Received: 30 June 2018 | Accepted: 27 August 2018
DOI: 10.1002/jcb.27698
RESEARCH ARTICLE
Daphnetin protects hippocampal neurons from oxygen‐
glucose deprivation–induced injury
Jin Zhi1 | Bin Duan2 | Jiwen Pei3
1
Department of Neurology, Xi’an NO.1
Hospital, Xi’an, China
2
Hemodialysis Centre of Nephrosis,
Shaanxi Provincial People’s Hospital,
Xi’an, China
3
Department of Traditional Chinese
Medicine, Xi’an City Hospital of TCM,
Xi’an, China
4
Department of Neurology, The Fourth
Hospital of Xi’an City, Xi’an, China
Correspondence
Junli Wei, Department of Neurology,
The Fourth Hospital of Xi’an City, No. 21
of Jiefang Road, Xi’an 710004, China.
Email: weijunli_neuro@163.com
1 | INTRODUCTION
Cerebral ischemia/reperfusion (I/R) is a medical
condition in which there is insufficient blood flow in
the brain followed by normal blood flow.1 Cerebral
I/R can lead to global cerebral ischemia and neuro-
logical dysfunction. It is associated with substantial
morbidity and mortality worldwide.1 Therefore, a
better understanding of the molecular mechanism is
helpful for the management of cerebral I/R injury. It
is well accepted that oxidative stress is one of the main
4132 | © 2018 Wiley Periodicals, Inc.
| Songdi Wu1 | Junli Wei4
Abstract
Daphnetin, a coumarin derivative extracted from Daphne odora var., was
reported to possess a neuroprotective effect. Recently, it has been demonstrated
that daphnetin attenuates ischemia/reperfusion (I/R) injury. However, the role
of daphnetin in cerebral I/R injury and the potential mechanism have not been
fully understood. The present study aimed to explore the regulatory roles of
daphnetin on oxygen‐glucose deprivation/reoxygenation (OGD/R)–induced cell
injury in a model of hippocampal neurons. Our results demonstrated that
daphnetin improved cell viability and reduced the lactate dehydrogenase
leakage in OGD/R–stimulated hippocampal neurons. In addition, daphnetin
inhibited oxidative stress and cell apoptosis in hippocampal neurons after OGD/
R stimulation. Furthermore, daphnetin significantly enhanced the nuclear
translocation of the nuclear factor erythroid 2‐related factor 2 (Nrf2) and heme
oxygenase‐1 (HO‐1) expression in hippocampal neurons exposed to OGD/R.
Knockdown of Nrf2 blocked the protective effect of daphnetin on OGD/
R–induced hippocampal neurons. In conclusion, these findings demonstrated
that daphnetin attenuated oxidative stress and neuronal apoptosis after OGD/R
injury through the activation of the Nrf2/HO‐1 signaling pathway in
hippocampal neurons. Thus, daphnetin may be a novel therapeutic agent for
cerebral I/R injury.
KEYWORDS
cerebral ischemia/reperfusion (I/R) injury, daphnetin, hippocampal neurons, neuroprotective,
Nrf2/HO‐1 pathway, oxidative stress
pathophysiological processes during I/R injury.2
Therapeutic strategies aimed at attenuating oxidative
stress are required for the prevention and treatment of
cerebral I/R injury.
Nuclear factor erythroid 2‐related factor 2 (Nrf2) is an
important transcription factor in the protection of cells
against oxidative damage.3 Activation of Nrf2 activates
the transcription of multiple antioxidant stress genes,
such as nicotinamide adenine dinucleotide phosphate
(NADPH) quinone oxidoreductase 1, glutamate‐cysteine
ligase, and heme oxygenase‐1 (HO‐1).4 Among these, the
wileyonlinelibrary.com/journal/jcb J Cell Biochem. 2019;120:4132-4139.
ZHI ET AL.
