Elsevier Editorial System(tm) for Free Radical Biology and Medicine Manuscript Draft
Manuscript Number: FRBM-D-11-00397R2
Title: Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress
Article Type: Original Contribution
Keywords: Butein; CCl4; ERK; Glutamate cysteine ligase; Heme oxygenase 1; Nrf2; Phloretin; tBHP Corresponding Author: Dr. Haw-Wen Chen,
Corresponding Author's Institution: China Medical University First Author: Ya-Chen Yang
Order of Authors: Ya-Chen Yang; Chong-Kuei Lii; Ai-Hsuan Lin; Yu-Wen Yeh; Hsien-Tsung Yao; Chien-Chun Li; Kai-Li Liu; Haw-Wen Chen
Abstract: Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butyl hydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The role of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression was determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride (CCl4)-induced hepatotoxicity. In conclusion, we demonstrated that butein and phloretin up-regulate HO-1 and GCL expression through the
Free Radical Biology and Medicine
Dr. Kevin Peter Moore
Manuscript No.: FRBM-D-11-00397R1
Dear Dr. Moore:
Upon the requirement of FRBM, we have uploaded a separate document with article highlights.
Thank you.
Sincerely,
Haw-Wen Chen, Ph.D. Department of Nutrition China Medical University Tel: 886-4-22053366 Ext. 7520 Fax: 886-4-22062891
Email: chenhw@mail.cmu.edu.tw
Butein and phloretin induce HO-1 and GCL expression. Enhanced GCL expression leads to increased hepatic GSH synthesis. ERK2/Nrf2 pathway plays a key role in the induction of HO-1 and GCL by butein. Induction of HO-1 and GCL protects liver against oxidative stress.
Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and
phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative
stress
Ya-Chen Yang a,1, Chong-Kuei Lii b,1, Ai-Hsuan Lin c, Yu-Wen Yeh b, Hsien-Tsung
Yao b, Chien-Chun Li c, Kai-Li Liu c, Haw-Wen Chen b,*
a
Department of Health and Nutrition Biotechnology, Asia University, Taichung,
Taiwan b
Department of Nutrition, China Medical University, Taichung, Taiwan c
Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan
*
Corresponding author. Department of Nutrition, China Medical University,
Taichung 404, Taiwan. Fax: +886 4 2206 2891.
E-mail address: chenhw@mail.cmu.edu.tw (H.-W. Chen). 1
These authors contributed equally to this work.
*Revised Manuscript (text UNmarked)
ABSTRACT
Butein and phloretin are chalcones that are members of the flavonoid family of
polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory
activities. In rat primary hepatocytes, we examined whether butein and phloretin
affect tert-butyl hydroperoxide (tBHP)-induced oxidative damage and the possible
mechanism(s) involved. Treatment with butein and phloretin markedly attenuated
tBHP-induced peroxide formation, and this amelioration was reversed by
l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc
protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced
both HO-1 and GCL protein and mRNA expression and increased intracellular
glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2
signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear
protein-DNA binding activity, and ARE-luciferase reporter activity. The role of the
ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit
(GCLC) expression was determined by using RNA interference directed against
ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC
protein expression. These results suggest the involvement of ERK2 and Nrf2 in the
induction of HO-1 and GCLC by butein. In animal study, phloretin was shown to
tetrachloride (CCl4)-induced hepatotoxicity. In conclusion, we demonstrated that
butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2
pathway and protect hepatocytes against oxidative stress.
Keywords: Butein; CCl4; ERK; Glutamate cysteine ligase; Heme oxygenase 1; Nrf2;
Introduction
The excessive production of reactive oxygen species (ROS) is associated with
cell damage and the development of chronic diseases in humans [1]. Therefore,
compounds with antioxidant properties may be useful for preventing oxidative
stress-mediated diseases [2]. Cardiovascular disease (CVD) is a common chronic
disease and is the leading cause of morbidity and mortality in the United States [3].
Atherosclerosis is one of the major causes of CVD, and it is well established that
oxidative stress and inflammation play a critical role in atherosclerosis [4]. From the
evidence, it is inferred that compounds with antioxidant and anti-inflammatory
properties may exert protective effects against CVD. In support of this, a large cohort
study demonstrated an inverse correlation between total fruit and vegetable intake and
risk of CVD [5].
Flavonoids are naturally occurring polyphenolic compounds and represent one of
the most prevalent classes in fruits, vegetables, nuts, and beverages such as tea, coffee,
and red wine [6] as well as in medical herbs [7]. Flavonoids are composed of flavones,
flavonols, flavanones, flavanols, chalcones, anthocyanins, and isoflavones.
Flavonoids are well known for their capacity as antioxidant and anti-inflammatory
agents and are reported to have health-promoting, disease-preventing, and
Glutathione (GSH) is an endogenously synthesized tripeptide thiol that is
involved in numerous basic cellular processes including maintaining redox status [9],
scavenging free radicals and electrophilic intermediates [10],
conjugation/detoxification reactions [11], apoptosis [12], and cell signaling [13].
Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in GSH synthesis; it
is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM)
subunits that are expressed by distinct genes [14]. Both in vivo and in vitro models in
broccoli seeds have shown that the induction of GCLC expression is dependent on
nuclear factor erythroid 2-related 2 (Nrf2) [15]. Nrf2 is a transcription factor that is
necessary for the induction of numerous phase II enzymes [16]. Under basal
conditions, Nrf2 is retained in the cytosol by binding to Klech-like ECH-associated
protein 1 (Keap1) [17]. The Keap1-Nrf2 complex is disrupted in response to several
electrophilic antioxidants, and free Nrf2 translocates to the nucleus, where it binds to
the antioxidant/electrophile response element (ARE) sequences of the target gene
promoter, in conjunction with small Maf proteins [18]. AREs are found in the
promoters of many antioxidant and detoxifying enzyme genes, including GCLC, GSH
S-transferase (GST), and heme oxygenase 1 (HO-1) [19].HO-1 is an inducible
enzyme and catalyzes the rate-limiting step of free heme degradation into Fe2+, carbon
by biliverdin reductase [20]. HO-1 is known for its cytoprotective effects against
oxidative stress [21] and plays an important role in the resolution of inflammation
[22].
Butein and phloretin are chalcones that are members of the flavonoid family and
that have well-known antioxidant and anti-inflammatory activities. Although these
antioxidant and anti-inflammatory properties of the chalcones are well known,
however, few studies have investigated the potential of the chalcones to activate
cellular antioxidant and anti-inflammatory genes such as HO-1 and GCL and the
specific transcription factor and upstream signaling kinases involved in HO-1 and
GCL expression. In this study, therefore, we investigated the role of Nrf2 and
upstream signaling kinases involved in the induction of HO-1 and GCL expression by
butein and phloretin in rat primary hepatocytes. Furthermore, we investigated the role
of HO-1 and GCL in protection against tert-butyl hydroperoxide (tBHP)- and
Materials and methods
Chemicals
Cell culture medium (RPMI-1640) and penicillin-streptomycin solution were
obtained from Gibco Laboratory (Grand Island, NY). TRIzol was purchased from
Invitrogen (Carlsbad, CA). Zinc protoporphyrin (ZnPP) was purchased from
Calbiochem (La Jolla, CA). Dexamethasone,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), HEPES,
l-buthionine-S-sulfoximine (BSO), butein, and phloretin were obtained from Sigma
Chemical (St. Louis, MO). ITS+ (insulin, transferrin, selenium, bovine serum albumin,
and linoleic acid) was obtained from BD Biosciences (San Jose, CA). Small
interfering RNAs (siRNAs) against Nrf2 and ERK2 were purchased from Dharmacon
(Lafayette, CO). Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic
transaminase (GPT) kits were obtained from Randox Laboratories Ltd, (UK).
Hepatocyte isolation and culture
BioLASCO Experimental Animal Center (Taipei, Taiwan). Hepatocytes were isolated
by a modification of the two-step collagenase perfusion method described previously
[23]. After isolation, hepatocytes (3x106 cells/dish) were plated on collagen-coated
60-mm plastic tissue dishes (Falcon, Franklin Lakes, NJ) in RPMI-1640 medium (pH
7.37) supplemented with 10 mM Hepes, 1% ITS+, 1 μM dexamethasone, 100 IU
penicillin/ml, and 100 μg streptomycin/ml. Cells were incubated at 37oC in a 5% CO2
humidified incubator. After 4 h, the cells were washed with phosphate-buffered saline
to remove any unattached or dead cells, and the same medium supplemented with 0.1 μM dexamethasone was added. Twenty hours after attachment, cells were treated with various concentrations of butein or phloretin. The protocol for each experiment is
described in the corresponding figure legend.
Cell viability assay
Cell viability was analyzed by MTT assay. The MTT assay measures the ability
of viable cells to reduce a yellow 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide to a purple formazan by mitochondrial succinate
dehydrogenase. After incubation with 10, 25, and 50 μM butein or phloretin for 24 h,
additional 12 h, the medium was removed, and hepatocytes were then incubated in
RPMI-1640 medium containing 0.5 mg/ml MTT for an additional 3 h. The medium
was then removed, and isopropanol was added to dissolve the formazan. After
centrifugation at 7,000×g for 5 min, the supernatant of each sample was transferred to
96-well plates, and absorbance was read at 595 nm in an ELISA reader. The
absorbance in cultures treated with 0.1% DMSO was regarded as 100% cell viability.
