Neuroregenerative Potential of Lion’s Mane Mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (Higher Basidiomycetes), in the Treatment of Peripheral Nerve Injury (Review

Neuroregenerative Potential of Lion’s Mane Mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (Higher Basidiomycetes), in the Treatment of Peripheral Nerve Injury (Review

Kah-Hui Wong,1,3 Murali Naidu,1,3 Pamela David,1,3 Robiah Bakar,1 & Vikineswary Sabaratnam*2,3
1Department of Anatomy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; 2Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; 3Mushroom Research Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia
*Address all correspondence to: Vikineswary Sabaratnam, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; Tel.: +603 79674349; Fax: +603 79674178; viki@um.edu.my.
ABSTRACT: We present a model case study of the activity of aqueous extract of Hericium erinaceus fresh fruit bodies in promoting functional recovery following crush injury to the peroneal nerve in adult female Sprague-Dawley rats. The aim was to explore the possible use of this mushroom in nerve repair. The activities of aqueous extract were compared to activities exhibited by mecobalamin (vitamin B12), which has been widely used in the treatment of peripheral nerve disorders. Analysis of walking track indicated that return of hind limb function and normal toe spreading occurred earlier in treated groups than in the negative control (non-treated) group. Regeneration of axons and reinnervation of motor endplates/neuromuscular junction in extensor digitorum longus muscle of rats in treated groups developed better than in the negative control group. Further, immunofluorescence studies also showed that dorsal root ganglia neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt and MAPK signaling pathways as well as c-Jun and c-Fos genes compared to the negative control group. Akt cascade plays a major role in mediating neurotrophin-promoted cell survival, while MAPK cascade is involved in mediating neurite outgrowth. Immediate early gene expression was also involved in the cascade of events leading to regeneration. Local axonal protein synthetic machinery was also enhanced in the distal segments of crushed nerves in treated groups. Therefore, daily oral administration of H. erinaceus could promote the regeneration of injured rat peroneal nerve in the early stage of recovery.
KEY WORDS: medicinal mushrooms, Hericium erinaceus, functional recovery, peripheral nerve, crush injury, toe-spreading reflex, extensor digitorum longus muscle, axonal regeneration, axonal reinnervation, dorsal root ganglia
ABBREVIATIONS: Akt: protein kinase B; Ca2+: calcium ion; CREB: cAMP response element-binding protein; CES-D: Center for Epidemiologic Studies Depression Scale; CNS: central nervous system; DRG: dorsal root ganglia; EDL: extensor digitorum longus; ERK: extracellular signal-regulated kinases; HDS-R: Hasegawa Dementia Scale; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; ICI: Indefinite Complaints Index; JNK: c-Jun N-terminal kinase; KMI: Kupperman Menopausal Index; MAPK: mitogen-activated protein kinase; MCA: middle cerebral artery; MMP-1: matrix metalloproteinase-1; mRNA: messenger RNA; MRSA: methicillin-resistant Staphylococcus aureus; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NF-200: neurofilament heavy; NGF: nerve growth factor; PFI: peroneal functional index; PLF: print length factor; PNS: peripheral nervous system; PSQI: Pittsburgh Sleep Quality Index; TIMP-1: tissue inhibitor of metalloproteinases 1; TSF: toe-spread factor; w/v: weight per volume
I. INTRODUCTION
Hericium erinaceus (Bull.: Fr.) Pers. (Hericiaceae, higher Basidiomycetes, also known as Lion’s Mane, Monkey’s Head, Hedgehog Mushroom, Satyr’s Beard, Pom Pom Blanc, Igelstachelbart, and Yamabushitake) is one of the edible and medicinal mushrooms distributed in Asia, Europe, and North America.1 It is a temperate mushroom that requires cool temperatures of 18°C to 24°C to produce fruit bodies. The nutritional and medicinal properties of H. erinaceus grown in low temperature conditions are well known and documented in Europe, China, and Japan.1 Since 2000, it has been successfully domesticated via adaptation to tropical climate in Ma428
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Peripheral Nerve Regeneration by Hericium erinaceus
these methods only provide a regenerative environment
for injured nerves. Recovery of function
depends on various local and systemic factors. Regeneration
of axons from the proximal stump of
an injured nerve to the distal nerve stump is one
of the most important factors in reinnervation of
peripheral tissue.
Studies have shown that locally applied neurotrophins
can enhance survival of damaged neurons
and regrowth of lesioned axons in the central
and peripheral nervous systems in rats.5 However,
in situ treatment is not an ideal treatment pattern.
The beneficial effect of systemically administered
neurotrophins on axonal regeneration is largely
limited by enzymatic degradation. In addition,
systemically delivered neurotrophins show unexpected
side effects such as the toxicity of the
circulating protein.6 Therefore, it is important to
explore substances that can produce neurotrophin-
like effects on axonal regeneration without
toxicity problems.
Mushrooms can be good candidates to induce
laysia. This mushroom grows and produces fruit
bodies in lowlands of tropical temperature (Fig. 1).
Peripheral nerve problems are common and
encompass a large spectrum of traumatic injuries,
diseases, tumors, and iatrogenic lesions. The incidence
of traumatic injuries is estimated as more
than 500,000 new patients annually.2 Injuries to
peripheral nerves result in partial or total loss of
motor, sensory, and autonomic functions in the
involved segments of the body. Nerve crush injury
lends itself to investigation of the intrinsic
cellular and molecular events that intervene in
peripheral nerve regeneration, and to assessment
of factors such as drugs that might enhance the
speed of regeneration and the effectiveness of reinnervation.
2 It is known that after the initial injury,
free oxygen radicals increase and cause further
tissue damage.3
Traditionally, functional nerve defects have
been remedied by many methods, including nerve
transfer, nerve grafts, artificial nerve conduit
bridging, and end-to-side neurorrhaphy.4 However,
FIGURE 1. Hericium erinaceus fresh fruit bodies grown in tropical climate of Malaysia.
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Wong et al.
neuronal differentiation and promote neuronal survival.
1,7 Recently, much attention has been given to
medicinal mushrooms’ neurotrophic effects8 and
stimulation of nerve growth factor (NGF) synthesis
in the brain.9 This raises the possibility that medicinal
mushrooms may promote peripheral nerve
regeneration after injury through the enhancement
of NGF production. They are expected to have a
regenerative action on the injured tissues in peripheral
nerve disorders.
