Experimental Gerontology 87 (2017) 2332

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Experimental Gerontology

journal homepage: www.elsevier.com/locate/expgero

Insulin-like growth factor-1 protects SH-SY5Y cells against

-amyloid-induced apoptosis via the PI3K/Akt-Nrf2 pathway
Zigao Wang a,b,1, Lu Xiong c,1, Guanqun Wang b,d, Wenbin Wan a, Chunjiu Zhong a, Hengbing Zu b,
a
Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai, PR China
b
Department of Neurology, Jingshan Hospital, Fudan University, Shanghai, PR China
c
Department of Anesthesiology, Tinglin Hospital, Shanghai, PR China
d
Department of Neurology, Jining No.1 People's Hospital, Shandong, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Insulin-like growth factor-1 (IGF-1) shows protective effect against A-induced cytotoxicity and apoptosis, but
Received 6 July 2016 the underlying mechanisms are poorly characterized. The present study was conducted to explore the mecha-
Received in revised form 31 October 2016 nisms involved in the benecial effect of IGF-1 against A-induced apoptosis in SH-SY5Y cells. We found that pre-
Accepted 21 November 2016
treatment with IGF-1 attenuated A2535-induced loss of cell viability and apoptosis in SH-SY5Y cells in a dose-
Available online 22 November 2016
dependent manner. In addition, IGF-1 inhibited the generation of reactive oxygen species (ROS) and increased
Section Editor: Christian Humpel the antioxidant activity in A2535-treated cells. Further, IGF-1 signicantly promoted the nuclear translocation
of Nrf2, and upregulated the expression of its downstream gene heme oxygenase-1 (HO-1). Moreover,
Keywords: LY294002, a specic PI3K inhibitor, was found to completely abolish the protective effect of IGF-1 on A2535-in-
IGF-1 duced apoptosis and ROS generation. Together, our ndings suggest that IGF-1 protects SH-SY5Y cells against
-amyloid A2535-induced cell injury by scavenging ROS via the PI3K/Akt-Nrf2 signaling pathway.
Apoptosis 2016 Published by Elsevier Inc.
Oxidative stress
PI3K/Akt
Nrf2

1. Introduction
Alzheimer's disease (AD) is a progressive and irreversible neurode-
generative disease characterized clinically by gradual deterioration of
intellectual function, and pathologically by extracellular senile plaques,
intracellular neurobrillary tangles, and loss of neurons and synapses in
the brain (Citron, 2010). Genetic, biochemical, and neuropathological
data suggest that aggregation and deposition of amyloid -protein
(A) are sufcient to initiate the cascade of pathological and clinical
changes associated with AD, including a great amount of neuronal
death in the brain (Hardy and Selkoe, 2002). Studies have demonstrated
that apoptosis induced by A was the predominant feature of neuronal
death in AD (Zhu et al., 2006). Although the precise mechanisms under-
lying A-mediated neuronal apoptosis have not been fully elucidated, it
has been suggested that oxidative stress plays a pivotal role in this pro-
cess. Increased protein oxidation, DNA oxidation, and lipid peroxidation
were found in the brains of AD patients (Behl et al., 1994; Buttereld,
1997). In addition, mouse models of AD showed increased urine and
Corresponding author at: Department of Neurology, Jinshan Hospital, Fudan
University, 1508 Longhang Road, Shanghai 201508, PR China.
E-mail address: hbzyy8@sina.com (H. Zu).
1
Zigao Wang and Lu Xiong contribute equally to this work.
plasma levels of markers of oxidative stress before plaque formation oc-
curred (Pratico and Sung, 2004). Moreover, an abundance of evidence
supports that A could induce an oxidative environment for neurons
(Behl, 1999; Buttereld, 1997). Therefore, oxidative stress is considered
the executor of A-induced neurotoxicity.
To maintain a redox balance, cells are equipped with a wide variety
of endogenous antioxidant enzymes such as superoxide dismutase
(SOD), catalase (CAT) and heme oxygenase-1 (HO-1) (Jung and Kwak,
2010; Shah et al., 2007). Transcription of these antioxidant proteins is
found to be under the control of nuclear transcription factor, NF-E2-re-
lated factor 2 (Nrf2). Upon exposure to reactive oxygen species (ROS),
Nrf2 dissociates from cytoplasmic Keap1 and translocates into the nu-
cleus, where it binds to antioxidant response elements (ARE) and in-
duces the production of these endogenous antioxidant enzymes.
Previous studies showed that nuclear Nrf2 expression and CAT activity
were decreased in AD patients (Ramsey et al., 2007). Moreover, HO-1
activity was negatively affected by amyloid precursor protein (APP) in
a mouse model of AD (Wojtunik-Kulesza et al., 2016). Therefore, en-
hancement of the activity of Nrf2-ARE antioxidant system may be a
promising target for exploring new drugs for AD.
Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone es-
sential for normal growth and developments (Bassil et al., 2014). In-
creasing evidence showed that IGF-1 was involved in the pathogenesis
of AD. Decreased levels of both IGF-1 and IGF-1 receptor were found

http://dx.doi.org/10.1016/j.exger.2016.11.009
0531-5565/ 2016 Published by Elsevier Inc.
