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Journal of Ethnopharmacology 140 (2012) 131– 140

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

ene expression profiling and pathway network analysis of hepatic metabolicnzymes targeted by baicalein

i Qina, Jihua Chena, Shunsuke Tanigawaa, De-Xing Houa,b,∗

Course of Biological Science and Technology, United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, JapanDepartment of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan

r t i c l e i n f o

rticle history:eceived 4 November 2011ccepted 21 December 2011vailable online 14 January 2012

eywords:aicaleinicroarrayepatic metabolic enzymesrf2RE

a b s t r a c t

Ethnopharmacological relevance: Baicalein is a flavone originally isolated from the roots of traditionalChinese medicinal herb, Scutellaria baicalensis, which has been proved as a promising chemopreventivecompound for many chronic human diseases.Aim of the study: The present study aimed to clarify the molecular mechanism targeted by baicalein.Materials and methods: Gene expression profiling of HepG2 cells treated with baicalein was carried out,using the Affymetrix 42K oligonucleotide microarray in the present study. Microarray data analyzed byIngenuity Pathway Analysis (IPA), further study performed by real time PCR, reporter gene assay, andWestern blot.Results: Among total 42K gene probes, baicalein treatment up-regulated the signals of 440 gene probes(1.04% of total gene probes) and down-regulated signals of 254 gene probes (0.6% of total gene probes)by ≥2-fold. These genes were categorized into 35 groups and hit for biological processes, molecular func-tions, and signaling pathways. The network and pathway analyses of these data further revealed thatan Nrf2 (nuclear factor-erythroid 2 p45-related factor 2)-mediated ARE (antioxidant response element)

pathway is involved in baicalein-induced gene expression of hepatic metabolic enzymes. The represen-tative enzymes involved in Nrf2/ARE pathway were further confirmed at mRNA level by real time PCRand at protein level by Western blot analysis. Moreover, the ARE-reporter gene assay demonstrated thatbaicalein stimulated Nrf2-mediated ARE transactivation.Conclusions: Our results provide a comprehensive data for understanding the hepatic metabolism, bioac-

ar me

tive role and the molecul

. Introduction

Baicalein (5,6,7-trihydroxyflavone) is a flavone originallysolated from the traditional Chinese medicinal herb, theoots of Scutellaria baicalensis, one of the most popular and

Abbreviations: AKR1C1/2/3, aldo-keto reductase family 1, member C1/2/3;LDH1L2, aldehyde dehydrogenase 1 family, member L2; ARE, antioxidantesponse element; CYPs, cytochrome P450 superfamily; DUSP1, dual specificityrotein phosphatase 1; GCLC/M, glutamate cysteine ligase catalytic/modifierubunit; GSR, glutathione reductase; HERPUD1, homocysteine-responsive endo-lasmic reticulum-resident ubiquitin-like domain member 1 protein; HO-1,eme oxygenase-1; Keap1, kelch-like ECH-associated protein 1; MTT, 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NQO1, NAD(P)H:quinonexidoreductase 1; Nrf2, nuclear factor-erythroid 2 p45-related factor 2; SLC7A11,ystine/glutamate transporter solute carrier family 7 member 11; SQSTM1,equestosome-1; SRXN1, sulfiredoxin-1; TXNIP, thioredoxin-interacting protein;XNRD1, thioredoxin reductase 1.∗ Corresponding author at: Department of Biochemical Science and Technology,

aculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-065, Japan. Tel.: +81 99 285 8649; fax: +81 99 285 8649.

E-mail address: hou@chem.agri.kagoshima-u.ac.jp (D.-X. Hou).

378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.12.046

chanisms of baicalein.© 2012 Elsevier Ireland Ltd. All rights reserved.

multi-purpose herbs used in China. Baicalein has been clini-cally used for anti-cardiovascular illness, and antitumor, anti-inflammatory, antioxidant, antiviral, and antibacterial purposes(Srinivas, 2010). Accumulated data showed that baicalein hadinhibitory effects on the adhesion, migration and invasion of MDA-MB-231 human breast cancer cells (Wang et al., 2010) and thetranscriptional activity of �-catenin/Tcf in HEK293 cells (Park andChoi, 2010). Baicalein also suppressed the growth of colon can-cer cells (Lea et al., 2010), lung cancer cells (Gao et al., 2011), andprostate cancer cells (Slusarz et al., 2010). Baicalein had inhibitoryeffects on LPS-induced NOs production (Wakabayashi, 1999), COX-2 gene expression (Woo et al., 2006), activation of NF-�B (Chenget al., 2007) and productions of many inflammatory cytokines(Kang et al., 2003; Hsieh et al., 2007; Yun et al., 2010). More-over, baicalein could serve as a free radical scavenger againsthydroxyl radicals, and had the ability to inhibit both lipoxyge-nase and xanthine oxidase enzymes (Shieh et al., 2000; Deschamps

et al., 2006; Van Leyen et al., 2006). Baicalein also could medi-ate P450 system (Si et al., 2009; Li et al., 2010) and induce theexpression of Nrf2 and downstream phase II genes such as HO-1(heme oxygenase-1) and NQO1 (NAD(P)H:quinone oxidoreductase

1 pharm

1bp

tcIi2mexflab(G(rteeamerohep2

TtTicpeai6O(g6aHca

itwkswonTembThb

the t-test p-value were imported into the IPA software. IPA wascarried out with p < 0.002 as the cutoff point. The genes were cat-

32 S. Qin et al. / Journal of Ethno

) (Hanneken et al., 2006; Chen et al., 2006), suggesting thataicalein may be involved in the activation of Nrf2-ARE antioxidantathway.

