Dry S. latifolia fruiting body were provided by Taihe edible fungus cultivation base in Shanxi Province of China, which were dried treatment in an oven at 35°C. The dry S. latifolia fruiting body were sliced into pieces, sieved into 200 mesh powder, and stored sealed at 4°C.

Macroporous resin HZ-830, DEAE-52 cellulose powder, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT), penicillin-streptomycin, dimethyl sulfoxide (DMSO), trypsin (Parenzyme), ethidium bromide, phenylmethyl sulfonyl fluoride (PMSF), and polyvinylidene difluoride (PVDF) were purchased from Solarbio Science & Technology Co. (Beijing, China). Dextran standard products of T-3, T-10, T-40, and T-70 were obtained from BioDee Biotechnology Co. (Beijing, China). Monosaccharide standards of D-glucose (Glc), D-mannose (Man), D-xylose (Xyl), D-galactose (Gal), L-arabinose (Ara), and D-fructose (Fru), and Lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO, United States).

RAW264.7 cells were purchased from Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified eagle medium (DMEM) high glucose medium were obtained from Zhejiang Tianhang Biotechnology Co. (Hangzhou, China) and HyClone (Utah, United States). The Micro NO Content Assay Kit, SDS-PAGE gel configuration kit, BCA protein concentration determination kit, high-sensitivity chemiluminescence detection kit, and RIPA cell lysate were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Assay kits for TNF-α, IL-6, and IFN-β were purchased from Shanghai Westang Biotechnology Co. (Shanghai, China). RNAiso Plus, SYBR^® Premix Ex TaqTM II (Tli RNaseH Plus), PrimeScriPt™ RT Master Mix (Perfect Real Time) were purchased from Takara Biomedical Technology Co., Ltd. (Dalian, China). The primary antibodies of TLR4 (bs-1021R), IRF3 (bs-2993R), JNK (bs-2592R), p-JNK (bs-1640R), ERK (bs-2637R), p38 (bs-0637R), and β-actin, and the secondary antibodies of HRP-labeled goat anti-mouse IgG (bs-0295G) were purchased from Bioss Biotechnology Co. (Beijing, China). And the primary antibodies of tumor necrosis factor receptor-related kinase 6 (TRAF6) (66498-1-lg) were purchased from Proteintech Group, Inc. (Wuhan, China).

The dry powder of SLNP was mixed with ultrapure water at a ratio of 1:40 (g:mL), and hydrolyzed at 75°C for 2 h. After being centrifuged at 4500 rpm for 10 min, the supernatant was concentrated, added ethanol at a ratio of 1:3 (mL:mL), and kept at 4°C for 12 h. The precipitate was collected by centrifugation at 5000 rpm for 5 min, and washed with acetone and ether for several times, dissolved in ultrapure water at 45°C for 8 h. Afterward, the supernatant was added Sevag solvent (Chloroform: N-butanol = 4:1), stirred by magnetic stirrer for 20 min, and centrifuged at 5000 rpm for 5 min. Next, the obtained supernatant was preliminarily decolorized and removed impurities by HZ-830 Macroporous Adsorption Resin to obtain crude polysaccharides. Then the 10 mg/mL crude polysaccharides were eluted in a DEAE-52 cellulose chromatography column (2.6 cm × 30 cm) with ultrapure water at a flow rate of 1 mL/min. The obtained polysaccharides were measured the content by the phenol sulfuric acid assay. Finally, the purified neutral polysaccharides were collected, dialyzed, and lyophilized.

The Mw of SLNP was evaluated by HPGPC (Agilent, United States) with a final concentration of 0.5 mg/mL and the injection volume of 20 μL using a detector differential refractive index detector and a TSK-GEL-4000 PWXL column (Agilent, United States) eluting with ultrapure water. The column temperature was 40°C, and the flow rate was 0.6 mL/min. T-dextran standards with different molecular masses were used for injection under the same conditions and the standard curve was drawn with the peak time as the abscissa and the relative Mw as the ordinate.

Twenty mg of SLNP was mixed with 4 mL of trifluoroacetic acid in a sealed test tube and was put into an oven to hydrolyze at 120°C for 6 h. Then the hydrolysate was added methanol to evaporate trifluoroacetic acid completely and dissolved in 1 mL of distilled water. Afterward, the obtained solution was diluted 10 times to measure the monosaccharide composition by ion chromatography (IC) with Dionex Carbopac PA10 column (250 mm × 4 mm) and Dionex pulsed amperometric detector (California, United States) with Au electrode. The detection conditions were as follows: the column temperature was 30°C, the injection volume was 25 μL, the flow rate was 0.45 mL/min, and elution mode was 10% 200 mmol/L NaOH and 90% ultrapure water. Glucose, fructose, mannose, xylose, galactose, and arabinose were selected as standard monosaccharide.

