Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites. Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit. Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P > 0.05). Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.


Introduction
Biological organisms are continually exposed to potentially harmful chemicals called xenobiotics, including prescriptions and over-the-counter (OTC) drugs, herbal medicines, and environmental contaminants. Lipophilic xenobiotics are particularly dangerous, because they are able to accumulate to toxic concentrations over a period of time [1]. Cytochrome P450 (CYP450, CYP) are phase I metabolic enzymes that can catalyze oxidative and reductive metabolism of a variety of drugs, precarcinogen, and other xenobiotics, and the synthesis and metabolism of steroid hormones and arachidonic acid [2]. Through regulating the expression of liver CYP gene which achieve the detoxification and removal of xenobiotics, the expression products of these genes catalyze xenobiotics to polarity, prompting foreign chemicals easily excreted from the body. Members of the CYP superfamily often catalyze the first step in the detoxification of lipophilic xenobiotics [3] because of their broad substrate specificity and abundance in the liver, intestine, and tissues that are constantly exposed to environmental chemicals [4,5]. Upregulation of CYP enzymes induced by xenobiotics is an integral component of the body's defense mechanism against chemical damage (chemoprotection) and enhances the detoxification of exogenous materials. In addition, CYP enzymes involve in the metabolism of multiple endogenous steroids bile acid and lipids.
The pregnane X receptor (PXR), which is a kind of ligandactivated transcription factors belonging to the nuclear receptor family, is an important part of the body's defense mechanism against toxic xenobiotics. In 1998, PXR was found in mice as a subfamily member of nuclear receptor family by 2 Evidence-Based Complementary and Alternative Medicine Kliewer et al. [6]. The receptor was named as PXR because it was originally discovered that endogenous pregnane was its ligand. And it is also known as steroid and xenobiotic receptor (SXR) based on its activation by both steroid hormones and xenobiotic. While PXR is activated by ligand, the conformation changes and combines with 9-cis-retinoic acid receptor which is also known as retinoid X receptor (RXR), forms heterodimer to control gene transcription and trigger subsequent biological effects, and regulates genes of toxic substances that are involved in detoxification and excretion [7]. PXR is widely expressed in various important tissues and organs such as liver, intestine, kidney, lung, brain, pancreas, and placenta [8,9].
Life and disease have very complex physiological and pathological processes, which involve multiple genes, multichannel process of molecular interaction networks function, to achieve the desired therapeutic effect being often more difficult to target through a single action. Thus, looking for modular drug targets will be an important development direction of drug research and development for multigene disorders associated "molecular group, " that is, network pharmacology [10]. Network pharmacology studies emphasize many effective drugs act via modulation of multiple proteins rather than single targets, highlighting overall thinking also shared by traditional Chinese medicine (TCM). Introducing network pharmacology in TCM will be easier to discover bioactive ingredients and endogenous/exogenous biomarkers, revealing molecular mechanisms and exploring scientific evidence of numerous herbs and herbal formulae in TCM on the basis of complex physiological and pathological processes [11][12][13].
CYP3A subenzymes are one of the most significant families involved in more than 50% of clinical drug metabolism. CYP3A gene can be induced by a lot of drugs, which is mediated by the PXR [14]. Many drugs can be used as ligand of PXR and activate the receptor PXR; these drugs directly combine with the ligand binding domain (LBD) forming heterodimers, then bind to the response element of the target gene promoter, upregulate the expression of transcription of target gene and the enzyme protein, and enhance the metabolic capacity of substrate. In addition to regulation of CYP3A gene, PXR also can regulate CYP2B and CYP2C genes, as well as the expression of phase II drug-metabolizing enzymes and drug transporters [15][16][17]. PXR receptor can mediate the formation of the enzyme CYP system; therefore, a drug may induce CYP enzymes by binding to the PXR receptor and activating it. The generated CYP enzymes will not only increase the metabolism of the drug itself, but also the coadministered drugs, which is called drug-drug interactions. As the metabolism of the drug accelerated, the clinical therapeutic effect is affected accordingly. Therefore, the PXR receptor can regulate the whole process of exogenous chemical reactions and drug metabolism.
The chemical name of helicid is 4-formylphenyl--Dallopyranoside, a kind of aromatic phenolic glycosides. It is an active ingredient extracted from the fruits from Yunnan Helicia Nilagirica Beed (synonyms Helicia Erratica Hook) in China (Figure 1). Thousands of years ago, the Jingpo people in Yunnan had already used it to reduce swelling and alleviate pain. The chemical structure of helicid is a kind of monomer glucoside (Figure 2(a)). Pharmacological and clinical research found that helicid can restore the imbalance between the cerebral cortex excitability and inhibition and people use it to alleviate pain and induce sleep or sedation [18][19][20]. Helicid tablets belong to the state OTC drugs, widely used for the adjuvant treatment of insomnia, neurasthenia, migraine, headache, and other symptoms; now as a novel treatment for depression and neuropathic pain, a series of research has been carried out to study the related pharmacological effects and mechanism on helicid and its derivatives. Studies showed that helicid has 2 kinds of metabolites in the human body: oxidized metabolites (Figure 2(b)) and the reduced metabolites (Figure 2(c)).
In the view of many drugs there exists induction or inhibition on hepatic drug metabolizing enzyme (especially CYP); this leads to serious drug-drug interactions (DDI) in clinical practice, especially during postmarketing stage. Sometimes abnormal plasma concentration and poisoning phenomenon even appeared. The Food and Drug Administration (FDA) of USA and China Food and Drug Administration (CFDA) have taken related measures, such that the drug's ability in inducing or inhibiting hepatic drug metabolizing enzyme must be studied in the process of new drug development in order to ensure the safety of the drug. Since the use of natural medicines has become more and more widely accepted, full attention should be paid to drug-induced adverse interactions.
At present, researches of helicid and its metabolites are mainly focused on clinical pharmacodynamics and less researches on the DDI. This paper was based on the molecular mechanisms in which drugs induced CYP450 enzymes, constructed luciferase reporter gene vectors containing CYP3A4, CYP2B6, and CYP2C9 gene promoter regulatory sequence with molecular and cell biology methods, established screening system in vitro of hPXR-mediated CYP3A4, CYP2B6, and CYP2C9 drugs inducers, and further used it to study helicid and metabolites on hPXR-mediated CYP3A4, CYP2B6, and CYP2C9 induced effects, to predict whether helicid can cause DDI in the human body or not.

