Sinomenine Protects PC12 Neuronal Cells against H2O2-induced Cytotoxicity and Oxidative Stress via a ROS-dependent Up-regulation of Endogenous Antioxidant System

Article abstract of DOI:10.1007/s10571-017-0469-1

Sinomenine (SN), a purified alkaloid from Chinese herb Sinomenium acutum that was used preferentially in the treatment of rheumatoid diseases, has exerted neuroprotective effects and anti-inflammatory properties in many previous studies. Some studies have revealed that the antioxidant property of SN, acting mainly through inhibiting NADPH oxidase activation, was involved in the beneficial effects of SN. However, SN belongs to the family of dextrorotatory morphinan analogues, which may initiate elevation of reactive oxygen species (ROS) levels. Thus in the present report, we conducted studies to examine its impact and mechanism on the resistance of PC12 neuronal cells to oxidative stress. Precondition with SN (0.1–5?μM) for 12?h significantly decreased H₂O₂-induced cytotoxicity and remarkably alleviated oxidative injury. However, SN exhibited little direct free radical scavenging property in vitro and induced “appropriate” production of ROS in PC12 cell. Interestingly, the SN-triggering ROS production served as a signal to activate the Nrf2 antioxidant system including Nrf2, HO-1, and NQO-1, which was inhibited by the antioxidant trolox. Furthermore, Nrf2 knockdown largely attenuated the beneficial effects of SN precondition on oxidative stress. In conclusion, our findings suggested that SN increased the resistance to oxidative stress in neuronal cells via a ROS-dependent up-regulation of endogenous antioxidant system, and this mechanism may be involved in the neuroprotection of SN.

Full text of DOI:10.1007/s10571-017-0469-1

Cell Mol Neurobiol
DOI 10.1007/s10571-017-0469-1
ORIGINAL RESEARCH
Sinomenine Protects PC12 Neuronal Cells against H2O2-induced
Cytotoxicity and Oxidative Stress via a ROS-dependent Up-
regulation of Endogenous Antioxidant System
Hua Fan1 Qing Shu2 Xinlei Guan3 Jiegang Zhao1 Junqiang Yan1
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•
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Xiangming Li1 Jiangbo Liu1 Zhaohui Jia1 Jian Shi1 Juan Li4
•
•
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•
Received: 17 August 2016 / Accepted: 25 January 2017
Ó Springer Science+Business Media New York 2017
Abstract Sinomenine (SN), a purified alkaloid from Chi-
nese herb Sinomenium acutum that was used preferentially in
the treatment of rheumatoid diseases, has exerted neuro-
protective effects and anti-inflammatory properties in many
previous studies. Some studies have revealed that the
antioxidant property of SN, acting mainly through inhibiting
NADPH oxidase activation, was involved in the beneficial
effects of SN. However, SN belongs to the family of dex-
trorotatory morphinan analogues, which may initiate eleva-
tion of reactive oxygen species (ROS) levels. Thus in the
present report, we conducted studies to examine its impact
and mechanism on the resistance of PC12 neuronal cells to
oxidative stress. Precondition with SN (0.1–5 lM) for 12 h
significantly decreased H2O2-induced cytotoxicity and
remarkably alleviated oxidative injury. However, SN
exhibited little direct free radical scavenging property
in vitro and induced ‘‘appropriate’’ production of ROS in
PC12 cell. Interestingly, the SN-triggering ROS production
served as a signal to activate the Nrf2 antioxidant system
including Nrf2, HO-1, and NQO-1, which was inhibited by
the antioxidant trolox. Furthermore, Nrf2 knockdown lar-
gely attenuated the beneficial effects of SN precondition on
oxidative stress. In conclusion, our findings suggested that
SN increased the resistance to oxidative stress in neuronal
cells via a ROS-dependent up-regulation of endogenous
antioxidant system, and this mechanism may be involved in
the neuroprotection of SN.
Keywords Sinomenine Á ROS-dependent neuroprotection Á
Oxidative stress Á Nrf2 antioxidant system
Introduction
Hua Fan, Qing Shu and Xinlei Guan have contributed equally to this
work.
Sinomenine (SN), a purified alkaloid from Chinese herb
Sinomenium acutum, has been used for the clinical treat-
ment of inflammatory diseases for many centuries in China
(Xu et al. 2008). Previous studies have demonstrated that
the pharmacological profiles of SN include immunosup-
pression, anti-inflammation, protection against acute lung
injury, anti-apoptotic properties, and so on (Bao et al.
2005; Feng et al. 2006; Li et al. 2013; Qian et al. 2007). In
recent 5 years, some studies have revealed that the SN
provided significant neuroprotection against neurological
diseases, including brain ischemia (Wu et al. 2011) and
Parkinson’s disease (PD) (Qian et al. 2007). Although
mechanistic studies raised some potential targets for SN,
much less is known about the molecular mechanism by
which SN exhibits neuroprotective effects.
& Hua Fan
fanhua19851229@126.com
& Juan Li
lijuan@tjh.tjmu.edu.cn
1
The First Affiliated Hospital, College of Clinical Medicine of
Henan University of Science and Technology,
Luoyang 471003, China
2
Department of Pharmacy, Ninth Hospital of Xi’an,
Xi’an 710054, China
3
Department of Pharmacy, Wuhan Puai Hospital, Tongji
Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
4
Department of Pharmacy, Tongji Hospital affiliated with
Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
Reactive oxygen species (ROS), including superoxide
(O2-•), hydrogen peroxide (H2O2), and hydroxyl radical
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Cell Mol Neurobiol
(OH•), can function either as signaling molecules or as
cellular toxicants that involved in a variety of physiological
and pathological processes (Carvalho et al. 2016; Kim et al.
2015; Lin and Beal 2006; Popa-Wagner et al. 2013). It is
well known that morphine generates cellular ROS in a
concentration- and time-dependent manner and ROS is
proverbially involved in the effect of morphine (Ma et al.