RNA isolation and RT-PCR
Total RNA of hepatocytes was extracted by using TRIzol reagent. After
treatment, cells were washed twice with cold PBS and scraped with 500 μl of TRIzol reagent. Cell samples were mixed with 100 μl of chloroform and centrifuged at 11,000×g for 15 min. The supernatant was collected and mixed with 250 μl of
isopropyl alcohol. After centrifugation at 12,000×g for 20 min, the supernatant was
discarded and the RNA precipitate was stored in 70% ethanol or dissolved in
deionized water for quantification. We used 0.2 μg of total RNA for the synthesis of
first-strand cDNA by using Moloney murine leukemia virus reverse transcriptase
(Promega Company, Madison, WI) in a 20-μl final volume containing 250 ng
reaction volume containing 20 μl of cDNA, BioTaq PCR buffer, 50 μM of each
deoxyribonucleotide triphosphate, 1.25 mM MgCl2, and 1 U of BioTaq DNA
polymerase (GENET BIO, Chungnam, Korea). Oligonucleotide primers of HO-1
(forward: 5’-AGCATGTCCCAGGATTTGTC-3’; reverse:
5’-AAGGCGGTCTTAGCCTCTTC-3’), GCLC (forward: 5’-CCTTCTGGCACAGCACGTTG-3’; reverse:
5’-TAAGACGGCATCTCGCTCCT-3’), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (forward: 5’-CCATCACCATCTTCCAGGAG-3’; reverse:
5’-CCTGCTTCACCACCTTCTTG-3’)were designed on the basis of published
sequences [24,25]. Amplification of HO-1 and GAPDH was performed by heating
samples to 95oC for 5 min and then immediately cycling 25 times through a 30-s
denaturing step at 94oC, a 45-s annealing step at 56oC, and a 45-s elongation step at
72oC. For GCLC amplification, the PCR cycle number was 32 times through a 60-s
denaturing step at 94oC, a 60-s annealing step at 60oC, and a 90-s elongation step at
72oC. The GAPDH cDNA level was used as the internal standard. PCR products were
resolved in a 1%-agarose gel and were scanned by using a Digital Image Analyzer
(Alpha Innotech) and quantitated with ImageGauge software (FUJIFILM Science
Measurement of intracellular reduced and oxidized GSH content
After treatment with butein or phloretin for 24 h, 100 μl of the cytosolic fraction of hepatocytes was reacted with 200 μl of 10 mM Ellman’s reagent by gentle mixing, and followed by adding 60 μl of 20% 5-sulfosalicylic acid to cause acid precipitation. After centrifugation at 10,000xg and 4oC for 10 min, 100 μl of supernatant was used
to analyze reduced and oxidized GSH content by use of a High-Performance Liquid
Chromatography-Mass Spectrophotometer (HPLC/MS, Hewlett Packard) [26].
Plasmids, transfection, and luciferase assays
A p2xARE/Luc fragment containing tandem repeats of double-stranded
oligonucleotides spanning the Nrf2 binding site, 5’-TGACTCAGCA-3’, was
introduced into the pGL3 promoter plasmid (Promega). All subsequent transfection
experiments were performed by using nanofection reagent (PAA, Pasching, Austria) according to the manufacturer’s instructions. For luciferase assays, the cell lysate was first mixed with a luciferase substrate solution (Promega), and the resulting luciferase
activity was measured by using a microplate luminometer (TROPIX TR-717, Applied
was normalized with β-galactosidase activity.
RNA interference by small interfering RNA
Hepatocytes were transfected with ERK2 or Nrf2 siRNA SMARTpool by using
DharmaFECT1 transfection reagent (Thermo, Rockford, IL) according to the
manufacturer’s instructions. The 4 siRNAs against the rat ERK2 gene are (1)
ACACUAAUCUCUCGUACAU, (2) AAAAUAAGGUGCCGUGGAA, (3)
UAUACCAAGUCCAUUGAUA, and (4) UCGAGUUGCUAUCAAGAAA. The 4
siRNAs against the rat Nrf2 gene are (1) GAACACAGAUUUCGGUGAU, (2)
AGACAAACAUUCAAGCCGA, (3) GGGUUCAGUGACUCGGAAA, and (4)
AGAAUAAAGUUGCCGCUCA. A nontargeting control-pool siRNA was also tested
and was used as the negative control. After 24 h of transfection, cells were treated
with butein or phloretin for another 24 h. Cell samples were collected for Western
blotting analysis.
Nuclear extract preparation
scraped from the dishes with 1000 μl of PBS. Cell homogenates were centrifuged at
2,000×g for 5 min. The supernatant was discarded, and the cell pellet was allowed to
swell on ice for 15 min after the addition of 200 μl of hypotonic buffer containing 10
mM HEPES, 1 mM MgCl2, 1 mM EDTA, 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet
P-40, 4 μg/ml leupeptin, 20 μg/ml aprotinin, and 0.2 mM PMSF. After centrifugation
at 6,000×g for 15 min, pellets containing crude nuclei were resuspended in 50 μl of
hypertonic buffer containing 10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 0.25 mM
EDTA, 0.5 mM DTT, 4 μg/ml leupeptin, 20 μg/ml aprotinin, 0.2 mM PMSF, and 10%
glycerol at 4oC for 30 min. The samples were then centrifuged at 10,000×g for 15 min.
The supernatant containing the nuclear proteins was collected and stored at -80oC
until the Western blotting and electrophoretic mobility shift assays.
