Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of

cytochrome P450 3A4 and pregnane X receptors

Yiqiang Xie1, Yuhua Li2, Mianqing Huang3, Qing Wu4, Qibing Liu3, Junqing Zhang3,
Huiming Deng3, Mi Liu3 and Ling Huang3*
1
The School of TCM of Hainan Medical University, Haikou, PR China
2
Department of Pharmacy, the First Affiliated Hospital of Nanchang University, Nanchang, PR China
3
School of Pharmaceutical Sciences of Hainan Medical University, Haikou, PR China
4
Department of Pharmacy, the Affiliated Children's Hospital of Chongqing Medical University, Chongqing, PR China

Abstract: Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of

Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids,
FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The
purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to
confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK
on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were
examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays,
respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter
assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was
examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity,
and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXR-
CYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on
CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the
PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with
other drugs.

Keywords: Feng-Liao-Chang-Wei-Kang, Cytochrome P450 3A4, Cytochrome P450 3A1, Pregnane X receptor, herb-
drug interaction.

INTRODUCTION flavonoids present in FLCWK (Zhang et al., 2011).

Keampferol is known to alter MAPK/ERK signaling at
As a traditional Chinese patent medicine, Feng-Liao- several key locations as well as the PI3K/AKT pathway
Chang-Wei-Kang (FLCWK) is consisted of Polygonum attenuating the risk of chronic conditions like
hydropiper and Daphniphyllum calycinum roots (Zhang et inflammation and cancer (Chen & Chen 2013). Dietary
al., 2011). Based on the clinical performance of FLCWK rutin can cut the production of pro-inflammatory genes,
in the treatment of chronic superficial gastritis, ulcerative for example, IL-6, interleukin-1β (IL-1β), and inducible
colitis, acute gastroenteritis, and mesenteric nitric oxide synthase (iNOS) (Hosseinzadeh & Nassiri
lymphadenitis (Mota et al., 2009; Tuñón et al., 2009), 2014).
FLCWK has been designated as one of China’s national
protected traditional medicines. Polygonum hydropiper is Herb-drug interactions occur when compounds induce or
known to contain sesquiterpenoids and flavonoids and has inhibit drug-metabolizing enzymes, which can lead to
antioxidant, anti-inflammatory and antimicrobial adverse effects and compromise the efficacy of the drugs
properties (Tao et al., 2016). Daphniphyllum calycinum (Dai et al., 2001). Cytochrome P450 3A4 (CYP3A4) is
has been shown to have potent antioxidant and anti- the most plentiful and significant isoform of the
inflammatory properties as well and contains alkaloids cytochrome P450 metabolizing enzymes in human
and flavonoids (Gamez et al., 1998). intestine and liver. CYP3A4 is involved in 60% of drug
metabolism and often plays a key role in drug interactions
According to our previous findings, flavonoids make up (Martínez et al., 2007). As a family of ligand-activated
no less than 12% of FLCWK by weight. Flavonoids have transcription factors, nuclear receptors (NRs) have been
demonstrated protective effects on the gastrointestinal identified as mediators of the drug-induced expression of
tract through anti-inflammatory and antioxidant pathways CYP3A4. In these receptors, the constitutive androstane
(Chen et al., 2016). Kaempferol and rutin are the major receptor (CAR) and the pregnane X receptor (PXR) are
the primary mediators of CYP3A4 activity (Cheng et al.,
*Corresponding author: e-mail: [email protected] 2011; Wonqanan et al., 2014) as both play a part in
Pak. J. Pharm. Sci., Vol.31, No.5(Special), September 2018, pp.2315-2321 2315
Regulation of CYP3A4 and PXR by Feng-Liao-Chang-Wei-Kang

CYP3A4 transcriptional regulation following activation Experimental Animal Ethics Review Committee of
by xenobiotics (Waxman 1999). Hainan Medical University.

