Journal of Pharmacological Sciences
©2004 The Japanese Pharmacological Society
Short Communication
J Pharmacol Sci 95, 294 – 298 (2004)
Polysaccharide Extract Isolated From Ganoderma lucidum Protects Rat
Cerebral Cortical Neurons From Hypoxia/Reoxygenation Injury
Hong-Bo Zhao1, Shu-Qian Lin2, Ji-Hong Liu3, and Zhi-Bin Lin1,*
1Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center,
38 Xueyuan Road, Beijing 100083, China
2Fuzhou Green Valley Institute of Bio-pharmaceutical Technology, Fuzhou 350002, China
3Confocal Microscope Laboratory, Peking University Health Science Center, Beijing 100083, China
Received January 8, 2004; Accepted April 16, 2004
Abstract. The effect of polysaccharide extract isolated from Ganoderma lucidum (Gl-PS) on
rat cortical neuronal cultures exposed to hypoxia /reoxygenation (H/R) was studied in vitro.
Gl-PS (1, 10, 100 g/ml) increased neuron viability following H/R as measured by the
inhibition of MTT reduction. Gl-PS also significantly reduced malondialdehyde content and
reactive oxygen species production and increased the manganese superoxide dismutase
(Mn-SOD) activity; furthermore, the translocation of nuclear factor-kappa B induced by H/R was
blocked. These findings suggest that Gl-PS might be useful in treating H /R-induced oxidative
stress and Mn-SOD might play a critical role in the neuroprotective effect of Gl-PS against
H/R injury.
Keywords: polysaccharide extract isolated from Ganoderma lucidum, cultured rat cortical neuron,
hypoxia/reoxygenation
Oxidative stress plays an important role in the
pathophysiological processes of damage during brain
ischemia and reperfusion (1). Oxidative stress can be
defined as the pathogenic outcome of the overproduction
of oxidants that overwhelms the antioxidant capacities
of free radical-quenching enzymes. Thus control over
the activities of these enzymes may protect against
free radical damage and may resist the development of
hypoxia/reoxygenation (H/ R) injury.
Ganoderma lucidum is one of the most popular
medicinal fungi with a long history in oriental countries.
In modern clinical trials, the preparations of Ganoderma
lucidum show therapeutic efficacy in treating cancer,
hyperlipidemia, diabetes, neurasthenia, insomnia, and
so forth. Polysaccharide and triterpene are two major
categories of compounds purified from Ganoderma
lucidum. It has been shown that Ganoderma lucidum
polysaccharides (Gl-PS) have antitumor and immunomodulatory
activities (2).
A recent study showed that Ganoderma lucidum
could limit neuronal loss after local cerebral ischemia
and also reduce infarct size in a rat model of focal
brain ischemia (3). Since many studies have reported
that Gl-PS possess anti-oxidant bioactivity (4, 5), we
hypothesized that polysaccharide may be useful in treating
H/R-induced oxidative stress injury. In the present
study, we examined the effect of Gl-PS on H/R injury
in cultured cortical neurons in vitro and characterized
the mechanisms involved in this activity.
Dissociated cortical cells from Spragure-Dawley 1-
day-old rats were grown in DMEM (Gibco/BRL,
Gaithersburg, MD, USA) with 5% fetal bovine serum
(FBS) and 5% horse serum. Cells were plated with a
density of 1 106 cells /ml in plates coated with
12.5 mg/l of poly-L-lysine (Sigma, St. Louis, MO,
USA). Cells were cultured for 7 days in an atmosphere
of 5% CO2 / 95% air at 37C. Cytosine arabinoside
10 mol/l (Sigma) was added 4 days after plating to
inhibit the replication of nonneuronal cells. Our neuronal
cultures consisted of approximately 83 – 92% neurons.
We confirmed the ratio of neurons by immunofluorescence
staining with antibody against neuronspecific
enolase in representative sister wells.