Research on the medicinal value of H. erinaceus
grown in Malaysia, a tropical country, is
minimal and has not been explored. In an earlier
study, Wong et al.10 had reported that aqueous extract
of H. erinaceus grown in a tropical environment
could stimulate neurite outgrowth of neural
hybrid clone NG108-15 cells. However, there is a
paucity of information on the nerve regeneration
and repair properties of the mushroom.
II. NATURAL HABITAT AND ORIGIN OF
H. ERINACEUS
H. erinaceus is a saprophytic inhabitant on
dead trunks of hardwoods, including oak, walnut,
beech, maple, sycamore, elm, and other broadleaf
trees. It is found most frequently on logs or
stumps, and is one of the wood-destroying fungi
that cause white rot.11 The first report on the cultivation
of H. erinaceus was published in China in
1988.12 It has been cultivated by artificial log using
bottles and polypropylene bags, making it possible
to constantly put this mushroom into market year
round.12
H. erinaceus has been well-known for hundreds
of years and treasured in traditional Chinese
and Japanese cookery and herbal medicine.
In China, it is called Houtou, as its fruit bodies
look like the head of a baby monkey, and Shishigashira
(Lion’s Head). It is one of the famous four
dishes in China (the other three are sea cucumber,
bear palm, and bird‘s nest).13 In Japan, it is
called Yamabushitake because it resembles the
ornamental cloth worn by Yamabushi—Buddhist
monks practicing asceticism in the mountains. It
is also called Jokotake (funnel-like), Usagitake
(rabbit-like), and Harisenbontake (porcupine
fish-like) according to its appearance. Japanese
scientists have studied and confirmed the biological
activities of H. erinaceus as a highly prized
medicinal mushroom.13
III. MEDICINAL PROPERTIES OF
H. ERINACEUS
The health benefits of H. erinaceus as a curative
for problems of digestive tract such as stomach
and duodenal ulcers are widely known among Chinese
doctors. The effectiveness of H. erinaceus
tablets in the treatment of ulcers, inflammations,
and tumors of the alimentary canal was proven in
the clinical trial subjects of Shanghai Third People’s
Hospital.14 Ingestion of this mushroom was
reported to have a remarkable effect in extending
the life of cancer patients. Pills were used in the
treatment of gastric and esophageal carcinoma.15
Further, sandwich biscuits supplemented with the
fruit bodies were used in the prevention and treatment
of nutritional anemia of preschool children.16
Hericenones A and B (cytotoxic phenols)17
and a novel fatty acid18,19 isolated from fruit bodies,
as well as erinapyrones A and B (γ-pyrones),20
hericenes A, B, and C (phenolic derivatives),21 and
erinapyrone C (γ-dihydropyrone)21 isolated from
mycelium, exhibited cytotoxicity against HeLa
cells. Mori et al.22 found that hericenone B had
strong antiplatelet activity, and it might be a novel
compound for antithrombotic therapy possessing a
novel mechanism. Hericenone B has been shown
to specifically inhibit collagen-induced platelet
aggregation through the inhibition of upstream of
arachidonic acid liberation in integrin α2/β1 signaling.
Fifteen polysaccharides were isolated from
hot-water extracts of H. erinaceus fruit bodies.
Five of them showed antitumor activity and prolonged
the longevity of the hosts. These highmolecular-
weight compounds were identified as
xylan, glucoxylan, heteroxyloglucan, and galactoxyloglucan.
1 Lectin, which inhibits erythrocyte
aggregation,23 and hericerins,24 with an inhibitory
effect on pine pollen germination and tea pollen
growth, were also isolated from H. erinaceus fruit
bodies. A rhamnoglucogalactan fraction known as
(1→3)-β-D-glucan, which inhibits the growth of
tumor, was isolated from alkaline extract of fruit
bodies.25 Xu et al.26 showed that polysaccharides of
H. erinaceus possess anti-skin-aging activities by
enhancing skin antioxidant enzymes, MMP-1 and
TIMP-1 activities, and collagen protein levels in
aged rats.
An ethanol extract of H. erinaceus promoted
an antimutagenic effect, as examined by the Ames
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Peripheral Nerve Regeneration by Hericium erinaceus
test.27 Methanol extract of fruit bodies was found
to have a hypoglycemic effect and reduce elevation
rates of serum triglyceride and total cholesterol
levels when administered to streptozotocin-induced
diabetic rats.28 An exo-biopolymer produced
from a submerged culture of H. erinaceus reduced
the level of plasma total cholesterol, low-density
lipoprotein cholesterol, triglyceride, phospholipids,
atherogenic index, and hepatic HMG-CoA reductase
activity. It also preserved the high-density
lipoprotein at a relatively high level in dietaryinduced
hyperlipidemic rats. These could help reduce
the risk of atherosclerosis.29
Chemotherapeutic resistance to drugs is a major
obstacle to the successful treatment of human
hepatocellular carcinoma. Lee and Hong30 demonstrated
that purified components of H. erinaceus
act as enhancers to sensitize doxorubicin (Dox)-
mediated apoptotic signaling, and this sensitization
can be achieved by reducing c-FLIP expression via
JNK activation and enhancing intracellular Dox
accumulation via the inhibition of NF-kB activity.
These findings suggest that H. erinaceus in combination
with Dox serves as an effective tool for
treating drug-resistant human hepatocellular carcinoma.
Methicillin-resistant Staphylococcus aureus
(MRSA) is currently one of the most prevalent
pathogens in nosocomial infections. Erinacines
A, B,31 and K32 were isolated as anti-MRSA compounds
from the mycelium. A clinical trial in Japan
showed that MRSA in some patients disappeared
after they consumed the mushroom. On top of that,
two novel and one known chlorinated orcinol derivatives
were also isolated from mycelium that
exhibited antimicrobial activities against Bacillus
subtilis, Saccharomyces cerevisiae, Verticillium
dahliae, and Aspergillus niger.33
It has been shown that cultivation conditions
did not affect selected bioactive properties of H.
erinaceus grown in tropical Malaysia.10,34–36 The
total phenolic content and total antioxidant activity
in the oven-dried fruit bodies was higher compared
to the freeze-dried or fresh fruit bodies.34 This may
be due to generation and accumulation of Maillard’s
reaction products during the heating process,
which are known to have antioxidant properties.