Hepatocytes are the main cells for flavonoid metabolizing withhe aid of drug metabolizing enzymes. Drug metabolizing enzymesonsisted of phase I, phase II metabolizing enzymes and phaseII transporters that play central roles in the metabolism, elim-nation and detoxification of xenobiotics and drugs (Xu et al.,005). Bioavailability of flavonoids is dependent on absorption,etabolism and elimination by the modulation of metabolizing

nzymes. In brief, phase I enzymes can modify the structure of theenobiotics, phase II enzymes then increase aqueous solubility ofavonoids and conjugate them with glutathione or glucuronate,nd finally phase III enzymes can either take flavonoids up fromlood to hepatocytes (SLC family) or efflux them into bile and bloodABC transporters). Examples of these enzymes include NQO1,SR (glutathione reductase), HO-1, SRXN1 (sulfiredoxin-1), GCLM

glutamate cysteine ligase modifier subunit), AKR1C2 (aldo-ketoeductase family 1, member C2) and TXNRD1 (thioredoxin reduc-ase 1) (Kaspar et al., 2009). Variations in the activity of thesenzymes and transporters affect drug activity and elimination andventually increased half-life and toxicity of xenobiotics (Buechlernd Weiss, 2011). Recently, several studies have focused on theechanisms that regulate the expression of the drug metabolizing

nzymes. Various nuclear factors including the aryl hydrocarboneceptor (AhR) (Miao et al., 2005; Anwar-Mohamed et al., 2011),rphan nuclear receptors (Xie and Evans, 2001; Shi, 2007), and Nrf2ave been identified to be the key mediators regulating the genexpressions of phase I, II metabolizing enzymes and phase III trans-orters in drug-induced changes (Enomoto et al., 2001; Miao et al.,005).

Flavonoids have been implicated as pharmaceutical agents.herefore, flavonoid metabolism has become more impor-ant subject in drug discovery and development (Testa, 2009).he modulation of chemopreventive enzymes by flavonoidss an important step for human health since these enzymesan inactivate carcinogens, which contributes to the cancerreventive properties of these compounds. Once baicaleinnters the human body, its bioavailability and bioactivityre also dependent on modulation of the drug metaboliz-ng enzymes. Five major metabolites including baicalein-O-beta-glucopyranuronoside (M1), 6-O-methyl-baicalein 7--beta-glucopyranuronosideoroxylin A 7-O-beta-glucuronide

M2), baicalein 7-O-beta-glucopyranuronoside (M3), 6-O-beta-lucopyranuronosyl-baicalein 7-O-sulfate (M4), and baicalein,7-di-O-beta-glucopyranuronoside (M5) were found in ratsdministered with baicalein or baicalin orally (Abe et al., 1990).owever, the whole-view for the influence of baicalein on hepato-ytes, especially on drug metabolizing enzymes and transportersre still not clear.

DNA microarray technology enables us to simultaneously exam-ne the expression of thousands of genes, and further profilehe bulk data of the gene expressions by using some path-ay analysis software (Chen et al., 2010, 2011). In order to

now the influence of baicalein on hepatocytes in genome-widecale, we carried out gene expression profiling of HepG2 cellsith or without baicalein treatment, using the Affymetrix 42K

ligonucleotide microarray, and subsequently analyzed the sig-aling pathways by using Ingenuity Pathways Knowledge Base.he results revealed that expressions of most hepatic metabolicnzymes genes were regulated by baicalein. Moreover, the Nrf2-ediated ARE pathway was demonstrated to be involved in

aicalein-induced gene expression of hepatic metabolic enzymes.he results provide a comprehensive data for understanding theepatic metabolism, bioactive role and molecular mechanism ofaicalein.

acology 140 (2012) 131– 140

2. Materials and methods

2.1. Materials, cell culture and cytotoxicity

Baicalein was purchased from Sigma (St. Louis, MO, USA).Human hepatoblastoma HepG2 cells were obtained from the Can-cer Cell Repository, Tohoku University, Japan, and cultured at37 ◦C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal bovine serum (FBS). Theantibodies against Nrf2 (C-20), Keap1 (E-20), HO-1, �-tubulin (B-7), rabbit IgG and horseradish peroxidase-conjugated anti-goatsecondary antibody were from Santa Cruz Biotechnology (SantaCruz, CA, USA). Horseradish peroxidase-conjugated anti-rabbitand anti-mouse secondary antibodies were from Cell SignalingTechnology (Beverly, MA, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sakao et al., 2009)was used to check the cytotoxicity of baicalein. In brief, HepG2cells were seeded onto 96-well plates at a density of 104 cells ofeach well and then were pre-incubated for 24 h at 37 ◦C. Baicaleinat the indicated doses (0, 10 �M, 20 �M, and 40 �M) were added,and incubated for another 24 h. MTT was then added to the plate(final concentration 0.5 mg/ml), and incubated for additional 4 h.The acidic isopropanol (0.04–0.1 N HCl in isopropanol) was addedto dissolve the formazan crystals and the optical density (OD) wasmeasured at 575 nm using a microplate reading spectrophotometer(Thermo Scientific, USA). Viability was determined by compar-ing the OD of baicalein-treated cells with those of the untreatedcells.

