Nerve Growth Factor-Inducing Activity of Hericium erinaceus in 1321N1 Human Astrocytoma Cells

Koichiro Mori,“ c Yutaro Obara,“’1 Mitsuru Hirota/ Yoshihito Azumi,c
Satomi Kinugasa,c Satoshi Inatomi,c and Norimichi Nakahata*“’*


a
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University; * 21st Century COE Program “CRESCENDO”, Graduate
School of Pharmaceutical Sciences, Tohoku University; Aoba 6-3, Aramaki, Aoba- ku, Sendai 980-8578, Japan: c Mushroom Laboratory, Hokuto
Corporation; 800-8 Shimokomazawa, Nagano 381-0008,


Japan: and dDepartment of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University; 8304 Minami- minowa, Kami-ina, Nagano
399-4598, Japan.

Received March 24, 2008; accepted June 20, 2008; published online June 23, 2008


Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic fac­tor-like substances or their inducers are
expected to be applied to the treatment of neurodegenerative diseases such as Alzheimer’s disease. In the present study, we firstly examined the
effects of ethanol extracts of four edible mushrooms, Hericium erinaceus (Yamabushitake), Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Agaricus blazei (Himematsutake), on nerve growth factor (NGF) gene expression in 1321N1 human
astrocytoma cells. Among the four mushroom extracts, only H. erinaceus extract promoted NGF mRNA expression in a con­centration-dependent
manner. In addition, secretion of NGF protein from 1321N1 cells was enhanced by H. eri­naceus extracts, and the conditioned medium of 1321N1
cells incubated with H. erinaceus extract enhanced the neurite outgrowth of PC12 cells. However, hericenones C, D and E, constituents of H. erinaceus, failed to pro­mote NGF gene expression in 1321N1 cells. The enhancement of NGF gene expression by H. erinaceus extracts
was inhibited by the c-jun N-terminal kinase (JNK) inhibitor SP600125. In addition, H. erinaceus extracts in­duced phosphorylation of JNK and
its downstream substrate c-Jun, and increased c-fos expression, suggesting that H. erinaceus promotes NGF gene expression via JNK
signaling. Furthermore we examined the efficacy of H. erinaceus in vivo. ddY mice given feed containing 5% H. erinaceus dry powder
for 7 d showed an increase in the level of NGF mRNA expression in the hippocampus. In conclusion, H. erinaceus contains active compounds that
stimulate NGF synthesis via activation of the JNK pathway; these compounds are not hericenones.

Key words nerve growth factor; Hericium erinaceus; astrocytoma; hericenone

Senile dementia is a serious social problem. In particular, Alzheimer’s disease, for which there is currently no effective therapy, is the most common
senile dementia. Alzheimer’s disease patients have notable abnormalities in cholinergic neurons in the basal forebrain.Neurotrophic factors have potent
biological activities, such as preventing neuronal death and promoting neurite outgrowth, and are essential to maintain and organize neurons functionally. 2-1 Glial cells sup­port neurons by releasing neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin 3, and glial-derived neurotrophic fac­tor (GDNF). In particular, it is assumed that functional defi­ciency of NGF is related to
Alzheimer’s disease and plays a part in the etiology of the disease process.3-

It is known that NGF levels are decreased in the basal forebrains of Alzheimer’s disease patients, and in the frontal cortices of undemented patients with
senile plaques.4,5- Fur­thermore, intracerebroventricular administration of NGF eliminates degeneration and resultant cognitive deficits in rats
after brain injury,6- and it enhances the retention of pas­sive avoidance learning in developing mice.7) In aged rats, intracerebral
infusion of NGF partly reverses cholinergic cell body atrophy and improves the retention of spatial memory.8) In addition, intranasal
administration of NGF ameliorates neurodegeneration and reduces the numbers of amyloid plaques in transgenic anti-NGF mice (AD11 mice), in which have a
progressive neurodegenerative phenotype resembling Alzheimer’s disease.9) Therefore, NGF is expected to be ap­plied to the treatment of
Alzheimer’s disease.10-1

