PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C from Peucedanum praeruptorum Dunn

We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on the CYP3A gene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level of CYP3a11 gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs.


Introduction
The root of Peucedanum praeruptorum Dunn. (Qianhu) is widely used in traditional Chinese medicine as antitussive, anti-inflammatory, and antiasthma component and as a remedy for angina. In China, Qianhu is an important ingredient in many kinds of famous traditional Chinese medicine preparations. Most traditional Chinese medicine prescriptions for antitussive contain Qianhu. In recent years, pharmacological evaluations have also revealed a wide variety of activities of Qianhu, including hypotensive [1], coronary dilatory [2], and myocardial dysfunction [3]. Praeruptorin C (PC) is the major active constituent isolated from Peucedanum praeruptorum Dunn. PC has been proved to possess multiple pharmacological activities such as prevention and treatment of vascular hyperplastic disease [4], relaxation of the smooth muscle of tracheas and pulmonary arteries [1], relaxation of coronary artery, and decreased contractility activity in left atria [5]. For the increasing wide use of Qianhu and its active component PC in the clinical practice, potential of clinical herb-drug interactions is strikingly increased, and thus, it is important to predict these potential herb-drug interactions.
The underlying mechanisms for most reported herb-drug interactions have not been clearly elucidated, but induction or inhibition of cytochrome P450 (CYP) enzymes is one of the most important risk factors for drug-drug interactions (DDIs). CYP3A4 is responsible for metabolic conversion of more than 50% of the currently clinical drugs to more polar metabolites for easier excretion [6]. Induction or inhibition of CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant DDIs or herb-drug interactions [7][8][9]. Clinically and preclinically relevant interactions have been hugely reported between herbs and drugs such as St. John's wort, pomelo, and grapefruit juice, and induction or inhibition of CYP3A4 by xenobiotics often results in these herbdrug interactions [10,11]. Therefore, CYP3A4-related DDIs have significant clinical impacts.
In recent years, important advances have been made in mechanisms involved in induction or inhibition of CYP3A4. A family of ligand-activated transcription factors, known as nuclear receptors (NRs), has been identified as mediators of drug-induced expression of CYP3A4. Among them, the pregnane X receptor (PXR, NR1i2) and the constitutive androstane receptor (CAR, NR1i3) are the mainly mediator of CYP3A4 [12][13][14].
To date, pregnane X receptor-(PXR-) mediated CYP3A4 induction has been well studied. PXR can be activated by a wide variety of small, hydrophobic endogenous and exogenous compounds. A number of naturally occurring compounds from herbs such as St John's wort [15], Ginkgo (Ginkgo biloba) [16,17], Gugulipid (Commiphora mukul) [18], Wu Wei Zi (Schisandra chinensis), Licorice (Glycyrrhiza uralensis) [19], and Dan Shen (Salvia miltiorrhiza) [20] have been reported to activate PXR. Upon activation by a ligand, PXR unites with RXR to bind and transactivate several specific elements, such as the everted repeat with a six-nucleotide spacer (ER6) or a direct repeat with a three-nucleotide spacer (DR3), in the 5 upstream regulatory region of the CYP3A4 gene [21,22], thus play a role in regulating transcription of CYP3A4. Therefore, over 64% pharmaceutical companies in the US have adopted cell-based PXR reporter assays routinely to assess the potential for DDIs due to CYP3A4 inductions [23]. In our most recent studies, we found that the active ingredients PA and PD of Qianhu could upregulate CYP3A4 expression by PXR [24,25], but whether and how PC could regulate CYP3A4 transcription through PXR pathway remains unclear.
The nuclear hormone receptor CAR is a sister xenobiotic receptor of PXR and plays a pivotal role in the induction of drug metabolism. CAR has been reported to synergistically regulate the transcriptional activity of the CYP3A4 with PXR [26]. According to our previous results [27], PC induction of CYP3A4 at the transcriptional level could be activated through CAR pathways. PC effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway in human LS174T cells. We also found that CYP3A4 luciferase expression was enhanced through PXR pathway by PC [28]. However, the results of luciferase reporter assay could not fully prove that PC could affect the CYP3A4 expression through PXR nuclear receptor pathway. Further study is needed to investigate whether PC could upregulate CYP3A transcription by activation of PXR pathway and whether PC could affect CYP3A4 activity by the cross-talk mediated effect of PXR and CAR.
Therefore, in this study, regulation of Cyp3a11 (CYP3A4's homolog in mouse) gene expression by PC through PXR pathway was confirmed in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA. On the other hand, the gene expression, protein expression, and catalytic activity of CYP3A4 were compared in LS174T cells and PXR overexpressed LS174T cells to further prove the effect of PC on PXR and CYP3A and thus to predict any potential herb-drug interactions between PC, Qianhu, and the coadministered drugs.  [30]. The pRL-TK R. reniformis control vector and pSG5-empty vector were obtained from Promega (Madison, WI, USA).