2.2. RNA extraction and microarray hybridization

HepG2 cells were pre-cultured in dishes for 24 h and thentreated with different concentrations of baicalein (the same asthat in Section 2.1) in 0.1% DMSO for 9 h. Total RNA was extractedusing an Isogen RNA Kit (Nippon Gene Co., Tokyo, Japan) followingthe manufacturer’s protocol. RNA quality was assessed by auto-mated capillary gel electrophoresis on an Agilent bioanalyzer 2100(Palo Alto, CA, USA) according to manufacturer’s instructions. Thesetotal RNA samples were labeled according to the standard one-cycle amplification and labeling protocol developed by Affymatrix(Santa Clara, CA, USA). The cRNAs were labeled at 40 ◦C for 2 h withcyanine 5 (Cy5) for samples and with cyanine 3 (Cy3) for the uni-versal human reference RNA (Affymatrix Technologies). After theamplification and labeling, the yields and dye incorporation effi-ciencies were determined using a spectrophotometer. AffymatrixGene Chip Human U133 plus 2.0 Array containing over 44K oligonu-cleotides was used for this study following microarray processingprotocol, the hybridized fluorescence was scanned in Affymatrixscanner.

2.3. Microarray data analysis

Microarray results were analyzed by Ingenuity Pathway Anal-ysis (IPA) System (http://www.ingenuity.com). The IngenuityKnowledge Base is the largest knowledge base of its kind, with mil-lions of findings created from the full text literature and is updatedon weekly basis (Helleman et al., 2010). Gene accession numbers,the fold change upon baicalein treatment vs. the control cells, and

egorized according to the molecular functions using the software.The analysis of canonical pathways identified the pathways fromthe IPA library of canonical pathways that were most significant tothe dataset (Li et al., 2007; Sethy et al., 2011).

S. Qin et al. / Journal of Ethnopharm

Table 1The primers used for real time PCR.

Gene primers Direction Sequences Tm (◦C)

HO-1

Fw cca gcg ggc cag caa caa agt gc60Re aag cct tca gtg ccc acg gta agg

AKR1C3Fw aag taa agc ttt gga ggt cac a

59Re gga cca act ctg gtc gat gaa

AKR1C1Fw atc cct ccg aga aga acc at

59Re aca cct gca cgt tct gtc tg

AKR1C2Fw gat ccc atc gag aag aac ca

59Re aca cct gca cgt tct gtc tg

GSRFw gat ccc aag ccc aca ata ga

59Re ctt aga acc cag ggc tga ca

TXNRD1Fw atc agg agg gca gac ttc aa

61Re ccc aca ttc aca cat gtt cc

DUSP1Fw cag ctg ctg cag ttt gag tc

59Re agg tag ctc agc gca ctg tt

SLC7A11Fw gtg tcc acc atc tcc aaa gg

60Re cgt cca gat ggt cag aga ca

GCLCFw gag ctg gga gga aac caa g

61Re tgg ttt ggg ttt gtc ctt tc

GCLMFw ggg aac ctg ctg aac tgg

61Re gca tga gat aca gtg cat tcc

NQO1Fw ctg gtt tga gcg agt gtt ca

60Re ttc cat cct tcc agg att tg

2

Psi3wOBOto(tM1((b

2

ao(tppeuopst

of 43 genes were changed by greater than or equal to 4 fold, ofwhich, expressions of 38 genes were upregulated while 5 geneswere downregulated. The expressions of 651 genes were changedbetween 2-fold and 4-fold, of which, expressions of 402 genes were

Table 2The number of genes that was regulated by baicalein treatment.

Fold of change Numbers of genes Regulation by baicalein

≥438 Up

5 Down

≤2 to <4402 Up249 Down

SRXN1Fw cat cga tgt cct ctg gat ca

61Re ctg caa gtc tgg tgt gga tg

.4. Real-time PCR

Validation of up-regulated expression was done by real timeCR. The primers specific to the genes used in the presenttudy are shown in Table 1, which were designed accord-ng to the NCBI sequence database using the software Primer. Reverse transcription and real-time PCR were performedith DyNAmoTM SYBR® Green 2-Step qRT-PCR Kit (Finnzymesy., Espoo, Finland) according to the manufacturer’s manual.riefly, RNA (200 ng) was reverse-transcribed to cDNA usingligo dT and M-MuLV RNase at 37 ◦C for 30 min, and the reac-

ion was then terminated at 85 ◦C for 5 min. The Tm-valuef PCR was determined according to each primer sequencehttps://www.finnzymes.fi/tm.determination.html). Each PCR con-ained 250 ng of reverse transcripts, 75 ng of each primer and 10 �l

aster mix. The thermal cycling conditions were held at 95 ◦C for5 min followed by 55 cycles of 30 s at 94 ◦C, 30 s at Tm-valuemelting temperature), and 30 s at 72 ◦C in Rotor-Gene-3000AKAACorbett Research Pty., NSW, Australia). The result was representedy the relative expression level normalized with control cells.

.5. Transient transfection and luciferase reporter gene assay

The pGL2-hQR41 luciferase reporter plasmid containing AREnd the expression plasmid pcDNA3-rNrf2 was described previ-usly (Tanigawa et al., 2007). In brief, human hepatoblastomaHepG2) cells were plated into each well of 12-well plates athe concentration of 1 × 105 and pre-cultured for 24 h in DMEMlus 10% FBS. The cells were then co-transfected with 0.1 �g AREromoter-encoding firefly luciferase plasmid and 0.1 �g pGL4-TK-ncoding Renilla luciferase plasmid (Promega, Madison, WI, USA)sing LipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA). For

ver-expression of Nrf2, cells were co-transfected with 0.05 �g ofcDNA3-rNrf2. The total amount of transfected DNA was kept con-tant at 0.25 �g/well by the addition of pcDNA3 control vector tohe DNA mixture. After 24 h incubation, the cells were treated by

acology 140 (2012) 131– 140 133

various concentrations (0, 10 �M, 20 �M, and 40 �M) of baicaleinin 0.1% DMSO, or 0.1% DMSO alone as a control and further incu-bated for 24 h. The activities of firefly and Renilla luciferase weremeasured in ARVOTMSX multilabel counter (Perkin Elmer, MA, USA)with the Dual-Luciferase Reporter Assay System (Promega, Madi-son, USA) according to the supplier’s recommendations. Luciferaseactivity values were normalized to transfection efficiency mon-itored by Renilla expression, and ARE transcription activity wasexpressed as fold induction relative to the control cells.