Nrf2/HO‐1 pathway has been well studied to play an
important neuroprotective role in cerebral I/R injury.5,6
Previous studies have demonstrated that the Nrf2/HO‐1
pathway is a potential target for alleviating the oxidative
damage during cerebral I/R injury.5,6
Daphnetin (7,8‐dihydroxycoumarin) is a natural
coumarin compound that possesses a variety of biological
activities such as anticancer,7 antioxidative,8 anti‐inflam-
matory,9 and neuroprotective effects.10 Daphnetin has
been found to protect the liver against I/R injury via
inhibiting the inflammation in an in vivo hepatic I/R
model.11 In addition, it was reported that daphnetin
exhibits neuroprotective and anti‐inflammatory effects on
cerebral I/R injury in mice.12 However, the mechanism of
the protective effect of daphnetin on cerebral I/R injury
has not been fully understood.
In this study, we investigated the effect of daphnetin on
cerebral I/R injury using a cell ischemia model in vitro.
We found that daphnetin protected hippocampal neurons
from I/R injury via regulating the Nrf2/HO‐1 pathway.
2 | MATERIALS AND MET HODS
2.1 | Cell culture
Ten Sprague‐Dawley rats (Charles River, Beijing Vital
River Laboratory Animal Technology Co., Ltd., China)
were used for the preparation of primary hippocampal
neurons as described previously.13 Briefly, cerebral
hippocampi were separated from brains and dissected
in HBS. After mechanical dissociation and trypsinization,
the cells were plated in dishes that were pretreated with
0.1% poly‐D‐lysine (Sigma‐Aldrich, St. Louis, MO). After
incubating for 2 hours, cells were cultured in neurobasal
medium supplemented with 2% B‐27, 1% penicillin‐
streptomycin, and 0.25% glumax for 7 days. Hippocampal
neurons were then purified by density gradient centrifu-
gation, and identified by neuron‐specific enolase immu-
nofluorescence (data not shown). All experiments were
carried out according to the National Institute of Health
Guide for the Care and Use of Laboratory Animals.
2.2 | Oxygen‐glucose deprivation/
reperfusion model and treatment
After 7 days, the primary hippocampal neurons were
subjected to oxygen‐glucose deprivation (OGD), through
incubating in serum and glucose‐free medium with 5%
CO2 and 95% N2 at 37°C for 3 hours. Then the cells were
incubated with normal medium containing serum and
glucose in normoxic conditions for 24 hours. For the
daphnetin treatment group, cells were pretreated with 10,
20, 40 μM daphnetin (≥97%, Sigma) for 12 hours before
| 4133
OGD/reperfusion (OGD/R) stimulation. The experiment
was repeated three times.
2.3 | Cell transfection
Small interfering RNA targeting Nrf2 (si‐Nrf2; Invitrogen,
Thermo Fisher Scientific, Waltham, MA) that can silence
the complementary target messenger RNA of Nrf2 was
transfected into the hippocampal neurons using Lipofec-
tamine 2000 (Invitrogen). After 24 hours, the transfection
efficiency was tested using Western blot analysis. The
experiment was repeated three times.
2.4 | Cell viability assay
Approximately 5 × 103 cells/well hippocampal neurons
were seeded in 96‐well plates and incubated for 12 hours
with or without the presence of daphnetin. Then, the
cells were treated with OGD/R stimulation, and then cell
viability was evaluated with the cell counting kit‐8 (CCK‐
8) (Dojindo Laboratories, Kumamoto, Japan) according
to the manufacturer’s instructions. Finally, the absor-
bance at 450 nm was measured with a microplate reader
(Bio‐Tek Instruments, Winooski, VT). The experiment
was repeated three times.
2.5 | Lactate dehydrogenase assay
The hippocampal neurons injury was assessed by
detecting the lactate dehydrogenase (LDH) activity
with a commercial LDH Cytotoxicity Assay Kit
(Jiancheng Bioengineering Institute, Nanjing, China).
Hippocampal neurons (5 × 103 cells/well) were plated
in 96‐well plates and subjected to OGD/R stimulation
with or without the presence of daphnetin. After
different treatments, 20 μL of cell supernatant was
collected for the detection of LDH activity according to
the manufacturer’s instructions. The absorbance was
measured spectrophotometrically at 450 nm using a
microplate reader (Bio‐Tek). The values are shown as
percentages of the total LDH. The experiment was
repeated three times.
