Neuroprotective effect of ligustilide against ischaemia-reperfusion injury via up-regulation of erythropoietin and down-regulation of RTP801.
ABSTRACT Ligustilide, the main lipophilic component of Danggui, has been reported to protect the brain against ischaemic injury. However, the mechanisms are unknown. Here, we investigated the roles of erythropoietin (EPO) and the stress-induced protein RTP801 in neuroprotection provided by ligustilide against ischaemia-reperfusion (I/R) damage to the brain.
The efficacy of ligustilide against I/R damage was assessed by neurological deficit, infarct volume and cell viability, using the middle cerebral artery occlusion model in rats in vivo and rat cultured neurons in vitro. EPO and RTP801 were analysed by Western blot. Over-expression of RTP801 was achieved by transfection of an expression plasmid.
Ligustilide decreased the neurological deficit score, infarct volume and RTP801 expression and increased EPO transcription in I/R rats, and increased cell viability and EPO and decreased LDH release and RTP801 in I/R neurons. Also, ligustilide increased ERK phosphorylation (p-ERK). The positive effects of ligustilide on p-ERK, cell viability and EPO were blocked by PD98059, but not LY294002 and SB203580. In addition, transfection of SH-SY5Y cells with RTP801 plasmid increased RTP801 and LDH release, while ligustilide inhibited the effects of transfection on RTP801 expression and also increased cell viability.
Ligustilide exerts neuroprotective effects against I/R injury by promoting EPO transcription via an ERK signalling pathway and inhibiting RTP801 expression, This compound could be developed into a therapeutic agent to prevent and treat ischaemic disorders.
- [show abstract] [hide abstract]
ABSTRACT: Ginkgo biloba extracts are now prescribed in several countries for their reported health benefits, particularly for medicinal properties in the brain. The standardized Ginkgo extract, EGb761, has been reported to protect neurons against oxidative stress, but the underlying mechanisms are not fully understood. To characterize the oral consumption of EGb761 in transient ischemia, we performed the middle cerebral artery occlusion (MCAO) filament model in wild-type and heme oxygenase 1 (HO-1) knockouts. Mice were pretreated for 7 days before the transient occlusion or posttreated acutely during reperfusion; then neurobehavioral scores and infarct volumes were assessed. Furthermore, primary cortical neuronal cultures were used to investigate the contribution of the antioxidant enzyme HO-1 in the EGb761-associated cytoprotection. Mice that were pretreated with EGb761 had 50.9+/-5.6% less neurological dysfunction and 48.2+/-5.3% smaller infarct volumes than vehicle-treated mice; this effect was abolished in HO-1 knockouts. In addition to the prophylactic properties of EGb761, acute posttreatment 5 minutes and 4.5 hours after reperfusion also led to significant reduction in infarct size (P<0.01). After our previous demonstration that EGb761 significantly induced HO-1 levels in a dose- and time-dependent manner in neuronal cultures, here we revealed that this de novo HO-1 induction was required for neuroprotection against free radical damage and excitotoxicity as it was significantly attenuated by the enzyme inhibitor. These results demonstrate that EGb761 could be used as a preventive or therapeutic agent in cerebral ischemia and suggest that HO-1 contributes, at least in part, to EGb761 neuroprotection.Stroke 10/2008; 39(12):3389-96. · 6.16 Impact Factor
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ABSTRACT: FOXO transcription factors are important regulators of cell survival in response to a variety of stress stimuli, among which are oxidative stress, DNA damage, and nutrient deprivation. Here we report a role for FOXO3a under conditions of hypoxic stress. In response to hypoxia, FOXO3a transcript levels accumulate in an HIF1-dependent way, resulting in enhanced FOXO3a activity. We show that transcription of CITED2, a transcriptional cofactor that functions in a negative feedback loop to control HIF1 activity, is induced by FOXO3a during hypoxia. In fibroblasts as well as in breast cancer cells, FOXO3a inhibits HIF1-induced apoptosis by stimulating the transcription of CITED2, which results in reduced expression of the proapoptotic HIF1 target genes NIX and RTP801. Thus, by fine-tuning HIF1 activity, FOXO3a plays an important role in the survival response of normal and cancer cells in response to hypoxic stress.Molecular Cell 01/2008; 28(6):941-53. · 15.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Radix Angelica sinensis, known as Danggui in Chinese, has been used to treat cardiovascular and cerebrovascular diseases in Traditional Chinese Medicine for a long time. Modern phytochemical studies showed that Z-ligustilide (LIG) is the main lipophilic component of Danggui. In this study, we examined whether LIG could protect ischemia/reperfusion-induced brain injury by minimizing oxidative stress and anti-apoptosis. Transient forebrain cerebral ischemia (FCI) was induced by the bilateral common carotid arteries occlusion for 30 min. LIG was intraperitoneally injected to ICR mice at the beginning of reperfusion. As determined via 2,3,5-triphenyl tetrazolium chloride (TTC) staining at 24 h following ischemia, the infarction volume in the FCI mice treated without LIG (22.1 +/- 2.6%) was significantly higher than that in the FCI mice treated with 5 mg/kg (11.8 +/- 5.2%) and 20 mg/kg (2.60 +/- 1.5%) LIG (P < 0.05 or P < 0.01). LIG treatment significantly decreased the level of malondialdehyde (MDA) and increased the activities of the antioxidant enzyme glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in the ischemic brain tissues (P < 0.05 or P < 0.01 vs. FCI group). In addition, LIG provided a great increase in Bcl-2 expression as well as a significant decrease in Bax and caspase-3 immunoreactivities in the ischemic cortex. The findings demonstrated that LIG could significantly protect the brain from damage induced by transient forebrain cerebral ischemia. The antioxidant and anti-apoptotic properties of LIG may contribute to the neuroprotective potential of LIG in cerebral ischemic damage.Brain Research 09/2006; 1102(1):145-53. · 2.88 Impact Factor
of ligustilide against
injury via up-regulation
of erythropoietin and
down-regulation of RTP801
Xiao-mei Wu1,2,3, Zhong-ming Qian2,3, Li Zhu3, Fang Du1,
Wing-ho Yung1, Qi Gong1and Ya Ke1
1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong,
Shatin, NT, Hong Kong,2Laboratory of Neuropharmacology and Department of Neurosurgery,
South-west Hospital, The Third Military Medical University, Chongqing, China, and3Institute for
Nautical Medicine and Jiangsu Key Laboratory of Neuroregeneration, Nantong University,
Ya Ke, School of Biomedical
Sciences, Faculty of Medicine,
The Chinese University of Hong
Kong, Shatin, N.T., Hong Kong.