24 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332
in the brains of AD patients (Moloney et al., 2010; Steen et al., 2005).
Lower serum levels of IGF-1 were associated with an increased risk of
developing AD (Westwood et al., 2014). Moreover, IGF-1 signicantly
enhanced cognitive function and ameliorated the pathology including
loss of neurons in transgenic model of AD (Carro et al., 2006). The ben-
ecial effect of IGF-1 in AD was explained partly by its ability to promote
the clearance of A from the brain (Carro et al., 2002) and partly by its
ability to protect neurons from the insult of A. Indeed, IGF-1 could pre-
vent A-induced apoptosis in lymphocytes in vitro (Jimenez Del Rio and
Velez-Pardo, 2006). Furthermore, IGF-1 could protect hippocampal
neurons from the death induced by A (Dore et al., 1997). These data
suggest that IGF-1 could function as a powerful inhibitor of cytotoxicity
induced by A. However, the mechanisms underlying this protective ef-
fect of IGF-1 remain unclear.
Previous studies showed that Nrf2/ARE pathway was involved in the
protective effect of IGF-1 against 6-hydroxydopamine (6-OHDA)-medi-
ated apoptosis in PC12 cells (Kim et al., 2012). IGF-1 decient mice
showed a decreased expression of Nrf2, an increased oxidative stress,
and apoptosis as well (Bailey-Downs et al., 2012). The present study,
therefore, was conducted to examine whether IGF-1 also protected neu-
rons against A-induced cell death through activation of the Nrf2/ARE
system. Our ndings demonstrate that IGF-1 protects against A2535-
induced neuronal injury by scavenging ROS via the PI3K/Akt-Nrf2 sig-
naling pathway.
2. Materials and methods
2.1. Reagents
A2535, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT), Hoechst 33,342, and Rhodamine 123 were purchased
from Sigma-Aldrich (St. Louis, MO, USA). IGF-1, LY294002, and U0126
were obtained from Cell Signaling Technology (Beverly, MA, USA).
Fetal bovine serum and Dulbecco's modied Eagle's medium (DMEM)
were purchased from Gibco (Carlsbad, CA, USA). The uorescent dyes,
2,7-dichlorodihydrouorescein diacetate (H2DCF-DA) were purchased
from Molecular Probes (Eugene, OR, USA). The kits for determining
MDA, SOD activity, and CAT activity were purchased from Abcam (Cam-
bridge, MA, USA). Rabbit polyclonal antibodies against Bcl-xL, Bax, HO-
1, phospho-Akt (Ser473), Akt, phospho-ERK1/2 (Thr202/Tyr204),
ERK1/2, COX IV, and rabbit monoclonal antibodies against cytochrome
c (Cyto c), cleaved caspase-3, Nrf2, GAPDH, and Lamin B1 were pur-
chased from Cell Signaling Technology (Beverly, MA, USA).
2.2. Cell culture and treatment
Human neuroblastoma SH-SY5Y cells, kindly provided by Stem Cell
Bank (Chinese Academy of Science, China), were cultured in DMEM
(Gibco) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL
penicillin, and 100 M streptomycin (Invitrogen) at 37 C and 5% CO2
as described previously (Wang et al., 2009). Cells were seeded at
a density of 1 10 4 /cm 2 for the study of Hoechst staining, and
2 10 5 /cm 2 for siRNA transfection assay. For the other experi-
ments, cells were seeded at a density of 1 10 5 /cm 2 . All experi-
ments were repeated three times independently to minimize the
errors of operation.
A2535 was initially dissolved in double-distilled water and incubat-
ed at 37 C for 710 days to allow aggregation. Different concentrations
(0, 5, 10, 25, 50 M) of aggregated A2535 were added to the cultures to
induce oxidative insult. Cells were kept in serum-free medium for 6, 12,
24, and 36 h. Preincubation with IGF-1 (0, 10, 50, 100, 200 ng/L) was
conducted 24 h before the addition of A2535. Pretreatment with
10 M of LY294002 and 20 M of U0126, was performed 1 h before
the addition of IGF-1.
2.3. Assessment of cell viability
Cell viability was measured by MTT assay as described previously
(Siqueira et al., 2009). After treatment, cells were incubated with MTT
(0.5 mg/mL) at 37 C for 2 h. Then supernatants were removed and
200 L of DMSO/well was added. Absorbance was measured at
550 nm using a SpectraMax Plus384 Microplate Reader (Molecular De-
vices, USA). Results were expressed as a percentage of the absorbance of
non-treated cells.
2.4. Hoechst staining
After treatment, cells were xed with 4% paraformaldehyde, and
stained with Hoechst 33,342 (10 g/mL) at 37 C for 10 min. Then
cells were washed with PBS and the apoptotic cells were observed
under a uorescence microscopy (Leica DM1000, Japan). Cells
exhibiting regular and round nuclei were considered normal cells,
while those with chromatin condensation and nuclear fragmentation
were regarded as apoptotic cells. Apoptotic cells were counted in 20
randomly chosen elds (using a 100 objective lens) of each slide.
