Molecular mechanism underlying chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide-induced immunosuppressed mice

journal of functional foods 15 (2015) 52–60

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Molecular mechanism underlying chemoprotective effects of Ganoderma atrum polysaccharide in cyclophosphamide-induced immunosuppressed mice Qiang Yu, Shao-Ping Nie *, Jun-Qiao Wang, Dan-Fei Huang, Wen-Juan Li, Ming-Yong Xie ** State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The molecular mechanism underlying the chemoprotective effects of Ganoderma atrum poly-

Received 29 September 2014

saccharide in cyclophosphamide-induced immunosuppressed mice was investigated. In Cy-

Received in revised form 9 March

treated mice, PSG-1 treatment significantly promoted the phagocytosis, and stimulated the

2015

production of NO and cytokines (TNF-α and IL-1β) in peritoneal macrophages. Moreover,

Accepted 9 March 2015

PSG-1 elevated the phosphorylation of MAPKs and Akt, as well as expression of NF-κB in

Available online

peritoneal macrophages. In addition, PSG-1 enhanced the recovery of T and B cell proliferation responses in Cy-treated mice. Furthermore, Ca2+ concentration and PKC activity of

Keywords:

spleen lymphocytes in PSG-1 groups dramatically increased as compared with that of the

Ganoderma atrum polysaccharide

model group. Finally, PSG-1 administration was found to dose-dependently improve the decline

Cyclophosphamide

of cAMP level and PKA activity caused by Cy. These findings indicated that the chemoprotective

Peritoneal macrophages

effects of PSG-1 may be attributed to its capacity to activate peritoneal macrophages and

Spleen lymphocytes

spleen lymphocytes in Cy-treated mice.

Immunosuppression

1.

Introduction

Cyclophosphamide (Cy) is the mainstay cancer chemotherapy agent, and extensively used to treat various types of cancer (Ehrke, 2003). However, numerous studies have reported that Cy usage has cytotoxic side effects, which may lead to

© 2015 Elsevier Ltd. All rights reserved.

significant morbidity and mortality (Pass et al., 2005). Immunosuppression caused by Cy administration represents a major limiting factor in clinical chemotherapy. Once the host immune system is impaired by the chemotherapy, incidence of secondary infections and immunodeficiency will increase (Ramioul & Zutterman, 1961). Therefore, great effort has been made to obtain immunomodulating agents which can reduce the

* Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel.: +86 791 88304452; fax: +86 791 88304452. E-mail address: [email protected] (S.-P. Nie). ** Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. Tel.: +86 791 83969009; fax: +86 791 83969009. E-mail address: [email protected] (M.-Y. Xie). Abbreviations: cAMP, cyclic adenosine monophosphate; Cy, cyclophosphamide; FBS, fetal bovine serum; LH, levamisole hydrochloride; MAPKs, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; NO, nitrite oxide; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C http://dx.doi.org/10.1016/j.jff.2015.03.015 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