Zhong-ming Qian, Laboratory of
Department of Neurosurgery,
Southwest Hospital, The Third
Military Medical University,
Chongqing 400030, China.
ischaemia-reperfusion in vivo and
in vitro; erythropoietin; RTP801;
neurological deficit score; infarct
volume; cell viability; ERK
5 October 2010
15 February 2011
24 February 2011
BACKGROUND AND PURPOSE
Ligustilide, the main lipophilic component of Danggui, has been reported to protect the brain against ischaemic injury.
However, the mechanisms are unknown. Here, we investigated the roles of erythropoietin (EPO) and the stress-induced
protein RTP801 in neuroprotection provided by ligustilide against ischaemia-reperfusion (I/R) damage to the brain.
The efficacy of ligustilide against I/R damage was assessed by neurological deficit, infarct volume and cell viability, using the
middle cerebral artery occlusion model in rats in vivo and rat cultured neurons in vitro. EPO and RTP801 were analysed by
Western blot. Over-expression of RTP801 was achieved by transfection of an expression plasmid.
Ligustilide decreased the neurological deficit score, infarct volume and RTP801 expression and increased EPO transcription in
I/R rats, and increased cell viability and EPO and decreased LDH release and RTP801 in I/R neurons. Also, ligustilide increased
ERK phosphorylation (p-ERK). The positive effects of ligustilide on p-ERK, cell viability and EPO were blocked by PD98059, but
not LY294002 and SB203580. In addition, transfection of SH-SY5Y cells with RTP801 plasmid increased RTP801 and LDH
release, while ligustilide inhibited the effects of transfection on RTP801 expression and also increased cell viability.
CONCLUSION AND IMPLICATIONS
Ligustilide exerts neuroprotective effects against I/R injury by promoting EPO transcription via an ERK signalling pathway and
inhibiting RTP801 expression, This compound could be developed into a therapeutic agent to prevent and treat ischaemic
EPO, erythropoietin; I/R, ischaemia-reperfusion; MCAO, middle cerebral artery occlusion; OGD, oxygen-glucose
deprivation; p-ERK, phosphorylated ERK; REDD1 (regulated in development and DNA damage responses 1)
British Journal of
332British Journal of Pharmacology (2011) 164 332–343
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
Radix Angelicae Sinensis, known as Danggui in Chinese, is
the root of Angelica Sinensis (Oliv.) Diels (Umbelliferae). It is a
popular traditional Chinese medicinal herb which has long
been used as a medicinal plant and is included in a number of
traditional Sino-Japanese herbal prescriptions (Hou et al.,
2004; Rhyu et al., 2005). The chemical constituents of the
Danggui extract are classified into essential oil and water
soluble components (Huang et al., 2004)and the essential oil
is believed to contain its main pharmacologically active com-
pounds (Huang and Song, 2001)and the major active agent is
ligustilide (Gijbels et al., 1982; Naito et al., 1992).
Ligustilide protected PC12 cells against injury-induced by
hydrogen peroxide (Yu et al., 2008), and also the brain from
damage induced by transient forebrain cerebral ischaemia in
male ICR mice (Swiss Hauschka strain) (Kuang et al., 2006)
and by permanent focal ischaemia in rats (Peng et al., 2007).
It has been suggested that the antioxidant and anti-apoptotic
properties of ligustilide might contribute to its neuroprotec-
tive role in cerebral ischaemic damage (Kuang et al., 2006)
and hydrogen peroxide-induced cell injury (Peng et al.,
2007). However, the precise molecular mechanisms underly-
ing the neuroprotective effect of ligustilide have not been
In the present study, we first tested whether ligustilide has
a protective effect against the damage induced by ischaemia-
reperfusion (I/R) in the cerebral circulation. Then we investi-
erythropoietin (EPO) and the stress-induced protein RTP801
(also known as REDD1, regulated in development and DNA
damage responses 1) in I/R rats in vivo and I/R neurons in
vitro. EPO was chosen in this study as a potential target of
ligustilide because the increased transcription of EPO, pro-
moted by hypoxia-inducible factor-1a (HIF-1a), plays a domi-
nant role in neuroprotection in ischaemic stroke and
intracerebral haemorrhage (Rabie and Marti, 2008; van der
Kooij et al., 2008; Fan et al., 2009). RTP801 was also measured
because this novel protein, the product of the Ddit4 gene, was
strongly up-regulated by hypoxia in vitro and in vivo (Shos-
hani et al., 2002) and an increase in RTP801 appeared to be
toxic to non-dividing neuron-like PC12 cells and increased
their sensitivity to ischaemic injury (Shoshani et al., 2002).
We proposed that ligustilide might increase transcription
of EPO, an endogenous protective factor, and might inhibit
expression of RTP801, a detrimental factor. We found that the
protection by ligustilide of the brain in vivo and neurons in
vitro, from damage induced by I/R was mediated by
up-regulation of EPO and down-regulation of RTP801, sup-
porting our original hypothesis. In addition, we demon-
strated that the increased EPO transcription induced by
ligustilide was mediated by activation of the ERK pathway.