Statistical Analysis.
The efficiency of transfection in each of the treated cells was corrected by the ratio of firefly luciferase activity to Renilla luciferase activity. Fold activation was defined as the ratio of sample activity value of the corrected transfection efficiency of each group to the same sample activity value of the corrected transfection efficiency of 0.1% DMSO vehicle control group. All results are presented as the mean ± standard deviation (SD). Data were analyzed using ANOVA analysis of variance, with a value of < 0.05 considered significant by SPSS 18.0, pairwise comparisons using LSD method. Each group was compared with the control group whether there is a statistically significant difference.  (Figures 4 and 5).

Dual Luciferase Reporter Gene Vector Construction.
Human gDNA as a template amplified, respectively, distal promoter regulatory sequences and proximal promoter regulatory sequences of CYP3A4, CYP2B6, and CYP2C9.1% agarose gel electrophoresis analysis showed suitable size DNA bands ( Figure 6). Insert distal and proximal regulatory sequences into the linearized pGL4.17 vector. CYP3A4 and CYP2B6 were verified by double restriction enzyme digestion analyses with Kpn I and Hind III; CYP2C9 was verified by single restriction enzyme digestion analyses with EcoR V (Figure 7). After verification by sequencing, promoter sequences connected with the vector PGL4.17 successfully; CYP3A4-promoter-Luc, CYP2B6-promoter-Luc, and CYP2C9-promoter-Luc vectors are successfully constructed.   II cannot significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.

