Mitochondria related pathway is essential for polysaccharides purified from Sparassis crispa mediated neuro-protection against glutamate-induced toxicity in differentiated PC12 cells

Article abstract of DOI:10.3390/ijms17020133

The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against L-glutamic acid (L-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1?3)-linked Rha in the backbone, and (1?2) linkages and (1?6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to L-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced L-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca²⁺ influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in L-Glu-explored DPC12 cells. These results suggested that SCWEA protects against L-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease.

Full text of DOI:10.3390/ijms17020133

International Journal of
Molecular Sciences
Article
Mitochondria Related Pathway Is Essential for
Polysaccharides Purified from Sparassis crispa
Mediated Neuro-Protection against
Glutamate-Induced Toxicity in Differentiated
PC12 Cells
Shuang Hu 1,†, Di Wang 1,†, Junrong Zhang 1, Mengyan Du 1, Yingkun Cheng 1, Yan Liu 1,
Ning Zhang 1, Di Wang 1,* and Yi Wu 2,
*
1
School of Life Sciences, Jilin University, Changchun 130012, China; summerhu1990@outlook.com (S.H.);
xiaowangdi@outlook.com (D.W.); archer707@163.com (J.Z.); jludumy@126.com (M.D.);
chengyk@jlu.edu.cn (Y.C.); liuyaorui@126.com (Y.L.); zhangning1313@mails.jlu.edu.cn (N.Z.)
School of Pharmaceutical Science, Jilin University, Changchun 130021, China
Correspondence: jluwangdi@outlook.com (D.W.); wuyi@jlu.edu.cn (Y.W.);
Tel.: +86-431-8516-8646 (D.W.); +86-431-8561-9705 (Y.W.) ; Fax: +86-431-8516-8637 (D.W.)
These authors contributed equally to this work.
2
*
†
Academic Editor: Katalin Prokai-Tatrai
Received: 30 October 2015; Accepted: 12 January 2016; Published: 26 January 2016
Abstract: The present study aims to explore the neuro-protective effects of purified Sparassis crispa
polysaccharides against L-glutamic acid (L-Glu)-induced differentiated PC12 (DPC12) cell damages
and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose
anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa
and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix
with (1
Ñ
3)-linked Rha in the backbone, and (1
Ñ
2) linkages and (1 6) linkages in the side bone.
Ñ
Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to L-Glu exposure
effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay) and reduced L-Glu-induced apoptosis (by Hoechst staining).
SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca2+ influx and
prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore,
SCWEA normalized expression of anti-apoptotic proteins in L-Glu-explored DPC12 cells. These
results suggested that SCWEA protects against L-Glu-induced neuronal apoptosis in DPC12 cells and
may be a promising candidate for treatment against neurodegenerative disease.
Keywords: Sparassis crispa; purified polysaccharides; neuro-protection; glutamate; mitochondria
1. Introduction
Neurodegenerative disorders, which include Alzheimer’s disease (AD), Parkinson’s disease (PD)
and amyotrophic lateral sclerosis (ALS), have received great attention due to their devastating nature
and limited treatment options. During the process of neurological disorders, neurons manifest common
pathological features, such as mitochondrial dysfunction and protein aggregation [1]. Although
the underlying mechanisms of neurodegenerative diseases remain largely unknown, the idea that
apoptosis of neurons plays a central role in the development of neurodegeneration has emerged as an
attractive theory [2].
As one of the pathological factors in cerebral ischemia disease, glutamate is able to induce
neuronal cell apoptosis via opening mitochondrial permeability transition pores (mPTP) and reducing
Int. J. Mol. Sci. 2016, 17, 133; doi:10.3390/ijms17020133
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mitochondrial membrane potential (MMP), which is initiated by the interaction between pro- and
anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members [ ]. As a primary downstream mediator of
phosphatidylinositol-3 kinase (PI3K), Protein Kinase B (AKT) preserves mitochondrial integrity by
regulating the Bcl-2 family [ ]. Reactive oxygen species (ROS) are responsible for later mitochondrial
events, which lead to full activation of the caspase cascade [
]. In addition, the Ca2+ influx is involved
3
4
5
in glutamate-induced cell apoptosis by mediating the opening of mPTP [6].
Extracts of herbs or fungi have been applied for centuries to elicit biological responses in patients.