2.5. Annexin V-FITC and propidium iodide double-staining assay
After treatment, cells were collected and resuspended in 200 L cold
binding buffer. Then cells were added with 10 L Annexin V-FITC and
5 L PI. After incubation for 15 min at room temperature in the dark,
cells was added with 300 L binding buffer to terminate the reaction,
and ow cytometric analysis was performed immediately on a
FACSCalibur Flow Cytometer (BD Biosciences, USA). Annexin-V+ cells
were considered apoptotic cells.
2.6. Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was analyzed using Rho123.
After treatment, cells were incubated with Rho123 (10 g/mL) at
37 C for 30 min. Then Rho123 was removed by washing with PBS,
and uorescence intensity was measured using a Gemini XPS Micro-
plate Reader (Ex/Em = 507/529 nm) (Molecular Devices, USA).
2.7. Preparation of cellular protein extracts
After treatment, cells were lysed in a buffer (50 mmol/L TrisHCl,
pH 8.0; 100 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L dithiothreitol;
1% TritonX-100; 0.1% sodium dodecyl sulfate; 50 mmol/L sodium uo-
ride, and 1 mmol/L sodium vanadate) containing a cocktail of protease
inhibitors for 30 min on ice. Then the lysate was centrifuged and the su-
pernatant consisting of total cellular protein extracts was collected for
protein analysis. For the measurement of mitochondrial Cyto c, the mi-
tochondrial proteins were isolated using a Cell Mitochondria Isolation
Kit (Abcam). Briey, following treatment, cells were frozen and thawed
to weaken membranes. Then cells were suspended in reagent A. After
homogenization, cells were centrifuged and the supernatant were col-
lected. Meanwhile, the pellet was resuspended in reagent B. After ho-
mogenization and centrifugation, the supernatant was saved. All the
supernatant was mixed thoroughly and centrifuged. Then the pellet
was collected, and added with 500 L reagent C supplemented with pro-
tease inhibitors. The aliquots were collected as mitochondrial fractions.
For the measurement of nuclear and cytoplasmic Nrf2, proteins were
extracted using the nuclear and cytoplasmic protein extraction kit
(Beyotime Institute of Biotechnology, China). In brief, following treat-
ments, cells were resuspended in PBS. Then cytoplasmic protein extrac-
tion agent A and B was subsequently added. The samples were
centrifuged and the supernatant was collected as the cytoplasmic frac-
tion. The nuclei pellet was resuspended in nuclear protein extraction
agent. After centrifugation, the supernatant was stored as the nuclear
 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332
fraction. Protein concentrations were determined by the BCA Protein
Assay kit (Beyotime Institute of Biotechnology, China).
2.8. Western blot analysis
Equal amounts of protein (20 g) were separated by 10% SDS-PAGE
and transferred to PVDF membrane. After blocked with 5% non-fat dry
milk in TBST for 1 h at room temperature, blots were incubated with pri-
mary antibodies overnight at 4 C. The following primary antibodies
were used: rabbit anti-cytochrome c (1:1000, CST); rabbit anti-COX IV
(1:1000, Abcam); rabbit anti-cleaved caspase-3 (1:500, CST); rabbit
anti-Bcl-xL (1:2000, CST); rabbit anti-Bax (1:2000, CST); rabbit anti-
Nrf2 (1:1000, CST); rabbit anti-HO-1 (1:1000, CST); rabbit anti-Lamin
B1 (1:2000, CST); rabbit anti-pAkt (1:1000, CST); rabbit anti-Akt
(1:1000, CST); rabbit anti-ERK1/2 (1:1000, CST); rabbit anti-pERK1/2
(1:1000, CST); and rabbit anti-GAPDH (1:10,000, CST). Then the blots
were washed with TBST and incubated with horseradish peroxidase-
conjugated secondary antibodies (1:1000) for 1 h at room temperature.
Finally, the blots were visualized by enhanced chemiluminescence (ECL
kit, Santa Cruz Biotechnology, CA, USA) and quantied using Chemi im-
ager 5500 V2.03 software.
2.9. Measurement of intracellular ROS and MDA
Intracellular ROS generation was evaluated by H2DCF-DA assay.
Following treatment, cells were incubated with H2DCF-DA
(10 M) for 30 min at 37 C in the dark. Then cells were rinsed
twice with PBS and the uorescence was measured using a Gemini
XPS Microplate Reader (Ex/Em = 532/553 nm) (Molecular Devices,
USA). Intracellular MDA level was measured with a respective
commercial kit. MDA level was determined by a method based on
the reaction with thiobarbituric acid. The absorbance was read at
532 nm by a SpectraMax Plus384 Microplate Reader (Molecular
Devices, USA).
2.10. Measurement of SOD and CAT activity
The activities of SOD and CAT were determined using commercially
available colorimetric assay kits (Abcam, USA) according to the proto-
cols provided by the manufacturer. Briey, protein was isolated using
a lysis buffer and 10 g of the total protein extract was used to detect
the antioxidant enzyme activities. Absorbance values were measured
by a SpectraMax Plus384 Microplate Reader (Molecular Devices, USA)
at 450 nm for SOD, and at 520 nm for CAT.
