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1、模版Effects of Ganoderma lucidum Polysaccharide on CYP2E1, CYP1A2 andCYP3A Activities in BCG-Immune Hepatic Injury in RatsXin WANG, Xuan ZHAO, Dan LI, Ya-Qing LOU, Zhi-Bin LIN, and Guo-Liang ZHANG*Department of Pharmacology, Basic Medical School, Beijing (Peking) University; 38 Xue-Yuan Road, Beijing

2、100083,P. R. China. Received February 12, 2007; accepted June 3, 2007The purpose of the present study was to investigate the effect of Ganoderma lucidum polysaccharide(GLPS), a major active component in Chinese medicinal fungus, on cytochrome P450 metabolic activity in BacillusCalmette Gurin (BCG)-i

3、nduced immune hepatic injury in rats. The enzyme kinetics of the probes includingchlorzoxazone (CYP2E1), phenacetin (CYP1A2) and nifedipine (CYP3A) were evaluated by HPLC. The resultsshowed that BCG-pretreatment (125 mg/kg) significantly increased serum levels of alanine transaminase (ALT),nitrite a

4、nd malondialdehyde (MDA), inhibited activities of superoxide dismutase (SOD) and decreased P450 totalcontent in microsomes (p_0.05). Administration of GLPS (50 and 200 mg/kg) reversed above hepatic injurystimulated by BCG in vivo. Moreover, GLPS dose-dependently inhibited activities of CYP2E1, CYP1A

5、2 andCYP3A in hepatic microsomes in vitro, suggesting that inhibition of GLPS on P450 oxidative metabolism mightparticipate in the hepatoprotective mechanism, and also suggested that pharmacokinetics might be changed bydrugherb interaction.Key words Ganoderma lucidum polysaccharide (GLPS); cytochrom

6、e P450; immune hepatic injury; Bacillus Calmette Gurin(BCG); HPLC; ratGanoderma lucidum (LEYSS, ex FR., G. lucidum) KARSThas been prescribed to improve health and longevity in thetraditional Chinese medicine, and it was used for the treatmentof neurasthenia, hypertension, hepatopathy and carcinomafo

7、r thousands of years.1) More than one hundredspecies of bioactive components have been isolated from G.lucidum,2) such as polysaccharides, triterpenoids and alkaloids,in which that Ganoderma lucidum polysaccharide(GLPS) is one of the major active components. Its multiplepharmacological effects, such

8、 as antitumor,3) antioxidation,4)immunomodulation5,6) and especially hepatoprotectionagainst chemical or immune hepatic damage,4,7) have beendemonstrated in many animal models in vivo and in vitro.However, the accurate protective mechanism of GLPS on theliver damage is still unknown.Cytochrome P450

9、(P450) monooxygenase superfamily isthe most important phase I metabolic enzyme system in liver.P450 is not only responsible for the oxidative metabolism ofnumerous exogenous compounds and endogenous hormones,but also plays a critical role involving in the activation ofvarious chemical toxicant and p

10、rocarcinogen.8) There areresearches which have confirmed that the expressions andactivities of some P450 isoenzymes may be down-regulatedunder the inflammatory or infecting condition, and thenresulting in change of the metabolic capability of theenzymes.9) However, whether P450 down-regulation is ah

11、omeostatic mechanism or a pathophysiological phenomenonhas not been elucidated. On the other hand, because G.lucidum is currently popularly used as self-medication inEast Asia,1) thus, it is needed to be investigated that whetherGLPS influences P450 metabolic activity.Bacillus Calmette Gurin (BCG),

12、a live attenuated Mycobacteriumbovis, is clinically used as a preventing vaccine fortuberculosis or therapeutic regimens for bladder cancer.10) Onthe other hand, some cases of disseminated BCG infectionhave been proven to induce granulomatous hepatitis, sepsisand multiple organ failure in these pati

13、ents.11) Several animalexperiments have confirmed that BCG infection could inducethe immune hepatic injury and production of inflammatorycytokines, active free radicals and nitric oxide (NO).12) Moreover,it was reported that microsomal P450 content and activitywere suppressed by NO from inducible ni

14、tric oxide synthase(iNOS) during BCG infection in rodent liver.13) Therefore,the pharmacokinetics of P450 metabolized substratesmay be changed by BCG in clinical patients, leading to enhancementof the therapeutic or adverse effects of drugs.In our previous experiment, it has been observed thatGLPS i

15、nhibits iNOS expression or NO production, and mitigatesimmune liver injury induced by BCG in mice in vivoand in vitro.7) The purpose of the present study was to investigatethe effect of GLPS on the metabolic activities of threeP450 isoenzymes including CYP2E1, CYP1A2 and CYP3A,which are abundantly e

16、xpressed in liver, and further to evaluatewhether GLPS modulating P450 activity involved in thehepatoprotective mechanism under the similar damaged conditionwith our previous study in rats.MATERIALS AND METHODSChemicals and Reagents Nifedipine, oxidized nifedipine,6-hydroxychlorzoxazone, b -nicotina