Western blotting analysis
Cells were washed twice with cold PBS and were harvested in 150 μl of lysis
buffer (10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 320 mM sucrose, 5 mM EDTA, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 2 mM dithiothreitol). Cell homogenates were centrifuged at 10,000×g for 20 min at 4oC. The resulting
preparation of nuclear extracts was described above. The total protein was analyzed
by use of the Coomassie Plus protein assay reagent kit (Pierce Biotechnology Inc.,
Rockford, IL). Equal amounts of proteins were electrophoresed in an
SDS-polyacrylamide gel, and proteins were then transferred to polyvinylidene
fluoride membranes. Nonspecific binding sites on the membranes were blocked with
5% nonfat milk in 15 mM Tris/150 mM NaCl buffer (pH 7.4) at room temperature for
2 h. Membranes were probed with anti-GCLC (Abcam, Cambridge, UK), anti-Nrf2
(Santa Cruz), anti-HO-1 (Calbiochem, San Diego, CA), anti-ERK, anti-phospho-ERK,
anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-Akt, anti-phospho-Akt,
anti-PARP (Cell Signaling Technology), anti-GAPDH (Millipore, Billerica, MA), and
anti-actin (Sigma) antibodies. The membranes were then probed with the secondary
antibody labeled with horseradish peroxidase. The bands were visualized by using an
enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA) and were
scanned by a luminescent image analyzer (FUJIFILM LAS-4000, Japan). The bands
were quantitated with ImageGauge software.
Electrophoretic mobility shift assay
previous study [27]. The LightShift Chemiluminescent EMSA Kit (Pierce Chemical
Company, Rockford, IL) and synthetic biotin-labeled double-stranded rHO-1 ARE
consensus oligonucleotides (forward: 5′-AACCATGACACAGCATAAAA-3′; reverse:
5′-TTTTATGCTGTGTCATGGTT-3′) were used to measure Nrf2 nuclear
protein-DNA binding activity. Five micrograms of nuclear extract, poly (dI-dC), and
biotin-labeled double-stranded ARE oligonucleotides were mixed with the binding
buffer (Chemiluminescent Nucleic Acid Detection Module, Thermo, Rockford, IL) to
a final volume of 20 μl, and the mixture was incubated at room temperature for 30
min. Unlabeled double-stranded rHO-1 ARE oligonucleotides and mutant
double-stranded oligonucleotides (5′- AACCAgtcCACAGCATAAAA -3′) were used
to confirm the protein-binding specificity. The nuclear protein-DNA complex was
separated by electrophoresis on a 6% TBE-polyacrylamide gel and was then
transferred to a Hybond-N+ nylon membrane. The membranes were cross-linked by UV light for 10 min and were then reacted with 20 μl of streptavidin-horseradish peroxidase for 20 min, and the nuclear protein-DNA bands were developed with a
Chemiluminescent Substrate (Thermo). The bands were scanned by a luminescent
image analyzer (FUJIFILM LAS-4000, Japan).
Detection of intracellular oxidative states was performed by using the probe
2,7-dichlorofluorescin diacetate (H2DCF-DA; Molecular Probes Inc., Eugene, OR).
Hepatocytes were pretreated with butein or phloretin for 24 h before being challenged
with 0.5 mM tBHP for 1 h. To assess the effects of the inhibitors BSO (a GCL
inhibitor) and ZnPP (a HO-1 inhibitor), cells were pretreated with the respective
inhibitor for 2 h before the addition of butein or phloretin. As a control, an equal
amount of DMSO was added to untreated cells. After treatment, cells were incubated with 5 μmol/l of H2DCF-DA for 10 min. The DCF fluorescence was then detected by a Confocal Microscope Detection System (Leica TCS SP2).
Animal study
Male Sprague-Dawley rats (weighing 200-220 g) received i.p. injection of 10 or
30 mg/kg phloretin for 5 consecutive days. At day 6, animals were sacrificed and liver
tissues were removed for HO-1 expression and GSH content determination as
described above. For the carbon tetrachloride (CCl4) experiment, rats were i.p.
administrated with 30 mg/kg phloretin for 5 consecutive days and then 1 ml/kg CCl4
(50% in olive oil) was i.p. injected at day 6. Animals were sacrificed after 24 h and
manufacturer’s instructions. The rats were treated in compliance with the Guide for
the Care and Use of Laboratory Animals [28].
Statistical analysis
Data were analyzed by using analysis of variance (SAS Institute, Cary, NC). The
significance of the difference among mean values was determined by one-way analysis of variance followed by the Tukey’s test and the difference between mean values was determined by Student’s t-test. P values <0.05 were taken to be
Results
Concentration changes in butein and phloretin during cell culture
To determine the concentration changes in butein and phloretin during cell
culture, the remaining percentage of butein and phloretin in the media was determined
by HPLC/MS during 24 h of treatment (Fig. 1). In the cell-free condition,
concentrations of butein (A) and phloretin (B) gradually decreased and 80% and 55%
were left, respectively, after 4 h. With hepatocytes, contents of chalcones dropped
quickly and the remaining percentages of both butein (A) and phloretin (B) at 4 h
were only 15% and 10%, respectively. The decreased remaining percentage in
presence of hepatocytes may implicate the uptake or degradation of butein and
phloretin by hepatocytes.
