Article Figures & Data
Figures
- Fig. 1.
Hepatic distribution of silybin in mice. Mice were orally administrated with silybin (50 mg/kg/d and 150 mg/kg/d, once daily) for a consecutive 14 days. The livers were collected at indicated time points (5 mice per time point) after the last administration. Exposure of silybin in livers was analyzed by a liquid chromatography tandem mass spectrometry system. The data are expressed as mean ± S.D.
- Fig. 2.
The mRNA levels of principal P450s (A) and UGTs (B) in mice liver samples. Mice were intragastrically treated with 50 mg/kg or 150 mg/kg silybin for 2 weeks once per day. Twenty-four hours after the last administration, mice were sacrificed and the livers were immediately removed. The mRNA levels of Cyp1a2, Cyp2c29, Cyp2e1, Cyp3a11, Ugt1a1, Ugt1a6, Ugt1a7, Ugt1a9, and Ugt2b were determined via reverse-transcriptase PCR analysis; glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples.
- Fig. 3.
Protein levels of principal P450s (A) and UGTs (B) in mice liver samples. Protein levels of CYP1A2, CYP2C, CYP2E1, CYP3A11, UGT1A1, UGT1A6, UGT1A, and UGT2B were determined via Western blot analysis; glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples.
- Fig. 4.
Mechanism-based inhibition of P450s by silybin in MLM. In NADPH-dependent inhibitory assays, 100 μM silybin was incubated with normal mouse liver microsomes in the presence or absence of NADPH-generating system. In time- and concentration-dependent inhibitory assays, silybin (0, 10, 50, and 100 μM) was preincubated for different periods (0, 2, 4, and 8 minutes) in the presence of NADPH, followed by incubation with each probe substrate. The remaining enzyme activities were then determined, as described in Materials and Methods. The kobs was obtained from the slop of the individual lines, and these slops were fit to a Kitz-Wilson plot (inset). (A) CYP3A11, (B) CYP2C, (C) CYP1A2.
- Fig. 5.
Mechanism-based inhibition of P450s by silybin in HLM. In NADPH-dependent inhibitory assays, 100 μM silybin was incubated with normal mouse liver microsomes in the presence or absence of NADPH-generating system. In time- and concentration-dependent inhibitory assays, silybin (0, 10, 50, and 100 μM) was preincubated for different periods (0, 2, 4, and 8 minutes) in the presence of NADPH, followed by incubation with each probe substrate. The remaining enzyme activities were then determined, as described in Materials and Methods. The kobs was obtained from the slop of the individual lines, and these slops were fit to a Kitz-Wilson plot (inset). (A) CYP3A4/5, (B) CYP2C9, (C) CYP1A2.
- Fig. 6.
Mechanism-based inactivation (A, B) and competitive inhibition (C, D) of silybin on UGT1A1. Silybin was preincubated in normal mouse (A) and human (B) liver microsomes for 0.5 hour before the enzyme activity assay of UGT1A1 in the presence or absence of NADPH-generating system. Data are expressed as percentage of control, and bars represent mean ± S.D. (n = 3). *P < 0.05, compared with control group (with or without NADPH, respectively). #P < 0.05, ##P < 0.01, compared with relative group treated with the same dose of silybin without NADPH. Silybin was coincubated with β-estradiol in normal mouse liver microsomes (C) and human liver microsomes (D). Double reciprocal plots for the kinetics of inhibition of β-estradiol glucuronidation by silybin in normal mouse liver microsomes.
- Fig. 7.
Effect of silybin on PPARα signal. (A) mRNA levels of Pparα and its target genes (L-Fabp and Acox1) in the liver of mice treated with silybin. Mice were treated with 50 mg/kg or 150 mg/kg silybin for 2 weeks. The mRNA levels of Pparα, L-Fabp, and Acox1 were determined via reverse-transcriptase PCR analysis. (B) The mRNA levels of PPARα and L-FABP and (C) luciferase activities of PPARα in HepG2 cells treated with silybin or fenofibrate. HepG2 cells were treated with dimethylsulfoxide (0.1%), silybin (25 and 50 μM), or fenofibrate (50 μM) for 24 hours. Cells were then collected for reverse-transcriptase PCR assay and reporter gene assays. *P < 0.05, **P < 0.01, ***P < 0.001 compared with respective control.
- Fig. 8.
