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International Journal of Biological Macromolecules 64 (2014) 395– 401

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

hemoprotective effects of Ganoderma atrum polysacchariden cyclophosphamide-induced mice

iang Yua, Shao-Ping Niea,∗∗, Jun-Qiao Wanga, Xiao-Zhen Liua, Peng-Fei Yina,an-Fei Huanga, Wen-Juan Lia, De-Ming Gonga,b, Ming-Yong Xiea,∗

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, ChinaSchool of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand

r t i c l e i n f o

rticle history:eceived 14 October 2013eceived in revised form 7 December 2013ccepted 18 December 2013vailable online 24 December 2013

a b s t r a c t

In this study, the chemoprotective effects of Ganoderma atrum polysaccharide (PSG-1) in cyclophos-phamide (Cy) treated mice were investigated. In Cy-treated mice, PSG-1 treatment accelerated recoverydose-dependently of peripheral red blood cells, white blood cells and platelets, enhanced splenic naturalkiller cell activity and cytotoxic T lymphocyte activity. In addition, PSG-1 elevated CD4+ T lymphocyte

+ +

eywords:anoderma atrum polysaccharideyclophosphamidehemoprotective effects

counts as well as the CD4 /CD8 ratio dose-dependently. Furthermore, PSG-1 restored the levels of IL-2, INF-�, IL-10, IgA, IgM and IgG, as well as hemolysin in the sera. Finally, PSG-1 can also significantlyincrease the total antioxidant capacity, activities of superoxidase dismutase, catalase and glutathioneperoxidase, and decrease the malondialdehyde level in vivo. These findings indicate that PSG-1 plays animportant role in the protection against myelosuppression and immunosuppression and oxidative stressin Cy-treated mice and could be a potential immunomodulatory agent.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Cancer is the second leading cause of death in developingountries [1]. However, it is known that most of the anti-cancerrugs currently used in chemotherapy bring an impairment ofhe host defense mechanisms, leading to immunosuppressivend cytotoxic effects, regardless of their great curative effects.yclophosphamide (Cy) has been the most widely used alkyl-ting agent in chemotherapy with a high therapeutic index

nd broad spectrum of activities against a variety of cancersince the late 1950s [2]. Cy is inactive and must first undergoetabolic activation, catalyzed by the hepatic cytochrome P450

Abbreviations: PSG-1, Ganoderma atrum polysaccharide; Cy, cyclophosphamide;H, levamisole hydrochloride; NC, normal control; MC, model control; Ig,mmunoglobulin; T-AOC, total antioxidant capacity; MDA, malondialdehyde; CAT,atalase; SOD, superoxidase dismutase; GSH-Px, glutathione peroxidise; WBC, whitelood cell; RBC, red blood cell; NK cell, natural killer cell; CTL, cytotoxic T lympho-yte.∗ Corresponding author at: State Key Laboratory of Food Science and Technology,anchang University, 235 Nanjing East Road, Nanchang 330047, China.el.: +86 791 83969009; fax: +86 791 83969009.∗∗ Corresponding author at: State Key Laboratory of Food Science and Technology,anchang University, 235 Nanjing East Road, Nanchang 330047, China.el.: +86 791 88304452; fax: +86 791 88304452.

E-mail addresses: spnie@ncu.edu.cn (S.-P. Nie), myxie@ncu.edu.cn,mync@163.com (M.-Y. Xie).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.12.029

to 4-hydroxy-cyclophosphamide and then to phosphoramide mus-tard and acrolein. The mustard component produces a cytotoxiceffect by preventing cell replication, while acrolein is linked withits toxic side effects [3]. Cy-induced myelosuppression, immuno-suppression and oxidative stress lead to significant morbidity andmortality, which is a major limiting factor in clinical chemotherapywithout efficacious remedies.

In recent decades, a wide range of polysaccharides from naturalproducts have attracted increasingly attention due to their broadspectrum of therapeutic properties and relatively low toxicity[4–6]. Mounting studies have reported that combining polysac-charides with chemotherapy may improve quality of life, tumorresponse and performance status, as well as reduce the toxicity ofchemotherapy, such as polysaccharides from Ganoderma lucidumand Sophora subprosrate [7,8].

