Dimethyl sulfoxide (DMSO), MTT and non-essential amino acids were obtained from Sigma-Aldrich (St. Louis, MO, USA). The plasmids, pcDNA3.1-hPXR, pGL3-PXRE and pRL-TK, were constructed as previously described (19). Mega Tran 1.0 transfection reagent was purchased from OriGene (Rockville, MD, USA). The dual-luciferase reporter assay system was provided by Promega Corporation (Madison, WI, USA). The E.Z.N.A.™Endo-free Plasmid mini kit was the product of Omega Bio-Tek, Inc. (Norcross, GA, USA). SYBR® Premix Ex Taq™ was purchased from Takara Bio, Inc. (Shiga, Japan). The primers used in qPCR were synthesized by Invitrogen Life Technologies (Shanghai, China). Rifampicin (RIF) was obtained from Sangon Biotech Co. Ltd. (Shanghai, China). CM and ZM were obtained from Huqingyutang Pharmaceutical Co., Ltd. (Zhejiang, China). All the other chemicals used were of analytical grade.

Human hepatic carcinoma cell line HepG2 and colon adenocarcinoma cell line LS174T were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). The cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% charcoal/dextran-treated fetal bovine serum (HyClone, Logan, UT, USA), 1% non-essential amino acids, 1% (v/v) penicillin and streptomycin (Corning Life Sciences, Oneonta, NY, USA). Cells were maintained routinely at 37°C in a 5% CO₂ humidified atmosphere.

Ethanol and aqueous extracts (ee- and ae-, respectively) of Maidong from two cultivation regions (ee-ZM, ee-CM, ae-ZM and ae-CM) were prepared with the heat reflux extraction method. To prepare the aqueous extracts of Maidong, the raw herbs were ground and soaked in an 8-fold volume of distilled water for 30 min and extracted by heat reflux extraction for 30 min in a boiling water bath. The herbs were heat reflux extracted three times, combined together and filtered, respectively. The filtrates were concentrated by a rotary evaporator and further dried by freeze drying, and the yield of dry extracts was calculated. Ethanol extracts were prepared in the same way as the aqueous extracts, except that the solvent was changed to 80% ethanol and the water bath temperature to 80°C. The dry extract yield was calculated in the same way.

HepG2 cells were seeded in 96-well plates at a density of 5,000 cells/well, allowed to attach overnight and were treated with Maidong extracts at indicated concentrations for 48 h. Medium containing herb extracts were renewed every 24 h. After treatment, 20 µl of the 5 mg/ml MTT was added to each well and incubated for 4 h at 37°C. The supernatant was removed carefully and 150 µl of DMSO was added to each well. Ten minutes after incubation at 37°C, the absorbance value of each well was read at 490 nm using an ELISA plate reader instrument (model 680; Bio-Rad, Tokyo, Japan).

Reporter gene assays were performed as described previously (19) with a slight modification. Briefly, plasmid DNA was prepared by the E.Z.N.A.™Endo-free Plasmid Mini kit. HepG2 cells were seeded in 24-well plates at a density of 2.5 × 10⁴ cells/well and allowed to attach overnight, and were transiently co-transfected with pcDNA3.1-hPXR, pGL3-PXRE and pRL-TK with the aid of Mega Tran 1.0 transfection reagent. Twenty-four hours later, the transfected cells were treated with medium containing aqueous extracts, ethanol extracts, 10 µM RIF (positive control) or 0.1% DMSO (blank control) for an additional 48 h. Medium containing drugs were renewed every 24 h. Following the treatment, the cells were rinsed twice with cold phosphate-buffered saline and were lysed. Firefly luciferase activity was determined and normalized against Renilla luciferase activity. Fold induction was calculated as the normalized reporter activity of a test drug divided by the solvent control (DMSO). Data are shown as the percentage of fold induction of a test extract relative to 10 µM RIF and represent the mean of three independent experiments. The test extracts were categorized into three groups: No response or negligible (<30%), weak to moderate (30–70%) and strong (>70%), as previously described (23,24).