Various extracts of H. erinaceus also inhibited the
growth of pathogenic bacteria but not of the tested
fungi.34 Besides that, freeze-dried fruit bodies
exhibited a cytoprotective effect against ethanolinduced
gastric mucosal injury in rats,35 and topical
application of aqueous extract of fruit bodies
accelerated the rate of wound healing enclosure in
rats.36
Two of our articles on H. erinaceus10,37 were
published in the International Journal of Medicinal
Mushrooms and one38 was published in
Evidence-based Complementary and Alternative
Medicine. These articles highlighted the ability of
H. erinaceus to stimulate neurite outgrowth and
promote regeneration after peripheral nerve injury.
The hot-water extract of dried fruit bodies
is used as healthy drink. It has been a practice to
extract the mushroom with water or pickle it in
brewed wine. A sport drink named Houtou was
employed in the 11th Asia Sport Festival (1990) in
China.13 Alcohol-based extracts prepared from pure
mycelium cultured on organic brown rice are produced
by Fungi Perfecti Co. (Olympia, WA, USA)
and tablets of polysaccharide are manufactured
by Shanghai Baixin Edible and Medicinal Fungi
of the Edible Fungi Institute (Shanghai, China). In
Malaysia, capsules containing 100% pure powder
of H. erinaceus are manufactured by Reishilab
(Selangor, Malaysia) and marketed under ANI Hericium
450 mg, and an essence is produced by Vita
Agrotech (Selangor, Malaysia) by combining three
types of mushrooms, namely Agaricus brasiliensis,
H. erinaceus, and Auricularia auricula-judae.
IV. NEUROPROTECTION AFTER PERIPHERAL
NERVE INJURY
Nerve regeneration is a complex phenomenon that
has gained growing interest among scientists for
many years. Neurons can be separated into central
nervous system (CNS) and peripheral nervous
system (PNS), which have different anatomical
structures and regenerative ability. In mammals,
the central neurons without myelin sheaths are difficult
to regenerate. In contrast to CNS, PNS with
myelin sheaths is capable of regeneration following
injury.39
The characterization of neurite formation,
maturation, and collapse/resorption is an area of
interest because these cellular processes are essential
for the connection between sensory and motor
neurons. Neurites are particularly interesting in
relation to neuropathological disorders, neuronal
injury/regeneration, and neuropharmacological
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Wong et al.
research and screening.40 Damage due to nerve
transection was once believed to be irreversible.
However, it has been proved that the regeneration
of damaged nerve fibers is an active process under
the control of molecules able to inhibit and repulse
growing neurites.41 Therefore, major efforts in nervous
system drug discovery research are focused
on the identification of compounds that affect the
growth of neurites.
Peripheral nerve injuries are encountered in
clinical practice due to accidental trauma, acute
compression, or surgery. Traffic crashes usually
induce traumatic nerve injury resulting in the disruption
of intraneural circulation.42 This condition
in turn induces demyelinization, remyelinization,
axonal degeneration, axonal regeneration, focal,
multifocal, or diffuse nerve fiber loss, and endoneural
edema.43 After injury, free radicals are elevated,
producing more tissue damage and retarding
the recovery process.3
Studies of neurotrophic factors are aimed
at finding new and more effective treatments for
nerve disorders. These substances, which are produced
naturally by the body, protect neurons from
injury and encourage their survival. Neurotrophic
factors also maintain normal function in mature
nerve cells and stimulate axonal regeneration. The
effects of these powerful chemicals on the PNS
may eventually lead to treatments that can reverse
nerve damage and cure peripheral nerve disorders.
44
Drug therapy is commonly used to promote
axonal regeneration in the treatment of nerve injuries,
45 including crush injury, transected injury,
or large nerve gap. Therefore, searching for effective
drugs, especially those of natural origin, has
gained extensive attention. Immunosuppressant
and anti-inflammatory drugs may accelerate the
rate of nerve regeneration following injury. However,
they are associated with severe side effects
such as high blood pressure, kidney problems, and
liver disorders.46 Therefore, it is important to search
for natural substances and possible new drug treatments
that could affect nerve regeneration.
V. NEUROLOGICAL ACTIVITIES OF
MUSHROOMS INCLUDING H. ERINACEUS
Much interest has been focused on the potential
of using medicinal mushrooms as neuroprotective
agents. Compounds that stimulate neurite outgrowth
and act as substitutes for NGF or exhibit
NGF-like activities that cause neurons and myelin
to regrow have been extensively studied.
Sarcodon scabrosus, a bitter mushroom, contains
diterpenoid compounds with a unique chemical
structure, such as the sarcodonins A-H.47 Further
investigations on the mushroom had led to the
isolation of new cyathane diterpenoids—scabronines
A,48 B, C, D, E, F,49 G,50 K, and L.51 They
have been tested for their ability to induce NGF
secretion from 1321N1 human astrocytoma cells
and induce neuritogenesis in PC12 cells (rat pheochromocytoma
cells). Scabronines increased the
expression of mRNA for NGF, and the secretion of
NGF from 1321N1 cells in a concentration-dependent
mechanism.50 Among different scabronines,
scabronine G methyl ester (SG-ME) was found
to be the most active, and the mechanism of action
was studied extensively. Cyrneines A, B,52 C,
and D53 from S. cyrneus and glaucopine C from S.
glaucopus54 were tested for their ability to induce
NGF production from 1321N1 cells, but they were
found to be much less active than scabronine G.53
Additionally, extract of Grifola frondosa was
found to induce neuronal differentiation of PC12
cells by phosphorylation of ERK1/2.55 An active
component was isolated from the extract and identified
as lysophosphatidylethanolamine. Lysophosphatidylethanolamine
is a phosphate-dylethanol
amine molecule lacking a fatty acid. Polysaccharides
in aqueous extract of Ganoderma lucidum
have been reported to induce neuronal differentiation
and prevent NGF-dependent apoptosis of rat
pheochromocytoma PC12 neuronal cells.56
Cordyceps is one of a growing number of traditional
Chinese medicines being considered as
cures for modern human diseases. Nucleosides,
especially adenosine, have been found to stimulate
axon growth in vitro and in the adult CNS.57 Cerebrosides
known as termitomycesphins A-D isolated
from Termitomyces albuminosus58 and aqueous
extract of Lignosus rhinoceros sclerotium59
were shown to stimulate neurite outgrowth and
induce neuronal differentiation of PC12 cells. L.
rhinocerus is a unique national treasure and is also
known as “cendawan susu rimau” in the Malay
language, or tiger’s milk mushroom in English.
