Xanthohumol, a polyphenol chalcone present in hops, activating nrf2 enzymes to confer protection against oxidative damage in pc12 cells

Article abstract of DOI:10.1021/jf505075n

Xanthohumol (2a?2,4a?2,4-trihydroxy-6a?2-methoxy-3a?2-prenylchalcone, Xn), a polyphenol chalcone from hops (Humulus lupulus), has received increasing attention due to its multiple pharmacological activities. As an active component in beers, its presence has been suggested to be linked to the epidemiological observation of the beneficial effect of regular beer drinking. In this work, we synthesized Xn with a total yield of 5.0% in seven steps and studied its neuroprotective function against oxidative-stress-induced neuronal cell damage in the neuronlike rat pheochromocytoma cell line PC12. Xn displays moderate free-radical-scavenging capacity in vitro. More importantly, pretreatment of PC12 cells with Xn at submicromolar concentrations significantly upregulates a panel of phase II cytoprotective genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, thioredoxin, and thioredoxin reductase. A mechanistic study indicates that the ?±,?2-unsaturated ketone structure in Xn and activation of the transcription factor Nrf2 are key determinants for the cytoprotection of Xn. Targeting the Nrf2 by Xn discloses a previously unrecognized mechanism underlying the biological action of Xn. Our results demonstrate that Xn is a novel small-molecule activator of Nrf2 in neuronal cells and suggest that Xn might be a potential candidate for the prevention of neurodegenerative disorders.

Full text of DOI:10.1021/jf505075n

Article
pubs.acs.org/JAFC
Xanthohumol, a Polyphenol Chalcone Present in Hops, Activating
Nrf2 Enzymes To Confer Protection against Oxidative Damage in
PC12 Cells
Juan Yao, Baoxin Zhang, Chunpo Ge, Shoujiao Peng, and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University,
Lanzhou, Gansu 730000, China
S
* Supporting Information
ABSTRACT: Xanthohumol (2′,4′,4-trihydroxy-6′-methoxy-3′-prenylchalcone, Xn), a polyphenol chalcone from hops (Humulus
lupulus), has received increasing attention due to its multiple pharmacological activities. As an active component in beers, its
presence has been suggested to be linked to the epidemiological observation of the beneficial effect of regular beer drinking. In
this work, we synthesized Xn with a total yield of 5.0% in seven steps and studied its neuroprotective function against oxidative-
stress-induced neuronal cell damage in the neuronlike rat pheochromocytoma cell line PC12. Xn displays moderate free-radical-
scavenging capacity in vitro. More importantly, pretreatment of PC12 cells with Xn at submicromolar concentrations significantly
upregulates a panel of phase II cytoprotective genes as well as the corresponding gene products, such as glutathione, heme
oxygenase, NAD(P)H:quinone oxidoreductase, thioredoxin, and thioredoxin reductase. A mechanistic study indicates that the
α,β-unsaturated ketone structure in Xn and activation of the transcription factor Nrf2 are key determinants for the cytoprotection
of Xn. Targeting the Nrf2 by Xn discloses a previously unrecognized mechanism underlying the biological action of Xn. Our
results demonstrate that Xn is a novel small-molecule activator of Nrf2 in neuronal cells and suggest that Xn might be a potential
candidate for the prevention of neurodegenerative disorders.
KEYWORDS: xanthohumol, oxidative stress, antioxidant, neuroprotection, Nrf2
from the inhibitory partner Keap1 and facilitate Nrf2 to
translocate into the nucleus, where Nrf2 binds to the
antioxidant-responsive element (ARE) consensus sequence to
initiate the transcription of phase II genes.6 In this sense,
activation of the Nrf2−ARE pathway is a promising therapeutic
approach in neurodegenerative diseases. Thus, the past years
have witnessed expanding endeavors in identifying and
developing naturally occurring or synthetic small-molecule
activators of the Nrf2−ARE pathway as potential neuro-
protective agents.7
Hops (Humulus lupulus L.), from the dried female clusters of
the hop plant, are widely used in beers and a few types of soft
drinks, such as Julmust and Malta. In traditional Chinese
medicine, hops have been used to treat a variety of ailments for
centuries. Xanthohumol (2′,4′,4-trihydroxy-6′-methoxy-3′-pre-
nylchalcone, Xn), a structurally simple polyphenol chalcone
that occurs only in the hop plants, is the principal prenylated
flavonoid in hops (up to 1% based on dry weight) and is
present in micromolar concentrations in beers.8,9 The presence
of a high concentration of Xn in beers might be linked to the
epidemiological observation of the beneficial effect of regular
beer drinking.10 Xn has attracted considerable interest because
of its multiple pharmacological functions,8,11 including
antioxidation,12 cadiovascular protection,13 anticancer and
INTRODUCTION
■
In aerobes, a small part of oxygen is physiologically metabolized
to reactive oxygen species (ROS), which generally serve as
messenger molecules to regulate diverse signaling pathways
involved in cell differentiation, proliferation, and death.1,2
Oxidative stress, arising from uncontrolled production of ROS
beyond the neutralizing capacity of the cellular antioxidant
defense system, causes deleterious effects and oxidative damage
to various cellular components, such as lipids, proteins, and
nucleic acids.1 As neuronal cells are particularly vulnerable to
oxidative stress and have limited replenishment during the
entire lifespan, increasing evidence has supported oxidative
stress as one of the pathogenic causes in the neuropathology of
adult neurodegenerative disorders such as Alzheimer’s disease
and Parkinson’s disease.1,3 Supplementation of antioxidants or
stimulation of the cellular endogenous antioxidant defense
system could efficiently block or retard the process of such
diseases. The latter approach is preferred as such action is
sustained and is regulated at the transcriptional level. A well-
deciphered and universal pathway to induce cellular intrinsic
antioxidant defense involves the transcription factor NF-E2-
related factor 2 (Nrf2)-mediated upregulation of a set of
cytoprotective genes (“phase II” genes).4 Under basal
conditions, the cytosolic regulatory protein Kelch-like ECH-
associated protein 1 (Keap1) binds tightly to Nrf2, retaining it
in the cytoplasm and directing it to proteasome degradation.5
The association of Keap1 with Nrf2 critically relies on the
cysteine residue(s) in the protein Keap1. Stimulants that could
modify such cysteine residue(s) cause the dissociation of Nrf2
Received: October 20, 2014
Revised: January 7, 2015
Accepted: January 14, 2015
© XXXX American Chemical Society
A
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cancer chemoprevention,14−18 antivirus,19 antiobesity,20,21 and
anti-inflammation.15,22 Nevertheless, the investigation of this
pleiotropic molecule on the neuronal system has only been
limitedly cultivated,23 and the underlying mechanism of the
action has not been well elucidated.
were mixed and incubated at room temperature. After 30 min, the
absorbance at 734 nm was determined. The absorbance of the free
radical solution without Xn was divided by that with 40 μM Xn to give
the percentage of the remaining free radicals.
