse.
AnimalsTreatmentandLiverDamageInduction
MaleSprague-Dawleyrats(bodyweight250_30g)were
purchasedfromtheDepartmentofExperimentalAnimals,
BeijingUniversity(Beijing,China).Theanimalstudieswere
approvedbytheAnimalEthicsCommitteeofBeijingUniversity,
andcarriedoutinaccordancewiththerequirements
ofChinanationallegislation.
Thirtyratswererandomlydividedintofivegroups:
control,
BCG,BCGplusGLPS(50or200mg/kg)andGLPS
(200mg/kg)group.Immunehepaticinjurywasinducedby
intravenousinjectionofBCG(125mg/kg)fortwoweeksin
ratsasdescribedinourpreviousreport.7)Thechoiceofdoses
andtimeperiodofGLPSwasalsobasedonourprevious
experimentandotherreports.7,16,17)AfterBCG-pretreatment
for7d,differentdosesofGLPSwereintragastricadministered
onceadayandcontinuedwithinthesuccedentone
week.Attheendoftwoweeksofimmunestimulation,rats
weresacrificedbydecapitation;
bloodsample(1ml)wascollected
forserumbiochemicalanalysis.Liverandspleenwere
removedrapidlyandweighted.Liverswerestoredat
_70°
Cforfurthermicrosomalstudyinvitro.
PreparationofHepaticMicrosomesHepaticmicrosomes
werepreparedbyreportedmethodswithmodification.18)
Afterhomogenizationandtwostepsofcentrifugation
(12500g_20min)andultracentrifugation(105000g_60
min),thepelletwasresuspendedin0.1Mpotassiumphosphate
buffer(containing1mMEDTA,pH7.4)at1ml/gliver
andstoredat_70°
Cuntiluse.Proteinconcentrationswere
estimatedusingtheBradfordassay.19)TotalP450contentwas
measuredbymethodofOmuraandSato.20)
BiochemicalAnalysisSerumalaninetransaminase
(ALT)andaspartateaminotransferase(AST)levelswereassessed
usingcommercialkitsobtainedfromBeijingBeiHua
KangTaiClinicalReagentCo.(Beijing,China).Theamount
ofNOproductionwasmeasuredbyserumnitritelevels
basedontheGriessreaction.21)Thelivermicrosomallevelof
malondialdehyde(MDA)andactivityofsuperoxidedismutase
(SOD)weredetectedusingcommercialanalysiskits
producedbyJianchengInstituteofBioengineering(Nanjing,
China).
MeasurementofP450IsozymeActivitiesUsingHPLC
TheactivitiesofCYP2E1,CYP1A2andCYP3Aisozymes
wereanalyzedusingdifferentprobedrugsbyHPLC.Liver
microsomeswereobtainedfromanothertwelveratsinabsence
orpresenceofBCGpretreatedgroupasabovedescribed
methods.TheHPLCsystemconsistedofaWaters
510pumpwith7725iinjector,Waters490UV-detectoranda
reversedphaseHPLCcolumn(AlltimaC18analyticalcolumn,
150_4.6mm,5mm,Alltech,U.S.A.).Theflow-ratesof
mobilephaseswereall1.0ml/min.
Chlorzoxazone6-HydroxylationCYP2E1activitywas
determinedviachlorzoxazone6-hydroxylationalmetabolism
inlivermicrosomesinvitro.22)Theincubationmixturein
0.5mltotalvolumecontained0.6mg/mlmicrosomalprotein,
0.1Mpotassiumphosphatebuffer(pH7.4),and500mM
chlorzoxazone(10mlof25mMstocksolution)
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