Study On The Effects Of Berberine On CYP3A And P-glycoprotein And Its Molecular Mechanism In Vivo And In Vitro

Background:Renal transplantation has become an effective clinical treatment of end-stagerenal failure,so,it is quite important for renal transplanted recipients long-termsurvival to make an effective and reasonable immunosuppression plan. CyclosporineA (CsA) has become the most important immunosuppressive drug used to preventorgan transplant rejection.The fact was observed in our clinical practice, the berberinechloride(BBR) could significantly increase whool-blood concentration of CsA inrenal transplanted recipients. It would be a useful means to substantially reduce thedosage of CsA and save its cost. By means of this new discovery,our lab has carriedout a series of experiments on human pharmacokinetics:we have conducted theexperiments on the pharmacokinetics of cyclosporine A in healthy volunteers andrenal transplanted recipients,and have confirmed the increasing in whool-bloodconcentration of CsA by BBR,besides, the experiments on animals: we havecomfirmed the effects of BBR or coadministration with CsA on microsomal enzymeactivity in rats and mouse,as well as the effects of BBR or Coadministration with CsAon the expression of CYP3A1,CYP3A2,CYP2E1,CYP1A1,MDR1a and MDR1b inrats and mouse. All above study has suggested that BBR could inhibit the expressionor activity of CYP3A and P-glycoprotein (P-gp) in rats and mouse liver and or smallintestine,resulting in the increased bioavailability of CsA, indirectly confirmed theeffects of BBR on CYP3A and P-gp. However, the mechanisms of regulation ofCYP3A4and P-gp by berberine is still unknown,if we can confirm the synergisticmechanisms of BBR on CsA through the study, it can provide the experiment basisfor its clinical application.Objectives:The aim of this project is to futher study the influence of berberine on CYP3Aand P-gp of rats in vivo.The effects of berberine on the mRNA and protein espressionof CYP3A4and P-gp in HepG2cells will be investigated. The effects of berberine on the mRNA expression of pregnane X receptor(PXR) and retinoid X receptor(RXR) inHepG2cells will also be investigated.All these can provide the experiment basis forclinical application of CsA.Methods:1Effects of Berberine on pharmacokinetics of midazolam and its metabolite inrats.We Created HPLC detection method to detect plasma samples of midazolam, aprobe drug of CYP3A, and its metabolite1′-hydroxy midazolam in rats, and thenassessed the effects of Berberine on the pharmacokinetics of midazolam, and itsmetabolite1′-hydroxy midazolam in rats of each group.2.Effects of Berberine on pharmacokinetics of Rhodamine123in rats.WeCreated HPLC detection method to detect Plasma samples of Rhodamine123, aprobe drug of P-gp in rats, and then assessed the effects of Berberine on thepharmacokinetics of Rhodamine123in rats of each group.3.The Cytotoxicity of Berberine on HepG2cells determined by MTT. HepG2cells were treated with different concentrations of BBR(1-100μg/mL)for48h, theneffects of BBR on the HepG2cells proliferation were detected by MTT assay. Theexperiments were taken when the cytotoxicity of BBR on HepG2cells was low.4.The effects of Berberine on protein expression of CYP3A4and P-gp in HepG2cells detected by Western blotting. The experiments were divided into the followinggroups: control group, groups administrated with different concentrations ofBBR(0.1-40μg/mL),10μM Rifampicin(Rif)group and groups administrated withdifferent concentrations of BBR (0.1-40μg/mL) and together with10μM Rif,incubated with the logarithmic phase of HepG2cells for48hours. Total protein wereextracted. The protein levels of CYP3A4,P-gp in HepG2cells were assayed withWestern blotting.5.The effects of Berberine on mRNA expression of CYP3A4,MDR1,PXR andRXR in HepG2cells were detected by Real-time quantitative PCR. According to theresults of Western blotting analysis of CYP3A4and P-gp in HepG2cells.Thefollowing experiments were divided into the following groups: control group, groupsadministrated with different concentrations of BBR(0.1-40μg/mL),10μM Rifampicin(Rif)group and groups administrated with different concentrations of BBR(0.1-40μg/mL) and together with10μM Rif, incubated with the logarithmic phase ofHepG2cells for48hours. Total RNA were extracted. The mRNA levels of CYP3A4,MDR1, pregnane X receptor (PXR） and retinoid X receptor(RXR） in HepG2cellswere assayed with Real-time quantitative PCR.To confirm whether the mRNA levelsof CYP3A4and MDR1was Associated with PXR and RXR mRNA.6.Data processing and statistical analysis: All datas were demonstrated bymean±standard deviation (x±s), calculate the Pharmacokinetic parameters ofmidazolam,1′-hydroxy midazolam and Rhodamine123of rats through DAS2.0pharmacokinetics software. Using SPSS19.0software for data processing, thedifference between the groups with the design of the t-test, the statistical results isP>0.05as no significant difference, the statistical