Epigallocatechin-3-gallate suppresses 1-methyl-4-phenyl-pyridine-induced oxidative stress in PC12 cells via the SIRT1/PGC-1α signaling pathway.

Abstract

Parkinson's disease is a high incidence neurodegenerative disease in elderly people, and oxidative stress plays an important role in the pathogenesis. Oxygen metabolism in the brain is high, which lacks an antioxidative protection mechanism. Recently, it has been found that polyphenols play an important role in antioxidation. (-)-epigallocatechin-3-gallate (EGCG) is an important component of tea polyphenols and its biological effects, such as strong antioxidation, scavenging of free radicals and anti-apoptosis, can pass through the blood brain barrier. The SIRT1/PGC-1α signaling pathway has not been reported in PC12 cells. Therefore, research of the protective mechanism of EGCG in PC12 cells damaged by -methyl-4-phenyl-pyridine (MMP+) may provide a new insight into protect against and treatment of Parkinson's disease.
MPP(+)-treated highly differentiated PC12 cells were used as the in vitro cell model. An MTT assay was used to investigate cell viability after EGCG treatment, a dichlorofluorescin diacetate assay was used to measure reactive oxygen species (ROS) production, western blot analysis was used to observe PGC-1α and SIRT1 protein expression, and real-time PCR to observe PGC-1α, SOD1 and GPX1 mRNA expression.
PC12 cell viability was significantly reduced after MPP(+) treatment by 11.46% compared with that of the control (P < 0.05). However, cell viability was unchanged by 10 μmol/L EGCG treatment. In co-treatments with EGCG and MPP(+), cell viability was significantly increased by 12.92% (P < 0.05) and MPP(+)-induced ROS production was markedly decreased. PGC-1α mRNA expression was obviously upregulated by 21.51% (P < 0.05), and SOD1 and GPX1 mRNA expression was slightly increased by 12.94% and 15.63% (P > 0.05), respectively, by treatment with EGCG and then MPP(+) for 12 h. The mRNA expression of PGC-1α, SOD1 and GPX1 was increased by 25.17%, 40% and 146% (all P < 0.05), respectively, by treatment with EGCG and then MPP(+) for 24 h. Such effects were not observed with MPP(+) treatment alone.
The SIRT1/PGC-1α pathway is one of the mechanisms of EGCG suppression of MPP(+)-induced injury of PC12 cells.

Full text

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RESEARCH ARTICLEOpen Access
Epigallocatechin-3-gallate suppresses 1-methyl-4
-phenyl-pyridine-induced oxidative stress in PC12
cells via the SIRT1/PGC-1α signaling pathway
Qinyong Ye*, Linfeng Ye, Xianjie Xu, Bixia Huang, Xiaodong Zhang, Yuangui Zhu and Xiaochun Chen
Abstract
Background: Parkinson’s disease is a high incidence neurodegenerative disease in elderly people, and oxidative
stress plays an important role in the pathogenesis. Oxygen metabolism in the brain is high, which lacks an
antioxidative protection mechanism. Recently, it has been found that polyphenols play an important role in
antioxidation. (−)-epigallocatechin-3-gallate (EGCG) is an important component of tea polyphenols and its biological
effects, such as strong antioxidation, scavenging of free radicals and anti-apoptosis, can pass through the blood
brain barrier. The SIRT1/PGC-1α signaling pathway has not been reported in PC12 cells. Therefore, research of the
protective mechanism of EGCG in PC12 cells damaged by -methyl-4-phenyl-pyridine (MMP+) may provide a new
insight into protect against and treatment of Parkinson’s disease.
Methods: MPP+-treated highly differentiated PC12 cells were used as the in vitro cell model. An MTT assay was
used to investigate cell viability after EGCG treatment, a dichlorofluorescin diacetate assay was used to measure
reactive oxygen species (ROS) production, western blot analysis was used to observe PGC-1α and SIRT1 protein
expression, and real-time PCR to observe PGC-1α, SOD1 and GPX1 mRNA expression.