2.11. siRNA transfection assay
siRNA transfection assay was performed as described previously (Xu
et al., 2015). Briey, cells were cultured in a six well tissue culture plate
(2 105 cells per well). Then the cells were transfected by Nrf2 siRNA
(50 nM) according to the manufacturer's instructions. Twenty-four
hours later, the infected cells were pretreated with 100 ng/L of IGF-1
for 24 h prior to exposure to A2535. A non-specic siRNA was used
as control. Specic silencing was examined by Western blot analysis
for Nrf2.
2.12. Statistical analysis
All data were expressed as mean SD. ANOVA was used to compare
multiple groups, followed by a post hoc Dunnett or Student-Newman-
Kuel test. P b 0.05 was considered statistically signicant.
25
3. Results
3.1. IGF-1 ameliorated A2535induced cytotoxicity in SH-SY5Y cells
Cells were incubated with A2535 with or without IGF-1, and cell vi-
ability was determined by MTT. The percentage of viable cells compared
with untreated controls was signicantly reduced to 88.33 2.88%
(5 M), 75.53 3.61% (10 M), 55.46 3.46% (25 M), 40.16
4.05% (50 M) by treatment with A2535 for 24 h (Fig. 1A). Further, ex-
posure of cells to 25 M of A2535 resulted in a time-dependent in-
crease in cell death (Fig. 1B). IGF-1 at a concentration ranging from
10 ng/L to 100 ng/L was used in the subsequent studies because IGF-1
at such a concentration alone did not lead to any apparent changes in
the viability of cells, in contrast to IGF-1 at 200 ng/L, which resulted in
a signicant decrease in cell viability (Fig. 1C). Next, cells were
pretreated with IGF-1 for 24 h before exposure to A2535. Compared
with untreated controls, cell viability decreased to 50.67 2.51% by in-
cubation with 25 M of A2535 for 24 h. However, the percentage of vi-
able cells was increased to 59.80 2.51% (10 ng/L), 68.33 5.02%
(50 ng/L) and 85.50 5.50% (100 ng/L) by pretreatment with IGF-1
(Fig. 1D). These ndings suggest that IGF-1 could ameliorate A2535-in-
duced cytotoxicity in SH-SY5Y cells.
3.2. IGF-1 inhibited A2535induced apoptosis in SH-SY5Y cells
To evaluate whether IGF-1 could prevent A-induced apoptosis,
Hoechst 33,342 staining was conducted. As shown in Fig. 2A, cells
pretreated with IGF-1 (100 ng/L) showed a decreased number of apo-
ptosis (9.10 1.05%) compared to those treated with A2535 alone
(29.85 2.52%). Annexin V-FITC/PI double staining was further per-
formed to conrm the inhibitive effect of IGF-1 on A-induced apopto-
sis. Administration with A2535 resulted in a signicant increase in
apoptotic cells (30.14 2.20%) compared to that of controls (4.76
0.68%). Conversely, pretreatment with IGF-1 blocked A2535-induced
apoptosis in a concentration-dependent manner, as evidenced by re-
duction of Annexin V-positive cells to 16.33 1.53% (10 ng/L),
10.25 1.05% (50 ng/L), and 6.08 0.95% (10 ng/L) (Fig. 2B). Given
that mitochondrial depolarization is considered an irreversible step in
the process of apoptosis, mitochondrial membrane potential (MMP)
was evaluated by Rhodamine (Rho123) staining. We found that A25
35 induced a 49 4.25% decrease in the Rho 123 uorescence intensity,
while pretreatment with IGF-1 reversed this effect of A2535 in a dose-
dependent manner (Fig. 2C). Release of Cyto c from mitochondria to cy-
toplasm is believed to be primary trigger leading to the onset of apopto-
sis. Incubation with 25 M of A2535 induced an 80 7.5% decrease in
the level of mitochondrial Cyto c and a 220.5 15.85% increase in cyto-
plasmic Cyto c. On the contrary, pretreatment with IGF-1 resulted in in-
creased mitochondrial Cyto c and decreased cytoplasmic Cyto c in a
concentration-dependent manner (Fig. 2D). Caspase-3 is one of the ex-
ecutioner caspases in apoptosis. We found that administration of IGF-1
signicantly inhibited the increase in cleaved caspase-3 induced by
A2535 in a dose-dependent manner (Fig. 2D). Bcl-2 family members
are major regulator of mitochondrial-initiated Cyto c release and cas-
pase activation. Pretreatment with IGF-1 signicantly blocked the up-
regulation of Bax and downregulaton of Bcl-xL induced by A2535
(Fig. 2D). These results demonstrate that IGF-1 could protect against
A2535-induced apoptosis in SH-SY5Y cells.