However, neurotrophic factors are proteins, and so are un­able to cross the blood-brain barrier; they are also easily me­tabolized by peptidases.
Therefore, their application as a medicine for the treatment of neurodegenerative disorders is assumed to be difficult. Alternatively, research has been
car­ried out on low-molecular weight compounds that promote NGF biosynthesis, such as catecholamines,11,12-1 benzo- quinones,13) fellutamides,14) idebenone,15) kansuinin, ingenol triacetate, jolkinolide B,16) dictyophorines, 17) scabronines,18) hericenones,18—21) erinacins,22—24) and cyrneines.25)

Hericium erinaceus
is a mushroom that grows on old or dead broadleaf trees. H. erinaceus is taken as a food in Japan and China without harmful effects. Hericenones
C—h2,19—21) and erinacines A—i22—24) were isolated from the fruit body and mycelium of H. erinaceus, respectively, all of
which pro­mote NGF synthesis in rodent cultured astrocytes. These re­sults suggest the usefulness of H. erinaceus for the treatment and prevention
of dementia. However, the detailed mecha­nism by which H. erinaceus induces NGF synthesis remains unknown.

In the present study, we examined the NGF-inducing activ­ity of ethanol extracts of H. erinaceus in 1321N1 human astrocytoma cells. The results
obtained indicate that H. eri­naceus has NGF-inducing activity, but that its active com­pounds are not hericenones. Furthermore, ICR mice given
feed containing 5% H. erinaceus dry powder for 7 d showed an increase in the level of NGF mRNA expression in the hip­pocampus.

© 2008 Pharmaceutical Society of Japan


MATERIALS AND METHODS

Materials
Dulbecco’s modified Eagle’s medium (DMEM) was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). FCS was from Biological Industries (Kibbutz Beit Haemek,
Israel). HS was from ICN Biochemicals, Inc. (Costa Mesa, CA, U.S.A.). Tri Pure Isolation Reagent was from Roche Di­agnostics (Indianapolis, U.S.A.).
Oligo(dT)primer and NGF ELISA Kit Emax® Immunoassay System were from Promega Co., Ltd. (Madison, WI, U.S.A.). Rever Tra Ace was from Toyobo Co.,
Ltd. (Tokyo, Japan). Syber Premix Ex Taq was from Takara Bio Inc. (Shiga, Japan). U0126 was from Sigma Aldrich Japan (Tokyo, Japan). SP600125 was from
BIOMOL (Plymouth Meeting, PA, U.S.A.). A23187, SB203580 and Gf109203X were from Calbiochem (San Diego, CA, U.S.A.). H89 was from Seikagaku Corporation
(Tokyo, Japan). Anti- NGF was from Boehringer Mannheim (Mannheim, Ger­many). Anti-phospho-ERK (Thr202/Tyr204) antibody, Anti- ERK antibody, anti-phosho-JNK
(Thr183/Tyr185) antibody, anti-JNK antibody, anti-phospho-c-Jun (Ser63) antibody, and anti-c-Jun antibody were obtained from Cell Signaling Tech­nology
(Beverly, MA, U.S.A.). Real-time PCR was carried out using an Opticon real-time PCR system (Bio-Rad Labo­ratories Inc., Japan). Dextrin was from Wako Pure
Chemical Industries Ltd. (Tokyo, Japan). ddY mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Hericium erinaceus, Pleurotus eryngii, Grifola frondosa, and Agaricus blazei were cultured by Hokuto Corporation in its facilities
(Nagano, Japan).

Cell Culture
1321N1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FCS, penicillin (50 units/ml), and streptomycin (50 mg/ml). PC-12
cells were grown in DMEM supplemented with 10% FCS, 5% HS, penicillin (50 units/ml), and streptomycin (50 mg/ml). The cells were cultivated in an incubator
contain­ing 5% CO2 at 37 °C.

Preparation of Mushroom Extracts
Fresh fruiting bod­ies of H. erinaceus, P eryngii, G. frondosa, and A. blazei were lyophilized and powdered. The dry powder (5 g) of
mushrooms was extracted with 150 ml of ethanol for 2h at room temperature, and H. erinaceus ethanol extract (499 mg), P eryngii ethanol
extract (386 mg), G. frondosa ethanol extract (328 mg) and A. blazei ethanol extract (426 mg) were obtained. Similarly,H. erinaceus was extracted with H2O and ethyl acetate, and H. erinaceus H2O extract (1734 mg), and H. erinaceus ethyl acetate extract (257 mg) were obtained. The extracts were stored at -30 °C before use.