The acquisition of bioactive single components from natural extracts has become one of the main aims of
drug research and development in biopharmaceutics. It has been found that polysaccharides extracted
from different species of mushrooms and some other fungi were neuro-protective [
the study, polysaccharides isolated from the flowers of Nerium indicum exerted protective effects in
cortical neurons induced by -amyloid (A ) peptides [ ]. Another polysaccharide fraction named J6,
isolated from the flowers of Nerium indicum, showed a neuro-protective effect, which was associated
with the inactivation of the c-Jun N-terminal kinase (JNK) signaling pathway [ ]. Molecular weight,
7]. According to
β
β
8
9
degree of branching, chemical composition and glycosidic linkages of polysaccharides are believed to
be associated with their bioactivities [10].
Sparassis crispa, an edible mushroom with various medicinal properties, has been studied for
years. As one of the main bioactive components, Sparassis crispa polysaccharides are reported to
possess anti-tumor effects in mice with strong vascular dilation and hemorrhage reactions [11]. Oral
administration of purified polysaccharides obtained from Sparassis crispa enhances the Th1 response
on tumor-bearing mice [12]. On the other hand, Sparassis crispa–purified polysaccharides stimulate
leukocytes to produce cytokines in whole-cell cultures of human peripheral blood and in mouse
splenocytes [13,14]. Through decreasing pro-inflammatory cytokine levels dependent on extracellular
signal-regulated kinase, Sparassis crispa polysaccharides exhibited antiallergic effect in both in vivo and
in vitro models [15]. Encouragingly, it has been successfully confirmed that Sparassis crispa extracts
show positive activity on stroke through the activation of the AKT/endothelial nitric oxide synthase
(eNOS) pathway in the brain of stroke-prone, spontaneously hypertensive (SHRSP) mice, which
suggests its neuro-protective potential [16].
In the present study, polysaccharides obtained from Sparassis crispa were purified and
characterized. Its neuro-protective effect and its underlying mechanisms were investigated on
L-Glutamic acid (L-Glu)-induced damages on differentiated rat pheochromocytoma (DPC12) cells. Our
data revealed that the purified polysaccharides improved cell viability and mitochondrial function,
and restored the abnormal expression of apoptosis-related proteins. All these findings demonstrated
that mitochondria-related pathways are essential for the neuro-protective effects of Sparassis crispa
polysaccarides against L-Glu-induced toxicity in DPC12 cells.
2. Results
2.1. Purification and Characterization of Sparassis crispa Polysaccharides
Polysaccharides obtained from the Sparassis crispa were separated by anion exchange
chromatography (DEAE-cellulose column) (Figure 1A). Two fractions were eluted by double-distilled
(D.D.) water and 0.1 M NaCl (Figure 1B), and they were named SCWE I and SCWE II, respectively.
SCWE I was further purified by a gel permeation chromatography system Sepharose G-100, and the
purified polysaccharides were named SCWEA (Figure 1C), which were applied in further experiments.
According to the high performance liquid chromatography (HPLC) data, rhamnose (Rha),
mannose (Man) and galactose (Gal) were contained in SCWEA with the molar ratio of 10:2:1, and the
molecular weight of SCWEA was 75 kDa (Table 1). The particle diameter of SCWEA was 88.9 nm
(Table 1).
According to the UV spectra, no absorbance peak was caught at the wavelengths of 260 nm
and 280 nm, which suggested no contamination of nuclear acid and proteins (Figure 2A). In the

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fourier transform infrared spectroscopy (FTIR) spectra, a strong hydroxyl absorption peak (3400 cm´1),
a weak C–H absorption peak (2930 cm´1), and a C=O characteristic absorption peak (1650 cm´1
were observed (Figure 2B). Absorption between 950~1200 cm´1 suggested the existence of C–O–C
and C–O–H. The red shift of SCWEA in compared with congo red solution suggested a triple
)
λ
max
helix conformation of SCWEA (Figure 2C). Periodate oxidation–Smith degradation was performed to
detect the linkage model of glucose within SCWEA (Figure 2D,E). The HPLC method was performed
to analyze the hydrolysates after Smith degradation. After hydrolysis, rhamnose and mannose
were observed inside the sack fraction while glycerol other than erythritol exists outside (Figure 2E).
In addition, 102 µmol periodate was consumed with 27.5 µmol of formic acid generated during NaIO4
oxidation on SCWEA.