Assay of Thiobarbituric Acid Reactive Substances (TBARSs). The
erythrocyte ghost, prepared following the published procedures,27 was
diluted to a final protein concentration of 0.96 mg/mL and incubated
at 37 °C in 0.1 M PBS (pH 7.4). The lipid peroxidation was initiated
by the addition of AAPH in the presence or absence of Xn (40 μM).
The reaction mixture was gently shaken at 37 °C for 30 min. The
reaction mixture was taken out, and a TCA−TBA−HCl stock solution
(15% (w/v) trichloroacetic acid, 0.375% (w/v) TBA, 0.25 M HCl)
was added, together with 0.02% (w/v) BHT. This amount of BHT
completely prevents the formation of any nonspecific TBARS from the
decomposition of AAPH during the subsequent boiling. The resulting
mixture was heated in a boiling water bath for 15 min. After cooling,
the precipitate was removed by centrifugation. TBARS in the
supernatant was quantified at 532 nm using an extinction coefficient
of 1.56 × 105 M−1 cm−1.27
Cell Cultures. The rat adrenal pheochromocytoma cell line PC12
was obtained from the Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences. The cells were cultured in
DMEM medium supplemented with 10% FBS, 2 mM glutamine, and
100 units/mL penicillin/streptomycin and maintained in a humidified
atmosphere with 5% CO2 at 37 °C. The generation of Nrf2-
knockdown PC12 cells is described in the following section.
Cytotoxicity Assays. MTT Assay. To investigate the cytotoxicity
of Xn on PC12 cells, the cells were seeded at a density of 1 × 104
cells/well in 96-well plates for 24 h followed by incubation with Xn or
other agents for 24 h at 37 °C in a final volume of 100 μL. For the
H2O2 or 6-OHDA injury model, PC12 cells (1 × 104 cells/well) were
plated in a 96-well plate and allowed to adhere for 24 h. After the cells
were further treated with the indicated concentrations of Xn or DMSO
(control) for 24 h, the medium was removed and replaced with fresh
medium containing 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24
h. At the end of the treatments, 10 μL of MTT (5 mg/mL) was added
to each well followed by incubation for an additional 4 h at 37 °C. An
extraction buffer (100 μL, 10% SDS, 5% isobutanol, 0.1% HCl) was
added, and the cells were incubated overnight at 37 °C. The
absorbance was measured at 570 nm using a microplate reader
(Thermo Scientific Multiskan GO, Finland).
Lactate Dehydrogenase (LDH) Release Assay. PC12 cells were
seeded in 12-well plates at a density of 2 × 105 cells/well. On the
following day, the cells were exposed to various concentrations of Xn
for 24 h, followed by addition of 600 μM H2O2 for an additional 12 h
or 200 μM 6-OHDA for an additional 24 h. The leakage of LDH from
cultured cells was quantified by measuring LDH activity in the culture
medium. Briefly, 50 μL of the culture medium was mixed with 110 μL
of 100 mM Tris−HCl buffer (pH 7.4), 20 μL of 2 mM NADH, and 20
μL of 20 mM pyruvate, and the change of absorbance at 340 nm due
to the consumption of NADH was monitored every 20 s for 5 min.
The content of LDH release was calculated by normalizing the LDH
activity of samples to that measured in culture medium from the
control cells.
Hoechst 33342 Staining. PC12 cells were plated in 12-well plates
at a density of 2 × 105 cells/well. On the following day, the cells were
incubated with the indicated concentrations of Xn for 24 h. After
replacement with the fresh medium containing 600 μM H2O2 or 200
μM 6-OHDA, the cells were allowed to continue growing for 12 h
(H2O2) or for 24 h (6-OHDA). After the treatments, Hoechst 33342
was subsequently added to a final concentration of 5 μg/mL to stain
the nuclei. The cells were visualized and photographed with a Leica
inverted fluorescent microscope.
As our continued efforts in discovering and developing novel
redox-active small molecules as potential therapeutic
agents,24−30 we synthesized Xn and investigated its cytopro-
tection and the underlying mechanism in a neuronlike rat
pheochromocytoma cell line, PC12. Xn was prepared from the
readily available starting materials in seven steps with a total
yield of 5.0%. As a polyphenolic compound, Xn moderately
scavenges free radicals in vitro. Moreover, Xn efficiently
activates the Nrf2−ARE signaling pathway at submicromolar
concentrations in PC12 cells, leading to upregulation of a panel
of phase II enzyme expression and remarkable protection of the
cells from hydrogen peroxide (H2O2)- or 6-hydroxydopamine
(6-OHDA)-induced neurotoxicity. We further clarified that the
potential neuroprotection of Xn is dependent on both the α,β-
unsaturated ketone moiety within Xn and the activation of
transcription factor Nrf2. Our results demonstrate, for the first
time, that Xn is a small-molecule activator of the Nrf2−ARE
pathway in neuronal cells and suggest that Xn might be a
potential candidate for the prevention of neurodegeneration.
The structural determinant of such cytoprotection sheds light
on designing and developing novel therapeutic agents.
MATERIALS AND METHODS
■
Materials. Dulbecco’s modified Eagle’s medium (DMEM), N-
acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), G418 disul-
fate salt, dimethyl sulfoxide (DMSO), yeast glutathione reductase
(GR), Hoechst 33342, and 2′,7′-dichlorfluorescein diacetate (DCFH-
DA) were obtained from Sigma-Aldrich (St. Louis, MO). NADPH was
obtained from Roche (Mannheim, Germany). 2,2′-Azinobis(3-ethyl-
benzthiazoline-6-sulfonic acid) (ABTS), 2,6-dichlorophenol indophe-
nol (DCPIP), 2,2′-azobis(2-methylpropionamidine) dihydrochloride
(AAPH), tert-butylhydroquinone (tBHQ), and β-NADH were
purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was
obtained from HyClone, Thermo Fisher Scientific (Waltham, MA).