2.6 | Measurement of intracellular
reactive oxygen species
Intracellular reactive oxygen species (ROS) was determined
using a fluorescence probe 2′,7′‐dichlorofluorescin‐diacetate
(DCFH‐DA; Sigma). As described by the manufacture’s
protocol, in the presence of ROS, DCFH‐DA can be
oxidized to the 2′,7′‐dichlorofluorescin, which is highly
fluorescent. After treatment, hippocampal neurons were
incubated with DCFH‐DA in the dark for 30 minutes. Then,
4134 | ZHI
the fluorescence intensity of the cells was analyzed using a
fluorospectrophotometer with the excitation/emission of
485/525 nm. The experiment was repeated three times.
FIGURE 1 Daphnetin ameliorates OGD/R–induced cell injury
in hippocampal neurons. A, Cell viability of hippocampal neurons
after incubation with daphnetin (0, 10, 20, 40, and 80 μM) for
48 hours. B, Cell viability of hippocampal neurons subjected to
OGD/R induction with or without the presence of daphnetin.
C, LDH release of hippocampal neurons subjected to OGD/R
induction with or without the presence of daphnetin. *P < 0.05
relative to control cells without OGD/R induction; #P < 0.05
relative to OGD/R–induced cells without daphnetin treatment.
LDH, lactate dehydrogenase; OGD/R, oxygen‐glucose deprivation/
reoxygenation
ET AL.
2.7 | Measurement of malondialdehyde,
superoxide dismutase, and glutathione
peroxidase
After treatments, the cell lysates of the hippocampal
neurons were prepared using cell lysis buffer (Beyotime
Biotechnology, Shanghai, China). The superoxide dis-
mutase (SOD) activity was measured using a SOD assay
kit with WST‐8 method (Beyotime). The O2‐ produced by
xanthine oxidase can react with WST‐8 to produce
formazan dye. The reaction can be blocked by SOD
because it can react with the O2‐; therefore, the
production of formazan dye is correlated with the SOD
activity. After incubation, the absorbance was measured
at 450 nm. The data are expressed as the fold activity
relative to the control. The measurement of malondial-
dehyde (MDA) level in cell lysates was measured using a
lipid peroxidation MDA assay kit (Beyotime). MDA can
react with thiobarbituric acid (TBA) in special conditions
to produce MDA‐TBA. The levels of MDA‐TBA can be
detected at 535 nm using a colorimetric method. The unit
of MDA concentration is nmol/mg protein.
The glutathione peroxidase (GPx) activity in cell
lysates was assessed by a reaction with the GPx detection
working solution from the kit (0.25 mM NADPH, 2.1 mM
reduced GSH, 0.5 unit/mL glutathione reductase, and
300 μM tert‐butyl hydroperoxide), in which the reduction
in NADPH absorbance was indicative of the GPx activity,
detected at 340 nm after an initial delay of 15 seconds and
monitored every 10 seconds for 1 minute at 25°C. The
data are expressed as the fold activity relative to the
control. The experiment was repeated three times.
2.8 | Cell apoptosis assay
The cell apoptosis of hippocampal neurons was analyzed
using an enzyme‐linked immunosorbent assay (ELISA)–
based cell death detection kit (Roche Molecular Bio-
chemicals, Mannheim, Germany) on the principle of the
quantification of apoptosis‐specific DNA fragmentation.
DNA fragmentation was measured by using a microplate
reader to measure the absorbance at 405 nm. The
experiment was repeated three times.
2.9 | Western blot assay
The cytosolic and nuclear proteins were prepared using a
celLytic nuclear extraction kit (Sigma). The protein concen-
tration was measured using a BCA Protein Assay Kit
(Thermo Fisher Scientific). After separation by 10% SDS‐
PAGE, the proteins were transferred to polyvinylidene
difluoride membranes (Thermo Fisher Scientific). The
membranes were blocked with 5% skim milk in tris‐buffered
ZHI ET AL. | 4135

saline containing 0.1% (v/v) Tween 20 (TBST) buffer for
1 hour at room temperature, and then incubated overnight at
4°C with the following antibodies: anti‐bax (1:1000 dilution;
Santa Cruz Biotechnology, Santa Cruz, CA), anti‐bcl‐2
(1:1,000 dilution, Santa Cruz), anti‐cleaved‐caspase‐3
(1:1000 dilution; Cell Signaling Technology, Boston, MA),
anti‐Nrf2 (1:500 dilution; Abcam, Cambridge, MA), anti‐HO‐
1 (1:500 dilution; Abcam), anti‐lamin B1 (1:1500 dilution;
Abcam), and anti‐GAPDH (1:1000 dilution; Santa Cruz). The
membranes were incubated with horseradish peroxidase–
conjugated secondary antibodies (1:3000 dilution; Cell
Signaling Technology) at room temperature for 1 hour. An
enhanced chemiluminescence detection system (Thermo
Fisher Scientific) was used to visualize the protein bands.