Results: PC12 cell viability was significantly reduced after MPP+treatment by 11.46% compared with that of the
control (P<0.05). However, cell viability was unchanged by 10 μmol/L EGCG treatment. In co-treatments with
EGCG and MPP+, cell viability was significantly increased by 12.92% (P<0.05) and MPP+-induced ROS production
was markedly decreased. PGC-1α mRNA expression was obviously upregulated by 21.51% (P<0.05), and SOD1 and
GPX1 mRNA expression was slightly increased by 12.94% and 15.63% (P>0.05), respectively, by treatment with
EGCG and then MPP+for 12 h. The mRNA expression of PGC-1α, SOD1 and GPX1 was increased by 25.17%, 40%
and 146% (all P<0.05), respectively, by treatment with EGCG and then MPP+for 24 h. Such effects were not
observed with MPP+treatment alone.
Conclusion: The SIRT1/PGC-1α pathway is one of the mechanisms of EGCG suppression of MPP+-induced injury of
PC12 cells.
Keywords: Parkinson’s disease, (−)-epigallocatechin-3-gallate, PC12 cells, PGC-1α, SIRT1
* Correspondence: unionqyye@163.com
Department of Neurology, Fujian Institute of Geriatrics, The Affiliated Union
Hospital of Fujian Medical University, 29 Xinquan Road, Fuzhou, Fujian
350001, China
© 2012 Ye et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ye et al. BMC Complementary and Alternative Medicine 2012, 12:82
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Background
Polyphenols are present in green tea extract, and many
studies have shown that polyphenols have strong anti-
oxidant and -apoptosis activities [1]. Most of the benefits of
green tea are due to the most prominent biological activity
of the green tea catechin (−)-epigallocatechin-3-gallate
(EGCG) [2]. Oxidative stress and cell apoptosis play an im-
portant role in the pathogenesis of Parkinson’s disease [3].
Therefore, we considered whether EGCG has a protective
effect against Parkinson’s disease. The role of the silent mat-
ing type information regulation 2 homolog (SIRT1)/peroxi-
somal proliferator-activated receptor- coactivator 1 (PGC-
1α) signaling pathway in oxidative stress induced by
1-methyl-4-phenyl-pyridine (MPP+) in PC12 cells and
whether EGCG is antagonized in PC12 cells with
MPP+-induced oxidative stress via the SIRT1/PGC-1α
cell signaling pathway have not been reported. In this
study, PC12 cells were used as a model of dopamin-
ergic neurons with MPP+-induced cell injury, and the
protective effect and mechanism of EGCG suppres-
sion of MPP+-induced cell injury were analyzed to
provide a biological basis for prevention and treat-
ment of Parkinson's disease.
Methods
Reagents
Cell culture reagents were purchased from Hyclone
(Logan, UT, USA). Acrylamide and western blot reagents
were purchased from Bio-Rad (Hercules,California, USA).
MPP+and EGCG were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Anti-PGC-1α and -SIRT1 anti-
bodies were purchased from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA). Anti-actin antibody, goat
anti-rabbit and goat anti-mouse IgG were purchased from
Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.
Superoxide dismutase 1 (SOD1) and glutathione peroxid-
ase 1 (GPX1) primers were synthesized by Shanghai Bio-
logical Engineering Company. 3-(4,5 dimethylthiazole-2yl
)-2,5-diphenyl tetrazolium bromide thiazolyl blue (MTT)
was purchased from Wuhan Ling fly Technology Co., Ltd.
Dichlorofluorescin diacetate (DCFH-DA) was purchased
from the Beyotime Institute of Biotechnology (Shanghai,
China). All other reagents were purchased from Chinese
suppliers.
Cell culture
Highly differentiated PC12 cells, obtained from the Chin-
ese Academy of Sciences Committee Type Culture Collec-
tion cell bank, were cultured in 100 mm plates in high-
glucose Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS),
100 U/ml penicillin and 100 U/ml streptomycin. Cells
were incubated at 37 °C in a humidified incubator (Model
No. 3130; Forma Scientific) supplemented with 5% CO2
and cultured to 80–90% confluency before being passa-
ging or use in experimentation.
MTT assay to evaluate cell survival
Cells were plated at 1×104cells/well in 96 well plates,
cultured, differentiated and treated as described above.
