Journal of

Translational Medicine

Kuo et al. J Transl Med (2016) 14:78

DOI 10.1186/s12967-016-0831-y

Open Access

RESEARCH

Hericium erinaceus mycelium

andits isolated erinacine A protection
fromMPTPinduced neurotoxicity throughthe
ER stress, triggering an apoptosis cascade
HsingChunKuo1,2, ChienChangLu3, ChienHengShen4, ShuiYiTung4,5, MengChiaoHsieh6, KoChaoLee3,
LiYaLee7, ChinChuChen7, ChihChuanTeng1, WenShihHuang5,6, TeChuanChen8 andKamFaiLee9*

Abstract
Background: Hericium erinaceus is an edible mushroom; its various pharmacological effects which have been investi
gated. This study aimed to demonstrate whether efficacy of oral administration of H. erinaceus mycelium (HEM) and its
isolated diterpenoid derivative, erinacine A, can act as an anti-neuroinflammatory agent to bring about neuroprotec
tion using an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinsons disease, which results
in motor disturbances, in addition to elucidating the mechanisms involved.
Methods: Mice were treated with and without HEM or erinacine A, after MPTP injection for brain injuries by the
degeneration of dopaminergic nigrostriatal neurons. The efficacy of oral administration of HEM improved MPTPinduced loss of tyrosine hydroxylase positive neurons and brain impairment in the substantia nigra pars compacta as
measured by brain histological examination.
Results: Treatment with HEM reduced MPTP-induced dopaminergic cell loss, apoptotic cell death induced by
oxidative stress, as well as the level of glutathione, nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE). Furthermore, HEM
reversed MPTP-associated motor deficits, as revealed by the analysis of rotarod assessment. Our results demonstrated
that erinacine A decreases the impairment of MPP-induced neuronal cell cytotoxicity and apoptosis, which were
accompanied by ER stress-sustained activation of the IRE1/TRAF2, JNK1/2 and p38 MAPK pathways, the expression of
C/EBP homologous protein (CHOP), IKB- and NF-B, as well as Fas and Bax.
Conclusion: These physiological and brain histological changes provide HEM neuron-protective insights into the
progression of Parkinsons disease, and this protective effect seems to exist both invivo and invitro.
Keywords: Hericium erinaceus mycelium, Erinacine A, Parkinsons disease, Endoplasmic reticulum stress
Background
Hericium erinaceus (Lions mane or Yamabushitake) is an
edible mushroom with medicinal properties; it grows on
old or dead broadleaf trees. It is used as a food and herbal
medicine in Japan and China without harmful effects [1].
The mushroom may be a good candidate for inducing
*Correspondence: lkf2758@gmail.com
9
Department ofPathology, Chang Gung Memorial Hospital, Chiayi,
Taiwan
Full list of author information is available at the end of the article

neuronal differentiation and promoting neuronal survival

[2]. Both the mycelium (erinacines A-I) and the fruiting
bodies (Hericenone C-H) are the source of many bioactive extracts with drug efficacy. Hericium erinaceus has
been extensively documented and possesses a range of
therapeutic properties, such as antioxidant activity [3],
hypolipidemic activity [4], hemagglutinating activity [5],
antimicrobial activity [6], antiaging activity [7], immune
modulation and anticancer activities [8, 9]. Erinacine
A has small molecular weight components that are the
major active agents isolated from the cultured mycelium

2016 Kuo etal. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided
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if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Kuo et al. J Transl Med (2016) 14:78

of H. erinaceus. These diterpenoid compounds also

play a role in varied functions, including neuroprotection through nerve growth factor (NGF) synthesis [10].
Therefore, H. erinaceus is attracting attention as a novel
resource, not only for medicinal drugs, but also for dietary phytochemicals for disease prevention and health
promotion through use of its biological properties [11].
Our previous study focused on exploring the biological
agent of erinacine A from H. erinaceus mycelium and its
structural elucidation by ethanol extraction and HPLC
analysis techniques [12, 13]. However, the mechanism by
which H. erinaceus mycelium and its isolated diterpenoid
derivative, erinacine A, promote neuron cell survival and
protection from MPTP-induced neurotoxicity remains
poorly understood, as does the mechanism by which H.
erinaceus mycelium and erinacine A initiate neuroprotection against MPTP injury to the brain.
Parkinsons disease (PD) involves a distinct sequence
of events behind the selective neuronal death that
occurs in PD, but these events are not fully understood
[1416]. Numerous diseases of the nervous system, such
as Parkinsons disease (PD) produce excessive free radical generation (reactive oxygen species [ROS] and reactive nitrogen species [RNS]), which then cause oxidative
damage. These include lipids, oxidative S-nitrosylation
proteins and nucleic acids, which have been linked to
apoptosis by the high levels of ROS in dopamine neurons
due to dopamine metabolism. Various disease models
for PD also show the involvement of the drug 1-methyl
4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) [17, 18].
Furthermore, the MPTP animal model is useful for the
study of neurodegeneration in PD. The neurotoxic effects
of MPTP are thought to be mediated by its metabolite
1-methyl-4-phenylpyridinium ion (MPP+) and monoamine oxidase-B (MAO-B) in neuron cells, leading to a
number of deleterious effects on cellular function, such
as impairing the dopaminergic nigrostriatal neurons,
generating free radicals from the mitochondria and a
neuroinflammatory response, similar to those seen in PD
[19, 20]. Our previous investigation focused on exploring the biological agent of erinacine A from H. erinaceus
mycelium, its structural elucidation by ethanol extraction and HPLC analysis techniques [12, 13]. However,
the mechanism by which H. erinaceus mycelium and
its isolated diterpenoid derivative, erinacine A, are able
to effectively improve the neuroprotective effects of the
endoplasmic reticulum (ER) stress pathway and apoptosis, as well as how the signal cascades become activated,
remain poorly understood.
Numerous studies have demonstrated that the ER
stress pathway might be crucial in various CNS degenerative diseases [21]. In fact, ER stress may be related to
neuronal death. In particular, the JNK/p38 MAPK/CHOP

