Journal of Ethnopharmacology 111 (2007) 219226

Ganoderma lucidum polysaccharides enhance the function of

immunological effector cells in immunosuppressed mice
Xiao-Ling Zhu
a,
, Alex-F. Chen
b
, Zhi-Bin Lin
a,
a
Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, PR China
b
Departments of Pharmacology and Neurology and the Neuroscience Program, Michigan State University, East Lansing, MI 48824-1317, USA
Received 18 April 2006; received in revised form 23 October 2006; accepted 16 November 2006
Available online 21 November 2006
Abstract
The present study was designed to determine in vivo efcacy of Ganoderma lucidum polysaccharides (Gl-PS) for enhancing the activity
of immunological effector cells in immunosuppressed mice. Mice were injected intraperitoneally (i.p.) once daily with low-dose (2.5 mg/kg),
intermediate-dose (25 mg/kg), and high-dose (250 mg/kg) of Gl-PS, respectively, for 7 consecutive days 24 h after i.p. injection of a immunosup-
pressing anti-tumor agent cyclophosphamide (Cy, 300 mg/kg). In Cy-treated mice, compared to vehicle, low-dose Gl-PS accelerated recovery of
bone marrow cells, red blood cells and white blood cells, as well as splenic natural killer cells and natural killer T cells, and enhanced T and B cell
proliferation responses on day 8, cytotoxic T lymphocyte activity on day 5, as well as NK cell and lymphokine activated killer cell activity on days
79. Furthermore, it promoted phagocytosis and cytotoxicity of macrophages on day 12. The above benecial effects induced by the low-dose
Gl-PS treatment did not result in any side effects. These results demonstrate the efcacious effects of low-dose Gl-PS treatment for enhancing
the activity of immunological effector cells in immunosuppressed mice, and may provide a basis for applying this herb as an efcacious adjacent
immunopotentiating therapy against cancer chemotherapy-induced immunosuppression.
2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ganoderma lucidum (Leyss. ex Fr.) Karst. (Ling Zhi); Polysaccharide; Cyclophosphamide; Cytotoxicity; Immunomodulation
1. Introduction
Ganoderma lucidum(Leyss. ex Fr.) Karst. (Ling Zhi) (Aphyl-
lophoromycetideae) (the family Polyporaceae) was rst indexed
in the Shen Nongs Materia Medica (206 BC8 AD) as a
longevity-promoting and tonic herb of the non-toxic superior
class, and has been used in traditional Chinese medicine (TCM)
for more than 2000 years to prevent and/or treat various human
diseases such as hepatitis, chronic bronchitis, gastritis, tumor
Abbreviations: Gl-PS, Ganoderma lucidum polysaccharides; i.p., injected
intraperitoneally; Cy, cyclophosphamide; Gl, Ganoderma lucidum; TCM,
traditional Chinese medicine; LH, levamisole hydrochloride; BMC, bone mar-
row cell; PBS, phosphate buffered saline; FCS, fetal calf serum; Con A,
concanavalin A; LPS, lipopolysaccharide; MTT, 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide; NK, splenic natural killer; CTL, cytotoxic
T lymphocytes; LAK, lymphokine-activated killer; IFN-, interferon-; LDH,
lactate dehydrogenase; mAb, monoclonal antibody; FITC, uorescein isothio-
cyanate; PE, phycoerythrin

Corresponding authors. Tel.: +86 10 8280 1686; fax: +86 10 8280 1686.
E-mail addresses: xiaolingzhu88@yahoo.com.cn (X.-L. Zhu),
chenal@msu.edu (A.-F. Chen), linzb@public3.bta.net.cn (Z.-B. Lin).
growth and immunological disorders. According to Fuzheng
Guben, one of the major TCM therapeutic principles, Gano-
derma lucidum (Gl) is capable of strengthening body resistance
and improving constitutive homeostasis in patients (Lin, 2001).