Then, PC12 cells were treated with various concentra-
tions of EGCG (2.5, 5, 10, 20 and 40 μmol/L) and/or
MPP+(125, 250, 500, 1000 and 2000 μmol/L) for 24 h.
Then, treatment solutions were discarded, and cells were
washed gently with PBS. MTT (20 μl) at 0.5 mg/ml and
medium (200 μl) were then added to each well. After in-
cubating for 4 h at 37 °C, with 5% CO2, the solution was
removed, and 150 μl dimethyl sulfoxide was added to
each well. The optical density (OD) was evaluated at
570 nm by a microplate reader after the precipitate in
each well was dissolved for 10 min.
Preparation of cell lysates
PC12 cells were washed several times with ice-cold
phosphate buffered saline (PBS), resuspended in RIPA
lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Tri-
ton X-100, 1% sodium deoxycholate, 0.1% sodium dode-
cyl sulfate (SDS), 2 mM sodium orthovanadate, 25 mM
sodium fluoride, 1 mM EDTA and 0.5 mM phenyl-
methanesulfonyl fluoride and incubated for 10 min on
ice. Lysates were then centrifuged at 12,000×g for
10 min at 4 °C.
Detection of intracellular reactive oxygen species (ROS)
To measure ROS in differentiated PC12 cells produced
by MPP+treatment, we used a DCFH-DA assay. DCFH-
DA is a fluorescent dye that crosses the cell membrane
and is enzymatically hydrolyzed by intracellular esterases
to non-fluorescent DCFH. Cells were plated at 4×105
cells per 6 well dish. Differentiated PC12 cells were pre-
treated with 10 μmol/L EGCG for 1 h and then treated
with 500 μM MPP+for 24 h. Cells were then incubated
with DCFH-DA at a final concentration of 10 μM in
high-glucose DMEM without FBS for 20 min at 37 °C,
washed three times with DMEM and analyzed using a
FACSCalibur flow cytometer (Becton Dickinson, San Jose,
CA, USA). For each analysis, 10,000 events were recorded.
Western blot analysis
Thirty micrograms of protein were loaded per lane and
resolved by 10% SDS-polyacrylamide gel electrophoresis
for 90 min at 80 V. Separated proteins were transferred
to polyvinylidene fluoride membranes (Millipore) for 2 h
at 140 V with a Bradford reagent (Bio-Rad, CA). Mem-
branes were blocked with 5% skim milk in PBS contain-
ing 0.05% Tween 20 for 1 h at room temperature. Then,
membranes were incubated with primary antibodies
against PGC-1α (Lot#: B2510, Santa Cruz), SIRT1 (Lot#:
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K0309, Santa Cruz) and β-actin. Proteins were detected
with a horseradish peroxidase-coupled secondary anti-
body. Specific bands were visualized using an enhanced
chemiluminescence detection kit (Cat#:: AR1170; Millipore,
Billerica, Massachusetts,America).
RNA isolation and real-time RT-PCR
Total RNA was isolated from PC12 cells using TRIzol
reagent (Invitrogen, CA) according to the manufacturer’s
protocol. Total RNA purity and integrity was confirmed
by an ND-1000 NanoDrop (NanoDrop Technologies)
and 2100 Bioanalyzer (Agilent). Real-time PCR was per-
formed using an ABI prism 7500 HT sequence detection
system (Applied Biosystems, Foster City, CA) based on
the 59-nuclease assay [4] for the genes indicated and the
housekeeping gene glyceraldehyde-3-phosphate dehydro-
genase. Relative expression was calculated using the ΔΔCt
method [5]. PCR amplification was carried out with cDNA
equivalent to 10 ng starting mRNA using specific oligo-
nucleotide primers for PGC-1α (forward, 5’-CAATGAAT
GCAGCGGTCTTA-3’, reverse, 5’-ACGTCTTTGTGGCT
TTTGCT-3’), SOD1 (forward, 5’-CGGCTTCTGTCGTC
TCCTTGCTT-3’, reverse, 5’-AACTGGTTCACCGCTTG
CCTTCT-3’), GPX1 (forward, 5’-CGGACATCAGGAGA
ATGGCAAGA-3’, reverse 5’-AGGAAGGTAAAGAGCG
GGTGAGC-3’) and β-actin (forward, 5’-GGAGATTACT
GCCCTGGCTCCTA-3’, reverse 5’-GACTCATCGTACTC
CTGCTTGCTG-3’).