Page 2 of 14

pathways involved in ER-stress-induced apoptosis in the

neurons are implicated in PD [22]. In addition, energy
metabolism with cultured neuronal cells, including
dopaminergic neurons, showed that MPP + triggers ER
stress and induces a number of genes [23]. Thus, extreme
oxidant and peroxide levels from the neurotoxicity of
MPTP suggest that inhibition of antioxidant defenses
results in inflammatory effects and generation of ROS
or RNS found in PD-related neuron damage [24, 25].
MPTP injury of the brain then induces oxidative stress,
which leads to activate the multiple-cellular-signaling
pathway, such as the IRE1 pathway. IRE1 binds TNF
receptor-associated factor 2 (TRAF2), apoptosis signalregulating kinase 1 (ASK1) and downstream kinases that
further activate Jun N-terminal kinase (JNK) and nuclear
factor-B (NF-B), which has also been linked to PD [26,
27]. In the present study, we explore the biological agent
of H. erinaceus mycelium that is associated with protection against ER stress and loss of dopaminergic nigrostriatal neurons.
In our previous study, we investigated the molecular
mechanisms underlying H. erinaceus that inhibit global
cerebral ischemic injury via inactivation of the iNOS/
RNS and p38 MAPK/CHOP pathways, which may be
among the possible pathways involved in stroke-related
neuron injury [12, 13]. In the present study, we assess
the neuroprotective effect of H. erinaceus mycelium and
its isolated compound erinacine A, as well as its relevance to idiopathic PD in the MPTP mouse model. We
were able to demonstrate that H. erinaceus mycelium, a
known antioxidant, is able to protect against the endoplasmic reticulum stress induced by the loss of dopaminergic neurons and disordered motor function by MPTP
injury. This results in its isolated compound erinacine A
promoting neuronal cell survival due to MPP+ -mediated induction expression of Fas and Bax via IRE1/
TRAF2 complex formation and phosphorylation of the
JNK1/2, p38 and NF-kB pathways. Moreover, developing
more effective dietary H. erinaceus mycelium for PD is an
important goal.

Methods
Hericium erinaceus extracts andanalysis oferinacine A

Fresh mycelium of H. erinaceus was extracted with ethanol. The extract was concentrated and fractionated by
solvent partition between ethylacetate and water. The
ethylacetate fraction was subjected to silica gel column
chromatography using n-Hexaneethylacetate as the eluent. The Hexaneacetone eluate was subjected to silica
gel column chromatography according to the previous
study [9, 12, 13]; HPLC analysis of erinacine A was executed according to the previous study with minor modifications. The analytical column used was a COSMOSIL

Kuo et al. J Transl Med (2016) 14:78

Page 3 of 14

5C18-AR-II (250 4.6 mm; particle size 5 m, Nacalai

USA, Inc., Kyoto, Japan). Separation was performed at
40C using two different gradients for the mobile phase,
which consisted of two solvents, methanol (A) and 2.0%
acetic acid in water (B). The gradient elution had the following profile: 020min, 6090% (A); 2025min, 90%
(A). The retention time of erinacine A was approximately
~17min at a flow rate of 1.0mL/min with a scanning UV
wavelength at 340nm. The 3mg/g erinacine A in the H.
erinaceus extracted with 85% ethanol was confirmed and
quantified by HPLC as shown in Fig.1 [12, 13]. Chemical
compounds studied in this article Erinacine A (PubChem
CID: 10410568).
Animals

C57BL/6 mice (810 weeks old, 2028 g) were kept

individually in a 12-h light/dark cycle cage and had free
access to water and food. Animal care and the general
protocols for animal use were approved by the Institutional Animal Care and Use Committee of Chang Gung
University of Science and Technology. Mice were operated on according to the modified MPTP-induced PDs
model, can be induced by the intraperitoneal injections
of MPTP-HCl (30mg/kg; Sigma, St. Louis, MO) or saline
in 5day. Four groups (six mice in each group) were randomly assigned to a sham control group, a MPTP group,
three H. erinaceus wet mycelia (HEM) groups (5.38,
10.76 and 21.52 mg) and erinacine A groups (1 mg/kg).
HEM was dissolved in water (H2O) and mice received
HEM oral administration indicated during the 25 days
before the onset of MPTP induction and HEM starting
after the first MPTP injection and continuing through 5

additional days. Erinacine A was dissolved in dimethyl

sulfoxide (DMSO) and administered intraperitoneally for
5 days before the MPTP induction. The sham-operated
group animals received an equivalent volume of saline.
Mice were killed 5days after MPTP injection and brains
were harvested, sectioned, and processed [28].
Chemical reagents andantibodies