2015). Based on its molecular structure, SN belongs to the
family of dextrorotatory morphinan analogues (as shown in
Fig. 1a) and therefore may also trigger the production of
ROS (Jin et al. 2008). However, some reports have indi-
cated that SN exhibited the antioxidant properties mainly
through inhibiting NADPH oxidase (NOX), which
contributed to its protective effects (Qian et al. 2007). Up
to now, very little is known about the mechanism under-
lying SN-mediated antioxidation. A recent report has
indicated that SN activated nuclear factor erythroid-derived
2-like 2 (Nrf2) signaling, a key antioxidant mechanism
maintains intracellular redox homeostasis underlying
oxidative stress (Qin et al. 2016a; b). Given the reported
antioxidant properties of SN, here, we conducted studies to
examine its impact and mechanism on the resistance of
PC12 cells to oxidative stress. Interestingly, it was
observed that SN protected PC12 neuronal cells against
H2O2-induced cytotoxicity through increased resistance to
oxidative stress which is mediated by a ROS-dependent up-
Fig. 1 SN protected PC12 cells against H2O2-induced cytotoxicity.
a Chemical structure of SN. b PC 12 cells were incubated with
different concentrations of H2O2 (25, 50, 100, and 200 lM) for 12 h,
the H2O2-induced cell toxicity was evaluated by MTT assay. c PC12
cells were incubated with indicated concentrations of SN (0.1, 0.5, 1,
5, 10, and 50 lM) for 24 h. The effects of SN on cell viability were
evaluated by MTT assay. d PC12 cells were incubated with 50 lM
SN for 24, 48, or 72 h. The effects of SN on cell viability were
evaluated by MTT assay. e–f PC12 cells (e) and primary cultured
cortical neurons (f) were pre-incubated 24 h with or without SN (0.1,
0.5, 1, and 5 lM) prior to H2O2 (200 lM) exposure for 12 h.
Protective effects of SN on H2O2-induced cell toxicity were evaluated
by MTT assay. g PC12 cells were co-incubated with H2O2 (200 lM)
and SN (0.1, 0.5, 1, and 5 lM) for 12 h. Protective effects of SN on
H2O2-induced cell toxicity were evaluated by MTT assay. Resveratrol
(Res) was used as a positive control. Results were expressed as a
relative change in comparison with controls, which was set to 100%.
Data are expressed as mean SEM. *p \ 0.05 or ***p \ 0.001
versus control group, #p \ 0.05, ##p \ 0.01 or ###p \ 0.001 versus
H2O2 treatment group
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Cell Mol Neurobiol
regulation of endogenous antioxidant system, and thus
might act as a novel neuroprotector for neurologic diseases
associated with oxidative stress.
accordance with guidelines for animal experiments estab-
lished by Henan University of Science and Technology.
Neonatal Sprague–Dawley (SD) rats (day 0–3) were
obtained from the Experimental Animal Center of Henan
University of Science and Technology. Briefly, cortex of
neonatal SD rats was carefully isolated and dissected, and
then tissue was incubated with 0.125% trypsin in PBS for
20 min at 37 °C. After collected by centrifugation at
800 9 g for 5 min, cells were resuspended in DMEM/F-12
(1:1) with 10% FBS, 5 U/ml penicillin, 5 g/ml strepto-
mycin, and 0.5 mM glutamine. Finally, cells were plated
onto poly-D-lysine coated 96-well (for the measurements of
cell viability) or 6-well plates (for the measurements of
gene expression) and kept at 37 °C in a 5% CO2 incubator.
After 24 h, the culture medium was changed to DMEM
medium supplemented with 2% B27, and the cortical
neurons were fed with fresh medium twice weekly. Glial
cells were minimized by treating the culture with cytara-
bine (10 M) on day 3, and experiments were performed on
day 7–9.
Materials and Methods
Materials
SN and resveratrol were obtained from the National Insti-
tutes for Food and Drug Control (Beijing, China). Dul-
becco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), heat-inactivated horse serum, streptomycin,
penicillin, glutamine, B27 supplements, and trizol reagent
were acquired from Gibco/Invitrogen Inc. (Carlsbad, CA,
USA). 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetra-
zolium bromide (MTT) and peroxide hydrogen (H2O2)
were purchased from Sigma-Aldrich Chemical Inc. (St.
Louis, MO, USA). The ApoGSHTMGlutathione (GSH)
Colorimetric Detection Kit was obtained from BioVision
Inc. (Milpitas, CA, USA). Malondialdehyde (MDA) assay
kit and total superoxide dismutase (SOD) assay kit were
acquired from Beyotime Biotechnology (Shanghai, China).
The lucigenin-enhanced chemiluminescence with a col-
orimetric assay kit was obtained from GENMED Scien-
tifics Inc. (Shanghai, China). The RevertAid-TM First
Strand cDNA Synthesis system was purchased from
Transgene Inc. (Beijing, China). SYBR Green I PCR Pre-
mix was obtained from TaKaRa Biotechnology (Dalian,
China). The anti-Nrf2 and anti-HO-1 polyclonal antibodies
were purchased from Cell signaling Technology (Beverly,
USA). The anti-NQO-1 polyclonal antibody and anti-
NOX2 monoclonal antibody were purchased from Abcam
(Cambridge, UK). The anti-b-actin and appropriate per-
oxidase-conjugated secondary antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, USA). All
other chemicals were of analytical grade.
SN was freshly prepared as stock solution in dimethyl
sulfoxide (DMSO) and diluted with phosphate-buffered
saline (PBS) before the experiment. The final concentration
of DMSO was\0.05%. No detectable effect of DMSO was
found in the experiments. To produce oxidative stress,
H2O2 was freshly prepared from 30% stock solution prior
to each experiment. PC12 cells or primary cultured cortical
neurons were pre-incubated with various concentrations of
SN for 1, 6, or 24 h before exposure to H2O2 (200 lM).
