Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro

Rifampicin (RIF), pregnenolone 16α-carbonitrile(PCN), phenobarbital (PB), dimethyl sulfoxide (DMSO), diethyl pyrocarbonate (DEPC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIF, PCN, and PB were dissolved in DMSO at concentrations appropriate for the specific studies in which they were used. Trypsin and Taq DNA polymerase were purchased from Gibco BRL (Grand Island, NY, USA). Reverse transcription system, dual-luciferase reporter assay system, pGL4.17-Luc and pGEM-T constructs were purchased from Promega (Madison, WI, USA).

Shaoyao (Paeoniae Radix Alba, the root of Paeonia lactiflora Pall.) and Gancao (Glycyrrhizae Radix et Rhizoma, the root and rhizome of Glycyrrhiza uralensis Fisch.) were purchased from Xiangya Hospital of Central south university (Changsha, China). The crude herbs were authenticated by Prof. SY Hu, Department of Chinese Herbal Medicine of Central South University. Voucher specimens (No. 20140505 and No. 20140506) were deposited at the Laboratory of Ethnopharmacology in Xiangya Hospital. Crude drugs of SGD (SY: GC, 1:1, w/w) 200 g were immersed in 400 ml distilled water for 30 min and boiled for 40 min, then filtered. The residue was boiled and filtered once more. The filtrates from each time were combined and centrifuged at 3000 rpm for 30 min at room temperature. The supernatant was filtered through a 0.22-μm filter. The concentration of the original SGD was adjusted to 500 mg crude drug per milliliter, and then diluted into 0.1, 1, 2 mg/ml. SY (Paeoniae Radix Alba 100 g) or GC (Glycyrrhizae Radix et Rhizoma 100 g) was immersed in 400 ml distilled water, and processed as SGD. The concentration of the original SY and GC was adjusted to 250 mg crude drug per milliliter, and then diluted into 0.05, 0.5, 1 mg/ml. Decoctions were maintained at 4 °C for further use.

Meanwhile, six key compounds (gallic acid, albiflorin, paeoniflorin, glycyrrhizin, benzoic acid and liquiritigenin) from SGD for quality control using a Waters Acquity ultra-performance liquid chromatography (UPLC) system have been reported in our previous studies [27‐29]. Among the six compounds, gallic acid, albiflorin, paeoniflorin and benzoic acid are for SY, while glycyrrhizin and liquiritigenin are for GC.

Three cell lines were used in this study including HepG2 (human Caucasian hepatocellular carcinoma), Caco2 (human colon adenocarcinoma) and LS174T (human colon adenocarcinoma). Three cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The HepG2 line was maintained in Dulbecco’s modified Eagle medium (DMEM) high glucose medium (HyClone), Caco2 and LS174T lines were maintained in DMEM/F12 medium (HyClone), at 37 °C in a humidified atmosphere containing 5% CO₂. All media were supplemented with 10% fetal bovine serum (FBS, Gibco), added with 100 U/ml penicillin and 100 U/ml streptomycin (Beyotime, Shanghai, China). The medium was replaced every 3 days.

The expression plasmid for the human PXR receptor pcDNA3.1-PXR was kindly provided by Prof. Guo Wang (Institute of Clinical Pharmacology, Central South University, Changsha, China), containing the full-length human PXR [30].

The CYP3A4-Luc reporter constructs containing the basal promoter (− 362 ~ + 53 bp) with proximal PXR response element and the distal xenobiotic responsive enhancer module (− 7836 ~ − 7208 bp) of the CYP3A4 gene 5′-flanking region inserted to pGL4.17-Basic reporter vector has been reported previously [30]. The construction of MDR1-Luc reporter gene vector has been described previously [21, 30]. Human MDR1 promoter fragment (− 7975 ~ − 7013 bp) containing the cluster of xenobiotic-responsive enhancer modules was amplified by PCR with primers 5′- TCTGCTAGCAGTGTTTCTTGT-3′, containing a natural NheI site, and 5′-AATCTCGAGCATATAAGGCAACTGTTTTGT-3′, introducing an XhoI site. The NheI/XhoI-digested PCR fragment was ligated between the NheI/XhoI sites of pGL4.17-Basic luciferase reporter vector.