The recombinant rat TrxR1, which has a specific activity of 50% of that
of the wild-type rat TrxR1 with the DTNB assay, was a gift from Prof.
Arne Holmgren at Karolinska Institute, Sweden. Escherichia coli Trx
was purchased from IMCO (Stockholm, Sweden, www.imcocorp.se).
Anti-TrxR1 antibody was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT), penicillin, and streptomycin were obtained
from Amresco (Solon, OH). Bovine serum albumin (BSA), phenyl-
methanesulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4),
and antilamin and antiactin antibodies were obtained from Beyotime
(Nantong, China). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was
obtained from J&K Scientific (Beijing, China). 6-Hydroxydopamine
hydrobromide (6-OHDA), anti-TrxR1 antibody, anti-Trx1 antibody,
and anti-Nrf2 antibody were from Santa Cruz Biotechnology. Anti-
HO-1 antibody and anti-NQO1 antibody were obtained from Sangon
Biotech (Shanghai, China). The shRNA plasmids targeting coding
regions of the rat Nrf2 gene (shNrf2) and the control nontargeting
shRNA (shNT) were purchased from GenePharma Co., Ltd.
(Shanghai, China). GeneTran III transfection reagent was obtained
from Biomiga (San Diego, CA). All other reagents were of analytical
grade.
Measurement of Caspase-3 Activity. PC12 cells were seeded in
60 mm dishes at a density of 2 × 106 cells/well for 24 h and then
treated with different concentrations of Xn or DMSO for another 24 h.
After replacement with the fresh medium containing 600 μM H2O2 or
200 μM 6-OHDA for 24 h, the cells were harvested, washed with PBS,
and lysed with RIPA buffer (50 mM Tris−HC,l pH 7.5, 2 mM EDTA,
0.5% deoxycholate, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM
In Vitro Antioxidant Activity of Xn. ABTS Free-Radical-
Scavenging Activity. The ABTS•+ scavenging capacity was
determined according to the published method.27 The ABTS•+ radical
cation solution, which was prepared from oxidation of ABTS (7 mM)
by potassium persulfate (2.5 mM) for 12−16 h in the dark at room
temperature, was diluted to an absorbance of 0.700 0.020 at 734 nm.
The ABTS•+ solution (2 mL) and the antioxidant solution (100 μL)
B
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Na3VO4, and 1 mM PMSF) for 30 min on ice. The protein
concentration was quantified using the Bradford procedure. A cell
extract containing 15 μg of total proteins was incubated with the assay
mixture (50 mM Hepes, 2 mM EDTA, 5% glycerol, 10 mM DTT,
0.1% CHAPS, 0.2 mM Ac-DEVD-pNA, pH 7.5) for 4 h at 37 °C in a
final volume of 100 μL. The absorbance at 405 nm was measured
using a microplate reader. The caspase-3 activity was expressed as the
percentage of the control cells that grow with DMSO treatment.
Assessment of the Intracellular ROS. PC12 cells (2 × 105 cells/
well) were seeded into 12-well plates and allowed to adhere for 24 h.
Then the cells were treated with Xn or DMSO for another 24 h,
followed by replacement with the fresh medium containing 600 μM
H2O2 for 3 h or 200 μM 6-OHDA for 6 h. After removal of the culture
medium, the ROS indicator DCFH-DA (10 μM) in fresh FBS-free
medium was added to each well, and incubation was continued for 30
min at 37 °C in the dark. The cells were visualized and photographed
under a fluorescent microscope. Intracellular ROS were quantified in
PC12 cells using the green fluorescent probe DCFH-DA. Cells were
seeded at 1 × 104 cells/well in black 96-well plates for 1 d. The cells
were exposed to 600 μM H2O2 for 3 h or 200 μM 6-OHDA for 6 h
after a 24 h pretreatment with or without Xn. After removal of the
medium and staining of the cells with DCFH-DA (10 μM) for 30 min,
the cells were washed with PBS and the fluorescence intensity at 525
nm excited at 488 nm in each well was read by an Infinite M200
(Tecan). The ROS levels are expressed as the percentage of treated
cells compared to the control.
Real-Time Reverse Transcription PCR (RT-PCR). PC12 cells
were seeded in 60 mm dishes at a density of 2 × 106 cells/well for 24
h, followed by treatment with 0.5 μM Xn for 0, 6, and 12 h. As a
positive control, the cells were treated with tBHQ (20 μM) for 6 h.
For the injury models, the cells were further exposed to 600 μM H2O2
for 6 h or 200 μM 6-OHDA for 12 h after the cells were pretreated
with Xn for 6 h. Total RNA was isolated from cells using an RNAiso
Plus (TaKaRa, Dalian, China) according to the manufacture’s protocol
and quantified through 260 nm/280 nm wavelength measurement.
Reverse transcription was performed using a Primescript RT reagent
kit according to the manufacture’s protocol (TaKaRa). RT-PCR was
performed on an Agilent Technologies Stratagene Mx3005P RT-PCR
system using SYBR Premix Ex Taq II (Tli RNaseH Plus). PCR
primers specific to each gene were as follows: glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), 5′-cagtgccagcctcgtctcat-3′ (for-
ward, F) and 5′-aggggccatccacagtcttc-3′ (reverse, R); heme oxygenase
1 (HO-1), 5′-gccctggaagaggagatagag-3′ (F) and 5′-tagtgctgtgtggct-
ggtgt-3′ (R); NAD(P)H:quinone oxidoreductase 1 (NQO1), 5′-
tcaccactctactttgctccaa-3′ (F) and 5′-ttttctgctcctcttgaacctc-3′ (R);
thioredoxin 1 (Trx1), 5′-ccttctttcattccctctgtgac-3′ (F) and 5′-cccaa-
ccttttgaccctttttat-3′ (R); thioredoxin reductase 1 (TrxR1), 5′-actgc-
tcaatccacaaacagc-3′ (F) and 5′-ccacggtctctaagccaatagt-3′ (R); catalytic
subunit of glutamate−cysteine ligase (GCLC), 5′-caaggacaagaacac-
accatct-3′ (F) and 5′-cagcactcaaagccataacaat-3′ (R); modifier subunit
of glutamate−cysteine ligase (GCLM), 5′-ggcacaggtaaaacccaatagt-3′
(F) and 5′-ttcaatgtcagggatgctttct-3′ (R). The GAPDH was used as an
internal control. Target gene expression was measured and was
normalized to the GAPDH expression level.
experiments, and the activity is expressed as a percentage of the
control.