Ganoderma atrum has been used as a traditional Chinesemedicine and healthy mushroom for thousands of years. Thepolysaccharides are regarded as the major bioactive substances inG. atrum [9]. We recently isolated and purified a polysaccharidefrom G. atrum, named as PSG-1, with a purity of >99.8%, whoseprimary structural features and molecular weight were character-ized [10,11]. Our previous results have demonstrated that PSG-1

had potent antioxidation [10], antitumor [12–14], immunomodu-latary [15,16] and cardiovascular protection [17,18] activities. Thepresent study was designed to investigate the protective effectsof PSG-1 on myelosuppression, immunosuppression and oxidative

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96 Q. Yu et al. / International Journal of Bi

tress induced by Cy treatment. The effects of PSG-1 were evaluatedrom hemopoietic function, lymphocyte activity, serum cytokine,emolysin, immunoglobulin (Ig) levels, as well as in vivo antioxi-ant activity in mice.

. Materials and methods

.1. Materials

Cy was purchased from Jiangsu Hengrui Medicine Co.Lianyungang, Jiangsu, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-iphenyltetrazolium bromide (MTT) was purchased from Amrescoo. Cell culture products were obtained from Life Technolo-ies (Paisley, Scotland, UK). ELISA kits were from R&D SystemsMinneapolis, MN, USA). Anti-CD4 and anti-CD8 antibodies wererovided by eBioscience (San Diego, CA, USA). The Ig kit wasurchased from Shanghai Sun Biological Products Co. Assay kitsor total antioxidant capacity (T-AOC), malondialdehyde (MDA),atalase (CAT), superoxidase dismutase (SOD), and glutathioneeroxidase (GSH-Px) were purchased from Nanjing Jiancheng Bio-ngineering Institute (Nanjing, China).

.2. Characteristics of G. atrum polysaccharide (PSG-1)

The polysaccharide from G. atrum (PSG-1) with a purity of99.8% was isolated and purified as described previously [10].riefly, the polysaccharide fractions were prepared from G. atrum,hich were collected from Ganzhou, Jiangxi Province, China. All

xtracts were finally pooled, and the polysaccharide-enriched frac-ions were precipitated by the addition of 80% (v/v) ethanol. Theolysaccharide fraction was further purified by gel filtration chro-atography. Its primary structural features and molecular weightere characterized by infrared spectrometry, gas chromatography,

ize exclusion chromatography, methylation analysis and 1D/2DMR spectroscopy [11]. Our previous study [16] showed that theuantity of endotoxin in PSG-1 was less than 0.015 EU/mg (nega-ive), as measured by LAL assay, which demonstrated that PSG-1 isree of lipopolysaccharide (LPS) contamination.

.3. Animals and experimental design

Female BALB/c mice (8 weeks old, 18–20 g) were purchased fromhanghai Slac Laboratory Animal Center, Chinese Academy of Sci-nces (Shanghai, China). The animals were provided with waternd mouse chow ad libitum, and were housed in a rodent facility at2 ± 1 ◦C with a 12 h light–dark cycle for acclimatization. The miceere randomly divided into 6 groups consisting of 10 mice each.ll animals were allowed one week to adapt to their environmentefore the treatment. One group of healthy mice was used as nor-al control (NC) group, and treated once daily with physiological

aline for 10 days. From Days 1 to 3, the other five groups of miceere given Cy at 80 mg/kg body weight (BW)/d via intraperitoneal

njection. From Days 4 to 10, the mice were administered as follows:odel control (MC) group, physiological saline; three PSG-1 groups,

t 25, 50, or 100 mg PSG-1/kg BW, levamisole hydrochloride (LH)10 mg/kg BW/d, an immunopotentiating agent) as positive control.y (0.2 mL) was administered via intraperitoneal injection. The oth-rs were administered via gavage in 0.2 mL solutions. Twenty-fourours after the last drug administration, the animals were weighednd then sacrificed via decapitation.

.4. Peripheral white blood cell, red blood cell and platelets counts

Blood was collected on the day of sacrifice by retro-orbital bleednto heparin tubes. White blood cell (WBC), red blood cell (RBC) and

l Macromolecules 64 (2014) 395– 401

platelets counts were analyzed using a Coulter LH755 HematologyAnalyzer.

2.5. Preparation of lymphocyte cells

The extirpated spleens were treated in germ-free condition.Single-cell spleen suspensions were pooled in serum-free RPMI-1640 medium by filtering the suspension through sieve mesh withthe aid of a glass homogenizer to exert gentle pressure on the spleenfragments. Samples were washed twice in PBS/0.1% bovine serumalbumin. After centrifugation (200 g, 5 min), the cells were resus-pended to a concentration of 2 × 106 cells/mL in RPMI 1640 mediumsupplemented with 10% fetal calf serum.