Assays for cell viability, MDA, GSH, SOD, NOX, quan-
titative analysis of gene expression, and western blotting
were performed at different time periods after H2O2 was
added.
Assay of Cell Viability
The cytoprotective activity of SN on H2O2-induced cell
injury was evaluated by MTT reduction assay. PC12 cells
or primary cultured cortical neurons were placed into a
96-well plate for 24 h and then pretreated with vehicle
alone or indicated concentrations of SN for 24 h. After the
pretreatment, the culture was added to H2O2 (200 lM) for
another 12 h and the MTT solution (dissolved in PBS) was
added to the medium with the final concentration of
0.5 mg/ml. Four h later, cells were lysed in DMSO and the
amount of MTT formazan was assessed by determining the
absorbance at 570 nm using a microplate reader (Spec-
traMax 250, Molecular Devices Inc., Sunnyvale, CA).
Survival of the control groups was defined as 100%, and
cell viability in the treated groups was expressed as a
percentage of the control groups.
Cell Culture and Experimental Treatment
The PC12 cell line was acquired from Cell Bank of Chi-
nese Academic of Science (Shanghai, China). PC12 cells
were grown in DMEM supplemented with 5% FBS, 10%
heat-inactivated horse serum, 100 U/ml streptomycin, and
100 lg/ml penicillin. Cultures were kept at 37 °C in a
humidified atmosphere incubator with 5% CO2/95% air.
The culture medium was replaced every two days. All
experiments were carried out 24 h after cells were seeded.
The isolation and culture of the cortical neurons were
slightly modified from our previous studies (Wu et al.
2011). All experimental procedures were performed in
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Cell Mol Neurobiol
Electron Spin Resonance (ESR) Measurement
The supernatants were removed to measure the activity of
SOD according to the manufacturer’s instructions. Briefly,
total activity of SOD was assessed by the scavenging of
superoxide anion generated by xanthine, while the
remained superoxide anion oxidizes WST-8 to WST-8
formazan. Therefore, the absorption of formed WST-8
formazan is negatively correlative to the total activity of
SOD, which can be evaluated by determining the absor-
bance at 450 nm using a microplate reader (Spec-
traMax250, Molecular Devices Inc., Sunnyvale, CA).
The ESR measurement was conducted as previously
described (Fan et al. 2015). N-tert-butyl-a-phenylnitrone
(PBN) was used as a free radical trapper and ESR signals
were detected with a Bruker e-scan ESR spectrometer
(Bruker, Karlsruhe, Germany). As a standard of the reactant
of OHÁ with PBN, we produced hydroxyl radical (OHÁ) by
the Fenton reaction in the mixture of H2O2 (0.5 mM) and
FeSO4 (0.2 mM) in the presence of PBN (30 mM). The spin
traps, SN (0.1, 0.5, 1, and 5 lM) were added before the Fe(II)
and H2O2. Samples (20 ll) were loaded into a quartz tube
and the ESR spectra were recorded at room temperature.
The ESR microwave power was set to 4.88 mW. The
modulation frequency was 9.76 GHz. The time constant
was 20.48 ms. The conversion time was 20.48 ms and a
sweep time of 10.49 s was used. Each sample was scanned
for a total of 20 times. A sweep width of 70 G was used for
experiments with PBN. The receiver gain was set to
3.17 9 103. Simulation and fitting of the ESR spectra were
performed using the Bruker WinEPR program.
Quantitative Analysis of Gene Expression
The procedure for quantitative real-time PCR (qPCR) was
performed as described in our previous studies (Fan et al.
2015). Total cellular RNA was extracted from PC12 cells
or primary cultured cortical neurons using trizol reagent
following the procedure described by the manufacturer.
The concentration and purity of RNA were determined
spectrophotometrically at ratio 260/280, respectively.
cDNA was synthesized from the total RNA using the
RevertAid-TM First Strand cDNA Synthesis system. The
Primers (Sangon, Shanghai, China) used for qPCR analysis
of Nrf2, HO-1, NQO-1, and b-actin were as follows: 50-
CCTCGCTGGAAAAAGAAGTG-30 and 50-GGAGAGGA
TGCTGCTGAAAG-30 for Nrf2; 50-CAGGTGATGCT
GACAGAGGA-30 and 50-TCTCTGCAGGGGCAGTA
TCT-30 for HO-1; 50-TTCTCTGGCCGATTCAGAGT-30
and 50-TCCAGACGTTTCTTCCATCC-30 for NQO-1; and
50-GCTCCTTCGTTGCCGGTCC-30 and 50-CTCTTG
CTCTGGGCCTCGTCA-30 for b-actin. qPCR reactions
were performed with SYBR Green I PCR Premix using an
ABI 7500 real-time PCR system (Applied Biosystems,
Carlsbad, USA). For each target mRNA, 2 ll cDNA was
used as a template in each 20 ll qPCR reactions. The
protocol was 2 min at 95 °C, 40 cycles of 15 s at 95 °C,
15 s at 62 °C, and 30 s at 72 °C. Each assay was run at
least in triplicate in separate experiment, and negative
controls were generated by substituting cDNA with dis-
tilled water. The purity of qPCR products was assessed by
melting curves, and the relative expression levels of target
genes were determined by comparative CT method by
normalizing to b-actin and relative to a calibrator
(2-44Ct).
Measurement of MDA
After the treatment, PC12 cells were lysed and then the cell
lysate was centrifuged at 1600 9 g for 10 min at 4 °C. The
supernatants were removed to measure the levels of MDA
according to the manufacturer’s instructions.
Measurement of GSH
The content of GSH was evaluated according to the manu-
facturer’s recommendations for the ApoGSHTM Glutathione
Colorimetric Detection Kit. Briefly, cells (5 9 105 cells)
were harvested by centrifugation at 700 9 g for 5 min at
4 °C and then the supernatants were removed. The cell pel-
lets were resuspended in 0.5 ml ice-cold PBS and lysed in
80 ll ice-cold glutathione buffer. Then the samples were
mixed with 20 ll 5-sulfosalicylic acid (SSA) and cen-
trifuged at 8000 9 g for 10 min. The supernatants (20 ll)
were added to the reaction mix (160 ll) at room temperature.