Measurement of NQO1 Activity. The NQO1 activity was
determined spectrophotometrically by monitoring the reduction of the
electron acceptor 2,6-DCPIP at 600 nm.27 In brief, PC12 cells were
seeded in 60 mm dishes at a density of 2 × 106 cells/well for 24 h and
treated with different concentrations of Xn or DMSO for another 24 h.
The cells were harvested and lysed in RIPA buffer. The total protein
concentration was quantified using the Bradford procedure. The
enzymatic reaction was initiated by the addition of cell lysate (5 μg of
total protein) to the reaction mixture (2 mM Tris−HCl, pH 7.4, 200
μM NADH, and 40 μM DCPIP), and the decrease in absorbance at
600 nm was measured every 15 s for 2 min at room temperature in the
presence or absence of 20 μM dicoumarol, a known NQO1 inhibitor.
The dicoumarol-inhibitable part of DCPIP’s reduction was used to
calculate the NQO1 activity. The same amounts of DMSO were added
to the control experiments, and the activity is expressed as a
percentage of the control.
Determination of Trx and TrxR Activity. After PC12 cells were
treated with different concentrations of Xn for 24 h, the cells were
harvested and washed twice with PBS. Total cellular proteins were
extracted by RIPA buffer for 30 min on ice. The total protein content
was quantified using the Bradford procedure. Trx and TrxR activity in
cell lysates was measured by the end point insulin reduction assay.32
Briefly, the cell extract containing 20 μg of total proteins was incubated
in a final reaction volume of 50 μL containing 100 mM Tris−HCl (pH
7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 15 μM E.
coli Trx (for assaying the activity of TrxR) or 250 nM recombinant rat
TrxR1 (for assaying the activity of Trx) for 30 min at 37 °C. The
reaction was terminated by adding 200 μL of 1 mM DTNB in 6 M
guanidine hydrochloride, pH 8.0. A blank sample, containing
everything except TrxR or Trx, was treated in the same manner.
The absorbance at 412 nm was measured, and the blank value was
subtracted from the corresponding absorbance value of the sample.
The same amounts of DMSO were added to the control experiments,
and the activity is expressed as a percentage of the control.
Western Blot Analysis. PC12 cells (4 × 106 cells/well) seeded in
100 mm dishes for 1 d and subsequently treated with Xn (0.5 μM) for
0, 2, 4, 6, 8 h were harvested, and different cell extracts were prepared
according to the published procedure.27 For the whole cell protein
extraction, PC12 cells were washed with ice-cold PBS three times and
then with RIPA buffer for 30 min on ice. Cell lysates were centrifuged
at 12000g for 20 min at 4 °C, and the supernatant was collected for
analysis or stored at −80 °C.
For the cytosolic and nuclear extracts, the cells were rinsed with ice-
cold PBS, and the pellet was dissolved in 100 μL of buffer A (10 mM
Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 μM
DTT, and 1 μM protease inhibitor cocktail freshly added to buffer A).
After 15 min of incubation, 10 μL of Nonidet-P40 (10%) was added,
and the lysate was vortexed for 15 s. Thereafter, the lysate was
repeatedly centrifuged for 10 min (1000g, 4 °C) to separate the nuclei
from the cytosolic fraction. The supernatant (cytosolic fraction) was
stored on ice. The pellet was resuspended in 100 μL of buffer B (20
mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 μM
DTT, and 1 μM protease inhibitor cocktail freshly added to buffer B)
and incubated on ice for 15 min with vortexing for 10−15 s every 2
min. After a final centrifugation step for 10 min (20000g, 4 °C), the
supernatant (nuclear extract) and the cytosolic extract were used for
analysis or stored at −80 °C. For Western blot analysis, equal amounts
of protein in each lysate sample were separated by SDS−PAGE and
electroblotted onto a PVDF membrane (Millipore, United States).
After blocking with 2.5% nonfat milk at room temperature for 2 h, the
membranes were incubated with primary antibodies at 4 °C overnight.
The membranes were washed with TBST three times and then
incubated with horseradish peroxidase-conjugated secondary antibod-
ies at room temperature for 1 h. The signal was detected using an
enhanced chemiluminescence (ECL) kit from GE Healthcare. The
band intensity was quantified by Image Pro Plus 6.0
Measurement of Total Glutathione. Quantification of the total
glutathione was based on the enzymatic recycling method.31 PC12
cells were seeded in 60 mm dishes at a density of 2 × 106 cells/well for
24 h and treated with different concentrations of Xn or vehicle for 24
h. For each sample, cells were collected and resuspended using ice-cold
extraction buffer containing 0.1% Triton X-100 and 0.6% sulfosalicyclic
acid in 0.1 M potassium phosphate buffer with 5 mM EDTA disodium
salt, pH 7.5 (KPE buffer) . The cell suspension was sonicated in ice
every 30 s for 2−3 min. After the cell suspension was centrifuged at
3000g for 4 min at 4 °C, the supernatant was immediately transferred
to a new Eppendorf tube. A 120 μL solution containing 0.33 mg/mL
DTNB and 1.66 units/mL glutathione reductase was added to each
sample (20 μL). β-NADPH (60 μL, 0.66 mg/mL) was then added
quickly, and the absorbance at 412 nm was measured every 15 s for 2
min. The same amounts of DMSO were added to the control
Generation of Nrf2-Knockdown PC12 Cells Using Short
Hairpin RNAs. The short hairpin RNAs specifically targeting the rat
C
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Scheme 1. Synthesis of Xn and TH-Xn
Nrf2 gene (shNrf2) and with a scrambled sequence (shNT) as the
control were used for Nrf2-knockdown experiments.27 The exponen-
tially growing PC12 cells were transfected with different shRNAs using
GeneTran III transfection reagent according to the manufacture’s
instructions. After 48 h of transfection, the cells were maintained in
DMEM, 10% FBS, 2 mM glutamine, and 100 units/mL penicillin/
streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and
selected by supplementation with 0.5 mg/mL G418. Knockdown of
the Nrf2 expression in the cells was analyzed by Western blotting.