2.6. Cytotoxicity assays of natural killer (NK) cell activity ofsplenocytes

Splenocytes prepared from the spleen were used as the effec-tor cells for splenic NK cell activity assay as described above [19].YAC-1 cells were used as the target cells. Briefly, effector cells(5 × 105 cells/well) in the 96-well round-bottom microplates intriplicate were co-cultured with target cells at 37 ◦C in a humid-ified atmosphere of 5% CO2 at a ratio of effector to target cells of50:1. The plates were then incubated for 20 h at 37 ◦C in 5% CO2atmosphere. 50 �L of MTT solution (2 mg/mL) was added to eachwell and the plate was incubated for another 4 h and subjected toMTT assay. Three kinds of control measurements were performed:target cells control, blank control and effector cells control. NKcell activity was calculated as the following equation: NK activity(%) = (ODT − (ODS − ODE))/ODT × 100%, where ODT is optical den-sity value of target cells control, ODS is optical density value of testsamples, and ODE is optical density value of effector cells control.

2.7. Assays of cytotoxic T lymphocyte (CTL) activity

The CTL activity was analyzed using MTT method as describedabove. Tumor (S180) cells and splenocytes were used as target cellsand effector cells, respectively. The ratio of effector cells to targetcells was 50:1. To determine the percentage of target cells killed,the following equation was used: % lysis = (ODT − (ODS − ODE))/ODT × 100.

2.8. Determination of CD4+ and CD8+ T lymphocytes in the spleen

Single-splenocyte suspension prepared as described above wasincubated with 10 �L of either anti-CD4 or anti-CD8 antibody for60 min at 4 ◦C. Cells were then washed twice with PBS and resus-pended in 1% paraformaldehyde (PFA). The counts of CD4+ and CD8+

T lymphocytes were determined by flow cytometry (Becton Dick-inson, NJ, USA) and expressed as percentages of total number ofT-lymphocytes.

2.9. Determination of IL-2, INF-� and IL-10 in serum

Serum was collected by enucleating eyeball 24 h after the lastadministration of PSG-1. The concentrations of IL-2, INF-� and IL-10 in the sera were determined using ELISA kits according to theinstruction of the manufacturer.

2.10. Measurements of serum Ig

Immunoturbidimetry was used to measure the Ig levels of serum

in mice. The eyes were removed, and venous blood samples (1 mL)were collected and centrifuged. The diluted blood sera were col-lected into test tubes, and 1 mL of IgA, IgG and IgM were separatelyadded to each tube and mixed. The samples were immersed in

Q. Yu et al. / International Journal of Biological Macromolecules 64 (2014) 395– 401 397

Table 1Effects of PSG-1 on numbers of red blood cells (RBC, 1012/L), white blood cells (WBC, 109/L), platelet (1011/L) in Cy treated mice (mean ± S.E.M., n = 10).

Group Dose (mg/kg) RBC (1012) WBC (109) Platelet (1011)

NC 0 4.24 ± 0.44 7.58 ± 1.13 5.63 ± 0.52MC 80 1.32 ± 0.26a 5.53 ± 0.55a 3.63 ± 0.33a

PSG-1 25 2.13 ± 0.42a,c 5.99 ± 0.75b,c 4.12 ± 0.48a,c

50 3.25 ± 0.66a,c 6.64 ± 0.76a,c 4.64 ± 0.38a,c

100 4.25 ± 0.79a,d 7.56 ± 0.74a,c 5.39 ± 0.53a,c

LH 10 4.06 ± 0.25a,c 7.53 ± 1.07b,c 5.58 ± 0.38a,d

NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).a P < 0.05 vs. normal mice.b P < 0.01 vs. normal mice.

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S180 tumor cells was investigated. As shown in Fig. 1, comparedwith the normal control group, NK cell and CTL cytotoxicity ofthe model control group was significantly decreased (P < 0.05),suggesting that the immunosuppressed model was successfully

c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

water bath at 37 ◦C for 15 min, a semiautomatic biochemistrynalyzer was adjusted to zero at 340 nm with NS, and the absorp-ion for each test was measured. The levels of IgA, IgG and IgMere calculated according to the absorption–concentration stan-ard curve.

.11. Measurement of serum hemolysin

The serum hemolysin level was determined using the methody [20]. The grouping and feeding of the mice were the sames described in Section 2.3, except that on the fifth day of PSG-

administration, each mouse was immunized by injection of.2 mL suspension of sheep erythrocytes (SRBC) (109/mL). Twenty-our hours after the last drug administration, blood samples wereollected. One hour later, these blood samples were centrifugedt 2000 rpm for 10 min and 20 �L of serum was diluted 500imes with saline. Approximately 0.5 mL of 10% SRBC and 1 mL ofresh guinea pig serum (1:10 dilution) was added to the reactionubes filled with 1 mL of diluted serum samples. After incuba-ion for 1 h at 37 ◦C, the reaction tubes were immediately movedo an ice bath and centrifuged again under the same conditions.bout 1 mL of the supernatant was mixed with 3 mL of Drabkin’solution. Ten minutes later, the absorbance at 540 nm was mea-ured.