The effect on the nervous system of some
compounds isolated from mushrooms, such as
muscarine, muscimol, and ibotenic acid, has been
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Peripheral Nerve Regeneration by Hericium erinaceus
studied.60 Neurotrophic effects of extracts of H.
erinaceus,61,62 Amanita sp.,63,64 Lentinus edodes,65,66
hallucinogenic mushroom Psilocybe cubensis,67
and other higher Basidiomycetes68,69 have also
been reported.
Preliminary studies suggest that H. erinaceus
may be useful in the treatment of a number
of neurological disorders. Kawagishi et al.9 reported
that the most promising activity of H. erinaceus is
the stimulation of NGF synthesis by hericenones
from fruit bodies and by erinacines from mycelium.
Phenol derivatives identified as hericenones C, D,
E,70 F, G, and H71 were isolated from fruit bodies of
H. erinaceus. Diterpene-xyloside possessing cyathan
skeletons derivatives identified as erinacines
A, B, C,72 D,73 E, F, G,74 H, I,75 P,76 Q,77 J, K,32 and
R,78 as well as erinacol,79 were isolated from mycelium
of H. erinaceus. These compounds accelerated
the synthesis of NGF and are observed to stimulate
neurons to regrow. Shimbo et al.80 showed that erinacine
A increased catecholamine and NGF content
in the CNS of rats. Rats treated with this compound
had increased levels of both noradrenaline and homovanillic
acid in the locus coeruleus at 4 weeks
of age and increased levels of NGF in both LC and
hippocampus at 5 weeks of age.
It has been reported that NGF has a protective
or inducible effect on neuronal cell death through
the Trk and p75 pathway.81 An exo-polysaccharide
of H. erinaceus could induce neuronal differentiation
and promote neuronal survival.82 An endoplasmic
reticulum stress-attenuating compound known
as 3-hydroxyhericenone F83 and dilinoleoyl phosphatidylethanolamine84
purified from an extract
of H. erinaceus dried fruit bodies were shown to
reduce endoplasmic reticulum stress-induced cell
death, which might reduce the risk of neurodegenerative
disease–induced cell death. Extracts of H.
erinaceus also induced phosphorylation of c-Jun
N-terminal kinase (JNK) and its downstream substrate
c-Jun, and increased c-Fos expression, suggesting
that H. erinaceus promotes NGF gene expression
via JNK signaling.85
Furthermore, the efficacy of H. erinaceus in
vivo has been examined. Mice that were given feed
containing 5% dry powder for 7 days showed an
increase in the level of NGF mRNA expression
in the hippocampus.85 Hazekawa et al.86 investigated
the neuroprotective effect of H. erinaceus
on ischemic brain damage in a middle cerebral artery
(MCA) occlusion model in mice. Infarct volumes
were markedly reduced in mice receiving 14
days of H. erinaceus treatment at a dose of 300
mg/kg prior to 4-hr MCA occlusion. Pre-treatment
significantly increased the levels of NGF in both
the cortex and striatum of mice subjected to 4-hr
MCA occlusion. Therefore, H. erinaceus provides
neuroprotection against focal cerebral ischemia by
increasing NGF levels and may be clinically useful
for preventing cerebral infarction.
A double-blind trial was performed on 50- to
80-year-old Japanese men and women diagnosed
with mild cognitive impairment in order to examine
the efficacy of oral administration of H. erinaceus in
improving cognitive functioning by using a cognitive
function scale based on the Revised Hasegawa
Dementia Scale (HDS-R).87 The subjects of the H.
erinaceus group took four 250-mg tablets containing
96% of dry powder three times a day for 16
weeks. Cognitive function scale scores increased
with the duration of intake. Laboratory tests showed
no adverse effect of H. erinaceus and it was effective
in improving mild cognitive impairment.87
Nagano et al.88 investigated the clinical effects
of H. erinaceus on menopause, depression, sleep
quality, and indefinite complaints, by means of a
questionnaire investigation. They detected a difference
between groups using the Kupperman Menopausal
Index (KMI), the Center for Epidemiologic
Studies Depression Scale (CES-D), the Pittsburgh
Sleep Quality Index (PSQI), and the Indefinite
Complaints Index (ICI). Their results showed that
H. erinaceus intake may reduce depression and
anxiety. It may also be relevant to frustration and
palpitation because H. erinaceus intake lowers
scores for the terms frustrating and palpitation.
For this reason, they suggested a mechanism that
is different from the NGF-enhancing action of H.
erinaceus. Mori et al.89 examined the effects of H.
erinaceus on amyloid β(25-35) peptide–induced
learning and memory deficits in mice. H. erinaceus
prevented impairments of spatial short-term and
visual recognition memory induced by intracerebroventricular
administration of amyloid β(25-35)
peptide and may be useful in the prevention of
cognitive dysfunction.
Neurotrophic effects of H. erinaceus dried fruit
bodies on the neurons of hippocampal slices in rats
(nerve cell spike activity) have been studied.61,62
They exert neurotrophic action or excitation of neuVolume
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Wong et al.
rons at concentrations that did not affect the growth
of nerve cells in vitro, and also did not evoke a toxic
effect or nerve cell damage. Neurons of the hippocampus
as part of the limbic system are closely
related to the regulation of motivation-emotional
responses, memory, and other mental functions.62 A
characteristic feature of this structure is an extreme
sensitivity to slight changes in the intercellular liquid
composition, which is considerably greater than
that of neocortex and cerebellum neurons.90 The
extract also promoted normal development of cultivated
cerebellar cells and demonstrated a regulatory
effect on the process of myelin genesis in vitro after
myelin damage.91 The myelin sheath is an important
component of neurons that is involved in the transmission
of nerve messages. Injury of myelin’s compact
structure leads to an impairment and severe illness
of the nerve system.