Statistics. Data are presented as the mean
SE. Statistical
differences between two groups were assessed by Student’s t test.
Comparisons among multiple groups were performed using one-way
analysis of variance (ANOVA), followed by a post hoc Scheffe test. P <
0.05 was used as the criterion for statistical significance.
RESULTS AND DISCUSSION
■
Figure 1. Scavenging of free radicals by Xn in vitro. (A) Neutralization
of ABTS free radicals by Xn. (B) Prevention of the free-radical-
mediated formation of TBARS in erythrocyte ghost by Xn. Key: **, P
< 0.01 vs the control group.
Chemical Synthesis. Xn and TH-Xn described in this
study were prepared according to the published proce-
dures.30,33 The synthetic routes are illustrated in Scheme 1
and afforded a total yield of 5.0% in seven steps. Analysis of the
chemical structure of Xn reveals it belongs to chalcones, the
structure of which could be easily constructed by the base-
catalyzed Claisen−Schmidt condensation of an aldehyde and
ketone in a polar solvent such as ethanol or methanol. The key
intermediate 4 was prepared from 3 via para-Claisen rearrange-
ment. The skeleton of Xn was afforded by reaction of 5 with p-
hydroxybenzaldehyde employing KOH as a catalyst in
anhydrous methanol. Removal of the methoxymethyl protect-
ing groups under acidic conditions furnishes the desired Xn.
TH-Xn was prepared by the Pt/C-catalyzed hydrogenation of
assay the antioxidant activity of Xn. Thermal decomposition of
AAPH in aqueous solution under aerobic conditions constantly
generates peroxyl radicals, which attack membrane unsaturated
lipids to induce lipid peroxidation. Xn remarkably protects the
erythrocyte ghost from AAPH-induced formation of TBARS
(Figure 1B), a biomarker of lipid peroxidation. Taken together,
Xn could directly scavenge free radicals in vitro.
Protecting PC12 Cells from H2O2- and 6-OHDA-
Induced Damage. As Xn was reported to have cytotox-
icity,16,18 we first determined this effect in PC12 cells. Xn shows
little cytotoxicity toward PC12 cells at submicromolar
concentrations, and significant cytotoxicity was observed only
at concentrations greater than 2 μM (Figure 2A). Thus, the
concentrations of Xn used in the following protection
experiments are no more than 1 μM. Next, we asked if Xn
has cytoprotection at nontoxic concentrations in PC12 cells.
Hydrogen peroxide, an endogenous cellular signaling molecule,
has been extensively used as an inducer of oxidative stress in
many cellular models. As shown in Figure 2B, PC12 cells
treated with H2O2 only showed about 50% cell death compared
with the control group. However, if the cells were pretreated
with Xn (0.5 μM) for 24 h followed by H2O2 insult, the
population of viable cells increased to about 75%. This
protection was significant even at 0.1 μM Xn. 6-OHDA is a
1
Xn. Xn and TH-Xn were fully characterized by H and 13C
NMR and MS (Figures S2−S6 in the Supporting Information).
The detailed chemical syntheses are described in the
Supporting Information. The stability of Xn was determined
in DMEM cell culture medium by scanning its absorbance
spectra, and >90% of the compound remained after 6 h (Figure
S1, Supporting Information).
Scavenging Free Radicals in Vitro. Xn is a polyphenol,
and thus, we first investigated its antioxidant activity in vitro.
ABTS radical cation is a stable radical with a characteristic
absorbance at 734 nm. Neutralization of ABTS free radicals
could be easily determined by following the decrease of optical
density at 734 nm. As shown in Figure 1A, Xn displays
moderate efficiency to scavenge ABTS free radicals. We also
employed a model of free-radical-mediated lipid peroxidation to
D
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Figure 2. Protection of Xn against H2O2- or 6-OHDA-induced PC12 cell damage. (A) Cytotoxicity evaluation of Xn toward PC12 cells. The cells
were treated with the indicated concentrations of Xn for 24 h, and the cytotoxicity of Xn was determined by the MTT assay. Protection against (B)
H2O2-induced and (C) 6-OHDA-induced PC12 cell damage. PC12 cells (1 × 104 cells/well) were plated in a 96-well plate for 1 d and subsequently
treated with Xn for another 24 h. After replacement with the fresh medium containing H2O2 and 6-OHDA and after incubation was continued for
the indicated times, the cell viability was measured by MTT assay. The content of LDH in the medium after H2O2 (D) or 6-OHDA (E) treatment
was determined as described in the Materials and Methods. All data represent the means SE of three independent experiments. Key: **, P < 0.01
#
vs the vehicle group; , P < 0.05 vs the H2O2- or 6-OHDA-treated group; ##, P < 0.01 vs the H2O2- or 6-OHDA-treated group.
potent neurotoxin and is widely used to generate an
experimental model of Parkinson’s disease. 6-OHDA-mediated
neurotoxicity is engendered, at least in part, by its ability to
generate ROS.34 As shown in Figure 2C, 6-OHDA treatment
reduced cell viability to about 60% of that of the control.
Similarly, addition of Xn, at either 0.1 or 0.5 μM, also markedly
promoted cell viability to more than 75%. Lactate dehydrogen-
ase (LDH) is a soluble cytosolic enzyme that is released into
the culture medium following loss of membrane integrity
resulting from cell damage. To confirm the cytoprotection of
Xn, we further determined the content of LDH leakage after
H2O2 or 6-OHDA insult. As shown in Figure 2D,E, H2O2 and
6-OHDA cause ∼4.7-fold and ∼2.4-fold increases of LDH
release, respectively. Pretreatment of PC12 cells with Xn
significantly reduces the leakage of LDH in a dose-dependent
manner. Taken together, Xn at nontoxic concentration can
significantly protect PC12 cells from H2O2- or 6-OHDA-
induced cell damage.