RT-PCR
1321N1 cells were seeded into 12-well plates and allowed to grow to confluence. Twenty-four hours before the incubation with mushroom extracts, the medium
was re­placed with serum-free DMEM. The mushroom extract was dissolved in DMSO at 50 mg/ml and then further diluted with serum-free DMEM to the appropriate
concentration. 1321N1 cells were incubated with the mushroom extracts for 3 h. Total RNA of 1321N1 cells was extracted using Tri Pure Isolation Reagent
according to manufacturer’s protocol. First-strand cDNA primed by Oligo(dT) primer was prepared from total RNA (1 mg) using Rever Tra Ace, and was diluted
with water by 5 times to use as a template for the real-time PCR analysis. The primers for amplification and the sizes of respective PCR products were as
follows: NGF (sense: 5′-

CCAAGGGAGCAGTTTCTATCCTGG-3′, and antisense: 5′- GGCAGTTGTCAAGGGAATGCTGAAGTT-3′, for 189bp), b-actin (sense: 5′-AGGGAAATCGTGCGTGACAT-3′, anti­sense: 5
‘-TCCTGCTTGCTGATCCACAT-3′, for 467bp), c­fos (sense: 5 ‘-GTTCTCGGGTTTCAACGCGGACTACGA- GGC-3′, antisense: 5 ‘-GGCACTAGAGACGGACAGATCT- GCGCAAAAGTCC-3′, for
922bp). Real-time PCR was car­ried out in a 20-ml solution volume containing SYBR Premix (10 ml), RT template (3 ml), water (6 ml) and primers (1 ml). The
amplification programs were as follows: NGF, 94 °C for 5 s, 61 °C for 20 s, and 72 °C for 15 s, for 35 cycles; b-actin, 94 °C for 10 s, 56 °C for 20 s, and
72 °C for 30 s, for 22 cy­cles; and c-fos, 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, for 39 cycles. The levels of NGF and c-fos mRNA were
normalized to that of the corresponding b-actin mRNA.

Assay for Neurite Outgrowth in PC12 Cells
1321N1 cells were seeded into 24-well plates and allowed to grow to confluence- Twenty-four hours before the incubation with H. erinaceus extracts
or hericenone D, the medium was replaced with serum-free DMEM. After the incubation with H. eri- naceus extracts or hericenone D for 48 h, the
medium was collected, and centrifuged to remove cells.

PC-12 cells were seeded in a 24-well plate at a density of 7X104 cells/well and cultivated for 24h. After aspiration of the medium, DMEM
containing 10% FCS and 5% HS (100 ml), and 1321N1 cell culture medium prepared as described above (400 ml) were added and incubated for 48 h. On the other
hand, PC-12 cells were stimulated with H. erinaceus extract or NGF directly for 48 h. PC-12 cells were observed under a phase-contrast microscope
and neurite outgrowth was regarded as a sign of differentiation. About one hundred PC-12 cells in each well were evaluated. Cells bearing neu- rites longer
than one cell diameter were regarded as differen­tiated cells. Data are expressed as means±S.E.M. of the val­ues for three wells.

Enzyme Immunoassay of NGF
1321N1 cells were seeded into 24 well plates and allowed to grow to confluence. Twenty-four hours before the incubation with mushroom ex­tracts, the medium
was replaced with serum-free DMEM. The mushroom extract was dissolved in DMSO at a concentration of 50 mg/ml, and then diluted with serum-free DMEM to the
appropriate concentration. 1321N1 cells were incubated with mushroom extracts for 24 h, and then the culture medium was collected. The NGF content in the
medium was meas­ured by ELISA (Emax® Immunoassay System, Promega) ac­cording to the procedures of the manufacturer, except using anti-NGF
secondary antibody.