Sparassis crispa
mycelium powder
B
A
Hot water (80 ℃,4 h, 2 times)
Centrifugation
Residue
Supernatant
Deproteinized by sevag reagent(4, v/v)
Precipitated by 95% ethanol(4, v/v)
Centrifugation
Supernatant
Residue
Redissolved by D.D water
Crude polysaccharides
C
Collection of D.D water
elution fraction
Collection of 0.1 mol/L
NaCl elution fraction
SCWEⅠ
SCWE Ⅱ
Isolated by Sepharose G-100 column
Eluted by D.D water, peak collection
SCWEA
Figure 1. The purification of polysaccharides isolated from Sparassis crispa. (
and isolation of polysaccharides from Sparassis crispa; ( ) DEAE-52 cellulose anion exchanged the
chromatogram of the crude polysaccharides. SCWE I and SCWE II were eluted by double-distilled
(D.D.) water and 0.1 mol/L NaCl at a flow rate of 1.0 mL/min, respectively; ( ) The crude
A) Scheme for extraction
B
C
polysaccharide SCWE I was further purified via Sepharose G-100, and SCWEA was obtained.
Table 1. Molecular weight and particle size.
Mr (kDa)
molar ratio, mol %
Molecular weight 1
Particle size 2
75
96.7
Z-Average (r.nm) 3
PdI 4
0.306
3
44.45
1
4
2
Molecular weight determined by HPLC; Particle size determined by DLS; r: radius of SCWEA;
PdI: polydispersity.
2.2. The Effect of SCWEA against L-Glu Induced Cell Damage in DPC12 Cells
After incubation with various concentrations of SCWEA and/or 25 mM L-Glu for 24 h, the
viability of DPC12 cells was determined by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay. Results showed that there was an approximately 60.8% reduction of cell viability
(p < 0.001; Figure 3A) in L-Glu-treated DPC12 cells, whereas SCWEA alone showed minimal affect on
cell viability. Pre-treatment with SCWEA at doses of 4 and 8 µg/mL improved nearly 28.9% (p < 0.001)
and 30.8% (p < 0.001) of the viability of L-Glu-explored cells (Figure 3A). SCWEA at doses of 4 and
8 mg/mL strongly reversed the apoptosis rate in 25 mM L-Glu-explored DPC12 cells, as indicated by
the decreasing blue fluorescence intensity of nuclear staining with Hoechst 33342 (Figure 3B).

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2.3. The Effects of SCWEA on Intracellular Calcium Concentration and Reactive Oxygen Species (ROS) Levels
Results obtained from 2,7-dichlorofluorescin diacetate (DCFH-DA) staining revealed that 12 h
L-Glu incubation led to an accumulation of intracellular ROS, as suggested by the increase of DCF
fluorescence. Lower green fluorescence noted in SCWEA–pre-treated DPC12 cells indicated that
SCWEA effectively reduced intracellular ROS levels after 12 h of co-incubation (Figure 4A).
Fluo-4-AM staining was applied to examine the intracellular calcium concentration in DPC12
cells. An extremely high green fluorescence was observed in 25 mM L-Glu-explored cells, which was
significantly relieved by 3 h of pre-treatment of SCWEA (4 and 8 µg/mL) (Figure 4B).
Figure 2.
SCWEA; (
degradation of SCWEA were detected using HPLC.
(
C
A
) Ultraviolet spectra of SCWEA; (
B
) Fourier transform infrared spectroscopy spectrum of
) Congo red test on SCWEA; ( ) Periodate oxidation time course; (E
D
) Products of Smith
CTRL
L-Glu
A
B
L-Glu + 4 μg/ml SCWEA
L-Glu + 8 μg/ml SCWEA
Figure 3. The neuro-protective effect of SCWEA against L-Glu-induced cell damage in DPC12 cells.
Cells were pretreated with SCWEA for 3 h, and then co-incubated with or without 25 mM L-Glu for 24 h.
Compared with L-Glu-treated cells, SCWEA enhanced cell viability (
rate (20 ; Bar: 100 m) ( ). Data were expressed as a percentage of corresponding control cells and
means S.D. (n = 6). ### p < 0.001 versus control cells (CTRL); *** p < 0.001 versus L-Glu-exposed cells.
A) and reduced nuclear apoptosis
ˆ
µ
B
˘

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2.4. The Effects of SCWEA on Mitochondrial Function
Mitochondrial membrane potential was known to play an important role in regulating the
intrinsic and1extrinsic apoptosis pathways in cells. Changes of MMP in DPC12 cells were monitored
1
1
1
by a JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide) molecular probe,
and the fluorescent shift from red to light green reflected the depolarization of MMP [17]. SCWEA
at doses of 4 and 8
a striking reduction in the expressions of Bcl-2 and B-cell lymphoma-extra large (Bcl-xL) was noted in
L-Glu-treated cells. The 8 g/mL of SCGA pre-treatment resulted in 24.7% and 46.1% enhancement of
Bcl-2 and Bcl-xL expression compared with that of L-Glu-treated cells (Figure 5B).