Mouse monoclonal antibodies against tyrosine phospho-Hydroxylase (Ser31), GAPDH, -actin, 4-Hydroxy2-nonenal (4-HNE), Nitro-tyrosine, CHOP, Fas, Bax,
NFkB p65, Histone H1, TRAF2, IRE1, and phosphoIKB- were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Rabbit monoclonal antibodies against phospho-p38 MAPK (Thr180/Tyr182) and
phospho-JNK1/2 (Thr183/Tyr185) were purchased from
Cell Signaling Technology (Beverly, MA, USA). The TdTmediated dUTP Nick End Labeling (TUNEL) kits were
purchased from Roche (Germany). MPP+ (1-methyl4-phenylpyridinium), SDS, NP-40, while sodium deoxycholate, protease inhibitor cocktail was purchased from
Sigma (St. Louis, MO, USA).
Behavioral testing

Behavioral assessments on mice were made 16 days

after MPTP injection. Motor performance was assessed
with a rotary rod apparatus using a protocol similar to
that described [29]. For the rotarod tests, the paradigms
were used: rockingdirection of rotation with each full
turn of the rod at 10rpm for 3min to a level just below
the bottom of the rod. The mice were placed on the rotating rod and the time until they fell off was recorded. This

Brain histological
examination

25 Days

MPTP, i.p.

Pretreatment
Hericium erinaceus
mycelium extract

MPTP disease
animal model

HO
CHO

5 Days

Sacrifice by
decapitation

Evaluation of
Rotarod test

OH
OH

Fig.1 Simplistic flow chart of neuroprotective activities of Hericium erinaceus mycelium (HEM) in a MPTP treated animal model

Kuo et al. J Transl Med (2016) 14:78

was repeated six times until the total time on the rod for
the control group was 3 min. Both the total time spent
on the rotating rod for each mice, five groups (Control,
MPTP, HEM), were recorded.
Immunohistochemistry

After the administration of a large dose of chloral

hydrate, the mice were killed by decapitation 8days after
MPTP treatment. The brains were quickly removed and
placed in ice-cold saline for 10 min. Next, the brains
were cut into seven 4 m-thickness slides transversely
from neuron impairment area using a mouse brain
matrix (Harvard Apparatus, MA, USA) and then immediately fixed in 10 % formalin overnight. The brain sections were then dehydrated with graded ethanol, passed
through chloroform, and embedded in paraffin, which
were assessed by hematoxylin and eosin (H and E) staining. Paraffin sections of the striatum and substantia nigra
were used for immunohistochemistry (IHC). Staining
was performed using a biotinylated secondary antibody
(Vectastain Universal Elite ABC Kit). Monoclonal rabbit
antibodies against tyrosine Hydroxylase, nitro-tyrosine,
4-HNE and Fas (CD95) were diluted in a ratio of 1:100.
The omission of primary antibodies was used as the negative control. Using the slides, the presence of cytoplasm
stained with brown was scored as positive. The protein
expression were quantitatively evaluated using an Olympus Cx31 microscope with an Image-pro Plus medical
image analysis system. The digital images were captured
using a digital camera (Canon A640). The positive area
and optical density of the positive cells were determined
by measuring three randomly selected microscopic fields
(100, 200 magnification) on each slide. The IHC index
was defined as average integral optical density (AIOD)
(AIOD=positive areaoptical density/total area) [30].

Page 4 of 14

incubated with MPP+for 24h. Thereafter the medium

was changed and incubated with MTT (0.5 mg/ml) for
4 h. The viable cell number is directly proportional to
the production of formazan following solubilization
with isopropanol, which can be measured spectrophotometrically at 563 nm [31]. Annexin V/propidium iodide
(Biosource International, USA) was used to quantify the
percentage of apoptosis cells. Flow cytometric analysis
was performed with a FACSCaliber using CellQuest software. Data were analyzed with CellQuest and WinMDI
software. The apoptotic cells (V+/PI-) were measured by
the fluorescence-activated cell sorter analysis in a FACS
analyzer (BectonDickinson). The data represented three
independent experiments [32].
Preparation oftotal cell extracts andimmunoblotting
analysis

Cells were lysed with a buffer containing 1 % NP-40,

0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS) and a protease inhibitor mixture (phenylmethylsulfonyl fluoride, aprotinin and sodium orthovanadate). The
total cell lysate (50g of protein) was separated by SDS
polyacrylamide gel electrophoresis (PAGE) (12 % running, 4% stacking) and analyzed by using the designated
antibodies and the Western-Light chemiluminescent
detection system (Bio-Rad, Hercules, CA), as previously
described [31].
Statistical analyses

Data were reported as the mean standard deviation (SD) of three independent experiments and were
analyzed by one-way analysis of variance (ANOVA).
The data were analyzed using the SAS software statistical package SigmaPlot, version 9.0 (SAS Institute Inc.,
Cary, NC, USA) [33].

