The fresh fruiting bodies of Maerkang L. deliciosus Gray were collected from Maerkang County, which lies in the Sichuan Aba Tibetan and Qiang Autonomous Prefecture. After vacuum freeze-drying, the bodies were crushed and stored at 4°C for use in the Key Laboratory of Southwest China Wildlife Resources Conservation, College of Life Sciences, China West Normal University, China. The fruiting bodies of Maerkang L. deliciosus Gray were identified by Professor Tang Zongxiang (Sichuan Agricultural University, China). The ethanol was purchased from Swancor Shanghai Fine Chemical Co., Ltd. (Shanghai, China). Sodium chloride was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. (Chengdu, China). Trifluoroacetic acid (TFA), standard monosaccharide and dextran of different molecular weight were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). DEAE-cellulose column, Sephacryl S-300 gel column and Sephadex G-200 column were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Cell counting kit (CCK)-8 (cat. no. CK04) was purchased from Dojindo Molecular Technologies, Inc. (Shanghai, China). PBS buffer, RPMI-1640 medium, phenol red free, 0.5% Trypsin-EDTA and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Mouse interleukin (IL)-6 (cat. no. M6000B) and tumor necrosis factor (TNF)-α (cat. no. MTA00B) ELISA kits were purchased from R&D Systems China Co., Ltd. (Shanghai, China). Neutral red and DMSO were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). MTT was purchased from Amresco, LLC (Solon, OH, USA). Cell cycle and apoptosis analysis kit (cat. no. C1052) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The inverted fluorescence microscope was purchased from Leica Microsystems (DMI4000B, Germany). The intelligent high-efficiency centrifuge was purchased from Beckman Coulter, Inc. (Brea, CA, USA; Avanti JXN-26). The refrigerated centrifuge was purchased from Eppendorf (Hanburg, Germany; cat. no. 5418R). All analytical reagents were of analytical grade.

The fresh fruiting bodies of Maerkang L. deliciosus Gray were washed with water, dried at 60°C and pulverized. The 300 g powder accurately weighed was boiled in water for 6 h and the ratio of powder and water was 1:3 (15,16). The supernatant was collected by centrifugation (16,670 × g for 20 min at 4°C; Avanti JXN-26, Beckman Coulter, Inc.) and the precipitate was boiled in water for 6 h (repeated three times). All supernatants were concentrated to 300 ml. Four volumes of absolute ethanol were added to precipitate crude polysaccharides. Flocculent precipitation was collected and dried. Crude polysaccharides were weighed and dissolved with distilled water, which was performed as previously described (17). After adding the supernatant to the activated DEAE cellulose-52 column (2×60 cm), it was allowed them to stand for 10 min to fully bind. Sodium chloride solutions with different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mol/l) were prepared as the mobile elution phase. After adding the mobile phase, the liquid was collected and concentrated. Phenol-sulfuric acid method was used to determine the polysaccharide as described previously (18). The eluate was purified on Sephadex G-200, then concentrated and centrifuged (12,400 × g for 20 min at 4°C; 5418R; Eppendorf). Small molecules in the supernatant were removed by dialysis (7 kDa) for 48 h. For lyophilization, Maerkang L. deliciosus Gray poly-saccharide (named LDG-M) was obtained for further analysis on its structure and bioactivities.

A total of 10 mg LDG-M was dissolved in 1 ml double distilled water, sonicated for 5 min and then filtered (0.22-µm pore). HPGPC was used to determine the molecular weight of LDG-M as previously described (19-21). The measured data were subjected to Empower Pro GPC software analysis (Agilent Empower Pro GPC Data Analysis Software for Agilent ChemStation; version B.01.02; Agilent Technologies Inc., Santa Clara, CA, USA) with a standard curve prepared from dextran to obtain molecular weight.

A total of 5 mg LDG-M and fully dry potassium bromide (KBr) were ground and mixed (22). Subsequently, the mixture was pressed and scanned in the Fourier transform infrared spectrometer at a range of 4,000-400 cm⁻¹ as described previously (23).

For NMR analysis, 20 mg LDG-M was weighed and dissolved in D₂O (24). The Varian Unity INOVA 400/45 (Varian Medical Systems, Inc., CA, USA) was used to detect the ¹H NMR spectra and ¹³C NMR spectra, and tetramethylsilane was used as an internal standard (11).

Methyl iodide was used to prepare methylated polysaccharide (25). The methylated product was dried and dissolved in 2 M TFA and hydrolyzed at 100°C for 6 h. Silane reagent was added and the derivatized product was used to perform the GC-MS experiment. The temperature program was set as follows: Initial temperature maintained at 80°C for 3 min, then raised to 200°C at a rate of 10°C/min and maintained at 200°C for another 10 min.

