Antioxidant and immunomodulatory effects of a α-glucan from fruit body of maitake (Grifola frondosa)

Preparation of MT-α-glucan

MT-α-glucan was extracted and purified from fruit body of maitake (G. frondosa) as previously described (Lei et al., 2007), which is a basically homogeneous polysaccharide fraction and its molecular weight is about 400,000–450,000 Da. MT-α-glucan was dissolved in 1% sodium carboxymethylcellulose (CMC-Na) and diluted to the concentration needed.

Animals

Healthy Kunming strain mice and C₅₇BL/6J mice, weighing 20±2 g were supplied by the Experimental Animal Center of Anhui Medical University (Hefei, China). They were housed in plastic cages and maintained under standard conditions (12-h light–dark cycle; 23–25°C; 35–60% r.h.). Before and during the experiment, mice were fed with a normal laboratory pellet diet and water was freely available. After randomisation into various groups, the mice were acclimatised in the new environment for two days before initiation of the experiment. The study complied with the current ethical regulations for the care and use of laboratory animals of Hefei University of Technology (Anhui, China), and all mice used in the experiment received humane care.

Main reagents

D-gal (Sigma Co.) was dissolved in 0.9% sterile saline at a concentration of 0.5% and stored at 4°C. Vitamin E (VE, Merck Co.) was dissolved in 1% CMC-Na with absolute ethanol (less than 0.2%, v/v). Various measuring kits were used during the study: superoxide dismutase (SOD) measurement kit, glutathione peroxidase (GSHpx), reduced glutathione (GSH) and malondialdehyde (MDA) measurement kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Concanavalin A (ConA) and lipopolysaccharides (LPS) were obtained from Sigma; RPMI 1640 medium was obtained from Gibco which was supplemented with HEPES buffer 25 mmol L⁻¹, sodium pyruvate 1 mmol L⁻¹, L-glutamine 2 mmol L⁻¹, penicillin 100 kU L⁻¹, streptomycin 100 mg L⁻¹ and 10% new born bovine serum and were adjusted to pH 7.2. Bovine serum was supplied by Hangzhou Sijiqing Bioengineering Co. (Hangzhou, China). All the other biochemicals and chemicals used in the experiment were of analytical grade.

Experimental design

In the experiment a total of 50 mice were used and were divided into 5 groups, each containing 10 mice as follows: normal control; D-gal model control; 3 treatment groups (given MT-α-glucan 450, 150 mg kg⁻¹ or VE 50 mg kg⁻¹). D-gal (40 mg kg⁻¹) was injected subcutaneously every other day for four weeks to induce senescent mice model (Wang, Sun, Zhang, Jiang, & Zhang, 2002). Mice were given drugs or dissolvent 0.1 mL/10 g orally by gavage once a day for four weeks, followed by body weight measured each week.

Rotating-rod test

The rota-rod apparatus consisted of a base, two poles and a rod. The rod is 60 cm long, 25 cm away from the base supported by two poles. At the beginning of the test, put the mice on the rod slightly. Then the rod is revolved by hand at the rotating speed of 10 rpm for one minute. The falling number of mice was recorded and the falling percent was calculated (Lei et al., 2003).

Step-down-type passive avoidance test

The apparatus consisted of an acrylic box with a stainless-steel grid floor. A wooden platform was fixed in the centre of the box. Electric shocks (40 mv) were delivered to the grid floor for three seconds with an isolated pulse stimulator. At the beginning of training, mice were placed in the box to adapt for three minutes. Then put the mice on the platform slightly. When the mice step down and place all its paws on the grid floor, it would jump to the platform as shock happened to be delivered. Step-down latency (time of staying on the platform, SDL) and the number of errors (NOE) were recorded within five minutes and repeated 24 h after training (Lei et al., 2003).

Biochemical measurements

At the end of the four-week treatment, mice were deprived of food overnight and killed by decapitation. The liver homogenate was used for estimation of the levels of SOD, GSHpx, GSH and MDA. The above biochemical parameters were determined using commercial kits according to the guidelines indicated.

