Oleanolic Acid and Ursolic Acid Induce UGT1A1 Expression in HepG2 Cells by Activating PXR Rather Than CAR.

Background: Oleanolic acid (OA) and its isomer ursolic acid (UA) have recently emerged as research foci based on their biologic activities. We previously demonstrated that UA can inhibit the activities of UGT1A3 and UGT1A4, and OA inhibits UGT1A3 activity in liver microsomes. However, whether OA and UA affect the expression of UGT1As in HepG2 cells and the underlying regulatory mechanism remain unclear. Purpose: The present study aimed to explore the effect of OA and UA on the expression of UGT1As in HepG2 cells and the regulatory mechanisms on UGT1A1 based on the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) signaling pathways. Methods: We analyzed the effect of OA and UA on UGT1A expression and on the PXR/CAR regulatory pathway in HepG2 cells, hPXR-silenced HepG2 cells, and hCAR-silenced HepG2 cells by Q-PCR, Western blotting, and dual-luciferase reporter gene assays. Results: In HepG2 cells, OA and UA both significantly induced the expression of UGT1A1, UGT1A3, UGT1A4, and UGT1A9 and upregulated the expression of PXR. However, OA and UA did not affect CAR expression. A dual-luciferase reporter assay showed that OA and UA could markedly promote PXR-mediated UGT1A1 luciferase activity, whereas OA and UA did not affect CAR-mediated UGT1A1 luciferase activity. In hPXR-silenced HepG2 cells, OA and UA did not elevate UGT1A1 activity compared to the control group. However, the expression of UGT1A1 in hCAR-silenced HepG2 cells was markedly elevated compared to the control group or with non-silenced HepG2 cells treated with OA (10, 20, and 40 µM) or UA (10, 20, and 40 µM). Conclusions: OA and UA significantly induce the expression of UGT1A1, UGT1A3, UGT1A4, and UGT1A9 in HepG2 cells, and their induction on UGT1A1 is mediated by PXR activation, not CAR.

Mechanism of Action of Panaxytriol on Midazolam 1'-Hydroxylation and 4-Hydroxylation Mediated by CYP3A in Liver Microsomes and Rat Primary Hepatocytes.

In our previous study, panaxytriol (PXT) was shown to enhance midazolam (MDZ) 1'-hydroxylation significantly but to inhibit MDZ 4-hydroxylation. To explore the underlying mechanism, we investigated the effects of PXT on cytochrome P450 3A (CYP3A)-mediated MDZ metabolic pathways using rat liver microsomes (RLM), human liver microsomes (HLM), and rat primary hepatocytes. In the presence of PXT, the Vmax of 4-OH MDZ decreased from 0.72 to 0.51 nmol/min·mg pro in RLM and from 0.32 to 0.12 nmol/min·mg pro in HLM, and the Km value increased from 5.12 to 7.26 µM in RLM and from 27.87 to 32.80 µM in HLM. But the presence of PXT reduced the Km and increased the Vmax values of MDZ 1'-hydroxylation in RLM and HLM. Interestingly, the differential effect of PXT on MDZ 4-hydroxylation and 1'-hydroxylation was also observed in primary rat hepatocytes after 45-min culture. PXT did not affect the expression levels of CYP3A1/2 mRNA in rat hepatocytes. With extension of the culture time to 6 h, however, PXT significantly inhibited both MDZ 4-hydroxylation and 1'-hydroxylation, and the expression level of CYP3A1/2 mRNA was decreased to 87% and 80% (CYP3A1) and to 89% and 85% (CYP3A2) of those in controls in the presence of PXT 4.0 and 8.0 µg/mL, respectively. These results suggest that PXT could activate MDZ 1'-hydroxylation but inhibit MDZ 4-hydroxylation by changing the CYP3A enzyme affinity and metabolic rate after a short-term intervention. However, long-term treatment with PXT could inhibit both the 4-hydroxylation and 1'-hydroxylation of MDZ by downregulating CYP3A1/2 mRNA expression.

Gene variation in IL10 and susceptibility to chronic hepatitis B.