Cell culture

Results

The mouse N2a (Neuro-2a) cells were purchased from

the American Tissue Culture Collection (ATCC, USA).
Cells were grown in Dulbeccos Modified Eagle Medium
(DMEM) (Gibco) supplemented with 10 % fetal calf
serum (Gibco), non-essential amino acids, 1mM sodium
pyruvate and 1% antibiotics (100units/mL of penicillin
and 100 g/mL of streptomycin). All experiments were
performed in plastic tissue culture flasks, dishes or in
microplates (Nunc, Naperville, Denmark). Incubation
was carried out at 37 C in a humidified atmosphere of
5% CO2 and 95% air [31].

Hericium erinaceus mycelium (HEM) inhibits the cytotoxic

effect ofneuron cells inthe MPTPtreatment animal model

Assessment ofcell viability andapoptosis assay

Cell viability, as previously reported by MTT quantitative

colorimetric assay, was capable of detecting viable cells.
The cells were seeded at 2 104 cells/ml density and

Our previous study demonstrated that H. erinaceus

mycelium and its structural analog erinacine A have
nerve-growth properties, allowing them to aid in the prevention of ischemic injury to neurons in the central nervous system of excessive oxidative stress subjects [12, 13].
Based on these studies, we assayed whether H. erinaceus
mycelium provides substantial therapeutic advantages
by suppressing brain impairment in a mouse model that
resembles PD. Figure 1 shows the neuroprotection of
HEM in an MPTP-treated animal model. Figure2 shows
the expressions of the results of TUNEL as well as tyrosine hydroxylase (dopaminergic neurons). There was also
a reduction in the number of positive TUNEL cells in
the striatum and substantia nigra, a marker of apoptosis,

Kuo et al. J Transl Med (2016) 14:78

Page 5 of 14

140
130
120

Positivel Numbers

110
100
90

Control
MPTP
10.76 mg /day
21.52 mg /day

80
70
60
50
40
30

*
*

20
*

10
0

TUNEL

Neuron

Kuo et al. J Transl Med (2016) 14:78

Page 6 of 14

(See figure on previous page.)

Fig.2 Effect of Hericium erinaceus mycelium (HEM) on brain histological examination in MPTP treated animal model. MPTP animal models (MPTP
injection and brains harvested) were performed on mice described in Methods section. a Histological examination of brain were revealed to stria
tum and substantia nigra zones as indicated by immunohistochemical staining of TUNEL. Representative brain sections stained as saline infusion
control group (I); Mice with MPTP injection (II); HEM treatment (10.76 and 21.52mg/day) (III, IV); Apoptotic cells were measured under microscopy
as described in Methods section. The MPTP group exhibited dopamine deficiency syndrome of the striatum, as indicated by apoptotic cells (filled
arrow). Magnification, 100. b Histological examination of brain were revealed in brain, as indicated by immunohistochemical staining of tyrosine
Hydroxylase p-Ser 31. Magnification, 200. The cells were counted from 10 fields (200magification) of each brain sample. c The results from
statistical analysis are the means of cells and were calculated per microscope field from six animals per group. Data are expressed as meanSD of
independent experiments. *p<0.05, MPTP group versus MPTP+HEM group. #P< 0.05, MPTP group versus MPTP+HEM group

when the MPTP mice treated with HEM were compared

with the untreated MPTP mice (MPTP=385; 10.76
and 21.52 mg/day, HEM = 24 3 and 7 2, * <0.05).
Tyrosine hydroxylase (TH) is expressed exclusively in
most dopaminergic neuronal cell types. TH is the enzyme
responsible for the conversion of the amino acid l-tyrosine to dopamine, while PD can be indicated as a THdeficiency syndrome of the striatum. Immunoreactivity
of phospho-Tyrosine hydroxylase was found to reside
mostly in the brain striatum area in the MPTP group
24 h after administering HEM. Quantification of the
dopaminergic neuron cells in terms of pathology showed
that the dosage of MPTP administration was inclined to
decrease the number of normal neurons compared to
the control group. However, a significant increase was
found between the MPTP and MPTP + HEM (10.76
and 21.52 mg/day) group (untreated MPTP = 32 5;
HEM treated MPTP=726 #<0.05 and 11613) (see
Fig.2c).

in a dose-dependent manner compared to the MPTP

group (p < 0.01, n = 6; Fig. 3). Based on these studies,
we tested whether HEM administration provided a protection of oxidative stress on densitometric analysis of
neurotrophic factors in the brain striatum. As shown in
Table1, infusion of MPTP had a pro-inflammatory effect
on brain injury, as shown through enzymatic analyses of
the MPTP treatment group compared to a healthy control group. Dopamine, NGF and glutathione (GSH) levels
were significantly lowered in the MPTP treatment group
(MPTP infusion vs. normal saline infusion, p < 0.05,
n=6). Neuron GSH levels have recently been established
as an indicator of oxidative stress in various diseases. The
administration of the HEM groups significantly (HEM
treatment vs. MPTP infusion, * <0.01, n = 6) elevated
dopamine, NGF and the GSH level in the MPTP group.