VI. A MODEL FOR THE STUDY OF
PERIPHERAL NERVE REGENERATION
FOLLOWING CRUSH INJURY
We investigated the effects of aqueous extract of H.
erinaceus fresh fruit bodies on the recovery of peroneal
nerve of the right hind limb following crush
injury in adult female Sprague-Dawley rats.37,38 The
study included: (1) functional recovery of the peroneal
nerve as assessed by walking track analysis and
toe-spreading reflex, (2) axonal reinnervation pattern
of the extensor digitorum longus (EDL) muscle
in unoperated and operated limbs, (3) expression of
the protein kinase B (Akt) and mitogen-activated
protein kinase (MAPK) pathways, and expression
of c-Jun and c-Fos in the ipsilateral DRG from injured
nerve, and (4) axonal regeneration and protein
synthesis in the injured nerves.
A. Preparation of Aqueous Extract
Mushrooms have always been prepared for medicinal
use by hot-water extraction, as in brewing
of teas or decoctions in traditional Chinese medicine
for prevention of oxidative stress-related diseases.
92 In our study, H. erinaceus fresh fruit bodies
were obtained from Vita Agrotech in Tanjung
Sepat, Selangor, Malaysia. Ten grams of fresh fruit
bodies were boiled with 10 mL of distilled water
[ratio of 1:1 (w/v)] for 30 min, cooled, and filtered.
8 In an earlier study of stimulation of neurite
outgrowth activity, aqueous extract of fresh fruit
bodies has been shown to be a potent enhancer of
neurite outgrowth activity in vitro.8 Neurotrophic
components of H. erinaceus dissolved readily in
water and retained their effectiveness even after
one month of storage at 4°C.8 The aqueous extract
was used within a week after preparation.
B. Induction of Peripheral Nerve Injury
The peroneal nerve is a branch of sciatic nerve,
which controls movement and sensation of the
lower leg, foot, and toes. The sciatic nerve contains
a larger number of fibers compared to the peroneal
nerve. Injury to the peroneal nerve normally has
a greater potential for regeneration and recovers
faster than injury to the sciatic nerve.93 Therefore,
we used peroneal nerve to achieve complete functional
recovery following crush injury.
Eighteen adult female Sprague-Dawley rats
weighing 200 ± 20 g were randomly assigned to
three groups of six rats each. A negative control
group received daily oral administration of distilled
water (10 mL/kg body weight/day); the experimental
group received aqueous extract of fresh
fruit bodies (10 mL/kg body weight/day); and the
positive control group received mecobalamin (130
μg/kg body weight/day). All treatments were administered
with an 18-gauge stainless steel feeding
needle for 14 days as pre-treatment before surgery.
After pre-treatment, the right sciatic nerve
and its two major branches were exposed through
a gluteal muscle-splitting incision. A crush injury
was created using a fine watchmaker forceps (no.
4) for 10 s on the peroneal nerve at 10 mm from the
EDL muscle, and complete crush was confirmed
by the presence of a translucent band across the
nerve (Fig. 2). After surgery, distilled water, aqueous
extracts, or mecobalamin was continuously fed
for another 20 days or until the complete return of
hind limb function.
Pre-treatment was used to examine the prevention
effect of the aqueous extract. There are no
hard rules regarding the period of pre-treatment
because medicinal mushrooms will not cause any
side effects. Pre-treatment was used to examine the
prevention effect of the aqueous extract. There are
no hard rules regarding the period of pre-treatment
because medicinal mushrooms will not cause any
side effects. However, pre-treatment must be done
at least 7 days before injury because oral drugs
take time to be absorbed and get into the general
blood circulation.
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Peripheral Nerve Regeneration by Hericium erinaceus
C. Assessment of Peripheral Nerve
Regeneration
The effects of aqueous extract of H. erinaceus on
the rate of hind limb recovery were assessed by
walking track analysis93 and toe-spreading reflex.94
Rats were allowed conditioning trials in a walking
track (8.2 × 42 cm) darkened at one end. The rat’s
hind limbs were dipped in Chinese ink, and the rat
was permitted to walk down the track, leaving its
hind footprints on the paper (Fig. 3). The peroneal
functional index (PFI) was determined based on
multiple linear regression analysis of factors derived
from measurements of walking tracks in rats with
peroneal nerve injury. The factors that contributed
to PFI were print length factor (PLF) and toe-spread
factor (TSF). As for toe-spreading reflex, activities
were classified according to the affected right hind
limb: no spreading, minimal spreading, average
spreading, and normal spreading.
As for microscopic investigation, frozen sections
(50 μm thick) of EDL muscle were cut longitudinally
in a cryostat microtome at –20°C and
stained for neuromuscular junction by a combined
silver-cholinesterase method.95 Expression of Akt
and MAPK signaling pathways as well as c-Jun
and c-Fos genes were also studied in DRG; axonal
regeneration and axonal protein synthesis96 were
studied in peroneal nerve by an immunohistochemical
method.
VII. THE POTENTIAL OF H. ERINACEUS IN
PERIPHERAL NERVE REGENERATION
Accumulating lines of evidence show that free
radicals generated after injury play the predominant
role in retarding functional recovery. Studies
on crush injury models in peripheral nerves have
shown better functional recovery when therapies
were directed against ischemia-reperfusion injury,
using antioxidants, lipid peroxidation inhibitors,
and anti-inflammatory agents.97,98 With this
in mind, medicinal mushrooms may be potential
alternatives to neurotrophic factors for peripheral
nerve repair. Although less effective than neurotrophins,
aqueous extract of H. erinaceus may be
used to assist in the application of neurotrophins
to enhance axonal regeneration in the nervous
system and cut down the dosage of neurotrophins
to reduce the toxic effects in humans. Our
findings showed that daily oral administration of
aqueous extract of H. erinaceus fresh fruit bodies
enhanced recovery of damaged peripheral nerve
in rats.37,38
A. The Effects of Aqueous Extract of
Hericium erinaceus on Functional
Recovery Following Crush Injury
It was observed that functional recovery in the
mecobalamin and aqueous extract groups was on
day 4, while the crushed limb in the negative con-
FIGURE 2. Complete crush of peroneal nerve is confirmed by presence of a translucent band (as indicated by an
arrow) across the nerve.
