Professional Documents
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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 efficacy 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 beneficial effects induced by the low-dose
Gl-PS treatment did not result in any side effects. These results demonstrate the efficacious 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 efficacious 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
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 2.2. Induction of immunosuppression and treatment
(Lei and Lin, 1993). However, the in vivo efficacy of Gl-PS protocols
on immunological effector cells, which play a key role against
tumor growth under immunosuppression, is poorly understood. Mice were injected intraperitoneally (i.p.) with a single sub-
The present study was thus designed to elucidate the effects of lethal dose of Cy (300 mg/kg) on day 0. Mice were divided into
Gl-PS on immunological effector cells in immunosuppressed five groups 24 h after Cy injection and received i.p injections
mice induced by Cy treatment. 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
2. Materials and methods immunopotentiating agent) as positive controls, and (5) vehicle
(i.e. sterile physiological saline) as negative controls.
2.1. Animals and drugs
2.3. Peripheral erythrocyte, leukocyte, splenocyte and bone
Inbred strain male (68 weeks old) C57BL/6j (H-2b ) mice marrow cell counts
were purchased from the Department of Experimental Ani-
mals, Health Science Centre, Peking University, Beijing, China. Blood was collected on the day of sacrifice by retro-orbital
Ganoderma lucidum (Leyss. ex Fr.) Karst. (Ling Zhi) (Aphyl- bleed into heparin tubes. Single-cell spleen suspensions were
lophoromycetideae) (Polyporaceae) cultivated with wood log pooled in serum-free RPMI-1640 medium (Gibco Laborato-
was obtained from The Ganoderma lucidum Production Base ries, NY, USA) by filtering the suspension through sieve mesh
in Taining County, Fujian Province in China. Gl has special with the aid of a glass homogenizer to exert gentle pressure on
characteristics such as umbrella like fungi composed of pileu the spleen fragments. Erythrocytes were lysed with an ammo-
and stipe, pileu semicircular, circular or reniform, dorsal sur- nium chloride solution (0.15 M NH4 Cl, 10 mM KHCO3 , 0.1 mM
face yellow-brown to red-brown, with annular ridges and radial EDTA, pH 7.2). Bone marrow cell (BMC) suspensions were pre-
veins, ventral surface nearly white to brownish, with many small pared by flushing a femur with serum-free RPMI-1640 media
pores in which there are numerous basidiospores, stipe mostly through syringe needles several times. At the time of assay, total
laterally located curved in zigzag way, yellow-brown, corky tex- numbers of circulating erythrocytes, leukocytes, splenocytes and
ture and light weight. The quality of Ganoderma lucidum fruit BMC were counted under light microscopy.
bodies was monitored by Dr. Xiaolan Mao, a senior researcher
of the National Institute of Microbiology and Microbiological 2.4. Phagocytosis of peritoneal macrophages
Institute, China Academy of Science. The selected suitable Gan-
Peritoneal macrophages were prepared as described (Berrebi
oderma lucidum strain for cultivation with wood log is Ga0801
et al., 2003). Phagocytosis of macrophages was measured by
(No. of strain) and was preserved by Profs. Shuqian Lin and
neutral red uptake method as described (Weeks et al., 1987).
Saizhen Wang of Fuzhou Institute of Green Valley Bio-Pharm
Briefly, peritoneal exudates were induced by intraperitoneal
Technology in China. Gl-PS were isolated from boiling water
injection of 2 ml 3% thioglycollate. Cells were harvested 96 h
extract of the fruit bodies of Ganoderma lucidum, followed by
later from exudates after wash with cold phosphate buffered
ethanol precipitation, dialysis, and protein depletion using the
saline (PBS) containing 5 U/ml heparin. Cells were then cul-
Sevag method, as we previously described (Cao and Lin, 2002;
tured overnight in RPMI-1640 with 10% fetal calf serum (FCS)
Lin et al., 2003). The component sugar and molecular weight
at 37 C in a humidified atmosphere of 5% CO2 . The follow-
distribution of the glycopeptides were determined by gel per-
ing day, all nonadherent cells were removed by washing with
meation chromatography (GPC) and high performance liquid
PBS. Adherent cells were detached using 10 mM EDTA in PBS
chromatography (HPLC). The structure of the glycopeptides
and seeded at a density of 1 105 cells/well in the 96-well
was detected by IR, 1 H NMR and 13 C NMR. As a polysaccharide
microplates with complete RPMI-1640 media. At 24 h after
peptide, the isolated Gl-PS has a molecular weight of 584,900,
culture, the cells were washed and neutral red (50 ng/ml) was
with a ratio of polysaccharides to peptides of 93.616.49%. The
added. The plates were incubated for 3 h and cells were then
polysaccharides consist of d-rhamnose, d-xylose, d-fructose, d-
washed to remove excess dye and blotted dry. The incorpo-
galactose, d-mannose, d-glucose, and uronic acid with a molar
rated dye was resuspended in ethanol (50%) containing glacial
ratio of 0.793:0.964:2.944:0.167:0.389:7.94:0.33. The glyco-
acetic acid (1%) and the absorbance was measured at 540 nm in
side linkage was major - with minor -bonding. The peptides
a microplate reader (Model 550 BIO-RAD). The absorbance (A)
contain 16 amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val,
was translated into phagocytosis ratio for comparison: phago-
Met, Ile, Leu, Phe, Lys, His, Arg, Pro) (Cao and Lin, 2002; Lin
cytosis ratio = testA /normal controlA 100%.