Our previous study demonstrated the basic FLCWK tablets were shattered and dissolved in PBS in at
pharmacokinetic characteristics of FLCWK (Zhang et al., concentrations of 25, 50, and 100mg/mL. Rats were
2011). However, the herb-drug interaction profile of randomly separated into five groups and every group
FLCWK has yet to be studied. Yu et al have shown that contained six rats. The first group was treated with DXM
the activities of CYP3A4 and NRs are affected by various (10 mg/kg) as a positive control, the second group was
flavonoid compounds including kaempferol and rutin (Yu treated with low-dose (0.25g/kg) FLCWK, the third was
et al., 2011; Lehmann et al., 1998), suggesting that treated with medium-dose (0.5g/kg) FLCWK, the fourth
FLCWK, as a complicated containing several kinds of with high-dose (1g/kg) FLCWK, and the fifth with a
flavonoids, has the potential to affect CYP3A4 and NRs saline control. Each rat was orally given a 1 mL/100 g
and influence drug metabolism. Here, we evaluate the solution for ten consecutive days. After the final dose, rats
efficacy of FLCWK on CYP3A4, CYP3A1 (the homolog were orally administered midazolam (MDZ) (15 mg/kg)
of CYP3A4 in rats), and the underlying mechanism of and then blood samples (approximately 0.2mL each) were
FLCWK’s influence on PXR/CAR- mediated collected from the right jugular vein in preheparinized
transactivation of CYP3A4. tubes pre-treatment and at 5, 10, 15, 30 and 45 min and at
1, 1.5, 2, 3, 4, 5 and 6 h post-treatment. Blood was then
MATERIALS AND METHODS centrifuged immediately to separate 100µL of plasma.
Rats were then sacrificed and liver tissues were harvested
Cells and chemicals and separated. Plasma and liver tissue samples were kept
The human liver cancer cell line HepG2 (The Chinese at -80 °C until analysis.
Academy of Sciences, Shanghai) was cultured in RPMI-
1640 medium containing 10% fetal bovine serum (FBS), Real-time PCR analysis
penicillin (100U/mL), and streptomycin (0.1 mg/mL) at For each liver sample, total RNA was extracted by TRIzol
37°C with 5% CO2. FLCWK was purchased from reagent from 30mg liver tissue according to the
Guangdong Guoyitang Pharmaceutical Co. Ltd. manufacturer’s guides and cDNA was synthesized for
(Guangzhou, China). Rifampicin (RIF), Dexamethasone further analysis by a PrimescriptTM RT Kit (Takara, Kyoto,
（ DXM), 6-(4-chloro phenyl) imidazo [2,1-b] [1,3] Japan). The 20µL reaction mixture consisted of 10µL of
SYBR Primix Ex Taq II (Takara, Kyoto, Japan), 1µL of
thiazole-5-carbaldehyde O-(3,4- dichloro benzyl) oxime the cDNA template, 8µL of RNase-free H2O, and 0.5µL of
(CITCO), midazolam (MDZ), and dimethyl sulfoxide each primer: CYP3A1 5'-TTCACCGTGATCC
(DMSO) were obtained from Sigma-Aldrich (St. Louis, ACAGCA-3' and 3'-TGCTGCCCTTGTTCTCCTT-5';
USA). The hPXR expression vector, hCAR2 expression CYP3A4 5'-TCAGCCTGGTGCTCCTCTATCTA T-3' and
plasmid, and pGL3-CYP3A4-XREM reporter plasmid 3'-AAGCCCTTATGGTAGGACAAAATATTT-5'; PXR
were generously provided by Doctor Min Huang (Sun 5'- ATGGCAGTGTC TG GAACTAC-3' and 5'-
Yat-sen University, China) (Ling et al., 2014). The dual- CAGTTGACACAGCT CGAAAG-3'. The amplification
luciferase reporter assay system and the pRL Renilla conditions were as follows: a denaturation step was
Luciferase control reporter vectors were bought from performed at 94°C for 60 s, followed by 45 cycles at
Promega (Madison, USA). Anti-CYP3A1 polyclonal 94 °C for 30 s, 58°C for 30s and 72°C for 30 s, then 95°C
antibody and β-actin antibody were bought from Cell for 10 s, 65°C for 45s and 40°C for 60 s. Relative
Signaling Technology (Danvers, USA). Anti-rabbit IG- expression levels were figured by Data Assist Software
HRP antibody was bought from R&D Systems version 3.0 (Applied Biosystems/Life Technologies) and
(Minneapolis, USA). the 2-∆∆ct ways. Each treatment was represented by three
replicates.
Animals and treatmnets
Female Sprague-Dawley (SD) rats weighing 220±5g were Western blotting analysis
bought from the Medical Experimental Animal Center of Total protein was extracted from rat liver tissue samples
Hunan Province and stored at 22-24°C with a light/dark after grinding with liquid nitrogen using 1% RIPA lysis
cycle of 12/12 h and relative humidity of 55%-60%. The buffer. The protein concentration was confirmed using a
rats can eat standard rodent chow and drink water freely BCA protein assay kit. 40µg supernatant protein samples
at the Experimental Animal Center of Hainan Medical were separated by 8% SDS-PAGE and semi-dry blotted
University. All animal experiments were performed onto PVDF (polyvinylidene fluoride) membranes
according to the Regulations of the Experimental Animal (Millipore, Bedford, MA, USA). After nonspecific
Administration released by the Ministry of Science and membranes were blocked with 5% nonfat milk, the blots
Technology of the People’s Republic of China. The design were incubated with a CYP3A1 and β-actin antibody
of the animal experiments was confirmed by the overnight at 4°C and with anti-rabbit Ig-HRP antibody for
2316 Pak. J. Pharm. Sci., Vol.31, No.5(Special), September 2018, pp.2315-2321
 Yiqiang Xie et al