journal of functional foods 15 (2015) 52–60

harmful side effects and enhance the curative effect of chemotherapy. Mounting studies have shown that polysaccharides isolated from fungi, plants and other natural sources can serve as immunomodulating agents and exhibit promising therapeutic potential without toxicity and significant side effects (Li, Nie, Xie, & Li, 2014; Meng et al., 2014; Xia et al., 2012; Zhang, Li et al., 2012; Zhang, Wu et al., 2012). Among them, polysaccharides from Ganoderma atrum, which have been safely used as ingredients of traditional medicines and functional foods for a long time in oriental countries, are attracting more and more attention (Nie, Zhang, Li, & Xie, 2013; Yu, Nie, Wang, Yin et al., 2014; Zhang, Nie, Yin, Wang, & Xie, 2014). We recently isolated and purified a polysaccharide from G. atrum, named as PSG-1, with a purity of >99.8%, whose primary structural features and molecular weight were characterized (Chen, Xie, Nie, Li, & Wang, 2008; Zhang, Li et al., 2012; Zhang, Wu et al., 2012). The data obtained indicated that PSG-1 contained 10.1% protein and 17 general amino acids, with an average molecular weight of about 1013 kDa. Sugar analysis revealed that PSG-1 was composed of glucose (Glc), mannose (Man), galactose (Gal) and galacturonic acid (GalA) in a mole ratio of 4.91:1:1.28:0.71. Methylation and GC-MS analysis indicated that the main linkage type was 1,3-linked-Glcp (21.08%), followed by T-Glcp (18.68%), 1,3,6-Glcp (12.97%), 1,4-Galp (12.70%), 1,6-Glcp (12.33%), 1,2Manp (8.06%), 1,4-GalpA (6.15%), 1,4-Manp (4.55%) and 1,4,6Glcp (3.24%). Our previous study demonstrated that PSG-1 possessed chemoprotective effects on immunosuppression caused by Cy treatment (Yu, Nie, Wang, Liu et al., 2014), but the underlying mechanism is still unclear. The immune system is composed of innate immunity and adaptive immunity. Macrophages play a vital role in the host innate defense against microbial invaders and external stimuli (Dunn, Barke, Ewald, & Simmons, 1987). Upon stimulation, macrophages will initiate phagocytosis and produce various effector molecules, such as nitride oxide (NO) and cytokines, which protect the host from damage (Adams & Hamilton, 1984; Aderem & Underhill, 1999). Lymphocytes are the major cellular components of the adaptive immune response. Lymphocytes are activated after a process known as antigen presentation by antigen-presenting cells. The activation of lymphocytes is a complex signaling transduction process that involves various second messengers and specific molecules, including Ca2+, cAMP, PKC and PKA (Feske, 2007; Weiss & Cambier, 2004). Therefore, in this study, we investigated the effects of PSG-1 on the innate and adaptive immune responses in Cy-induced immunosuppressed mice, especially focused on elucidating the molecular mechanism involved in PSG-1-mediated peritoneal macrophages and spleen lymphocyte activation.

2.

Materials and methods

2.1.

Animals

Female BALB/c mice (8 weeks old, 18–20 g) were purchased from Shanghai Slac Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). Mice were housed in a rodent facility at a constant temperature (25 °C) with a 12 h light–dark cycle. All animals used in this study were cared for in accordance

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with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health, Bethesda, MD (NIH Publication 85-23, 1996). All procedures were approved by the Animal Care Review Committee, Nanchang University, China.

2.2.

Establishment of immunosuppressed mice model

Briefly, after 1 week of acclimatization, the mice were randomly divided into 6 groups (n = 10 per group). Besides the normal control (NC) group that was treated once daily with physiological saline for 10 consecutive days, the other five groups were administered by 80 mg/kg body weight (bw)/d Cy (0.2 mL) via intraperitoneal injection for 3 days. From Days 4 to 10, the mice were administered as follows: model control (MC) group was gavaged with 0.2 mL physiological saline; PSG-1 groups (low, medium, high) were gavaged with 0.2 mL PSG-1 (25, 50 or 100 mg/kg bw/d); positive control group was gavaged with 0.2 mL levamisole hydrochloride (LH) (10 mg/kg bw/d). Twenty-four hours after the last treatment, the animals were sacrificed via cervical dislocation.

2.3.

Materials and reagents

Cy was purchased from Jiangsu Hengrui Medicine Co. (Lianyungang, Jiangsu, China). ELISA kits were from R&D Systems (Minneapolis, MN, USA). Nitric oxide (NO) assay kit was obtained from Beyotime Biotech Inc. (Haimen, Jiangsu, China). The compound 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolim bromide (MTT) was from Sigma (St. Louis, MO, USA). Fluo-3/AM was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Antibodies against phospho-ERK1/2 (p-ERK1/2), phosphoJNK1/2 (p-JNK1/2), phospho-p38 (p-p38), phospho-Akt (p-Akt), and NF-κB and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were purchased from Cell Signaling (Beverly, MA, USA). Cell culture products were obtained from Life Technologies (Paisley, Scotland, UK).

2.4.

Isolation of mouse peritoneal macrophages

Each mouse was intraperitoneally injected with 5 mL cold PBS; peritoneal macrophages were aseptically harvested from peritoneal exudates by peritoneal lavage. Cells were resuspended in RPMI-1640 containing 10% heat-inactivated FBS, 1% penicillinstreptomycin at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h incubation, non-adherent cells were removed by washing with PBS. Adherent cells were collected as peritoneal macrophages.

2.5.

Neutral red uptake

Peritoneal macrophages were seeded at a density of 5 × 105 cells/ well in the 96-well plate with complete RPMI-1640 media and cultured for 48 h. The cells were washed and neutral red (50 mg/mL) was added and incubated for 3 h. Then, cells were washed with PBS to remove excess dye. The cells were resuspended in 50% ethanol containing 1% glacial acetic acid, and the absorbance (A) values at 540 nm were measured in a microplate reader. Phagocytic rate was calculated as follows: phagocytic rate = test A/normal control A × 100%, where A is the absorbance value.