All animal care and experimental procedures complied with
the guidelines developed by the Health Department of Local
Government and Experimentation Ethics Committee of the
Universities and approved by the Department of Health of
the Hong Kong Government and the Animal Ethics Commit-
tee of The Chinese University of Hong Kong. Male Sprague-
Dawley rats (3-month-old, 250 to 270 g, n = 51) were
provided by the Animal Centers of The Chinese University of
Hong Kong and Nantong University. The animals were
housed in pairs in stainless steel cages at 21 ? 2°C and had
free access to food and water. Rooms were in a cycle of 12 h
of light (7:00 to 19:00) and 12 h of darkness (from 19:00 to
Pharmacological treatments and
Danggui was purchased from the Danggui Cultivating Base of
Good Agricultural Practice in Nin Xian County, Gansu Prov-
ince, China and ligustilide was prepared by a well-established
procedure as described previously (Qian et al., 2005). Its
purity was over 98% based on the percentage of total
peak area in the high performance liquid chromatography
To test whether ligustilide has a protective effect against
the damage induced by I/R, this compound (suspended in
0.5% sodium carboxymethylcellulose) was administered by
oral gavage at a dose of 20, 40 or 80 mg·kg-1, at 3 and 0.5 h
before the middle cerebral artery occlusion (MCAO) proce-
dure, according to Zhang et al. (2009). Rats in the sham group
received volume-matched vehicle. Nimodipine was used as a
positive control as it is known that nimodipine pretreatment
attenuated infarct volumes after MCAO, by reducing Ca2+
entry (Mcculloch, 1992). Nimodipine in 0.5% sodium car-
boxymethylcellulose solution was given by oral gavage once
at a dose of 12 mg·kg-1at 1 h prior to onset of the ischaemia.
To investigate the effect of ligustilide on cell viability and
LDH in cultured neurons, ligustilide was freshly prepared as a
stock solution in dimethyl sulphoxide and diluted with neu-
ronal culture medium before the experiments. Neurons were
pretreated with ligustilide (0, 0.625, 1.25, 2.5, 5, 10 or
20 mmol·L-1) for 2 h before being exposed to oxygen-glucose
deprivation (OGD) for 4 h (Peng et al., 2007). The concentra-
tion of dimethyl sulphoxide in the final culture medium was
0.1% (v/v) which had no effect on cell viability.
To study the effects of ligustilide on EPO and RTP801
expression, neurons were pretreated with ligustilide (0, 1.25,
5 and 10 mmol·L-1) before exposing to OGD. The effects of
inhibitors of PI3K, ERK and p38 MAPK were investigated by
pre-incubating neurons with 0 or 50 mmol·L-1of LY294002, 0
or 25 mmol·L-1of PD98059, or 0 or 50 mmol·L-1of SB203580
for 1 h, respectively, before being treated with 5 mmol·L-1of
ligustilide for 2 h followed by OGD.
The rats were randomized to treatment or placebo group.
The investigators who induced the stroke, analysed the
infarct volumes and assessed the neurological deficits were
unaware of the group identity of each animal.
Ischaemia-reperfusion model in vivo and
determination of neurological deficit and
Ischaemia-reperfusion was induced in rats by MCAO followed
by reperfusion as described by Saleem et al. (2008). Three out
of a total of 45 rats subjected to MCAO surgery died before
Ligustilide and neuroprotection
British Journal of Pharmacology (2011) 164 332–343 333
the endpoint of reperfusion. Post-mortem examination
revealed that these were caused by trauma, such as internal
bleeding, caused during the surgical procedure and these
animals were therefore excluded from any data analysis. The
animals were fasted overnight but allowed free access to
water. They were then anesthetized with chloral hydrate
(400 mg·kg-1, i.p.). Throughout the surgery, rectal tempera-
ture was maintained at 37°C by a heating pad. A 4-0 silicon-
coated monofilament nylon suture with a round tip was
advanced from the right external carotid artery into the
lumen of the internal carotid artery to occlude the origin of
the middle cerebral artery and maintained for 2 h. Reperfu-
sion was introduced by withdrawing the monofilament after
occlusion. As a control, sham-operated rats underwent iden-
tical surgery but did not have the suture inserted. After recov-
ery from anaesthesia, the rats were returned to their cage with
free access to water and food. Twenty-four hours after reper-
fusion, rats were scored for neurological function based on a
modified Longa EZ test, which employs a six-point scale
(Longa et al., 1989) with some modifications: 0 = no apparent
deficits; 1 = failure to extend left forepaw fully; 2 = circling to
the left; 3 = falling to the left; 4 = did not walk spontaneously
and had a depressed level of consciousness; 5 = death (exclud-
ing causation in operation).
The brains of rats were then removed, sliced coronally
into five 2 mm thick sections starting at 1 mm from
thefrontal pole,and incubated
triphenyltetrazolium chloride for 30 min at 37°C followed by
4% formaldehyde solution overnight. Infarct volume was
then measured as described previously (Kuang et al., 2006).
The normal area of brain was stained dark red based on intact
mitochondrial function whereas infarct tissue remained
unstained (white). The infarct area from each section was
measured using the Pro-plus 6.0 image analysis software
(Image J, Bethesda, MD, USA). The total volume of infarction
was calculated as the sum of the infarct areas (all sections) ¥
thickness (2 mm). The infarct volume was expressed as a
percentage of contralateral hemisphere.
Neuronal culture and a model of
oxygen-glucose deprivation (OGD) in vitro
The primary cortical neuronal culture was prepared using a
method described by Ho et al. (2003) and Wu et al. (2009). In
brief, the cortex was aseptically removed from 15–16 day-old
mouse embryos, minced with sterile surgical blades, incu-
bated in 0.125% trypsin and dissociated by trituration in
DNase/trypsin inhibitor solution. The cortical neurons were
suspended in complete DMEM containing 2.5 mmol·L-1
glucose and 10% fetal bovine serum, and plated in poly-L-
lysine-coated six-well or 96-well plates (Corning) at a density
of 2.0 ¥ 105cells·cm-2. Cultures were kept in an incubator at
5% CO2(NAPCO 5400) at 37°C. The medium was replaced by
Neurobasal medium with supplemental B27 (Invitrogen) 4 h
after cells were seeded. After 5 days in culture, purity of the
neurons was assessed by staining with neuron-specific anti-
body against microtubule associated protein 2 (Millipore). In
our case, over 98% of cells were positively stained. The cul-
tures were therefore used for the following experiments.