HEM treatmentattenuated brain protein ROS oxidants

andbehavioral impairment inmice withMPTP intoxication

Our previous findings demonstrated the effects of the

biological agent of erinacine A from HEM fractionation and its effect on ischemia reperfusion injury. This
suggests that the inactivation of the iNOS/RNS and p38
MAPK/CHOP pathways may contribute to the neuroprotective effects for both cortical and subcortical
infarctions. MPTP exerts its neurotoxic effect through
biological activation by MPP+, leading to mitochondrial energy loss, generation of toxic ROS and neuron cell death [34]. To clarify whether erinacine A has
an inhibitory effect on the MPP+ model of neuronal
injury that is cytotoxic to N2a cells and the molecular
mechanisms underlying the MPP+ -mediated activation of IRE1a-inducing Jun N-terminal kinase (JNK1/2)
and p38 MAP Kinase (MAPK), we induced N2a cells to
MPP+ for 24 h, performed MTT assays and examined
the protein levels of the ER stress-signaling pathway.
Significantly decreased cell death was found in the erinacine A group compared to the MPP+ group (Fig.5a).
As shown in Fig. 5b, the extent of apoptosis of MPP+
induction was quantified as a percentage of annexin
V-positive cells and shown as 20 %. Erinacine A treatment of N2a cells also resulted in increased neuron

In a previous study, an animal model of Parkinsons

disease showed the result of the presence of excessive
free radicals after brain tissue damage; these have been
reported to damage dopamine neurons due to dopamine
formation for PD [23]. These findings demonstrated that
HEM was able to inhibit oxidative nitrotyrosine proteins
and lipid peroxidation 4-HNE, resulting in the dopaminergic nigrostriatal neurons apoptosis. This suggests
that the ER stress effectors of CHOP via p38 MAPK
may contribute to the neuroprotective effects by MPTP
administration (Fig.3, p<0.05). Figure4 shows the neuroprotective activities of HEM in an MPTP-treated animal model. Motor function of MPTP injection of HEM
was also determined by a rotary rod apparatus, in comparison to an MPTP group. Five days after MPTP treatment, the mice showed significant motor function, as
indicated by a decrease in the time periods on a rotary
rod at 6 days, as compared to the control group MPTP
mice (p < 0.05, n = 6). Significant differences were
found between the HEM-treated and MPTP groups.
The HEM group showed reduced motor dysfunction

Inhibition effects oferinacine A treatment

onMPP+induced ROSrelated apoptosis andthe ER
stresssignaling mechanisms

Kuo et al. J Transl Med (2016) 14:78

Page 7 of 14

Percentage of positive area (%)

60

#
Nitro-tyrosine
4-HNE

50

40

#
*

30

*
*#

20

10

Control

MPTP

10.76 mg /day 21.52 mg /day

Hericium erinaceus mycelium

Kuo et al. J Transl Med (2016) 14:78

Page 8 of 14

(See figure on previous page.)

Fig.3 Effect of Hericium erinaceus mycelium (HEM) on brain histological nitrotyrosine and 4-HNE protein expression in MPTP animal model.
Histological examination of brain were revealed to somata of dopamine neurons by immunohistochemical staining. a Mice were sacrificed and the
brains were separated. Representative brain sections stained control group (I); Mice treated with MPTP at 6days (I); HEM administration [10.76 (III)
and 21.52 (IV) mg/day] staining of nitrotyrosine (a) and 4-HNE (b) protein in brain tissue. c Evaluation of nuclear protein expression and nitrotyros
ine and 4-HNE expression were quantitative in the striatum. The area of dopamine neurons positive stained area from the SN was evaluated from
three randomly selected observation fields of each brain section. Quantitative of immunohistochemical proteins by average integrated optical
density (AIOD). Positive stained area was evaluated from three randomly selected observation fields of each liver section. Data were expressed as
meanSD (n=6/group). *p<0.05, nitrotyrosine expression, MPTP group versus MPTP+HEM group. #P<0.05, 4-HNE expression, MPTP group
versus MPTP+HEM group

survival by 6 and 3 %, respectively. Erinacine A might

have neuroprotection effects on neuron injury caused
by oxidative stress of MPP+.