1h at room temperature. Protein levels were detected and total RNA was extracted and investigated by qPCR,
using the Supersignal® West Pico Chemiluminescent (Ling 2013). Each treatment was represented by three
Substrate Kit (ThermoFisher Scientific; Waltham, MA, replicates.
USA). Each treatment was represented by three replicates.
STATISTICAL ANALYSIS
Analysis of CYP3A1 activity using HPLC for MDZ
A Waters Alliance 2695 HPLC apparatus was purchased Significant differences were confirmed using the unpaired
from Waters Science and Technology Co. Ltd, TD. The Student's t-test or one-way analysis of variance (one-way
chromatographic column was DiamonsilTM C18 (200 mm ANOVA) followed by Dunnett's t-test with the software
× 4.6 mm, 5 m), the mobile phase consisted of acetonitrile SPSS 13.0. P<0.05 was considered to indicate a
and 0.01 mol/ L potassium dihydrogen phosphate buffer statistically important difference.
solution (pH 5.0) (58:42). The flow rate was 1.0 mL/min
and the detected wavelength was 220 nm. RESULTS
Diphenytriazol-methanol solution was added into clean FLCWK increases CYP3A1 expression in rats
centrifuge tubes as an internal standard. After volatilizing As illustrated by qPCR (fig. 1), Cyp3a1 mRNA
the methanol to dryness, 0.1mL of plasma sample was expression level increased by 23.1-fold compared to the
added to the tubes. Next, 1mL of ethyl acetate extraction control group after DXM treatment for 10 days. FLCWK
was added and the tubes were vortexed for three minutes administration at dosages of 0.25, 0.5, and 1 g/mg
followed by centrifugation at 13,000r/min for 5 min. Then increased Cyp3a1 mRNA expression in rat livers by 3.93,
0.9 mL of the upper organic solvent was obtained for 5.94, and 8.57-fold (P<0.05), respectively, showing that
vacuum drying, the residue was dissolved with 50µL of FLCWK had a vital effect on Cyp3a1 mRNA expression
the mobile phase, and 20µL of sample was evaluated at a in a dose-dependent manner.
wavelength of 220 nm.