Transient transfections of HepG2 and Caco2 cells were performed as described previously [30]. In brief, prior to cell transfection for 24 h, HepG2 or Caco2 cells were seeded in 24-well and 6-well plates at a density of 1 × 10⁵ cells per well. Transfections were performed in triplicate using 600 ng CYP3A4/MDR1-Luc, 100 ng pcDNA3.1-PXR (or 100 ng pcDNA3.1), 10 ng internal control PRL-SV40, and 5 μl lipofectamine 2000 (Invitrogen) in a well. Four to six hours after transfection, HepG2 cells were washed in DMEM, replaced with DMEM + 10% FBS medium and treated with drugs 24 h later, while Caco2 cells were washed in DMEM/F12, replaced with DMEM/F12 + 10% FBS medium and treated with drugs 24 h later.

LS174T cells (1.2 × 10⁵ per well) were seeded into 24-well and 6-well plates and cultivated for 24 h. Then LS174T cells were transfected with 100 ng/well PXR expression plasmids or pcDNA3.1 vector or weren’t transfected. Four to six hours after transfection, LS174T cells were washed in DMEM/F12, replaced with DMEM/F12 + 10% FBS medium and treated with drugs 24 h later.

After transfection, we firstly detected expression of PXR protein in non-transfected, vector-transfected, and PXR-transfected cells by western blot. As for HepG2 or Caco2 cells in 6-well plates, we set non-transfected cells as the blank group, cells transfected with 600 ng pGL4.17-CYP3A4 (or MDR1) + 100 ng pcDNA3.1 + 10 ng PRL-SV40 as the vector group, cells transfected with 600 ng pGL4.17-CYP3A4 (or MDR1) + 100 ng pcDNA3.1-PXR + 10 ng PRL-SV40 as the trans-PXR group. As for LS174T cells in 6-well plates, we set non-transfected cells as the blank group, cells transfected with 100 ng pcDNA3.1 + 10 ng PRL-SV40 as the vector group, cells transfected with 100 ng pcDNA3.1-PXR + 10 ng PRL-SV40 as the trans-PXR group.

Western blot analysis was performed according to the standard protocol. Following 48 h transfection, transfected cells were washed with cold phosphate buffer saline once and lysed using 50 μl radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. After 30 min on ice, lysates were clarified by centrifugation at 12000 × rpm for 15 min at 4 °C. Amounts of protein 50–100 μg were loaded on a 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. Following a blocking incubation with TBST containing 5% non-fat dried milk at room temperature for 2 h, membranes were incubated at 4 °C overnight with primary antibody (1:1000 dilution) to PXR (rabbit, no. 15607–1-AP, Proteintech) or β-actin (mouse, no. 60008–1-Ig, Proteintech), followed by incubation at room temperature for 1 h with the goat anti-mouse or anti-rabbit horseradish peroxidase conjugated secondary antibody (1,3000 dilution; cat no. ab97051, Proteintech). The membranes were analyzed using ECL detection reagent (Thermo). β-actin was used as an internal control. All experiments were conducted in triplicate.

Co-transfected HepG2 or Caco2 cells were treated with rifampicin (10 μM), PCN (10 μM), SGD (0.1, 1, and 2 mg/ml), SY (0.05, 0.5, and 1 mg/ml), GC (0.05, 0.5, and 1 mg/ml), or the solvent control (0.1% DMSO) for 24 h, respectively. Vector control (VC) group was transfected with pcDNA3.1-PXR + PRL-SV40 + pGL4.17 (or pcDNA3.1 + PRL-SV40 + pGL4.17-CYP3A4) and treated with 0.1% DMSO. The blank group was co-transfected with pcDNA3.1-PXR + PRL-SV40 + pGL4.17-CYP3A4 and treated with nothing. The firefly luciferase activity was detected using the dual-luciferase reporter assay (Promega) according to the manufacturer’s protocol. The Renilla luciferase activity was used as an internal control. Each experiment was repeated three times in triplicate. The efficiency of transfection in each treatment groups was adjusted by the ratio of firefly luciferase activity to Renilla luciferase activity.