µg/mL strongly restored MMP in L-Glu-treated cells (Figure 5A). In addition,
µ
A
CTRL
CTRL
L
-Glu + 4 μg/mL SCWEA
L
-Glu + 8 μg/mL SCWEA
L
-Glu
B
L-Glu + 8 μg/mL SCWEA
L-Glu
L-Glu + 4 μg/mL SCWEA
Figure 4. SCWEA suppressed Ca2+ influx (
accumulation ( ) in L-Glu-exposed DPC12 cells (20
8 µg/mL SCWEA for 3 h, followed by exposure to 25 mM L-Glu for another 12 h. The intracellular levels
of Ca2+ and ROS were detected by Fluo-4-AM and DCFH-DA staining, respectively. The experiments
were repeated three times.
A
) and intracellular reactive oxygen species (ROS)
; Bar: 100 m). Cells were pretreated with 4 and
B
ˆ
µ
L-Glu (25 mM)
B
A
SCWEA
4 μg/mL
8 μg/mL
CTRL
Bcl-2
CTRL
L-Glu
Bcl-xL
GADPH
L-Glu + 8 μg/mL SCWEA
L-Glu + 4 μg/mL SCWEA
Figure 5. The positive regulatory effects of SCWEA on mitochondrial function. DPC12 cells were
pretreated with 4 and 8 g/mL SCWEA for 3 h, followed by exposure to 25 mM L-Glu for 12 or 24 h.
) SCWEA restored L-Glu-induced MMP loss. The changes of MMP were determined by JC-1 staining
(20 ; Bar: 100 m). Red fluorescence indicates healthy cells with high MMP, whereas green fluorescence
indicates apoptotic or unhealthy cells with low MMP. The experiments were repeated three times;
) SCWEA enhanced the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large
(Bcl-xL) in L-Glu-exposed DPC12 cells. Quantification data were normalized by GAPDH. Data were
µ
(A
ˆ
µ
(
B
expressed as a percentage of corresponding control cells and means
control cells; * p < 0.05 and *** p < 0.001 versus L-Glu-exposed cells.
˘
S.D. (n = 3). ### p < 0.001 versus

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2.5. Protein Kinase B (AKT) Contributing to SCWEA-Mediated Neuro-Protective Effect
Reduced phosphorylation of AKT (P-AKT) and glycogen synthase kinase 3
β (P-GSK-3β) was
observed in L-Glu-exposed DPC12 cells after 24 h of treatment (p < 0.001; Figure 6A). SCWEA 3 h
pre-treatment replenished the activation of AKT and GSK-3 in L-Glu-challenged DPC12 cells
β
(p < 0.001; Figure 6A). No significant changes in the expressions of total AKT (T-AKT) and total
GSK-3β (T-GSK-3β) were observed (Figure 6A).
Furthermore, the neuro-protective effect of SCWEA was significantly abrogated by 30 min of
pre-treatment with 10
µ
M LY 294002, an AKT inhibitor (73.8%
˘
4.1% versus 55.7%
˘
4.6%; p < 0.001;
Figure 6B). Taken together, these findings suggest the activation of AKT mediates neuro-protection of
SCWEA in DPC12 cells.
L-Glu (25 mM)
SCWEA
4 μg/mL 8 μg/mL
B
A
CTRL
P-AKT
T-AKT
P-GSK
T-GSK
GADPH
Figure 6. AKT/GSK3
were pre-treated with 4 and 8
for 24 h. ( ) The activation of AKT and GSK-3
were normalized by corresponding T-AKT and T-GSK3
against L-Glu on cell viability were abolished by LY294002 pre-treatment. DPC12 cells were pre-treated
with 10 M LY294002 for 30 min, followed by treatment with 4 g/mL SCWEA for 3 h and exposure
to 25 mM L-Glu for 24 h. Data were expressed as a percentage of corresponding control cells and
means S.D. (n = 6). ### p < 0.001 versus control cells; *** p < 0.001 versus L-Glu-exposed cells;
ˆˆˆ p < 0.001 versus SCWEA and L-Glu co-treated cells.