Luciferase assay
HepG2 cells were seeded at 70%-80% confluence into 96-
well plates at a density of 0.2 × 105 cells/well for 24h.
Transfection mixtures were created with 50ng of hPXR or
50 ng of pcD-hCAR2 expression vector, 100 ng of
CYP3A4-XREM luciferase reporter, and 15ng of pRL-TK
Renilla luciferase reporter. After the transfection mixtures
were incubated with Lipofectamine 2000 (Invitrogen,
Carlsbad, USA) for 6 h, HepG2 cells were exposed to
10µM RIF or CITCO, which are known hPXR and hCAR
activators, or FLCWK solution at 10, 20, and 40 mg/mL
for 24h. Cells were then lysed, and Firefly and Renilla
luciferase activity was measured using the dual-luciferase Fig. 1: Effects of FLCWK on CYP3A1 mRNA expression
reporter assay system according to the manufacturer’s in rat liver. SD rats were administered saline (control
instructions with a TD 20/20n single-tube luminometer group), DXM (10 mg/kg), 0.25g/kg (FLCWK-L), 0.5g/kg
(Promega, Madison, WI USA). The transfection (FLCWK-M) or 1g/kg (FLCWK-H) FLCWK for ten
efficiency was showed as the fold induction of Firefly to consecutive days before liver tissues were removed for
Renilla luciferase activities relative to the empty or real-time PCR studies. Values are expressed as the means
vehicle control. ± SEM, n = 6, *P<0.05, **P<0.01.

Transient transfection To investigate the effect of FLCWK on CYP3A1 protein

In the RNAi experiment, HepG2 cells were plated in 6- expression, western blotting was performed on rat liver
well plates at a density of 5×106 and PXR siRNA or NC tissues. As is shown in fig. 2, CYP3A1 protein expression
siRNA was transfected with siRNA transfection reagent was significantly increased 3.38-fold after DXM
(Roche, Branford, CT USA) according to the administration and FLCWK at 0.5g/kg and 1 g/kg
manufacturer’s instructions. Primers for hPXR were as increased CYP3A1 protein expression by 1.89 and 2.44-
follows: 5′ - GCA CCT GCT GCT AGG GAA TA-3′ and fold (P<0.05), respectively.
5′ - CTC CAT TGC CCC TCC TAA GT-3′. The target
sequences of siPXR were as follows: 5′- FLCWK increases the catalytic activity of the CYP3A1
GAUGGACGCUCAGAUGAAATT-3′ and 3′-UUUCAU enzyme in SD rats
CUGAGCGUCCAUCTT -5’. Cells were further The concentration of MDZ, a probe used to determine
incubated with 10 µM RIF or 10, 20, or 40mg/mL CYP3A activity in vivo, was measured using HPLC to
FLCWK for 72 h. After incubating, cells were harvested observe the inductive capacity of FLCWK on CYP3A1.
Pak. J. Pharm. Sci., Vol.31, No.5(Special), September 2018, pp.2315-2321 2317
Regulation of CYP3A4 and PXR by Feng-Liao-Chang-Wei-Kang

The average plasma concentration-time profile of MDZ is transfected with the hPXR expression plasmid, FLCWK
expressed in fig. 3 and table 1. Compared to control group, at concentrations of 20 and 40 g/L significantly increased
the AUC0-360min and the AUC0→∞ of DXM group were CYP3A4 luciferase activity by 4.72 and 5.37-fold,
significantly decreased (P<0.01), the AUC 0→∞ of MDZ respectively (P<0.01). However, FLCWK did not induce
of the rats administrated FLCWK for 10 days at dosages CYP3A4 luciferase activity when the CYP3A4-XREM
of 0.5g/kg and 1g/kg were decreased to 105.54±9.35 reporter plasmid was co-transfected with the CAR
(P<0.05) and 92.00±7.68 (P<0.05), respectively. It expression plasmid, indicating that FLCWK upregulation
suggests that FLCWK can significantly up-regulate the of CYP3A4 is mediated by the PXR pathway rather than
catalytic activity of CYP3A1. by the CAR pathway.