Discussion
There are almost more than 90% of clinical drugs metabolized through CYP3A4, CYP2C9, or CYP2B6. The inducers or inhibitors of hepatic drug metabolizing enzymes can speed up or slow down the metabolism of the substrates or other drugs. These constitute the material basis of the drug metabolism in the body and interactions between the drugs [24]. Therefore, the impact of drugs on the activity of CYP enzymes is one of the main reasons leading to clinical DDI. CYP450 enzymes are composed of the enzyme protein encoded by the gene superfamily and involved in the biotransformation of almost all endogenous and exogenous substances. CYP gene can be induced by a lot of drugs; studies have shown that this induction is mediated via the nuclear receptor PXR [14]. Many clinical drugs can be ligand of PXR to activate the receptor, to regulate the expression of CYP gene, such as antibiotic, psychotropic drugs, and antidiabetic drugs. Metabolism's study of traditional Chinese medicine is becoming one of the hot areas of the traditional Chinese medicine research in recent years; more and more reports appeared. For example, ginkgo, coumarin, and Hypericum perforatum can inhibit the expression of multiple isoforms of CYP450, and glycyrrhizic acid and angelica polysaccharide can induce some of them [25]. Helicid used as a kind of traditional medicine has a long history, and it is a chemical monomer with potential medicinal value whose activity is similar to hyperoside and has the basis of long-term clinical applications and new use patent. Helicid used for the treatment of depression was authorized by the State Patent Office in January 2005 [26]. Therefore, there is a very important clinical significance for predicting DDI through the research of helicid act on CYP enzymes.
This paper was based on the molecular mechanisms in which drugs induced CYP450 enzymes and constructed luciferase reporter gene vectors containing CYP3A4, CYP2B6, and CYP2C9 gene promoter regulatory sequence with molecular and cell biology methods. Reporter gene vectors were cotransfected with PXR expression vector into HepG2 cells in vitro, were treated with hPXR agonist rifampicin, and regulated CYP3A4, CYP2B6, and CYP2C9 promoter regulatory sequences by activating the exogenous expression vector hPXR to significantly increase the expression of luciferase. Nuclear receptor PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter  T  T  T  T  T  T  T  T T  T  C  C  C  C  C C  C  C  C  C  C C  C  C C  C  C  C  G  350  360  370  380  390  400  410  420  G  gene platforms were successfully established and used the reporter gene platforms to detect helicid and its metabolites I and II. At the three concentrations, no significant difference was found between helicid or its metabolites I, II and 0.1% DMSO vehicle control group ( > 0.05); it is shown that helicid and its metabolites I, II cannot significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9 and prompt the probability that clinical DDI of helicid and its metabolites induce CYP enzymes mediated by hPXR which is small. This study laid the theoretical foundation for predictions of the development and DDI of helicid. This study successfully established a cell platform which can screen CYP3A4/CYP2C9/CYP2B6 inducers and used it to verify helicid. The results show that the effect of the positive drug is obvious; helicid displays a negative result.
Combination drugs of helicid are proven to be safe. The screening system in vitro provides useful information for clinical drugs, especially the development of new drugs and other therapeutic agents. Discover AhR, PXR, and CAR which are closely relevant to drug-metabolizing enzymes and transcription activation [27,28] (Figure 9). AhR is the ligand-activated transcription factor of bHLH PAS family; CAR and PXR are members of nuclear hormone receptor superfamily. The two receptors CAR and PXR are closely related to each other in the regulation of drug-metabolizing enzymes and have overlapping functions [29,30]. Mueller et al. [31] found that the induction of CYP2b10, CYP3a11, CDC20, and Cdk1 depend on both CAR and PXR, but AACS enzyme is only inducted by the PXR. The PXR also regulates   the expression of drug metabolizing enzymes which include UDP-glucuronosyltransferase enzyme (UGT), sulfotransferase (SULT), and glutathione-S-transferase (GST) [32] and transporter proteins like P-glycoprotein (P-gP) [33] and organic anion transporter 2 (OATP2) [34]. This paper focuses on hPXR-mediated drug metabolizing enzymes CYP3A4, CYP2B6, and CYP2C9 inducing activity screening, studies are lacking in other areas, and follow-up will continue to be studied.

Conclusion
A convenient, economical, and new high-throughput in vitro screening system is established and validated to investigate the CYP enzyme-inducing activity and was successfully applied to investigate helicid and its metabolites. The screening system is a useful tool for the prediction of potential drugdrug interactions of clinical therapeutic agents.