To investigate the effect of ligustilide, cultured neurons
were pretreated with this compound for 2 h before being
exposed to OGD. OGD was achieved by placing cells in
DMEM without glucose and in a dedicated chamber (NAPCO
7101FC-1) with 1% O2, 94% N2and 5% CO2for 4 h at 37°C as
previously described (Wu et al., 2009). After OGD, neurons
were incubated with the original medium in a normoxic
incubator for 24 h.
Assessment of cell viability
The cell viabilities were measured using a MTT (3-(4,5-
assay as described previously (Du et al., 2008; He et al., 2008).
Briefly, a total of 25 mL MTT (1 g·L-1in PBS) was added to each
well before the conduction of incubation at 37°C for 4 h. The
reaction was stopped by the addition of a 100 mL lysis buffer
(20% SDS in 50% N’Ndimethylformamide, pH 4.7). Optical
density was measured at the 570 nm wavelength by the use of
an ELX-800 microplate assay reader (Bio-tek, USA) and the
results were expressed as a percentage of absorbance mea-
sured in the control cells.
The intracellular enzyme LDH leaks into the culture medium
when cell membranes are damaged (Zhu et al., 2007) and the
quantity of LDH (unit·mL-1·min-1) released into the medium
was determined by the decrease in absorbance at 340 nm for
NADH disappearance within 3 min (Zhu et al., 2008). Briefly,
the cells were treated, 500 mL of supernatant was then
collected from each well and mixed with 1.3 mL of
NADH (0.217 mmol·L-1) and 1.3 mL of sodium pyruvate
(1.77 mmol·L-1) in the modified Krebs-Henseleit buffer
(118 mmol·L-1NaCl, 4.8 mmol·L-1KCl, 1 mmol·L-1KH2PO4,
NaHCO3, 3 mmol·L-1
MgPO4, pH 7.4) for 30 s at 37°C. The activity was spectropho-
tometrically measured (Mod-756) at optical density of
CaCl2, 0.8 mmol·L-1
After being deeply anesthetized, the rats were transcardially
perfused with normal saline solution, followed by 4%
paraformaldehyde in 0.1 M PBS 24 h after I/R. Brains were
removed and post-fixed in 4% paraformaldehyde for 4 h,
then transferred into 30% sucrose solution until the brains
sank to the bottom of the container. Coronal sections
(25 mm) were made using a Leica CM3050S cryostat (Leica
Microsystems, Germany). Sections were blocked with 3%
normal goat serum (diluted in PBS containing 0.3% Triton
X-100) for 1 h and incubated with primary antibodies (anti-
EPO and anti-RTP801, 1:200) overnight at 4°C. After rinsing
with PBS, sections were incubated with FITC-conjugated
donkey anti-goat IgG (for RTP801) or Alexa 586-conjugated
goat anti-rabbit IgG (for EPO) as secondary antibodies (1:200)
for 2 h at room temperature. The specificity of antibody was
tested using a negative control which was performed by
adding only second antibody without primary antibody pre-
incubation in brain slices. The immunofluorescence images
from at least four fields each from three rats were examined
using the same brightness and exposure settings. Image-Pro
Plus software (Media Cybernetics, Silver Spring, MD, USA)
was used to analyse the intensity of immunofluorescence for
EPO or RTP801 in each photograph. All the sections were
X Wu et al.
334British Journal of Pharmacology (2011) 164 332–343
stained within the same immunohistochemistry run and the
results were shown as the percentage of the control.
The pcDNA3.1-RTP801 construct was prepared by subcloning
the open reading frame of human RTP801 cDNA (Genebank,
NM_019058) into the HidIII/XhoI sites of expression plasmid
pcDNA3.1 (Invitrogen Co., Carlsbad, CA, USA). The cDNA
fragment was obtained by PCR amplification using RTP801-
specific primer 5′-CCCAAGCTTATGCCTAGCCTTTGGGAC-3′
and 5′-CCGCTCGAGTCAACACTCCTCAATGAG-3′ with the
added HindIII and XhoI restriction sites (indicated by under-
lines respectively). SH-SY5Y cells grown to 80% confluence in
24-well plates were transfected with 0.8 mg of pcDNA3.1-
RTP801 plasmid DNA using Lipofectamine 2000 reagent. The
empty vector was used as a control.
Western blot analysis
Neurons receiving different treatments were washed with ice-
cold PBS, homogenized in ice-cold lysis buffer [50 mM Tris-
HCl (pH 7.4), 1 mM EDTA, 1% SDS, 1% Nonide P-40, 1 mM
Na3VO4, 1 mM NaF, 5% b-mercaptoethanol, 400 uM phenyl-
methysulphonyl fluoride, 2 mg·mL-1each of pepstatin, apro-
tinin and leupeptin]. After centrifugation at 12 000¥ g for
10 min at 4°C, the supernatant was collected, and protein
concentration in the extracts was determined by Bradford
assay kit (Bio-Rad). Aliquots of the cell extract containing
equal amounts of protein was boiled in a protein loading
buffer for 5 min, separated on a 10% SDS-polyacrylamide
gels, and transferred to PVDF membranes. Transfer mem-
branes were blocked with 5% non-fat milk in Tris-buffered
saline for 1 h and incubated in primary antibodies (anti-EPO
and anti-RTP801, 1:500; anti-ERK42/44 and phospho-ERK42/
44, 1:5000) overnight at 4°C (He et al., 2008; Li et al., 2008).
Then, the membranes were washed three times and incu-
bated with appropriate secondary antibodies (1:2000) for 2 h
followed by three washes. Signal detection was performed
with an enhanced chemiluminescence kit (Pierce Biotechnol-
ogy). b-Actin monoclonal antibody (1:20 000) was used as
internal protein controls.
Data are presented as mean ? SEM. The differences among
neurological deficit scores were determined by Kruskall–
Wallis test followed by Mann–Whitney test for multiple com-
parisons. Analysis of other parametric assay was performed
using one-way or two-way ANOVA in appropriate experiments
followed by Newman–Keuls post hoc test. A probability value
of P < 0.05 was taken to be statistically significant.