The intensity of the bands was analyzed using quantity one
software (Bio‐Rad, Hercules, CA). The experiment was
repeated three times.
2.10 | Statistical analysis
The quantitative data are shown as the mean ± SEM. A
one‐way analysis of variance followed by a Dunnett’s post
hoc test were applied for analyzing the multiple
comparisons. The statistical analysis was performed
using the SPSS 18.0 (SPSS, Chicago, IL), and the
statistical significance was set at P < 0.05.
3 | RE SU L TS
3.1 | Daphnetin ameliorates
OGD/R–induced neuron injury
We first evaluated the effect of daphnetin on the cell
viability of hippocampal neurons. CCK‐8 assay showed
that daphnetin did not affect cell viability until the
concentration up to 40 μM (Figure 1A). Thus, the
concentrations of 10, 20, 40 μM were selected for
the following study. Next, we investigated the role of
daphnetin in the OGD/R–induced neuron injury.
Figure 1B shows that daphnetin inhibited OGD/
R–induced inhibition of hippocampal neurons viability
in a dose‐dependent manner. Figure 1C revealed that
daphnetin dose‐dependently reduced OGD/R–induced
LDH release in hippocampal neurons.

FIGURE 2 Daphnetin represses OGD/R–induced oxidative stress in hippocampal neurons. Hippocampal neurons were subjected to
OGD/R induction with or without the presence of daphnetin. The oxidative stress was evaluated through detecting the levels of ROS and
MDA, as well as the activities of SOD and GPx. A, ROS production. B, MDA production. C, SOD activity. D, GPx activity. *P < 0.05 relative to
control cells without OGD/R induction; #P < 0.05 relative to OGD/R–induced cells without daphnetin treatment. GPx, glutathione
peroxidase; MDA, measurement of malondialdehyde; OGD/R, oxygen‐glucose deprivation/reoxygenation; ROS, reactive oxygen species;
SOD, superoxide dismutase
4136 | ZHI
FIGURE 3 Daphnetin suppresses OGD/R–induced cell
apoptosis in hippocampal neurons. A, The cell apoptosis of
hippocampal neurons was analyzed by the ELISA‐based cell death
detection kit. B, The expressions of apoptotic related proteins
including bax, bcl‐2, and cleaved caspase‐3 were measured by
Western blot analysis. *P < 0.05 relative to control cells without
OGD/R induction; #P < 0.05 relative to OGD/R–induced cells
without daphnetin treatment. ELISA, enzyme‐linked
immunosorbent assay; OGD/R, oxygen‐glucose deprivation/
reoxygenation
3.2 | Daphnetin represses
OGD/R–induced oxidative stress
in hippocampal neurons
The effect of daphnetin on OGD/R–induced oxidative
stress was investigated by detecting the levels of ROS and
MDA, as well as the activities of SOD and GPx. ROS and
MDA production was significantly increased in the OGD/
R–induced cells relative to the control cells. However, the
ET AL.
induction caused by OGD/R was markedly inhibited
by daphnetin in a dose‐dependent manner (Figures 2A
and 2B). The activities of SOD and GPx were lower than
that in the control cells. Pretreatment with daphnetin
increased the activities of SOD and GPx when compared
with the OGD/R–induced cells (Figures 2C and 2D).
