| Objectives: To study the effects of breviscapine on the transcriptional regulation of drug metabolizing enzymes CYP3A4,CYP2C19 and upstream regulatory factors PXR and CAR in Chang liver cells and preliminary study the molecular mechanisms of Breviscapine on CYP3A4 and CYP2C19.Methods:1.The cytotoxicity effect of breviscapine to Chang liver,Hep G2 and Caco-2 was detected by MTT assay.2.The expression of PXR,CAR and CYP3A4 and CYP2C19 m RNA in Chang liver cells were detected by RT-q PCR.The protein expression of CYP2C19 in Chang liver cells was detected by Western blot analysis.3.The dual-luciferase reporter gene assay was used to assess the effects of breviscapine on human PXR/CAR mediated CYP3A4 and CYP2C19 transcription.Results:1.MTT results showed,as to Chang liver cells breviscapine showed a concentration dependency and time dependency.The IC50 of breviscapine to Chang liver cells after24 h,48 h and 72 h was 9768.8,1372.8,1422.8 μmol/L respectively.Breviscapine displayed inhibitory effect at high concentration on Hep G2.As to Caco-2 cells breviscapine showed a concentration dependency.The IC50 of breviscapine to Hep G2 cells after 24 h,48 h and 72 h was 24320.5 μmol/L,30667.7 μmol/L and 15586.1μmol/L,respectively;While to Caco-2 cells its IC50 after 24 h,48 h and 72 h was11417.0 μmol/L,832.2 μmol/L and 1369.2 μmol/L,respectively.2.RT-q PCR results showed that the expression of CYP3A4 m RNA displayed promoted effect when the breviscapine concentration was 160,320 μmol/L.However,the expression of CYP3A4 m RNA displayed inhibitory effect when the concentration was 10,20,40,80 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 24 h.Compared with the control group,the relative expression of CYP3A4 m RNA decreased by 0.24(P<0.05),0.51,0.80,0.34(P<0.01)fold,respectively,when the concentration was 10,20,40,80 μmol/L.The relative expression of CYP3A4 m RNA increased by 0.08(P>0.05),0.54(P<0.01)fold,respectively,when the concentration was 160,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 48 h.The relative expression of CYP3A4 m RNA decreased by 0.09(P>0.05),0.61(P<0.01),0.50(P<0.05)and 0.00(P>0.05)fold,respectively,when the concentration was 10,20,40,80 μmol/L.The relative expression of CYP3A4 m RNA increased by 0.41 and 0.19 fold,respectively,when the concentration was 160,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 72 h.The relative expression of CYP3A4 m RNA decreased by 0.12(P>0.05),0.57(P<0.05),0.49(P>0.05),0.32(P>0.05)fold,respectively,when the concentration was 10,20,40,80 μmol/L.The relative expression of CYP3A4 increased by 1.39(P<0.05),0.9(P>0.05)fold,respectively,when the concentration was 160,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 24 h.Compared with the control group,the relative expression of PXR m RNA decreased by0.45(P<0.05),0.13(P>0.05),0.60(P<0.01),0.49(P<0.01)fold,respectively,when the concentration was 10,20,40,80 μmol/L.The relative expression of PXR m RNA increased by 0.02、0.03(P>0.05)fold,respectively,when the concentration was 160,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 48 h.The relative expression of PXR m RNA decreased by0.91(P<0.01),0.73(P<0.01),0.55(P<0.05)fold,respectively,when the concentration was 10,20,40 μmol/L.The relative expression of PXR m RNA increased by0.56(P>0.05),1.56(P<0.01),2.06(P<0.01)fold,respectively,when the concentration was 80,160,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 72 h.The relative expression of PXR m RNA decreased by 0.22,0.09,0.06(P>0.05)fold,respectively,when the concentration was10,20,40 μmol/L.The relative expression of PXR m RNA increased by 0.46,0.29,0.17(P>0.05)fold,respectively,when the concentration was 80,160,320 μmol/L.The expression of Breviscapine on CYP2C19 and CAR m RNA displayed inhibitory effect.Chang liver cells were treated with the corresponding concentration of Breviscapine for 24 h,48 h and 72 h.Compared with the control group,the relative expression of CYP2C19 m RNA increased by 0.97,0.75,1.07,0.64,1.65,1.02(P<0.01)fold;1.11(P<0.01),0.04,0.09(P>0.05),1.05,2.08,1.91(P<0.01)fold;2.25,1.47,2.10,2.77,2.24,4.40(P<0.01)fold,respectively,when the concentration was 10,20,40,80,160,320 μmol/L.Compared with the control group,the relative expression of CAR m RNA increased by 0.72,1.81,1.02,1.02,1.23,3.06(P<0.01)fold;1.48(P<0.05),1.02(P>0.05),1.52(P<0.05),2.58(P<0.01),2.59(P<0.01),2.03(P<0.05)fold;1.59(P<0.01),1.13(P<0.05),1.69(P<0.01),0.64,0.58,0.84(P>0.05)fold,respectively,when the concentration was 10,20,40,80,160,320 μmol/L.3.Western-blot results showed that breviscapine promoted the expression of CYP-2C19 protein.Chang liver cells were treated with the corresponding concentration of Breviscapine for 24 h.Compared with the control group,the relative expression of CYP2C19 protein increased by 12%(P<0.05),27%(P<0.01),8%(P<0.05),respectively,when the concentration was 10,20,320 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for 48 h.The relative expression of CYP2C19 protein increased by 28%,35%,38%,36%(P<0.05),respectively,when the concentration was 10,20,40,80 μmol/L.Chang liver cells were treated with the corresponding concentration of Breviscapine for72 h.The relative expression of CYP2C19 protein increased by 20%(P<0.05),46%,50%(P<0.05),respectively,when the concentration was 40,80,160 μmol/L.4.Transient transfection double fluorescent reporter gene tested results showed that Breviscapine had a significant induced effect on the activity of CYP3A4 promoter when the concentration was 40,80,160,320 μmol/L and there was no significant effect on CYP2C19 promoter activation after co-transfection of p SG5-h PXR,p GL3-XREM-CYP3A4/p GL3-h CYP2C19-pro and ph RL-TK in Hep G2 and Caco-2cells.The effect of Breviscapine on CYP2C19 promoter activation showed promoting effect and there was no significant effect on CYP3A4 promoter activation after co-transfection of p CMX-h CAR,p GL3-XREM-CYP3A4/p GL3-h CYP2C19-pro and ph RL-TK in Hep G2 and Caco-2 cells.Conclusion:1.Breviscapine inhibited m RNA expression of CYP3A4 and PXR when the concentration was 10,20,40,80 μmol/L and promoted m RNA expression of CYP3A4 and PXR when the concentration was 160,320 μmol/L.Breviscapine promoted the expression of CYP2C19 and CAR m RNA when the concentration was 10,20,40,80,160,320 μmol/L.2.The effect of Breviscapine on CYP3A4 was mediated by the nuclear receptor PXR pathway rather than the CAR pathway.The promoting effect of CYP2C19 was mediated by CAR rather than the PXR pathway. |
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