3.3. IGF-1 attenuated A2535induced oxidative stress in SH-SY5Y cells
Oxidative stress is believed to play a pivotal role in neuronal apopto-
sis induced by A. Intracellular ROS generation was measured by
H2DCF-DA assay. We found that the increased intracellular DCF uores-
cence intensity induced by A2535 was ameliorated by pretreatment
with IGF-1 in a dose-dependent manner (Fig. 3A). Further, the intracel-
lular levels of malondialdehyde (MDA), a widely used marker of cellular
26 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332
Fig. 1. IGF-1 protected against A2535-induced neurotoxicity in SH-SY5Y cells. A. Cells were incubated with indicated concentrations of A2535 for 24 h. B. Cells were treated with 25 M of
A2535 for the indicated time. C. Cells were treated with indicated concentrations of IGF-1 for 24 h. D. Cells were pretreated with IGF-1 (0, 10, 50, 100 ng/L) for 24 h prior to exposure to
25 M of A2535. Cell viability was assessed by MTT assay. Values were expressed as mean SD. *P b 0.05, compared to group treated with vehicle; #P b 0.05, ##P b 0.01, compared to group
treated with A2535 alone.
oxidative damage, were measured. Compared to controls, A2535 led to
a 3.9 fold increase in the levels of intracellular MDA. On the contrary,
pre-incubation with IGF-1 ameliorated the increased MDA induced by
A2535 in a concentration-dependent manner (Fig. 3B). SOD and CAT
are two important endogenous antioxidant enzymes. Treatment with
A2535 resulted in a 75 5.6% and 52.5 4.8% decrease in the activity
of SOD and CAT, respectively. By contrast, pretreatment with IGF-1 re-
versed the A2535-induced decreased enzyme activity of both SOD
and CAT (Fig. 3CD). These data indicate that IGF-1 inhibits oxidative
stress triggered by A2535 in SH-SY5Y cells.
3.4. IGF-1 protected against A2535induced neurotoxicity by activating
Nrf2/HO-1 pathway in SH-SY5Y cells
Since Nrf2/HO-1 pathway plays a key role in neuronal resistance to
A-induced oxidative stress, we further examined whether Nrf2/HO-1
pathway was involved in the protective effect of IGF-1 against A-in-
duced cytotoxicity. Compared to controls, treatment with IGF-1 for
24 h resulted in an increase in the protein levels of nuclear Nrf2 to
150.85 10.85% (10 ng/L), 213.35 18.64% (50 ng/L) and 443.35
35.80% (100 ng/L), associated with a decrease in the cytoplasmic Nrf2
to 71.35 6.68% (10 ng/L), 58.55 5.50% (50 ng/L) and 23.80
2.55% (100 ng/L). Meanwhile, the expression of HO-1 was increased to
152.80 12.50% (10 ng/L), 240.55 18.6% (50 ng/L) and 428.60
29.60% (100 ng/L) (Fig. 4A), indicating that IGF-1 induced the nuclear
translocation of Nrf2 and subsequently promoted the expression of
HO-1 in a concentration-dependent manner. Similarly, IGF-1 also led
to a time-dependent increase in the nuclear translocation of Nrf2 and
expression of HO-1(Fig. 4B). Interestingly, exposure of cells to A2535
alone also potentiated the nuclear translocation of Nrf2 and the expres-
sion of HO-1 (Fig. 4C). Next, Nrf2 was knocked down by an Nrf2-specic
siRNA to further examine the role of Nrf2/HO-1 pathway in the protec-
tive effect of IGF-1. As expected, protein levels of Nrf2 and that of its
downstream gene HO-1 were markedly inhibited by the Nrf2 specic
siRNA (Fig. 4D). More importantly, the protective effect of IGF-1 against
A2535-induced oxidative stress and apoptosis was also signicantly at-
tenuated, as evidenced by a 175.85 15.60% increase in levels of MDA
and a 115.50 11.48% increase in cleaved caspase-3 in siRNA-treated
cells compared to those in IGF-1-pretreated cells (Fig. 4EF). These re-
sults support the notion that activation of Nrf2/HO-1signaling pathway
is involved in the protective effect of IGF-1 against A2535-induced
neurotoxicity.
3.5. PI3K/Akt pathway was involved in the protective effect of IGF-1 against
A2535induced oxidative stress and apoptosis in SH-SY5Y cells
IGF-1 has been reported to exert its biological effects by binding to
IGF-1 receptors and subsequently activating PI3K/Akt and MEK/ERK sig-
naling pathways (Parrizas et al., 1997). To determine which signaling
pathway was involved in the protective effects of IGF-1 against A25
35-induced oxidative stress and apoptosis, cells were pretreated with
10 M of LY294002 or 20 M of U0126 for 1 h prior to incubation with
IGF-1. As expected, pretreatment with IGF-1 could signicantly activate
both PI3K/Akt and MEK/ERK signaling pathways, as evidenced by a
210.85 19.56% increase in phosphorylated Akt and a 110.80
18.85% increase in phosphorylated ERK1/2 (Fig. 5A). In addition,
LY294002 and U0126 could signicantly inhibit the activation of Akt
and ERK1/2 induced by IGF-1, respectively (Fig. 5A). Importantly, the
nuclear translocation of Nrf2 and upregulation of HO-1 induced by
IGF-1 were blocked by LY294002, but not U0126 (Fig. 5A). Further, it
was LY294002, rather than U0126, that could reverse the inhibitive ef-
fect of IGF-1 on A2535-induced oxidative stress as measured by in-
creased DCF uorescence intensity and levels of MDA (Fig. 5BC).