After 10 min incubation, the substrate solution (20 ll) was
added to the mixture for a further 10 min and the content of
GSH was quantified via determining the absorbance at
415 nm using
a microplate reader (SpectraMax250,
Molecular Devices Inc., Sunnyvale, CA). The standard curve
was obtained from the absorbance of the diluted GSH stan-
dard which was incubated in the mixture as in samples.
Western Blotting
After treatments, PC12 cells were washed three times with
PBS and lysed in ice-cold lysis buffer (1 mM EDTA,
1 mM phenylmethanesulfonyl fluoride, 3 mM Na3VO4,
20 mM NaF, 50 mM Tris–HCl, 100 mM NaCl, 1% Non-
idet P-40 (v/v), 2.0 l g/ml aprotinin, 2.0 l g/ml leupeptin,
and 2.0 l g/ml pepstatin A). Equal amounts of protein
Measurement of SOD Activity
After the treatment, PC12 cells were lysed and then the cell
lysate was centrifugated at 1600 9 g for 10 min at 4 °C.
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Cell Mol Neurobiol
(20 lg) from above lysates were separated in 10–15%
SDS-PAGE gels and transferred onto nitrocellulose mem-
branes by using a transfer cell system (Bio-Rad, California,
USA). The membranes were blocked with 5% bovine
serum albumin (BSA) diluted in TBS/0.1% Tween-20 for
1 h at room temperature. After blocking, the membranes
were incubated with the following primary antibodies
(anti-Nrf2 at 1 : 1000 dilution, anti-HO-1 at 1 : 1000
dilution, anti-NQO-1 at 1 : 1000 dilution, anti-NOX2 at 1:
2000 dilution, and anti-b-actin at 1 : 2000 dilution) over-
night at 4 °C. Then the membranes were washed three
times with TBS/0.1% Tween-20 and appropriate secondary
antibodies were used to detect the proteins. Immunoblots
were developed on microchemi (DNR, Jerusalem, Israel)
by using the enhanced chemiluminescence technique
(ECL, Pierce, Rockford, USA). All assays were performed
at least three times. An image analysis system and ImageJ
1.49 were used to determine the densitometry of immu-
noblots relative to the loading control.
Results
SN Pre-conditioning Protected PC12 Cells
and Primary Cultured Neurons Against H2O2-
Induced Cytotoxicity
Initially, the cytotoxic potential of H2O2 was measured. As
shown in Fig. 1b, the rate of cell viability was dose-de-
pendently inhibited when PC12 cells were incubated with
indicated concentrations of H2O2 (25, 50, 100, and
200 lM) for 12 h (n = 10, *p \ 0.05 or ***p \ 0.001 vs.
control). When PC12 cells were exposed to different con-
centrations of SN (0.1, 0.5, 1, 5, 10 or 50 lM) alone for
24 h, no statistically significant difference in the viability
of PC12 cells was observed (Fig. 1c). However, PC12 cells
incubated with 50 lM SN for 72 h exhibited obvious toxic
effects (n = 10, *p \ 0.05 vs. control, Fig. 1d). To further
evaluate the neuroprotective effects of SN, PC12 cells and
primary cultured cortical neurons were pretreated with
indicated concentrations of SN for 24 h and then exposed
to H2O2 (200 lM). After 12 h, the H2O2-induced cell
injury was determined by MTT assay. As shown in
Fig. 1e–f, incubation with 200 lM H2O2 significantly
decreased the rate of cell viability by 52.67 5.60%
(n = 10, ***p \ 0.001 vs. control, Fig. 1e) in PC12 cells
and 43.43 6.38% (n = 10, ***p \ 0.001 vs. control,
Fig. 1f) in primary neurons. When cells were pretreated
with SN (0.1, 0.5, 1, and 5 lM) for 24 h, H2O2-induced
cell toxicity was efficiently attenuated in a dose-dependent
manner. SN at a concentration of 5 lM rescued the H2O2-
induced decrease in viability rate by about 92.95 3.45%
(n = 10, ###p \ 0.001 vs. H2O2, Fig. 1e) in PC12 cells and
77.37 7.59% (n = 10, ##p \ 0.01 vs. H2O2, Fig. 1f) in
primary neurons, respectively. Finally, the protective
effects of SN were also assessed when PC12 cells were co-
treated with H2O2 and SN, and similar but less effective
results of SN were obtained. As shown in Fig. 1g,
cotreatment with 5 lM SN rescued the H2O2-induced
decrease in rate of cell viability by 78.43 6.51%
(n = 10, ##p \ 0.01 vs. H2O2).
Nrf2 Gene Silencing Experiments
siRNA oligonucleotides targeting Nrf2 was purchased from
Santa Cruz Biotechnology. Cells were transfected with
25 nM of Nrf2 or scrambled-siRNA oligonucleotides using
Lipofectamine RNAiMAX according to the manufacturer’s
instructions (Invitrogen).
Measurement of NADPH Oxidase Activity (NOX
Activity)
The activity of NOX was evaluated by lucigenin-enhanced
chemiluminescence with a colorimetric assay kit following
the manufacturer’s instructions. Briefly, the supernatant of
cell lysates was incubated with oxidative cytochrome c in a
quartz cuvette at 30 °C for 3 min, and then NADPH which
served as the NOX substrate was added into the reaction
mixture for another 15 min. The change of absorbance at
550 nm was recorded by a spectrophotometer and the
activity of NOX was measured by calculating cytochrome
c reduction in per minute. The relative NOX activity was
expressed as a percentage of the control value.
SN pre-conditioning remarkably alleviated H2O2-
induced oxidative stress in PC12 cells
Statistics
Oxidative stress is considered to play an essential role in
H2O2-dependent cytotoxicity; therefore, the effect of SN
on ROS was studied by evaluating the levels of malondi-
aldehyde (MDA), the content of glutathione (GSH), and
superoxide dismutase (SOD) activity.