Fig. 3: Effects of FLCWK on CYP3A1 enzyme activity in

SD rats. Plasma concentration-time curves of MDZ after
saline, DXM (10mg/kg), 0.25g/kg (FLCWK-L), 0.5g/kg
(FLCWK-M) or 1g/kg (FLCWK-H) treatment. Values are
expressed as the means ± SEM, n = 6, *P<0.05, **P<
Fig. 2: Effects of FLCWK on CYP3A1 protein expression 0.01.
in SD rat liver. SD rats were administered saline (control
group), DXM (10mg/kg), 0.25g/kg (FLCWK-L), 0.5g/kg
(FLCWK-M) or 1g/kg (FLCWK-H) FLCWK for ten
consecutive days before liver tissue was removed for
western blotting. Values are expressed as the means ±
SEM, n = 3, *P<0.05, **P<0.01.

Effect of FLWCK on PXR/CAR-CYP3A4 luciferase

reporter assay
To further understand the mechanism underlying the
inductive effect of FLCWK on CYP3A4, a dual-luciferase
reporter assay was utilized. The CYP3A4-XREM
luciferase reporter plasmid and pRL-TK Renilla luciferase
reporter were co-transfected together with hPXR or Fig. 4: The effect of FLCWK on CYP3A4 reporter gene
hCAR over-expression plasmids into HepG2 cells. construct transactivation in transiently transfected HepG2
FLCWK, at concentrations of 10, 20 and 40g/L, had cells. Values are expressed as the means of the fold
negligible cytotoxicity towards native and transiently increases in activity compared to the control vehicle-
transfected HepG2 cells. Compared to the control group, treated cells from more than three independent
CYP3A4 luciferase activity was significantly induced experiments. Values are expressed as the means ± SEM, n
6.67 or 5.69-fold by 10µM RIF or 10µM CITCO (fig. 4), = 6, ##P<0.01, **P<0.01.
indicating that the dual-luciferase reporter assay was
successfully established. PXR is required for FLCWK regulation of CYP3A4
expression
When the CYP3A4-XREM reporter plasmid was co- To further investigate the up-regulation of CYP3A4 by
2318 Pak. J. Pharm. Sci., Vol.31, No.5(Special), September 2018, pp.2315-2321
 Yiqiang Xie et al

Table 1: The AUC of MDZ in SD rats.

Parameter Control DXM FLCWK-L FLCWK-M FLCWK-H

AUC0-360min (mg/L·h) 148.14±10.21 65.29±5.16** 115.91±11.16 100.44±9.28* 88.71±7.55*
AUC0→∞ (mg/L·h) 153.29±12.83 68.61±6.23** 118.52±15.27 105.54±9.35* 92.00±7.68*
SD rats were administered with saline, 10mg/kg DXM, 0.25g/kg (FLCWK-L), 0.5g/kg (FLCWK-M) or 1g/kg (FLCWK-H)
FLCWK. Values are expressed as the means ± SEM, n = 6, *P < 0.05, **P < 0.01.
FLCWK through the PXR pathway, the effect of FLCWK study, we demonstrated that Cyp3a1 mRNA (the rat
on CYP3A4 mRNA levels in HepG2 cells with PXR homolog for human Cyp3A4) and protein expression
knockdown was measured. fig. 5(a) shows that PXR was levels were significantly increased in rat liver tissues after
down-regulated by PXR siRNA. As is shown in fig. 5(b), FLCWK treatment. This suggests that the compounds
after HepG2 cells were transfected with NC siRNA and have the potential to affect CYP3A1 activity during
treated with FLCWK at 20mg/mL, 40mg/mL for 72 h, clinical application.
Cyp3a4 mRNA was increased 3.76 fold and 3.51-fold,
respectively (P<0.05). However, the induction of
CYP3A4 mRNA by FLCWK (40mg/mL) in PXR-
silenced cells was significantly suppressed to 35.7% and
57.2% compared to the unsilenced PXR group (P<0.05),
suggesting that FLWCK up-regulates CYP3A4 expression
via PXR.