2.6. Western blotting

HepG2 cells (1 × 106) were pre-cultured in 6 cm dishes for 24 h,and then treated with baicalein at the concentration ranges of0–40 �M for 9 h. After being washed with phosphate-bufferedsaline (PBS) twice, the cells were lysed with 100 �l of sodiumdodecyl sulfate (1× SDS). The lysates were homogenized in an ultra-sonicator with ice water for 12.5 min, and immediately heated at100 ◦C for 5 min. The whole cell lysates were applied to 10–15%SDS PAGE and electrophoretically transferred to PVDF membrane(Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blotting,the membrane was incubated with specific antibody overnight at4 ◦C and further incubated for 1 h with secondary antibody. Boundantibodies were detected using the ECL system (GE Healthcare,Buckinghamshire, UK) and the relative amounts of proteins asso-ciated with specific antibody were quantified using Lumi VisionImager software (TAITEC Co., Koshigaya-shi, Japan).

2.7. Statistical analysis

All the experimental data shown were repeated at least threetimes, unless otherwise indicated. Differences between treated andcontrol cells were analyzed by Student’s t-test. A statistical proba-bility of p < 0.05 was considered significant.

3. Results

3.1. Gene expression profiling

According to the results of our initial experiments, HepG2 cellswere treated with or without 20 �M baicalein for 12 h. Underthese conditions, HepG2 cells did not show cytotoxicity in responseto treatment with baicalein. For instance, an increased Nrf2 pro-tein was observed after such treatment (data not shown). CellularmRNA was prepared and processed for hybridization to the humanoligonucleotide DNA microarray, as described in Section 2. Com-paring the hybridization signals from baicalein-treated mRNAwith those of the control mRNA revealed that the expressions

≤1.5 to <21382 Up1485 Down

Total1822 Up1739 Down

134 S. Qin et al. / Journal of Ethnopharmacology 140 (2012) 131– 140

Table 3The differential expressions of drug metabolizing enzymes by baicalein treatment.

Gene symbol Gene description Accession no. Fold change Regulation

Phase I metabolismALDH1L2 Aldehyde dehydrogenase 1 family, member L2 AI654224 1.27 DownCYP19A1 Cytochrome P450, family 19, subfamily A, polypeptide 1 NM 000103 1.04 UpCYP11A1 Cytochrome P450, family 11, subfamily A, polypeptide 1 NM 000781 1.09 UpCYP1A1 Cytochrome P450, family 1, subfamily A, polypeptide 1 NM 000499 2.81 UpCYP24A1 Cytochrome P450, family 24, subfamily A, polypeptide 1 NM 000782 1.52 UpCYP2B6 Cytochrome P450, family 2, subfamily B, polypeptide 6 NM 000767 1.04 UpCYP7A1 Cytochrome P450, family 7, subfamily A, polypeptide 1 NM 000780 1.00 DownCYP3A4 Cytochrome P450, family 3, subfamily A, polypeptide 4 J04449 1.32 UpCYP2C19 Cytochrome P450, family 2, subfamily C, polypeptide 19 X65962 1.13 UpCYP51A1 Cytochrome P450, family 51, subfamily A, polypeptide 1 U40053 1.21 UpCYP2U1 Cytochrome P450, family 2, subfamily U, polypeptide 1 AL359563 1.23 UpCYP4A22 Cytochrome P450, family 4, subfamily A, polypeptide 22 AL135960 1.11 DownCYP26B1 Cytochrome P450, family 26, subfamily B, polypeptide 1 NM 019885 1.12 DownCYP2S1 Cytochrome P450, family 2, subfamily S, polypeptide 1 AF335278 1.28 UpCYP26B1 Cytochrome P450, family 26, subfamily B, polypeptide 1 AC007002 1.33 UpCYP4V2 Cytochrome P450, family 4, subfamily V, polypeptide 2 BE326857 1.39 DownCYP39A1 Cytochrome P450, family 39, subfamily A, polypeptide 1 BC010358 1.06 UpDPYD Dihydropyrimidine dehydrogenase NM 000110 1.44 DownESD Esterase D/formylglutathione hydrolase AA193515 1.41 UpFMO4 Flavin containing monooxygenase 4 NM 002022 1.45 UpHERPUD1 Homocysteine-inducible, endoplasmic reticulum stress-inducible,

ubiquitin-like domain member 1AF217990 1.02 Down

FMO6P Flavin containing monooxygenase 6 pseudogene AL021026 1.03 Down

Phase II metabolismCES4 Carboxylesterase 4-like NM 016280 1.42 UpCES4 Carboxylesterase 4-like NM 016280 1.42 UpGSTA1 Glutathione S-transferase A1 NM 000846 1.23 UpGSTM3 Glutathione S-transferase M3 (brain) AI459140 1.59 UpGSTP1 Glutathione S-transferase pi NM 000852 1.37 UpMGST1 Microsomal glutathione S-transferase 1 AV705233 1.54 UpSULT2B1 Sulfotransferase family, cytosolic, 2B, member 1 NM 004605 1.35 UpSULT1C2 Sulfotransferase family, cytosolic, 1C, member 2 AI307799 1.34 UpHMGCL 3-Hydroxymethyl-3-methylglutaryl-Coenzyme A lyase

