Activation of the phase II enzymes for neuroprotection by ginger active constituent 6-dehydrogingerdione in PC12 cells

Article abstract of DOI:10.1021/jf405553v

The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of many neurodegenerative diseases. Molecules with the ability to enhance the antioxidant defense thus are promising candidates for neuroprotective drugs. 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received increasing attention due to its multiple pharmacological activities. However, how this pleiotropic molecule works on the neuronal system has not been studied. This paper reports that 6-DG efficiently scavenges various free radicals in vitro and displays remarkable cytoprotection against oxidative stress-induced neuronal cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-DG significantly up-regulates a panel of phase II genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, and thioredoxin reductase. Mechanistic study indicates that activation of the Keap1-Nrf2-ARE pathway is the molecular basis for the cytoprotection of 6-DG. This is the first revelation of this novel mechanism of 6-DG as an Nrf2 activator against oxidative injury, providing the potential therapeutic use of 6-DG as neuroprotective agent.

Full text of DOI:10.1021/jf405553v

Article
pubs.acs.org/JAFC
Activation of the Phase II Enzymes for Neuroprotection by Ginger
Active Constituent 6‑Dehydrogingerdione in PC12 Cells
Juan Yao, Chunpo Ge, Dongzhu Duan, Baoxin Zhang, Xuemei Cui, Shoujiao Peng, Yaping Liu,
and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University,
Lanzhou, Gansu 730000, China
ABSTRACT: The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of
many neurodegenerative diseases. Molecules with the ability to enhance the antioxidant defense thus are promising candidates
for neuroprotective drugs. 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received
increasing attention due to its multiple pharmacological activities. However, how this pleiotropic molecule works on the neuronal
system has not been studied. This paper reports that 6-DG efficiently scavenges various free radicals in vitro and displays
remarkable cytoprotection against oxidative stress-induced neuronal cell damage in the neuron-like rat pheochromocytoma cell
line, PC12 cells. Pretreatment of PC12 cells with 6-DG significantly up-regulates a panel of phase II genes as well as the
corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, and thioredoxin
reductase. Mechanistic study indicates that activation of the Keap1-Nrf2-ARE pathway is the molecular basis for the
cytoprotection of 6-DG. This is the first revelation of this novel mechanism of 6-DG as an Nrf2 activator against oxidative injury,
providing the potential therapeutic use of 6-DG as neuroprotective agent.
KEYWORDS: 6-dehydrogingerdione, Nrf2, oxidative stress, neuroprotection, antioxidant
neuropathology of several adult neurodegenerative disorders
such as Alzheimer’s disease (AD) and Parkinson’s disease
(PD).8,9 Besides the counteraction of various ROS by
exogenous small molecule antioxidants, targeting the preven-
tion of oxidative stress could be best achieved by stimulation of
endogenous cytoprotective molecules known to serve this
purpose because their actions are more sustained and are
amplified by transcription-mediated signaling pathways. In this
sense, activation of the Nrf2-ARE pathway is a promising
therapeutic approach in neurodegenerative diseases.3,10−12
Thus, the past years have witnessed expanding endeavors in
identifying naturally occurring and synthetic neuroprotective
small molecule activators of the Nrf2-ARE pathway,13,14
including carnosic acid,15 resveratrol,16 curcumin,17 sulfor-
aphane,18 quercetin,19 epicatechin,20 plumbagin,21 luteolin,22
and tert-butylhydroquinone.18
Ginger (rhizome of the dietary plant Zingiber officinale) has
been commonly used as a popular spice or food supplement
and has been equally reputed for its medicinal properties for
centuries.23,24 6-Dehydrogingerdione (6-DG), one of the major
components of dietary ginger, has received extensive attention
due to its multiple pharmacological activities, such as inhibition
of lipid peroxidation,25 enhancement of skin cell proliferation,26
and induction of cancer cell apoptosis.27,28 However, how this
pleiotropic molecule works on the neuronal system has not
been elucidated. As part of our continuing effort in the
discovery and development of novel redox active small
INTRODUCTION
■
The cellular endogenous antioxidant system is crucial for
counteracting reactive oxygen species (ROS)-mediated oxida-
tive stress and preventing cell death. A well-elucidated universal
pathway to induce intrinsic antioxidant defense involves
transcriptional regulation through activation of the antiox-
idant-responsive element (ARE).1,2 Under this circumstance,
stimulants induce a set of genes encoding antioxidant and
detoxifying enzymes (“phase II” enzymes), including heme
oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1
(NQO1), thioredoxin reductase 1 (TrxR1), thioredoxin (Trx),
glutathione (GSH), γ-glutamylcysteine synthetase (γGCS), and
γ-glutamyl cysteine ligase (γ-GCL), which provide efficient
cytoprotection against oxidative stress and electrophilic
assault.3,4 The transcription factor NF-E2-related factor 2
(Nrf2) mediates transcription of phase II genes by binding to
the ARE within nuclear DNA and subsequently initiates
antioxidant genes transcription. Under basal conditions, the
cytosolic regulatory protein Kelch-like ECH-associated protein
1 (Keap1) binds tightly to Nrf2, retaining it in the cytoplasm.2
Molecules, such as electrophiles or oxidants, which could
modify the critical cysteine residue(s) in the regulatory protein
Keap1, would cause the release of Nrf2 and facilitate its
translocattion into the nucleus and thus activate the tran-
scription of phase II genes.4−7
In healthy cells, the level of ROS is tightly regulated by the
antioxidant defense system. However, upon environmental
stress or cellular damage, cells cannot readily detoxify the ROS
generated and may thereby suffer from oxidative stress. As
neuronal cells are particularly sensitive to oxidative stress, an
increasing amount of experimental evidence has indicated that
oxidative stress is a causal, or at least an ancillary, factor in the
Received: December 11, 2013
Revised: May 26, 2014
Accepted: May 28, 2014
Published: May 28, 2014
© 2014 American Chemical Society
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a
Scheme 1. Synthesis of 6-DG, M-6-DG, and DH-6-DG
a
The active motif of 6-DG is highlighted in circles. Reagents and conditions: (a) NaH, Et2O, 64%; (b) boron trioxide, 78%; (c) boron trioxide, 71%;
(d) 10% Pd−C, H2, 70%.