Immunoblot Analysis
After drug incubation, the medium was removed and cells were lysed in lysis buffer (75 mM Tris-HCl, 2% SDS, 10% glycerol, 3% 2-mercapto-ethanol, and 0.003%
bromophenol blue) for 15 min at room tempera­ture and scraped. Lysates were subjected to SDS-PAGE on a 10% polyacrylamide gel. Separated proteins were
transferred to a polyvinylidene difluoride membrane (Millipore, Biller­ica, MA, U.S.A.) using a semidry blot apparatus. The mem­brane was blocked 3% skim
milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20 (TBST) for 40 min at room temperature. The membrane was washed with TBST and in­cubated with
primary antibody overnight at 4 °C. Then, the membrane was washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2

h at room temperature. The immunoreactive proteins were detected using an ECL Western blotting detection system.

Fig. 1 . Effects of Mushroom Extracts on NGF mRNA Expression in 1321N1 Cells

Isolation and Analysis of Hericenones C, D, and E

1321N1 cells were stimulated with ethanol extract (100 mg/ml) of H. erinaceus (H), P. eryngii (P), G. frondosa (G), A. blazei (A), and A23187 (1 mM, as a positive control) for 3 h at 37 °C, and then NGF gene expression was examined by RT-PCR. Values rep­resent
the means±S.E.M. (n=3). *p<0.05 vs. control (Cont.).

Fig. 2. Effects of Mushroom Extracts on the Secretion of NGF from 1321N1 Cells

Fresh fruiting bodies of

H. erinaceus

(6.0 kg) were extracted with ethanol. The extract was concentrated and fractionated by solvent partition between chloroform and water. The chlo­roform
fraction (25.0 g) was subjected to silica gel column chromatography using toluene-acetone as the eluent. The toluene-acetone (9:1) eluate was subjected to
silica gel col­umn chromatography using hexane-ethyl acetate. The hexane-ethyl acetate (10:1) eluate was further purified by ODS column chromatography
using methanol to give heri- cenone C (210.9 mg), D (35.4 mg), and E (48.0 mg). Their spectral data (

1

H-NMR,

13

C-NMR, IR, and HR-FAB-MS) completely agreed with the data reported.

20

Hericenone C, D, and E in the extract of H. erinaceus were analyzed using HPLC (Shimadzu LC-10 series, SPD-6AV UV-VIS detector Shimadzu set at 260
nm, with a mbondapak C18 3.9X300mm, 10mm column (Waters)). The solvent used for separation was acetonitrile and the flow rate was 1.0 ml/min.

1321N1 cells were stimulated with ethanol extract (50, 100, 150 mg/ml) of H. eri­naceus (H), ethanol extract (100 mg/ml) of P eryngii
(P), G. frondosa (G), A. blazei (A), and A23187 (1 mM, as a positive control) for 24 h at 37 °C. The amount of NGF protein in the culture
media of 1321N1 cells was measured by ELISA. Values represent the means±S.E.M. (n=3). *m<0.05 vs. control (Cont.).

Fig. 3. Effects of H. erinaceus Extract on NGF mRNA Expression in 1321N1 Cells

NGF mRNA Expression in Mouse Brain Tissue

Male ddY mice aged 5 weeks and weighing 33—35 g at the begin­ning of the experiments, were used. Mice were divided into a control group and a

H. erinaceus

group, so that the average of their body weights was equalized. While the control group was given normal diet (MF, Oriental Yeast, Tokyo, Japan) containing
5% dextrin, the

H. erinaceus

group was given MF containing 5% freeze-dried powder of

H. erinaceus.

Animals were given feed and water freely and maintained under con­trolled conditions at a temperature of 24 ±1 °C, relative hu­midity of 45 ±5%, and 12-h
light cycle (09:00—21:00). Mice were fed an experimental diet for 1 or 7 d. At the end of the experiment, mice were killed by cervical dislocation, and the
cerebral cortex and hippocampus were dissected out. The tis­sues were rapidly homogenized in Tri Pure Isolation Reagent (Takara, Japan) and extracted
according to the manufacturer’s protocol. Then, NGF mRNA expression was examined by RT-PCR.

Statistical Methods
Data are expressed as means±S.E.M. Significant differences (^<0.05) were determined by one­way ANOVA, followed by a Tukey’s test.