Effects of butein and phloretin on cell viability
We used the MTT assay to test whether the concentrations of butein and
phloretin used in the present study caused cell damage. The cell viabilities of rat
108.0±5.1%, 100.2±5.2%, 96.1±7.9%, 100.2±6.7%, 93.3±0.6%, and 92.9±5.0%,
respectively, compared with the unstimulated controls (100%). As indicated, there
were no adverse effects on the growth of rat primary hepatocytes up to a
concentration of 50 μM butein or phloretin. Thereafter, the highest concentration of
butein and phloretin used in this study was 25 μM.
Butein and phloretin alleviate tBHP-induced oxidative damage through the induction
of HO-1 and GCLC
Oxidative stress plays a key role in the pathogenesis of several chronic diseases.
Oxidative stress can be induced in vitro by use of numerous oxidants, including
hydrogen peroxide, tBHP, 4-hydroxynonenal, sodium arsenite, and UV irradiation
[29]. In the present study, we used tBHP to induce oxidative damage and to
investigate the protective effects of butein and phloretin. As shown in Fig. 2A, cell
viability was significantly decreased in cells treated with 0.5 mM tBHP for 12 h. In
cells pretreated with 25 μM butein or phloretin, damage was significantly attenuated
(p < 0.05). Cellular peroxide formation was also measured in cells, as shown in Fig.
2B, peroxide formation was significantly greater in cells treated with 0.5 mM tBHP
and then challenged with 0.5 mM tBHP for an additional 1 h showed significantly less
peroxide formation than did cells treated with tBHP alone (pictures c and d vs. b). In
cells pretreated with BSO or ZnPP for 2 h, the protective effect of butein on peroxide
formation was abolished (pictures e and f). Thus, the induction of GCLC and HO-1 at
least partially explains the antioxidant activity of butein and phloretin.
Butein and phloretin increase GSH, total GSH, GSH/oxidized GSH (GSH/GSSG), and
GCLC expression in hepatocytes
Compared to control hepatocytes, hepatocytes treated with 5, 10, or 25 μM
butein and phloretin for 24 h had significantly greater cellular GSH, total GSH, and
GSH/GSSG (p<0.05) (Table 1). It is reported that GCL catalyzes the rate-limiting
step in GSH synthesis [14]. Because of the increased cellular GSH content with butein
and phloretin treatments, we determined the effects of butein and phloretin on GCLC
protein expression. As shown in Fig. 3A, treatment with both butein and phloretin for
24 h induced GCLC protein expression in a dose-dependent manner. These results
suggest that the enhanced GCLC protein expression contributes to the increased
cellular GSH content. In addition to protein expression, GCLC mRNA expression was
and phloretin significantly induced GCLC mRNA expression (p<0.05) (Fig. 3B).
Butein and phloretin induce HO-1 expression in hepatocytes
HO-1 is an inducible enzyme that catalyzes the rate-limiting step in the
degradation of free heme. Because of the well-known antioxidant and
anti-inflammatory activities of HO-1, compounds with HO-1-inducing activity are
recognized to have cytoprotective properties. Butein (5, 10, and 25 μM) and phloretin
(25 μM) significantly induced HO-1 protein expression (p<0.05) (Fig. 3C).We also
observed the induction of HO-1 mRNA expression by butein and phloretin (Fig. 3D).
ERK and Nrf2 activation in butein and phloretin induction of HO-1 and GCLC
expression
Nrf2 activation can be under the regulation of protein kinase C,
phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase [30].
The effect of chalcones on kinase signaling pathway was performed with butein but
not with phloretin. To clarify the importance of the kinase signaling pathway in the
μM butein for 0.5, 1, 2, 3, 6, and 12 h. As shown in Fig. 4A, ERK1/2 phosphorylation occurred 0.5 h after treatment (p<0.05), and phosphorylation declined at 6 h after
treatment. However, no effect of butein on the activation of the other
mitogen-activated protein kinases and Akt was observed (data not shown). The role of
ERK2 in butein-induced HO-1 and GCLC protein expression was further investigated.
As shown in Fig. 4B, silencing ERK2 attenuated the butein-induced HO-1 and GCLC
protein expression. In addition, silencing ERK2 abolished the butein-induced nuclear
accumulation of Nrf2 (Fig. 4C). These data imply that ERK1/2 may be the candidate
kinase for Nrf2 activation and nuclear translocation and subsequent HO-1 and GCLC
induction by butein.