Gl polysaccharides (Gl-PS), a glycopeptide isolated from the
water-soluble polysaccharides of Gl, is a major effective com-
ponent of Gl (Lin, 2001). Our and some others previous studies
have demonstrated that Gl-PS exhibit immunomodulatory and
anti-tumor effects (Houet al., 1995; Zhanget al., 2002; Baoet al.,
2002; Lin and Zhang, 2004; Gao et al., 2005; Sung et al., 2005;
Zhu and Lin, 2005; Zhu and Lin, 2006). Cyclophosphamide (Cy)
is the most widely used alkylating agent in cancer chemotherapy
to date. The anti-tumor effect of Cy is in proportion to the dose
of Cy administered, often resulting in immunosuppressive and
cytotoxic effects (Singh et al., 1993). Chemotherapy-induced
leukopenia leads to signicant morbidity and mortality, a major
limiting factor in clinical chemotherapy without efcacious
remedies. As a tumor grows progressively, the immune systemof
tumor-bearing hosts is frequently impaired (Bear, 1986; Hoover
et al., 1990). Our previous studies have shown that Gl-PS signif-
icantly ameliorate the inhibitory effects in lymphocytes induced
0378-8741/$ see front matter 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2006.11.013
220 X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226
by anti-tumor agents such as mitomycin and etoposide in vitro
(Lei and Lin, 1993). However, the in vivo efcacy of Gl-PS
on immunological effector cells, which play a key role against
tumor growth under immunosuppression, is poorly understood.
The present study was thus designed to elucidate the effects of
Gl-PS on immunological effector cells in immunosuppressed
mice induced by Cy treatment.
2. Materials and methods
2.1. Animals and drugs
Inbred strain male (68 weeks old) C57BL/6j (H-2
b
) mice
were purchased from the Department of Experimental Ani-
mals, Health Science Centre, Peking University, Beijing, China.
Ganoderma lucidum (Leyss. ex Fr.) Karst. (Ling Zhi) (Aphyl-
lophoromycetideae) (Polyporaceae) cultivated with wood log
was obtained from The Ganoderma lucidum Production Base
in Taining County, Fujian Province in China. Gl has special
characteristics such as umbrella like fungi composed of pileu
and stipe, pileu semicircular, circular or reniform, dorsal sur-
face yellow-brown to red-brown, with annular ridges and radial
veins, ventral surface nearly white to brownish, with many small
pores in which there are numerous basidiospores, stipe mostly
laterally located curved in zigzag way, yellow-brown, corky tex-
ture and light weight. The quality of Ganoderma lucidum fruit
bodies was monitored by Dr. Xiaolan Mao, a senior researcher
of the National Institute of Microbiology and Microbiological
Institute, China Academy of Science. The selected suitable Gan-
oderma lucidum strain for cultivation with wood log is Ga0801
(No. of strain) and was preserved by Profs. Shuqian Lin and
Saizhen Wang of Fuzhou Institute of Green Valley Bio-Pharm
Technology in China. Gl-PS were isolated from boiling water
extract of the fruit bodies of Ganoderma lucidum, followed by
ethanol precipitation, dialysis, and protein depletion using the
Sevag method, as we previously described (Cao and Lin, 2002;
Lin et al., 2003). The component sugar and molecular weight
distribution of the glycopeptides were determined by gel per-
meation chromatography (GPC) and high performance liquid
chromatography (HPLC). The structure of the glycopeptides
was detectedbyIR,
1
HNMRand
13
CNMR. As a polysaccharide
peptide, the isolated Gl-PS has a molecular weight of 584,900,
with a ratio of polysaccharides to peptides of 93.616.49%. The
polysaccharides consist of d-rhamnose, d-xylose, d-fructose, d-
galactose, d-mannose, d-glucose, and uronic acid with a molar
ratio of 0.793:0.964:2.944:0.167:0.389:7.94:0.33. The glyco-
side linkage was major - with minor -bonding. The peptides
contain 16 amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val,
Met, Ile, Leu, Phe, Lys, His, Arg, Pro) (Cao and Lin, 2002; Lin
et al., 2003). As a water-soluble powder, Gl-PS was dissolved in
physiological saline, ltered through a 0.22 m lter and stored
at 4

C before use. Cy (Shanghai Hualian Pharmaceutical Co.

Ltd., Shanghai, China) was dissolved in sterilized saline prior to
its injection to mice. Levamisole hydrochloride (LH) was pur-
chased from Hunan Dongting Pharmaceutical Co. Ltd. (Hunan,
China).