RESULTS

Firstly, we investigated the NGF-inducing activity of four edible mushrooms, H. erinaceus, P eryngii, G. frondosa, and A. brazei, in
1321N1 human astrocytoma cells. The NGF mRNA level in 1321N1 cells was significantly increased by the calcium ionophore A23187 at a concentration of 1 mM (positive control), and by the ethanol extract of H. erinaceus at a concentration of 100 mg/ml. However, no significant in­crease in
NGF mRNA levels was induced by the ethanol ex­tracts of the other mushrooms (Fig. 1). In addition, the NGF protein level in the culture medium was also
increased by the ethanol extract of H. erinaceus, while the ethanol extracts of other three mushrooms failed to induce such an increase (Fig. 2).
Moreover, we investigated the effects of H2O and ethyl acetate extracts of H. erinaceus on NGF mRNA expres­sion compared with the
effects of the ethanol extract. The ethanol and ethyl acetate extracts promoted NGF mRNA ex-

1321N1 cells were stimulated with H2O extract (0.4, 2.0, 4.0 mg/ml), ethanol extract (50, 100, 150 mg/ml), and ethyl acetate extract (50, 100,
150 mg/ml) for 3h at 37 °C, and then NGF gene expression was examined by RT-PCR. Values represent the means±S.E.M. (n=3). *m<0.05 vs. control (Cont.).

pression in a concentration-dependent manner with similar potency. However, the H2O extract did not increase NGF mRNA expression (Fig. 3).

Next, we examined the NGF mRNA-inducing activity of hericenone C, D and E, components of H. erinaceus reported to be stimulators of NGF
biosynthesis in mouse astroglial cells at 33 mg/ml.20) However, hericenones C, D and E did not increase NGF mRNA expression at 10—100 mg/ml in
1321N1 cells (Fig. 4). Furthermore, we failed to demonstrate that hericenones C, D, and E stimulated NGF mRNA expression in primary cultured rat astroglial
cells (data not shown). Moreover, we analyzed the levels of hericenones C, D, and E

Fig. 4. Effects of Hericenones C, D, and E on NGF mRNA Expression in 1321N1 Cells




1321N1 cells were stimulated with hericenones C, D, and E (10, 30, 100 mg/ml) for 3h at 37 °C, then NGF gene expression was examined by RT-PCR. Values
represent the means±S.E.M. (n=3). *p<0.05 vs. control (Cont.).

in the ethanol extract of H. erinaceus by HPLC, and their concentrations in 100 mg/ml ethanol extract of H. erinaceus were 20 ng/ml for
hericenone C, 4 ng/ml for hericenone D, and 2 ng/ml for hericenone E.

To investigate the physiological effects of H. erinaceus on neurite outgrowth via NGF, PC-12 cells were cultivated for 2d in the
conditioned medium of 1321N1 cells that had been incubated with the ethanol extract of H. erinaceus for 24 h. Considering that NGF significantly
promoted neurite out­growth in PC-12 cells at concentrations of 10 and 100 ng/ml, but not at 0.1 and 1 ng/ml, NGF level in the conditioned medium of 1321N1
cells with H. erinaceus extract (Fig. 2) was too low to promote neurite outgrowth in PC-12 cells. Furthermore, the ethanol extract of H. erinaceus did not pro­mote neurite outgrowth in PC12 cells directly. However, neu- rite outgrowth was significantly promoted by the conditioned
medium of 1321N1 cells incubated with H. erinaceus ethanol extract at concentrations of 125 and 250 mg/ml for 24 h. On the other hand, hericenone
D did not show the effect such as ethanol extract of H. erinaceus (Fig. 5).

To clarify the mechanism underlying NGF induction by H. erinaceus, 1321N1 cells were pretreated with various kinase inhibitors, and then
stimulated with ethanol extract of H. eri- naceus. The promotion of NGF mRNA expression by the ethanol extract of H. erinaceus was
significantly inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125, but not by the MEK inhibitor U0126, the p38 MAPK inhibitor SB203580, the
PKA inhibitor H89, or the PKC inhibitor GF109203X (Fig. 6). In fact, the ethanol extract of H. eri- naceus induced phosphorylation of JNK and its
downstream substrate c-Jun in a time-dependent manner (Fig. 7). Further­more, the ethanol extract enhanced c-fos gene expression

(Fig. 8).