.12. Biochemical assays

To investigate the influence of the PSG-1 on the antioxi-ant status of the Cy-induced mice with immunosuppression, theiochemical parameters were analyzed in various tissues. Theomogenate of liver, heart or kidney was prepared in 0.1 g/mLet weight of ice-cold isotonic physiological saline. The sam-les were centrifuged at 2000 × g at 4 ◦C for 10 min, and theupernatants were then subjected to the measurements of pro-ein, T-AOC, SOD, CAT, GSH-Px and MAD levels. All biochemicalarameters were measured with assay kits: the method of ferriceducing/antioxidant power assay for T-AOC, the xanthine oxidaseechnique at 550 nm for SOD activity, the ammonium molybdate

ethod for CAT, 5, 50-dithiobis-p-nitrobenzoic acid method forSH-Px activity and thiobarbituric acid method for MDA levels

n the cytosolic fraction of these organs. Enzyme activity wasxpressed as nanomoles per milligram protein.

.13. Statistical analysis

Values are expressed as means ± SEM. One-way analysis of vari-nce followed by the Student–Newman–Keuls test was used toetermine the statistical significance between various groups. Aalue of P < 0.05 was considered to be statistically significant.

3. Results

3.1. Effect of PSG-1 on hemopoietic function in Cy-treated mice

To evaluate the protective effect of PSG-1 on the myelosuppres-sion induced by Cy, the RBC, WBC and platelets from peripheralblood in Cy-treated mice were counted. It was found that periph-eral RBC, WBC and platelets counts in Cy-treated mice decreasedsignificantly (Table 1). However, as shown in Table 1, the countsof RBC, WBC and platelets were elevated remarkably by PSG-1 ina dose-dependent manner. Treatments with PSG-1 at 100 mg/kg/drestored WBC and RBC counts to normal levels, even higher thanLH treatment. These results suggested that PSG-1 can restore themyelosuppression induced by Cy.

3.2. Effects of PSG-1 on splenic NK and CTL cytotoxicities inCy-treated mice

Tumor cell elimination is known to be mediated in part bythe cytotoxic activity of NK cells and CTL. Therefore, the cytotoxicactivity of splenocytes against NK cell-sensitive YAC-1 cells and

Fig. 1. Effects of PSG-1 on splenic natural killer cell (NK) and cytotoxic T lym-phocytes (CTL) cytotoxicities in Cy-treated mice. Splenocytes were prepared andassayed for NK cell and CTL activity by the MTT method as described in the text. NC,normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH,levamisole hydrochloride (positive control). Data were expressed as mean ± S.E.M.of 10 mice. aP < 0.05 and bP < 0.01 vs. normal mice. cP < 0.05 and dP < 0.01 vs. Cy-treated mice administrated saline vehicle.

398 Q. Yu et al. / International Journal of Biological Macromolecules 64 (2014) 395– 401

Table 2Effects of PSG-1 on T lymphocyte subsets from spleen in Cy-induced mice (mean ± S.E.M., n = 10).

Group Dose (mg/kg) CD4+ (%) CD8+ (%) CD4+/CD8+

NC 0 27.62 ± 2.11 15.43 ± 1.56 1.79 ± 0.12MC 80 19.33 ± 2.35a 13.65 ± 1.43a 1.42 ± 0.21a,c

PSG-1 25 21.49 ± 2.45b,c 14.02 ± 1.78a,c 1.53 ± 0.16a,c

50 24.64 ± 2.16a,c 14.67 ± 1.55a,c 1.68 ± 0.17b,d

100 27.54 ± 2.54a,c 15.37 ± 1.34a,c 1.79 ± 0.19a,d

LH 10 26.53 ± 2.87b,c 15.78 ± 1.35a,d 1.68 ± 0.11a,c

NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).a P < 0.05 vs. normal mice.

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b P < 0.01 vs. normal mice.c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

stablished. Treatment with low-, intermediate- and high-doseSG-1 enhanced dose-dependently NK cell and CTL cytotoxicityompared with model control group, suggesting that PSG-1 couldmprove the cell immune function in these mice. The treatment ofH also promoted recovery of NK cell activity of splenocytes, but itailed to compete with normal group.