Unless otherwise stated, all chemicals were obtained from
Sigma Chemical Company, St. Louis, MO, USA. Neurobasal
medium with supplemental B27, Dulbecco’s modified Eagle’s
medium (DMEM), Trizol and Lipofectamine 2000 were pur-
chased from Invitrogen Co., Carlsbad, CA, USA. Fetal bovine
serum was obtained from Hyclone, Logan, UT, USA. Primary
polyclonal rabbit anti-ERK42/44 and phospho-ERK42/44
were bought from Cell Signalling Technology, Beverly, MA,
USA. Primary polyclonal goat anti-RTP801, rabbit anti-EPO
antibody and FITC-labelled secondary donkey anti-goat IgG
were bought from Santa Cruz Biotechnology Inc., Santa Cruz,
CA, USA. Alexa 586-labelled goat anti-rabbit IgG was pur-
chased from Molecular Probes, Leiden, the Netherlands.
Nimodipine was provided by Zhenzhou Rikang Pharmaceu-
tical Company Limited, China. Drug and molecular target
nomenclature follow Alexander et al., (2009).
In vivo study
Ligustilide protected brain from injury induced by I/R.
investigated whether ligustilide could protect the brain
against damage induced by I/R. Our results based on the
modified Longa EZ test clearly showed that I/R induced a
significant increase in neurological deficit score, compared
with rats treated with sham procedures only. Pretreatment
with 20, 40 or 80 mg·kg-1of ligustilide significantly reduced
neurological deficit score in a dose-dependent manner in I/R
rats. The scores in all ligustilide groups were lower than those
in the vehicle group (all P < 0.01, Figure 1A). Nimodipine
pretreatment, as a positive control, at a dose of 12 mg·kg-1,
also decreased the neurological deficit score (Figure 1A).
We also assessed the effects of ligustilide pretreatment on
infarction induced by I/R and found that this compound (20,
40 or 80 mg·kg-1) dose-dependently reduced infarct volume.
Also, infarct volume in the rats pretreated with nimodipine
(12 mg·kg-1) was significantly lower than that in the vehicle-
treated rats (Figure 1B,C). These findings demonstrated that
ligustilide protected the brain against I/R-induced damage.
Ligustilide increased EPO and decreased RTP801 expression in rats
treated with I/R.
To find out whether the protective effects of
ligustilide were associated with changes in EPO and RTP801,
we then investigated EPO and RTP801 expression in the brain
of I/R rats. Figure 2A shows representative photographs of
EPO and RTP801 expression by immunohistochemistry in
the rats received different treatments (I. sham; II. 0; III. 20; IV.
40; and V. 80 mg·kg-1ligustilide). Immunofluorescence analy-
sis (Figure 2B,C) shows that EPO expression within the infarct
region of cortex in the vehicle-treated group was slightly
increased compared with that of the sham-operated group.
Prior administration of ligustilide enhanced EPO expression
dose-dependently. The EPO contents in the rats of all ligustil-
ide groups were significantly higher than those in the rats
treated without ligustilide (P < 0.05 or 0.01) (Figure 2A,B). In
the case of RTP801, I/R induced a significant increase, while
pretreatment with ligustilide resulted in a significant decrease
in expression of the protein in the infarct region of cortex,
also in a dose-dependent manner. The levels of RTP801 in the
rats of all ligustilide groups were significantly lower than
those in the rats without ligustilide treatment (P < 0.01)
In vitro study
The above in vivo studies suggested that the protective effects
of ligustilide on brain I/R damage were associated with
up-regulation of EPO and down-regulation of RTP801 expres-
sion. To provide more direct evidence of the involvement of
Ligustilide and neuroprotection
British Journal of Pharmacology (2011) 164 332–343335
EPO and RTP801 and to elucidate the underlying molecular
mechanisms, we performed a series of in vitro studies.
Ligustilide increased cell viability and decreased LDH release in
neurons exposed to OGD.
We first investigated the effects of
ligustilide on cell viability and LDH release in cultured
neurons exposed to OGD. The neurons were pre-incubated
with ligustilide (0, 0.625, 1.25, 2.5, 5, 10 or 20 mmol·L-1) for
2 h before undergoing OGD for 4 h followed by normal con-
ditions for 24 h. As expected, OGD induced a significant
decrease in cell viability (Figure 3A) and a marked increase in
LDH release (Figure 3B) in neuron cultures. In OGD neurons
pretreated with ligustilide, the cell viabilities increased and
LDH decreased progressively with the increase of ligustilide
concentrations, attaining the highest (cell viability) and
lowest (LDH release) at 5 mmol·L-1. Above this concentration,
cell viability decreased again and LDH release increased (10
and 20 mmol·L-1ligustilide; Figure 3). These data suggested
that ligustilide had protective effects on neurons against the
injury induced by OGD in vitro, at the lower concentrations
but at concentrations greater than 5 mM may cause cytotox-
icity in cultured neurons. The findings also confirmed the
results of our in vivo studies and provided the basis for using
the in vitro model to further study the mechanisms underly-
ing the effect of ligustilide.
Ligustilide up-regulated EPO and down-regulated RTP801 expres-
sion in neurons exposed to OGD.
different concentrations on EPO and RTP801 expression were
investigated by pre-incubating neurons with ligustilide (1.25,
5 and 10 mmol·L-1) before undergoing OGD. Western blot
analysis showed that ligustilide at a dosage of 1.25 or
5 mmol·L-1could induce a significant increase in EPO expres-
sion in neurons exposed to OGD, while 10 mmol·L-1of
ligustilide produced a much weaker effect (Figure 4A,B). The
EPO content of neurons pretreated with 1.25 or 5 mmol·L-1of
ligustilide were significantly higher than those in the controls
Exposure to OGD also induced a significant increase
(about three-fold control) in the expression of RTP801 in
neurons, as was found in the cortex in vivo. The increased
RTP801 induced by OGD could be significantly inhibited by
pretreatment with ligustilide in a dose-dependent manner.