molecules,29−32 we determined herein the antioxidant activity
literature with minor modifications. Briefly, acetone reacts with ethyl
hexanoate under basic condition (Claisen condensation) and gives 2,4-
of 6-DG in vitro and employed a neuron-like rat pheochro-
nonanedione in 64% yield. The dione reacting with vanillin and
mocytoma cell line, PC12, to assess the molecular mechanisms
methylvanillin via Claisen−Schmidt condensation affords 6-DG and
responsible for the neuroprotective effect of 6-DG. 6-DG,
M-6-DG in 71 and 78% yields, respectively. Hydrogenation of 6-DG
prepared from the readily available starting materials in two
catalyzed by palladium on carbon furnished DH-6-DG in 70% yield.
steps, efficiently scavenges free radicals and prevents eryth-
All compounds were fully characterized by NMR and MS.
2,4-Nonanedione.33 To a stirred mixture of sodium hydride (1.2 g,
50 mmol) and ethyl hexanoate (7.2 g, 50 mmol) was added acetone
(1.51 g, 26 mmol) in 7 mL of dry diethyl ether over a period of 30
min. The temperature of the reaction mixture was kept at 30−40 °C
by occasional cooling with ice water. After the acetone was added, the
reaction mixture was stirred at 40−50 °C for 1 h. Diethyl ether was
continuously added to maintain a fluid reaction mixture. After
continuous stirring at room temperature overnight, the reaction was
quenched by sequential addition of 8 mL of ethanol and 80 mL of 0.75
M HCl. Extraction with diethyl ether (50 mL × 3) and removal of the
solvent under vacuum gave a pale yellow oil, which was added to a hot
solution of CuSO4·5H2O (12.4 g, 40 mmol) in 60 mL of water. After
cooling, crystals were collected and washed with 100 mL of hexane
and further dissolved in 50 mL of 10% H2SO4. The resulting solution
was extracted with diethyl ether and dried over Na2SO4. After removal
of the solvent, the crude product purified by column chromatography
on silica gel gave 2,4-nonanedione (64%). 1H NMR (400 MHz,
CDCl3), δ 5.49 (s, 1H), 2.23−2.27 (t, 2H), 2.05 (s, 3H), 1.58−1.62
(m, 2H), 1.30−1.32 (m, 4H), 0.88−0.91 (t, 3H); ESI-MS (M + 1)+,
157.2.
rocytes from free radical-induced hemolysis. Importantly, 6-DG
activates the Nrf2-ARE signaling pathway, leading to up-
regulation of phase II enzyme expression and remarkable
protection of PC12 cells from hydrogen peroxide (H2O2)- or 6-
hydroxydopamine (6-OHDA)-induced neurotoxicity. We clari-
fied that the hydroxyl group and the α,β-unsaturated ketone
moiety are determinants of the biological function of 6-DG.
Our results demonstrate that 6-DG is a novel small molecule
activator of Nrf2-ARE pathway and suggest that 6-DG might be
a potential candidate for the prevention of neurodegeneration.
Targeting the Nrf2-ARE signaling pathway by 6-DG thus
discloses a previously unrecognized mechanism underlying the
biological action of 6-DG and provides a deep insight into
understanding the neuroprotective effects of 6-DG.
MATERIALS AND METHODS
■
Chemicals and Enzymes. Dulbecco’s modified Eagle’s medium
(DMEM), N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA),
dimethyl sulfoxide (DMSO), yeast glutathione reductase (GR),
Hoechst 33342, 2′,7′-dichlorfluorescein diacetate (DCFH-DA), 2,2′-
azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-
1-picrylhydrazyl (DPPH), 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB), 2,6-dichlorophenol-indophenol (DCPIP), NADH, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), pen-
icillin, and streptomycin were obtained from Sigma-Aldrich (St. Louis,
MO, USA). NADPH was obtained from Roche (Mannheim,
Germany). Fetal bovine serum (FBS) was obtained from HyClone.
6-Hydroxydopamine (6-OHDA) and antibodies against TrxR1, Trx1,
actin, and Nrf2 were from Santa Cruz Biotechnology. Antibodies
against HO-1 and NQO1 were 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,
USA). Bovine serum albumin (BSA), phenylmethanesulfonyl fluoride
(PMSF), and sodium orthovanadate (Na3VO4) were from Beyotime
(Nantong, China). All other reagents were of analytical grade.
6-DG.34 Vanillin (6.3 g, 42 mmol), 2,4-nonanedione (19.3 g, 124
mmol), and boron trioxide (11.5 g, 165 mmol) were mixed with 50
mL of N,N-dimethylformamide (DMF) and warmed to 90 °C. A
solution of isobutylamine (40 mg, 0.4 mmol) in 40 mL of DMF was
added dropwise for 2 h. After stirring at 85−90 °C for 1 h, ∼60 mL of
the solvent was evaporated in vacuo, and 250 mL of water was added.