3.3 | Daphnetin suppresses
OGD/R–induced cell apoptosis
in hippocampal neurons
We next tested whether daphnetin affects the cell apoptosis
of hippocampal neurons after OGD/R stimulation with an
ELISA‐based cell death detection kit. Figure 3A shows that
the percentage of apoptotic cells was higher in the OGD/
R–induced cells as compared to the normal cells. After
daphnetin pretreatment, the percentage of apoptotic cells
was decreased relative to the OGD/R‐induced cells. More-
over, we detected the expressions of bax, bcl‐2, and cleaved
caspase‐3 with Western blot analysis. Figure 3B shows that
OGD/R induction caused significant increases in the
expressions of bax and cleaved caspase‐3, while it led to a
reduction in the expression of bcl‐2. Daphnetin pretreat-
ment mitigated the changes in the expressions of bax, bcl‐2,
and cleaved caspase‐3, which were caused by OGD/R
induction.
3.4 | Daphnetin induces the activation
of Nrf2/HO‐1 pathway in hippocampal
neurons induced by OGD/R
To explore the effect of daphnetin on the activation of the
Nrf2/HO‐1 pathway, the expressions of Nrf2 in nuclear
fraction, and the HO‐1 in the whole cell lysates were
measured by Western blot analysis. As indicated in
Figure 4A, the expression of Nrf2 nucleus was increased
in the OGD/R–induced hippocampal neurons, indicating
that OGD/R slightly induced the nuclear translocation of
Nrf2. The HO‐1 expression in the whole cell lysates was
increased in the OGD/R–induced hippocampal neurons
compared with the control cells. However, daphnetin
elevated the nuclear translocation of Nrf2 and HO‐1
expression relative to the OGD/R–induced hippocampal
neurons.
To further evaluate the role of the Nrf2/HO‐1 path-
way, the Nrf2 was silenced by si‐Nrf2. Western blot
analysis showed that the expressions of Nrf2 and HO‐1
were dramatically decreased after transfection with
si‐Nrf2 (Figure 4B). Si‐Nrf2 attenuated the inhibitory
effects of daphnetin on cell viability, ROS production,
and cell apoptosis (Figures 4C‐E). The results indicated
that the Nrf2/HO‐1 pathway was implicated in the
neuroprotective effects of daphnetin.
ZHI ET AL. | 4137

FIGURE 4 Daphnetin induces the activation of the Nrf2/HO‐1 pathway in OGD/R OGD/R–induced hippocampal neurons. A, The
expressions of Nrf2 and HO‐1 were measured by Western blot analysis. B, Transfection efficiency was tested using Western blot analysis
after transfection with si‐Nrf2 or control siRNA (si‐con). C, Cell viability. D, ROS production. E, Cell apoptosis. *P < 0.05 relative to control
cells without OGD/R induction; #P < 0.05 relative to OGD/R–induced cells without daphnetin treatment; &P < 0.05 relative to
OGD/R–induced cells transfected with si‐con. HO‐1, heme oxygenase‐1; Nrf2, nuclear factor erythroid 2‐related factor 2; OGD/R,
oxygen‐glucose deprivation/reoxygenation; ROS, reactive oxygen species; si‐Nrf2, small interfering RNA targeting Nrf2

4 | DISCUSSION oxidative‐related pathway.14 Reperfusion of the is-

I/R injury is a pathological phenomenon in which
the blood supply returns to normal levels after a period
of ischemia.14 Ischemia leads to the lack of oxygen
and nutrients, and the following restoration of circula-
tion results in the cell injury or death through the
chemic tissues produces a large amount of reactive
oxygen and nitrogen species, leading to redox signaling
disruption and permanent mitochondrial lesion, and
finally resulting in cell damage and apoptosis.15
Oxidative stress has been considered to be a major
contributor to I/R injury. Accumulating evidence
4138 | ZHI
proves that inhibition of oxidative stress may contribute
to the attenuation of I/R injury.16,17 In the present
study, we used an OGD/R model to simulate cerebral
I/R injury in primary hippocampal neurons. As
expected, OGD/R stimulation caused cytotoxicity,
oxidative stress, and apoptosis in hippocampal neurons.
Daphnetin is a coumarin derivative extracted from
Daphne odora var., which is a traditional Chinese
medicine commonly used for diminishing inflammation.