17、mide-adenine dinucleotidephosphate, reduced form (b -NADPH), and 8-chlortheophylline were purchased from Sigma-Aldrich Co.St. Louis, MO, U.S.A.), and phenacetin from Fluka ChemieGmbH (Buchs, Switzerland). Mycobacterium bovis BCGvaccine, acetaminophen and chloramphenicol were orderedfrom National Ins

18、titute for the Control of Pharmaceutical andBiological Products (Beijing, China). Chlorzoxazone waskindly provided by Professor Xiumei Zhang, School of Medicine,Shandong University (Shandong, China). All otherchemicals and solvents used were of analytical grade.Preparation of GLPS GLPS was provided

19、by FuzhouInstitute of Green Valley Bio-Pharm Technology (Fuzhou,China). The preparation, purification and identification ofGLPS were performed as previously described.14,15) Briefly,GLPS was extracted by hot water from the G. lucidum fruit-ing body (No. of strain Ga0801), followed by ethanol precipi

20、tation,dialysis, and protein depletion using the Sevagmethod. The total yield of GLPS was 0.82% (w/w) in termsof the G. lucidum fruiting body. The purity and molecularweight distribution of GLPS were determined by gel permeationchromatography (GPC) and HPLC. Monosaccharidecomposition was determined

21、by gas chromatography (GC).The structure of the glycopeptides was detected by IR, 1Hand13C-NMR. The GLPS was a peptide-bound polysaccharideconsisting of approximately 93.61% polysaccharide and6.49% peptides. GLPS had a molecular weight of 584900,consisting D-rhamnose, D-xylose, D-fructose, D-galacto

22、se, Dmannose,D-glucose, and uronic acid with a molar ratio of0.793 : 0.964 : 2.944 : 0.167 : 0.389 : 7.94 : 0.33. The glycosidelinkage was major b - with minor a -bonding. The peptidesconsisted of 16 amino acids (Asp, Thr, Ser, Glu, Gly, Ala,Cys, Val, Met, Ile, Leu, Phe, Lys, His, Arg, Pro). As a ha

23、zelcoloredwater-soluble powder, GLPS was dissolved in distilledwater and stored at 4 C before use.Animals Treatment and Liver Damage InductionMale Sprague-Dawley rats (body weight 250_30 g) werepurchased from the Department of Experimental Animals,Beijing University (Beijing, China). The animal stud

24、ies wereapproved by the Animal Ethics Committee of Beijing University,and carried out in accordance with the requirementsof China national legislation.Thirty rats were randomly divided into five groups: control,BCG, BCG plus GLPS (50 or 200 mg/kg) and GLPS(200 mg/kg) group. Immune hepatic injury was

25、 induced byintravenous injection of BCG (125 mg/kg) for two weeks inrats as described in our previous report.7) The choice of dosesand time period of GLPS was also based on our previousexperiment and other reports.7,16,17) After BCG-pretreatmentfor 7 d, different doses of GLPS were intragastric admi

26、nisteredonce a day and continued within the succedent oneweek. At the end of two weeks of immune stimulation, ratswere sacrificed by decapitation; blood sample (1 ml) was collectedfor serum biochemical analysis. Liver and spleen wereremoved rapidly and weighted. Livers were stored at_70 C for furthe

27、r microsomal study in vitro.Preparation of Hepatic Microsomes Hepatic microsomeswere prepared by reported methods with modification.18)After homogenization and two steps of centrifugation(12500 g_20 min) and ultracentrifugation (105000 g_60min), the pellet was resuspended in 0.1 M potassium phosphat

28、ebuffer (containing 1mM EDTA, pH 7.4) at 1 ml/g liverand stored at _70 C until use. Protein concentrations wereestimated using the Bradford assay.19) Total P450 content wasmeasured by method of Omura and Sato.20)Biochemical Analysis Serum alanine transaminase(ALT) and aspartate aminotransferase (AST

29、) levels were assessedusing commercial kits obtained from Beijing Bei HuaKang Tai Clinical Reagent Co. (Beijing, China). The amountof NO production was measured by serum nitrite levelsbased on the Griess reaction.21) The liver microsomal level ofmalondialdehyde (MDA) and activity of superoxide dismu

30、tase(SOD) were detected using commercial analysis kitsproduced by Jiancheng Institute of Bioengineering (Nanjing,China).Measurement of P450 Isozyme Activities Using HPLCThe activities of CYP2E1, CYP1A2 and CYP3A isozymeswere analyzed using different probe drugs by HPLC. Livermicrosomes were obtained

31、 from another twelve rats in absenceor presence of BCG pretreated group as above describedmethods. The HPLC system consisted of a Waters510 pump with 7725i injector, Waters 490 UV-detector and areversed phase HPLC column (Alltima C18 analytical column,150_4.6 mm, 5m m, Alltech, U.S.A.). The flow-rat

32、es ofmobile phases were all 1.0 ml/min.Chlorzoxazone 6-Hydroxylation CYP2E1 activity wasdetermined via chlorzoxazone 6-hydroxylational metabolismin liver microsomes in vitro.22) The incubation mixture in0.5 ml total volume contained 0.6 mg/ml microsomal protein,0.1 M potassium phosphate buffer (pH 7.4), and 500m Mchlorzoxazone (10m l of 25mM stock solution)