Volume 14, Number 5, 2012 435
Wong et al.
trol group remained dysfunctional (Fig. 4). Rats
in the negative control group showed clumping
of toes and dragging of the injured foot. On the
other hand, the mecobalamin and aqueous extract
groups demonstrated toe spreading (Fig. 5) and
clear footprints on the walking tracks. The peroneal
functional index in the aqueous extract group
returned to pre-surgery values 4 to 7 days earlier
than for rats in the negative control group as measured
by walking track analysis, and normal toe
spreading was achieved 5 to 10 days earlier in the
aqueous extract group than in the negative control
group.
After peripheral nerve lesions, a simple, precise,
and inherently meaningful measure is the return
of toe spreading.99 Toe spreading requires reinnervation
of the most distal muscles in the sciatic
distribution, the intrinsic muscles of the foot including
the interossei. Its absence provided visible
evidence of interruption of the nervous pathway
FIGURE 3. Walking track apparatus. Rat in an 8.2 x 42 cm walking track apparatus lined with white office paper. After
the hind limbs of the rat are dipped in Chinese ink, the rat walks towards the darkened end of the corridor.
436 International Journal of Medicinal Mushrooms
Peripheral Nerve Regeneration by Hericium erinaceus
and its reappearance indicated the regeneration of
the nerve and the reestablishment of the nervous
circuit.
Functional data show that the toe-spreading reflex
appeared 2 to 3 days earlier than recovery of
motor function as observed in walking track analysis.
Toe spreading precedes recovery of locomotion
following peroneal nerve crush injury because proper
walking requires coordinated function involving
sensory input, motor response, and cortical integration.
Locomotion includes the return of ankle dorsiflexion
and toe spreading during walking.
B. The Effects of Aqueous Extract of H.
erinaceus on Axonal Reinnervation of
Motor Endplates in EDL Muscle Following
Crush Injury
Adult mammalian muscles recover virtually completely
from temporary denervation provided
that the reinnervation is allowed to proceed unhindered.
100,101 Axonal reinnervation of the EDL
muscle was more enhanced in the aqueous extract
group than in the negative control group after 14
days of crush injury as demonstrated by combined
silver-cholinesterase staining (Fig. 6).
An important difference between the muscles
of the groups occurs with respect to the regenerating
axons. Extensor digitorum longus muscles
of the negative control group contained a mixture
of degenerating and regenerating axons, and migration
of macrophages to remove degenerated
myelin and axon fragments, a process called Wallerian
degeneration.2 The functional connection
between motor neuron and EDL muscle fibers
has not been reestablished at this stage. In rats
treated with mecobalamin and aqueous extract,
loose axon bundles indicate that the regeneration
process is ongoing. Axon bundles are more
compact and the regeneration process is more
advanced after 14 days of crush injury compared
to 7 days after injury. In treated groups, a high
density of regenerating axons reinnervating motor
endplates can be observed. This indicates reestablishment
of connection between motor neuron
and EDL muscle fibers, leading to functional
recovery.38
FIGURE 4. Walking tracks of footprints after 4 days of peroneal nerve crush injury. Arrows indicate footprints of
the operated limb. (A) Footprints in negative control group—distilled water (10 mL/kg body weight/day). The palsy
after interruption of the peroneal nerve is characterized by flexion contracture of the paws (“footdrop”), absence
of toe-spreading reflex, and some dragging of the operated limb. (B) Footprints in positive control group—mecobalamin
(130 μg/kg body weight/day). Clear footprints of the operated limb can be seen. (C) Footprints in aqueous
extract group—H. erinaceus fresh fruit bodies (10 mL/kg body weight/day). Aqueous extract group demonstrated toe
spreading and clear footprints of the operated limbs on the walking tracks.
Volume 14, Number 5, 2012 437
Wong et al.
C. The Effects of Aqueous Extract of H.
erinaceus on Expression of Akt and MAPK
Signaling Pathways as well as c-Jun and
c-Fos Genes in the DRG Following Crush
Injury
Expression of Akt and MAPK pathways as well
as expression of c-Jun and c-Fos in the ipsilateral
DRG from injured nerve was more enhanced in the
aqueous extract group than in the negative control
group after 7 days of crush injury as demonstrated
by an immunohistochemical method. There was
bright immunofluorescence for Akt, MAPK, c-Jun,
and c-Fos in small neurons of ipsilateral DRG from
injured nerve in the aqueous extract group (Fig. 7).
The population of large neurons is defined by the
expression of NF-200 as green fluorescence. Akt,
MAPK, c-Jun, and c-Fos staining as orange fluorescence
was targeted to small neurons. Up-regulation
of Akt, MAPK, c-Jun, and c-Fos is crucial
in facilitating axonal regeneration and subsequent
reinnervation of target muscle, which brings a return
of hind limb function.
Injury to neurons results in complex sequences
of molecular responses that play an important
role in the successful regenerative response and
the eventual recovery of function. In the injured
neurons, the rapid arrival of injury-induced signals
is followed by the induction of transcription factors,
adhesion molecules, growth-associated proteins,
and structural components needed for axonal
regrowth.102 Injury to adult peripheral neurons,
but not to CNS neurons, reactivates the intrinsic
growth capacity and allows regeneration to occur.
Primary sensory neurons with cell bodies in the
DRG provide a useful model system to study the
mechanisms that regulate regeneration.
Peripheral nerve injury induced peripheral
sensitization, causing activation of Akt, MAPK, c-
Jun, and c-Fos in small DRG neurons. These contributed
to pain hypersensitivity found at the site of
tissue damage and inflammation. In general, DRG
neurons can be divided into large (>1200 μm2),
medium (600–1200 μm2), and small (<600 μm2)
neurons. Small neurons respond to thermal, mechanical,
and chemical nociceptive stimulations,
whereas large neurons transmit touch and proprioceptive
sensations.103 Akt, MAPK, c-Jun, or c-Fos
did not co-localize with NF-200 in large DRG
neurons. It is possible that aqueous extract of H.
erinaceus could trigger the expression of protein
kinases and early genes that regulate nociceptive
function and inflammation associated with nerve
recovery.