.3. Effects of PSG-1 on immunophenotypes of splenocytes iny-treated mice

To investigate the effect of PSG-1 on the cellular immunity,mmunophenotypes of splenocytes were evaluated by determin-ng the counts of CD4+ and CD8+ T lymphocytes by flow cytometry.s shown in Table 2, the percentages of CD4+ and CD8+ T lympho-ytes, and the rate of CD4+/CD8+ were lower in the model control,hen compared with the normal control. In comparison, the per-

entage of the CD4+ T-lymphocytes was increased in PSG-1-treatedroups (25, 50 or 100 mg/kg/d), compared to the model group.oreover, PSG-1 (50 or 100 mg/kg/d) significantly up-regulated the

D4+/CD8+ ratio up to 1.68 or 1.79.

.4. Effects of PSG-1 on serum IL-2, IFN-� and IL-10 levels

As shown in Fig. 2, Cy injection caused significant reduction in

he levels of IL-2 and IFN-�. PSG-1 at a dose of 50 or 100 mg/kg/dmproved the decline of IL-2 level, but no significant change wasbserved in administration of PSG-1 at a low dose. Similarly, PSG-125, 50 or 100 mg/kg/d) induced elevation in serum IFN-� against

ig. 2. Effects of PSG-1 on IL-2, IFN-� and IL-10 levels in the serum of Cy-treatedice. NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysac-

haride; LH, levamisole hydrochloride (positive control). Data were expressed asean ± S.E.M. of 10 mice. aP < 0.05 and bP < 0.01 vs. normal mice. cP < 0.05 and

P < 0.01 vs. Cy-treated mice administrated saline vehicle.

Cy and restored IFN-� to near normal levels. Furthermore, signif-icant increases in levels of IL-10 were observed in serum of micetreated with Cy alone as compared with normal control. The admin-istration of PSG-1 decreased the levels of IL-10 significantly in adose-dependent manner.

3.5. Effects of PSG-1 on serum Ig contents

To determine the effects of PSG-1 on humoral immunity, thelevels of IgM, IgG and IgA in the sera of Cy-treated mice were deter-mined by immunoturbidimetry. As shown in Table 3, the serumIgA, IgG and IgM levels were significantly decreased by Cy (P < 0.01).However, significant enhancements in total serum IgA, IgG, and IgMlevels were observed in mice treated with PSG-1 at the three dosescompared with model control group (P < 0.05, or P < 0.01) (Table 3).Especially at the dose of 100 mg/kg/d, the levels of IgA, IgG, and IgMwere increased to the level of normal control group level, but lowerthan LH group.

3.6. Effect of PSG-1 on serum hemolysin formation

To further investigate the effect of PSG-1 on the humoralimmune response, the content of serum hemolysin in response tocellular antigen was measured. The results (Table 3) showed thatthe production of serum hemolysin was noticeably suppressed inthe model control compared with the normal control (P < 0.05). Incomparison, the serum hemolysin level was significantly increasedin all three PSG-1 groups and LH group compared to the modelgroup.

3.7. Antioxidant activities of PSG-1

T-AOC reflects or represents the capacity of the non-enzymaticantioxidant defense system. As shown in Tables 4–6, T-AOC levelsin all the three organs were significantly decreased in the Cy groupcompared to the NC group (P < 0.01). In comparison, PSG-1 admin-istration greatly elevated the T-AOC in heart, liver and kidney. Thelevel of T-AOC at the PSG-1 group at 100 mg/kg/d had the maximalincrease in all the organs tested, and reached to the level of the nor-mal group. Meanwhile, the LH treatment also significantly raisedthe activity of T-AOC compared with the model control in heart,liver and kidney (P < 0.05). The enzymes (SOD, CAT and GSH-Px)protect against oxidative stress and tissue-damage by convertingactive oxygen molecules into non-toxic compounds [21]. The treat-ment with Cy resulted in a severe decrease of SOD, CAT and GSH-Pxin the heart, liver and kidney (Tables 4–6), which was significantlyincreased by PSG-1 treatments at 25, 50 and 100 mg/kg/d. The activ-

ities of SOD, CAT and GSH-Px in PSG-1 group (100 mg/kg/d) andpositive group were almost recovered to normal level. MDA is amain marker of the endogenous lipid peroxidation. MDA levels inall the organs were found to increase when the mice were treated

Q. Yu et al. / International Journal of Biological Macromolecules 64 (2014) 395– 401 399

Table 3Effect of PSG-1 on serum Ig levels and hemolysin formation in Cy-treated mice (mean ± S.E.M., n = 10).