Statistical analysis
Data were analyzed and expressed as the mean±stand-
ard error of the mean. Comparisons were made using
analysis of variance and the Newman-Keuls multiple
comparisons test. A value of P<0.05 was considered
statistically significant.
Results
EGCG suppresses decreased PC12 cell viability induced by
MPP+
To evaluate the cell viability of differentiated PC12 cells
after exposure to oxidative injury, PC12 cells were treated
with various concentrations of MPP+(125–2000 μmol/L)
for 24 h. Different concentrations of MPP+significantly
decreased cell viability in a dose-dependent manner
(Figure 1A). As an optimal MPP+
500 μmol/L MPP+was selected for subsequent experi-
ments, because cell viability was deceased by 38.2%
(P=0.011) (Figure 1A). We investigated the neuropro-
tective effects of EGCG, which showed no cytotoxic ef-
fect at concentrations ranging from 2.5 to 10 μmol/L.
concentration,
C
80
85
90
95
100
105
control 500 1.252.5510
percent survival
MPP+(uM)
EGCG (uM) +500 (uM) MPP+

MPP (uM)
A
0
20
40
60
80
100
120
control 125 25050010002000
Percent survival
0
20
40
60
80
100
120
control
2.55510 10 2040
EGCG(u)M
B
Percent survival
Figure 1 EGCG and MPP+affect PC12 cell viability. (A) MPP+-induced PC12 cell viability. MPP+concentrations were 0, 125, 250, 500, 1000 and
2000 μmol/L (*P<0.05;**P<0.01 vs. vehicle control). (B) PC12 cell viability after EGCG treatment. EGCG concentrations were 0, 2.5, 5, 10, 20 and
40 μmol/L (**P<0.01 vs. vehicle control). (C) Treatment with EGCG and then MPP+affects PC12 cell viability. Groups were: control, 500 μmol/L
MPP+, 500 μmol/L MPP++ EGCG (1.25, 2.5, 5 or 10 μmol/L) (#P<0.05 vs. vehicle control, **P<0.01 vs. MPP+treatment alone).
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However, at 20–40 μmol/L EGCG, PC12 cell viability
was significantly attenuated (Figure 1B). PC12 cells
were pretreated with 10 μmol/L EGCG for 1 h and then
treated with 500 μmol/L MPP+for 24 h. An MTT assay
indicated that the cell viability of PC12 cells treated
with EGCG and then MPP+was significantly increased
by 12.92% (P<0.05) compared with that of MPP+treat-
ment alone. However, there was no significant differ-
ence compared with the cell viability of the control
(P>0.05) (Figure 1C).
EGCG attenuates intracellular ROS production by MPP
+-induced oxidative stress
To measure intracellular ROS production in PC12 cells
after MPP+treatment and the effect of EGCG on ROS
production, we used a DCFH-DA assay. As shown in
Figure 2, MPP+increased ROS production by 19.77%,
compared with that in the control. Pretreatment with
EGCG (10 μM) resulted in intracellular ROS production
decreasing by 13.95%, compared with that of MPP+treat-
ment alone. EGCG alone also attenuated ROS production
by 2.6%, compared with that in the control. These results
indicated that EGCG treatment significantly suppressed
intracellular ROS production in PC12 cells, compared
with that in the control and MPP+treatment alone.
Therefore, EGCG showed a significant protective effect
against MPP+-induced oxidative stress (Figure 2).
PGC-1α and SIRT1 protein expression is enhanced by
EGCG pretreatment of MPP+-treated PC12 cells
Western blot showed that there were protein bands at
120, 90 and 42 kDa (Figure 3). PGC-1α and SIRT1
Figure 2 EGCG attenuates intracellular ROS production after MPP+-induced oxidative stress. PC12 cells were treated as indicated by the
various groups. Data. 01: negative control, control group without DCFH-DA; Data.02: positive control, control group with DCFH-DA and rosup;
Data.03: control group with DCFH-DA; Data.04: 500 μM MPP+; Data.05: 10 μM EGCG; Data.06: 500 μM MPP+and EGCG 10 μM. Intracellular ROS
production was determined by DCFH-DA fluorescence.