2.2. Induction of immunosuppression and treatment
protocols
Mice were injected intraperitoneally (i.p.) with a single sub-
lethal dose of Cy (300 mg/kg) on day 0. Mice were divided into
ve groups 24 h after Cy injection and received i.p injections
once daily for 7 consecutive days of (1) low-dose Gl-PS (2.5 mg/
kg), (2) intermediate-dose Gl-PS (25 mg/kg), (3) high-dose
Gl-PS (250 mg/kg), (4) levamisole hydrochloride (10 mg/kg, an
immunopotentiating agent) as positive controls, and (5) vehicle
(i.e. sterile physiological saline) as negative controls.
2.3. Peripheral erythrocyte, leukocyte, splenocyte and bone
marrow cell counts
Blood was collected on the day of sacrice by retro-orbital
bleed into heparin tubes. Single-cell spleen suspensions were
pooled in serum-free RPMI-1640 medium (Gibco Laborato-
ries, NY, USA) by ltering the suspension through sieve mesh
with the aid of a glass homogenizer to exert gentle pressure on
the spleen fragments. Erythrocytes were lysed with an ammo-
niumchloride solution(0.15 MNH
4
Cl, 10 mMKHCO
3
, 0.1 mM
EDTA, pH7.2). Bone marrowcell (BMC) suspensions were pre-
pared by ushing a femur with serum-free RPMI-1640 media
through syringe needles several times. At the time of assay, total
numbers of circulatingerythrocytes, leukocytes, splenocytes and
BMC were counted under light microscopy.
2.4. Phagocytosis of peritoneal macrophages
Peritoneal macrophages were prepared as described (Berrebi
et al., 2003). Phagocytosis of macrophages was measured by
neutral red uptake method as described (Weeks et al., 1987).
Briey, peritoneal exudates were induced by intraperitoneal
injection of 2 ml 3% thioglycollate. Cells were harvested 96 h
later from exudates after wash with cold phosphate buffered
saline (PBS) containing 5 U/ml heparin. Cells were then cul-
tured overnight in RPMI-1640 with 10% fetal calf serum (FCS)
at 37

C in a humidied atmosphere of 5% CO
2
. The follow-
ing day, all nonadherent cells were removed by washing with
PBS. Adherent cells were detached using 10 mM EDTA in PBS
and seeded at a density of 1 10
5
cells/well in the 96-well
microplates with complete RPMI-1640 media. At 24 h after
culture, the cells were washed and neutral red (50 ng/ml) was
added. The plates were incubated for 3 h and cells were then
washed to remove excess dye and blotted dry. The incorpo-
rated dye was resuspended in ethanol (50%) containing glacial
acetic acid (1%) and the absorbance was measured at 540 nm in
a microplate reader (Model 550 BIO-RAD). The absorbance (A)
was translated into phagocytosis ratio for comparison: phago-
cytosis ratio =test
A
/normal control
A
100%.
2.5. Assay of splenocyte proliferation induced by T cell and
B cell mitogens concanavalin A and lipopolysaccharide,
respectively
Splenocytes were placed into the 96-well at-bottomed
microplates in triplicate at 5 10
5
cells/well, then 2.5 g/well
X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226 221
of concanavalin A (Con A) or 10 g/well of lipopolysac-
charide (LPS, both from Sigma, St. Louis, MO, USA)
was added to the wells. The cells were then incubated
in a total volume of 200 l/well. Serum free RPMI-1640
medium was used as control. Cell proliferation was mea-
sured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay 72 h after culture (Wang et al.,
2002). MTT (Sigma, St. Louis, MO, USA) solution of
20 l (5 g/l) was added to each well. After 4 h incuba-
tion, the cells were lysed and the purple formazan crystals
were solubilized for detection at 570 nm. The absorbance
(A) was translated into lymphocyte proliferation ratio
for comparison: lymphocyte proliferation ratio =test
A
/normal
control
A
100%.