Maximum time on Rod (sec)

200
180

160
140

120
100

40
20

&

&

&

80
60

#
#

Control group (CL)

MPTP treatment (MPTP)
5.38 mg/day
10.76 mg/day
21.52 mg/day

&

0
1

Days at testing
Fig.4 Effect of Hericium erinaceus mycelium (HEM) treatment on
motor function by the rocking rotarod assessment of coordina
tion. Mean duration (in seconds) of the time spent on the rocking
rotarod as a function of increasing motor function for MPTP and HEM
administration mice. At 5days after the MPTP injection, the mice
exhibited a significant motor dysfunction as indicated by a decrease
in the time period on rotarod. The mice with MPTP treated with
HEM(III) and assessed by rotarod apparatus. Data were expressed as
meanSD (n=6/group). *p<0.05, 4days, control group versus
MPTP and MPTP+HEM groups. #P<0.05, 5days, MPTP group
versus MPTP+HEM groups. & P<0.05, 5days, MPTP group versus
MPTP+HEM groups

Erinacine A prevented cell death andwas involved inthe

modulation ofIRE1/RF2 complex levels, aswell
asERassociated protein expression byMPP+ inneuron
cells

Our results clearly showed that MPP+ resulted in neuron

cell death and that oxidative damage was relative to the
expression of Fas, Bax and CHOP, as well as to activation
of the ER stress-signaling pathway, such as the p-JNK1/2,
p38 MAPK and NF-B triggering pathways, which may
function downstream of the ER stress to induce apoptosis in response to neuronal damage, as an index of excessive oxidative damage with Western blotting (Fig. 6a).
Conversely, treatment with erinacine A also resulted in a
significant inhibition of JNK1/2 and p38 phosphorylation
and ER-associated protein expression for 24 h. In addition, erinacine A was also found to induce effects on the
IRE1/RF2 association (Fig.6). In order to verify the
effects of oral administration of HEM and erinacine A on
the relationships between neuroprotection, inflammation and ER stress in the brain, Fas protein was selected
for further examination using immunohistochemistry
assays in vivo after MPTP injury and HEM treatment.
As shown in Fig. 7, mice with MPTP impairment demonstrated increased expression of Fas (CD95) in the
brain tissue samples. In particular, HEM and relative
erinacine A treatment resulted in a significant reduction
of Fas expression. These results are consistent with the
results in vitro, indicating that HEM is applicable when
protecting MPTP-induced brain neuron injury. Taken
together, the results and the data showed that H. erinaceus mycelium and relative erinacine A administration
in neuron cells is essential to involvement in oxidative

Table1 Biochemical effects ofMPTP administration animal model byHericium erinaceus mycelium
Neurotoxic effects ofthe striatum
Control (n=6)

MPTP (n=6)

MPTP+HEM
5.38mg/day (n=6)

MPTP+HEM
10.76mg/day (n=6)

Dopamine (ng/ml)

3.60.15

1.70.1

3.10.1*

3.80.1*

NGF (ng/ml)

2.80.5

1.20.1

1.90.3*

2.30.4*

14011

626

948

GSH/mg

*P<0.05, when compared with MPTP group

13410

MPTP+HEM
21.52mg/day (n=6)
4.10.1*
2.70.3*
1388*

Kuo et al. J Transl Med (2016) 14:78

Page 9 of 14

120

Percentage of positive area (%)

100

80

60

40

20

Control

MPP

EA 1.0M EA 2.0M EA 5.0M

Fig.5 Effect of MPTP on assessment of cell viability in N2a cells for 24h. a after 24h their viability was measured using an MTT assay. b After an
indicated treatment for 24h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Methods
section. The percentages presented in each frame depicted the apoptotic cells. *p<0.05, indicates significant difference compared to an untreated
group of N2a

Kuo et al. J Transl Med (2016) 14:78

N
6

24

MPP
3 6 24

Page 10 of 14

MPP+EA
3 6 24

Fas
33 kDa
Bax
19 kDa

p-JNK 1/2
48 kDa
p-p38
21 kDa
-actin
42 kDa
CHOP
19 kDa
NF-kB p65
65 kDa
p-IKB-

65 kDa
Histone H1
IP:TRAF2
WB: IRE1
120 kDa

Protein level
(Fold of control)

*
*

* *

Control
MPP
MPP+EA

**

* **

* *

*
*

Fas

p-JNK1/2

p-p38

CHOP

NFkBp65 IRE1

Fig.6 Erinacine A mediated and ER-associated proteins expres

sion between untreated (control) and treated (MPP+) in N2a cells. a
Nuclear and whole cell lysate proteins were prepared and ana
lyzed by Immunoblotting, using antisera to detect Fas, Bax, p-JNK,
p-p38,CHOP, NF-kB p65, p-IKB-b and IRE1a (b-actin and Histone H1
served as the internal control). The association of TRAF2 with IRE1-a
was determined by immunoprecipitation with TRAF2 followed by
western blot with anti-IRE1-a antibody. b Protein levels were quanti
fied by densitometric analysis with the control being set at 100%.
Data presented in western blot are derived from a representative
study, and comparisons of protein expression are calculated from
three independent experiments. *P<0.05, when compared with
control untreated group

stress-signaling MPP+ -mediated cell death along both

IRE1 and JNK1/2/NF-B-related pathways.