Nrf2 is a transcription factor necessary for the induction of numerous antioxidant
and detoxification phase II enzymes [16], and it binds to the ARE sequences of target
gene promoters in conjunction with small Maf proteins. Nrf2 undergoes nuclear
translocation and induces gene expression in response to stress. As shown in Fig. 5A,
cells treated with 25 μM butein had a higher level of Nrf2 accumulation in the nuclear
fraction as early as 1 h, and this accumulation was sustained until 12 h. However, the
level of cytosolic Nrf2 was relatively stable throughout the 12 h of treatment with 25 μM butein (Fig. 5A). Our results are consistent with the finding reported by Kim et al. [31] that phytochemical-induced HO-1 expression is mediated by Nrf2.
Furthermore, we used EMSA to determine the effects of butein and phloretin on
Nrf2 nuclear protein-DNA binding activity. As shown in Fig. 5B, 25 μM butein and
phloretin increased the DNA binding activity of Nrf2. We further used cells
transfected with luciferase reporter constructs harboring the ARE to determine the
specificity of butein and phloretin for this sequence. As shown in Fig. 5C, both 25 μM
butein and phloretin significantly increased ARE-luciferase activity (p<0.05).
Next, we examined the role of Nrf2 in the induction of HO-1 and GCLC protein
expression by butein by using an siRNA SMARTpool system to create an
Nrf2-knockdown model. Hepatocytes were transfected with an siNrf2 construct for 24
h, followed by treatment with 25 μM butein for an additional 24 h. Control cells were
transfected with a nontargeting siRNA construct. The efficiency of the siRNA
SMARTpool system in knocking down Nrf2 was assayed by Western blot (Fig. 6).
Knockdown of Nrf2 expression, the induction of HO-1 and GCLC protein expression
by butein was apparently abolished (Fig. 6).
Phloretin increases rat liver GSH content and HO-1 expression and decreases
CCl4-induced hepatotoxicity
for 5 consecutive days had significantly greater GSH content in liver. The GSH
contents of rats treated with 0, 10 or 30 mg/kg phloretin were 5.0±0.8, 7.3±0.7, and
7.2±1.2 μmol/g liver (n=4). We also found that the induction of liver HO-1 expression
by phloretin treatment (Fig. 7). Next, we determined whether phloretin treatment
decreases CCl4-induced hepatotoxicity. As shown in Table 2, rats receiving 30 mg/kg
phloretin for 5 consecutive days followed by i.p. injection of CCl4 (1 ml/kg, 50% in
olive oil) had significantly less plasma GOT and GPT activities. Thus, the inhibition
of CCl4-induced hepatotoxicity by phloretin might be attributed to its increased
Discussion
In this study, we demonstrated that both butein and phloretin effectively
suppressed tBHP-induced oxidative damage and that this inhibition was likely
associated with an up-regulation of HO-1 and GCLC expression through the
ERK2/Nrf2 pathway. We also found that rats receiving phloretin treatment had
significantly less CCl4-induced hepatotoxicity.
Oxidative stress is a state wherein the balance between the free radicals
generated and the free radical or oxidant scavenging capacity of the endogenous
antioxidant system is disrupted. Oxidative stress is documented to be involved in the
pathogenesis of several chronic diseases [1]. Cellular antioxidants are composed of
oxidant scavengers and antioxidant enzymes that convert free radicals to more benign
molecules. Thiol-containing compounds such as GSH and thioredoxin are oxidized by
free radicals and rapidly regenerated. GSH is an endogenously synthesized tripeptide
thiol, and GCL catalyzes the rate limiting step in GSH synthesis [14].
HO-1 is an inducible enzyme that catalyzes the rate-limiting step of free heme
degradation into Fe2+, carbon monoxide, and biliverdin, the last of which is
subsequently catabolized into bilirubin by biliverdin reductase [20]. Accumulating
diseases, including atherosclerosis, diabetes, obesity, cardiovascular disease, and
hypertension [21,32]. In a previous review, Ryter et al. [33] described that increased
HO-1 expression is associated with increased antioxidant protection. The antioxidant
function of bilirubin has been demonstrated both in vivo and in vitro. For example, the
addition of bilirubin in micromolar concentrations in cell culture media protects
against cytotoxicity induced by H2O2 in bovine vascular smooth muscle cells [34]. In
addition, increased HO-1 expression and bilirubin accumulation are involved in
heme-conferred resistance to H2O2-mediated cytotoxicity in vascular smooth muscle
cells. The antioxidant activity of bilirubin was demonstrated in vivo in the jaundiced
Gunn rat model [35]. Jaundiced Gunn rats with hyperbilirubinemia display a reduced
manifestation of plasma biomarkers of oxidative stress in response to hyperoxia
compared with their nonjaundiced counterparts.
Hyperglycemia has been linked to oxidative stress and leads to endothelial cell
dysfunction. Streptozotocin-induced diabetic rats have significantly greater levels of
circulating endothelial cells and urinary output of 8-epi-isoprostane PGF2α, an
indicator of overall oxidative stress, than do their normal counterparts [36]. Diabetic
rats receiving daily i.p. injection of CORM-3, a CO donor, exhibit a greater than
6-fold increase in plasma CO levels compared with that in rats receiving vehicle. CO
8-epi-isoprostane PGF2α in diabetic animals (p<0.05).