The resulting mixture was stirred at 60 °C for 1 h and at room
temperature for 12 h. The reaction mixture was extracted with ethyl
acetate (50 mL × 3), and the solvents were evaporated. The crude
product was purified by column chromatography on silica gel to afford
1
6-DG (71%). mp, 84 °C; H NMR (400 MHz, CDCl3), δ 7.51−7.55
(d, J = 16 Hz, 1H), 7.09−7.11 (d, J = 8 Hz, 1H), 7.03 (s, 1H), 6.92−
6.94 (d, J = 8 Hz, 1H), 6.33−6.37 (d, J = 16 Hz, 1H), 5.83 (s, 1H),
5.63 (s, 1H), 3.95 (s, 3H), 2.36−2.40 (t, 2H), 1.63−1.70 (m, 2H),
1.33−1.36 (m, 4H), 0.90−0.93 (t, 3H); ESI-MS (M + 1)+, 291.2.
M-6-DG. The synthesis of M-6-HG exactly followed the procedure
described for 6-DG except for the use of methylvanillin (6.9 g, 42
1
mmol) instead of vanillin. Yield, 78%; mp, 45 °C; H NMR (400
MHz, CDCl3), δ 7.53−7.57 (d, J = 16 Hz, 1H), 7.11−7.13 (m, 1H),
7.06 (s, 1H), 6.87−6.89 (d, J = 8 Hz, 1H), 6.35−6.39 (d, J = 16 Hz,
1H), 5.64 (s, 1H), 3.89 (s, 6H), 2.37−2.41(t, 2H), 1.26−1.34 (m, 6H),
0.9−0.93 (t, 3H); ESI-MS (M + 1)+, 305.2.
Synthesis of 6-DG, Methylated 6-DG (M-6-DG), and Hydro-
genated 6-DG (DH-6-DG). 6-DG, M-6-DG, and DH-6-DG
described in this study were prepared using a straightforward
chemistry (Scheme 1) by following the method described in the
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DH-6-DG.31 To a mixture of 10 mg of Pd/C (10%) in 5 mL of ethyl
acetate under a hydrogen atmosphere was added 10 mg of 6-DG. The
reaction was kept at room temperature for 12 h. After the reaction, the
Pd/C was removed by filtration, and the filtrate was concentrated and
purified by silica gel column chromatography to afford DH-6-DG
(70%). 1H NMR (400 MHz, CDCl3), δ 6.85−6.83 (d, J = 8 Hz, 1H),
6.70−6.68 (d, J = 8 Hz, 2H), 5.51 (s, 1H), 5.46 (s, 1H), 3.87 (s, 3H),
2.89−2.83 (m, 2H), 2.59−2.55 (t, 2H), 2.28−2.24 (t, 2H), 1.32−1.26
(m, 6H), 0.92−0.87 (m, 3H); ESI-MS (M + Na)+, 315.3.
Free Radical-Scavenging Capacities. The various concentra-
tions of antioxidants were mixed with the DPPH solution (50 μM),
and the mixture was shaken vigorously and left to stand at room
temperature for 30 min in the dark. The absorbance of the reaction
solution was measured spectrophotometrically at 517 nm. The
ABTS•+ scavenging ability was determined according to the published
method.35 The ABTS•+ 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) were mixed, and the absorbance at 734 nm was
recorded after 30 min.
followed by the 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 culture
media. Briefly, 50 μL of culture medium was mixed with 110 μL of 100
mM Tris-HCl (pH 7.4) containing 20 μL of 2 mM NADH and 20 μL
of 20 mM pyruvate, and the A340 nm change 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 and Assessment of the Intracellular
ROS.29,30 PC12 cells (2 × 105 cells/well) were seeded into 12-well
plates. After 24 h, the cells were treated with 6-DG for another 24 h
followed by replacement with fresh medium containing 600 μM H2O2
for 12 h or 200 μM 6-OHDA for 24 h. Hoechst 33342 was
subsequently added to a final concentration of 5 μg/mL to stain the
nuclei. The cells were visualized and photographed under a Leica
inverted fluorescent microscope. For assaying the ROS, the medium
was removed, and DCFH-DA in fresh FBS-free medium was added
(10 μM); incubation was continued for 30 min at 37 °C in the dark.
The cells were visualized and photographed under fluorescent
microscopy.
Measurement of Caspase-3 Activity. PC12 cells (1 × 106 cells/
well) were seeded in 60 mm dishes for 24 h and then treated with
different concentrations of 6-DG for another 24 h. After replacement
with fresh medium containing 600 μM H2O2 or 200 μM 6-OHDA for
24 h, the cells were lysed with RIPA buffer. The protein content was
quantified using the Bradford procedure. The caspase-3 activity in the
lystae was determined by following the published procedure.38 Briefly,
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 same amounts of DMSO were added to
the control experiments, and the activity was expressed as the
percentage of the control.
Measurement of Total Glutathione, NQO1 Activity, Trx
Activity, and TrxR Activity. PC12 cells (1 × 106 cells/well) were
seeded in 60 mm dishes for 24 h and treated with different
concentrations of 6-DG for another 24 h. After replacement with fresh
medium containing H2O2 (600 μM) for 12 h or 6-OHDA (200 μM)
for 24 h, cells were collected and resuspended in extraction buffer
containing 0.1% Triton X-100 and 0.6% sulfosalicyclic acid in 0.1 M
PBS with 5 mM EDTA, pH 7.5 (KPE buffer). The suspension was
sonicated on ice for 2−3 min with vortexing every 30 s. The cells were
centrifuged at 3000g for 4 min at 4 °C, and the supernatant was ready
for the total GSH assay by enzymatic detection as described in the
published protocols.39 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 of 0.66 mg/mL) was then added
quickly, and the absorbance at 412 nm was measured every 15 s for 2
min. The rate of chromophore production at 412 nm is proportional
to the concentration of glutathione within the sample. The total
glutathione content in the samples was determined by comparison
with the predetermined glutathione standard curve. For measuring
NQO1, Trx, and TrxR activity, the cells were lysed in RIPA buffer.