Preparation of Primary Cultures of Mice Hepatocytes.
Liver cells were isolated from BALB/c male mice at 6-8 weeks of age by the two-step collagenase perfusion technique previously described [31] with slight modifications [32]. A density of 2-3 × 10 6 mice primary hepatocyte per g liver tissue with more than 70% cell viability was obtained. Standard culture conditions were as follows: the cells were dispersed in William's medium E containing 10% FBS, insulin (0.5 U/mL), and hepatocytes were seeded into 24-well collagen-coated plate at a density of 1 × 10 5 cells/well. The hepatocytes anchored to the collagen-precoated plates within 8 h and subsequently formed a monolayer, and only hepatocytes with viability greater than 90% were used for this study. The 24well collagen-coated plate was maintained at 37 ∘ C in 5% CO 2humidified incubator.  the equation 2 −ΔΔCt , where ΔCt represents the differences in cycle threshold numbers between the target gene and -actin, and ΔΔCt represents the relative change in the differences between control group and treatment group. The effect of PA on CYP3A4 mRNA levels is presented as fold mRNA expression to vehicle control.

PXR Expression Plasmid Transient Transfection and
Western Blotting Analysis. LS174T cells (1.0 × 10 6 per well) were seeded into 6-well plates, cultivated for 24 h, and then transfected with hPXR expression plasmids (1 g/well). Appropriate cell samples were exposed to DMSO (0.1%), RIF at 10 M, and PC at 2.5, 10, and 40 M for 72 h. All the proteins were extracted by high-speed centrifugation and quantified using Coomassie Protein Assay Kit (Pierce, Rockford, IL,USA). CYP3A4 protein levels were measured by Western blot analysis, and the Western blot analysis was conducted as described in our previous study [27]. The data were expressed as relative folds over vehicle controls.

PXR Expression Plasmid Transient Transfection and
Functional Analysis of CYP3A4 Activity. LS174T cells (1.0 × 10 6 per well) were seeded into 6-well plates and cultivated for 24 h. Cells were transfected or untransfected with hPXR expression plasmids (1 g/well) and then exposed to PC at a concentration of 2.5, 10, and 40 M for 72 h. The cells were lysed using radio immunoprecipitation assay (RIPA) buffer plus phenylmethylsulfonyl fluoride (PMSF); total protein was extracted by high-speed centrifugation and quantified using Coomassie Protein Assay Kit (Pierce, Rockford, IL,USA). The protein pretreatment was performed as described in our previous report [27], and the concentration of the nifedipine metabolite was determined using an established LC-MS/MS method [33].

Statistical Analysis.
One-way ANOVA followed by Dunnett's multiple comparison post hoc test or unpaired Student's -test was used for statistical analysis of data using SPSS version 13.0 software (SPSS Inc, Chicago, IL, USA). Probability values <0.05 were considered to be statistically significant.