(hydroxymethylglutaricaciduria)NM 000191 1.14 Down

GCLC Glutamate-cysteine ligase, catalytic subunit NM 001498 3.50 UpHMGCLL1 3-Hydroxymethyl-3-methylglutaryl-Coenzyme A lyase-like 1 AF131827 1.07 UpGCLM Glutamate-cysteine ligase, modifier subunit AI753488 2.59 UpHMGCLL1 3-Hydroxymethyl-3-methylglutaryl-Coenzyme A lyase-like 1 BC024194 1.58 UpAKR1B10 Aldo-keto reductase family 1, member B10 (aldose reductase) NM 020299 2.29 UpAKR1C1 Aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase

1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)BF508244 1.83 Up

AKR1CL2 Aldo-keto reductase family 1, member C-like 2 AI243406 1.19 UpAKR1C2 Aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase

2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase,type III)

CA425039 1.17 Up

SQSTM1 Sequestosome-1 N30649 2.45 UpCBR3 Carbonyl reductase 3 NM 001236 1.49 UpHMOX1 Heme oxygenase (decycling) 1 NM 002133 3.00 UpATF4 Activating transcription factor 4 (tax-responsive enhancer element

B67)NM 001675 1.46 Up

NFE2L2 Nuclear factor (erythroid-derived 2)-like 2 AF323119 1.59 UpFTH1 Ferritin, heavy polypeptide 1 AA083483 1.09 UpSOD1 Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1

(adult))NM 000454 1.24 Down

SOD3 Superoxide dismutase 3, extracellular NM 003102 1.41 UpSOD2 Superoxide dismutase 2, mitochondrial W46388 1.21 UpTXN Thioredoxin AF065241 1.15 UpTXNIP Thioredoxin interacting protein NM 006472 2.06 UpTXNRD1 Thioredoxin reductase 1 NM 003330 1.92 UpGSR Glutathione reductase AI888037 1.03 Up

Phase III metabolismATP6V1E2 ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E2 NM 080653 2.24 UpLOC100133772///SLC16A5 Solute carrier family 16, member 5 (monocarboxylic acid transporter

6)///similar to MCTNM 004695 2.63 Up

SLC16A6 Solute carrier family 16, member 6 (monocarboxylic acid transporter7)

NM 004694 2.27 Up

SLC6A9 Solute carrier family 6 (neurotransmitter transporter, glycine),member 9

NM 006934 2.36 Up

SLC7A11 Solute carrier family 7 (cationic amino acid transporter, y+ system)member 11

AB040875 3.77 Up

SLC7A1 Solute carrier family 7 (cationic amino acid transporter, y+ system),member 1

AA148507 2.27 Up

SLC1A4 Solute carrier family 1 (glutamate/neutral amino acid transporter),member 4

W72527 3.79 Up

S. Qin et al. / Journal of Ethnopharmacology 140 (2012) 131– 140 135

Table 3 (Continued )

Gene symbol Gene description Accession no. Fold change Regulation

SLC2A14///SLC2A3 Solute carrier family 2 (facilitated glucose transporter), member3///solute carrier family 2 (facilitated glucose transporter), member 14

AL110298 2.46 Up

SLC2A10 Solute carrier family 2 (facilitated glucose transporter), member 10 NM 030777 2.14 UpSLC20A1 Solute carrier family 20 (phosphate transporter), member 1 AI671885 2.06 UpABCA11 ATP-binding cassette, sub-family A (ABC1), member 11 (pseudogene) NM 024903 1.70 UpABCA17P ATP-binding cassette, sub-family A (ABC1), member 17 (pseudogene) AI570450 1.23 UpABCA9 ATP-binding cassette, sub-family A (ABC1), member 9 AI284184 1.23 UpABCC9 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 NM 005691 1.38 UpABCC4 ATP-binding cassette, sub-family C (CFTR/MRP), member 4 AI248055 1.30 UpATP7A ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome) NM 000052 1.36 UpATP4B ATPase, H+/K+ exchanging, beta polypeptide NM 000705 1.27 UpATP2B2///LOC100134286 ATPase, Ca++ transporting, plasma membrane 2///similar to ATPase,

Ca++ transporting, plasma membrane 2M97260 1.71 Up

ATP11B ATPase, class VI, type 11B AB023173 1.29 UpATP10A ATPase, class V, type 10A N35112 1.29 Up

a2

subun

uoedg1sswlsp

3a

tepepw1(PupSpamiAsdtp

3

carLA

ATP11A ATPase, class VI, type 11A

ATP6V0A2 ATPase, H+ transporting, lysosomal V0 subunitATP6V1E2 ATPase, H+ transporting, lysosomal 31 kDa, V1

pregulated and 249 genes were downregulated. The expressionsf 2867 genes were changed between 1.5-fold and 2-fold, of which,xpressions of 1382 genes were upregulated and 1485 genes wereownregulated. Taken together, there were expressions of 3561enes out of the total 44K genes (8.09%) showing fold changes above.5-fold (Table 2). To validate the accuracy of the microarray data,ome representative genes with changes in expression were cho-en, and their expression levels were detected by real-time PCRith the same RNA. The real-time PCR results exhibited a simi-

ar expression pattern with that of the DNA microarray (data nothown here); suggesting the DNA microarray data obtained in theresent study is valid.