It is reported that acute administration of iron to intact rats [37] or to rat
hepatoma cells [38] induces the synthesis of the iron-storage protein ferritin. The
cytoprotective antioxidant role of ferritin has been demonstrated in porcine aorta
endothelial cells [39]. There is an inverse correlation between endothelial cell ferritin
and H2O2/hemin-mediated cytotoxicity.
Enhancement of intracellular antioxidant capacity is believed to reduce the risk
of oxidative stress-mediated diseases. Chalcones are one kind of flavonoids that are
recognized to have health-promoting, disease-preventing, and chemopreventive
activities because of their antioxidant and anti-inflammatory properties. Compared
with the other flavonoids, however, few studies have investigated the antioxidant
potential of chalcones including butein and phloretin. In the present study, we found
that butein and phloretin significantly suppressed tBHP-induced peroxide formation
(Fig. 2B), and that this inhibition was attenuated by BSO (a GCL inhibitor) and ZnPP
(a HO-1 inhibitor). Our results imply that the antioxidant activity of butein and
phloretin is associated with their induction of GCLC and HO-1. Butein and phloretin
were shown to induce GCLC and HO-1 protein and mRNA expression in hepatocytes
(Figs. 3A, 3B, 3C, and 3D), and phloretin was shown to induce HO-1 protein
cells treated with butein and phloretin had significantly greater cellular GSH, total
GSH, and GSH/GSSG than did control cells (Table 1), and the GSH-enhancing effect
of phloretin in rat liver was demonstrated as well.
It is well established that Nrf2 activation is under the regulation of protein kinase
C, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinase
[30]. Sulforaphane induces Nrf2 activity, induces GCL activity, and increases cellular
GSH content, and these actions are mediated by the PI3K/Akt pathway [40]. Nrf2 is
reported to be phosphorylated at Ser-40 by protein kinase C, and this phosphorylation
leads to Nrf2 dissociation from Keap1 [41]. Subsequently, free Nrf2 transactivates
ARE-mediated transcription. It has been demonstrated that activation of p38 MAPK
and an increase in the nuclear level of Nrf2 are involved in gallic acid-induced P-form
phenol sulfotransferase expression in human hepatoma HepG2 cells [42]. In addition,
Nrf2 has also been shown to play a critical role in the ARE-driven gene expression of
HO-1 [43]. Supporting this idea, we examined the effect of butein on the protein
expression of Nrf2 as well as the nuclear accumulation of Nrf2. In the present study,
butein was found to activate ERK (Fig. 4A), and to increase the nuclear accumulation
of Nrf2 (Fig. 5A), increase Nrf2 nuclear protein-DNA binding activity (Fig. 5B), and
increase ARE-luciferase reporter activity (Fig. 5C). To further determine the signaling
expression, we used RNA interference directed against ERK2 and Nrf2. Both siERK2
and siNrf2 abolished butein-induced HO-1 and GCLC protein expression (Figs. 4B
and 6).
Our results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1
and GCLC by butein. The findings of this study are schematically presented in Fig. 8.
In conclusion, we have shown that butein and phloretin inhibit tBHP-induced
oxidative damage by up-regulating HO-1 and GCLC expression through the
ERK2/Nrf2 pathway and that phloretin suppresses CCl4-induced hepatotoxicity via
the enhancement of hepatic GSH content and HO-1 expression. The antioxidant
property of butein and phloretin confers them with protective activity against
Acknowledgements
Abbreviations
ARE, antioxidant/electrophile response element; BSO, l-buthionine-S-sulfoximine;
CCl4, carbon tetrachloride; CVD, cardiovascular disease; EMSA, electrophoretic
mobility shift assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCL,
glutamate cysteine ligase; GCLC, glutamate cysteine ligase catalytic subunit; GCLM,
glutamate cysteine ligase modifier subunit; GOT, glutamic oxaloacetic transaminase;
GPT, glutamic pyruvic transaminase; GSH, glutathione; GST, glutathione
S-transferase; HO-1, heme oxygenase 1; ITS+, insulin, transferrin, selenium, bovine
serum albumin, and linoleic acid; Keap1, Klech-like ECH-associated protein 1; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nrf2, nuclear factor
erythroid 2-related 2; ROS, reactive oxygen species; siRNA, small interfering RNA;
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Figure legends
Fig. 1. Concentration changes in butein and phloretin during cell culture. Butein and
phloretin (25 μM) were added to the cell culture medium in absence of hepatocytes (◆)
or in presence of hepatocytes (■). Changes in butein (A) and phloretin (B)
concentrations in the medium over 24 h were determined by HPLC-MS. Values are
means ± SD of three independent experiments.
Fig. 2. Effects of butein and phloretin on tBHP-induced cell damage and peroxide
formation. Cells were pretreated with 25 μM butein or phloretin for 24 h followed by
challenge with 0.5 mM tBHP for an additional 12h (A). Cells were pretreated with
butein and phloretin for 24 h before being challenged with tBHP for an additional 1 h.