2.6. Preparations of anti-tumor effector cells
Splenocytes were prepared as the effector cells for splenic
natural killer (NK) activity assay as described previously (Li
et al., 1998). Splenic cytotoxic T lymphocytes (CTL) were
prepared as described (Li et al., 1998). Briey, splenocytes
(1 10
7
cells/well) isolated from C57 BL/6j mice (H-2
b
) as
responders were cultured with 25 mg/l mitomycin C-treated
P815 sensitizer cells (stimulator; H-2
d
) at a 50:1 ratio in 24-well
tissue culture plates. On day 5 after incubation, the responder
cells were harvested as CTL. We conrmed that the positive
rate of T cells was greater than 94% and the positive ratio
of NK cells was less than 1% after incubation for 120 h, as
determined by ow cytometry, which corresponded with the
previous report (Li et al., 2000). To generate lymphokine-
activated killer (LAK) cells, splenocytes at a concentration
of 1 10
9
cells/l were incubated with 300 IU/ml rIL-2 and
cells were harvested after 3 days. The peritoneal macrophages
were isolated as described (Wilbanks et al., 1999). They
were incubated overnight (1824 h) with interferon- (IFN-
, 100 U/ml nal concentration) (PeproTech EC Ltd. London,
UK), and the adherent cells were washed twice and incubated
overnight in the presence of LPS (2 g/ml) prior to cytotoxicity
assay.
2.7. Cytotoxicity assays
Tumoricidal activity of the effector cells was assayed by
measuring lactate dehydrogenase (LDH) released from the tar-
get cells using a cytotoxicity assay kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) according to the man-
ufactures protocol. Briey, effector cells (2 10
5
per well) in
the 96-well microplates were co-cultured with target cells at
an E/T ratio of 20:1. After 24 h of culture, supernatants were
evaluated for LDHactivity released by the damaged cells. Spon-
taneous LDHrelease fromeffector cells, cell-free culture media,
and target cells lysed with Triton X-100 were served as con-
trols. The percentage of specic cell release was calculated as
percentage-specic cytotoxicity (% C). All assays were per-
formed in triplicate. YAC-1 cells were used as target for NK
cells and CTL, and P815 cells were used as target for LAK cells
and macrophages.
2.8. Cell staining for phenotype analysis
Effector cells were freshly harvested and washed twice
with ice-cold FACScan buffer (PBS containing 2% FCS and
0.1% sodium azide). Twenty percent of mixed mouse and rat
sera were used to block non-specic antibody binding before
cells were then stained with the monoclonal antibody (mAb)
against CD3 coupled to uorescein isothiocyanate (FITC)
(Santa Cruz, CA, USA) or mAbs against NK1.1 coupled to
phycoerythrin (PE) (Pharmingen, CA, USA) for 30 min at
4

C in the dark. The stained cells were washed twice, xed

with 1% paraformaldehyde in FACScan buffer, and then ana-
lyzed by a FACScan ow cytometer (Becton Dickinson, NJ,
USA).
2.9. Statistical analysis
Data were analyzed by one-way analysis of variance
(ANOVA), followed by Dunnetts t-test. Results were presented
as mean S.E.M. Values of P<0.05 were considered to be a
statistically signicant nding.
Table 1
Effects of Gl-PS on numbers of red blood cells (RBC, 10
12
l
1
), white blood cells (WBC, 10
9
l
1
), bone marrow cells (BMC, 10
6
/a femur) and splenocytes
(SC, 10
7
) on days 3 and 9 after Cy treatment
Groups (mg/kg) Day 3 Day 9
RBC WBC BMC SC RBC WBC BMC SC
Gl-PS
250.0 1.8 0.3
c
2.0 0.4
b
3.5 0.5
c
0.3 0.1
c
3.3 0.5
b
3.1 0.8
b
6.4 0.8
b
1.0 0.1
b
25.0 1.9 0.3
c
1.9 0.4
b
4.4 0.6
c
0.8 0.1
c
4.0 0.6
b
3.5 0.6
b
9.0 0.8 1.4 0.4
b
2.5 2.5 0.4
c
2.9 0.6
b
4.6 0.7
c
1.0 0.2
c
4.3 0.6
b
3.3 0.7
b
8.8 0.8 1.6 0.5
b
LH
10.0 2.5 0.4
c
2.8 0.5
b
4.3 0.7
c
0.7 0.1
c
3.8 0.5
b
2.8 0.5
b
6.2 1.0
b
1.4 0.3
b
Vehicle 1.5 0.6
c
2.7 0.6
b
4.1 0.6
c
0.4 0.1
c
3.5 0.5
b
2.6 0.4
b
6.5 0.8
b
1.3 0.2
b
Normal 7.5 0.8 6.8 1.5 9.9 1.0 4.3 0.5 9.0 1.9 6.9 1.7 9.6 0.6 4.0 0.6
The data are expressed as mean S.E.M. of six mice. Gl-PS: Ganoderma lucidumpolysaccharide; LH: levamisole hydrochloride.
b
P<0.05 and
c
P<0.001 vs. normal
mice.