.2. Expression of hepatic drug metabolizing enzymes amongntioxidant response and transporters caused by baicalein

To know the influence of baicalein on hepatocytes ando predict its metabolism in liver, we investigated the genexpression changes of drug metabolizing enzymes and trans-orters in baicalein-treated cells. As shown in Table 3, the genexpression changes of 75 drug metabolizing enzymes and trans-orters were observed. Among them, 22 genes were associatedith phase I such as CYP1A1 (cytochrome P450 superfamily

A1), CYP24A1 (cytochrome P450 superfamily 24A1), ALDH1L2aldehyde dehydrogenase 1 family, member L2), and HER-UD1 (homocysteine-responsive endoplasmic reticulum-residentbiquitin-like domain member 1 protein); 30 genes associated withhase II such as GCLC (glutamate cysteine ligase catalytic), GCLM,QSTM1 (sequestosome-1), and TXNIP (thioredoxin-interactingrotein); 23 genes associated with phase III such as ATP family, LOC,nd SLC family. The expressions of genes that were upregulatedore than 2-fold included ALDH1L2, CYP1A1, CYP24A1, HERPUD1

n phase I, GCLC, GCLM, SQSTM1, TXNIP in phase II, and ABC, SLC,TP families in phase III (Table 3). On the other hand, the expres-ions of CYP26B1, CYP4V2, ESD, DPYD, GSTA1 and SOD1 genes wereownregulated. Thus, treatment with 20 �M baicalein upregulatedhe expressions of hepatic drug metabolizing enzymes, especiallyhase II and phase III genes.

.3. Canonical pathway analysis

The pathway analysis of these genes identified five canoni-al pathways with top significant changes in gene expressions

s following: NDR/RXR activation, Nrf2-mediated oxidative stressesponse, tyrosine metabolism, methionine metabolism, andPS/IL-1 mediated inhibition of RXR function (data not shown here).mong them, Nrf2-mediated pathway was identified as the second

AL161996 1.26 UpBG106215 1.68 Up

it E2 NM 080653 2.24 Up

category with the p-value of 4.75E-04 as shown in Fig. 1, whichmeans that the expressions of 12 genes in this pathway were reg-ulated above 2-folds out of the total 185 genes (the ratio = 0.065).The expressions of genes marked with colors were over 2-folds,which include transcriptional factors such as c-Fos, FRA1, smallMAF, and Jun; chaperone and stress response proteins such as HSP(heat shock protein) family and HERPUD1, phase II metabolizingenzymes or antioxidant proteins such as GCLC, GCLM, and SQSTM1.To confirm the results, the expression of Nrf2-mediated down-stream genes, such as GCLC/M, AKR1C1/2/3, HO-1, TXNRD1, NQO1,GSR, DUSP1 (dual specificity protein phosphatase 1), SLC7A11 (cys-tine/glutamate transporter) and SRXN1, was further detected byreal-time PCR as shown in Fig. 2. Most of these genes revealeda similar expression pattern between microarray and real-timePCR data. For example, the expression level of GCLM increased3.47-fold in real-time PCR, and 2.19-fold in DNA microarray. Thesedata demonstrated that an Nrf2-mediated pathway was involvedin baicalein-treated HepG2 cells.

3.4. Baicalein stimulates Nrf2-mediated ARE transcriptionactivity

Accumulating data have shown that these enzymes contain spe-cific nucleotide sequences in their gene promoters, defined as ARE,with the consensus sequence 5′-TA/CANNA/GTGAC/TNNNGCA/G-3′ (Wasserman and Fahl, 1997; Wruck et al., 2011). ARE has beenreported to contribute to the protection of cells against carcinogensand oxidative stress. Several molecules, such as nuclear factor-E2-related factor 2 (Nrf2), c-Jun, ATF2, and ATF4, have been proposedas potential modulators of ARE (Venugopal and Jaiswal, 1996; Itohet al., 1997, 1999; Nguyen et al., 2000; Hayes and McMahon, 2001).Of these, Nrf2, a member of the CNC family of bZIP proteins, isextensively proven to be a strong activator of ARE-mediated geneexpression (Wasserman and Fahl, 1997; Jonathan and Yamamoto,2010). Thus, we postulated that the mRNA expressions of thesegenes induced by baicalein were due to Nrf2-mediated ARE tran-scriptional regulation. The consensus sequences of ARE are found inthe promoter regions of genes of some hepatic drug metabolizingenzymes such as NQO1, AKR1C2, GCLM, GST, TXNRD1, SRXN1 andHO-1 (Fig. 3). To evaluate whether baicalein induces Nrf2-mediatedARE activity, we performed reporter gene assay by transfectingan ARE-luciferase reporter plasmid into HepG2 cells. As shown inFig. 4A, baicalein induced ARE-driven activity in dose dependent

manner. Next, we co-transfected Nrf2 expression plasmid withARE-luciferase plasmid in HepG2 cells. Over-expression of Nrf2stimulated ARE activation, and baicalein enhanced the activationof ARE in a dose-dependent manner (Fig. 4B). To further confirm

136 S. Qin et al. / Journal of Ethnopharmacology 140 (2012) 131– 140

Fig. 1. Effects of baicalein on Nrf2-mediated oxidative stress response pathway. Analysis of canonical pathway was performed by Ingenuity Pathway Analysis System. Coloredgenes (red and green) were identified by microarray analysis as differentially upregulated or downregulated in HepG2 cells treated by baicalein (20 �M) compared withcontrol. Other uncolored nodal genes are directly or indirectly associated with the differentially expressed genes but were not found to be regulated significantly by baicaleintreatment. Genes are linked by their sub-cellular location, just nucleus part shown here due to the space limited. Node (gene) and edge (gene relationship) symbols weredescribed in the bottom part of the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