Data from experiments were analyzed with the statistical
program SPSS 18.0 software (SPSS, Chicago, IL). Com-
parison between two groups was evaluated by an unpaired
and two-sided Student’s t test. ANOVAs and post hoc tests
(Fisher’s LSD) were used to analyze differences in dif-
ferent treatment groups. Data are presented as mean
SEM. Differences between experimental conditions were
considered statistically significant when p \ 0.05.
MDA has been extensively studied as an indicator of
lipid peroxidation which is one of the earliest recognized
and most widely studied manifestations of oxygen toxicity
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Cell Mol Neurobiol
in biology. As shown in Fig. 2a, a significant increase of
MDA levels (2.09 0.16 nm/mg pro) compared to control
group (0.77 0.06 nm/mg pro) was observed in PC12
cells exposed to H2O2 (n = 5, ***p \ 0.001 vs. control).
Pretreatment with SN at a concentration of 1 and 5 lM
remarkably decreased the content of MDA by
of SN (0.1, 0.5, 1, or 5 lM) could not significantly
decrease the signal intensity of PBN-OH adducts (n = 5),
which suggested that SN exhibited little direct effects on
scavenging ROS.
#
SN Pre-conditioning Dose-Dependently Increased
the Expression of Nrf2-related Antioxidant Genes,
Which Mediated the Protective Effects of SN
1.57 0.12 nm/mg pro (n = 5, p \ 0.05 vs. H2O2) and
1.28 0.13 nm/mg pro (n = 5, ##p \ 0.01 vs. H2O2),
respectively. Furthermore, antioxidant agents, such as
GSH, and antioxidant enzymes, such as SOD, are the pri-
mary defense systems to protect biological systems from
oxidative stress. As shown in Fig. 2b, c, exposure of PC12
cells to H2O2 significantly reduced the content of GSH
(6.44 0.46 lm/mg pro) and the activity of SOD
(31.68 3.40 U/mg pro) to 3.40 0.28 lm/mg pro and
12.03 1.45 U/mg pro, respectively, as compared to
control group (n = 5, **p \ 0.01 vs. control). However,
the decrease of GSH levels and SOD activity induced by
H2O2 was dose-dependently attenuated when PC12 cells
were pre-incubated with SN at a concentration of 1 and
5 lM for 24 h (n = 5, ##p \ 0.01 or ###p \ 0.001 vs.
H2O2). These findings indicated that SN protected PC12
cells against H2O2-induced cell injury mainly via increas-
ing resistance to oxidative stress.
Nrf2 antioxidant system is considered as good candidate
molecules for modulating intracellular redox state. We
hypothesized that SN pre-conditioning may increase the
expression of Nrf2-related antioxidant genes in PC12 cells.
Firstly, we analyzed the relative expression of Nrf2-related
antioxidant genes including Nrf2, HO-1, and NQO-1 by
quantitative real-time PCR. As shown in Fig. 4a–c, com-
pared to the mRNA expression in the control group, the
Nrf2 (n = 5, *p \ 0.05 or ***p \ 0.001 vs. control,
Fig. 4a), HO-1 (n = 5, ***p \ 0.001 vs. control, Fig. 4b),
and NQO-1 mRNA (n = 5, **p \ 0.01 or ***p \ 0.001
vs. control, Fig. 4c) expression was significantly increased
in a dose-dependent manner when PC12 cells were pre-
incubated with SN at a concentration of 1, 5, 10, and
50 lM for 6 h. Next, we investigated the effect of SN on
the protein expression of Nrf2 antioxidant system. It was
found that pretreatment with SN (5 lM, 24 h) obviously
elevated the protein expression of Nrf2, HO-1, and NQO-1
to 215.12 33.43% (n = 5, **p \ 0. 01 vs. control,
Fig. 4d), 289.23 42.70% (n = 5, ***p \ 0.001 vs.
control, Fig. 4d), and 172.23 15.32% (n = 5, *p \ 0.05,
Fig. 4d), respectively, when compared with the control
group. Moreover, in order to confirm the role of Nrf2 in the
neuroprotective effects of SN, we compared its actions on
the rate of cell viability following H2O2 exposure in control
and Nrf2-silenced PC12 cells. Expectedly, the protective
effects of SN were largely abolished when Nrf2 was
SN Exhibited Little Free Radical Scavenging
Property In Vitro
Then, we investigated the mechanism underlying the pro-
tection of SN against oxidative stress. One possible
explanation is that the intracellular SN exerts direct free
radical scavenging property. To confirm this hypothesis,
we studied the antioxidant effect of SN in vitro. As shown
in Fig. 3, the hydroxyl radical (OH•) was produced by the
Fenton reaction and the levels of OH• were evaluated by
electron spin resonance (ESR) measurement. As compared
to control group, cotreatment with different concentrations
Fig. 2 SN remarkably alleviated H2O2-induced oxidative stress in
PC12 cells. PC12 cells were pre-incubated 24 h with or without SN (1
and 5 lM) prior to H2O2 (200 lM) exposure for 12 h. The levels of
MDA (a), the content of GSH (b), and the activity of SOD (c) were
evaluated. Res was used as a positive control. Results were expressed
as a relative change in comparison with controls, which was set to
100%. Data are expressed as mean SEM. **p \ 0.01 or
***p \ 0.001 versus control group, #p \ 0.05, ##p \ 0.01 or
###p \ 0.001 versus H2O2 treatment group
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Cell Mol Neurobiol
Fig. 3 SN exhibited little direct free radical scavenging property
in vitro. ESR spectra of the spin adduct of OH• radical were observed
during the reaction of 0.5 mM H2O2 containing 30 mM PBN and
0.2 mM FeSO4 with or without indicated concentrations of SN (0.1,
0.5, 1, and 5 lM). The levels of ESR peak intensity were expressed as
a relative change in comparison with control, which was set to 100%.