Alleviating H2O2- and 6-OHDA-Induced PC12 Cell
Apoptosis. Nuclear condensation and caspase-3 activation are
hallmarks of cell apoptosis. The morphologic changes of the
nuclei were revealed by Hoechst staining. As shown in Figure
3A,B, both H2O2 and 6-OHDA elicit apoptosis in PC12 cells
evidenced by the appearance of apoptotic nuclei as highly
fluorescent, condensed bodies (indicated by arrows), while no
apparent apoptotic nuclei were observed in control cells.
Pretreatment of PC12 cells with nontoxic concentrations of Xn
remarkably lowers the population of apoptotic nuclei.
Activation of caspase-3 is a biochemical character in all
apoptotic cells. We thus further quantified the level of
caspase-3 activation. Both H2O2 and 6-OHDA activate the
cellular caspase-3, but with different potencies (Figure 3C,D).
Again, Xn significantly alleviates the extent of caspase-3
activation in a concentration-dependent manner. Collectively,
Xn alleviates H2O2- and 6-OHDA-induced apoptotic cell death
in PC12 cells.
Preventing ROS Accumulation in PC12 Cells. DCFH-
DA is a cell-permeable, nonfluorescent probe. After diffusing
into cells, DCFH-DA is deacetylated by cellular esterases to
nonfluorescent 2′,7′-dichlorodihydrofluorescin (DCFH), which
is rapidly converted to highly fluorescent 2′,7′-dichlorofluor-
escein (DCF) upon reacting with ROS. Stimulation of the cells
with H2O2 or 6-OHDA leads to intracellular burst of ROS,
while the control cells display a negligible ROS level, as
revealed by DCFH-DA staining (Figure 4A,B), and the
quantifying results are shown in Figure 4C,D. Pretreatment
of the cells with Xn remarkably reduces the ROS accumulation
induced by either H2O2 or 6-OHDA. As oxidative stress, arising
from the overproduction of ROS beyond the cellular capacity
to remove them, has been implicated in a common pathway of
neurotoxicity in a wide variety of acute and chronic neurologic
disorders, the inhibition of ROS accumulation in neuronal cells
might be the molecular basis of the neuroprotective action of
Xn.
Inducing Phase II Gene Expression. Xn belongs to the
chalcone family, which bears an α,β-unsaturated ketone
structure. Due to the electron-deficient character of the double
bond, this structure is renowned as a Michael acceptor moiety,
which is a core structure of many reported small-molecule ARE
inducers.7 In analogy to known ARE activators, we thus
hypothesized that Xn might activate the cellular ARE response.
We measured the expression of Nrf2-driven antioxidant/
detoxifying genes (HO-1, NQO1, Trx1, TrxR1, GCLC, and
GCLM) at 0, 6, and 12 h after treatment with Xn (0.5 μM) in
PC12 cells (Figure 5). Although the positive control tBHQ
displays more potency, the induction of these genes by Xn is
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Figure 4. Prevention of ROS accumulation in PC12 cells by Xn.
Pretreatment of the cells with Xn significantly alleviates ROS
accumulation induced by H2O2 (A, C) or 6-OHDA (B, D) determined
by DCFH-DA staining as described in the Materials and Methods. The
fluorescence images (A, B) were acquired by inverted fluorescence
microscopy. The ROS level was quantified in (C) and (D) by a
fluorescence microplate reader, Infinite M200 (Tecan). Key: **, P <
0.01 vs the vehicle group; #, P < 0.05 vs the H2O2- or 6-OHDA-treated
group; ##, P < 0.01 vs the H2O2- or 6-OHDA-treated group.
Figure 3. Inhibition of H2O2- or 6-OHDA-induced apoptosis in PC12
cells. PC12 cells were treated as described in the Materials and
Methods, and the morphological changes of the nuclei were
determined by Hoechst 33342 staining (A, B). Activation of the
cellular caspase-3 (C, D) was determined by a colorimetric assay as
described in the Materials and Methods. The data represent the means
SE of three independent experiments. Key: **, P < 0.01 vs the
vehicle group; #, P < 0.05 vs the H2O2- or 6-OHDA-treated group; ##
P < 0.01 vs the H2O2- or 6-OHDA-treated group.
,
significant. The time-course studies showed the highest
induction of the genes at 6 h after stimulation with Xn. The
HO-1 and TrxR1 mRNAs were highly transcribed by ∼2.8- and
2.3-fold increases, respectively, after treatment with Xn for 6 h.
The induction of other genes is moderate but sustainable, and
this induction could last for 12 h. The phase II genes were also
induced if the cells were pretreated with Xn followed by the
oxidant insults. Our data suggest that Xn treatment can
effectively promote the transcription of Nrf2-driven antiox-
idant/detoxifying genes.
Upregulating the Antioxidant Defense System in
PC12 Cells. The induction of phase II enzymes is an important
defense mechanism against exogenous stress. As a series of
phase II genes have been upregulated at different levels, we
then examined the corresponding gene products. Xn dose-
dependently elevated HO-1, NQO1, TrxR1, and Trx1 protein
expression (Figure 6). The glutamate cysteine ligase, composed
of a GCLC and a GCLM subunit, is a rate-limiting enzyme for
cellular GSH synthesis. As a consequence of the elevation of
GCLC gene expression, the total GSH level in PC12 cells was
increased by ∼1.6-fold (Figure 6C). By adopting the
Figure 5. Induction of cytoprotective genes in PC12 cells by Xn. The
cells were treated, and the mRNA levels of different genes were
analyzed and normalized using GAPDH as an internal standard by
qRT-PCR as described in the Materials and Methods. Pretreatment of
the cells with Xn significantly elevates phase II gene expression under
H2O2 or 6-OHDA conditions determined by qRT-PCR. PC12 cells
were pretreated with Xn for 6 h or cotreated with H2O2 or 6-OHDA
for another 6 or 12 h. tBHQ was used as a positive control. The data
represent the means SE of three independent experiments. Key: *, P
< 0.05 vs the control group (0 h); **, P < 0.01 vs the control group (0
h); #, P < 0.05 vs the H2O2- or 6-OHDA-treated group; ##, P < 0.01 vs
the H2O2- or 6-OHDA-treated group.