*Corresponding author. FAX: +86-10-82801686 At day 8, neuronal cell cultures in serum-free medium
E-mail: linzb@public3.bta.net.cn
Gl-PS Protects Neurons From H/R Injury 295
without glucose were moved into modular waterjacketed
incubator chambers [12 h at 37C, humidified
95% N2 / 5% CO2 (hypoxia)]. Cultures were then
returned for the next 24 h to a normoxic atmosphere at
37C (deoxygenating), whereas controls were constantly
maintained under standard conditions.
Gl-PS was extracted by hot water from the fruiting
body of Ganoderma lucidum (Leyss. ex Fr.) Karst,
followed by ethanol precipitation, reserve dialysis, and
protein depletion. The yield of Gl-PS was 0.82% (w/w)
in terms of the fruiting body of Ganoderma lucidum.
It is a polysaccharide peptide with 17 amino acids. The
ratio of polysaccharides to peptides is 93.51%:6.49%.
The polysaccharides consist of rhamnose, xylose, fructose,
galactose, mannose, and glucose with molar ratios
of 0.793:0.964:2.944:0.167:0.384:7.94 and are linked
by -glycosidic linkages. Gl-PS (1, 10, 100 g/ml) was
added prior to H/R, and the neurons were treated for
36 h.
Total cell lysates were prepared as follows: Briefly,
treated cells were lysed in 50 l of lysis buffer (20 mM
Tris-HCl, pH 8.0; 137 mM NaCH; 10% (w/ v) glycerol;
1% (w/ v) Triton X-100; 1 mM Na3VO4; 20 mM sodium
fluoride; 14 mM -mercaptoethanol; 2 mM ethylenediaminetetraacetic
acid (EDTA); 1 mM phenylmethanesulfonyl
fluoride (PMSF); 2 g/ml leupeptin and
2 g/ml aprotinin) for 20 min at 4C with intermittent
shaking. Detergent insoluble material was pelleted by
centrifugation (13,000 g, 15 min, 4C). The protein
concentration in the detergent-soluble fraction was
determined by the Bradford method.
Cell viability was monitored by the spectrophotometric
method using 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) according to
Yamamuro et al. (6).
The concentration of malondialdehyde (MDA) in
the cell lysate was used as a biochemical marker for
lipid peroxidation and was evaluated by the assay of
thiobarbituric acid-reacting compounds.
Intracellular reactive oxygen species (ROS) production
was measured with a confocal microscope (TCSSP2;
Leica, Mannheim, Germany) by using a fluorescent
dye, 2',7'-dichlorofluorescin diacetate (DCF-DA)
(Molecular Probes, Eugene, OR, USA), which fluoresces
green when oxidized. DCF-DA is a membranepermeable
compound. After intracellular deacetylation,
DCF-DA forms a nonfluorescent product (DCF),
which upon oxidation is transformed into fluorescent
DCF. Neurons were preincubated with 10 mol/ l
DCF-DA for 30 min at 37C prior to measurement.
Neurons were imaged via the epifluorescence mode
with a 40 immersion lens at Exl 488 nm and Eml
530 nm. The cell images were stored on a computer
and transformed to the cell fluorescence intensities
using Leica confocal software.
Superoxide dismutase (SOD) activity in cell lysate
was determined by inhibition of NADH oxidation. In
order to discriminate between Cu/Zn-SOD and
Mn-SOD activity, the assay was also performed after
5 min of incubation in the presence of 5 mM NaCN
to inhibit Cu /Zn-SOD and thus measure the residual
Mn-SOD activity.
For immunofluorescence localization of nuclear
factor-kappa B (NF-B) by confocal miscroscopy, after
H/R (12 h/45 min), fixed cells were reacted with a
rabbit monoclonal antibody against p65 subunit (Santa
Cruz Biotechnology, Santa Cruz, CA, USA), followed
by FITC-conjugated goat anti-rabbit IgG antibody
(Santa Cruz Biotechnology). The nuclei were stained
with propidium iodide (Sigma). NF-B translocation
from the cytosol to the nucleus was used as an index
of activation. A change in color of the coleus from red
to yellow (due to co-localization of green FITC
fluorescence and red propidium iodide fluorescence)
was indicative of NF-B translocation.