DISCUSSION

CYP3A4 is a major player in drug metabolism that may

be influenced by a series of compounds. Changes in
CYP3A4 activity can result in altered systemic drug
concentrations and even toxicity (Yang et al., 2012;
Brantley et al., 2013), so it is important to clarify how
traditional Chinese medicines affect drug metabolism to
improve the safety and efficacy of these medications
(Hsin et al., 2013; Fasinu et al., 2012). In recent years, the
effects of Chinese patent medicines on drug-metabolizing
enzymes have become an increasingly important area of
interest.

FLCWK is a popular traditional Chinese patent medicine

with proven therapeutic effects on gastrointestinal Fig. 5: Effects of FLCWK on CYP3A4 mRNA expression
diseases such as chronic superficial gastritis and in in HepG2 cells. (a) HepG2 cells were transfected with
ulcerative colitis. Several studies have shown that negative control siRNA or PXR siRNAs. PXR mRNA
flavonoid compounds can affect the activity of CYP3A4 levels were analyzed by real-time PCR. **P<0.01. (b)
via the NR pathway (Cheng et al., 2011). Kaempferol and HepG2 cells were transfected with negative control or
rutin are the main effective components of FLCWK; PXR siRNA and were treated with 10µM RIF or FLCWK
Kaempferol has been reported to inhibit CYP3A for 72 h. The CYP3A4 mRNA expression was analyzed
expression in rats and further cell-based gene assays have using real-time quantitative PCR. *P<0.05, **P<0.01
revealed that kaempferol can activate the expression of compared to control in NC siRNA transfected groups.
PXR and CAR (Chang 2009; Yao et al., 2010). Rutin can Values are expressed as means ± SEM, n = 6.
reduce the bioavailability of immunosuppressants by
activating CYP3A4 and P-glycoprotein (Martínez et al., To further confirm the inductive effect of FLCWK on
2007). Therefore, it is assumed that FLCWK can affect CYP3A1, an HPLC assay was used to detect rat CYP3A1
the expression of CYP3A4 and NRs and may therefore catalysis activity. In an in vivo study, we found that
interfere with drug metabolism. CYP3A1 activity was increased after FLCWK treatment
for ten consecutive days. The decreased AUC between 0
Rat liver tissue is an ideal model for observing the effects and 6 h after MDZ treatment observed in FLCWK-treated
of FLCWK on Cyp3a1 mRNA and protein expression due rats also indicated that FLCWK enhanced CYP3A1
to its higher endogenous expression of many CYP activity. The DXM-treated rat model has been widely
enzymes (Li et al., 2012; Choi et al., 2010). In the present used to model the induction of CYP3A1 activity. In this
Pak. J. Pharm. Sci., Vol.31, No.5(Special), September 2018, pp.2315-2321 2319
Regulation of CYP3A4 and PXR by Feng-Liao-Chang-Wei-Kang

model, the plasma concentration of MDZ is dependent administered or combined with other drugs. These
upon CYP3A1 activity. The observed effects of FLCWK findings should be considered due to the specific herb-
on the AUC of MDZ are consistent with our results drug interactions that may occur during FLCWK
obtained from real-time PCR and Western blotting administration.
analyses, suggesting that FLCWK can significantly up-
regulate the metabolic activity of CYP3A1. ACKNOWLEDGEMENTS

PXR and CAR are the most important NRs regulating This study was supported by the National Natural Science
CYP3A4 transcription activity. A key event after ligand Foundation of China (Grant No. 81760674).
binding is the alteration of nuclear receptor conformation
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