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2.6.

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Measurement of NO production

We placed 5 × 105 cells/well mouse peritoneal macrophages in a 96-well plate. After incubating in complete RPMI 1640 medium for 48 h, the isolated supernatants were mixed with an equal volume of Griess reagent, and incubated at room temperature for 10 min. The absorbance was read at 540 nm, and the concentrations of NO2− were determined by using a standard curve derived from known concentrations of NaNO2.

ratio for comparison: lymphocyte proliferation ratio = test A/normal control A × 100%.

2.11.

Spleen lymphocytes were seeded at a density of 1 × 106 cells/ well in the 24-well plate, and incubated with the fluorescent probe Fluo-3/AM at 37 °C for 45 min. After washing twice with PBS, fluorescent signals were then measured by flow cytometry.

2.12. 2.7.

Western blot analysis

Protein was prepared from peritoneal macrophages using Cell lysis kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Forty micrograms denatured protein were loaded onto 10% SDS polyacrylamide gel by electrophoresis and transferred to nitrocellulose membrane. Five percent nonfat milk (prepared in TBS containing 0.1% Tween 20) was used to block non-specific binding for 1 h at 37 °C. Then the membrane was incubated with specific primary antibodies at 4 °C overnight. After washing three times with TBST, the membrane was incubated at room temperature for 1 h with secondary antibody and the signal was analyzed by using ECL chemiluminescence detection kit (Amersham Biosciences, Uppsala, Sweden). Densitometry was performed using the software Quantity One (Bio-Rad, Hercules, CA, USA).

2.9.

Preparation of spleen lymphocytes

The extirpated spleens were minced into small pieces in a germ-free condition. Single cell suspension was prepared by filtering the suspension through with a sterile sieve mesh. The cells were treated with lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3 and 0.1 mM disodium salt of ethylenediaminetetraacetic acid (Na2EDTA), pH 7.4) to get rid of red blood cells, followed by washing twice with cold PBS. Then the cells were adjusted to the concentration of 5 × 106 cells/mL in RPMI 1640 medium supplemented with 10% fetal calf serum, and incubated for 3 h in Petri dishes. The suspended cell populations were collected.

2.10.

Measurement of PKA and PKC activities

Cytokine measurement by ELISA

Peritoneal macrophages were seeded at a density of 5 × 105 cells/ well in the 96-well plate and cultured in complete RPMI 1640 medium for 48 h. The supernatants were harvested, and levels of TNF-α and IL-1β were determined by the ELISA kits (R&D Systems, Minneapolis, MN, USA).

2.8.

Measurement of intracellular [Ca2+]

Determination of lymphocyte proliferation

Cells were placed into the 96-well plate at 5 × 104 cells/well, and then ConA (final concentration, 5 µg/mL) or LPS (final concentration, 10 µg/mL) was added to the wells. After incubation for 48 h, 20 µL MTT (5 mg/mL) were added to each well for additional 4 h incubation. The plate was centrifuged at 268 × g for 5 min and the supernatant was discarded, then 150 µL dimethyl sulfoxide (DMSO) were added to each well. The absorbance at 570 nm was read with a microplate reader. The absorbance (A) was translated into lymphocyte proliferation

The PKA and PKC activities were measured using PepTag Assay kits for Non-Radioactive Detection of PKC or cAMP-Dependent PKA (Promega, Madison, WI, USA). Briefly, spleen lymphocytes were resuspended and homogenized in 0.5 ml of cold PKC extraction buffer, and centrifuged at 14,000 g for 5 min. Then the supernatant was purified on 1 ml column of DEAE cellulose and eluted by PKC extraction buffer. After incubating with PKC reaction mixture, the resultant mixture was separated by agarose gel. The bands were quantized by spectrophotometry.

2.13.

Measurement of cAMP concentration

A total of 1 × 106 cells/well was seeded in the 24-well plate and lysed with repeated freezing and thawing method. The plate was centrifuged at 720 g for 5 min and the supernatants were collected. The cAMP concentration was measured by the ELISAbased cAMP assay kit (IBL, Hamburg, Germany).

2.14.