Moreover, the protective effect of IGF-1 on A2535-induced apoptosis
measured by increased Annexin V-positive cells and cleaved caspase-3
protein level was abolished by LY294002, but not by U0126 (Fig. 5D
E). These results suggest that the PI3K/Akt pathway plays a crucial
role in the protective effects of IGF-1 against A2535-induced oxidative
stress and apoptosis.
 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332 27

Fig. 2. IGF-1 protected against A2535-induced apoptosis in SH-SY5Y cells. Cells were pretreated with indicated concentrations of IGF-1 for 24 h prior to exposure to 25 M of A2535. A.
Hoechst 33,342 staining showed apoptotic cells characterized by chromatin condensation and nuclear fragmentation (arrows). Bar = 50 m. B. Apoptotic cells were evaluated by ow
cytometry. Annexin-V+ cells were regarded as apoptotic cells. C. Mitochondrial membrane potential was determined by Rho 123 staining. D. Representative image of immunoblots
for cytochrome c (Mito = mitochondrial, Cyto = cytoplasmic), cleaved caspase-3, Bcl-xL and Bax. Values were presented as mean SD. *P b 0.05, compared to group treated with
vehicle; #P b 0.05, ##P b 0.01, compared to group treated with A2535 alone.
28 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332
Fig. 3. IGF-1 inhibited A2535-induced oxidative stress in SH-SY5Y cells. Cells were pretreated with indicated concentrations of IGF-1 for 24 h prior to exposure to 25 M of A2535. A.
Intracellular ROS was evaluated by the oxidation of H2DCF-DA to DCF. B. MDA level was measured with a lipid peroxidation (MDA) assay kit. C. SOD activity was detected with
superoxide dismutase activity colorimetric assay kit. D. CAT activity was detected with catalase specic activity assay kit. Values were presented as mean SD. P b 0.05, compared to
control; #P b 0.05, ##P b 0.01, compared to group treated with A2535 alone.
4. Discussion
Oxidative stress has been found to be implicated in the neurotoxicity
of A and in the pathogenesis of AD (Behl et al., 1994). Previous studies
have shown that IGF-1 had neuroprotective effect against A-induced
cell death in vitro (Dore et al., 1997; Jimenez Del Rio and Velez-Pardo,
2006). IGF-1 has also been shown to have benecial effects in AD trans-
genic models (Carro et al., 2006). However, the mechanisms by which
IGF-1 protects against A-induced neurotoxicity remain unknown.
Here, we demonstrated that IGF-1 reversed the A2535-induced injury
in SH-SY5Y cells, a process marked by a decrease in cell viability and in-
creases in apoptosis. In addition, IGF-1 inhibited intracellular ROS and
MDA generation, and increased the enzyme activity of both SOD and
CAT. Further, IGF-1 promoted the nuclear translocation of Nrf2 and
the expression of its downstream antioxidant gene HO-1. However, all
these effects of IGF-1 were blocked by a specic Nrf2 siRNA. Moreover,
a specic PI3K inhibitor, LY294002, was found to completely abolish
the effect of IGF-1 on Nrf2/HO-1 expression, while inducing increased
oxidative stress and apoptosis. Taken together, these ndings suggest
that IGF-1 protects against A2535-induced neurotoxicity by scaveng-
ing ROS via activation of PI3K/Akt-Nrf2 signaling pathway in SH-SY5Y
cells.
It is now well established that A plays a causal role in the pathogen-
esis of AD. Aggregated A is toxic to many cell types, including neurons
both in vitro and in vivo (Hardy and Selkoe, 2002). It has been reported
that IGF-1 could prevent A-induced apoptosis in lymphocyte (Jimenez
Del Rio and Velez-Pardo, 2006). It has also been shown that IGF-1 could
inhibit A-induced toxicity in rat primary hippocampus neurons (Behl
et al., 1994). Our results reinforced these ndings and found that
A2535 was highly toxic to SH-SY5Y cells as exemplied by the reduc-
tion in MTT values in a time- and concentration-dependent manner.
Further, A2535 signicantly induced cell apoptosis in SH-SY5Y cells,
as evidenced by morphological and biochemical alterations associated
with apoptosis that was condensed chromatin, reduced Annexin-V pos-
itive cells, decreased MMP, increased release of mitochondrial Cyto c,
and activation of caspase-3. However, these toxic effects induced by
A2535 were signicantly attenuated by pretreatment with IGF-1 in a
dose-dependent manner. Bcl-2 family is the best-characterized protein
family involved in the regulation of apoptosis, consisting of anti-apopto-
tic and pro-apoptotic members. It has been suggested that the anti-ap-
optotic members of this family, such as Bcl-XL, prevented apoptosis by
preventing the release of mitochondrial apoptogenic factors such as
Cyto c into the cytoplasm. In contrast, pro-apoptotic members of this
family, such as Bax, triggered the release of caspases and induced the re-
lease of mitochondrial apoptogenic factors into the cytoplasm (Gross,
2016). We showed that treatment with A2535 resulted in an increase
in Bax and decrease in Bcl-xL. Nevertheless, pretreatment with IGF-1
abrogated this effect of A in a dose-dependent manner. These data in-
dicate that IGF-1 could protect SH-SY5Y cells from A-induced
apoptosis.