Daphnetin has been demonstrated to have strong anti‐
inflammatory and antioxidative properties. Yang et al10
reported that daphnetin prevents N‐methyl‐D‐aspartate
induced cell loss and apoptosis in primary cultured
cortical neurons. Daphnetin significantly increases neur-
ite outgrowth and promotes neuronal survival in primary
cultured rat cortical neurons.18 Daphnetin attenuates
hydrogen peroxide‐induced cell apoptosis and improves
cell morphology in neuronal‐like rat pheochromocytoma
PC12 cells.8 These findings suggest that daphnetin has
neuroprotective effects. In addition, daphnetin has been
proved to prevent cerebral ischemic injury in mice.10
In a middle cerebral artery occlusion and reperfusion
(MCAO/R) model, daphnetin treatment improved neu-
rological score and infarct size in mice.12 Daphnetin also
inhibited the inflammation response and neural cells
apoptosis in MCAO/R mice.12 In another in vivo study,
daphnetin reduces infarct volume and improves neuro-
logical deficits in MCAO/R mice.19 Besides, daphnetin
also reduces infarct volume in a hypoxia/ischemia
neonatal rat model.19 Additional studies indicate that
daphnetin executes its neuroprotective effect via elevat-
ing cellular antioxidative activity.19 In accordance with
previous studies, in the current study, we found that
daphnetin attenuated OGD/R–caused oxidative stress,
cell damage, and apoptosis in hippocampal neurons,
which suggested that daphnetin might ameliorate cere-
bral I/R injury.
Nrf2 is a basic leucine zipper transcription factor that
controls the expressions of many proteins including
antioxidant proteins, drug transporters, detoxifying en-
zymes, and numerous cytoprotective proteins.20 Nrf2 is
normally bound to Kelch‐like erythroid Cap “n” collar
homologue (ECH)‐associated protein 1 (Keap1), which is a
cytoskeleton binding protein.3 Under specific conditions,
the Nrf2‐Keap1 complex is disrupted. Then the dissociative
Nrf2 translocates to the nucleus, and binds to the promoter
sequence “antioxidant responsive element (ARE),” thereby
leading to the upregulation of target genes. The Nrf2/HO‐1
pathway is a crucial pathway that is involved in oxidative
stress.20 Daphnetin exposure suppresses tert‐butyl hydro-
peroxide‐induced oxidative damage and cell apoptosis.21
Daphnetin upregulated the Keap1‐Nrf2/ARE signaling
pathway, and the protective effects are mostly blocked
ET AL.
by Nrf2 knockout.21 Mohamed et al22 report that daphneti
n induces the nuclear translocation of Nrf2, thereby
inducing the expression and activity of HO‐1. These results
suggest that daphnetin ameliorates oxidative injury
via activating the Nrf2/HO‐1 pathway.22 Our results
showed that daphnetin enhanced the activation of the
Nrf2/HO‐1 pathway in OGD/R–induced hippocampal
neurons. We further demonstrated that the Nrf2/HO‐1
pathway was implicated in the neuroprotective effects of
daphnetin. However, the molecular mechanism of activat-
ing the Nrf2/HO‐1 pathway in response to daphnetin
pretreatment remains unclear, and further investigation is
needed.
In summary, we explored the role of daphnetin in an
in vitro OGD/R model in primary hippocampal
neurons. The results showed that daphnetin attenuated
OGD/R–caused oxidative stress, cell damage, and apop-
tosis in hippocampal neurons. Daphnetin enhanced the
activation of the Nrf2/HO‐1 pathway in OGD/R–induced
hippocampal neurons. Transfection with si‐Nrf2 abol-
ished the protective effect of daphnetin. These data
indicated that daphnetin protected hippocampal neurons
from OGD/R–induced cell damage through the activation
of the Nrf2/HO‐1 pathway.
CON F L I C T S OF I N T E RE S T
The authors declare that they have no conflicts of
interest.
ORCID
Junli Wei http://orcid.org/0000-0002-3759-5015
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How to cite this article: Zhi J, Duan B, Pei J,
Wu S, Wei J. Daphnetin protects hippocampal
neurons from oxygen‐glucose deprivation–induced
injury. J Cell Biochem. 2019;120:4132‐4139.
https://doi.org/10.1002/jcb.27698