222 X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226
3. Results
3.1. Effects of Gl-PS on peripheral red blood cells (RBC),
white blood cells (WBC), splenocytes and bone marrow
cells (BMC) in Cy-treated mice
The numbers of peripheral RBC and WBC, splenocytes and
BMC in Cy-treated mice receiving various treatment protocols
were examined at different time points, and the results were
shown in Tables 1 and 2. Cy treatment markedly reduced the
numbers of RBC and WBC, splenocytes and BMC on day 3
(Table 1). Treatments with Gl-PS of low- or intermediate-dose
restored BMC and RBC counts to normal levels in Cy-treated
mice on days 9 and 24, respectively. Low-dose Gl-PS treat-
ment also resulted in partial but signicant recovery of WBC
counts compared with saline vehicle group on day 24 (Table 2).
While levamisole hydrochloride (LH) treatment also increased
RBC counts on day 24, it failed to augment other cell counts
compared to Gl-PS treated mice. No evident toxic or side
effects were observed in Gl-PS treated mice during these
experiments.
3.2. Effects of Gl-PS on concanavalin A or
lipopolysaccharide-induced lymphocyte proliferation
The normal murine lymphocyte proliferation ratio induced
by Con A or LPS and treated with RPMI medium 1640 was
regarded as 100%. The proliferative responses of lymphocytes
to both Tcell and Bcell mitogens (Con Aand LPS, respectively)
were reduced markedly on day 3 in Cy-treated mice, when com-
pared to the normal lymphocyte proliferation ratio (Fig. 1A).
Treatments with low- and intermediate-dose of Gl-PS or lev-
amisole hydrochloride (LH) promoted recovery of both T and B
cell proliferation responses on day 8, whereas the lymphocytes
proliferation ratio in high-dose of Gl-PS and the vehicle groups
was still signicantly lower than the normal (Fig. 1B).
3.3. Effects of Gl-PS on macrophage phagocytosis and
anti-tumor activity in Cy-treated mice
The phagocytosis of macrophages from normal mice was
regarded as 100% by neutral red uptake method. As shown in
Table 2
Effects of Gl-PS on numbers of red blood cells (RBC, 10
12
l
1
) and white
blood cells (WBC, 10
9
l
1
) on day 24 after Cy treatment
Groups RBC WBC
Gl-PS (mg/kg) 250.0 4.1 0.3
b
3.6 0.4
c
25.0 6.2 0.4 4.3 0.4
b
2.5 7.8 0.3 5.0 0.3
be
LH (mg/kg) 10.0 6.7 0.7 3.9 0.8
b
Vehicle 4.4 0.6
b
3.7 0.3
b
Normal 8.2 0.9 6.1 0.4
The data were expressed as mean S.E.M. of six mice. Gl-PS: Gano-
derma lucidum polysaccharide; LH: levamisole hydrochloride.
b
P<0.05 and
c
P<0.001 vs. normal mice.
e
P<0.05 vs. Cy-treated mice administrated saline
vehicle.
Fig. 1. Effects of Gl-PS on concanavalin A and lipopolysaccharide-induced
lymphocyte proliferation on day 3 (A) and day 8 (B) following Cy treatment.
Data were expressed as mean S.E.M. of six mice. Gl-PS: Ganoderma lucidum
polysaccharide; LH: levamisole hydrochloride.
*
P<0.05 and
**
P<0.01 vs. nor-
mal mice.