(Figure 4C, D). The RTP801 content of neurons treated with
1.25, 5 and 10 mmol·L-1of ligustilide were all significantly
lower than those in the neurons without ligustilide treatment
(all P < 0.01) (Figure 4C,D). The effects of ligustilide on EPO
and RTP801 expression were consistent with the protection
by ligustilide of neurons against I/R injury, suggesting
correlation of the protective effect of ligustilide with the
up-regulation of EPO and down-regulation of RTP801
The effects of ligustilide at
Ligustilide (LIG) protected brain from injury induced by ischaemia-
reperfusion in rat. (A) Neurological deficit scores. (B) Representative
photographs of brain slices stained with 2,3,5-triphenyltetrazolium
chloride. (C) Infarct volume expressed as the percentage of brain
volume. Animals were subjected to sham operation (I), administra-
tion of vehicle only (II), nimodipine at a dose of 12 mg·kg-1(III),
Ligustilide at a dose of 20 (IV), 40 (V) or 80 mg·kg-1(VI) at 3 h and
0.5 h before undergoing MCAO for 2 h followed by 24 h reperfu-
sion. Neurological deficit scores were analysed using Kruskall–Wallis
test followed by Mann–Whitney test for multiple comparisons to
identify which was different from the ‘vehicle’ group. The infarct
volume was analysed by one-way ANOVA followed by Newman–Keuls
post hoc test. Parametric data are presented as mean ? SEM (n = 6)
and non-parametric data as box and whisker plots with the minimum
and maximum values (n = 9). *P < 0.05, **P < 0.01 significantly
different from ‘vehicle’.
X Wu et al.
336 British Journal of Pharmacology (2011) 164 332–343
Inhibition of ERK reduced significantly the effects of ligustilide on
cell viability and EPO expression in neurons exposed to OGD.
understand the signalling pathway involved in the protective
effect of ligustilide on neurons, we then investigated the
effects of inhibitors of PI3K, ERK and p38 MAPK on cell
viability and LDH release. The neurons were pre-incubated
with 0 or 50 mmol·L-1of LY294002 (a specific inhibitor of
PI3K), 0 or 25 mmol·L-1of PD98059 (a specific inhibitor of
ERK), or 0 or 50 mmol·L-1of SB203580 (a specific inhibitor
of p38 MAPK) (Harada et al., 2001; Wu et al., 2009) for 1 h
before being treated with 5 mmol·L-1of ligustilide for 2 h and
exposed to OGD. As shown above, treatment with ligustilide
increased cell viability and decreased LDH release in neurons
exposed to OGD. These effects of ligustilide, however, could
be blocked by PD98059 but not LY294002 and SB203580
(Figure 5A,B). In control experiments, LY294002, PD98059
and SB203580 did not affect the cell viability of neurons, in
the absence of ligustilide (data not shown).
To further explore the relationship between ligustilide,
EPO and ERK, we studied the effects of PD98059, LY294002
Effects of ligustilide (LIG) on erythropoietin (EPO) and RTP801 expression in cerebral cortex after ischaemia-reperfusion in rats. Rats were subjected
to ligustilide treatment (0 (vehicle), 20, 40 or 80 mg·kg-1) given at 3 h and 0.5 h before undergoing MCAO for 2 h followed by 24 h reperfusion.
(A) Representative photographs of EPO and RTP801 expression, assayed immunohistochemically. (B,C) Data were normalized to the sham group,
which was taken as 100% and presented as mean ? SEM. Statistical significance was assessed by two-way ANOVA followed by a Newman–Keuls
post hoc test. #P < 0.05, ##P < 0.01 significantly different from sham (control) group; *P < 0.05, **P < 0.01 significantly different from vehicle
group. Three independent experiments were carried out in each group.
Ligustilide and neuroprotection
British Journal of Pharmacology (2011) 164 332–343337
and SB203580 on EPO expression and ERK phosphorylation
(p-ERK) in neurons exposed to OGD. The positive effects of
ligustilide on EPO expression were inhibited by PD98059 but
not LY294002 and SB203580 (Figure 5C,D). Furthermore,
ligustilide increased the amount of p-ERK and this effect was
also blocked by PD98059 (Figure 5E,F). In the absence of
ligustilide, LY294002, PD98059 and SB203580 had no effect
on EPO expression.
Ligustilide reduced RTP801 expression and LDH release in
SH-SY5Y cells transfected with pcDNA3.1-RTP801 plasmid
To provide further evidence for the involvement of
RTP801 in ligustilide-induced protective effects on I/R
neurons, we next investigated the effects of ligustilide on
RTP801 expression and LDH release in SH-SY5Y cells, trans-
fected with 0.8 mg of pcDNA3.1-RTP801 plasmid DNA. The
transfection increased RTP801 expression and LDH release in
the cell cultures (P < 0.01, Figure 6), implying that the
increased expression of RTP801 is associated with increased
cellular damage, as indicated by the increased LDH release.
Treatment of cells with ligustilide inhibited the effects of the
transfection on RTP801 expression, and also increased cell
Effect of ligustilide (LIG) on cell viability and LDH release in neurons
treated with OGD. Neurons were pretreated with ligustilide (0, (OGD
alone) 0.625, 1.25, 2.5, 5, 10 or 20 mmol·L-1) for 2 h before under-
going OGD for 4 h followed by a return to normal media and
oxygenation for 24 h. Statistical significance was assessed by two-way
ANOVA followed by a Newman–Keuls post hoc test. (A) Cell viability by
MTT assay. (B) LDH release. Data are presented as mean ? SEM
(percentage of control) (n = 8), ##P < 0.01 significantly different from
control; **P < 0.01 significantly different from OGD alone group.
Effect of ligustilide (LIG) on erythropoietin (EPO) and RTP801 expres-
sion in neurons treated with OGD. Neurons were pretreated with
ligustilide (0 (OGD alone), 1.25, 5 or 10 mmol·L-1for 2 h before
undergoing OGD for 4 h followed by a return to normal media and
oxygenation for 24 h. (A) A representative Western blot of EPO. (B)
Quantification of expression of EPO. (C) A representative Western
blot of RTP801. (D) Quantification of expression of RTP801 (n = 3 in
all cases). Data are presented as mean ? SEM. Statistical significance
was assessed by one-way ANOVA followed by a Newman–Keuls post
hoc test. **P < 0.01 significantly different from OGD alone group.