LS174T cells were seeded in 24-well plates at a density of 5×10⁴ cells/well and allowed to attach overnight. Cells were subsequently treated with Maidong extracts at indicated concentrations for 72 h and 0.1% DMSO and 10 µM RIF were used as the blank and positive control, respectively. Medium containing drugs were renewed every 24 h. After treatment, total RNA was extracted from monolayers of LS174T cells using the Ultrapure RNA kit (CWBio, Beijing, China) according to the manufacturer's instructions. The RNA quality and quantity of samples was assessed by the 260/280 nm absorbance ratios and the 260 nm absorbance, respectively, using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Beijing, China). Total RNA (2 µg) from each sample was used to synthesize cDNA using the Hifi-MMLV cDNA kit (CWBio) according to the manufacturer's instructions. All the PCR reactions were carried out using SYBR® Premix Ex Taq™ on an ABI PRISM 7500 qPCR system. Oligonucleotide primers for CYP3A4 and GAPDH were 5′-GTGGGGCTTTTATGATGGTCA-3′, 5′-ACATCTCCATACTGGGCAATGA-3′; and 5′-CTTAGCACCCCTGGCCAAG-3′, 5′-GATGTTCTGGAGAGCCCCG-3′, respectively. The experiments were run in triplicate as follows: Initial denaturation step of 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Quantitation of CYP3A4 mRNA in treated samples versus vehicle control samples was calculated by correcting for GAPDH in each sample (ΔCt) using the equation: Fold induction = 2^−ΔΔCt.

All the statistical analyses were carried out using the SPSS 19.0 statistical software package (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation from one of three independent experiments. One-way analysis of variance was used for the statistical comparisons. P<0.05 was considered to indicate a statistically significant difference.

In the present study, HepG2 cells with an extremely low level of PXR and CYP3A4 expression and LS-174T cells with a high level of PXR and CYP3A4 expression were used as cell models in the reporter gene assay and RT-qPCR analysis to investigate PXR activation and CYP3A4 induction, respectively. First, RT-qPCR analysis was performed to compare and verify the PXR and CYP3A4 mRNA expression of these two cell lines. The data showed that the PXR mRNA expression level was extremely low (mean value, 6.06×10⁻⁶ vs. 1) and the CYP3A4 mRNA level was also extremely low (mean value, 0.074 vs. 1) in HepG2 cells compared to LS-174T cells (Fig. 1). The results demonstrate that the HepG2 and LS-174T cells were qualified for further analysis.

To compare the cytotoxicity of the CM and ZM extracts, the relative viability of HepG2 cells treated with ae-CM, ee-CM, ae-ZM and ee-ZM at various concentrations for 48 h was evaluated by the MTT assay. As shown in Fig. 2, ee-ZM inhibited cell viability in a significant dose-dependent manner and showed a significant inhibition effect even at the concentration of 0.1 mg/ml, while ee-CM had no clear effect on cell viability when the concentration was not >1 mg/ml. Furthermore, at every concentration level used, ee-ZM demonstrated higher toxicity to HepG2 cells than ee-CM. As for the aqueous extracts of two Maidong strains, overall they showed a much lower cytotoxicity than their ethanol extract counterparts and ae-CM only showed a slight or moderately more cytotoxicity than ee-CM (Fig. 2). These data indicate that ee-ZM possesses a markedly higher cytotoxicity than ee-CM, while ae-ZM and ae-CM have no evident difference on cytotoxicity.

The reporter gene assay was carried out to evaluate and compare the activation of human PXR by the ethanol and aqueous extracts of CM and ZM. As presented in Fig. 3, overall the extracts of ZM and CM activate human PXR in a dose-dependent manner at the range of 5 mg/ml. The ee-ZM and ee-CM showed no response or negligible effect (<30%) only at the concentration of 0.1 mg/ml. The ee-CM had a weak to moderate effect, while ee-ZM also had a negligible effect when the dose was <1 mg/ml. Furthermore, ee-ZM and ee-CM demonstrated a strong activation effect on human PXR at the dose of 5 mg/ml. The ee-CM exhibited a significantly higher percentage of maximum induction relative to RIF compared to ee-ZM at each concentration (Fig. 3). The ae-ZM and ae-CM showed a similar pattern to their ethanol counterparts. The ae-CM activated human PXR much stronger than the ee-ZM in the reporter gene assay (Fig. 3).

To further investigate and compare the induction effect on CYP3A4 mRNA by the ethanol and aqueous extracts of CM and ZM, RT-qPCR analysis was performed. DMSO (0.1%; vehicle) and 10 µM RIF were used as a blank and positive control, respectively. Compared to the vehicle-treated group, only the 1 and 5 mg/ml ee-ZM and 5 mg/ml ee-CM groups induced CYP3A4 mRNA expression significantly. Additionally, ee-ZM demonstrated a markedly higher induction of CYP3A4 mRNA expression than ee-CM at the concentration of 1 and 5 mg/ml (Fig. 4). The ae-ZM and ae-CM did not exhibit an evident induction effect on CYP3A4 mRNA expression at all the used concentrations (Fig. 4).