Fig. 2, compared to vehicle and normal, on day 12 in Cy-treated
mice, injection with low-dose Gl-PS or levamisole hydrochlo-
ride (LH) signicantly enhanced whereas intermediate-dose
Gl-PS treatment restored impaired macrophage phagocytosis,
and furthermore, treatment with low-dose Gl-PS signicantly
increased the cytotoxicity (an indicator of their anti-tumor activ-
ity) against P815 cells (NK-resistant) in activated macrophages
Fig. 2. Effects of Gl-PS on macrophage phagocytosis indicated by Neutral Red
assay in Cy-treated mice on day 12. Data were expressed as mean S.E.M. of six
mice. Gl-PS: Ganoderma lucidum polysaccharide; LH: levamisole hydrochlo-
ride.
*
P<0.05 and
**
P<0.01 vs. normal mice;
#
P<0.001 vs. Cy-treated mice
administrated saline vehicle.
X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226 223
Fig. 3. Effects of Gl-PS on macrophage cytotoxicity in Cy-treated mice on day
12. The cytotoxicity of activated macrophage was determined by LDH assay of
the supernatants from cultured P815 target cells. Effectors:targets =20:1. Data
were expressed as mean S.E.M. of six mice. Gl-PS: Ganoderma lucidum
polysaccharide; LH: levamisole hydrochloride.
*
P<0.05 and
**
P<0.01 vs.
normal mice;
#
P<0.001 vs. Cy-treated mice administrated saline vehicle.
fromCy-treated mice compared to the vehicle and normal, while
intermediate-dose Gl-PS or LH treatment restored impaired
macrophage cytotoxicity, but cytotoxicities in high-dose Gl-PS
and vehicle groups were lower than the normal (Fig. 3).
3.4. Effects of Gl-PS on splenic natural killer cell (NK),
lymphokine-activated killer cell (LAK) and cytotoxic T
lymphocytes (CTL) cytotoxicities in Cy-treated mice
A single sublethal dose of Cy inhibited NK cell, LAK cell
and CTL cytotoxicity signicantly by day 5 in vehicle group,
compared with normal mice as shown in Fig. 4A. In Cy-treated
mice, only low-dose Gl-PS as well as levamisole hydrochloride
(LH) restored the CTLcytotoxicity to normal on day 5 (Fig. 4A).
LH, intermediate- and low-dose Gl-PS had similar effect on NK
cell and LAK cell cytotoxicity on day 7, but that cytotoxicities
in high-dose Gl-PS and vehicle groups still were lower than the
normal (Fig. 4B). On day 9, above parameters in all groups
restored normal, injection with low-dose Gl-PS signicantly
augmented LAK cytotoxicity further compared to the vehicle
and the normal, and NK cell cytotoxicities of LH, intermediate-
and low-dose Gl-PSgroup in Cy-treated mice were stronger than
that of normal and vehicle (Fig. 4C).
3.5. Effects of Gl-PS on immunophenotypes of splenocytes
in Cy-treated mice
Immunophenotypes were evaluated by determining the abso-
lute numbers of splenocytes in Cy-treated mice on day 9.
Compared to normal mice, the numbers of both CD3
+
T lym-
phocytes and CD3
+
NK1.1
+
(NKT) cells in Cy-treated mice
were decreased to a similar extend in all experimental groups
(Table 3). Treatment with low- and intermediate-dose of Gl-PS
rescued NK1.1
+
cell numbers to the normal levels, an effect
that was not mimicked in other treatment groups. In addi-
tion, treatment with low-dose Gl-PS signicantly increased the
number of NKT cells as compared to the saline vehicle group
(Table 3).
Fig. 4. Effects of Gl-PSonsplenic natural killer cell (NK), lymphokine-activated
killer cell (LAK) andcytotoxic Tlymphocytes (CTL) cytotoxicities inCy-treated
mice on day 5 (A), day 7 (B) and day 9 (C). Effectors:targets =20:1. Gl-PS:
Ganoderma lucidum polysaccharide; LH: levamisole hydrochloride. Data were
expressed as mean S.E.M. of six mice.
*
P<0.05 and
**
P<0.001 vs. normal
mice.
#
P<0.05 vs. Cy-treated mice administrated saline vehicle.
