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EffectsofGanodermalucidumPolysaccharideonCYP2E1,CYP1A2and

CYP3AActivitiesinBCG-ImmuneHepaticInjuryinRats

XinWANG,XuanZHAO,DanLI,Ya-QingLOU,Zhi-BinLIN,andGuo-LiangZHANG*

DepartmentofPharmacology,BasicMedicalSchool,Beijing（Peking）University;38Xue-YuanRoad,Beijing100083,

P.R.China.ReceivedFebruary12,2007;acceptedJune3,2007

ThepurposeofthepresentstudywastoinvestigatetheeffectofGanodermalucidumpolysaccharide

（GLPS）,amajoractivecomponentinChinesemedicinalfungus,oncytochromeP450metabolicactivityinBacillus

CalmetteGuérin（BCG）-inducedimmunehepaticinjuryinrats.Theenzymekineticsoftheprobesincluding

chlorzoxazone（CYP2E1）,phenacetin（CYP1A2）andnifedipine（CYP3A）wereevaluatedbyHPLC.Theresults

showedthatBCG-pretreatment（125mg/kg）significantlyincreasedserumlevelsofalaninetransaminase（ALT）,

nitriteandmalondialdehyde（MDA）,inhibitedactivitiesofsuperoxidedismutase（SOD）anddecreasedP450total

contentinmicrosomes（p_0.05）.AdministrationofGLPS（50and200mg/kg）reversedabovehepaticinjury

stimulatedbyBCGinvivo.Moreover,GLPSdose-dependentlyinhibitedactivitiesofCYP2E1,CYP1A2and

CYP3Ainhepaticmicrosomesinvitro,suggestingthatinhibitionofGLPSonP450oxidativemetabolismmight

participateinthehepatoprotectivemechanism,andalsosuggestedthatpharmacokineticsmightbechangedby

drug–herbinteraction.

KeywordsGanodermalucidumpolysaccharide（GLPS）;cytochromeP450;immunehepaticinjury;BacillusCalmetteGuérin

（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.

BacillusCalmetteGuérin（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）