The total protein content was quantified using the Bradford procedure.
The NQO1 activity was determined spectrophotometrically by
monitoring the reduction of the electron acceptor, DCPIP, at 600
nm.40 The enzymatic reaction was initiated by the addition of cell
lysate (5 μg of total protein) to the reaction mixture (20 mM Tris-
HCl, pH 7.4, 200 μM NADH, and 40 μM DCPIP), and the decrease
in absorbance at 600 nm was measured every 8 s for 2 min at room
temperature in the presence or absence of 20 μM dicoumarol. 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 was expressed as the
percentage of the control. The Trx and TrxR activities in the lysate
were determined by following the published protocols.41 To determine
the activity of TrxR, the cell extract containing 20 μg of total proteins
was incubated in a final reaction volume of 50 μL containing 100 mM
Assay for Hemolysis of Red Blood Cells (RBCs) and
Thiobarbituric Acid Reactive Substances (TBARS). Human
RBCs were separated from heparinized blood that was drawn from a
healthy donor. The 5% suspension of RBC in PBS (pH 7.4) was
incubated under air at 37 °C for 5 min into which a PBS solution of
AAPH was added to initiate hemolysis. The extent of hemolysis was
determined spectrophotometrically as described previously.36 Briefly,
aliquots of the reaction mixture were taken out at appropriate time
intervals, diluted with 0.15 M NaCl, and centrifuged at 3000 rpm for 5
min to separate the RBCs. The percentage hemolysis was determined
by measuring the absorbance of the supernatant at 540 nm and
compared with that of complete hemolysis by treating the same RBC
suspension with distilled water. For measurements of TBARS, the
erythrocyte ghost was incubated at 37 °C in 0.1 M PBS (pH 7.4) and
diluted to a final protein concentration of 0.96 mg/mL. The
peroxidation was initiated by the addition of AAPH. The reaction
mixture was gently shaken at 37 °C, and aliquots of the reaction
mixture were taken out at specific intervals to which 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
solution was heated in a boiling water bath for 15 min. After cooling,
the precipitate was removed by centrifugation. TBARS in the
supernatant were quantified at 532 nm using the extinction coefficient
of 1.56 × 105 M−1 cm−1.37
Cell Cultures. The rat adrenal pheochromocytoma cell line, PC12
cells, was obtained from Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences, and was cultured in DMEM
supplemented with 10% FBS, 2 mM glutamine, and 100 units mL−1
penicillin/streptomycin and maintained in an atmosphere of 5% CO2
at 37 °C.
MTT Assay and Lactate Dehydrogenase (LDH) Release
Assay. To evaluate the cytotoxicity, PC12 cells (1 × 104 cells/well)
were seeded in 96-well plates for 1 day followed by incubation with 6-
DG or other agents for 24 h at 37 °C. 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 and then treated with the drugs
for 24 h. After replacement with fresh medium containing 600 μM
H2O2 for 12 h or 200 μM 6-OHDA for 24 h, cell viability was
determined by MTT assay. At the end of the treatment, 10 μL of
MTT (5 mg/mL) was added to each well and incubated for an
additional 4 h at 37 °C. One hundred microliters of extraction buffer
(10% SDS, 5% isobutanol, 0.1% HCl) was added, and the cells were
incubated overnight at 37 °C. Cells treated with DMSO alone were
used as controls. The absorbance was measured at 570 nm on a
Multiskan GO (Thermo Scientific). For the LDH release assay, the
cells (2 × 105 cells/well) were plated in 12-well plates. The following
day, the cells were exposed to various concentrations of 6-DG for 24 h,
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Figure 1. Antioxidant activity of 6-DG in vitro: (A) DPPH and (B) ABTS•+ scavenging abilities measured as described under Materials and
Methods; (C) inhibition of TBARS formation during the AAPH-induced peroxidation of erythrocyte ghost by 6-DG; (D) protection of 6-DG
against AAPH-induced hemolysis of RBC. Data represent the mean SD of three independent experiments.
Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA,
and 15 μM Escherichia coli Trx for 30 min at 37 °C. To determine the
activity of Trx, 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
60 nM recombinant rat TrxR1 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
Trx (for TrxR assay) or TrxR (for Trx assay), 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 was expressed as the percentage of the
control.
Real-Time Reverse Transcription-PCR (RT-PCR). PC12 cells (1
× 106 cells/well) were seeded in 60 mm dishes for 24 h and treated
with 10 μM 6-DG for 0, 6, 12, and 24 h. Total RNA was isolated from
cells using the RNAiso plus (TaKaRa, Dalian, China) according to the
manufacturer’s protocol and quantified through 260/280 absorbance.