S. Qin et al. / Journal of Ethnopharmacology 140 (2012) 131– 140 137

Fig. 2. Comparison of gene expressions obtained by real time PCR with that obtained by DNA microarray. Real time PCR was performed with SYBR Green 2-Step qRT-PCRKit as shown in Section 2. The result was expressed as the relative expression level. Each value represents the mean ± SD of three separate experiments, *p < 0.05 vs. control.CTL, control; AKR1C, aldo-keto reductase family 1, member C; NQO1, NAD(P)H:quinone oxidoreductase; HO-1, heme oxygenase-1; TXNRD1, thioredoxin reductase 1; GSR,glutathione reductase; DUSP1, dual specificity protein phosphatase 1; GCLC, glutamate cysteine ligase, catalytic subunit; GCLM, glutamate cysteine ligase, modifier subunit;SLC7A11, solute carrier family 7, member 11; SRXN1, sulfiredoxin-1.

Fig. 3. Promoter sequence assignment of selected human phase II genes. All thepromoter sequences are selected from human genes. Nucleotides at essential posi-tions for ARE marked in bold in its consensus, and elliptical frame showed the samentc

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Fig. 4. Baicalein stimulated Nrf2-mediated ARE transcription activity in HepG2 cells.(A) Effects of baicalein on the transcriptional activity of ARE in HepG2 cells. HepG2cells were co-transfected with the pGL2-ARE Firefly and pGL4-TK-Renilla luciferaseplasmids for normalization. After 5 h, cells were maintained in 10% serum mediumfor 20 h and then stimulated with 0–40 �M baicalein for an additional 24 h. Cells

ucleotides of all the genes. The abbreviation follow standard IUPAC nomencla-ure (n = A, T, C or G). The promoter sequences begin with 5′ are shown as reverseomplements, and the sequences begin with 3′ are shown as frontal complements.

he obtained results or previous results at protein level, we treatedepG2 cells with various concentrations of baicalein for 9 h and

hen detected the protein levels by Western blotting. As shown inig. 5, baicalein treatment increased Nrf2, NQO1, AKR1C1 and HO-1ut decreased Keap1 level significantly. According to these resultsrom microarray, real time PCR, reporter gene assay and Westernlotting analysis, we concluded that an Nrf2-mediated ARE activa-ion is involved in baicalein-induced expressions of these hepaticrug metabolizing enzymes including phase I, phase II metabolizingnzymes and phase III transporters.

. Discussion

Baicalein is a major active compound distributed in the rootsf Scutellaria baicalensis which has been utilized as medicinal herb

n traditional Chinese medicine. Although several lines of studiesave showed that baicalein has a number of pharmaceutical actionsSrinivas, 2010), the effect of baicalein on hepatocytes, especiallyn the phase I, II metabolizing enzymes and phase III transporters

were lysed and analyzed for Firefly and Renilla luciferase activities. (B) Effects ofbaicalein on Nrf2-mediated ARE activity. HepG2 cells were co-transfected with0.5 �g of pGL2-ARE-luciferase and 0.05 �g of the Nrf2 expression plasmid. Othersteps are the same as shown in (A). Each value represents the mean ± SD of fourseparate experiments, *p < 0.05; **p < 0.01; ***p < 0.005 vs. control.

138 S. Qin et al. / Journal of Ethnopharm

Fig. 5. Baicalein increased the levels of typical antioxidant proteins. HepG2 cellswere treated with 0–40 �M baicalein for 9 h. Nrf2, Keap1, NQO1, HO-1, AKR1C1 and�-tubulin were detected by Western blot analysis with their respective antibod-i*

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Nrf2-inducers with low toxicity to enhance the Nrf2-mediated

es. Each value represents the mean ± SD of three separate experiments, *p < 0.05;*p < 0.01 vs. control.

s still not clear. In this study, we profiled the gene expressions andegulation of baicalein-treated HepG2 cells by DNA microarray andathway analysis technologies.

Toxicological and pharmacological studies usually pay mainttention on ADME enzymes, but the best target such as humanepatocyte is restricted to gain due to the ethical consideration,hich leads to many other immortalized cell lines emerging,

uch as HepG2 and primary human hepatocytes. Both HepG2nd primary human hepatocytes are widely used in ADME, tox-cological, and other basic research, but they still have theirwn insufficiencies. HepG2 cells are deficient in the expres-ion of phase I drug-metabolizing enzymes such as P450 systemRodriguez-Antona et al., 2002; Liguori et al., 2008). Human primaryepatocytes tend to dedifferentiate over time, eventually losingheir drug-metabolizing capability, and are subject to various stim-li such as exposure to wide assortment of drugs, disease, diet,lcohol and so on (LeCluyse, 2001). Some reports used microar-ay to compare the gene expression differences between HepG2nd primary human hepatocytes treated by chemicals such astyrene, aflatoxin B1, and food promutagens (Wilkening et al.,003; Harris et al., 2004; Diodovich et al., 2006; Liguori et al.,008), it seems that human hepatocytes are the preferred modelor biotransformation in human liver, whereas HepG2 cells maye still useful to study regulation of drug metabolizing enzymes.n the present paper, we paid more attention on phase II metab-lizing enzymes especially antioxidant proteins, so we choseepG2 cells.