Activation of Akt, MAPK, c-Jun, and c-Fos
persisted for 1 week after injury until axonal
regeneration occured. The findings are in accordance
with a study by Naidu et al.104 In their
study, immunoreactivity for phospho-Akt was detected
in ipsilateral DRG after 2, 4, and 7 days of
FIGURE 5. Recovery of toe-spreading reflex after 7
days of peroneal nerve crush injury. Arrows indicate the
operated limb. (A) Minimal toe spreading on right limb in
negative control group—distilled water (10 mL/kg body
weight/day). (B) Normal toe spreading on right limb in
treated groups—mecobalamin (130 μg/kg body weight/
day) and aqueous extract of H. erinaceus fresh fruit bodies
(10 mL /kg body weight/day).
438 International Journal of Medicinal Mushrooms
Peripheral Nerve Regeneration by Hericium erinaceus
sciatic nerve crush by using western blot analysis,
whereas clear up-regulation of both phospho p44
MAPK and phospho p42 MAPK was detected after
7 days of injury. In a study using in situ hybridization,
Ito et al.105 reported enhanced gene
expression for PI3K in the hypoglossal motor
neurons following axonal crush in the first week
after injury. Phospho-Akt was clearly expressed
in the normal DRG and nerve, and was up-regulated
during nerve regeneration in the DRG neurons.
The expression of Akt in the normal DRG
and nerve may be required to maintain day-to-day
cell survival, and an up-regulation could be essential
to prevent mass cell death as a result of
nerve injury.
MAPK pathway activation in neurons has
been demonstrated to be important for neurite
outgrowth, regeneration, synaptic plasticity, and
memory functions in mature neurons. Naidu et
al.104 showed that phospho-MAPK was expressed
in normal DRG but confirmed its presence in
the rat sciatic nerves even after injury. This was
probably needed for axonal regeneration. Further,
Cheung et al.56 utilized a model system, PC12
cells, to demonstrate the presence of neuroactive
compounds in G. lucidum. Incubation with the
mushroom extract resulted in a reduction of cell
proliferation and induction of neuronal differentiation
of PC12 cells. The ability of the extract to
induce phosphorylation of ERK1/2 in PC12 cells
suggests that it may be mediated via the ras/ERK
signaling pathway.
Downstream events influenced by crush injury–
activated kinases include up-regulation or activation
of several transcription factors. Activated
Akt and MAPK induce up-regulation and phosphorylation
of the transcription factors c-Jun and
c-Fos into the nucleus, leading to formation of
AP-1 complexes that activate many downstream
genes. In this study, up-regulation of c-Jun and
c-Fos occurred in ipsilateral DRG neurons after
7 days of crush injury. Similar to the study on
signaling pathways, activation of c-Jun and c-Fos
also persisted for 1 week after injury until axonal
FIGURE 6. The morphology of silver-cholinesterase-stained longitudinal section of EDL muscle in normal unoperated
limb and operated limb after 14 days of peroneal nerve crush injury. Yellow arrows indicate the axons. Violet
arrows indicate the degenerating axons. Red arrows indicate polyneuronal innervation. Asterisks indicate the motor
endplates. Scale bar = 500 μm. (A) Normal unoperated limb. Axon bundles are clear and compact. (B) Operated
limb in negative control group—distilled water (10 mL/kg body weight/day). Wallerian degeneration can be detected.
Degenerated axons are being phagocytosed by the cooperative action of denervated Schwann cells and infiltrating
macrophages. (C) Operated limb in positive control group—mecobalamin (130 μg/kg body weight/day). Loose
axon bundles indicate regeneration process is ongoing. (D) Operated limb in aqueous extract group—H. erinaceus
fresh fruit bodies (10 mL/kg body weight/day). Axon bundles are more compact and regeneration process is more
advanced compared to positive control group.
Volume 14, Number 5, 2012 439
Wong et al.
regeneration took place.
c-Jun is a protein that may be important for
successful axonal regeneration. Studies by Broude
et al.106 on adult rat DRG showed that c-Jun was
substantially up-regulated in DRG neurons following
a peripheral axotomy, but after a central
axotomy, only 18% of the neurons expressed c-
Jun. In a study by Chi et al.,107 c-Fos expression in
the lumbar spinal cord increased in the superficial
dorsal horn and deep dorsal horn within hours, and
lasted for at least 4 weeks following sciatic nerve
transection in rats.
FIGURE 7. Expression of Akt, MAPK, c-Jun, and c-Fos in the DRG after crush injury. Double immunofluorescence
staining between expression of signaling pathways or genes (orange) and NF-200 (green) in ipsilateral DRG from
injured nerve. The expressions did not co-localize with large neurons. Although all small DRG neurons are stained
brightly with Akt, MAPK, c-Jun, or c-Fos, immunoreactivity was higher in the ipsilateral DRG from injured nerve in
rats treated with aqueous extract or mecobalamin than in negative control. White arrows indicate large neurons
whereas yellow arrows indicate small neurons. Scale bar = 100 μm. (A-C): Akt activation. (D-F): MAPK activation.
(G-I): c-Jun activation. (J-L) : c-Fos activation.
440 International Journal of Medicinal Mushrooms
Peripheral Nerve Regeneration by Hericium erinaceus
D. The Effects of Aqueous Extract of H.
erinaceus on Axonal Regeneration and
Axonal Protein Synthesis Following Crush
Injury
Crushing of peripheral mammalian axons initiates
the process of axonal regeneration. During
this process, fine-caliber axonal sprouts emerge
from the parent stump of the axon, which remains
attached to the neuronal cell body, and the new
sprouts elongate distally. Provided that environmental
conditions are supportive, regeneration
continues until the new axons elongate sufficiently
to reconnect with appropriate target tissues. The final
stage of the regeneration process involves the
radial growth of the new axons. The cytoskeletal
polymers, microfilaments, microtubules, and neurofilaments
are well known to have a central role
in the regeneration process since they provide the
basis for structure, motility, and final stability of
the new axons.108
On injury to peripheral nerve, the neuronal cell
bodies contributing to the damaged nerve respond
to the insult in various ways. One of the most noted
changes is a decrease in the production and anterograde
transportation of neurofilament protein.109
Consequently, axonal continuity and transport are
disrupted by crush, and the portion distal to the
site of injury is no longer supplied with neurofilament
protein. Neurofilaments function chiefly as
architectural elements that operate by displacing
volume within the neuron and thereby increasing
the volume of the neuron.110 Specifically, neurofilament
proteins are assembled into neurofilaments in
the nerve cell body, and are then transported into
the axon, where they contribute to the radial dimensions
of the axon.