Reverse transcription was performed using Primescript RT reagent kit
according to the manufacturer’s protocol (TaKaRa). RT-PCR was
performed on an Mx3005P RT-PCR System (Agilent Technologies)
using SYBR Green PCR Master Mix (Takara) . The GAPDH was used
as an internal control. Target gene expression was measured and was
normalized to the GAPDH expression level. PCR primers specific to
each gene are as follows: GAPDH, 5′-cagtgccagcctcgtctcat-3′ and 5′-
aggggccatccacagtcttc-3′; HO-1, 5′-gccctggaagaggagatagag-3′ and 5′-
tagtgctgtgtggctggtgt-3′; NQO1, 5′-tcaccactctactttgctccaa-3′ and 5′-
ttttctgctcctcttgaacctc-3′; Trx1, 5′-ccttctttcattccctctgtgac-3′ and 5′-
cccaaccttttgaccctttttat-3′; TrxR1, 5′-actgctcaatccacaaacagc-3′ and 5′-
ccacggtctctaagccaatagt-3′; GCLC, 5′-caaggacaagaacacaccatct-3′ and 5′-
cagcactcaaagccataacaat-3′; GCLM, 5′-ggcacaggtaaaacccaatagt-3′ and
5′-ttcaatgtcagggatgctttct-3′. GAPDH was used as an internal control.
Target gene expression was measured and was normalized to the
GAPDH expression level.
Preparation of Different Protein Extracts for Western Blot
Analysis.42 For the whole cell protein extraction, PC12 cells were
lysed with RIPA buffer. For the cytosolic and nuclear extracts, the cells
were rinsed with ice-cold PBS and resolved 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 mixture was vortexed for 15 s. Thereafter, the lysate
was repeatedly centrifuged for 10 min (1000g, 4 °C) to separate the
nuclear from the cytosolic fraction. 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 were
freshly added to buffer B) and incubated on ice for 15 min, and the
mixture was vortexed for 10−15 s every 2 min. After a final
centrifugation step for 10 min (20000g, 4 °C), the supernatant was
collected as the nuclear extract. The band intensity was analyzed and
quantified by Image pro plus (IPP) software.
Knockdown of Nrf2 Expression Using Short Hairpin RNAs.
Three shRNAs (shNrf2-842, shNrf2-184, and shNrf2-136) targeting
rat Nrf2 gene and one shRNA with scrambled sequence (shNT) as
control were used for Nrf2 knockdown experiments. Exponentially
growing cells were transfected with different shRNAs using GeneTran
III transfection reagent according to the manufacturer’s instruction.
After 48 h of transfection, the cells were maintained in DMEM, 10%
FBS, 2 mM glutamine, 100 units/mL penicillin/streptomycin at 37 °C
in a humidified atmosphere of 5% CO2 and selected by
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Figure 2. Protection of 6-DG against (A) H2O2- and (B) 6-OHDA-induced PC12 cell damage. PC12 cells (1 × 104 cells/well) were plated in a 96-
well plate for 1 day and subsequently treated with 6-DG for another 24 h. After replacement of the medium containing H2O2 or 6-OHDA and
continued incubation for indicated times, cell viability was measured by MTT assay. The content of LDH in the medium after H2O2 (C) or 6-
OHDA (D) treatment was determined as described under Materials and Methods. All data represent the mean
SD of three independent
experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group.
supplementation with 0.5 mg/mL of G418. Knockdown of the Nrf2
expression in the cells was analyzed by Western blotting.
Statistics. Data are presented as the mean standard deviation
(SD). 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.
activity in vitro, we next investigated if 6-DG has a protective
effect against oxidative stress-induced neuronal cell death. 6-DG
shows weak cytotoxicity toward PC12 cells, and significant
cytotoxicity was observed only at high concentrations (>50 μM,
data not shown). As shown in Figure 2A, compared with the
control group, PC12 cells treated with H2O2 for 12 h showed
about 60% cell death. However, pretreatment of cells with 6-
DG at nontoxic concentrations (1−10 μM) for 24 h followed
by H2O2 challenge significantly decreased the population of
dead cells to about 30%. This protection was observed even at 1
μM 6-DG. However, hydrogenation of 6-DG to DH-6-DG
almost completely blocked the protection, indicating the
importance of the α,β-unsaturated ketone structure in 6-DG.
The neurotoxin 6-OHDA has been widely used to generate an
experimental model of PD. 6-OHDA-mediated neurotoxicity is
engendered, at least in part, by generation of ROS.43 6-OHDA
treatment reduced cell viability to about 60% of control (Figure
2B). Similarly, addition of 6-DG also markedly promoted cell
viability to >80% of control. 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
neuroprotection of 6-DG, we determined the content of LDH
leakage after H2O2 or 6-OHDA insult (Figure 2C,D). Both
H2O2 and 6-OHDA cause ∼3-fold increase of LDH release.
Pretreatment of PC12 cells with 6-DG significantly reduces the
leakage of LDH. Taken together, 6-DG at nontoxic
concentration can significantly protect PC12 cells from H2O2-
and 6-OHDA-induced cell damage.
RESULTS
■
Scavenging of Free Radicals in Vitro. Previous studies
have indicated that 6-DG is an effective antioxidant.25 We
further investigated herein the potency of 6-DG in scavenging
free radicals in vitro. As shown in Figure 1A,B, 6-DG dose-
dependently neutralizes DPPH and ABTS free radicals. To
address the role of the hydroxyl group in scavenging radicals,
we prepared M-6-DG, the methylated form of 6-DG. M-6-DG
completely lost the ability to quench DPPH, whereas it retained
partial capacity to scavenge ABTS. The remaining activity
might be due to the 1,3-diketone structure in 6-DG as this
moiety could also quench radicals.25 Our results demonstrated
the critical role of the hydroxyl group in scavenging free
radicals. Thermal decomposition of AAPH in aqueous solution
under aerobic condition constantly generates peroxyl radicals,36
which attack membrane lipids to induce lipid peroxidation. We
found 6-DG efficiently protects the erythrocyte ghost from
AAPH-induced formation of TBARS, a biomarker of lipid
peroxidation (Figure 1C). In the model of AAPH-induced
hemolysis of intact human RBC, 6-DG remarkably retards the
hemolysis with a clear concentration dependence (Figure 1D).