β
pathway contributes SCWEA-mediated neuro-protective effect. DPC12 cells
g/mL SCWEA for 3 h, followed by exposure to 25 mM glutamate
was detected via Western blot. Quantification data
, respectively; ( ) Protective effects of SCWEA
µ
A
β
β
B
µ
µ
˘
3. Discussion
Our study demonstrated, for the first time, the neuro-protective effect of purified polysaccharides
separated from Sparassis crispa (SCWEA). The characteristic structure of SCWEA was analyzed by
FTIR, congo red and periodate oxidation-Smith degradation. Generation of 27.5
during NaIO4 oxidation suggested the existence of (1 6) linkages; meanwhile, the low yield of formic
acid suggested that these (1 6) linkages do not belong to the main chain. Minimal consumption of
periodate (0.85 mol per glycosyl) indicated the existence of (1 3) linkages, which shall not consume
any periodate during oxidation. The remaining rhamnose and mannose in the hydrolysates after
Smith degradation further confirmed the existence of (1 3) linkages. No (1 4) linkages existed
within SCWEA, as indicated by the absence of erythritol in hydrolysates. SCWEA may structuralize
µmol formic acid
Ñ
Ñ
Ñ
Ñ
Ñ

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with (1
was observed. Collectively, SCWEA contains (1
linkages and (1 6) linkages in the side bone. Combined with the results obtained from congo red test,
Ñ
2) linkages since over two more folds of periodate consumption than formic acid generation
Ñ
3)-linked Rha in the backbone, as well as (1 2)
Ñ
Ñ
the conformation of SCWEA is a triple helix rather than a random coil.
The present data has successfully confirmed the neuro-protective effect of SCWEA against
L-Glu-induced cell damage via improving cell viability and reducing nuclear apoptotic rate.
Preliminarily, data also displayed that SCWEA alone successfully induced neurogenesis and
differentiation in PC12 cells (Figure A1). The changed parameters caused by central nervous system
stress, such as the shutdown of nerves, result in high levels of glutamate, which may in turn lead
to cell apoptosis [18]. Mitochondrial dysfunction is involved in L-Glu-caused neurotoxicity. As the
major sources of cellular ATP and a hub for Ca2+ signaling, mitochondria are considered as a center of
intracellular energy metabolism [19]. Mitochondrial Ca2+ is a positive effector of ATP synthesis, yet
Ca2+ overload leads to the production of free radicals and the opening of mPTP, which, taken together,
result in mitochondrial depolarization, matrix solute loss and cytochrome c release [20]. On the other
hand, the overproduction of ROS is also linked to the opening of mPTP [21]. SCWEA pre-treatment
significantly prevented intracellular Ca2+ and ROS accumulation, and mitigated MMP dissipation.
Our data further revealed that SCWEA enhanced L-Glu-suppressed expressions of Bcl-2 and Bcl-xL,
the anti-apoptotic members of the Bcl-2 family that maintain normal mitochondrial function and
prevent mitochondrial outer membrane permeabilization. Bcl-2 is an integral membrane protein, and
the expression of Bcl-2 is known as a hallmark of mitochondria dysfunction–associated cell death [22].
Altogether, the mitochondrial apoptotic pathway may be mainly involved in SCWEA-mediated
neuro-protection against L-Glu-caused DPC12 cell damage.
As reported, liquiritin modulated AKT and its downstream element GSK-3
β to relieve
L-Glu-induced cell damage in DPC12 cells. The AKT signaling molecule is involved in cell
differentiation, proliferation, survival and apoptosis [23]. The activation of AKT can suppress several
pro-apoptotic proteins such as Bcl-2 family members and some signal molecules such as GSK-3β [24].
Micro-vesicles released by mouse bone marrow-derived mesenchymal stem cells were reported to
protect PC12 cells from L-Glu-induced excitotoxicity via upregulating AKT phosphorylation and Bcl-2
expression [25]. GSK-3
regulator of programmed cell death. GSK-3
is related to mitochondrial dysfunction [26]. In addition, GSK-3
mediating cytosolic leakage of cytochrome c from mitochondria, and phosphorylated GSK-3
the opening of mPTP [27]. Furthermore, 25-hydroxycholesterol could induce mitochondria-dependent
apoptosis via the activation of GSK-3 in PC12 cells [28]. L-Glu-induced suppression of AKT and
subsequent dephosphorylation of GSK-3 , as we demonstrated, would be expected to provide a
β
participates in apoptosis in several cell types, and it is known as an upstream
promotes activation and translocation of Bax, which
is considered a critical factor in
inhibits
β
β
β
β
β
mitochondria-related pro-apoptotic stimulus. Combining literature study with our present results,
we proposed that the neuro-protective effect of SCWEA in L-Glu-treated neurons is mainly dependent
on the restoration of AKT and the subsequent shutdown of the mitochondrial apoptotic pathway.
Concerns were particularly taken with the concentration of glutamate in the present study.
Although the highest concentration of glutamate in the brain is 10–12 mM, the potential glutamate level
for excitotoxicity in normal cerebrospinal fluid (CSF) is at the remarkably low level of 100–200 nM [29].