Group Dose (mg/kg) IgA (g/L) IgM (g/L) IgG (g/L) Hemolysin level (OD)

NC 0 3.53 ± 0.42 4.22 ± 1.11 5.73 ± 0.32 0.763 ± 0.032MC 80 0.85 ± 0.23a 1.42 ± 0.25a 2.33 ± 0.34a 0.425 ± 0.021a

PSG-1 25 2.93 ± 0.42a,c 2.34 ± 0.45b,c 3.52 ± 0.44a,c 0.531 ± 0.022a,c

50 2.32 ± 0.66a,c 3.27 ± 0.36a,c 4.47 ± 0.78a,c 0.592 ± 0.026a,d

100 3.35 ± 0.79a,d 4.34 ± 0.74a,c 5.89 ± 0.53a,c 0.734 ± 0.009a,d

LH 10 4.01 ± 0.211a,c 4.53 ± 0.27b,c 5.92 ± 0.38a,d 0.745 ± 0.021a,d

NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).a P < 0.05 vs. normal mice.b P < 0.01 vs. normal mice.c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

Table 4Effects of PSG-1 on activities of T-AOC, SOD, CAT and GSH-Px, and level of MDA in the heart of Cy-treated mice (mean ± S.E.M., n = 10).

Group Dose (mg/kg) T-AOC (U/mg pro) SOD (U/mg pro) CAT (U/mg pro) GSH-Px (U/mg pro) MDA (nmol/mg pro)

NC 0 4.34 ± 0.42 217.20 ± 22.12 45.22 ± 1.56 55.23 ± 5.35 2.52 ± 0.11MC 80 2.34 ± 0.65a 119.37 ± 9.35a 23.39 ± 4.44a 25.53 ± 6.46a 4.71 ± 0.32a,c

PSG-1 25 3.37 ± 0.64a,c 141.42 ± 8.45b,c 26.02 ± 4.75a,c 34.04 ± 5.76a,c 4.69 ± 0.16a,c

50 3.85 ± 0.62a,c 184.48 ± 12.12a,c 34.67 ± 6.51a,c 44.45 ± 6.54a,c 3.62 ± 0.14b,d

100 4.25 ± 0.39a,d 197.12 ± 18.37a,c 42.32 ± 4.34a,c 53.27 ± 8.54a,c 2.83 ± 0.26a,d

LH 10 4.44 ± 0.22a,c 226.46 ± 25.82b,c 48.43 ± 5.32a,d 55.78 ± 4.73a,d 2.78 ± 0.24a,c

NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).a P < 0.05 vs. normal mice.b P < 0.01 vs. normal mice.c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

Table 5Effects of PSG-1 on activities of T-AOC, SOD, CAT and GSH-Px, and MDA level in the liver of Cy-treated mice (mean ± S.E.M., n = 10).

Group Dose (mg/kg) T-AOC (U/mg pro) SOD (U/mg pro) CAT (U/mg pro) GSH-Px (U/mg pro) MDA (nmol/mg pro)

NC 0 3.22 ± 0.42 158.23 ± 12.13 57.38 ± 2.54 72.45 ± 6.47 3.24 ± 0.32MC 80 1.64 ± 0.33a 89.33 ± 6.36a 29.33 ± 3.24a 39.53 ± 5.83a 5.96 ± 0.62a,c

PSG-1 25 2.27 ± 0.26a,c 111.31 ± 6.62b,c 35.36 ± 4.67a,c 47.75 ± 3.77a,c 4.58 ± 0.56a,c

50 2.55 ± 0.24a,c 134.43 ± 8.12a,c 44.53 ± 3.75a,d 62.45 ± 8.58a,c 4.23 ± 0.62b,d

100 3.25 ± 0.53a,c 157.13 ± 12.34a,d 55.36 ± 7.84a,d 73.27 ± 8.98a,c 3.33 ± 0.27a,d

LH 10 3.41 ± 0.16a,d 167.43 ± 23.54b,d 59.45 ± 5.32a,d 75.42 ± 4.53a,d 3.43 ± 0.25a,d

NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).a P < 0.05 vs. normal mice.b P < 0.01 vs. normal mice.