Proliferative response of splenocytes

The splenocytes suspension (1×10¹⁰ L⁻¹) was prepared in a general way. Splenocytes suspension 100 µL was seeded on a 96-well microtiter plate in the presence of ConA (final concentration 3 mg L⁻¹). The splenocytes were incubated at 37°C in 5% CO₂ atmosphere for 48 h. The supernatant, 150 µL, was collected and stored at 20°C until tested for interleukine-2 (IL-2) activity. Then RPMI-1640 medium containing 150 µL 10% fetal calf serum (FCS) and 20 µL MTT (5 mg/mL) was added to each well and splenocytes were cultured for another six hours. Then the supernatant was discarded and 150 µL DMSO was added to each well. After shaking the plate gently, the optical density (OD) values were measured at 570 nm (Song et al., 2007). The results were expressed as means of OD value of triplicate wells.

IL-2 assay

The supernatant containing IL-2 was diluted by 40 times. Activated splenocytes suspension (2×10⁹ L⁻¹), 100 µL, was seeded on a 96-well microtiter plate in the presence of the 100 µL dilution. The splenocytes were incubated at 37°C in 5% CO₂ atmosphere for 24 h (Song et al., 2007). MTT assay was done according to the methods stated above. The results were expressed as means of OD value of triplicate wells.

Proliferative response of macrophages and NO, NOS and iNOS assay

Mouse peritoneal macrophages were collected by lavage with cold D-Hank’s solution (pH 7.4) and suspended in RPMI-1640 medium containing 10% FCS. One millilitre cell suspension (2×10⁹ L⁻¹) was seeded in a 24-well microtiter plate and was cultured at 37°C in 5% CO₂ atmosphere for two hours. After removing the culture supernatant, the adherent cells were washed with 37°C PBS and were cultured in RPMI-1640 medium containing 10% FCS in the presence of LPS (final concentration 6 mg L⁻¹) at 37°C in 5% CO₂ atmosphere for six hours. Then the cells were washed again with PBS and adherent cells were cultured in RPMI-1640 medium containing 10% FCS at 37°C in 5% CO₂ atmosphere for 42 h (Andras, Gyongyi, Laszlo, Tamas, & Gyorgy, 2006). The supernatant in each well was collected and was used for NO, NOS and iNOS assay and IL-1 assay. NO, NOS and iNOS assays were determined by using commercial kits according to the guidelines indicated. Cell activity was assayed by MTT method according to the methods stated above. The results were expressed as means of OD value of triplicate wells.

IL-1 assay

The supernatant containing IL-1 was diluted by 30 times. Mouse thymocytes were prepared in a general way which was used for measuring IL-1 activity. Thymocytes suspension (2×10¹⁰ L⁻¹), 50 µL, was seeded in a 96-well microtiter plate in the presence of 100 µL dilution and ConA (final concentration 3 mg L⁻¹). The thymocytes were incubated at 37°C in a 5% CO₂ incubator for 48 h (Song et al., 2007). Cell activity was assayed by MTT method according to the methods stated above. The results were expressed as means of OD value of triplicate wells.

Statistical analysis

Data were expressed as means ± s.d. Statistical analysis was evaluated by one-way analysis of variance, followed by the Student–Newman–Keuls test for multiple comparisons, which was used to evaluate the difference between two groups. P < 0.05 was considered significant.

Effect of MT-α-glucan on the body weight of of D-gal-induced ageing mice

The procedure of preparation of MT-α-glucan was studied in our laboratory. The purity of the compound estimated by high performance gel permeation chromatography (HPGPC) demonstrated that the molecule was basically homogeneous, which had the molecular weight about 400,000–450,000 Da. Results of structural analyses (IR, ¹H-NMR and ¹³C-NMR) and monosaccharide analysis (TLC and GC) demonstrated that the molecule is an α-glucan (Lei et al., 2007), rather than a β-glucan, molecules hitherto reported to be most commonly produced by this mushroom strain (Kubo, 1994, 1997). So this molecule is unique to maitake among mushrooms according to the references we have searched. Another five groups of polysaccharides having diverse molecular mass (470–1650 kDa) were prepared from mycelium extract and submerged culture of G. frondosa, which had antioxidant and free radical scavenging activities (Lee et al., 2003).