For in vitro experiments, 20 to 25 mM glutamate were used to induce damage of DPC12 cells [30,31].
Our preliminary experiments tested the cytotoxic effects of L-Glu ranging from 10 to 50 mM, and
eventually 25 mM glutamate was utilized to establish the neuro-toxic DPC12 cell model (Figure A2).
In summary, SCWEA purified from Sparassis crispa exhibits neuro-protective activity against
L-Glu-induced DPC12 cell damage, which is, at least partially, related to the activation of AKT and
the mitochondrial pathway. Our study suggests that SCWEA could be a potential therapeutic or
modulating agent for neurodegenerative damage.

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4. Materials and Methods
4.1. Submerge Fermentation of Sparassis crispa and Crude Extract Preparation
Sparassis crispa (SC031; CCTCC No.M2015275) was cultured in a hundred-liter full-automatic
fermentor (BaoXing Bioscience Company, Shanghai, China) by using a defined liquid medium which
contains: 20 g/L sucrose, 5 g/L yeast extract powder, 1 g/L MgSO4 7H2O and 0.25 g/L vitamin B1.
¨
˝
Sparassis crispa mycelium obtained from submerged fermentation was extracted at 80 C for 4 h in D.D.
water and the process was repeated two times. After being centrifuged, the supernatant (SCWE) was
sequentially concentrated and freeze-dried for further experiments.
4.2. Polysaccharide Purification and Characterization
4.2.1. Purification of Sparassis crispa Polysaccharides
Sevag reagent (v(n-butanol):v(chloroform) = 1:4, 50 mL) was used to remove proteins in SCWE.
Four-fold ethanol was added to the supernatant and placed at 4 ˝C overnight [32]. Furthermore, the
dissolved precipitation in D.D. water was subjected to DEAE-52 cellulose anion exchange column
(2.6 cm
ˆ
35 cm; Whatman, Bucks, UK). The column was eluted with D.D. water followed with 0.1 and
0.2 mol/L NaCl at a flow rate of 1 mL/min. The polysaccharide fraction was collected and detected
using phenol-sulfuric acid method. Briefly, samples (1 mL) mixed with 1 mL phenol solution (5%)
were added into 20 mL tube. Ten minutes after 5 mL sulfuric acid addition, the tubes were cooled
down at 30 ˝C for 20 min. The absorbance was spectrophotometrically measured at a wavelength
of 490 nm [33]. Further purification was performed via a gel permeation chromatography system
Sepharose G-100 (Pharmacia, Uppsala, Sweden). The column was eluted with D.D. water at a flow rate
of 0.5 mL/min. The fractions (10 mL each) were collected, detected and then freeze-dried. The purified
polysaccharides were named SCWEA (Figure 1A).
4.2.2. Molecular Weight and Particle Size Measurements
The molecular weight was analyzed by LC-10ATvp HPLC system (Shimadzu, Tokyo, Japan),
which is equipped with a TSK-GEL G4000PWXL column (Tosoh Co., Tokyo, Japan) and an Alltech
2000ESELSD (Shimadzu, Tokyo, Japan). D.D. water was served as the mobile phase, the flow rate
˝
was 0.45 mL/min, aerosol level was 60%, drift tube temperature was 120 C and nebulizing nitrogen
pressure was 25 psi. The dextran standards were used to create a calibration curve [34]. The particle
size of polysaccharides in deionized water was detected by Dynamic light scattering (DLS) using a
Malvern Zetasizer Nano (ZS90, Malvern Instruments Ltd., Malvern, UK). The sample was dissolved in
the D.D. water at a concentration of 1 mg/mL, and there were 10 runs performed in each procedure.
4.2.3. Ultraviolet (UV) Spectra Measurement
UV-2401PC UV-Vis recording spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was
applied to obtain the spectra of purified polysaccharide. Each spectrum was at the average of
five parallel scanning from 200 to 400 nm. The scan interval was 0.2 nm and the width of entrance slit
was 12 nm.
4.2.4. Fourier Transform Infrared Spectroscopy (FTIR) Determination
A 4 mg sample was ground thoroughly with 150 mg KBr, and its average transmission spectra
(n = 100) were recorded via a IRPrestige-21 FTIR spectrometer (Shimadzu, Tokyo, Japan) at the
´1
wavelength ranging from 900 to 4000 cm
.