Results
The primary hepatocytes used in the study were isolated from wild-type mice and PXR knockdown mice primary hepatocytes were further obtained by transient transfection of PXR siRNA. A density of 2-3 × 10 6 cells per g liver tissue was obtained. Approximately 3-5 × 10 6 cells per mouse were harvested with viability and attachment efficiency above 90%. The silencing effects of siRNA transfection on PXR gene expression were confirmed by quantitative real-time PCR. Figure 2(a) shows that the PXR mRNA level were significantly decreased to 26% in mice primary hepatocytes after transfection of PXR siRNA at 50 nM. Furthermore, no inhibitory effect on PXR gene expression was observed in mice primary hepatocytes after transfection of the negative control siRNA.
The effect of PC on the Cyp3a11 mRNA levels in mice primary hepatocytes with or without PXR knockdown was measured. Figure 2 significantly suppressed 41.8% and 46.3% when PXR siRNA was transfected ( < 0.05). Previously, we found that PC significantly transactivated CYP3A4 reporter gene construct in PXR transiently transfected LS174T cells [27]. But CYP3A4 mRNA level was not significantly increased in the untransfected LS174T cells after administered PC. In order to determine whether or not PC induces CYP3A4 expression by PXR-mediated pathway, LS174T cells were transfected with pSG5-hPXR expression plasmids, and cells were exposed to PC at 2.5, 10, and 40 M for 48 h, then the mRNA levels of PXR and CYP3A4 were detected by real-time PCR. As shown in Figure 3(a), compared with the untransfected LS174T cells, the PXR expression had significantly increased after transfection with pSG5-hPXR expression plasmids, indicating that the PXR overexpression cells model was successfully established. Figure 3(b) shows that significant increase of CYP3A4 mRNA by RIF (as the positive control) was found to compare with the vehicle control (5.36-fold at 10 M, < 0.01). At 10 M and 40 M, PC can significantly induce CYP3A4 mRNA expression in transfected LS174T cells to 3.6-and 3.85-fold ( < 0.05, < 0.01), respectively. However, in our previous study, we did not observe significant increase of CYP3A4 mRNA level in the untransfected LS174T cells which was relatively low expression of PXR [28].
To confirm that PC induces CYP3A4 through PXRmediated pathway, CYP3A4 protein expression in PXR overexpressed LS174T cells were measured by Western blot analysis. After transfection with pSG5-hPXR expression plasmids, cells were exposed to PC at 2.5, 10, and 40 M for 72 h; CYP3A4 protein expression was subsequently measured by Western blot assay. As shown in Figure 4(a), LS174T cells transfected with PXR plasmid yielded significant increase to 2.28-fold ( < 0.05) in CYP3A4 protein expression after 72 h exposure to RIF at 10 M. Compared with the vehicle control, the expression was significantly induced to 2.19-and 2.22fold by PC at 10 M and 40 M, respectively ( < 0.05). These results were generally consistent with those observed in the real-time PCR assay. Also, Figure 4(b) shows that the CYP3A4 protein expression was not increased by PC in untransfected LS174T cells.
Furthermore, effect of PC on CYP3A4 enzyme activity was measured in PXR-overexpressed cells by LC-MS/MS assay. After transfection with PXR expression plasmids, cells were exposed to PC at 2.5, 10, and 40 M for 72 h; CYP3A4 activity was subsequently determined by LC-MS/MS assay based on measurement of specific CYP3A4mediated Nifedipine dehydrogenation. Figure 5 shows that LS174T cells transfected with plasmid encoding PXR plasmids yielded significant increase to 2.4-fold in CYP3A4 catalytic activity after 72 h exposure to RIF at 10 M ( < 0.01). LS174T cells transfected with plasmid encoding PXR plasmids yielded significant increase to 1.81-and 2.1-fold in CYP3A4 catalytic activity after 72 h exposure to PC at 10 M and 40 M, respectively, compared with control group ( < 0.05, < 0.01). At the same time, we can also observe that CYP3A4 catalytic activity was not increased by PC in untransfected LS174T cells.