To investigate the effect of H. erinaceus in vivo, we meas­ured NGF mRNA expression in the cortex and hippocampus of mice administered H. erinaceus. The mice in the H. eri- naceus group were fed a diet containing 5% dried powder of H. erinaceus, and the mice of
control group were fed a diet containing 5% dextrin instead of H. erinaceus for 1 or 7 d. The level of NGF mRNA in the hippocampus of mice in the H. erinaceus group was significantly increased compared with that of mice in the control group at the 7th day. On the other hand, NGF mRNA
expression in cortex was not in­creased by H. erinaceus during the test period (Fig. 9).

Fig. 5. Morphological Differentiation of PC 12 Cells Induced by the Medium of 1321N1 Cells Conditioned with Ethanol Extract of H. erinaceus




(A) Morphological changes of PC12 cells. PC12 cells were directly stimulated with ethanol extract (100 mg/ml) of H. erinaceus (H) or NGF (100
ng/ml) for 2 d (upper fig­ures). PC12 cells were stimulated for 2d with 20% DMEM plus 80% 1321N1 culture medium conditioned with 125 or 250 mg/ml of
ethanol extract of H. erinaceus (lower figures). Scale bar: 100 mm. (B) Evaluation of neurite outgrowth. PC12 cells were di­rectly stimulated with
ethanol extract (100 mg/ml) of H. erinaceus (H) or NGF (0.1— 100 ng/ml) for 2 d (left). After 1321N1 cells were incubated for 2d in DMEM
contain­ing 125 or 250 mg/ml of ethanol extract of H. erinaceus (H) or 30 mg/ml of hericenone D (He.D), PC12 cells were cultivated for an
additional 2d in 20% DMEM plus 80% 1321N1 culture medium (right). Values represent the means±S.E.M. (n=3). * ,tp< 0.05.

Fig. 6. Effects of U0126, SP600125, SB203580, H89, and GF109203X on H. erinaceus-Induced NGF mRNA Expression




1321N1 cells were preincubated with U0126 (10 mM), SP600125 (30 mM), SB203580 (3 mM), H89 (10 mM), and GF109203X (5 mM for 20 m before the
addition of H. eri­naceus extract. After incubation with H. erinaceus ethanol extract (100 mg/ml) for 3 h, NGF mRNA expression was
examined by RT-PCR. Values represent the means ± S.E.M. (n=3). *,tp<0.05.

DISCUSSION

In the present study, we demonstrate that the ethanol ex­tract of H. erinaceus promotes the synthesis of NGF in 1321N1 human astrocytoma cells. H. erinaceus alone had NGF-inducing activity among the four mushrooms exam­ined. However, 100 mg/ml of the H. erinaceus ethanol extract

1321N1 cells were stimulated with ethanol extract of H. erinaceus (100 mg/ml) for 3h at 37 °C, and then c-fos gene expression was examined by
RT-PCR. Values repre­sent the means±S.E.M. (n=3). *p<0.05 vs. 0min.

Fig. 9. NGF Gene Expression in the Brains of Mice Fed H. erinaceus

Mice were fed a diet containing 5% H. erinaceus or dextrin (control), and then NGF mRNA expression in their hippocampus and cortex was analyzed by
RT-PCR. Values represent the means±S.E.M. (Day 1 and Day 7: n=8; Day 21: n=3). * p<0.05 vs. Control (- ).




significantly increased NGF mRNA expression but not NGF protein synthesis. Therefore, it is possible that effective con­centrations of H. erinaceus ethanol extract to induce NGF protein synthesis and secretion differs from that to induce NGF mRNA expression, because protein
synthesis/secretion is regulated by several factors.

Fig. 7. H. erinaceus–Induced Phosporylation of JNK and c-Jun in 1321N1 Cells

1321N1 cells were incubated with ethanol extract of H. erinaceus (100 mg/ml) for the indicated times, and then JNK and c-Jun phosphorylation were
analyzed by Western blotting. (A) Time-dependent increase in the level of phosphorylation of JNK induced by H. erinaceus. (B) Time-dependent
increase in the level of phosphorylation of c-Jun induced by H. erinaceus.