et al., 2003). As a water-soluble powder, Gl-PS was dissolved in
physiological saline, filtered through a 0.22 m filter and stored 2.5. Assay of splenocyte proliferation induced by T cell and
at 4 C before use. Cy (Shanghai Hualian Pharmaceutical Co. B cell mitogens concanavalin A and lipopolysaccharide,
Ltd., Shanghai, China) was dissolved in sterilized saline prior to respectively
its injection to mice. Levamisole hydrochloride (LH) was pur-
chased from Hunan Dongting Pharmaceutical Co. Ltd. (Hunan, Splenocytes were placed into the 96-well flat-bottomed
China). microplates in triplicate at 5 105 cells/well, then 2.5 g/well
X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226 221
Table 1
Effects of Gl-PS on numbers of red blood cells (RBC, 1012 l1 ), white blood cells (WBC, 109 l1 ), bone marrow cells (BMC, 106 /a femur) and splenocytes
(SC, 107 ) on days 3 and 9 after Cy treatment
Groups (mg/kg) Day 3 Day 9
Gl-PS
250.0 1.8 0.3c 2.0 0.4b 3.5 0.5c 0.3 0.1c 3.3 0.5b 3.1 0.8b 6.4 0.8b 1.0 0.1b
25.0 1.9 0.3c 1.9 0.4b 4.4 0.6c 0.8 0.1c 4.0 0.6b 3.5 0.6b 9.0 0.8 1.4 0.4b
2.5 2.5 0.4c 2.9 0.6b 4.6 0.7c 1.0 0.2c 4.3 0.6b 3.3 0.7b 8.8 0.8 1.6 0.5b
LH
10.0 2.5 0.4c 2.8 0.5b 4.3 0.7c 0.7 0.1c 3.8 0.5b 2.8 0.5b 6.2 1.0b 1.4 0.3b
Vehicle 1.5 0.6c 2.7 0.6b 4.1 0.6c 0.4 0.1c 3.5 0.5b 2.6 0.4b 6.5 0.8b 1.3 0.2b
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 lucidum polysaccharide; 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
Table 2
Effects of Gl-PS on numbers of red blood cells (RBC, 1012 l1 ) and white
blood cells (WBC, 109 l1 ) on day 24 after Cy treatment
Groups RBC WBC
Table 3
Effects of Gl-PS on immunophenotypes of splenocytes on day 9 after Cy treatment
The data were expressed as mean S.E.M. of six mice. Values were expressed as the absolute number per spleen 106 . 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) cyclophosphamide-treated groups were under normal levels by
activity as well as NK cell and lymphokine activated killer day 24.
(LAK) cell activity, (3) augmented macrophage phagocytosis The proliferation of T and B lymphocytes is known as a
and anti-tumor cytotoxicity, and (4) achieved the greatest effect response to the stimulation induced by antigen or mitogens.