Protection of PC12 Cells from H2O2- and 6-OHDA-
Induced Damage. As 6-DG demonstrates potent antioxidant
Alleviation of H2O2- and 6-OHDA-Induced PC12 Cell
Apoptosis by 6-DG. Apoptosis is a precisely regulated
process of cell destruction with specific defining morphological
and molecular features. Two known apoptotic signaling
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Figure 3. Inhibition of H2O2- or 6-OHDA-induced apoptosis in PC12 cells. PC12 cells were treated as described under Materials and Methods, and
the morphological changes of nuclei were determined by Hoechst 33342 staining (A, C). Activation of the cellular caspase-3 was determined by a
colorimetric assay as described under Materials and Methods. Data represent the mean SD of three independent experiments. (∗∗) P < 0.01
versus the vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group.
Figure 4. Prevention of ROS accumulation in PC12 cells by 6-DG. Pretreatment with 6-DG significantly alleviates ROS accumulation induced by
H2O2 (A) or 6-OHDA (B) determined by DCFH-DA staining as described under Materials and Methods. The phase contrast (top panel) and
fluorescence (bottom panel) images were acquired by inverted fluorescence microscopy.
pathways, the extrinsic cell surface receptor pathway44 and the
intrinsic mitochondrial pathway,45 converge on the caspase
activation.46 As shown in Figure 3A,C, 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), whereas no apoptotic nuclei were
observed in control cells. Pretreatment of PC12 cells with 6-
DG remarkably lowers the population of apoptotic nuclei.
Activation of caspase-3 is a biochemical character in all
apoptotic cells.47 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 3B,D).
Again, 6-DG significantly alleviates the extent of caspase-3
activation in a concentration-dependent manner. Collectively,
6-DG, at nontoxic concentrations, can alleviate H2O2- and 6-
OHDA-induced apoptosis in PC12 cells.
Inhibition of ROS Accumulation in PC12 Cells. DCFH-
DA is a cell membrane permeable dye. After diffusing into cells,
DCFH-DA is deacetylated by cellular esterases to non-
fluorescent 2′,7′-dichlorodihydrofluorescin, which is rapidly
converted to highly fluorescent 2′,7′-dichlorofluorescein upon
reacting with ROS. Stimulation of the cells with H2O2 or 6-
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OHDA leads to an intracellular burst of ROS, whereas the
control cells display negligible ROS level (Figure 4). Pretreat-
ment of the cells with 6-DG dose-dependently reduces the ROS
accumulation. As oxidative stress, arising from the over-
production of ROS, has been implicated in a final common
pathway for neurotoxicity in a wide variety of acute and chronic
neurological disorders, the inhibition of ROS accumulation in
neuronal cells might be the molecular basis of the neuro-
protective action of 6-DG.
Induction of Phase II Gene Expression. Analysis of the
chemical structure of 6-DG reveals that there is one Michael
acceptor moiety (highlighted in a circle in Scheme 1), which is
a core structure of many ARE inducers.5,10,13 In analogy to
known ARE activators, we thus hypothesized that 6-DG might
activate the cellular ARE response. We measured the expression
of Nrf2-driven antioxidant genes (HO-1, NQO1, Trx1, TrxR1,
GCLC, and GCLM) after treatment with 6-DG (10 μM) in
PC12 cells (Figure 5). The time course studies showed the
activities were significantly increased (Figure 6E,H,K). After
6-DG (10 μM) treatment, the total GSH level (Figure 6L),
NQO1 activity (Figure 6E), Trx activity (Figure 6H), and TrxR
activity (Figure 6K) were increased by ∼1.6-, ∼2-, ∼1.7-, and
∼1.3-fold, respectively. H2O2 and 6-OHDA treatments caused
serious GSH depletion (Figure 6M,N), whereas pretreatment
with 6-DG gradually restored the total GSH level.
Promotion of Nrf2 Nuclear Localization by 6-DG.
Induction of antioxidant gene expression via the Nrf2-
dependent cytoprotective pathway requires translocation of
Nrf2 from cytosol to nucleus. We therefore examined whether
6-DG could activate the Nrf2 signaling pathway in PC12 cells.
Upon 6-DG treatment, immunoblotting results revealed that
the Nrf2 expression slightly increased with peaking at 4 h and
gradually decreased to the basal level (Figure 7A). Notably, the
cytosolic Nrf2 decreased progressively, in coincidence with its
continuous rise in the nucleus (Figure 7B,C), indicating the
translocation of Nrf2 from cytosol to nucleus. The accumu-
lation of Nrf2 in nuclei thus facilitates its binding to ARE and
subsequently initiates the transcription process.
Involvement of Nrf2 in Neuroprotection of 6-DG. To
further clarify whether 6-DG protects PC12 cells via the Nrf2
pathway, we conducted Nrf2 knockdown experiments in PC12
cells. The knockdown efficiency of different shRNAs (shNrf2-
842, shNrf2-184, shNrf2-136, and shNT) was validated by
Western blots (Figure 8A). The expression of Nrf2 protein in
all cells transfected with shNrf2s was drastically decreased.