Results obtained are expressed as the mean S.E.M.
Statistical analysis was performed by one-way ANOVA
followed by least-significant difference (LSD). P values
below 0.05 were considered significant.
H/R caused neuronal death, as shown by a decrease
in MTT reduction; pretreatment with Gl-PS significantly
attenuated the decline of MTT reduction (Fig. 1).
We performed confocal analysis with a ROS-specific
fluorogen, DCF-DA, to examine the changes in ROS in
rat cortical neurons. Exposing cortical neurons to H/R
(12 h/24 h) significantly increased the fluorescent
intensity, indicating that H /R increases ROS production.
Fig. 1. Effect of Gl-PS on inhibition of MTT reduction induced
by H/R in cortical cultures. The values represent the means S.E.M.
obtained in five to six independent experiments. *P0.05, **P0.01;
compared to H/R.
296 H-B Zhao et al
The increase in the formation of DCF-DA was
attenuated by pretreating cortical neurons with Gl-PS
(10,100 g/ ml) (Fig. 3A).
MDA content in neuron lysates was estimated in
neurons to determine the development of oxidative
stress and free radical generation. MDA content of
neurons treated with H/R was increased significantly,
while Gl-PS significantly reduced MDA content.
(Fig. 2A).
To understand the mechanism underlying the neuroprotective
effect of Gl-PS against H/R injury, the
changes in the SODs’ activities were investigated. We
found that addition of Gl-PS to neurons cultures
increased total SOD and Mn-SOD activities, whereas
the activity of Cu /ZnSOD was not affected. The result
demonstrates that Mn-SOD is involved in Gl-PSinduced
neuroprotection against H/R (Fig. 2B).
Representative immunofluorescent labeling for NF-
B p65 (green) and dye labeling for propidium iodide
(red) in cortical neurons are presented in Fig. 3B.
Exposure to H/R (12 h/45 min) induced NF-B
activation in rat cortical neurons as indicated by the
translocation of NF-B into the nucleus (Fig. 3Bb).
However, low levels of NF-B were observed in
untreated cells nucleus (Fig. 3Ba). NF-B translocation
into the nucleus was blocked by the pretreatment with
Gl-PS (100 g/ ml) (Fig. 3Bc).
In this study, we confirmed that H/R exerts neurotoxic
effects on rat cultured cortical neurons. These
effects were accompanied by free radical generation,
lipid peroxidation production, and NF-B activation.
Gl-PS significantly reduced MDA content and the
production of ROS and increased the Mn-SOD activity;
furthermore, the translocation of NF-B induced by
H/R was blocked.
The results demonstrate that H /R causes cell damage
to cortical neurons, as deduced by the inhibition of
MTT reduction. In addition, pretreatment with Gl-PS
significantly reduced the dimension of neuronal damage
following H/R.
Oxidative stress may play a pivotal role in the process
of cell injury following hypoxia/reoxygenation (1).
In our study, exposing cortical neurons to H / R significantly
increased the fluorescent intensity of DCF-DA, a
ROS-specific fluorogen, and the production of MDA.
The results demonstrated that ROS plays an important
role in the mechanisms of H/R injury in our research.
Additionally, the finding that pretreatment with Gl-PS
could reduce the ROS and MDA production demonstrated
that the neuroprotective effect of Gl-PS may be
associated with its antioxidant activity.
With respect to the detoxifying systems, SODs
constitute the first defense step by preventing superoxide
anion from forming singlet oxygen. Cu/Zn-SOD is
present in the cytosolic compartment, whereas Mn-SOD
is located in mitochondria (7). There is much evidence
that SOD has a protective role against hypoxic-ischemic
insults (8), and Mn-SOD conferred cell resistance to
H/R-induced cell injury (9). In our study, at 24 h
after reoxygenation, there was no significant difference
between H/R-treated neurons and Gl-PS-treated
Fig. 2. Effect of Gl-PS on cellular production of ROS and the
ability of cells to defend against them. A: Effect of Gl-PS on lipid
peroxidation in neurons lysate. Lipid peroxidation is expressed as the
concentration of lipid peroxide byproducts MDA. B: Effect of Gl-PS
on SOD activities in neurons lysate. Data are expressed as mean
percentages of change from matched controls. The values represent
the means S.E.M. obtained in five to six independent experiments.