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protein levels were not obviously affected in PC12 cells
treated with EGCG and then MPP+, compared with
those in the control. PGC-1α protein expression was sig-
nificantly elevated by 25.91% (P<0.05), and SIRT1
protein expression was increased by 20.55% (P<0.05)
after treatment with EGCG and then MPP+, compared
with that of MPP+and EGCG treatments alone (Figure 3).
PGC-1α, SOD1 and GPX1 mRNA levels are elevated in a
time-dependent manner by EGCG pretreatment of MPP
+-treated PC12 cells for 12 and 24 h
Real-time PCR showed that PGC-1α, SOD1 and GPX1
mRNA expression was not significantly affected by MPP
+and EGCG treatments alone, compared with those in
the control. PGC-1α mRNA expression was obviously
upregulated by 21.51% (P<0.05), SOD1 and GPX1
mRNA expression was slightly increased by 12.94% and
15.63% (P>0.05), respectively, by treatment with EGCG
and then MPP+for 12 h. PGC-1α, SOD1 and GPX1
mRNA expression was increased by 25.17%, 40% and
146% (all P<0.05), respectively, by treatment with
EGCG and then MPP+for 24 h, compared with those by
MPP+and EGCG treatments alone (Figure 4).
Discussion
Drinking at least two cups of green tea a day can reduce
the risk of Parkinson's disease [6]. Green tea extract con-
tains polyphenols that have strong biological effects such
as anti-oxidization, free radical scavenging, anti-apoptosis
and inhibition of monoamine oxidase B. In addition, poly-
phenols can pass through the blood brain barrier and may
be a drug candidate for treating neurodegenerative dis-
eases [7]. Most of the benefits of green tea are due to the
catechin EGCG, which is the most abundant and active
catechin in green tea [8,9].
In drug-induced nerve degeneration research, MPP+
has been widely used to study dopamine neurotoxicity,
0.0
0.5
1.0
1.5
2.0
2.5
3.0
contrl500um
Mpp+
10um
EGCG
Relative expression
SIRT1SIRT1
PGC-1
EGCG10um
Mpp+500um
-actin-
PGC- 1
- 90KD
-
SIRT1
- 42KD
-
- 120KD
-
ABCD
-
Figure 3 Effect of MPP+and EGCG on PGC-1α and SIRT1
protein expression in PC12 cells. (A) vehicle-only control, (B) MPP
+alone, MPP+500 μmol/L, (C) EGCG alone, 10 μmol/L EGCG, (D)
10 μmol/L EGCG+500 μmol/L MPP+(treatment with EGCG for
30 min, followed by MPP+treatment). Results show that EGCG
pretreatment could enhance PGC-1α and SIRT1 protein expression,
and this up-regulation was higher than respective treatments with
MPP+and EGCG alone. β-actin was used as an internal reference.
*P<0.05 vs. vehicle-only control.
Figure 4 Effect of MPP+and EGCG on PGC-1α, GPX1 and SOD1 mRNA expression in PC12 cells. Groups were: control, 500 μmol/L MPP+,
10 μmol/L EGCG, 10 μmol/L EGCG +500 μmol/L MPP+for 12 h (treatment with EGCG for 30 min, followed by MPP+for 12 h), 10 μmol/L
EGCG+500 μmol/L MPP+for 24 h (treatment with EGCG for 30 min, followed by MPP+for 24 h). Results show that PGC-1α mRNA expression was
obviously upregulated by 21.51% (P<0.05), and SOD1 and GPX1 mRNA expression was slightly increased by treatment with EGCG and then MPP
+for 12 h. PGC-1αz SOD1 and GPX1 mRNA expression were increased by 25.17%, 40% and 146% (all P<0.05), respectively, by treatment with
EGCG and then MPP+for 24 h,.*P<0.05 vs. vehicle-only control.
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