The mouse L929, B and macrophage RAW264.7 cell lines, purchased from the cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Science (Shanghai, China), were cultured in RPMI-1640 medium with 10% FBS, 1% penicillin (100 IU/ml) and streptomycin (100 mg/l) in an incubator at 37°C with 5% CO ₂.

The pharmacological evaluation of B and RAW264.7 cell stimulation was examined using a CCK-8 assay (26). Cells were cultured in RPMI-1640 medium. When cells were in the logarithmic growth period, the medium was added to dilute cells. A density of 1×10⁵ cells/ml were added to 96-well plates at 100 µl/well and incubated in an incubator for 24 h at 37°C with 5% CO₂ (11). Different concentrations of LDG-M prepared using cell culture medium (0.625, 1.25, 2.5, 5, 10, 20 µg/ml) were added to the 96-well plates at 100 µl/well. Furthermore, 5 µg/ml lipopolysaccharide (LPS) was used as positive control (27) and the cell culture medium without LDG-M was used as blank control. After incubated at 37°C for 24 h, 5 µl of CCK-8 reagent was added to each well and the cells were cultured for an additional 2 h. A microplate reader was used to detect the optical density (OD) at 450 nm. The formula used to calculate cell viability (%) was as follows: [(Ac-As)/(Ac-Ab)] ×100%, where Ac was the absorbance of the control group, Ab was the absorbance of the blank group, and as was the absorbance of the experimental groups.

RAW264.7 cells (1×10⁵ cells/ml) were seeded into 96-well plates at 100 µl/well and incubated for 24 h. Subsequently, 100 µl cell culture medium (blank control), LPS (final concentration 5 µg/ml, positive control) and LDG-M solutions (0.625, 1.25, 2.5, 5, 10, 20, 40 µg/ml) were added to the 96-well plates. After incubation at 37°C for 24 h, neutral red reagent (0.075 g/l) was added to the 96-well plates. After 30 min, the neutral red reagent was discarded and the cells were washed with PBS thrice, followed by the addition of 200 µl lysis buffer (ethanol:glacial acetic acid, 1:1). The 96-well plates were placed in an incubator for 2 h. The OD was measured at 540 nm.

RAW264.7 cells (effector cells, 1×10⁵ cells/ml) and L929 cells (target cells, 1×10⁶ cells/ml) in the logarithmic growth period were cultured on 96-well plates and incubated for 24 h. Effector cells (RAW264.7 cells) and target cells (L929 cells) were added to 96-well plates at a ratio of 1:2 with 100 µl/well. Simultaneously, separate effector and target cell groups were set at 200 µl/well. When cells were adherent, supernatants were discarded. The followings groups were used: Blank control (cell culture medium), positive control (LPS 5 µg/ml) and experimental (LDG-M 5 µg/ml). Following an incubation at 37°C for 24 h, supernatants were removed and MTT (5 mg/ml) was added at 10 µl/well. The plate was then cultured in incubator at 37°C for 4 h. The supernatants were discarded, and DMSO was added at 100 µl/well for 15 min. The absorbance value was measured at 570 nm. The inhibitory activity (%) of L929 tumor cells in the presence of macrophage RAW264.7 cells in vitro was calculated according to the following formula: [1-(OD value of experimental group-OD value of individual effector cell)/OD value of individual target cell] ×100%.

Following treatment with LDG-M as aforementioned, cells were observed under a Leica inverted fluorescence microscope directly without staining (magnification, ×100). Leica Application Suite X software (Leica Microsystems, Inc.; version 3.0.0.15697) was used for the assessment of cell morphology.

In order to understand the alterations in the level of cytokines, TNF-α and IL-6 secreted by RAW264.7 cells, ELISA kits were used according to the manufacturers' protocols.

Cell cycle and apoptosis analysis kit were used to evaluate the effects of LDG-M on the cell cycle of B cells. The following groups were used: Blank control, experimental (LDG-M, 2.5 and 5 µg/ml) and positive control (LPS, 5 µg/ml). A total of 1 ml 70% ethanol was added to cell plates at 4°C for 2 h in order to immobilize cells. Subsequently, 0.5 ml propidium iodide staining solution was added to each sample in a 37°C water bath for 30 min. The percentages of cells at the G₀/G₁, S and G2/M phases were analyzed using a flow cytometer as previously described (28).

Data are presented as the mean ± standard deviation. Significant differences between the experimental and control groups were analyzed using one-way ANOVA followed by Student-Newman-Keuls test with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) (27). There were six replicates in each group. P<0.05 was used to indicate a statistically significant difference.