In control neurons, basal activities during the whole experimental
period corresponded to 284.927 26.23 nU/100 g protein for
SOD, 40.745 3.02 nU/100 g protein for Mn-SOD; #P0.05,
compared to normal control; *P0.05, **P0.01, compared to H/R.
Gl-PS Protects Neurons From H/R Injury 297
neurons, which is consistent with previous reports
(10, 11). At this point in the experiment, Gl-PS increased
total SOD and Mn-SOD activities in cortical neurons,
but did not affect Cu /Zn-SOD. The same enhancement
of total SOD and Mn-SOD activity could be observed
even in normoxic neurous treated with Gl-PS. Treatment
with Gl-PS (100 g/ml) for 12, 24, 48, and 72 h could
increase Mn-SOD activity by 10.524%, 16.921%,
30.258%, and 53.035% respectively, suggesting that
amplifying the activity of Mn-SOD might be an
important channel for the effect of Gl-PS.
It is reported that transcription factor NF-B is
activated in response to oxidative stress like that caused
by H/R (12). In addition, it is becoming clear that
NF-B is a principle mediator of the response to H/R
injury. NF-B binds to the promotor region of many
genes including tumor necrosis factor (TNF-) and
interleukin 1 (IL-1). These findings suggest that
inhibition of NF-B activity may prevent H/R injury.
Our finding is consistent with those of previous studies
(13), which demonstrated that in rat cortical neurons,
exposure to H/R (12 h/45 min) induced NF-B activation
as indicated by its translocation into the nucleus.
Gl-PS (100 g/ml) treatment efficiently blocked NF-
B activation. It is hypothesized that Gl-PS blocked
activation of NF-B, possibly by changing cell redox
balance. Furthermore, recent studies demonstrated that
significant attenuation in the activation of NF-B
occurred in cells cultures treated with SOD (14, 15),
so we hypothesize that Mn-SOD may play a critical
role in the neuroprotective effect of Gl-PS against H/R.
In summary, this is the first report showing that Gl-PS
exhibits a remarkable protection against H /R injury in
rat cortical neurons. As a plant drug, Ganoderma
polysaccharide has fewer undesirable side effects than
other agents. Hence, our findings could provide novel
therapeutic targets to prevent neuronal death following
stroke. Further studies should include the application
of Gl-PS to an in vivo model.
Fig. 3. Typical conforcal photographs of the neurons subjected to H/R. A: Effect of Gl-PS on ROS production in cortical
neurons subjected to H/R. a: A culture treated with vehicle; b: neurons exposed to H/R for 12 h/24 h increased intracellular
ROS levels as revealed by fluorescent intensities of DCF (green spots); c: a culture pretreated with 100 g/ml Gl-PS and
subsequently exposed to H/R for 12 h/24 h showed lower ROS level compared to H/R treated cultures. B: Effect of Gl-PS
(100 g/ml) on H/R-induced NF-B activation in cortical neurons. a: Untreated cortical neurons showing no nuclear NF-B
translocation; b: H /R (12 h/45 min)-treated neurons showing a mixture of red and yellow colors (due to co-localization of
green FITC fluorescence and red propidium iodide fluorescence) in the nuclei, indicative of partial NF-B translocation; c: Gl-PS
(100 g/ml)-treated cortical neurons, in which NF-B translocation into the nucleus was blocked. Scale bar: Aa, 40 m; Ab,
40 m; Ac, 40 m; Ba, 16 m; Bb, 8 m; Bc, 20 m.
298 H-B Zhao et al
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