X Wu et al.
338 British Journal of Pharmacology (2011) 164 332–343
viability. In the plasmid-transfected cells, RTP801 expression
and LDH release were both significantly lower in the
ligustilide-treated group than the group without ligustilide
treatment (Figure 6).
In spite of a long history of clinical application of Danggui in
the treatment of ischaemic disorders of the cardiovascular
and cerebrovascular systems in traditional Chinese medicine,
very little is known about which of the compound(s) in
Danggui is the active ingredient(s) that improves syndromes
associated with the diseases. In previous studies, we demon-
strated the neuroprotective effects of ligustilide, a major com-
ponent of the Danggui extract, in different models including
hydrogen peroxide-induced neuronal damage in PC12 cells
as well as brain damage induced by transient forebrain cere-
bral ischaemia and permanent focal ischaemia in mice and
rats respectively (Kuang et al., 2006; Peng et al., 2007; Yu
Effects of inhibitors of PI3K, ERK and p38 MAPK on cell viability, LDH release and erythropoietin (EPO) expression in neurons treated with ligustilide
(LIG) and then exposed to OGD. Neurons were pre-incubated with or without LY294002 (LY, a specific inhibitor of PI3K, 50 mmol·L-1), PD98059
(PD, a specific inhibitor of ERK, 25 mmol·L-1) or SB203580 (SB, a specific inhibitor of p38 MAPK, 50 mmol·L-1) for 1 h before being treated with
ligustilide (5 mmol·L-1) for 2 h and then exposed to OGD for 4 h followed by a return to normal media and oxygenation for 24 h. (A) Cell viability
by MTT assay (n = 8). (B) LDH release (n = 8). (C) A representative Western blot of EPO. (D) Quantification of expression of EPO (n = 3). (E) A
representative experiment of Western blot of p-ERK. (F) Quantification of expression of p-ERK (n = 3). Statistical significance was assessed by
two-way ANOVA followed by a Newman–Keuls post hoc test. Data (mean ? SEM) are expressed as a percentage of control or fold increase over
control. ##P < 0.01 significantly different from control group; **P < 0.01 significantly different from OGD alone;@@P < 0.01 significantly different
from 5 mmol·L-1of ligustilide with OGD.
Ligustilide and neuroprotection
British Journal of Pharmacology (2011) 164 332–343339
et al., 2008). In the present study, we found that pretreatment
with ligustilide reduced neurological deficit scores and infarct
volume in a dose-dependent manner in I/R rats in vivo and
increased cell viability with a corresponding decrease in LDH
release, in neurons exposed to OGD in vitro. These findings
provide further evidence that ligustilide is one of the active
ingredients of Danggui and also suggest that ligustilide might
have a protective effect against I/R-induced damage to the
brain. However, it should be pointed out that because infarct
volume was assessed at 24 h in the present study, the com-
pound might slow down the evolution of damage, rather
than actually reduce final magnitude of the infarct. To rule
out that possibility, further observation until at least 48 h in
this model is needed.
Unravelling the precise molecular mechanisms of ligustil-
ide is important in order to design neuroprotective therapies
for hypoxic-ischaemic brain injury based on this compound.
Ligustilide induced a significant increase in the activities of
glutathione peroxidase and superoxide dismutase, and Bcl-2
expression in the ischaemic brain tissues, implying that the
antioxidant and anti-apoptotic properties of ligustilide may
also contribute to its neuroprotective role in cerebral
ischaemic damage (Kuang
peroxide-induced cell injury (Peng et al., 2007). The precise
molecular mechanisms underlying the neuroprotective effect
of ligustilide have not been fully elucidated. In the present
study, we investigated whether ligustilide had any effects on
the expression of EPO (an endogenous protective factor) and
RTP801 (an endogenous detrimental factor) in I/R rats in vivo
and OGD neurons in vitro and whether these effects induced
by ligustilide had any connections with its neuroprotective
The protein EPO was originally recognized as a humoral
mediator involved in the maturation and proliferation of
erythroid progenitor cells (van der Kooij et al., 2008; Fan
et al., 2009). Recent studies have found EPO mRNA and
protein in the brain of a variety of mammals including
humans (Fan et al., 2009) and the EPO receptor was widely
expressed in most cerebral cell types, including astrocytes,
neurons, endothelial cells and microglial cells (Nagai et al.,
2001; Marti, 2004). The increased expression of EPO induced
by HIF-1 has a dominant role in neuroprotection after
ischaemic stroke and intracerebral haemorrhage (Yatsiv et al.,
2005; Rabie and Marti, 2008; van der Kooij et al., 2008; Fan
et al., 2009). Consistent with this, application of EPO
increased survival and function of retinal ganglion cells in
rats suffering from optic neuritis (Sattler et al., 2004). The
presence of EPO and its receptor in the brain and the function
of EPO in neuroprotection led us to propose that EPO might
be one of pharmacological targets of ligustilide and that
ligustilide may play a role in promoting EPO transcription.
An increase in EPO might be one of the mechanisms for the
neuroprotection by ligustilide. In the present study, a signifi-
cant increase in EPO was found in I/R rats in vivo and also in
cultured neurons pretreated with ligustilide and exposed to
OGD. These findings were accompanied by a significant
reduction in neurological deficit score and infarct volume in
vivo as well as an increase in cell viability and a decrease in
LDH release in vitro. These results suggested that EPO might
be one of the essential mediators in ligustilide-induced neu-
roprotection. In a recent study, Zheng et al. (2010) demon-
et al.,2006) and hydrogen
LDH Release (% of Control)
Ligustilide (LIG) reduced RTP801 expression and LDH release in
SH-SY5Y cells transfected with pcDNA3.1-RTP801 plasmid. SH-SY5Y
cells were transfected with empty pcDNA3.1 plasmid (Empty) or
recombinant pcDNA3.1-RTP801 plasmid respectively. The cells that
were transfected with recombinant pcDNA3.1-RTP801 plasmid were
treated with 0 or 5 mmol·L-1of ligustilide as described in Methods.