4. Discussion
In the present study, we determined, for the rst time, the ef-
cacyproles of chronic, low-dose Gl-PStreatment for enhancing
the activity of immunological effector cells and haematopoiesis
in immunosuppressed mice. The major new ndings are that in
cyclophosphamide-induced immunosuppressed mice, chronic
treatment with low-dose Gl-PS resulted in (1) accelerated recov-
ery of bone marrow cells (BMC), red blood cells (RBC) and
white blood cells (WBC), as well as splenic natural killer (NK)
cells and natural killer T (NKT) cells, (2) enhanced T and
224 X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226
Table 3
Effects of Gl-PS on immunophenotypes of splenocytes on day 9 after Cy treatment
Groups CD3
+
(10
6
) NK1.1
+
(10
6
) CD3
+
NK1.1
+
(10
5
)
Gl-PS (mg/kg) 250.0 4.3 0.3
c
0.5 0.1
c
1.7 0.1
c
25.0 4.8 0.6
c
1.2 0.3 2.3 0.2
c
2.5 5.3 0.6
c
1.3 0.3 2.8 0.2
ce
LH (mg/kg) 10.0 4.4 0.9
c
0.8 0.2
b
2.1 0.1
c
Vehicle 4.8 0.3
c
0.7 0.1
b
1.3 0.1
c
Normal 18.1 0.9 1.3 0.1 4.4 0.3
The data were expressed as mean S.E.M. of six mice. Values were expressed as the absolute number per spleen 10
6
. Gl-PS: Ganoderma lucidum polysaccharide;
LH: levamisole hydrochloride.
b
P<0.05 and
c
P<0.001 vs. normal mice;
e
P<0.05 vs. Cy-treated mice administrated saline vehicle.
B cell proliferation responses, cytotoxic T lymphocyte (CTL)
activity as well as NK cell and lymphokine activated killer
(LAK) cell activity, (3) augmented macrophage phagocytosis
and anti-tumor cytotoxicity, and (4) achieved the greatest effect
on promoting granulocytopoiesis. None of mice treated with
Gl-PS died, nor did their body weights change signicantly
(P>0.05, data not shown) during the experiment period. The
above benecial effects induced by the low-dose Gl-PS treat-
ment were not paralleled with any evident toxic or side effects.
Thus, in vivo treatment with low-dose Gl-PS accelerates the
recovery of immunosuppressed mice from leukopenia, myelo-
suppression and immunosuppression, the common conditions
associated with cancer chemotherapy.
In sublethal dose (300 mg/kg, i.p.) of cyclophosphamide
treated mice, the immunosuppression manifest in markedly
reduced the numbers of peripheral RBC, WBC, splenocytes and
bone marrowcells, inhibited lymphocyte proliferative responses
to both T and B mitogens, and activity of NK cell, LAK
cell and CTL. These data are consistent with previously pub-
lished studies (Ballas, 1986; Gazit and Kedar, 1994; Shen et al.,
1994; Yang, 1994). Although cyclophosphamide is the main-
stay cancer chemotherapy agent, its immunosuppressing activity
represents a major clinical challenge and is a main limiting
factor for sustained clinical use. One of the few remedies is
the use of levamisole, an immunopotentiating agent. Although
levamisole has been shown to improve the function of immuno-
logical effector cells, it causes a number of severe side effects
including serious neurological symptoms, gastric haemorrhage
and vomiting of blood, colic, anaemia and vasculitis (Bagga
and Hari, 2000; Grohn et al., 1984; Palcoux et al., 1994; Joly et
al., 1998; Tenbrock et al., 1998). For the past several decades,
attempts have been made to search for safer immunomodulating
agents, and one of the special focuses has been on the biological
response modier (BRM) derived from natural products. The
results of the present study demonstrate that low-dose Gl-PS,
when injected intraperitoneally in mice, affords the greatest pro-
tection for immune effector cells and hematopoietic progenitors
against the sublethal effects induced by cyclophosphamide.
Our ndings showed that a single sublethal dose of
cyclophosphamide reduced the counts of peripheral blood cells
on day 3, which corresponded with other reports (Cox, 2000;
Yeager et al., 1982), and low-dose Gl-PS restored the bone mar-
row cells to the normal levels on day 9, had the same effect
on RBC on day 24, and markedly increased WBC counts com-
pared with the saline vehicle group, despite that the WBC in all
cyclophosphamide-treated groups were under normal levels by
day 24.
The proliferation of T and B lymphocytes is known as a
response to the stimulation induced by antigen or mitogens.