OBJECTIVES: To evaluate the correlation between IL-10 gene polymorphisms and hepatitis B infection. METHODS: Tag single nucleotide polymorphisms (SNPs) were used to investigate the relationship between IL-10 gene polymorphisms and susceptibility to chronic hepatitis B virus (HBV) infection by comparing 996 chronic HBV infection cases to 301 acute infection controls. RESULTS: This study found that rs3024490 G/T allele, located in the intron 1 region and highly prevalent in Chinese populations, was significantly different between the chronic HBV infection cases and the acute infection controls in single allele analysis, genetic models analysis, and haplotypes analysis. CONCLUSIONS: This suggested that the rs3024490 within IL-10 was associated with susceptibility to chronic hepatitis B in a Chinese Han population.

Contribution of cytochrome P450 isoforms to gliquidone metabolism in rats and human.

1. Gliquidone, a second generation sulfonylurea, is a widely used oral antidiabetic drug. Due to the differences in its rate of metabolism, gliquidone shows inter-subject variability in pharmacokinetic and pharmacodynamic profiles. 2. Cytochrome P450 (CYP450) isoforms are involved in the metabolism of a majority of drugs in clinical use and plays a significant role in reducing possible drug interactions. This research aimed to systematically study the contribution of various human CYP450 isoforms to gliquidone metabolism in vitro in rats and human. 3. In rat liver microsomes, gliquidone was metabolized mainly by the most abundant CYP2C. The other isoforms involved in the metabolism included CYP3A, CYP2D, CYP1A and CYP2E. 4. Further investigation of rat recombinant enzymes showed that CYP3A1 and CYP2C11 played a major role in gliquidone metabolism in vitro, while CYP2D1, CYP1A2 and CYP2E1 were also involved. 5. But the metabolism of gliquidone in the human liver microsomes was mainly mediated by CYP3A4. The other isoforms involved in this process were CYP2C9, CYP2C19 and CYP2D6. 6. The further study of human recombinant enzymes demonstrated that CYP3A4 was the principal isoform enzyme for the metabolism of gliquidone. The intrinsic clearance (Vmax/Km) of CYP3A4 during gliquidone metabolism was 3-12 times greater than that of other CYP450 isoforms including CYP2C9, CYP2D6 and CYP2C19. 7. These findings may assist in valuable prediction of potential interactions of gliquidone with other drugs that are CYP3A4 inhibitors or inducers and help to design more efficacious and safer pharmacotherapy for patients of diabetes mellitus.

Identification of the active components in Shenmai injection that differentially affect Cyp3a4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam.

Shenmai injection (SMI) is a popular herbal preparation that is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis. In our previous study, SMI was shown to differentially affect CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam (MDZ). The present study was conducted to identify the active components in SMI responsible for the differential effects on MDZ metabolism, using in vitro incubation systems (rat and human liver microsomes and a recombinant CYP3A4 system) to measure 1'-hydroxylation and 4-hydroxylation of MDZ. First, different fractions of SMI were obtained by gradient elution on an solid phase extraction system and individually tested for their effects on MDZ metabolism. The results demonstrated that lipid-soluble constituents were likely to be the predominant active components of SMI. Second, the possible active components were gradually separated on an high-performance liquid chromatography system under different conditions and individually tested in vitro for their effects on MDZ metabolism. Third, the active component obtained in the above experiment was collected and subjected to structural analysis, and identified as panaxytriol (PXT). Finally, it was validated that PXT had significant differential effects on 1'-hydroxylation and 4-hydroxylation of MDZ in various in vitro systems that were similar to those of SMI. We conclude that PXT is the constituent of SMI responsible for the differential effects on CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of MDZ.

[Research progress of the atypical kinetic profiles of cytochrome P450 enzymes].

Cytochrome P450 enzymes are composed of many isozymes and involved in the biotransformation of both exogenous and endogenous substances. A growing number of studies have found that the P450 enzymes do not always follow the classical Michaelis-Menten kinetics, but show atypical kinetic behavior, which is also the current research hotspot. In this paper, the category and mechanisms of atypical kinetics of the P450 enzyme were reviewed, providing theoretical basis for the research of enzyme kinetics.