The RTP801 expression and cell viability in the cells in different
groups were measured by Western blot and LDH assay, respectively,
after transfection for 48 h. (A) A representative Western blot of
RTP801. (B) Quantification of expression of RTP801. (C) LDH release.
Statistical significance was assessed by two-way ANOVA followed by a
Newman–Keuls post hoc test. Data are presented as mean ? SEM (n
= 6). ##P < 0.01 significantly different from control group (trans-
fected with empty pcDNA3.1 plasmid). **P < 0.01 significantly dif-
ferent from 5 mmol·L-1ligustilide group transfected with pcDNA3.1-
X Wu et al.
340British Journal of Pharmacology (2011) 164 332–343
strated that Danggui Buxue Tang (DBT), a Chinese herbal
decoction prepared from Radix Astragali and Radix Angelicae
Sinensis, triggered EPO expression in cultured HEK293T cells.
Based on the findings in the present study, it is reasonable to
speculate that the DBT-induced increase in EPO expression
might be partly associated with ligustilide and this com-
pound could be one of the major active ingredients of DBT.
A previous study in vitro (Siren et al., 2001) demonstrated
that inhibition of MAPK and PI3K blocked EPO-mediated
protection of rat hippocampal neurons against hypoxia.
Using ERK-1/-2 and Akt inhibitors, Kilic et al. (2005) showed
that activation of these proteins was essential for EPO-
mediated neuroprotection in an animal model of focal cere-
bral ischaemia. These findings implied that PI3K, ERK and
p38 MAPK are all associated with EPO expression and EPO-
mediated neuroprotection in hypoxia and ischaemia (Rabie
and Marti, 2008). To understand how ligustilide increased
EPO expression and EPO-mediated neuroprotection in our
experiments, we investigated the effects of inhibitors of PI3K,
ERK and p38 MAPK on cell viability, EPO expression and
p-ERK in neurons pretreated with ligustilide and exposed to
OGD. Our data showed that ligustilide increased the level of
p-ERK, along with increased cell viability and EPO. EPO-
mediated neuroprotection was significantly blocked by inhi-
bition of ERK using PD98059, while the specific inhibitors of
PI3K and p38 MAPK had no such roles. These data indicated
that the ligustilide-induced increase in EPO might be medi-
ated only by ERK and was not associated with PI3K and p38
MAPK as seen in other neuroprotective models (Asomugha
et al., 2010). We would propose that ligustilide promotes the
phosphorylation of ERK and this increased p-ERK then
increased the phosphorylation of HIF-1. The latter change, in
turn, increased the transactivating activity of HIF-1 and
hence, EPO expression (Figure 7). This scheme is consistent
with the findings of previous studies which had revealed that
the transactivating activity of HIF-1 is increased by phospho-
rylation of HIF-1 (Berra et al., 2000; Minet et al., 2001) and
that the PI3K/Akt and the ERK pathway have been proposed
to be involved in HIF-1 phosphorylation (Jewell et al., 2001;
Minet et al., 2001; Stolze et al., 2002).
RTP801 is the product of a recently cloned gene and is
strongly up-regulated by hypoxia in vitro and in vivo (Shos-
hani et al., 2002). It appears to contribute to apoptotic cell
death in several contexts (Shoshani et al., 2002; Bakker et al.,
2007) although the molecular mechanisms are largely
unknown. We therefore speculated that RTP801 might be
another potential target of ligustilide such that this com-
pound might inhibit the expression of RTP801 and thus
protect neurons against I/R damage. Our findings showed
that transfection of SH-SY5Y cells with pcDNA3.1-RTP801
plasmid DNA increased RTP801 expression as well as LDH
release, implying that the increased expression of RTP801 was
associated with the increased cellular damage as indicated by
increased LDH release. The results are in line with the earlier
studies of Shoshani et al. (2002) and Bakker et al. (2007) as
mentioned above. We also demonstrated that pretreatment
of the cells with ligustilide inhibited the effects of transfec-
tion on RTP801 expression and also increased cell viability. In
addition, ligustilide reduced RTP801 expression in both rat
brain and cultured neurons in I/R or OGD respectively. These
results imply that neuroprotection of ligustilide might be
partly mediated by its ability to inhibit RTP801 expression in
brain following I/R in vivo and following OGD in neurons in
vitro (Figure 7).
In conclusion, the present study demonstrated for the
first time that ligustilide pretreatment protected brain against
I/R injury and neurons in vitro against OGD injury by pro-
moting EPO transcription via an ERK signalling pathway and
inhibiting RTP801 expression. Our findings also provide
further evidence that ligustilide is one of the active ingredi-
ents of Danggui. Thus ligustilide has the potential to be
developed into an effective therapeutic agent in preventing
and treating ischaemic disorders of the cardiovascular and
The studies in our laboratories were supported by Key Project
Grant of Jiangsu Province (BG2007607), the Applied Science
Foundation of Nantong City (K2007021), National 973 pro-
(N-CUHK433/08), Direct Grant of CUHK (2009.2.036),
National Natural Science Foundation of China (30971197)
Proposed mechanisms for the neuroprotective role of ligustilide
(LIG). The neuroprotective effect of ligustilide against ischemia-
reperfusion injury is mediated by up-regulation of erythropoietin
(EPO) and down-regulation of RTP801. Ligustilide might promote
the phosphorylation of ERK (p-ERK). The increased p-ERK then
induces an increase in the phosphorylation of hypoxia-inducible
factor-1a (HIF-1a). The latter leads to an increase in the trans-
activating activity of HIF-1 and then EPO expression. In addition,
ligustilide also protects the cells by inhibiting the expression of the
pro-apoptotic protein RTP801.
Ligustilide and neuroprotection
British Journal of Pharmacology (2011) 164 332–343 341
and grants from South-west Hospital of The third Military
Medical University and Shenzhen-Hong Kong Innovation
Circle Programa (2008, 2009).
Conflict of interest
The authors state no conflict of interest.
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British Journal of Pharmacology (2011) 164 332–343 343