Statistical analysis

Values are expressed as mean ± SEM. One-way analysis of variance followed by the Student–Newman–Keuls test was used to determine the statistical significance between various groups. A value of P < 0.05 was considered to be statistically significant.

3.

Results and discussion

3.1. Effect of PSG-1 on macrophage phagocytosis in Cy-treated mice Macrophages are the key participants in the innate immune response, and play an essential role in host defense against pathogen infection and microbial invasion (Katsiari, Liossis, & Sfikakis, 2010). The phagocytosis capability of macrophages is one of the important indexes in evaluating innate immune function. Phagocytosis of peritoneal macrophages was measured by devouring neutral red. As shown in Fig. 1A, there was a significant reduction of macrophage phagocytosis in the model group compared with the normal group (P < 0.05). However, the phagocytic rates of peritoneal macrophages were remarkably evoked by PSG-1 treatment (25, 50 and 100 mg/kg) in a dosedependent manner. The treatment of LH also promoted recovery of macrophage phagocytosis.

3.2. Effect of PSG-1 on NO production of peritoneal macrophages in Cy-treated mice NO synthesized by activated macrophages is an important major effector molecule, which is responsible for cytostatic and

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Fig. 1 – Effect of PSG-1 on phagocytosis, NO production and cytokine release in peritoneal macrophages of Cy-treated mice. Peritoneal macrophages were seeded at a density of 5 × 105 cells/well in the 24-well plate and cultured for 48 h. (A) Phagocytosis of peritoneal macrophages was measured by devouring neutral red. (B) NO production was measured by Griess method. (C) Levels of TNF-α and IL-1β were measured by ELISA method. 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.

cytotoxic activities against infectious organisms and tumor cells in non-specific immunity (Brüne, Zhou, & Von Knethen, 2003). As shown in Fig. 1B, the level of NO in the model control group was obviously lower than the normal group (P < 0.05). However, the administration of PSG-1 (25, 50 and 100 mg/kg) and LH treatment significantly increased the levels of NO as compared with the model control group (P < 0.05, or P < 0.01). These results show that Cy treatment depresses the NO production, while PSG-1 treatment evokes dose-dependently NO release in peritoneal macrophages in immunosuppressed mice.

3.3. Effect of PSG-1 on cytokines production of peritoneal macrophages in Cy-treated mice It is well known that macrophages are highly secretory cells. Upon stimulation, macrophages orchestrate the immune response via secreting numerous cytokines. Our previous study found that PSG-1 significantly stimulated TNF-α and IL-1β production in macrophages in vitro (Yu et al., 2013). In line with this finding, the present study showed that PSG-1 administration restored the release of TNF-α and IL-1β decreased by

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Cy treatment. Specifically, the levels of cytokines (TNF-α and IL-1β) secreted by peritoneal macrophages in the model control group were significantly reduced (P < 0.05) as compared to the normal control group. Treatment with PSG-1 (25, 50 and 100 mg/kg) dose-dependently enhanced production of cytokines compared with the model control group (Fig. 1C). PSG-1 at a dose of 100 mg/kg increased the concentrations of TNF-α and IL-1β to 221.72 and 23.01 pg/mL (P < 0.01).

3.4. Effect of PSG-1 on MAPKs, PI3K/Akt and NF-κB pathways in Cy-treated mice Previous studies have demonstrated that macrophages were activated via a variety of intracellular signal transduction

pathways (Kim et al., 2012; Zhao et al., 2014). Thus we sought to determine whether these pathways were also activated by PSG-1 administration in Cy-treated mice. MAPKs are a family of serine/threonine protein kinases that include three major groups in mammalian cells, i.e. ERK1/2, JNK1/2, and p38 (Rao, 2001). PI3K and the downstream serine/threonine kinase Akt control several cellular responses, including cell growth, survival, cytoskeletal remodeling and the trafficking of intracellular organelles (Fruman & Cantley, 2002; Fruman, Meyers, & Cantley, 1998). Our previous in vitro study demonstrated that MAPKs and PI3K/Akt signaling pathways were simultaneously activated in PSG-1-stimulated macrophages (Yu et al., 2012). To study the putative effect of PSG-1 on MAPKs and PI3K/Akt signaling pathways in Cy-treated mice, western blot was performed to

Fig. 2 – Effect of PSG-1 on the phosphorylation of ERK1/2, JNK1/2, p38 and Akt, as well as expression of NF-κB in peritoneal macrophages of Cy-treated mice. (A) The cytosolic and nuclear proteins isolated from peritoneal macrophages were analyzed by western blot with anti-p-ERK1/2, anti-p-JNK1/2, anti-p-p38, anti-p-Akt and anti-NF-κB antibodies. (B) Histogram represents quantification of protein expression levels using Quantity One software (levels of NC group defined as 1). NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide. Data were expressed as mean ± S.E.M. of 10 mice.