HPGPC was used to evaluate the relative molecular weight of the polysaccharide, using the Z+1-average molecular weight (Mz+1), Z-average molecular weight (Mz), weight-average molecular weight (Mw), peak molecular weight (Mp) and number-average molecular weight (Mn). The peaks of LDG-M on HPGPC were broadly symmetrical. The Mz+1 of LDG-M was 33391519 Da, Mz was 2626727 Da, Mw was 573689 Da, Mp was 16707 Da, and Mn was 4480 Da (Fig. 1A).

The working principle of infrared spectroscopy is due to different vibration levels. According to the results for LDG-M, no absorption peak at wavelengths of 280 and 260 nm were present, indicating no protein and nucleic acid in the LDG-M sample. The FT-IR spectra of LDG-M displayed typical polysaccharide absorption peaks in the range of 4,000-400 cm⁻¹ (Fig. 1B). A broad absorption peak at 3,443.46 cm⁻¹ was designated as a OH stretching vibration peak, 2,920.91 cm⁻¹ was designated as a CH stretching vibration peak, 1,639.88 cm⁻¹ was designated as a CO stretching vibration peak, 1,401.54 cm⁻¹ was designated as a bending vibration peak of CH₂, CH and OH, 1,093.48 cm⁻¹ was designated as a CO stretching vibration peak and 6,18.19 cm⁻¹ was designated as a CH rocking vibration peak (29,30).

The proton spectrum of LDG-M is shown in Fig. 1C. In the ¹H NMR (400 Hz) spectrum, δ 5.03, δ 4.92 and δ 4.92 ppm indicated that LDG-M had three anomeric protons, suggesting that LDG-M consisted of at least three monosaccharides. The overlapping proton signal at δ 4.92 ppm was assigned to β-pyranose unit, whereas another signal at δ 5.03 ppm was attributed to α-pyranose forms (21). The proton signal for water was δ 4.70 ppm. The signals in δ 3.49-4.14 ppm were the proton signal of H2-H6 of glucose and H2-H5 of lyxose.

In the ¹³C NMR spectra of LDG-M (Fig. 1D), the signals of δ 102.69, δ 102.67 and δ 100.79 ppm were anomeric carbon peaks, which indicated that LDG-M had α and β anomeric configurations (11). It was in accord with the results of FT-IR and ¹H NMR. In virtue of inexistence of signals among δ 160-180 ppm, accordingly, no carboxyl group was present in LDG-M. Due to the absence of a chemical shift at δ 106-109 ppm, no furan ring was present in LDG-M. According to the literature (31), the chemical shift at δ 102.69 ppm could belong to C1 of residue A (1,4,6-linked β-D-Glcp), the chemical shift at δ 102.67 ppm could belong to C1 of residue B (1,6-linked β-D-Glcp) and the chemical shift at δ 100.79 ppm could belong to C1 of residue C (1-linked α-D-Lyxp), respectively. The strong signals among δ 80-δ 69 ppm may be assigned to the carbon signal of C2-C6 of glucose and C2-C5 of lyxose. Signals of every carbon atom are listed in Table I.

The proton chemical shift at δ 5.03, δ 4.92, δ 4.92 ppm and a cross peak at 5.03/3.89, 4.92/3.92, 4.92/3.79 ppm were readily obtained from H-H correlated spectroscopy (Fig. 2A), which implied that the chemical shift of H2 were δ 3.89, δ 3.92 and δ 3.79 ppm, respectively.

Long-range ¹H-¹³C heteronuclear multiple bond correlation (HMBC) spectrum was used to analyze the association between monosaccharide residues of LDG-M (Fig. 2B). A H1/C3 association was identified in the ¹H-¹³C HMBC spectrum, such as δ 4.92/δ 70.88 ppm corresponding to residue A, δ 4.92/δ 79.50 ppm corresponding to residue B and δ 5.03/δ 72.19 ppm corresponding to residue C, respectively, which was consistent with the GC-MS analysis.

The GC-MS results on LDG-M methylation are presented in Table II. According to the GC-MS results, it may be suggested that the fragment ion peaks of LDG-M were consistent with the data of the D-configuration monosaccharide fragment ion peaks. Therefore, the D-configuration was the configuration of glucose residues that could be determined (Fig. 2C-E). In the present study, it may be suggested that the β-D-glucosepyranose residues were 2,3,4-tri-O-methyl-1,6-bis-O-trimethysilyl-substitued and 2,3-di-O-methyl-1,4,6-tris-O-trimethysilyl-substituted and the α-D-lyxopyranose residues were 2,3,4-tri-O-methyl-1-O-tri-methysilyl-substitued (Fig. 2C-E). According to the LDG-M methylation results, the amount of (1→6)-linked-β-D-glu cosepyranose and (1→4,6)-linked-β-D-glucosepyranose was the highest, which constituted the main chain of the structure. In addition, the side chain was comprised of the α-D-lyxopyranose residues. LDG-M had repeating units of a backbone of (1→6)-β-D-glucosepyranose and (1→4,6)-β-D-glucosepyranose, and a branch of a (1→4)-α-D-lyxopyranose residue.