Next, we evaluated the protective effect of 6-DG against
oxidative insult toward the transfected cells. In line with the
cytoprotective function of Nrf2, silence of Nrf2 expression
slightly increased the cytotoxicity of H2O2 and 6-OHDA
(Figure 8B,C). 6-DG displays a similarly protective pattern in
PC12-shNT cells as observed in normal PC12 cells. However,
this protection drops remarkably in PC12 cells transfected with
different shNrf2 plasmids, and the most significant results were
observed in PC12-shNrf2-842 cells, where the protection of 6-
DG is almost completely abolished upon either H2O2- or 6-
OHDA-induced cell death. In PC12-shNrf2-184 and PC12-
shNrf2-136 cells, the protection of 6-DG is only partially lost.
This could be due to the variation of the silence efficiency in
cells. Although the protection of 6-DG declines to variable
levels in different Nrf2 knockdown cells, the extents of all the
decrease are significant. Taken together, these results indicate
that the cytoprotection of 6-DG in PC12 cells was mediated
through the action of Nrf2.
Figure 5. Induction of cytoprotective genes in PC12 cells by 6-DG.
The cells were treated as described under Materials and Methods, and
the mRNA levels of different genes were analyzed and normalized
using GAPDH as an internal standard by qRT-PCR as described. Data
represent the mean SD of three independent experiments. (∗∗) P <
0.01 versus the control group (0 h).
highest induction of all genes at 6 h after stimulation with 6-
DG. A >10-fold increase of the HO-1 mRNA was observed
after 6 h of treatment, consistent with HO-1 being a sensitive
marker for oxidative/electrophilic stress. The expression of the
genes decreased after 6 h, but was still significantly higher at 12
h compared to the control. After treatment with 6-DG for 24 h,
the mRNA levels of HO-1 and TrxR1 were back to the control
level, whereas the others remained significantly elevated. Taken
together, these results indicate that 6-DG is a potent activator
of Nrf2-regulated antioxidant defenses.
Up-regulation of the Antioxidant Defense System in
PC12 Cells. The induction of phase II detoxifying enzymes is
an important defense mechanism against exogenous insult. As a
series of phase II genes has been up-regulated at different levels,
we then examined the corresponding gene products. 6-DG
dose-dependently elevated HO-1, NQO1, Trx1, and TrxR1
protein expression and cellular GSH level in PC12 cells (Figure
6). By adopting the convenient assays, we further determined
the cellular activity of NQO1, Trx1, and TrxR1. In line with the
up-regulation of protein expression, all of the enzymes’
DISCUSSION
■
ROS are constantly produced under physiological metabolisms,
and the intricate balance between their production and
neutralization is controlled dynamically by the cellular
antioxidant defense system. Oxidative stress, arising from
excessive production of ROS, causes damage to proteins, lipids,
and nucleic acids and eventually entails cell death. Neuronal
cells in the brain are particularly vulnerable to oxidative stress as
they possess high concentrations of oxidizable unsaturated fatty
acids and high levels of pro-oxidants (e.g., iron) and show high
oxygen consumption and low levels of antioxidant defense
capacities.48 Consistent with the above-described sense, there
has been increasing evidence underpinning oxidative stress as a
major cause in a number of neurodegenerative disorders such as
AD and PD.8,9 Treatment through replacing the dying or dead
neurons in these diseases has been demonstrated with limited
success.49 Therefore, pharmacological interventions aiming at
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Figure 6. Up-regulation of antioxidant enzymes by 6-DG in PC12 cells. (A) Induction of HO-1 expression by 6-DG. (B) Quantification of the band
intensity in panel A. (C) Induction of NQO1 expression and enzyme activity (E) by 6-DG. (D) Quantification of the band intensity in panel C. (F)
Induction of Trx1 expression and total Trx enzyme activity (H) by 6-DG. (G) Quantification of the band intensity in panel F. (I) Induction of
TrxR1 expression and total TrxR enzyme activity (K) by 6-DG. (J) Quantification of the band intensity in panel I. (L) 6-DG induces up-regulation
of GSH in PC12 cells. (M, N) Prevention of H2O2- or 6-OHDA-induced GSH depletion by 6-DG. The total intracellular GSH was measured as
described under Materials and Methods. Data are expressed as the mean SD of three experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the
vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group.
alleviating the oxidative stress are promising to confer
protection to neuronal cells, eventually leading to retardation
or blockage of the progression of these neurodegenerative
diseases. Tactics against oxidative stress include direct
antioxidant action by ROS neutralization, as well as indirect
antioxidant defense by awakening the pathway(s) involved in
the expression of endogenous cytoprotective molecules. In this
regard, accumulating attention has been paid to discovering
antioxidants or molecules that can induce endogenous
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Figure 7. Promotion of Nrf2 nuclear accumulation by 6-DG. After the cells were treated with 6-DG (10 μM) for 0, 2, 4, 6, or 8 h, the cells were
harvested and different cell extracts were prepared as described. Total Nrf2 (A), cytosolic Nrf2 (B), and nuclear Nrf2 (C) were analyzed by Western
blotting with an antibody against Nrf2. Actin was used as a loading control. Quantification of the blots is presented in the right-hand panel. Data are
expressed as the mean SD of three experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the control group (0 h).
antioxidant defense as potential therapeutic agents for neuro-
degenerative diseases.13,16,50,51
low as 1 μM, could significantly protect neuronal cells from
oxidative insult. This concentration could be easily reached by
daily intake of ginger or ginger products. The contribution of 6-
DG to the observed activities of ginger and the physiological
significance of the neuroprotective action of 6-DG require
further investigation.