on promoting granulocytopoiesis. None of mice treated with While cellular multiplication induced by concanavalin A is
Gl-PS died, nor did their body weights change significantly commonly used to detect T lymphocyte immunity in vitro, the
(P > 0.05, data not shown) during the experiment period. The LPS-induced activation of B cells and subsequent immunoglob-
above beneficial effects induced by the low-dose Gl-PS treat- ulin synthesis reflect B lymphocyte immunity (Quakyi et al.,
ment were not paralleled with any evident toxic or side effects. 1997). Previous reports have shown that cyclophosphamide sup-
Thus, in vivo treatment with low-dose Gl-PS accelerates the presses both humoral and cellular immune responses (Balow et
recovery of immunosuppressed mice from leukopenia, myelo- al., 1975; Rondinone et al., 1983; Morato et al., 1996; Masnaya
suppression and immunosuppression, the common conditions and Ratner, 2000). Indeed, a single dose of cyclophosphamide
associated with cancer chemotherapy. (200 mg/kg) inhibited the proliferative response to both B and
In sublethal dose (300 mg/kg, i.p.) of cyclophosphamide T cells (Quakyi et al., 1997). Although the suppressed antibody
treated mice, the immunosuppression manifest in markedly productions are reconstituted gradually after 36 weeks, inhibi-
reduced the numbers of peripheral RBC, WBC, splenocytes and tion of mitogen-stimulated lymphocyte proliferation is sustained
bone marrow cells, inhibited lymphocyte proliferative responses even after 5 weeks (Moynihan and Cohen, 1989). In the present
to both T and B mitogens, and activity of NK cell, LAK study, the proliferative responses to both T and B mitogens were
cell and CTL. These data are consistent with previously pub- reduced markedly on day 3 in all cyclophosphamide-treated
lished studies (Ballas, 1986; Gazit and Kedar, 1994; Shen et al., groups due to the exposure of the mice to maximal tolerated
1994; Yang, 1994). Although cyclophosphamide is the main- dose of cyclophosphamide (i.e. 300 mg/kg). Treatment with
stay cancer chemotherapy agent, its immunosuppressing activity low- or intermediate-dose of Gl-PS promoted the recovery of
represents a major clinical challenge and is a main limiting T and B cell proliferation responses on day 8, to a similar
factor for sustained clinical use. One of the few remedies is extends to the effect induced by levamisole. Our results also
the use of levamisole, an immunopotentiating agent. Although showed that cyclophosphamide treatment impaired macrophage
levamisole has been shown to improve the function of immuno- phagocytosis, consistent with the previously reported find-
logical effector cells, it causes a number of severe side effects ings of such toxicity induced by cyclophosphamide in vivo
including serious neurological symptoms, gastric haemorrhage (Marcinkiewicz et al., 1994). Treatment with low-dose Gl-PS
and vomiting of blood, colic, anaemia and vasculitis (Bagga resulted in enhanced phagocytosis and anti-tumor activities of
and Hari, 2000; Grohn et al., 1984; Palcoux et al., 1994; Joly et macrophages. As shown previously, CTL, NK cell and LAK
al., 1998; Tenbrock et al., 1998). For the past several decades, cell activities are significantly reduced following treatment with
attempts have been made to search for safer immunomodulating low dose (150100 mg/kg) or sublethal dose (300 mg/kg) of
agents, and one of the special focuses has been on the biological cyclophosphamide, which are not recovered until day 9 (Ballas,
response modifier (BRM) derived from natural products. The 1986; Gazit and Kedar, 1994; Shen et al., 1994; Yang, 1994).
results of the present study demonstrate that low-dose Gl-PS, Our data showed that treatment with low-dose Gl-PS acceler-
when injected intraperitoneally in mice, affords the greatest pro- ated recovery of CTL on day 5, NK cell and LAK cell activity
tection for immune effector cells and hematopoietic progenitors on day 7, and significantly enhanced NK cell and LAK cell
against the sublethal effects induced by cyclophosphamide. activity on day 9. NKT cells are a novel subpopulation of T cells
Our findings showed that a single sublethal dose of that express NK markers (e.g. NK1.1 in certain mouse strain)
cyclophosphamide reduced the counts of peripheral blood cells (Maruoka et al., 1998). They have been shown to play a role in
on day 3, which corresponded with other reports (Cox, 2000; immunoregulation and tumor surveillance as anti-tumor effec-
Yeager et al., 1982), and low-dose Gl-PS restored the bone mar- tor cells (Brutkiewicz and Sriram, 2002; Smyth et al., 2002).
row cells to the normal levels on day 9, had the same effect Treatment with low-dose Gl-PS also accelerated the recovery of
on RBC on day 24, and markedly increased WBC counts com- splenic NK cell and NKT cell numbers in cyclophosphamide-
pared with the saline vehicle group, despite that the WBC in all treated mice. Recent clinical studies suggested that subgroups
X.-L. Zhu et al. / Journal of Ethnopharmacology 111 (2007) 219226 225
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