The initial event leading to A-induced neurotoxicity is unknown at
present, but may involve induction of oxidative stress (Chen and Zhong,
2013). Accumulating data indicate that oxidative stress and A were
linked to each other since A aggregation induced oxidative stress in
vivo and in vitro (Bezprozvanny and Mattson, 2008; Harkany et al.,
2000), and oxidants increased the production of A (Misonou et al.,
2000; Paola et al., 2000). While A peptides favored Ca2+ inux into
neurons by inducing membrane-associated oxidative stress, rendering
neurons vulnerable to excitotoxicity and apoptosis (Bezprozvanny and
Mattson, 2008), oxidative stress may also be the cause of A accumula-
tion. Oxidant agents and oxidative products promoted APP expression,
and increased intracellular and secreted A levels in neuronal and
non-neuronal cells (Misonou et al., 2000; Paola et al., 2000). Excessive
ROS accumulation would cause damage to DNA and induce lipid perox-
idation, subsequently leading to cell death and apoptosis. In agreement
with previous ndings (Bezprozvanny and Mattson, 2008), our results
showed that treatment with A2535 resulted in a signicant increase
of intracellular ROS and MDA production. Furthermore, A2535 led to
a signicant decrease in the activity of SOD and CAT, two important en-
zymes involved in the degradation of ROS. IGF-1 has been demonstrated
 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332 29

Fig. 4. Nrf2-HO-1 pathway was involved in the protective effect of IGF-1 against A2535-induced neurotoxicity. A. Cells were incubated with various concentrations of IGF-1 for 24 h. B.
Cells were incubated with IGF-1 (100 ng/L) for the indicated time. C. Cells were pretreated with IGF-1 for 24 h prior to exposure to 25 M of A2535 for another 24 h. AC. Expression of
nuclear (Nuc) and cytoplasmic (Cyt) Nrf2 protein, as well as HO-1, was analyzed by Western blot. D. Immunoblot of whole protein extracts from control cells, cells expressing Nrf2 siRNA
and cells transfected with vector. E. MDA level was measured with a lipid peroxidation (MDA) assay kit. F. Representative image of immunoblots for cleaved caspase-3. Values were
presented as mean SD. *P b 0.05, **P b 0.01, compared to control; #P b 0.05, ##P b 0.01, compared to group treated with A2535 alone; &P b 0.05, &&P b 0.01, compared to group
treated with both A2535 and IGF-1.

to have antioxidant properties. IGF-1 could improve mitochondrial
function and reduce mitochondrial ROS caused by mutant huntingtin
(Ribeiro et al., 2014). IGF-1-decient mice were associated with in-
creased oxidative stress (Bailey-Downs et al., 2012). In accordance
with these data, we demonstrated that oxidative stress induced by
A2535 was markedly alleviated by pretreatment with IGF-1, as evi-
denced by decrease in intracellular ROS and MDA levels, and increase
in the activity of SOD and CAT. Together, these results support the
notion that IGF-1 protects against A2535-induced injury by eliminat-
ing oxidative stress in SH-SY5Y cells.
Nrf2 is a basic leucine zipper transcription factor that regulates the
expression of antioxidant proteins that protect against oxidative dam-
age triggered by various injuries and inammations (Lu et al., 2016).
Under physiological conditions, Nrf2 exists in the cytoplasm in an inac-
tive state as a consequence of binding to Keap 1. Once activated by
redox-dependent stimuli, Nrf2 translocates into the nucleus, where it
30 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332

Fig. 5. IGF-1 protected SH-SY5Y cells against A2535-induced oxidative stress and apoptosis via PI3K/Akt pathway. Cells were pretreated with 10 M of LY294002 or 20 M of U0126 for 1 h
prior to incubation with IGF-1 (100 ng/L) for 24 h, and then cells were incubated with A2535 (25 M) for another 24 h. A. Protein levels of Akt, ERK1/2, Nrf2 and HO-1 were detected by
Western blot. B. Intracellular ROS was evaluated by the oxidation of H2DCF-DA to DCF. C. MDA level was measured with a lipid peroxidation (MDA) assay kit. D. Apoptotic cells were
evaluated by ow cytometry. Annexin-V+ cells were considered apoptotic cells. E. Representative image of immunoblots for cleaved caspase-3. Values were presented as mean SD.
*P b 0.05, compared to control; #P b 0.05, compared to group treated with A2535 alone; &P b 0.05, compared to group treated with both A2535 and IGF-1.