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determine the phosphorylation levels of MAPK and Akt proteins in peritoneal macrophages. As shown in Fig. 2, compared with the normal group, phosphorylation of ERK1/2, JNK1/2, p38 and Akt was significantly decreased in the model control group. However, PSG-1 administration dose-dependently increased the phosphorylation of ERK1/2, JNK1/2, p38 and Akt. Furthermore, NF-κB is a ubiquitous transcription factor that regulates the transcription of numerous genes related to innate immunity (Baeuerle & Henkel, 1994). Our data demonstrated that PSG-1 also enhanced NF-κB expression in the peritoneal macrophages in Cy-treated mice. These results indicated that PSG-1 may restore the immunosuppression induced by Cy treatment via activating peritoneal macrophages, which is mediated, at least partially, by the MAPKs, PI3K/Akt and NF-κB signaling pathways.

3.5. Effect of PSG-1 on spleen lymphocyte proliferation in Cy-treated mice The proliferation of T and B lymphocyte response to mitogens or antigens has been widely used as an immune parameter to investigate lymphocyte responsiveness. Normally, Con A-induced cellular proliferation is used to detect T lymphocyte immunity, while LPS-induced activation of B cells reflects B lymphocyte immunity (Quakyi, Carter, Tsai, & Marti, 1997). In the present study, the effect of PSG-1 on Con A- and LPSinduced spleen lymphocyte proliferation in Cy-treated mice was shown in Fig. 3. The spleen lymphocyte proliferation of the model group remarkably decreased when compared with that of the normal group (P < 0.05). PSG-1 enhanced Con A- or LPS-stimulated lymphocytes in a dose-dependent manner

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compared with the model group. Treatment with PSG-1 at 100 mg/kg/d enhanced the levels of proliferation response 2.6and 1.7-fold in Con A- and LPS-stimulated spleen lymphocytes, respectively, when compared to the model control. These results indicated that PSG-1 could improve the immune response of the spleen lymphocytes in Cy induced immunosuppressed mice.

3.6. Effect of PSG-1 on Ca2+ concentration and PKC activity in spleen lymphocytes of Cy-induced mice Calcium ions, known as the most widely used intracellular messengers, play a pivotal role in a number of physiological processes (Clapham, 2007). In response to diverse stimuli, elevation in the concentration of Ca2+ in the cytosol triggers many types of events, such as the activation and proliferation of lymphocytes (Oh-hora & Rao, 2008). In our study, the concentration of Ca2+ was noticeably decreased in the model control group compared with the normal control group (Fig. 4). However, PSG-1 administration greatly elevated the Ca2+ concentration. In particular, the level of Ca2+ in the PSG-1 group at 100 mg/kg/d reached the level of the normal group. These results showed that PSG-1 was capable of reversing the decrease of the concentration of Ca2+ in Cy-treated mice to the normal level. Protein kinase C (PKC), a calcium-dependent kinase, exists in many different cell types (Nishizuka, 1986). PKC-regulated signaling pathways play a significant role in a wide variety of aspects of immune responses, including development, differentiation, activation and survival of lymphocytes (Newton, 1995). As shown in Table 1, PKC activity in spleen lymphocytes was significantly decreased in the model control group compared to the normal control group (P < 0.05). In comparison, PKC activities were significantly increased in PSG-1 groups (25, 50 and 100 mg/kg/d) and LH group compared to the model control group. Our result showed that PKC activities were dramatically increased by PSG-1 treatment, indicating that Ca2+/PKC signaling pathway may be involved in PSG-1 mediated lymphocyte activation in Cy-induced immunosuppression.

3.7. Effect of PSG-1 on cAMP production and PKA activity in spleen lymphocytes of Cy-induced mice

Fig. 3 – Effect of PSG-1 on Con A- and LPS-induced spleen lymphocyte proliferation in Cy-treated mice. Cells were placed into the 96-well plate at 5 × 104 cells/well with ConA (5 µg/mL) or LPS (10 µg/mL). Spleen lymphocyte proliferation was measured by MTT method. 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. a P < 0.05 and bP < 0.01 vs. normal mice. cP < 0.05 and d P < 0.01 vs. Cy-treated mice administrated saline vehicle.