Previous studies showed that chronic injections of D-gal subcutaneously into mice could induce changes which resembled accelerated ageing (Wang et al., 2002). The ageing model shows neurological impairment, decreased activity of antioxidant enzymes and poor immune responses (Zhang & Li, 1990). Thus, we exploited D-gal-treated mice as experimental senile model to investigate the anti-ageing effect of MT-α-glucan and its mechanism of action. Results showed that all D-gal-treated mice had the signs of senescent manifestation, such as low body weight, hair pull-off, slow behaviour and lassitude. The success percentage of senescent mice model was 100%.

Figure 1 shows the effect of MT-α-glucan on body weight of D-gal-treated mice. The body weight of D-gal model mice was much lower than that of the normal control. After four weeks of treatment, MT-α-glucan (450, 150 mg kg⁻¹) markedly increased the body weight of D-gal-treated mice. This suggested that MT-α-glucan could alleviate the lowered body weight of mice induced by D-gal.

Figure 1. Effect of MT-α-glucan on body weight of D-gal-induced ageing mice.

Effects of MT-α-glucan on the behaviour of D-gal-induced ageing mice

The motor function and memory would decline with age. This might be related to the declining of advanced function of the brain (Zhan, Liu, & Zhou, 1990). In this study, step-down-type avoidance and rotating-rod tests were used to examine the long-term memory and motor function, which are as index of senescence of mice.

Table 1 shows that D-gal (40 mg kg⁻¹, sc every other day for four weeks) increased the falling rate of mice determined by rotating-rod test (P<0.01), shortened SDL (P<0.01) and increased NOE (P<0.01) determined by step-down test. The results of our study showed that the functions of motor and memory of D-gal-treated mice were all lower than that of the normal young mice. MT-α-glucan (450, 150 mg kg⁻¹, ig once a day for four weeks) and VE (50 mg kg⁻¹) markedly improved motor and memory dysfunctions of D-gal-treated mice (Table 1). This suggested that MT-α-glucan could improve equilibrium ability and brain function of aged mice induced by D-gal, which resulted in delay in senility.

Effect of MT-α-glucan on levels of liver SOD, GSHpx, GSH and MDA in D-gal-induced ageing mice

There are several assumptions concerning ageing, of which the well-known theories are free radical theory and immune theory. The free radical theory states that oxygen free radicals are the important factors involved in the phenomenon of biological ageing (Nohl, 1993). The immune theory states that the decreased immune function, especially cellular immune function, is the main reason of ageing (Miller, 1991), in which the most obvious change is the decrease of IL-2) production and IL-2 receptor expression (Duan, Wang, & Shen, 1993). It has been demonstrated that polysaccharides derived from maitake mushrooms possess antioxidative properties in vitro (Li et al., 2003) and immune regulatory activity (Inoue et al., 2002). In this study, the anti-ageing effect of MT-α-glucan and its mechanism of action concerning oxidative stress and immune response were investigated, exploiting D-gal-induced senile mice model.

The changes in the levels of SOD, GSHpx, GSH and MDA in the liver of normal mice, D-gal-induced ageing mice and experimental groups are shown in Table 2. Results showed that the activity of SOD, GSHpx and GSH content in the livers of D-gal model mice markedly decreased compared with the normal control (P < 0.01). Treatment with MT-α-glucan (450, 150 mg kg⁻¹) and VE (50 mg kg⁻¹) markedly increased SOD, GSHpx activity and GSH content. MDA content was markedly increased in D-gal model mice compared with the normal control (P < 0.01). MT-α-glucan (450, 150 mg kg⁻¹) and VE (50 mg kg⁻¹) markedly decreased MDA. This suggested that MT-α-glucan has an antioxidative effect on D-gal-induced ageing mice.