To determine CYP3A4 and MDR1 mRNA induction, LS174T cells were transfected with 100 ng/well PXR expression plasmids or pcDNA3.1 vector or weren’t transfected. Following 24 h transfection, appropriate cell samples were exposed to phenobarbital (1 mM), SGD (1 mg/ml), SY (1 mg/ml), GC (1 mg/ml) or the solvent control (0.1% DMSO) for 48 h, respectively. Then cells were harvested and the mRNA levels of CYP3A4, MDR1 were measured by real-time qPCR.

Total RNA isolation was performed using TRIzol reagent (Invitrogen) and converted into cDNA using the Fermentas RT kit according to the manufacturer’s instructions. The primers designed using Primer Premier 5.0 are presented in Table 1. PCR was performed in a total reaction volume of 30 μL, containing 15 μL 2 × SYBR Green PCR Master Mix (Applied Biosystems), 3 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 10 μL double-distilled water. Real-Time qPCR analysis was performed using the Applied Biosystems 7500 real-time PCR system. The temperature profile was 95 °C for 10 min; 40 cycles of 95 °C for 10 s, 59 °C for 50 s; melting curve program 60–95 °C. Real-time qPCR for each gene of interest was performed in triplicate, and the expression values were calculated as their average. Gene expression was evaluated in at least three independent experiments and each performed in triplicates. The expression of the target genes were normalized to the reference gene β-actin and then processed using the 2^−ΔΔCt method [31]. The data are expressed as the fold changes in the activation of gene expression relative to the non-transfected group with 0.1% DMSO treatment (set to be 1).

First, we examined whether SGD and its constituent prescriptions (SY, GC) affect the activation of CYP3A4-Luc reporter constructs through hPXR. Since CYP3A4 is mainly distributed in the liver and intestine organs, we used the two human tumor cell lines [32]. We successfully transfected PXR-CYP3A4 plasmid constructs to HepG2 and Caco2 cells (Fig. 2). Twenty-four hours following co-transfection, HepG2 and Caco2 cells were separately treated with rifampicin (10 μM, hPXR agonist), PCN (10 μM, rodent PXR agonist), 0.1, 1, 2 mg/ml SGD or 0.05, 0.5, 1 mg/ml constituent prescriptions (SY and GC) for 24 h, then detected dual luciferase activity.

Results of specific activity values of various concentrations groups are presented in Fig. 3. In HepG2 and Caco2 cells co-transfected with hPXR expression plasmid, rifampicin, SGD and its constituent prescriptions significantly increase the luciferase activities of CYP3A4 when compared with 0.1% DMSO vehicle control group (p < 0.05). In HepG2 cells transfected with PXR expression plasmid, rifampicin (10 μM), SGD (0.1 and 2 mg/ml), SY (0.05, 0.5, and 1 mg/ml) and GC (0.05, 0.5, and 1 mg/ml) markedly increase CYP3A4 luciferase activity by 24.80, (8.27, 12.05), (5.24, 5.74, 7.12), (12.68, 13.86, 16.85)-fold compared with DMSO, respectively. While 1 mg/ml SGD increase 10.00-fold CYP3A4 luciferase activity to the DMSO group without a statistical significance. In Caco2 cells transfected with PXR expression plasmid, CYP3A4 luciferase activity is significantly increased by rifampicin (22.26), SGD (8.62, 10.75, 13.40), 1 mg/ml SY (9.24), GC (13.87, 14.53, 17.83) when compared with DMSO, respectively. The increases in CYP3A4 luciferase activities by three decoctions show a concentration-dependent trend.