It is well established that cyclic nucleotides act as second messengers of intracellular signaling pathways (Friedman, 1976). cAMP is an important cyclic nucleotide in eukaryotic cell, which is synthesized from ATP by the action of adenylyl cyclases. As a secondary messenger, cAMP can regulate a series of cell functions, from cell growth and differentiation, kinase activation to gene expression (Serezani, Ballinger, Aronoff, & Peters-Golden, 2008). As shown in Table 1, Cy injection caused significant decrease in the level of cAMP. However, significant enhancementsin cAMP level were observed in mice treated with PSG-1 at all three doses and LH compared with the model control group (P < 0.05 or P < 0.01), suggesting that treatments with PSG-1 could improve the decline of cAMP level caused by Cy in a dose dependent manner. Protein kinase A (PKA) is an important cAMP-dependent Ser/ Thr protein kinase that is involved in regulation of various signal transduction pathways in lymphocytes (Walsh & Van Patten, 1994). The rise in cAMP leads to activation of PKA, which in

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Fig. 4 – Effect of PSG-1 on Ca2+ concentration in spleen lymphocytes of Cy-treated mice. The concentration of intracellular [Ca2+]i was measured with Fluo-3/AM by flow cytometry. NC, normal control; MC, model control; PSG-1, Ganoderma atrum polysaccharide; LH, levamisole hydrochloride (positive control).

turn phosphorylates target proteins and mediates gene transcription (Skalhegg & Tasken, 2000). Given that PSG-1 treatment increased cAMP level, we further investigated the capacity in the activation of PKA. Our results showed that PKA activity was significantly decreased by Cy (P < 0.01), but the administration of PSG-1 (25, 50 and 100 mg/kg/d) recovered PKA activities significantly in a dose-dependent manner. These findings suggested that PSG-1 may act against the immunosuppression induced by Cy via cAMP/PKA pathway.

4.

Conclusions

The PSG-1 treatment significantly stimulated the phagocytosis, elevated NO, TNF-α and IL-1β production, and activated MAPKs, PI3K/Akt and NF-κB signaling pathways in peritoneal macrophages in Cy-treated mice. In addition, PSG-1 enhanced the recovery of T and B cell proliferation responses. Ca2+/PKC and cAMP/PKA signaling pathways may be involved

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Table 1 – Effect of PSG-1 on cAMP production, PKA and PKC activities in spleen lymphocytes of Cy-induced mice. Group

Dose(mg/kg)

cAMP(nmol/L)

PKA(U/L)

PKC(U/L)

NC MC PSG-1

– –

7.67 ± 0.21 3.51 ± 0.31a 4.62 ± 0.31ac 5.77 ± 0.81bc 6.21 ± 0.41bd 7.43 ± 0.31ad

10.31 ± 0.15 5.12 ± 0.31b 5.97 ± 0.17ac 6.84 ± 0.42bd 7.92 ± 0.34ad 8.71 ± 0.21ac

0.44 ± 0.01 0.23 ± 0.04a 0.25 ± 0.02 ac 0.37 ± 0.05 ad 0.40 ± 0.02bc 0.42 ± 0.02ac

LH

25 50 100 10

Note: aP < 0.05 and bP < 0.01 vs. normal mice. cP < 0.05 and dP < 0.01 vs. Cy-treated mice administrated saline vehicle. NC, normal control; MC, model control; LH, levamisole hydrochloride (positive control).

in the lymphocyte activation in Cy-treated mice. In conclusion, we have elucidated the mechanism of PSG-1-mediated chemoprotection in Cy-induced immunosuppressed mice, and provided a theoretical basis for the potential of PSG-1 as a candidate in lessening chemotherapy-induced immunosuppression.

Acknowledgements The financial support for this study by the National Key Technology R & D Program of China (2012BAD33B06), Key Program of National Natural Science Foundation of China (No: 31130041), the Program for New Century Excellent Talents in University (NCET-12-0749), Research Program of State Key Laboratory of Food Science and Technology (SKLF-ZZA-201301), the Project of Science and Technology of Jiangxi Provincial Education Department (KJLD13004) and the Key Project of International Cooperation of Jiangxi Provincial Department of Science and Technology (20141BDH80009) is gratefully acknowledged.

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