On the basis of the aforementioned experimental data, the conceivable structure of LDG-M was confirmed, composing of a backbone of 1,6-linked-β-D-glucose and 1,4,6-linked-β-D-glucose and one (1→4)-linked-α- D-lyxopyranose residue as a branch (Fig. 2F).

The results demonstrated that the LDG-M group (0.625, 1.25, 2.5, 5, 10, 20 µg/ml) significantly promoted the proliferation of RAW264.7 cells compared with the blank control group (P<0.01), as shown in Fig. 3A. The proliferation efficiency reached the maximum with an increase of 87.8% at 5 µg/ml compared with the blank control group.

Neutral red is a fluorescent reagent with a large molecule and strong absorption peak at 540 nm. As a macromolecular substance, neutral red enters macrophages only through endocytosis. Therefore, the phagocytosis of macrophages can be measured using neutral red (32). Compared with the blank control group, LDG-M significantly promoted the phagocytosis of RAW264.7 cells (Fig. 3B). At a concentration of 2.5 µg/ml LDG-M, the phagocytic activity reached the maximum, which was increased by 61.4% compared with that of the blank control group.

Compared with the blank control group, LDG-M significantly promoted the secretion of TNF-α from RAW264.7 cells (Fig. 3C). When the concentration of LDG-M was 2.5 µg/ml, the secretion of TNF-α reached the maximum, which was 14.4 times higher compared with that of the blank control group.

Compared with the blank control group, LDG-M significantly promoted the secretion of IL-6 from RAW264.7 cells (Fig. 3D). When the concentration of LDG-M was 5 µg/ml, the secretion of IL-6 reached the maximum, which was 24.5 times higher compared with that of the blank control group.

The results demonstrated that the inhibitory activity of RAW264.7 cells on L929 cells reached 49.36% at an LDG-M concentration of 5 µg/ml, while it was 32.94% in the blank control group (Fig. 3E and F). Cell morphology observation revealed that RAW264.7 cells grew well and L929 cells grew weakly under the stimulation of LDG-M compared with the blank control group. This was consistent with the above statistical data, which confirmed that LDG-M could enhance the inhibitory activity of RAW264.7 cells on L929 cells.

B lymphocytes that can secrete antibodies were derived from bone marrow pluripotent stem cells and was used as a model of humoral immunity. Compared with the blank control group, the LDG-M and LPS groups significantly promoted the proliferation of B cells (Fig. 4A). At a concentration of 2.5 µg/ml LDG-M, the proliferation efficiency reached the maximum at 39.9%. Using the Leica Microsystems inverted fluorescence microscope to observe the cell morphology and the alterations to cell numbers (Fig. 4B), it is evident that compared with the blank control group, the number of B cells in the LDG-M group significantly increased and were grouped together more.

Cell cycle is a process involving cell division and involves cell interphases and the cell division phase. Cells are dormant when they are in the G0 phase. Cell interphase is divided into the G1, S and G2 phases. The G1 phase involves the synthesis of RNA and ribosomes. The S phase includes the synthesis of DNA and histones, and in general, once the cells enter the S phase, cell division continues until the G1 phase of the next cycle. The G2 phase involves the mitosis preparation period when protein synthesis is completed, and the M phase is the cell division period. The results for cell cycle analysis are shown in Fig. 4C-D. LDG-M was demonstrated to promote cell cycle progression in B lymphocytes, inducing cell division. In Fig. 4D, compared with the blank control group, when the concentration of LDG-M was 2.5 µg/ml, the number of B lymphocytes in the G₀/G₁ and G2/M phases decreased significantly from 66 to 58.2%, and from 17 to 11.6%, respectively, while the number of cells in S phase increased significantly from 17.6 to 30.8%. The percentage of cells in the G₀/G₁ phase was the lowest at 2.5 µg/ml. However, when the concentration of LDG-M was 5 µg/ml, the number of B lymphocytes in the G₀/G₁ phase did not change significantly. The B cell cycle assay showed that when the concentration of LDG-M was 2.5 µg/ml, the number of B cells in the S phase was the highest, but whether it is associated with the proliferation of B cells remains to be investigated.