Fig. 8. Effects of H. erinaceus Ethanol Extracts on c-fos mRNA Expres­sion in 1321N1 Cells

Morphological differentiation of PC12 cells was promoted by the conditioned media of 1321N1 cells incubated with

H. erinaceus

ethanol extract, suggesting that

H. erinaceus

stim­ulates neuronal differentiation

via

an increment in the release of neurotrophic factors, including NGF, from glial cells. However, the NGF concentration elevated by

H. erinaceus

extract is assumed to be too low to promote neurite out­growth in PC-12 cells. Therefore, other factors might be in­volved in neurite outgrowth. In fact,
astrocytes secrete other neurotrophins, cytokines and growth factors, some of which influence morphological change or facilitate NGF-induced
differentiation in PC12 cells.

226—28)

It has been reported that phorbol esters enhance PKC-de- pendent NGF synthesis in primary mice astrocytes, and AP- 1, one of the targets for PKC, was
assumed to regulate NGF gene expression.20 In fact, there is an AP-1 consensus se­quence (TRE: TPA-response element) downstream of the TATA box
at the junction of the exon I/intron I region of the NGF gene.30) AP-1 consists of homo or hetero dimers of Jun/ Jun or Fos/Jun, which bind to
DNA at the AP-1 site TRE. In the present study, the enhancement of NGF gene expression by H. erinaceus was inhibited by the JNK inhibitor
SP600125, and H. erinaceus caused phosphorylation of JNK. These re­sults suggest that JNK is involved in the enhancement of NGF gene expression
induced by H. erinaceus. JNK is the predominant kinase to phosphorylate c-Jun.31) Furthermore, H. erinaceus enhanced c-Jun
phosphorylation and c-fos gene expression as well as JNK phosphorylation. The activation of AP-1 by H. erinaceus is assumed to participate in NGF
gene expression downstream of JNK, but PKC is not involved in this signaling pathway, because of a lack of inhibitory action with the PKC inhibitor
GF109203X.

It has been reported that the active components of H. eri- naceus are the hericenones C—H, which stimulate NGF pro­tein synthesis in mouse or rat
astrocytes.19—21) However, heri­cenones C, D, and E did not exhibit NGF-promoting activity at all under the present experimental condition using
1321N1 human astrocytoma cells. In addition, the concentrations of hericenones in the ethanol extract were very low (the concen­trations of hericenones C,
D, and E in the 100 mg/ml ethanol extract of H. erinaceus were 20, 4, and 2 ng/ml, respectively) compared to their effective concentration (33
mg/ml) as shown in a previous report.20) These results, therefore, raise the pos­sibility that H. erinaceus has unknown active
compounds that promote NGF expression, other than hericenones, which are lipid-soluble (soluble in ethanol and/or ethyl acetate).

Furthermore, the oral administration of H. erinaceus in­creased NGF mRNA expression in the mouse hippocampus. This result suggests the possibility
that the active compound could be absorbed into blood and delivered into the central nervous system through the blood-brain barrier. The hip­pocampus is
postulated to encode working memory.32) The increase in the level of NGF mRNA in the hippocampus sug­gests the potential of H. erinaceus to act on the central ner­vous system in vivo. However, we could not elucidate why

H. erinaceus
increased NGF mRNA expression in the hip­pocampus but not in the cortex. This difference might result from variations of expression level of signaling
molecules re­lated to JNK signaling pathway or kinetic difference of active components of H. erinaceus such as brain distribution and metabolism
in these tissues.

On the other hand, the mycelia of H. erinaceus are known to contain erinacines, which also stimulate NGF synthe- sis.22 24) It has been
reported that oral administration of eri- nacine A significantly increases the level of NGF in the rat locus coeruleus and hippocampus, but not in the
cerebral cortex.33) However, it has not yet been reported that the fruit body of H. erinaceus contains erinacines. Thus, it is
neces­sary to reevaluate whether fruit bodies contain erinacines, and to examine the existence of unknown derivatives with NGF-inducing activity in the
fruit bodies of H. erinaceus.

In conclusion, H. erinaceus contains active compounds that stimulate NGF synthesis via activation of the JNK path­way; these compounds
are not hericenones.

Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 18790039 to Y. O. and No.
19659011 to N. N.), from the Ministry of Education, Culture, Sports, Science and Technology (No. 18058002 to N. N.) of Japan, and from Hokuto Life Science
Foundation.

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1
To whom correspondence should be addressed. e-mail: nakahata@mail.pharm.tohoku.ac.jp

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