Ganoderma Lucidum Polysaccharides Enhance The Function Of: Immunological Effector Cells in Immunosuppressed Mice
Ganoderma Lucidum Polysaccharides Enhance The Function Of: Immunological Effector Cells in Immunosuppressed Mice
Ganoderma Lucidum Polysaccharides Enhance The Function Of: Immunological Effector Cells in Immunosuppressed Mice
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 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