Effect of MT-α-glucan on proliferative response and IL-2 production of splenocytes induced by ConA in D-gal-induced ageing mice

Proliferation and activation of whole spleen cells and of macrophages, combined with immunocytokines secreted from these immunocytes, were investigated in this study. IL-2 is a kind of the major and well-known protective immunocytokines secreted from splenocytes. IL-2 has critical actions on the immune system and has extensive immuno-enhancing activity. IL-2 remains the most effective cytokine for T lymphocyte cell expansion and regulation of many critical functions in T-cell biology. Moreover, IL-2 can augment B-cell proliferation and increase immunoglobulin synthesis, boost the cytolytic activity of natural killer (NK) cells, and exert actions on neutrophils and monocytes (Ruth, Carlos, & Gershwin, 2008). The thymus index, proliferative response and IL-2 production of splenocytes induced by ConA are shown in Figure 2 and Table 3. The results showed that the thymus index, levels of proliferative response and IL-2 production of splenocytes were significantly lower in D-gal-induced ageing mice as compared with normal control (P<0.01 or P<0.05). Treatment with MT-α-glucan (450, 150 mg kg⁻¹) increased the thymus index, the proliferative response and IL-2 production markedly. This suggested that MT-α-glucan has the effect of improving cellular immunity in D-gal-induced ageing animal model.

Figure 2. Effect of MT-α-glucan on spleen proliferative response and IL-2 production of D-gal-induced ageing mice. Data are the mean ± s.d., n=10. In each vertical column, *P < 0.05 and **P < 0.01, compared with the D-gal group (analysis of variance followed by the Student–Newman–Keuls test).

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Effect of MT-α-glucan on macrophage proliferative response and IL-1 and NO production by macrophage in D-gal-induced ageing mice

Macrophages are considered to be one of the effectors that cause the immunity disruption by producing IL-1 and NO (Hirotada, Noriko, & Hiroaki, 2000). IL-1 is primarily produced by cells of the mononuclear phagocytic lineage. The biologic effects and function of IL-1 involve systemic and local effects that have influence on immunologic properties, including T-cell activation, lymphocyte activating factor, increasing antibody production and inducing the synthesis of other cytokines. IL-1 also has profound effects on endothelial, smooth muscle, vascular and myocardial cells. Because of its many, varied, and multiple biologic effects, IL-1 may play a significant role in the mediation of a number of inflammatory diseases. Cell death can be the physiologic response to macrophage activation and overwhelming expression of IL-1. Nitric oxide (NO), synthesised and secreted by macrophages, is a message for a wide variety of physiological functions which are synthesised through the L-arginine pathway by the enzyme NO synthase (NOS) (H.P. Ji, H.K. Ji, & Won, 2006). NOS has three different isoforms: endothelial constitutive NOS (ecNOS), neural NOS and inducible NOS (iNOS). However, NO is produced in a large amount by iNOS. Increased NO generation damages DNA, resulting in destruction of cells. The levels of macrophage proliferative response and IL-1 and NO production by macrophage are shown in Figure 3 and Table 3.

Figure 3. Effect of MT-α-glucan on macrophage proliferative response and IL-1 production of D-gal-induced-ageing mice. Data are the mean ± s.d., n=10. In each vertical column, *P < 0.05 and **P < 0.01, compared with the D-gal group (analysis of variance followed by the Student–Newman–Keuls test).

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The levels of macrophage proliferative response and IL-1 and NO production, and the activity of NOS and iNOS were significantly higher in D-gal model mice as compared with normal control (P<0.01). Treatment with MT-α-glucan (450, 150 mg kg⁻¹) decreased the macrophage proliferative response and IL-1 and NO production, NOS and iNOS activities markedly. This suggested that MT-α-glucan has the effect of suppressing production of destructive immune factors in D-gal-induced ageing animal model. MT-α-glucan could adjust the immunity, which has the effects of promoting beneficial immune reaction and inhibiting destructive immune response, and consequently exerting immunomodulatory effect.

In conclusion, MT-α-glucan had an anti-ageing effect on D-gal-treated mice, which was related to its antioxidative and immunomodulatory effects. Our work can be further developed for a healthy food having the effect of delaying senescence, antioxidation or immunomodulation, which has potential industrial or consumer application. Further mechanism of action concerning antioxidation and immunomodulation will be done in the future study.