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c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

ith Cy (Tables 4–6). PSG-1 administration significantly decreasedhe content of the MDA in the heart, liver and kidney of the Cy-reated mice. When the dose of PSG-1 reached 100 mg/kg/d, theontents of MDA in heart, liver and kidney were recovered to the

ormal levels. The activities of MDA of the positive group were

ower than those of normal group in heart, liver and kidney. Theesults suggested that PSG-1 can enhance anti-oxidative activitiesn the three organs.

able 6ffects of PSG-1 on activities of T-AOC, SOD, CAT and GSH-Px, and MDA level in the kidne

Group Dose (mg/kg) T-AOC (U/mg pro) SOD (U/mg pro)

NC 0 3.83 ± 0.53 178.34 ± 14.53

MC 80 1.88 ± 0.38a 96.53 ± 7.46a

PSG-1 25 2.47 ± 0.16a,c 132.45 ± 8.57b,c

50 2.69 ± 0.22a,c 154.15 ± 14.45a,c

100 3.95 ± 0.33a,c 177.53 ± 16.84a,d

LH 10 4.01 ± 0.23a,d 187.43 ± 15.74b,d

C, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levaa P < 0.05 vs. normal mice.b P < 0.01 vs. normal mice.c P < 0.05 vs. Cy-treated mice administrated saline vehicle.d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

4. Discussion

It is well known that Cy is an important chemotherapeuticdrug in tumor treatment, but it is adverse to healthy cells and

cause side effects such as myelosuppression, immunosuppres-sion and oxidative stress, which sometimes are life-threatening[22]. In the study, mice treated with Cy were used as an animalmodel of a weakened immune system. As expected, Cy markedly

y of Cy-treated mice (mean ± S.E.M., n = 10).

CAT (U/mg pro) GSH-Px (U/mg pro) MDA (nmol/mg pro)

77.35 ± 5.78 86.25 ± 6.58 3.78 ± 0.3239.57 ± 3.25a 57.35 ± 8.85a 6.64 ± 0.42a,c

53.33 ± 6.78a,c 64.68 ± 5.78a,c 5.54 ± 0.54a,c

64.34 ± 7.35a,d 75.79 ± 7.55a,c 4.64 ± 0.24b,d

75.35 ± 6.75a,d 89.24 ± 7.57a,c 3.76 ± 0.21a,d

79.75 ± 8.36a,d 91.37 ± 6.76a,d 3.65 ± 0.15a,d

misole hydrochloride (positive control).

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educed RBC, WBC and platelets counts, inhibited splenic NK cellnd CTL activities, decreased CD4+ T lymphocyte counts and theD4+/CD8+ ratio. Besides, the levels of cytokines (IL-2, INF-� and

L-10), immunoglobulins (IgA, IgM and IgG), as well as hemolysinn the sera were decreased by Cy. Moreover, Cy impaired thentioxidant system by reducing the T-AOC, SOD, CAT and GSH-Pxevels, and increasing the MDA levels. These data are consistent

ith previous reports [23–27]. The protective effects of PSG-1 ony-induced myelosuppression, immunosuppression and oxidativetress were investigated in the study.

Myelosuppression is an important limiting factor on the out-ome and recovery of tumor patients receiving chemotherapy.ematopoietic stem cells possess multi-potentiality, enabling

hem to self-renew and also to produce mature blood cells, suchs erythrocytes, leukocytes and platelets [28]. Our findings showedhat Cy reduced WBC, RBC and platelets amounts, consistent withther reports [29,30]. The administration of PSG-1 significantlyestored WBC, RBC and platelets counts in a dose-dependentanner, suggesting that PSG-1 could provide protection againstyelosuppression induced by Cy.NK cells and CTL are two major populations of cytotoxic lym-

hocytes [31,32], and play important roles in the defense againstumors and viruses [33,34]. NK cells and CTL are capable of killingutologous cells infected with intracellular pathogens, as well asumor cells. NK cells and CTL share a lot of similarity in term ofunction, however, unlike CTL, the killing by NK cells is non-specific.K cells can skip the step of recognizing antigen/MHC on the tar-et cells, directly react against and destroy target cells withoutrior sensitization to it. In this study, treatment with PSG-1 dose-ependently accelerated the recovery of NK cells and CTL numbers

n Cy-treated immunosuppressed mice.CD4+ and CD8+ are T helper (Th) and T cytotoxic (Tc) lym-

hocytes, respectively, which are two common T lymphocytesmportant for adaptive immunity [35]. Since they are responsi-le for releasing pro-inflammatory cytokines that recruit differentffector cells, including macrophages, neutrophils, eosinophils,tc., many studies have reported the cytotoxic effects of them36]. Previous studies have shown that the ratio of CD4+/CD8+

n immunosuppressed mice was lower than normal mice [7]. Inine with literatures, the percentage of CD4+ or CD8+ cells andhe ratio of CD4+/CD8+ treated by PSG-1 increased substantiallyhen compared with the model group (Table 2), indicating that