Computational molecular docking of silybin and fenofibrate to the ligand binding domain of PPARα. (A) Chemical structure of silybin and fenofibrate. The conformer with least energy for silybin or fenofibrate was chosen for subsequent docking. Crystal structure of human PPARα was obtained from Protein Data Bank, and the docking analysis was conducted by DISCOVERY STUDIO. Repeated dockings were carried out until no further refinement in clustering or binding energy of conformer was achieved. Based on population size and binding energy, the best conformation was chosen for further analysis. Amino acid residues predicted to interact with the ligands are shown (B).
- Fig. 9.
Effect of silybin on PPARα activation in the presence or absence of fenofibrate in HepG2 cells. HepG2 cells were treated with dimethylsulfoxide (0.1%) or silybin (25 and 50 μM) in the presence or absence of fenofibrate (50 μM) for 24 hours. Cells were then collected to detect the mRNA level of PPARα (A) and L-FABP (B), as well as the luciferase activities of PPARα (C). ***P < 0.001 compared with dimethylsulfoxide (vehicle) group; ###P < 0.001 compared with fenofibrate group.
- Fig. 10.
Effect of silybin on PPARα activation in the presence or absence of fenofibrate in mice. C57BL/6 mice were treated with vehicle or silybin (50 and 150 mg/kg) together with or without fenofibrate (100 mg/kg) for a consecutive 14 days. Twenty-four hours after the last administration, the mice were sacrificed and their livers were immediately removed. mRNA levels of Pparα (A), L-Fabp (B), and Acox1 (C) were determined via reverse-transcriptase PCR analysis; glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples. ***P < 0.001 compared with dimethylsulfoxide (vehicle) group; ##P < 0.01, ###P < 0.001 compared with fenofibrate group.
- Fig. 11.
Effect of coadministration of silybin and fenofibrate on the expression and activity UGT1A6. (A) mRNA levels of UGT1A6 in HepG2 cells. After being treated with dimethylsulfoxide (0.1%) or silybin (25 and 50 μM) in the presence or absence of fenofibrate (50 μM) for 24 hours, HepG2 cells were collected for reverse-transcriptase PCR assay. (B) mRNA levels and (C) activities of Ugt1a6 in the livers of mice. C57BL/6 mice were treated with vehicle or silybin (50 and 150 mg/kg) together with or without fenofibrate (100 mg/kg) for a consecutive 14 days. Twenty-four hours after the last administration, the mice were sacrificed and their livers were immediately removed to detect the mRNA levels and activities of UGT1A6. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples in reverse-transcriptase PCR analysis. *P < 0.05 compared with dimethylsulfoxide (vehicle) group; #P < 0.05 compared with fenofibrate group.
Tables
- TABLE 1
Enzyme activities of main DMEs
Mice were treated with silybin (50 mg/kg or 150 mg/kg, once daily) for a consecutive 2 weeks. Twenty-four hours after the last administration, the mice were sacrificed and their livers were immediately removed to prepared microsomes. Enzyme activities of main P450s and UGTs were tested using the typical probe substrate approach. Data are shown as mean ± S.D. (n = 3).
Substrates Activity of DMEs (pmol/min/mg microsome protein) Vehicle Silybin (50 mg/kg) Silybin (150 mg/kg) Phenacetin 516.23 ± 79.93 440.59 ± 33.02 319.10 ± 27.71a Diclofenac 159.06 ± 4.39 168.32 ± 8.92 126.97 ± 1.51a Chlorzoxazone 1446.31 ± 29.98 1553.31 ± 59.82 1332.36 ± 81.52 Midazolam 1291.11 ± 98.69 1305.59 ± 29.69 907.76 ± 60.01b β-estradiol 718.71 ± 42.12 621.71 ± 16.36a 419.42 ± 21.95b 4-MU 75,459.46 ± 9478.70 109,474.65 ± 2324.02 83,348.31 ± 8070.37 Naloxone 286.74 ± 38.05 295.84 ± 31.74 289.04 ± 29.78
Data Supplement
Files in this Data Supplement:
- Supplemental Data -
Supplemental Table 1 - List of primer sequences used for RT-PCR
Supplemental Figure 1 - Effects of co-administration of silybin and fenofibrate on the mRNA levels of UGTs in HepG2 cells
Supplemental Figure 2 - mRNA levels of Ugt isozymes in the livers of mice treated with silybin and/or fenofibrate
Supplemental Figure 3 - Activities of Ugt isozymes in the livers of mice treated with silybin and/or fenofibrate
Supplemental Figure 4 - Activities of main P450s in the livers of mice treated with silybin for only once
- Supplemental Data -
In this issue
Regulatory Effects of Silybin on DMEs