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Figure 6. Upregulation of antioxidant enzymes by Xn in PC12 cells. (A) Induction of HO-1 protein expression by Xn. (B) Quantification of the
band intensity in (A). (C) Xn induces upregulation of GSH in PC12 cells. The total intracellular GSH was measured as described in the Materials
and Methods. Induction of NQO1 expression (D) and enzyme activity (F) by Xn. (E) Quantification of the band intensity in (D). Induction of
TrxR1 expression (G) and total TrxR1 enzyme activity (I) by Xn. (H) Quantification of the band intensity in (G). Induction of Trx1 protein
expression (J) and total Trx enzyme activity (L) by Xn. (K) Quantification of the band intensity in (J). Data are expressed as the means SE of
three experiments. Key: *, P < 0.05 vs the vehicle group; **, P < 0.01 vs the vehicle group.
convenient assays, we further determined the cellular activity of
NQO1, TrxR1, and Trx1. In line with the upregulation of
protein expression, all the enzymes’ activities were significantly
increased (Figure 6F,I,L). After Xn (0.5 μM) treatment, the
NQO1 activity (Figure 6F), TrxR1 activity (Figure 6I), and
Trx1 activity (Figure 6L) were increased by ∼1.7-, ∼1.2-, and
∼1.5-fold, respectively. Our data demonstrate that Xn at
submicromolar concentration could efficiently boost various
endogenous cytoprotective molecules.
Promoting Nrf2 Nuclear Localization. Induction of
antioxidant gene expression via the Nrf2-dependent cytopro-
tective pathway requires translocation of Nrf2 from the cytosol
to the nucleus. We therefore examined whether Xn could
promote Nrf2 to accumulate in the nuclei. After isolation of the
nuclei from the cells, the total Nrf2, the cytosolic Nrf2, and the
nuclear Nrf2 were determined by immunoblots. Upon Xn
treatment, immunoblotting results revealed that the total Nrf2
expression increased, peaking at 4 h, and decreased to the basal
level at 8 h (Figure 7A). Notably, the cytosolic Nrf2 decreased
gradually, coincident with its continuous accumulation in the
nuclei (Figure 7B,C), indicating the translocation of Nrf2 from
the cytosol to the nuclei. We also analyzed the change of
nuclear Nrf2 under H2O2 or 6-OHDA treatment conditions. As
shown in Figure 7D, H2O2 or 6-OHDA alone has a marginal
effect on the Nrf2 nuclear accumulation. However, after
pretreatment with Xn followed by the oxidant insult, the
amount of Nrf2 in the nucleus remarkably increased. The
presence of Nrf2 in the nuclei would facilitate its binding to
ARE and thus subsequently initiates the transcription process
to induce the phase II gene expression.
Requiring the α,β-Unsaturated Ketone Structure in Xn
and Nrf2 for Cytoprotection. As aforementioned, we
speculated that the α,β-unsaturated ketone moiety in Xn and
the transcription factor Nrf2 are key determinants for the action
of Xn in PC12 cells. We prepared TH-Xn, a molecule without
the α,β-unsaturated ketone structure, and generated Nrf2-
knockdown PC12 cells to validate the molecular mechanism
underlying the cytoprotection of Xn. Hydrogenation of Xn to
TH-Xn almost completely abolishes the protective effect
(Figure 8A), indicating the importance of the α,β-unsaturated
ketone structure in Xn. Next, we compared the protection of
Xn in PC12 cells with different Nrf2 expressions. After
confirmation of the knockdown efficiency of Nrf2 (Figure
8B), we evaluated the effect of Xn against oxidative insult
toward different cells. As shown in Figure 8C,D, Xn displays a
protective pattern in PC12 cells transfected with shNT similar
to that observed in wild-type PC12 cells. However, this
protection is drastically lost in PC12 cells transfected with
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Figure 8. Involvement of Nrf2 in the cytoprotection of Xn in PC12
cells. (A) Protection of TH-Xn against H2O2-induced PC12 cell
damage. (B) Evaluation of Nrf2-knockdown efficiency by different
shRNAs. PC12 cells were transfected with shRNAs specifically
targeting the rat Nrf2 gene (shNrf2s) or nontargeting shRNA
(shNT), and the efficiency of silencing Nrf2 was evaluated by Western
blots. (C, D) Impairment of cytoprotection of Xn in Nrf2-knockdown
cells. Different PC12 cells were treated with Xn for 24 h followed by
treatment with 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24 h.
The cell viability was determined by MTT assay. The results are
expressed as the means SE. Key: **, P < 0.01; *, P < 0.05.
Figure 7. Promotion of Nrf2 nuclear accumulation by Xn. After the
cells were treated with Xn (0.5 μM) for 0, 2, 4, 6, and 8 h, they were
harvested, and different cell extracts were prepared as described in the
Materials and Methods. The total Nrf2 (A), cytosolic Nrf2 (B), and
nuclear Nrf2 (C) were analyzed by Western blotting. (D) Pretreat-
ment of the cells with Xn promotes nuclear Nrf2 accumulation under
H2O2 or 6-OHDA treatment conditions. PC12 cells were pretreated
with Xn for 6 h and cotreated with H2O2 or 6-OHDA for another 6 or
12 h. Actin or lamin B was used as a loading control. Quantification of
the blots is presented in the right panel. The data are expressed as the
means SE of three experiments. Key: *, P < 0.05 vs the control
group (0 h); **, P < 0.01 vs the control group (0 h). #, P < 0.05 vs the
H2O2- or 6-OHDA-treated group; ##, P < 0.01 vs the H2O2- or 6-
OHDA-treated group.