SG-1 can restore the immunosuppression induced by Cy treat-ent.It is well documented that following antigen recognition, the

ctivated Th cells are divided into Th1 and Th2 according to theunction and their difference in secretion of cytokines [37]. The dif-erent cytokine patterns secreted by Th1 and Th2 cells are majoreterminants of the differences in cellular function [38]. Th1 cellsecrete IL-2, IFN-� and TNF-�, the main body of the cell-mediatedmmune response, whereas Th2 cells secrete IL-4, IL-6 and IL-10,

ainly mediated by the humoral immune response. Under normalircumstances, the function of Th1 and Th2 cells is in a dynamicquilibrium state, in order to maintain normal cellular and humoralmmune function. IL-2 is an important cytokine produced by acti-ated T cells, and is necessary for the growth, proliferation andifferentiation of T cells to become effector T cells. IFN-� promoteshe differentiation of Th1 cells and therefore a predominantly cell-ased immune response. It functions principally on macrophages tonhance antimicrobial properties [39]. IL-10 has been reported tonhibit the activity of T helper Th1 cells, NK cells and macrophages,ll of which are required for optimal pathogen clearance [40].

ur results showed that PSG-1 was able to significantly reverse

he increase of serum IL-10 level in Cy-treated mice in a dose-ependent manner to a normal scope, and improved the decline in

evels of serum IL-2 and IFN-�. This indicates that PSG-1 possesses

l Macromolecules 64 (2014) 395– 401

the capacity of modulating the secretion of Th1/Th2 cytokines forenhancement of immunity.

IgA, IgG and IgM are the major immunoglobulins which areinvolved in the complement activation, opsonization, neutraliza-tion of toxins, etc. [41]. There are increasing evidences indicatingthat many kinds of polysaccharides can enhance humoral immuneresponse by promoting the production of specific IgA, IgM andIgG [42]. Furthermore, the formation of serum hemolysin withSRBC immunization reflects humoral immunologic function [43].Our results demonstrated that PSG-1 significantly increased serumhemolysin formation, as well as the levels of IgA, IgG and IgM in Cy-immunosuppressed mice. These suggested that PSG-1 can enhancehumoral immunity.

In recent years, “oxidative stress” has become a subject of con-siderable interest of research. It is well known that reactive oxygenspecies (ROS) are frequently generated spontaneously in the livingcell during metabolism, however, aberrant production or regula-tion of reactive oxygen species (ROS), such as superoxide anion,hydrogen peroxide and hydroxyl radical, has been demonstratedto contribute to tissue damage and loss of function in a number oftissues and organs [44]. Antioxidant enzymes are considered to be aprimary defense against ROS-induced damage to lipids, protein andDNA. The antioxidant enzymes in the tissues can protect againstoxidative stress and tissue damage by converting active oxygenmolecules into non-toxic compounds [21], such as SOD, CAT andGSH-Px.

Cy is known to generate free radicals in the biological system andthereby cause oxidative stress [45]. In the present study, the oxida-tive stress was induced in the mice by intraperitoneal injection ofCy at a dose of 80 mg/kg/d. Cy treatment resulted in suppressedT-AOC, which reflects total anti-oxidative ability in a living organ-ism or a specific organ, in the heart, liver and kidney. Activities ofthree antioxidant enzymes, SOD, CAT and GSH-Px in the heart, liverand kidney were also decreased by Cy treatment. Moreover, MDA,which is considered a bio-marker of oxidative stress, was increasedin all three organs.

The accumulating evidence strongly suggests that many kindsof polysaccharides have potential and potent capabilities of pre-venting oxidative damage in living organisms from free radicalscavenging [46,47]. In the present study, the administration ofPSG-1 (25, 50 and 100 mg/kg/d) caused significant increases inthe T-AOC levels and SOD, CAT and GSH-Px activities as well asa decrease in the MDA levels in heart, liver and kidney. Thesefindings showed that PSG-1 can be effective in scavenging vari-ous types of oxygen free radicals and their products, indicatingthat PSG-1 was able to protect against oxidative stress inducedby Cy.

In conclusion, the present study has demonstrated thatPSG-1 not only improved the immune functions, but alsoraised the antioxidant activities of immune organs in the Cy-treated mice. Our results also suggest that PSG-1 is a potentimmunomodulating agent and may be applied to antineo-plastic immunotherapy in combination with chemotherapeuticagents.

Acknowledgements

The financial support for this study by the Key Program ofNational Natural Science Foundation of China (No. 31130041),National Key Technology R & D Program of China (2012BAD33B06),National Natural Science Foundation of China (Nos. 21265011

and 31071532), the Program for New Century Excellent Talentsin University (NCET-12-0749), and Research Project of State KeyLaboratory of Food Science and Technology (SKLF-ZZA-201301,SKLF-TS-201107) is gratefully acknowledged.

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