However, in both transfected HepG2 and Caco2 cells, PCN treatment doesn’t cause a significant increase in CYP3A4 luciferase activities when compared with the DMSO group. Previous studies have found rifampicin activates human but not rodent PXR, while PCN activates rodent but not human PXR [33, 34]. Consistent with the previous report [35], similar activation of hPXR and inductions of CYP3A4 and MDR1 by rifampicin are observed in HepG2, Caco-2 cells, except PCN.

Then, we examined whether the three decoctions induce the activation of MDR1-Luc reporter constructs through hPXR in HepG2 and Caco2 cells. We successfully transfected PXR-MDR1 plasmid constructs in HepG2 and Caco2 cells (Fig. 2). Twenty-four hours following transfection, HepG2 and Caco2 cells were separately treated with rifampicin (10 μM, hPXR agonist), PCN (10 μM, rPXR agonist), SGD (0.1, 1, and 2 mg/ml), SY (0.05, 0.5, and 1 mg/ml) and GC (0.05, 0.5, and 1 mg/ml) for 24 h, detected dual luciferase activity.

Results are shown in Fig. 4. In hPXR-transfected HepG2 and Caco2 cells, rifampicin, SGD and its constituent prescription GC significantly increase the luciferase activities of MDR1 when compared with 0.1% DMSO group (p < 0.05). However, SY only shows the significant increase in luciferase activities of transfected HepG2 cells. The increases of the three decoctions in MDR1 luciferase activities also show a concentration-dependent trend. In PXR-HepG2 cells, MDR1 luciferase activity is significantly increased by rifampicin (22.90), 0.1 mg/ml SGD (8.10), 2 mg/ml SGD (12.80), SY (4.60, 5.25, 6.04), GC (13.83, 15.14, 17.10) when compared with DMSO, respectively. In PXR-Caco2 cells, the luciferase activity of MDR1 is significantly increased by rifampicin (24.10), 1 mg/ml SGD (11.71), 2 mg/ml SGD (12.19) and GC (14.86, 15.43, 17.33) when compared with DMSO. However, in HepG2-hPXR and Caco2-hPXR cells, PCN treatment doesn’t cause a significant increase in MDR1 luciferase activities when compared with the DMSO group.

In a word, in HepG2 and Caco2 cells, SGD and its constituent prescription GC significantly increase promoter activities of CYP3A4 and MDR1 via PXR in a concentration-dependent manner. While SY doesn’t significantly increase promoter activities of MDR1 in transfected Caco2 cells. Like rifampicin, 1 mg/ml GC is a strong agonist of PXR, followed by SGD, and SY ranges the weakest.

Finally, to verify effects of SGD and its constituent herbs on CYP3A4 and MDR1 mRNA expression levels by PXR activation, we used PXR-transfected LS174T cell lines for real-time qPCR (Fig. 5). LS174T cells express endogenous hPXR with inducible activity [32], which was supported by our observation of a low PXR protein expression in LS174T (Fig. 2B). Twenty-four hours following transfection, LS174T cells were exposed to phenobarbital (1 mM, PXR agonist), 1 mg/ml SGD, SY and GC for 48 h, separately. In order to avoid the effect of herbal concentration, the same concentration was adopted. In LS174T cells with non-transfected or blank vector-transfected, phenobarbital, SGD, SY and GC significantly up-regulate level of CYP3A4 and MDR1 mRNA compared with corresponding DMSO groups (p < 0.05). And GC has a significant up-regulation when compared to SY (p < 0.01). Significantly enhancements of CYP3A4 and MDR1 mRNA expression are also observed in PXR-transfected cells. This indicates phenobarbital, SGD, SY and GC may activate endogenous and exogenous PXR and then significantly up-regulate CYP3A4 and MDR1 mRNA expression in LS174T cells. In PXR-transfected groups, increases of CYP3A4 and MDR1 mRNA expression are significantly higher by phenobarbital, SGD, SY, GC than them in non-transfected groups or vector-transfected groups (p < 0.01). All of that means SGD and its constituent prescriptions up-regulate expression of CYP3A4 and MDR1 mRNA in LS174T via PXR.