Preceding studies have suggested that Xn could promote
neuronal differentiation as well as neurite outgrowth23 and
prevent middle cerebral artery occlusion-induced cerebral
ischemia in rats38 to confer neuroprotective function. We
revealed in this work for the first time that Xn displayed potent
cytoprotection against H2O2- or 6-OHDA-mediated cell
damage in PC12 cells via the induction of phase II
antioxidant/detoxifying enzymes. Treatment of PC12 cells
with Xn significantly upregulates a set of Nrf2-driven phase II
genes as well as the corresponding gene products, including the
antioxidant enzymes (HO-1, NQO1, Trx1, and TrxR1) and
small peptide antioxidant GSH. Activation of Nrf2 by Xn in
different types of cells, including microglial BV2 cells,22 human
hepatocytes,39 and Hepa 1c1c7 cells,40 and even in vivo,41 has
been reported. Besides, Xn has been demonstrated to possess
diverse functions in a variety of cellular assays, such as
anticancer14−18 and anti-inflammation.15,22 However, there has
been no study on induction of Nrf2 in neuronal cells prior to
this work, and most of the activity in these cellular assays
requires higher concentrations of Xn (>1 μM). We
demonstrated that Xn, as low as 0.1 μM, could significantly
protect neuronal cells from oxidative insult. This concentration
could be easily reached in vivo by daily intake of Xn-containing
products, such as beers. The pharmacokinetics and metabolites
of xanthohumol in rats have been evaluated by giving the rats
intravenous injection or oral administration.20,42 The highest
plasma concentration of 2.97 mg/L (∼8 μM) could be reached
after intravenous injection at a dose of 1.86 mg/kg. Oral
administration of Xn also leads to a significant amount of
plasma circulation. One major challenge for most neuro-
protective agents is their ability to pass through the blood−
brain barrier (BBB). There are no available BBB penetration
data for the flavonoid Xn. However, there is strong evidence
showing that structurally different flavonoids are able to
traverse the BBB.43 The prenyl group further increases the
shNrf2 (Figure 8C,D), demonstrating the definite involvement
of Nrf2 in cytoprotection of Xn in PC12 cells.
Common targets and mechanisms of neurodegenerative
diseases have been elegantly summarized in a recent paper.35
Among various pathogenic hypotheses, increasing evidence has
supported that oxidative stress is a causal, or at least an
ancillary, factor in the progressive degeneration of a subset of
neurons, which is the pathologic hallmark of adult-onset
neurodegenerative diseases.1,36 Surgical operation to replace the
dying or dead neurons in these diseases has been demonstrated
only with limited success.37 Therefore, pharmacological
management intended to assuage the oxidative stress is one
promising strategy to protect loss of neurons, which is expected
to eventually lead to retardation or blockage of the neuro-
degenerative progression. Tactics against oxidative stress
include direct antioxidative action by supplying exogenous
antioxidants and indirect defense by activating the pathway(s)
involved in expression of endogenous cytoprotective molecules.
In this regard, accumulating enthusiasm has been devoted to
discovering and developing novel antioxidants or molecules
that may prompt endogenous antioxidant defense as potential
therapeutic agents for neurodegenerative disorders.7
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lipophilicity of Xn, which favors BBB penetration. It is much
like Xn having the ability to pass through the BBB in vivo.
Thus, our finding, i.e., the induction of endogenous
cytoprotective enzymes by Xn to confer potential neuro-
protection, is of physiological significance.
Subsequently, Nrf2 is translocated into the nucleus, where it
binds to ARE and initiates the transcription of a variety of
cytoprotective genes to confer protection against oxidative
stress.
In summary, we conclude that Xn is effective in preventing
oxidative-stress-induced neuronal cell damage. Xn displays a
moderate capacity to directly neutralize ROS in vitro. More
importantly, this cytoprotection is mediated by the induction of
endogenous antioxidant defense molecules, which relies on the
α,β-unsaturated ketone structure in Xn and the transcription
factor Nrf2 in PC12 cells as removal of such a chemical
structure or knockdown of Nrf2 abolishes the protection. Our
discovery discloses that Xn is a novel small-molecule activator
of the Keap1−Nrf2 pathway and provides deep insights into
understanding the molecular mechanism underlying the
cytoprotection of Xn. This mechanism of action could lead to
the design and development of novel small molecules as
potential neuroprotective agents.
The Keap1−Nrf2−ARE regulatory pathway plays a central
role in protecting cells against oxidative or electrophilic insult.
Under unstressed conditions, Nrf2 is bound to the repressor
protein Keap1, which anchors Nrf2 in the cytoplasm and
targets it for ubiquitination and proteasome degradation. Upon
exposure to stressors, the Nrf2−Keap1 complex dissociates and
Nrf2 translocates to the nucleus. The mechanisms underlying
the activation of the Keap1−Nrf2−ARE pathway are not well
understood but may involve oxidation/alkylation of key thiol(s)
in Keap1 and/or phosphorylation of Nrf2. Upon exposure to
electrophiles or oxidants, the Cys-rich protein Keap1 is
modified by forming covalent adduct or disulfide bond(s)
within the protein, and it is this Cys-based modification of
Keap1 that allows dissociation of Nrf2 from Keap1 and
subsequent nuclear translocation of Nrf2. Analysis of the
chemical structure of Xn reveals that the molecule contains one
α,β-unsaturated ketone moiety, a thiol-reactive electrophilic
center that may covalently modify the Cys thiols. Many natural
or synthetic molecules bearing such a structure are known
effective inducers of phase II genes.6,44 Thus, alkylation of
certain Cys residue(s) might be the molecular basis for the
activation of the Keap1−Nrf2−ARE pathway by Xn, which was
evidenced by the liquid chromatography−tandem mass
spectrometry analysis40 and our observation that removal of
the electrophilic center from Xn blocks such protection (Figure
8A). Abrogating the protection of Xn by silencing Nrf2
expression in PC12 cells further supports the involvement of
Nrf2 in the cellular action of Xn. Our findings suggest a novel
neuroprotective mechanism of Xn shown schematically in
Figure 9. The lipophilic character of Xn allows it to easily
ASSOCIATED CONTENT
■
S
* Supporting Information
Stability of Xn determined by its absorbance spectrum and
synthesis and original spectra of Xn and TH-Xn. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: fangjg@lzu.edu.cn. Fax: +86 931 8915557.
Funding
Financial support from Lanzhou University (Fundamental
Research Funds for the Central Universities, Grant lzujbky-
2014-56) and Natural Science Foundation of Gansu Province
(Grant 145RJZA225) is greatly acknowledged.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express heartfelt appreciation to Prof. Arne Holmgren for
recombinant rat TrxR1 and E. coli Trx.
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