Data are expressed as mean SEM
$
The antioxidant trolox significantly prevented
the SN-induced up-regulation of Nrf2-related
antioxidant genes
silenced (n = 5, p \ 0.05 vs. H2O2 ? scrambled-siRNA,
Fig. 4e).
SN Increased Intracellular ROS Levels at its High
Concentration
To further confirm that SN increased the expression of
Nrf2-related antioxidant genes via a ROS-dependent
mechanism, we studied the effect of the antioxidant trolox
on the SN-induced activation of Nrf2 signaling system. As
shown in Fig. 6a–c, pretreatment with SN (1 and 5 lM,
6 h) obviously increased the mRNA expression of Nrf2,
HO-1 and NQO-1 (n = 5, *p \ 0.05, **p \ 0.01 and
***p \ 0.001 vs. control) in a dose-dependent manner.
Similar results were obtained in primary neurons (n = 5,
*p \ 0.05, **p \ 0.01 and ***p \ 0.001 vs. control
Fig. 6d–f). However, when the antioxidant trolox was co-
treated with SN in PC12 cells, the SN-induced dose-de-
pendent augment in the expression of Nrf2 (n = 5,
#p \ 0.05 vs. 1 lM SN and $p \ 0.05 vs. 5 lM SN,
Fig. 6a), HO-1 (n = 5, ##p \ 0.01 vs. 1 lM SN and
$$p \ 0.01 vs. 5 lM SN, Fig. 6b), and NQO-1 (n = 5,
#p \ 0.05 vs. 1 lM SN and $p \ 0.05 vs. 5 lM SN,
Fig. 6c) was significantly attenuated. These data indicated
that SN activated Nrf2 antioxidant system via triggering
ROS generation, followed by the increase of cell resistance
to oxidative stress.
We therefore asked the underlying mechanism that how SN
increased the expression of Nrf2-related antioxidant genes.
Although overproduction of ROS causes cell injury, ROS
at low concentration may provide beneficial effects via
triggering cellular endogenous antioxidant system (Four-
quet et al. 2010). It is well accepted that morphine con-
centration and time dependently generate cellular ROS,
which is proverbially involved in the effect of morphine
(Ma et al. 2015). SN belongs to the family of dextrorota-
tory morphinan analogues; therefore, we speculated that
SN activated the Nrf2 signaling system through induction
of intracellular ‘‘appropriate’’ production of ROS. As
shown in Fig. 5, the levels of MDA, the content of GSH
and the activity of SOD were assessed when PC12 cells
were treated with higher concentrations of SN (1, 5, 10 and
50 lM). SN at a concentration of 1, 5 or 10 lM did not
efficiently alter the levels of MDA (Fig. 5a), the content of
GSH (Fig. 5b) and SOD activity (Fig. 5c). However, the
content of MDA was efficiently elevated (n = 5, *p \ 0.05
vs. control, Fig. 5a) and the activity of SOD was signifi-
cantly reduced (n = 5, *p \ 0.05 vs. control, Fig. 5c)
when PC12 cells were treated with 50 lM SN for 1 h.
These data suggested that SN increased intracellular ROS
levels at its high concentration, which indicated that the
SN-induced activation of Nrf2-antioxidant system may be
ROS-dependent.
SN Pre-conditioning Inhibited the Activation
of NOX
Previous studies have showed that SN exhibits anti-in-
flammatory effects through inhibiting the activation of
NADPH oxidase (NOX) in the microglia; therefore, we
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Cell Mol Neurobiol
Fig. 4 SN dose-dependently increased the expression of Nrf2-related
antioxidant genes, which mediated the protective effects of SN. a–
c PC12 cells were treated with SN (1, 5, 10, or 50 lM) for 6 h. The
mRNA expression of Nrf2 (a), HO-1 (b), and NQO-1 (c) was
evaluated by quantitative real-time PCR. Res was used as a positive
control. d PC12 cells were treated with SN (5 lM) for 24 h. The
protein expression of Nrf2, HO-1, and NQO-1 was evaluated by
western blotting. e PC12 cells were transfected with scrambled-
siRNA or Nrf2-siRNA and incubated with H2O2 (200 lM) for 12 h.
The effects of Nrf2 silencing on the protective effects of SN were
evaluated by MTT assay. Results were expressed as a relative change
in comparison with controls, which was set to 100%. Data are
expressed as mean SEM. *p \ 0.05, **p \ 0.01 or ***p \ 0.001
versus control group, ###p \ 0.001 versus H2O2 treatment group,
$p \ 0.05 versus H2O2 ? scrambled-siRNA group
Fig. 5 SN increased intracellular ROS levels at its high concentra-
tion. PC12 cells were incubated with different concentrations of SN
(1, 5, 10, and 50 lM) for 1 h. The levels of MDA (a), the content of
GSH (b), and the activity of SOD (c) were evaluated. Results were
expressed as a relative change in comparison with controls, which
was set to 100%. Data are expressed as mean SEM. *p \ 0.05
versus control group
asked if there exist other mechanisms involved in the
protective role of SN against H2O2-induced cytotoxicity
and oxidative stress. As shown in Fig. 7, the NOX2
expression (n = 5, *p \ 0.05 vs. control, Fig. 7a) and the
activity of NOX (n = 5, **p \ 0.01 vs. control, Fig. 7b)
were markedly increased in the H2O2-treated PC12 cells
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Cell Mol Neurobiol
Fig. 6 The antioxidant trolox significantly prevented the SN-induced
up-regulation of Nrf2-related antioxidant genes. a–c PC12 cells were
co-incubated with SN (1 or 5 lM) and Trolox (0 or 0.5 mM) for 6 h.