Discussion
As for the increasing wide use of herbal medicines, the potential of clinical herb-drug interactions are dramatically increased, which is frequently caused by induction or inhibition of metabolizing enzymes. CYP3A4 is the most abundant and important CYP isoform expressed in human liver. Important advances have been made in the understanding of the mechanisms involved in induction or inhibition of CYP3A4 [34]. Nuclear receptors, especially the PXR and CAR, are the most significant regulator of CYP3A4 gene expression. Xenobiotics metabolism research has found that CYP3A4 expression and activity were affected by many ligands through both PXR and CAR pathways. PXR and CAR could generate the "cross talk" regulation effect on CYP3A4 transcription [35,36].
Our previous results demonstrate that PC effectively transactivated mRNA expression, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway in human LS174T cells [27]. However, the effect of PC on the transactivation of CYP3A4 via PXR pathway is not fully understood, and it will be of clinical significance to elucidate the effect of PC on CYP3A4 through the PXR-mediated pathway, and then to find whether PC has "cross-talk" effect on PXR and CAR pathways. Therefore, in this study, we investigated the effect of PC on the Cyp3a11 mRNA expression in PXR knockdown or PXR unknockdown mice primary hepatocytes, and regulations of CYP3A4 mRNA, protein expression, and catalytic activity by PC through PXR pathway were further investigated in human LS174T cells with overexpression of PXR.
Mice primary hepatocyte is an ideal model used for PXR mRNA knockdown for its higher endogenous PXR expression and easier obtain than human primary hepatocyte. PXR knockdown mice primary hepatocytes were successfully obtained by transient transfection of PXR siRNA and further validated by measuring the PXR mRNA level 72 h after transfection. We found that PC could significantly increase the level of CYP3a11 gene expression in negative transfection groups, and the induction of Cyp3a11 mRNA by PC was significantly suppressed in the PXR knockdown mice primary hepatocytes. These results demonstrate that knockdown of PXR suppresses the upregulation of Cyp3a11 mRNA by PC, indicating that PXR pathway is causally involved as a contributing mediator.
On the other hand, LS174T cells has lower endogenous expression of PXR; thus, it is more ideal to transfect PXR into LS174T cells to produce the PXR high expression cell model. To confirm that PC induces CYP3A4 directly through PXR-mediated pathway, CYP3A4 mRNA, protein expression, and catalytic activity in PXR-overexpressed LS174T cells were measured by real-time PCR, western blot analysis, and LC-MS/MS assay. According to our previous results [28], CYP3A4 mRNA expression could not be induced by PC in the LS174T cells without transfection with PXR plasmid. However, in the current study, after transfection with PXR plasmid, PC can significantly induce the level of CYP3A4 mRNA. These results suggest that PC-mediated transactivation of CYP3A4 gene via interaction with PXR pathway. Finally, we analyzed CYP3A4 protein expression and enzymatic activity in LS174T cells exposed to PA for 72 h after PXR transfection. Significant increase of CYP3A4 activity was observed in LS174T cells transfected with PXR plasmid. In order to further confirm the impacts of PC on the untransfected LS174T cells, the cells were treated with PC directly. The results show that increase of CYP3A4 protein expression could not be observed in untransfected LS174T cells, which was consistent with the results of mRNA expression and catalytic activity. The results indicate that PC can upregulate CYP3A4 protein level and its catalytic activity through PXR pathway. As mentioned above, PXR and CAR are critical determinants of xenobiotics-induced CYP3A4 expression and they can generate "cross-talk" regulation on CYP3A4 transcription. It is not a surprise that compounds that regulate CYP3A4 via PXR pathway might also interact with CAR to activate CYP3A4. We recently reported that PC can effectively transactivate luciferase activity and mRNA and protein expression of CYP3A4 via CAR-mediated pathway in LS174T cells; the results demonstrate that CAR also plays a role in the activation of CYP3A4 by PC [27]. In current results, it was elucidated for the first time that PC can effectively transactivate CYP3A4 mRNA expression, protein expression, and catalytic activity via the PXR-mediated pathway in human LS174T cells. Combined with current results, PC can coactivate the CAR-mediated and PXR-mediated pathway to coregulate CYP3A expression. Therefore, PC could regulate CYP3A4 gene expression, protein expression, and activity through both PXR-and CAR-mediated pathways and thus accelerate detoxification and metabolism of CYP3A4 substrates. Further mechanistic studies are needed to investigate how the herbal compounds interact with PXR, CAR, and their coactivators and corepressors.
Besides PC, Praeruptorin A (PA) and Praeruptorin D (PD) are the other two major active constituents isolated from Peucedanum praeruptorum Dunn (Qianhu). For the increasing wide use of Qianhu and its active components such as PA, PC, and PD in the clinical practice, it is important to predict any potential herb-drug interactions between PA, PC, PD, Qianhu, and the other coadministered drugs. Most recently, we reported that PA and PD can also significantly upregulate CYP3A level via the PXR-mediated pathway [24,25]. From the results of this study and our published data, we found that PD has the stronger regulation on CYP3A4 mRNA, protein, and enzyme activity via PXR pathway than PA and PC. However, there is no significant difference in the regulation of CYP3A by these three compounds. Very recently, we also found that PA and PC can significantly upregulate the expression of MRP2 and UGT1A1 via CAR-mediated pathway (data not shown). Taken together, all these findings suggest that PA, PC, and PD have varieties of effects on metabolizing enzymes or transporters via PXR or CAR mediated pathways; thus, this information should be taken into consideration to predict any potential herb-drug interactions between PA, PC, PD, Peucedanum praeruptorum Dunn, and the other coadministered drugs.

Conclusion
In summary, this study found that the level of CYP3a11 gene expression in mouse primary hepatocytes was significantly increased by PC, but such an induction was suppressed after knockdown of PXR by its siRNA. PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway in PXR-overexpressed LS174T cells; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs. More attentions should be paid to ensure the safety in clinical utilization of Qianhu and PC.