4.2.5. Congo Red Test
Congo red test was performed to detect the chain conformation of polysaccharides in aqueous
solution, in which a red shift in the light absorption maximum was attributed to the triple helices
λ
max

Int. J. Mol. Sci. 2016, 17, 133
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of polysaccharide chains [35]. Sample aqueous solution at 1 mg/mL containing 81
µM Congo red was
treated with 1 M NaOH at various concentrations ranging from 0 to 0.5 M. Visible light absorption
spectrum was obtained over the range from 200 to 900 nm at room temperature with a UV-2401PC
UV-Vis spectrophotometer and deionized water which were considered as the blank control.
4.2.6. Monosaccharides Analysis
˝
The polysaccharides (20 mg) were hydrolyzed with 1 M H2SO4 (1 mL) at 105 C for 6 h in a
sealed glass tube, and then the pH was adjusted to be neutral with BaCO3. The solution was then
centrifuged at 4000
the HPLC/ELSD (Evaporative Light-Scattering Detector) system. The chromatograph was fitted with
a Prevail ES carbohydrate analysis column (Alltech Associates, Inc., Deerfield, IL, USA), which was
ˆ
g for 10 min to separate the hydrolysates, which were further analyzed by using
™
eluted with 75% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) at a flow rate of 1.0 mL/min. The
results were compared with the monosaccharide standards: D-glucose, L-rhamnose (Rha), D-xylose,
D-galactose (Gal), D-mannose (Man) and L-arabinose (Sigma-Aldrich, USA) [34].
4.2.7. Periodate Oxidation-Smith Degradation Reaction of Polysaccharides
Similar to the previous study [36], 20 mg polysaccharide was dissolved in 15 mM NaIO4 (25 mL,
pH 4) at 4 ˝C in darkness. NaIO4 in different concentrations were used to create a calibration curve
to calculate the HIO4 consumption, while the formic acid production was determined by titration.
After 48 h dialyzing against D.D. water, the dialysate was concentrated and incubated with potassium
borohydride (70 mg) overnight at room temperature. After adjusting pH value to 7.0 by acetic acid,
the solution was dialyzed against D.D. water for another 24 h. The 3 mL sample was detected by
HPLC/ELSD system. The rest of product was hydrolyzed with 1 M H2SO4 at 25 ˝C for 40 h, and the
pH value was adjusted to 7.0 by BaCO3. The solution was centrifuged at 4000
the hydrolysates which were further analyzed by HPLC/ELSD system.
ˆ
g for 10 min to separate
4.3. Cell Culture
PC12 cells (obtained from ATCC, Manassas, VA, USA; CRL-1721; passages < 10) grew in
Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Grand Island, NY, USA) which was
supplemented with 5% horse serum (HS; Invitrogen, USA), 10% fetal bovine serum (FBS; Invitrogen,
USA), penicillin (100 units/mL), and streptomycin (100 µg/mL) (Invitrogen, USA), under a humidified
˝
atmosphere containing 5% CO2 at 37 C. The culture medium was changed every three days. PC12 cells
were differentiated for 48 h with 50 ng/mL nerve growth factor (NGF; Sigma-Aldrich, USA) which was
dissolved in DMEM medium containing 1% FBS, 1% HS and 100 U/mL of penicillin/streptomycin.
4.4. Cell Viability Analysis
A quantitative colorimetric assay with MTT (Sigma-Aldrich, USA) was applied to measure cell
viability. DPC12 cells were seeded in 96-well plates at 2
SCWEA (1–8 g/mL) alone for 24 h, or pretreated with SCWEA (1–8
25 mM L-Glu for another 24 h. In another separated experiment, cells were pretreated with 10
LY294002 for 30 min, and exposed to 4 g/mL SCWEA for 3 h, followed with another 24 h co-incubation
with 25 mM L-Glu. After incubation with MTT solution (0.5 mg/mL) for 4 h at 37 ˝C in darkness,
100 L dimethyl sulfoxide (DMSO) was added to dissolve crystals. A micro-plate reader (Bio-Rad,
ˆ
104 cells/well. Cells were treated with
µ
µg/mL) for 3 h and exposed to
µM
µ
µ
Hercules, CA, USA) was used to detect the absorbance at 540 nm. Viability values of treated cells were
expressed as a percentage of that of corresponding control cells.
4.5. Cellular Morphology Analysis
Nucleus morphological alterations were analyzed by Hoechst 33342. PC12 cells (1
seeded into each well in a six-well plate. After differentiation, cells were pre-treated with 4 and
ˆ
105) were

Int. J. Mol. Sci. 2016, 17, 133
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8 µg/mL SCWEA for 3 h, followed with 12 h co-incubation with 25 mM L-Glu. Then cells were
˝
incubated with Hoechst 33342 (5 µg/mL; Sigma-Aldrich, USA) for 15 min at 37 C in darkness. After
being washed with PBS, the fluorescence intensity in the nucleus was photographed by a fluorescent
microscope (20ˆ; CCD camera, TE2000, Nikon, Tokyo, Japan).