The mRNA expression of Nrf2 (a), HO-1 (b), and NQO-1 (c) was
evaluated by quantitative real-time PCR. d–f Primary cultured
cortical neurons were treated with SN (1 and 5 lM) for 6 h. The
mRNA expression of Nrf2 (d), HO-1 (e), and NQO-1 (f) was
evaluated by quantitative real-time PCR. Results were expressed as a
relative change in comparison with controls, which was set to 100%.
Data are expressed as mean SEM. *p \ 0.05, **p \ 0.01 or
***p \ 0.001 versus control group, #p \ 0.05 or ##p \ 0.01 versus
$
1 lM SN treatment group, p \ 0.05 or $$p \ 0.01 versus 5 lM SN
group
Fig. 7 SN pre-conditioning inhibited the activation of NOX. PC12
cells were pre-incubated 24 h with or without SN (5 lM) prior to
H2O2 (200 lM) exposure for another 24 h. a The protein expression
of NOX2 was evaluated by western blotting. b The NOX activity was
detected by lucigenin-enhanced chemiluminescence with colorimetric
assay kit. Results were expressed as a relative change in comparison
with controls, which was set to 100%. Data are expressed as
mean SEM. *p \ 0.05 or **p \ 0.01 versus control group,
#p \ 0.05 versus H2O2 treatment group
compared to control cells. Although pretreatment with SN
at a concentration of 5 lM had no significant effect on the
H2O2-induced protein expression of NOX2 (n = 5,
Fig. 7a), the increase of NOX activity induced by H2O2
was obviously attenuated (n = 5, #p \ 0.05 vs. control,
Fig. 7b).
Discussion
In this work, we report for the first time that pretreatment
with SN, a natural alkaloid extracted from Chinese
medicinal plant S. acutum extensively used in treatment of
rheumatoid arthritis and various inflammatory disorders,
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Cell Mol Neurobiol
up-regulated the endogenous antioxidant system including
Nrf2, HO-1, and NQO-1, in PC12 cells via a ROS-de-
pendent mechanism. This mechanism was mainly respon-
sible for the protective effects of SN pretreatment against
cellular oxidative stress, for SN exhibited little direct
scavenging effect in a Fenton reaction system. Evidence
collected in this study provided a new mechanism under-
lying the neuroprotective effects of SN.
activity of NOX in PC12 cells exposure to oxidative stress.
Little effect of SN pretreatment on the expression of NOX2
in PC12 cells was observed. The activity of NOX was
reduced by SN pretreatment at high concentration (5 lM).
But at low concentration (1 lM), SN pretreatment exhib-
ited significant protective effects against oxidative stress,
without inhibition on NOX activity in PC12 cells. Thus, the
effects of SN pretreatment at low concentration against
oxidative stress might not be attributed to inhibition of
NOX activity. There may exist other mechanisms under-
lying its antioxidant effects. This mechanism was not due
to its direct radical scavenging property, for SN was
removed from the medium before the exogenous hydrogen
peroxide was added. Furthermore, we observed that SN
exhibited little direct effects on scavenging the exogenous
ROS. Nrf2 is known to be an important transcription factor
that is involved in the cellular antioxidant response, as it
binds to antioxidant response elements (ARE) in the genes
encoding antioxidant enzymes, such as HO-1 and NQO-1
(Buendia et al. 2016; Hybertson and Gao 2014). Moreover,
it has also been reported that SN activated Nrf2 in the
kidney (Qin et al. 2016a). Thus, we observed the effects of
SN on the Nrf2 system in PC12 cells. We found that after
incubation with SN, the mRNA level and the protein level
of Nrf2 system in PC12 cells, including Nrf2, HO-1, and
NQO-1, were up-regulated in a concentration-dependent
manner. Moreover, the protective effects of SN pretreat-
ment on PC12 cells were largely attenuated when Nrf2 was
silenced. Our findings mainly explained the antioxidant
effects of SN pretreatment on cells.
Previous studies have showed that SN displays adverse
spectrum of biological activities, including anti-inflamma-
tory, neuroprotective, and immune suppressive potential
(Chen et al. 2011; Feng et al. 2006; Mark et al. 2003; Qian
et al. 2007). Considering its ability to come across blood–
brain barrier (Liu et al. 2005; Long et al. 2010), and
increasing evidence for its beneficial effects on central
nerve system, SN emerged as a new pharmacological agent
to rescue neurobiological diseases, including stroke and PD
(Qian et al. 2007; Wu et al. 2011). The generation of ROS
is an essential metabolic process for maintaining home-
ostasis. A series of antioxidant mechanisms maintain
intracellular redox homeostasis. When they deficit, over-
production of ROS causes DNA mutations and protein
dysfunction, which eventually leads to neuronal injury and
neurobiological diseases (Espinosa-Diez et al. 2015;
Schieber and Chandel 2014; Zhang et al. 2011). We used
PC12 neuronal cells, which share many properties with
primary sympathetic neurons and chromaffin in cell cul-
tures, to evaluate the effect of SN on ROS. We found that
SN exhibited little effects on scavenging the exogenous
ROS, but pre-incubation with SN significantly increased
the resistance to oxidative stress in PC12 cells. The indirect
antioxidant effect of SN may underlay the beneficial effects
of SN in the central nerve system.