 Z. Wang et al. / Experimental Gerontology 87 (2017) 2332
binds to ARE and initiates the transcription of antioxidative genes, such
as SOD, CAT and HO-1 (Jung and Kwak, 2010; Shah et al., 2007). The es-
sential role of Nrf2 in combating oxidative stress has been demonstrated
by the increased sensitivity of Nrf2/ mice to oxidative damage
(Johnson et al., 2008). Nrf2-disrupted mice were much more suscepti-
ble to toxicities mediated by environmental chemicals and stresses
than wild-type mice (Kang et al., 2012). So Nrf2/ARE system is consid-
ered one of the most important endogenous defenses against oxidative
stress.
In the present study, we showed that exposure of SH-SY5Y cells to
A2535 resulted in the nuclear translocation of Nrf2, and an increase
in the expression of its downstream gene HO-1. Similar ndings were
observed previously (Wang et al., 2010). Kim et al. reported that the ex-
pression of Nrf2 and HO-1 was also increased in oxidative insult induced
by 6-OHDA (Kim et al., 2012). It can be concluded from these observa-
tions that an increase in nuclear translocation of Nrf2 and expression
of its downstream gene induced by A2535 may represent the endoge-
nous antioxidant responses in the SH-SY5Y cells. Moreover, pretreat-
ment with IGF-1 could further promote Nrf2 translocation into the
nucleus, and increase the expression of HO-1 in A2535-treated cells.
Notably, the nuclear translocation of Nrf2, as well as the expression of
HO-1, was promoted by IGF-1 in a dose-dependent manner. In agree-
ment with our results, Nrf2-HO-1 pathway had been proven to be in-
volved in the protective effect of IGF-1 against 6-OHDA-mediated
apoptosis in PC12 cells (Kim et al., 2012). These data suggest that IGF-
1 could profoundly activate the endogenous antioxidant system in re-
sponse to A2535 insult. To further conrm that rf2-mediated antiox-
idant responses were involved in the protective effect of IGF-1, a specic
Nrf2 siRNA was constructed. As expected, Nrf2 siRNA signicantly
inhibited the expression of Nrf2 and its downstream gene HO-1. More
importantly, Nrf2 siRNA was sufcient to abolish the protective effect
of IGF-1 against A-induced oxidative stress and apoptosis, as evi-
denced by increased MDA and cleaved caspase-3 protein levels. These
results suggest that activation of Nrf2 may mediate, at least partly, the
protective effect of IGF-1 against A2535-induced cell death in SH-
SY5Y cells.
Studies have demonstrated that Nrf2-regulated antioxidant re-
sponse contributed to proliferative and mitogenic signals downstream
of Akt (Mitsuishi et al., 2012). Indeed, deletion in the phosphatase and
tens in homolog gene PTEN potentiated the induction of antioxidant
gene expression by Nrf2 (Mitsuishi et al., 2012). It has also been
shown that Nrf2 was a target of the MAPK, and ERK1/2 facilitated the
dissociation of Nrf2 from Keap1, which led to translocation of Nrf2
into the nucleus, thereby activating the Nrf2/ARE pathway (Nguyen et
al., 2004). Interestingly, it is well known that IGF-1 exerts its biological
function by binding to IGF-1 receptor and subsequently activates both
PI3K/Akt and MAPK/EKR signal pathways (O'Neill et al., 2012; Ren and
Anversa, 2015). Previous studies showed that both PI3K and MAPK
pathways were involved in the preventive effect of IGF-1 against apo-
ptosis induced by serum-deprivation in PC12 cells (Parrizas et al.,
1997). On the other hand, IGF-1 prevented A2535/H2O2-induced apo-
ptosis in lymphocytes via PI3K-dependent pathway (Jimenez Del Rio
and Velez-Pardo, 2006). In the present study, we conrmed that pre-
treatment with IGF-1 resulted in a signicant phosphorylation of both
Akt and ERK1/2. More importantly, we found that LY294002,a specic
inhibitor of PI3K, could abolish the nuclear translocation of Nrf2 and ex-
pression of HO-1 induced by IGF-1, while increasing intracellular ROS
generation. Moreover, LY294002 attenuated the protection of IGF-1 on
A2535-induced apoptosis, as evidenced by increased Annexin V-posi-
tive cells and cleaved caspase-3 protein levels. However, U0126, a spe-
cic inhibitor of MEK, had no effect on the protective effect of IGF-1
against A2535-evoked cell injury. These results indicate that IGF-1 in-
hibits A2535-induced oxidative stress and apoptosis in SH-SY5Y cells
via PI3K/Akt-Nrf2 pathway.
In conclusion, our ndings demonstrate that IGF-1 exerts a neuro-
protective effect against A2535-induced oxidative stress and apoptosis.
31
Activation of PI3K/Akt-Nrf2 signaling pathway contributes to this pro-
tective effects of IGF-1.
Funding source
This study was funded by Jinshan Municipal Population and Family
Planning Commission (Grant No. JSKJ-KTQN-2014-08) and Science
and Technology Commission of Shanghai Municipality (Grant No.
20164Y0193).
Disclosure
The authors declare that they have no conict of interest in the cur-
rent study.
Acknowledgments
We thank Prof. Tang Wei for her excellent advice in manuscript
writing.
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