Discussion
ER stress-induced apoptosis is implicated in various
pathological conditions, but the mechanisms of H. erinaceus mycelium on the suppression of oxidative stress

and JNK/p38 MAPK as well as Fas, Bax and CHOP protein expression, which confer neuroprotection in PD
involving ER stressmediated signaling to neuron apoptotic pathways, remain unclear. It has been previously
reported that a number of H. erinaceus extracts, used
as a medicinal mushroom, can improve sleep quality,
ameliorate depression and neurodegenerative disease,
and improve cognitive impairment [35]. Moreover, it
was reported that polysaccharides purified from the liquid culture broth of HEM and erinacine A enhanced the
growth of mice adrenal nerve cells, improved the extension of neurite extension and expanded catecholamine
in the brain of mice. Recently, studies were conducted
on the synergistic effects of HEM extracts and exogenous NGF on neurite outgrowth in a glioma cell line.
These studies demonstrate that the HEM extracts contain
certain neuroactive components that induce NGF synthesis and promote neurite outgrowth, which has been
shown to be a mycelium- growth-associated metabolite
that stimulates NGF synthesis in cultured astrocytes [7].
Using an in vivo MPTP animal model and mouse neuron cell culture MPP+ -induced apoptosis (Fig. 1), our
invivo data demonstrated that the dopaminergic lesions
and oxidative stress in the striatum and substantia nigra,
as well as motor disorder, were significantly decreased
after treatment with HEM (Figs.2, 3, 4). In addition, we
deployed several neuron cell death assays to confirm that
the MPP+ damage, as measured by brain neuron death,
is significantly decreased with erinacine A treatment at
the concentration of 5M for 24h in N2a cells (Fig.5).
Studies have shown that ER stress- and inflammationinduced apoptosis is a key pathogenic event in disease
processes as divergent as metabolism disease, cancer,
hepatitis, heart disease and neurological diseases [5, 8,
22, 36]. The overall objective of this study made it necessary to develop more effective drugs from natural compounds of edible mushrooms to prevent neuronal death
at high MPP+ exposures related to the mechanism of
ER stress-induced apoptosis. The specific goal was linking upstream ER stress-mediated events to downstream
apoptosis execution pathways in a model involving the
JNK1/2, p38, NF-kB and CHOP pathways. Of current
interest is the concept that prolonged CHOP expression
leads to the release of ER calcium stores resulting from
ER stress-induced expression of the Fas death receptor
and Bax mitochondrial pathways of apoptosis through
a pathway involving JNK and p38. Figure 6 shows that
treatment of N2a cells with erinacine A increased the
level of phosphorylated JNK and p38 MAPK as a result
of an upregulation of CHOP and NFB at 24 h, as well
as a decrease in the Fas/Bax expression and IRE1/
TRAF2 complex interaction as early as 3 h. Our results
showed that erinacine A inhibited MPP+ -induced

Kuo et al. J Transl Med (2016) 14:78

Page 11 of 14

II

III

IV

VI

Average integral optical density (AIOD, %)

70
60
50
40

30
20

10

Control

MPTP II

HEM III HEM IV

HEM V

EA VI

Fig.7 Effect of Hericium erinaceus mycelium (HEM) on brain histological Fas protein expression in MPTP treated animal model. Histological exami
nation of brain were revealed to striatum and substantia nigra zones as indicated by immunohistochemical staining. Representative brain sections
stained as saline infusion control group (I); Mice with MPTP injection (II); HEM treatment (5.38, 10.76 and 21.52mg/day) (III, IV, V); Erinacine A (i.p. VI);
The results from statistical analysis are the means of cells and were calculated per microscope field from six animals per group. Data are expressed
as meanSD of independent experiments. *p<0.05, MPTP group versus MPTP+HEM group and MPTP+EA group

neuron damage by inactivating oxidative stress-dependent CHOP expression. We also showed that both the Fas
and Bax pathways, through induction of the Fas receptor
itself and the mitochondrial pathway by MPP+ , and then
specifically erinacine A, reduced the production of the
ER stress-signaling pathway in dose-relative inhibition.
Moreover, our histopathological and immunohistochemical assays provide unique evidence suggesting that HEM

is able to suppress neuron impairment in MPTP mice

at doses below the safe starting-dose range (Conversion
of Animal Doses), and that this involves the significant
inhibition of neuron death and Fas expression (Fig. 5).
These findings provide an integrated, unique mechanism linking the ER stress to apoptosis, and suggest
that it is feasible to determine whether H. erinaceus and
relative erinacine A are able to effectively improve the