An interesting question is that how could SN activate
Nrf2 system? Based on its molecular structure, SN belongs
to the family of dextrorotatory morphinan analogues (Jin
et al. 2008). It is well known that morphine generates
cellular ROS in a concentration- and time-dependent
manner and ROS is proverbially involved in the effect of
morphine (Ma et al. 2015). Although overproduction of
ROS causes cell injury, ROS at low concentration may
provide beneficial effects via triggering cellular endoge-
nous antioxidant system (Fourquet et al. 2010). Interest-
ingly, we found that SN increased intracellular ROS levels
at its high concentration, indicating that SN may up-regu-
late endogenous antioxidant system via a ROS-dependent
pathway, which is further confirmed by the result that
incubation with antioxidant trolox prevented the up-regu-
lation of Nrf2 system induced by SN. This point was in
according with previous reports that morphine protected
SH-SY5Y human neuroblastoma cells against 6-OHDA-
induced cell injury via increasing cellular antioxidant
(Elyasi et al. 2014). It should be noted that although it
seemed that the lower concentrations of SN did not
increase ROS level but activated Nrf2 in PC12 cells, the
effects of SN pretreatment on PC12 cells were abolished by
A serious of studies has found that SN exhibited
antioxidant effects in different cells, including neuronal
cells (Li et al. 2008; Qin et al. 2016b; Wang et al. 2016;
Yang et al. 2014). However, to our knowledge, there are
fewer reports about the mechanism underlying the antiox-
idant effect. Previous studies have revealed that SN
inhibited the activation of NADPH oxidase (NOX) in the
microglia, which may confer to the anti-inflammation of
SN (Qian et al. 2007). In our study, we used an oxidative
stress model by addition of exogenous hydrogen peroxide,
which is not entirely dependent on NOX. Interestingly, it
was observed that pre-incubation with SN exhibited a good
protection against cellular oxidative stress. Meanwhile, we
also found that the beneficial effects of SN at low con-
centration (1 lM) were abolished when SN was co-incu-
bated with H2O2. However, 1 lM SN pretreatment
exhibited significant protective effects against oxidative
stress, indicating that SN may trigger the resistance to
oxidative stress in PC12 cells. We further observed the
effects of SN pretreatment on the NOX2 expression and the
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Cell Mol Neurobiol
Elyasi L, Eftekhar-Vaghefi SH, Esmaeili-Mahani S (2014) Morphine
protects SH-SY5Y human neuroblastoma cells against 6-hy-
droxydopamine-induced cell damage: involvement of anti-oxi-
dant, calcium blocking, and anti-apoptotic properties.
Rejuvenation Res 17:255–263. doi:10.1089/rej.2013.1473
Espinosa-Diez C, Miguel V, Mennerich D, Kietzmann T, Sanchez-
Perez P, Cadenas S, Lamas S (2015) Antioxidant responses and
cellular adjustments to oxidative stress redox biology 6:183–197.
doi:10.1016/j.redox.2015.07.008
Fan H et al (2015) Methionine sulfoxide reductase A negatively
controls microglia-mediated neuroinflammation via inhibiting
ROS/MAPKs/NF-kappaB signaling pathways through a catalytic
antioxidant function. Antioxid Redox Signal 22:832–847. doi:10.
1089/ars.2014.6022
Feng H, Yamaki K, Takano H, Inoue K, Yanagisawa R, Yoshino S
(2006) Suppression of Th1 and Th2 immune responses in mice
by Sinomenine, an alkaloid extracted from the chinese medicinal
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Fourquet S, Guerois R, Biard D, Toledano MB (2010) Activation of
NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide
formation. J Biol Chem 285:8463–8471. doi:10.1074/jbc.M109.
051714
Hybertson BM, Gao B (2014) Role of the Nrf2 signaling system in
health and disease. Clin Genet 86:447–452. doi:10.1111/cge.
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Jin HZ, Wang XL, Wang HB, Wang YB, Lin LP, Ding J, Qin GW
(2008) Morphinane alkaloid dimers from Sinomenium acutum.
J Nat Prod 71:127–129. doi:10.1021/np0704654
Kim GH, Kim JE, Rhie SJ, Yoon S (2015) The role of oxidative stress
in neurodegenerative diseases. Exp Neurobiol 24:325–340.
doi:10.5607/en.2015.24.4.325
Li L, Gao XL, Ding BX (2008) Inhibitory effect of sinomenine on
H2O2-induced apoptosis in neonatal rat cardiomyocytes. Zhong-
guo Zhong yao za zhi = Zhongguo zhongyao zazhi = China
journal of Chinese materia medica 33(939–941):961
Li J, Zhao L, He X, Zeng YJ, Dai SS (2013) Sinomenine protects
against lipopolysaccharide-induced acute lung injury in mice via
adenosine A(2A) receptor signaling. PLoS ONE 8:e59257.
doi:10.1371/journal.pone.0059257
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative
stress in neurodegenerative diseases. Nature 443:787–795.
doi:10.1038/nature05292
Liu ZQ, Chan K, Zhou H, Jiang ZH, Wong YF, Xu HX, Liu L (2005)
The pharmacokinetics and tissue distribution of sinomenine in
rats and its protein binding ability in vitro. Life Sci
77:3197–3209. doi:10.1016/j.lfs.2005.05.054
trolox, a ROS scavenger. We hypothesized that the increased
ROS level of SN pretreatment with low concentration was
not observed because of the detection limitation. Altogether,
these evidences strongly supported that SN increased the
resistance to oxidative stress in PC12 cells via triggering
beneficial levels of ROS and activating the cellular antioxi-
dant response. However, there may exist other potential
mechanisms. For instance, SN shows a Michel acceptor on
the structure that might react with the cysteine residues
present at keap1 that might be involved in the induction
capability. This possibility required further investigation.
In summary, this study showed that pre-incubation with
SN at low concentration induced cytoprotection against
oxidative stress-induced neuronal cell death via triggering
an ‘‘appropriate’’ ROS signal, followed by activation of
cellular defense system. As a clinic-used drug in the ther-
apy of inflammatory disorders, SN may be used not only in
the treatment but also in the prevention of neurological
diseases in the future.
Acknowledgement This work was supported by the National Nature
Science Foundation of China (Grant Nos. U1504808 and 81501075).
Author Contributions Hua Fan, Qing Shu, and Juan Li conceived
and designed the experiments; Xinlei Guan, Jiegang Zhao, Junqiang
Yan, Xiangming Li, Jiangbo Liu, Zhaohui Jia, and Jian Shi performed
the experiments; Xinlei Guan and Qing Shu analyzed the data; Hua
Fan and Juan Li wrote the paper.
Compliance with Ethical Standard
Conflicts of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.
Informed consent Informed consent was obtained from all individ-
ual participants included in the study.
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