results is P<0.05as significantdifference. the statistical results is P<0.01indicated that there as a very significantdifference.Results:1.The established HPLC detection method of midazolam and1′-hydroxymidazolam in rats was developed to meet the requirements of plasma samplesanalysis and testing requirements, besides, the other established HPLC detectionmethod of Rhodamine123in rats was also developed to meet the requirements ofplasma samples analysis and testing requirements.The two methods with highsensitivity and specificity were applicable in modeling and description of the possiblepharmacological interactions between the medicines and CYP3A enzymes or P-gptransporter.2.The Pharmacokinetic Parameters of midazolam after coadministration withberberine were as follows: AUC（0-t）(μg/mL·h):（1.25±0.29）（negative control）,（1.69±0.31）（BBR50mg/kg） and （2.03±0.44）（BBR100mg/kg） and（2.12±0.66）（BBR200mg/kg） and （2.47±0.49）（ketoconazole75mg/kg）;Cmax（μg/mL）:(1.13±0.25)（negative control）and (1.69±0.23)（BBR50mg/kg） and （1.84±0.29）（BBR100mg/kg） and （2.12±0.53）（BBR200mg/kg）and（2.21±0.29）（ketoconazole75mg/kg）. The Pharmacokinetic Parameters of1′-hydroxy midazolam after coadministration with berberine were asfollows:AUC（0-t）(μg/mL·h):（0.66±0.28）（negative control）and（0.46±0.19）（BBR50mg/kg）and（0.42±0.11）（BBR100mg/kg）and（0.36±0.09）（BBR200mg/kg） and （0.37±0.10）（ketoconazole75mg/kg）;Cmax（μg/mL）:（0.51±0.16）（negative control） and （0.44±0.13）（BBR50mg/kg） and（0.40±0.08）（BBR100mg/kg）and（0.32±0.07）（BBR200mg/kg）and（0.33±0.09）（ketoconazole75mg/kg）. Statistical analysis by SPSS19.0softwareshowed （BBR50、100、200mg/kg） and （ketoconazole75mg/kg） groupsincreased in AUC（0-t）,AUMC（0-t）and Cmax（P＜0.05）of midazolam and （BBR100、200mg/kg）and（ketoconazole75mg/kg）groups significantly decreased inAUC（0-t）,AUMC（0-t）and Cmax（P＜0.05） of1′-hydroxy midazolam dosedependent.But （BBR50mg/kg） group, the Pharmacokinetic Parameters of1′-hydroxy midazolam were not significantly altered(P>0.05).3.The Pharmacokinetic Parameters of Rhodamine123after coadministrationwith berberine were as follows: AUC（0-t）(μg/L·h):（48.36±6.4）（negativecontrol） and （59.58±13.37）（BBR50mg/kg） and （77.51±6.84）（BBR100mg/kg） and （95.49±15.99）（BBR200mg/kg） and （93.01±13.07）（verapamil4mg/kg）;Cmax（μg/L）:（4.41±0.45）（negative control）and（10.18±5.59）（BBR50mg/kg）and（11.78±3.19）（BBR100mg/kg）and（16.25±8.65）（BBR200mg/kg）and（11.39±2.76）（verapamil4mg/kg）. Statisticalanalysis showed that（BBR100、200mg/kg）and（verapamil4mg/kg）groupscould significantly increase the AUC（0-t）and Cmaxof Rhodamine123（P＜0.05）.4.When the concentration of BBR was during1μg/mL to40μg/mL, thecytotoxicity of BBR on HepG2cells was low, besides, the living rates of HepG2cellswere all above80%.5.Western blotting analysis of CYP3A4and P-gp in HepG2cells. Westernblotting analysis showed that comparing with the control group,groups administratedwith0.1,0.5and2μg/mL BBR and10μM Rif significantly induced the expression ofCYP3A4and P-gp proteins in HepG2cells(P<0.05). However, the10and40μg/mLBBR groups could markedly inhibit the expression of CYP3A4and P-gp proteins in HepG2cells(P<0.01). Compared with the Rif group,10μg/mL BBR+10μM Rif、40μg/mL BBR+10μM Rif groups inhibitted the expression of CYP3A4and P-gpproteins in HepG2cells(P<0.01).6.Real-time PCR analysis of CYP3A4mRNA in HepG2cells. Real-timequantitative PCR analysis indicated that compared with the control group, groupsadministrated with10,20and40μg/mL BBR could strongly down-regulated theCYP3A4, PXR, and MDR1mRNA expression in HepG2cells(P<0.05) but10μg/mLBBR group had no significant inhibitory effects on the RXR mRNAexpression(P>0.05),20and40μg/mL BBR groups markedly inhibited the RXRmRNA expression(P<0.05). Compared with the control group,Rif group up-regulatedthe CYP3A4,MDR1, PXR and RXR mRNA expression in HepG2cells(P<0.01),butcompared with the Rif group,10μg/mL BBR+10μM Rif、20μg/mL BBR+10μMRif、40μg/mL BBR+10μM Rif groups could strongly down-regulated the CYP3A4,MDR1, PXR and RXR mRNA expression in HepG2cells(P<0.05).Conclusions：This study firstly confirmed the effects of berberine on CYP3A and P-gp invivo from the animal level firstly,and then investigated the mechanisms of regulationof CYP3A and P-gp by berberine through PXR pathways from the molecular biologylevel.1.The current results provided direct and explicit evidence that the metabolismof midazolam (a probe drug of CYP3A) and Rhodamine123(a probe drug of P-gp)was controlled by berberine in comparison with vehicle-treated rats. Both of theresults were probably due to the inhibition of berberine on CYP3A enzymes or P-gptransporter.Thus, the berberine could be used to increase whool-blood concentrationof CsA in renal transplanted recipients to save its cost.2.The results suggested that, berberine had a dual role on the expression ofCYP3A4and P-gp. Induction was leaded by low doses of berberine, inhibition wasleaded by high doses of berberine. The mechanisms of regulation of CYP3A4and P-gp by berberine was probably through PXR pathways.