Promotion of axonal regeneration and protein
synthesis in the injured peroneal nerves was more
accelerated in the aqueous extract group than in the
negative control group after 7 days of crush injury.
Anti-neurofilament immunohistochemistry was
used to compare the peroneal nerve regeneration.
FIGURE 8. Representative photomicrographs of longitudinally sectioned peroneal nerves distal to the injury site
after 7 days of peroneal nerve crush injury and the pathologic scale used for depicting these extents of injury or
axonal loss. The green fluorescent strands represent individual axon fibers stained with anti-neurofilament 200 and
blue fluorescent patches indicate DNA. Scale bar = 100 μm. (A): 0 = normal nerve of unoperated limb. (B): 1 = mild
axonal damage of operated limb. (C): 2 = moderate axonal damage of operated limb. (D): 3 = severe axonal damage
of operated limb.
Volume 14, Number 5, 2012 441
Wong et al.
Microscopic evaluation of the axon was performed
in a double-blind fashion by three individuals.
Each peroneal nerve was graded for damage on a
qualitative 4-point scale.111 Normal nerve received
a score of 0. Mild, moderate, and severe axonal
damage received a score of 1, 2, and 3, respectively
(Fig. 8). Axons from each group were evaluated to
determine the proportion of nerves with an injury
greater than or equal to the moderate (≥2) level. In
a normal peroneal nerve section from an unoperated
limb, axons appeared to have normal morphology,
were arranged more densely, and had uniform
neurofilament immunostaining. In crushed peroneal
nerve sections, axonal regeneration distal to the
site of injury can be observed. Regenerating axons
sprouted aberrantly and formed tangled masses or
neuromas. Nerve fibers are nonparallel. Five rats in
the negative control group and two rats in the mecobalamin
or aqueous extract groups demonstrated
moderate or severe axonal damage after 7 days of
crush injury.38
Protein synthesis has been shown to be involved
in axonal regeneration. There was bright
immunofluorescence for nuclear ribonucleoprotein
along the regenerating axons distal to the injury
site after 7 days of crush injury, coincident with the
synthesis of neurofilament proteins. Local protein
synthesis occurred in regenerating axons and was
up-regulated by aqueous extract of H. erinaceus
after injury (Fig. 9). The original dogma was that
necessary components for axonal growth and regeneration
are usually synthesized by the cell body
and sent along the axons by fast or slow axonal
transport to their respective targets, which are usually
hundreds of micrometers away from the cell
body. These processes are crucial in facilitating
axonal regeneration and subsequently reinnervation
of target muscle.
The pool of newly synthesised cytoskeletal
proteins is likely to act as a source of structural
proteins for growth cone reformation and axonal
growth,112 as well as to regulate cytoskeletal dynamics.
113 Axonal regeneration has been thought
to recapitulate development in many aspects. The
injured axon must transition to a growth state, and
this transition distinguishes protein synthesis in
adult axons from that in developing axons. The
old arguments that adult axons do not synthesize
proteins were based largely on the absence of
polysomes in axons compared with those in dendrites.
114 This scarcity of protein synthetic machinery
suggests that the mature axon must recruit
(and activate) ribosomes and mRNAs to the injury
site. It is not clear what triggers this recruitment
of protein synthetic machinery after injury, but an
increase in axoplasmic [Ca2+] is a likely candidate.
Adult vertebrate axons have the capacity to synthesize
many different proteins.115
VIII. CONCLUSIONS
This is the first study revealing the relationship between
activation of Akt and MAPK signaling pathways,
c-Jun and c-Fos genes, axonal regeneration,
protein synthesis and degradation, and peripheral
nerve regeneration following crush injury in an in
vivo experiment using H. erinaceus, a medicinal
mushroom known for its neurological effects. Upregulation
of Akt, MAPK, c-Jun, and c-Fos in ipsilateral
DRG neurons, as well as axonal protein
synthesis and degradation after 7 days of crush
injury, are consistent with the beginning of functional
recovery of injured right hind limb.
Aqueous extract of H. erinaceus enhanced
nerve regeneration and accelerated motor functional
recovery after crush injury. Patients who receive
H. erinaceus may experience a more expeditious
improvement in the quality of life after injury. The
functional evaluation combined with morphological
examination of regenerated nerves, ipsilateral
DRG, and target EDL muscle revealed that the
aqueous extract promoted peripheral nerve regeneration
with significant functional recovery. It was
also noted that the neurotherapeutic effects of the
aqueous extract were comparable to those elicited
by mecobalamin, a CNS drug used in peripheral
nerve disorders. However, taking mecobalamin for
the treatment of nerve injury gives rise to side effects
such as gastrointestinal and dermatological
problems.116
It will be of interest to examine the potential
of different signaling pathways that underlie peripheral
nerve regeneration. There is a possibility
that H. erinaceus may mediate neuronal functions
by modulating the activities of different signaling
pathways such as activation of cAMP response element-
binding (CREB). The importance of CREB
signaling on learning and memory, as well as hyperphosphorylation
of CREB, plays an important
role in the long-term potentiation of the hippocampus.
442 International Journal of Medicinal Mushrooms
Peripheral Nerve Regeneration by Hericium erinaceus
At the present time, many of the medicinal
properties attributed to mushroom products are
based on data obtained from in vitro and animalbased
experiments. The protective effects of H. erinaceus
in humans require further research, as the
in vivo trial in rats has limited scope. Much more
advanced science is required to demonstrate that
claims of enhanced function and reduced disease
risks are also applicable in the human context.117
Long-term in vivo trials may need to be assessed
as there may be a cumulative effect.
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