6-DG both acts as a direct antioxidant in scavenging free
radicals in vitro and confers protective effects against oxidative
stress-induced neuronal cell damage by activating the cellular
antioxidant defense systems. As a phenolic compound, the
direct antioxidant action depends primarily on the ability of 6-
DG to donate its phenolic H atom to different radicals via a
hydrogen abstraction reaction. Our result that M-6-DG almost
completely lost free radical-scavenging activity (Figure 1A,B)
supports that the hydroxyl group in 6-DG plays a key role in
the direct antioxidant activity. More importantly, 6-DG, via
This study demonstrated for the first time that 6-DG, an
active constituent from dietary plant ginger, was a promising
neuroprotective agent. Although 6-gingerol and 6-shogaol are
two major components of ginger, the amount of 6-DG in ginger
is also significantly high (up to 20 mg/100 g ginger).52
Compared to 6-shogaol and 6-DG, 6-gingerol appears to have
lower biological activity in different model systems.53−57 6-DG
has a larger conjugated system than 6-shogaol and contains one
additional active 1,3-diketone moiety, which could explain why
6-DG displays better antioxidant activity than 6-shogaol
does.53,54 Furthermore, 6-DG shows lower cytotoxicity than
6-shogaol.55 The high antioxidant activity and low cytotoxicity
make 6-DG an ideal molecule for further development as a
therapeutic agent. In our present work, we found that 6-DG, as
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Figure 8. Involvement of Nrf2 in the cytoprotection of 6-DG in PC12 cells. (A) Evaluation of Nrf2 knockdown efficiency by different shRNAs.
PC12 cells were transfected with shRNAs specifically targeting rat Nrf2 gene (shNrf2s) or nontargeting shRNA (shNT), and the efficiency of
silencing Nrf2 was evaluated by Western blots. (B, C) Impairment of cytoprotection of 6-DG in Nrf2-knockdown cells. Different PC12 cells were
treated with 6-DG for 24 h followed by treatment with 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24 h. Cell viability was determined by MTT
assay. Results are represented as the mean SD (n = 6). (∗∗) P < 0.01 and (∗) P < 0.05.
Figure 9. Dual neuroprotective mode of 6-DG in PC12 cells.
activation of the Keap1-Nrf2-ARE pathway, awakens the
endogenous antioxidant defense system, including stimulation
of GSH synthesis and up-regulation of diverse antioxidant
enzyme expression and activity. The nuclear translocation of
Nrf2 is a prerequisite for induction of the endogenous
antioxidant defense. However, the mechanisms underlying
such a process are not well understood but may involve
oxidation/alkylation of key thiols in Keap1 and/or phosphor-
ylation of Nrf2.58 Upon exposure to electrophiles or oxidants,
the Cys-rich protein Keap1 is modified by forming a covalent
adduct or disulfide bonds within the protein, and it is this Cys-
based modification of Keap1 that allows dissociation,
accumulation, and subsequent nuclear translocation of Nrf2,
where it initiates the transcription of cytoprotective genes
(phase II genes).58 Analysis of the chemical structure of 6-DG
reveals that the molecule contains one α,β-unsaturated ketone
moiety (highlighted in Scheme 1), a thiol-reactive electrophilic
center that may covalently modify the Cys thiols. Many natural
or synthetic molecules bearing such a structure are known to be
effective inducers of phase II genes.5,10,59 Thus, we speculated
that alkylation of certain Cys residues might be the molecular
basis for the activation of the Keap1-Nrf2-ARE pathway by 6-
DG, which was supported by the observation that removal of
the electrophilic center from 6-DG severely blocks the
protection (Figure 2A). The observation that knocking down
Nrf2 expression abolishes the effect of 6-DG suggests that the
cytoprotection of 6-DG is mediated, at least in part, via Nrf2
activation.
Our findings suggest a dual neuroprotective mechanism of 6-
DG shown schematically in Figure 9. The lipophilic character of
6-DG allows it to easily pass the plasma membrane and
accumulate within cells. As a phenolic compound, 6-DG may
directly counteract against various ROS. Furthermore, 6-DG,
like many other electrophiles,5,10,59 could selectively interact
with the Cys residue(s) on the cytosolic protein Keap1, which
is an adaptor protein for ubiquitinating Nrf2 and, thus, drives
the continuous degradation of Nrf2. The binding of 6-DG to
Keap1 perturbs the Keap1-Nrf2 complex and stabilizes Nrf2,
causing the liberation of Nrf2 and enabling it to be translocated
into nucleus, where it binds to ARE and stimulates the
transcription of cytoprotective genes. Thus, 6-DG provides dual
protection against oxidative stress-induced neuronal cell
damage.
In conclusion, we have demonstrated that 6-DG was effective
in preventing oxidative stress-induced neuronal cell damage.
This neuroprotection may involve its capacity in directly
neutralizing free radicals and activating endogenous cellular
antioxidant defense. The hydroxyl group is critical for the direct
antioxidant activity, whereas the α,β-unsaturated ketone
structure is indispensable for activation of the Keap1-Nrf2-
ARE pathway in cells. Our discovery will provide deep insights
in understanding 6-DG effects in vivo, and this protective
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AUTHOR INFORMATION
■
Corresponding Author
*(J.F.) E-mail: fangjg@lzu.edu.cn. Fax: +86 931 8915557.
Funding
Financial support from Lanzhou University (Fundamental
Research Funds for the Central Universities, lzujbky-2014-56),
the National Natural Science Foundation of China for
Fostering Talents in Basic Research of the (J1103307), and
the 111 project is gratefully acknowledged.
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Notes
The authors declare no competing financial interest.
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