Among total 44K genes, treatment with baicalein-enhanced822 gene signals (4.14% of total genes), and baicalein-reduced739 gene signals (3.95% of total) by ≥1.5-fold (Table 1). Theseesults suggested that baicalein could regulate gene expressionsn HepG2 cells. Since the drug metabolizing enzymes play central

oles on the metabolism, detoxification and elimination of xeno-iotics and drugs, we next listed out drug metabolizing enzymesased on the other publications (Lee et al., 2008; Guo et al.,

acology 140 (2012) 131– 140

2009). In total, the expression changes of 75 drug metabolizingenzymes were observed (Table 2). Especially, the gene expres-sion of CYP1A1, CYP24A1, ALDH1L2, HERPUD1 (phase I enzymes),GCLC, GCLM, SQSTM1, TXNIP (phase II enzymes), and ATP fam-ily, LOC, and SLC family (phase III enzymes) were over 2-foldchange by baicalein. CYP family was well recognized as impor-tant metabolizing enzymes for toxic and carcinogenic xenobiotics.Flavonoids have been shown to modulate the CYP family, includingthe induction of specific CYP isozymes, the activation or inhibitionof enzymes in this family (Wood et al., 1986). In animal stud-ies, inducers of CYP family usually decrease the carcinogenicityof chemical carcinogens, suggesting that induction of CYP familyplays more important role in detoxification rather than formationof carcinogenic metabolites (Conney, 2003). Our DNA microarraydata showed that baicalein induced the expression of CYP1A1 genewhich is supported by the previous report (Zhang et al., 2003).CYP24A1 plays a role in regulating the level of vitamin D3 (Sakakiet al., 2005). The induction by baicalein suggests the possibilityof baicalein in calcium homeostasis and the vitamin D endocrinesystem. Previous study has suggested that flavonoid with ortho-hydroxyl substituent(s) on the aromatic ring(s) can induce theexpression of phase II enzymes such as NQO1, HO-1, GCLC andGCLM (Dinkova-Kostova et al., 2001). Baicalein contains severalortho-hydroxyl substituents on its aromatic rings, which may raisethe reactivity and facilitate addition of other substituent such asmercaptans, thereby raising its inducer potencies on expression ofphase II enzyme genes. Phase III transporters such as solute carri-ers group (SLC family) and ATP-binding cassette transporters (ABCfamily) have a substantial impact on systemic drug exposure andtoxicity (Hesselson et al., 2009; Franke et al., 2010). On the otherhand, we also observed that the expressions of ESD, DPYD, GSTA1and SOD1 genes are decreased.

In order to know the regulation of these genes in signal networklevel, we performed signal pathway analyses by IPA software. Theresults showed that an Nrf2-mediated pathway was involved in theregulation of these gene expressions (Fig. 2). Moreover, the consen-sus sequences of ARE are found in the promoter regions of somehepatic drug metabolizing enzyme genes such as NQO1, AKR1C2,GCLM, GSR, TXNRD1, SRXN1 and HO-1 (Fig. 3). Reporter gene assaydemonstrated that baicalein induced ARE-driven activity in HepG2cells in a dose dependent manner (Fig. 4A). Over-expression of Nrf2stimulated ARE activation, and baicalein enhanced the activation ofNrf2-mediated ARE in a dose-dependent manner (Fig. 4B). Finally,we confirmed them at protein level. Baicalein treatment increasedthe levels of Nrf2, NQO1 and HO-1, but decreased Keap1 level sig-nificantly (Fig. 5). Therefore, we conclude that Nrf2-mediated AREactivation is involved in baicalein-induced expressions of thesehepatic drug metabolizing enzymes.

Nrf2 is a redox-sensitive transcription factor (Lee and Johnson,2004). Nrf2-mediated ARE activation has become a target forchemoprevention since Nrf2 binds to ARE located in the promoterregion of many phase II detoxifying or antioxidant enzymes such asNQO1, GSR, HO-1, SRXN1, GCLC/M, AKR1C2 and TXNRD1 (Kasparet al., 2009). Induction of these cytoprotective enzymes via Nrf2-ARE signaling hence provides an effective means for achievingcellular protection against a variety of electrophilic carcinogensand other reactive toxicants as well as ROS (Kensler et al., 2007).Recent studies have shown that the activation of this pathway isimportant in preventing human diseases, such as cancer, neurode-generative disease, diabetes, pulmonary fibrosis, cardiovasculardiseases, ischemia, and inflammatory diseases (Zhang, 2010). Asa result many researchers have focused on identifying potent

adaptive response for disease prevention. In the present study, wehave found that baicalein can activate Nrf2-ARE signaling path-way, which is not only supplying the molecular mechanism of this

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idely used traditional Chinese medicine, but also implying theromising potency in low-toxicity drug development.

In summary, our DNA microarray data, for the first time,evealed gene expression profiles of baicalein in HepG2 cells. More-ver, signaling pathway analysis demonstrated that Nrf2-mediatedRE activation is involved in baicalein-induced expressions of mostepatic drug metabolizing enzyme genes. These results provide

comprehensive data for understanding the hepatic metabolism,ioactive role and the molecular mechanisms of baicalein.

onflict of interest

We have no direct or indirect commercial financial incentivessociated with publishing this article. Additionally, the authorsave no conflict of interest, and the source of extra-institutional

unding is indicated in the manuscript.

cknowledgments

This work was partially supported by grant-in-aid for scientificesearch of the Ministry of Education, Culture, Sports, Science andechnology (MEXT) of Japan (18580125), and by the fund of Frontiercience Research Center of Kagoshima University to D. X. Hou.

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