While cellular multiplication induced by concanavalin A is
commonly used to detect T lymphocyte immunity in vitro, the
LPS-induced activation of B cells and subsequent immunoglob-
ulin synthesis reect B lymphocyte immunity (Quakyi et al.,
1997). Previous reports have shown that cyclophosphamide sup-
presses both humoral and cellular immune responses (Balow et
al., 1975; Rondinone et al., 1983; Morato et al., 1996; Masnaya
and Ratner, 2000). Indeed, a single dose of cyclophosphamide
(200 mg/kg) inhibited the proliferative response to both B and
T cells (Quakyi et al., 1997). Although the suppressed antibody
productions are reconstituted gradually after 36 weeks, inhibi-
tion of mitogen-stimulated lymphocyte proliferation is sustained
even after 5 weeks (Moynihan and Cohen, 1989). In the present
study, the proliferative responses to both T and Bmitogens were
reduced markedly on day 3 in all cyclophosphamide-treated
groups due to the exposure of the mice to maximal tolerated
dose of cyclophosphamide (i.e. 300 mg/kg). Treatment with
low- or intermediate-dose of Gl-PS promoted the recovery of
T and B cell proliferation responses on day 8, to a similar
extends to the effect induced by levamisole. Our results also
showed that cyclophosphamide treatment impaired macrophage
phagocytosis, consistent with the previously reported nd-
ings of such toxicity induced by cyclophosphamide in vivo
(Marcinkiewicz et al., 1994). Treatment with low-dose Gl-PS
resulted in enhanced phagocytosis and anti-tumor activities of
macrophages. As shown previously, CTL, NK cell and LAK
cell activities are signicantly reduced following treatment with
low dose (150100 mg/kg) or sublethal dose (300 mg/kg) of
cyclophosphamide, which are not recovered until day 9 (Ballas,
1986; Gazit and Kedar, 1994; Shen et al., 1994; Yang, 1994).
Our data showed that treatment with low-dose Gl-PS acceler-
ated recovery of CTL on day 5, NK cell and LAK cell activity
on day 7, and signicantly enhanced NK cell and LAK cell
activity on day 9. NKT cells are a novel subpopulation of T cells
that express NK markers (e.g. NK1.1 in certain mouse strain)
(Maruoka et al., 1998). They have been shown to play a role in
immunoregulation and tumor surveillance as anti-tumor effec-
tor cells (Brutkiewicz and Sriram, 2002; Smyth et al., 2002).
Treatment with low-dose Gl-PS also accelerated the recovery of
splenic NK cell and NKT cell numbers in cyclophosphamide-
treated mice. Recent clinical studies suggested that subgroups
X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226 225
of advanced-stage caner patients treated with Gl-PS 5.4 g/day
orally for 12 weeks might be responsive, which resulted in
immune-modulation, such as signicant increase in NKactivity,
phytohemagglutinin response, counts of CD3
+
, CD4
+
, CD8
+
,
CD56
+
cells, and plasma concentration of interleukin 2, 6 and
interferon-, whereas plasma concentrations of interleukin 1 and
tumor necrosis factor- decreased. The results of the clinical
studies were signicantly variable (Gao et al., 2003; Huang et
al., 2005), but our experiment results from mice were less uc-
tuant comparatively. So the number of patients enrolled need to
be increased to evaluate clinical response and toxicity.
In summary, the present study demonstrate, for the rst time,
that chronic treatment withlow-dose Gl-PSresults inaccelerated
recovery of immuno-suppression in cyclophosphamide-treated
mice, without evident side effects. Our ndings may provide
a mechanistic basis for using Gl-PS as an alternative means
in lessening chemotherapy-induced immunosuppression in can-
cer patients via its actions of immunopotentiation. Taken the
efcacious and safe proles together, our experimental results
also provide support for future clinical studies of applying this
ancient Chinese herb medicine in cancer patients undergoing
chemotherapy.
Acknowledgements
Gl-PSwas kindlyprovidedbyProfs. ShuqianLinandSaizhen
Wang of Fuzhou Institute of Green Valley Bio-Pharm Technol-
ogy. Research Fund of Shanghai Green Valley Holding Co. Ltd.
supported this study.
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