BSO and ZnPP were added 2 h before butein treatment. Cells treated with DMSO
were used as the control (a). tBHP induced peroxide formation (b). Both butein (c)
and phloretin (d) suppressed tBHP-induced peroxide formation. ZnPP (a HO-1
activity inhibitor) (e) and BSO (a GCL activity inhibitor) (f) reversed the inhibition of
tBHP-induced peroxide formation by butein.
Fig. 3. Butein and phloretin induce GCLC and HO-1 protein and mRNA expression in
protein expression. Total RNA was isolated from cells and was subjected to RT-PCR
with specific GCLC (B), HO-1 (D), and GAPDH primers as described in Materials
and Methods. Values are means ± SD of three independent experiments. Values not
sharing the same letter are significantly different (p < 0.05). One representative
immunoblot from three independent experiments is shown.
Fig. 4. Effect of butein on ERK activation in rat primary hepatocytes, and the role of
ERK2 in butein-induced HO-1 and GCLC expression. After attachment, hepatocytes
were treated with 25 μM butein for various time periods and ERK phosphorylation
was determined (A). After attachment, hepatocytes were transiently transfected with
non-targeting control siRNA or siERK2 for 24 h, followed by treatment with 25 μM
butein for an additional 24 h. siERK2 attenuated the butein-induced HO-1 and GCLC
protein expression (B). Aliquots of nuclear protein (20 μg) were used for Western blot
analysis. siERK2 abolished butein-induced Nrf2 nuclear accumulation (C).
Fig. 5. Effect of butein on Nrf2 activation. Cells were prepared after treatment with
25 μM butein for the indicated time periods. Immunoblots of nuclear, cytosolic, and
total extracts from treated cells were then probed with the Nrf2-specific antibody (A).
used for Nrf2 nuclear protein DNA-binding activity (B). Aliquots of nuclear extracts
(5 μg) were used for EMSA. To confirm the specificity of the nucleotide, 200-fold
cold probe (biotin-unlabeled ARE binding site) and biotin-unlabeled double-stranded
mutant ARE oligonucleotide (2 ng) were included in the EMSA. One representative
experiment out of three independent experiments is shown. Cells were transfected
with the ARE-luciferase construct for 12 h and were then stimulated with 25 μM
butein and phloretin for an additional 24 h (C). The cells were then lysed and
analyzed for luciferase activity. Values not sharing the same letter are significantly
different (p < 0.05).
Fig. 6. Effect of siNrf2 on butein-induced HO-1 and GCLC expression in rat primary
hepatocytes. After attachment, hepatocytes were transiently transfected with
non-targeting control siRNA or siNrf2 for 24 h, followed by treatment with 25 μM
butein for an additional 24 h. Nrf2 siRNA inhibited butein-induced HO-1 and GCLC
expression.
Fig. 7. Effect of phloretin treatment on HO-1 expression in rat liver. Male
Sprague-Dawley rats received i.p. injection of 30 mg/kg phloretin in 100 μl DMSO
HO-1 protein expression in pooled liver samples was determined.
Fig. 8. Scheme summarizing the inhibition of tBHP-induced oxidative damage by
butein and phloretin via the up-regulation of HO-1 and GCLC expression through the
ERK2/Nrf2 pathway and the suppression of CCl4-induced hepatotoxicity by phloretin
Table 1
Effects of butein and phloretin on cellular GSH content*.
Treatment Dose (μM) GSH (nmol/mg protein) Total GSH (nmol/mg protein) GSH/GSSG Control 115.2±21.8a 116.7±22.1a 156.6±22.1a butein 5 154.5±18.1ab 157.1±16.0ab 254.7±59.0b 10 171.1±15.2b 172.3±15.3b 276.5±16.3b 25 199.0±25.1b 200.5±25.4b 303.6±33.8b phloretin 5 188.8±38.9ab 190.4±38.9ab 281.0±22.8b 10 200.5±19.5b 201.9±19.4b 369.4±19.8b 25 210.2±28.7b 211.6±28.6b 361.9±71.5b *
Values are means ± SD, n = 3. Twenty hours after attachment, hepatocytes were
incubated with various concentrations of butein and phloretin for 24 h. Values not
sharing a same letter are significantly different (p < 0.05).
Table 2
Effect of phloretin on carbon tetrachloride-induced hepatotoxicity*.
CCl4 treatment control phloretin
GOT (U/L) before 24.1±5.9 25.1±9.4
after 252±71.6 165.0±50.6＊
GPT (U/L) before 22.0±4.9 15.2±4.8＊
after 231.3±87.8 107.5±56.3＊
Values are means ± SD, n =7-8. Male Sprague-Dawley rats received i.p. injection of
30 mg/kg phloretin for 5 consecutive days followed by i.p. injection of 1 ml/kg carbon
tetrachloride (CCl4) (50% in olive oil). Rats were sacrificed after 24 h, and plasma
was collected before and after 24 h CCl4 treatment for GOT and GPT determinations. *
Means significant difference between control and phloretin groups (p < 0.05).
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Supplementary Material