Kuo et al. J Transl Med (2016) 14:78

neuroprotective effects for intervention directed against

PDs ER stress-induced apoptosis.
Studies have shown that natural phytochemicals from
certain plants have the capability to exert neuroprotective effects in various experimental models of neurological disorders, and also to have cytotoxic effects on
cancer cells [13, 37]. Furthermore, reducing oxidative
stress-induced free radicals via an antioxidant effect
has been shown to protect against the neuronal damage
caused by neurotrophin deficiencies and toxin-induced
degenerative diseases in response to chemopreventive drugs, such as dietary phytochemicals, phenols,
alkaloids, flavonoids and mushrooms [38]. Moreover, finding a suitable neuroprotective agent is needed
since this is a very important property with regard to
its ability to cross the bloodbrain barrier (BBB). To
validate these findings, further study of H. erinaceus
is needed to determine whether there are mediated
actions to reach the target sites of the CNS in mice.
However, phytochemicals that regulate neurodegenerative disease by targeting neurotrophins might provide a
promising future. Both NGF and BDNF are expressed
in dopaminergic neurons, suggesting that these act as a
survival factor for the trophic support of neurons. Phytochemicals that potentiate neurotrophins and activate
Trk receptors may serve to prohibit the onset of neurodegenerative PD [39]. On the other hand, it has been
shown that JNK and p38 MAPK stress protein kinases
are involved in inflammation and oxidative stress in
neurodegenerative neuronal death in PD [36]. Our
results showed that the upregulation of NGF protein
and dopamine affected the striatum neuron cell with
an increase of the antioxidative indicator GSH; these
resulted in inhibitors targeting JNK and p38MAPK,
preceded by the administration of H. erinaceus mycelium (Table1; Fig.6).

Conclusion
In this study, we evaluated the molecular mechanisms
underlying the roles of H. erinaceus against MPTPinduced neurotoxicity. Consequently, based on MPTPtreated mice, our results imply the possibility of a toxic
effect of MPTP, a food provided by the mushroom components of H. erinaceus, on N2a cells and enable the
evaluation of the protective ability and the underlying
mechanisms against MPP+ cytotoxicity. In vivo study
demonstrated that H. erinaceus mycelium decreased
the brain dopamine neuronal loss and motor function.
Our study showed that erinacine A inhibited the neuron cell apoptosis as a result of oxidative stress signaling and the JNK/p38/NF-B/CHOP/Fas/Bax pathways
(Fig. 8). These results provide insights into the neuroprotective activity of erinacine A of the Hericium

Page 12 of 14

Fig.8 Schematic presentation of the ER Stress-signaling pathways

involved in Hericium erinaceus mycelium and erinacine A suppressed
MPP-induced apoptosis in N2a cells. Hericium erinaceus mycelium
has neuroprotective potential through blocking oxidative stress
MPTP-induced neuron damage and that erinacine A contribute to
the protective action. The inhibition of effect of MPP on the ER stress
triggering production of ROS activates IRE1a/TRAF2 complex forma
tion and phosphorylation of JNK1/2, p38 and NF-kB pathways, which
inhibits the Fas and Bax up-regulation and decreases an apoptosis
cascade

erinaceus mycelium, which thus may be promising candidates for the treatment of neurodegenerative diseases
such as PD.
Authors contributions
K-FL Provision of study material, collection and assembly of data and
histopathological evaluation, C-CL Design, collection, assembly of data and
manuscript writing, M-CH Conception, collection, and assembly of data, C-HS
Provision of study material or animals, K-CL Provision of study material or ani
mals, C-CT Provision of study material, collection, and assembly of data, K-CL
Administrative support, collection, and assembly of data (flow cytometry),
L-YL, L-YL and C-CC Provision of study material or animals, W-SH and H-CK
Conception and design, financial support, administrative support, manuscript
writing, final approval of manuscript. All authors read and approved the final
manuscript.
Author details
Department ofNursing, Chang Gung University ofScience andTechnology,
Chiayi, Taiwan. 2Research Center forIndustry ofHuman Ecology, Chang Gung
University ofScience andTechnology, Taoyuan, Taiwan. 3Division ofColorectal
Surgery, Department ofSurgery, Chang Gung Memorial Hospital, Kaohsiung
Medical Center, Chang Gung University College ofMedicine, Kaohsiung, Tai
wan. 4Department ofHepatoGastroenterology, Chang Gung Memorial Hos
pital, Chiayi, Taiwan. 5Chang Gung University College ofMedicine, Taoyuan,
Taiwan. 6Division ofColon andRectal Surgery, Department ofSurgery, Chang
Gung Memorial Hospital, Chiayi, Taiwan. 7Grape King Biotechnology Inc.,
ZhongLi, Taiwan. 8Division ofNephrology, Kaohsiung Chang Gung Memorial
Hospital andChang Gung University College ofMedicine, Kaohsiung, Taiwan.
9
Department ofPathology, Chang Gung Memorial Hospital, Chiayi, Taiwan.
1

Acknowledgements
Funding for this study was provided in part by research Grants from the
Tomorrow Medical Foundation, Taiwan. This study was supported by grants
BMRPD42, CLRPG8D0112, CMRPG6E0181, CMRPF6E0011, CMRPF6E0041,
CMRPF6E0021, CMRPG8C1261, CMRPG8C1262, CMRPG6C0013,

Kuo et al. J Transl Med (2016) 14:78

CMRPG6C0302, and CMRPF6D0072 from Chang Gung Memorial HospitalKaohsiung Medical Center, Chang Gung Memorial Hospital, and Chang Gung
University of Science and Technology, Chia-Yi Campus, Taiwan and by the
Ministry of Science and Technology, Taiwan (MOST 103-2622-B-255-001-CC3
and MOST 103-2313-B-255-001 and MOST 104-2320-B-255 -003 -MY3).
Competing interests
The authors declare that they have no competing interests.
Received: 5 January 2016 Accepted: 5 March 2016

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