4.6. Measurement of ROS
The production of intracellular ROS was measured via DCFH-DA (Nanjing Jiancheng, Nanjing,
China). The DCFH-DA passively entered the cell where it reacted with ROS to form the highly
fluorescent compound dichlorofluorescein (DCF). Briefly, DPC12 cells were exposed to 4 and 8 µg/mL
SCGA, and co-incubated with L-Glu (25 mM) for 12 h. Treated cells were incubated with 10 µM
DCFH-DA at 37 ˝C for 30 min. The fluorescent color was photographed by a fluorescent microscope
(20ˆ; CCD camera) after being washed with PBS three times.
4.7. Measurement of Intracellular Calcium Concentration ([Ca2+]i)
DPC12 cells were seeded in a six-well plate (1
ˆ
105 cells/well). On the next day, cells were treated
with 4 and 8
µ
g/mL SCGA for 3 h prior to exposure to 25 mM L-Glu for 12 h. Then, the supern˝atant
was removed, and cells were incubated with 5 µM Fluo-4-AM (Invitrogen, USA) for 30 min at 37 C in
darkness. After three washes, moderate PBS was added to cover the cells, and then cells were observed
through fluorescence microscope (20ˆ; CCD camera).
4.8. Mitochondrial Membrane Potential (MMP) Analysis
DPC12 cells (1
pre-treated with 4 and 8
Treated cells were incubated with 2
ˆ
105) were seeded into each well in a six-well plate. Overnight cells were
g/mL SCWEA for 3 h, followed with 12 h co-incubation with 25 mM L-Glu.
M JC-1 (Sigma-Aldrich, USA) at 37 ˝C for 10 min. Fluorescent
µ
µ
microscope (20ˆ; CCD camera) was applied to record the fluorescent color in each group.
4.9. Western Blot
DPC12 cells were pre-treated with 4 and 8 µg/mL SCWEA for 3 h, and followed with 24 h
co-incubation with 25 mM L-Glu. Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer
(Sigma-Aldrich, USA) which contains 2% phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich,
USA) and 1% protease inhibitor cocktail (Sigma-Aldrich, USA). Proteins were separated via 12%
SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Amherst,
NY, USA). The transferred membranes were then blotted with antibodies as follows: Bcl-2, Bcl-xL,
T-AKT, P-AKT, T-GSK-3β, P-GSK-3β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(1:1000; Abcam, Cambridge, UK) at 4 ˝C overnight, followed by incubation with horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz, CA, USA). Chemiluminescence was detected
by using ECL detection kits (GE Healthcare, Bucks, UK). The intensity of the bands was quantified by
scanning densitometry using software Image J (National Institutes of Health, Bethesda, MD, USA).
4.10. Statistical Analysis
All data were presented as mean
˘
standard deviation (S.D.). Data were evaluated by
one-way analysis of variance (ANOVA) to detect statistical significance, followed by post-hoc multiple
comparisons (Dunn’s test) by using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Values of p < 0.05
were considered to be significant.
Acknowledgments: This work was supported by the national Natural Science foundation of P.R.China
(Grant No. 81402955), Science and Technology Key Project in Jilin Province of China (Grant No. 20130201006ZY,
20140301003YY and 20150203002NY).

Int. J. Mol. Sci. 2016, 17, 133
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Author Contributions: Di Wang * and Yi Wu conceived and designed the experiments; Shuang Hu, Di Wang,
Junrong Zhang, Mengyan Du, Yingkun Cheng, Yan Liu, Ning Zhang performed the experiments; Shuang Hu,
Di Wang and Di Wang * wrote the paper. * is used to differ the two author Di Wang, in which Di Wang * is
referring to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix
Figure A1. The regulatory effects of SCWEA on the morphology of PC12 cells. PC12 cells were exposed
to 4 and 8
scale bar: 50 µm.
µ
g/mL SCWEA for 24 h and the morphologies were detected via microphotograph. 10 ,
ˆ
Figure A2. Effects of various concentrations of L-Glu on the viability of DPC12 cells. DPC12 cells were
exposed to 10–40 mM of L-Glu for 24 h. Data were expressed as a percentage of corresponding control
cells and means ˘ S.D. (n = 6). ** p < 0.01, *** p < 0.001 versus control cells.
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