The functional chemistry of SLNP (2 mg in KBr pellets) was detected with a Bruker Tensor 27 IR instrument (Karlsruhe, German) at a frequency range of 400–4000 cm^–1.

Sparassis latifolia neutral polysaccharide was treated with deuterium (D₂O, 99.9%) and lyophilized with D₂O for three times to exchange protons. Afterward, SLNP was placed in a 5 mm NMR tube and dissolved in 0.5 mL of D₂O. NMR-spectra was recorded with a Brucker AVANCE III 850 MHz NMR (Karlsruhe, Germany). The analysis included a 1D spectrogram (¹H, ¹³C).

The RAW264.7 macrophages were cultured in DMEM high glucose medium containing 10% fetal bovine serum in a 5% CO₂ incubator at 37°C. Then the cells at the logarithmic growth phase and under stable growth conditions were selected for further experiments.

The effects of SLNP on the viability of RAW264.7 cells were measured by MTT method. The RAW 264.7 macrophages were planted into 96-well plates at a density of 1 × 10⁴ cells per well and cultured overnight in a CO₂ cell incubator. Next the cells were cultured with 100 μL of SLNP solution with 12 different concentrations between 1.95 μg/mL and 4000 μg/mL for 24 h, respectively, during which LPS (1 μg/mL) was used as a positive control group, and the culture medium was used as a negative control group. There were 6 replicates in each group. After that, cells in each well were added 20 μL MTT solution to continuously incubate in dark for 4 h. After discarding MTT and adding 150 μL of Dimethyl sulfoxide (DMSO) in each well, the cells were gently shaken on the shaker for 15 min and the cell viability was determined with a SpectraMax i3X microplate reader (Sunnyvale, United States) at 490 nm.

The RAW264.7 cells (2.5 × 10⁵ cells/well) under good growth conditions were seeded in 24-well plates and cultured overnight, and the old medium was discarded after adherent. Then the cells were treated with various concentrations of SLNP (62.5, 125, 250, 500 μg/mL), LPS (1 μg/mL), and the medium for 24 h. There were 4 replicates in each group. Next the supernatants in each well were collected and centrifuged to determine the levels of NO and IL-6, TNF-α, IFN-β by Micro NO Content Assay Kit and the corresponding ELISA kits.

To further study the effects of TLR4 antibody on the secretion of NO and cytokines, the RAW264.7 cells (2.5 × 10⁵ cells/well) in each well were treated with 20 μg/mL of TLR4 antibody for 1 h. Afterward, the cells were treated with 250 μg/mL of SLNP, 1 μg/mL of LPS, and the medium for 24 h. Each group had 4 replicates. After incubation, the supernatants were collected to measure the concentrations of NO and cytokines of IL-6, TNF-α, IFN-β by Micro NO Content Assay Kit and the corresponding ELISA kits.

The RAW264.7 cells were planted into 6-well plates at a density of 2 × 10⁶ cells per well for 12 h. After discarding the old medium, cells were incubated with 125, 250, 500 μg/mL of SLNP, 1 μg/mL of LPS, and new medium for another 24 h, respectively. Total RNA was extracted from RAW264.7 cells using the RNAiso Plus according to the manufacturer’s protocols. After that, the purity and content of RNA were measured by Nanodrop 2000c (Thermo Fisher, Delaware, United States). Next, cDNA was synthesized from the total RNA with the PrimeScript^® RT reagent Kit. Afterward, the mRNA expression levels of TLR4, TRAF6, IRF3, JNK, ERK, p38, and β-actin in RAW264.7 macrophages were detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) under the following conditions: 95°C for 90 s, 40 cycles of 95°C for 5 s, 60°C for 30 s, 72°C for 30 s, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The levels of the target genes were calculated according to 2^–ΔΔCt method with the β-actin gene as the internal reference. The primers used for qPCR were listed in Table 1.

After treating with different concentrations of SLNP (125, 250, and 500 μg/mL), 1 μg/mL of LPS and new medium for 24 h, the RAW264.7 cells in each well were added 150 μL of RIPA cell lysate containing PMSF and protein phosphatase inhibitor to lyse for 30 min. After centrifugation at 12,000 r/min for 15 min, the supernatants were collected and stored at −80°C for further use. Afterward, the total protein was quantified using the BCA method, subjected to SDS-PAGE, and transferred onto PVDF membranes. Next, the PVDF membranes were blocked with 5% non-fat milk at room temperature for 2 h, added the specific primary antibody of TLR4 (diluted 1:500), β-actin (1:1000), IRF3 (1:1000), JNK (1:1000), ERK (1:1000), p38 (1:1000), P-JNK (1:1000), and TRAF6 (1:1000), respectively, and shaken overnight at 4°C. After being rinsed 3 times with TBST, the PVDF membrane was added HRP-labeled goat anti-mouse IgG (diluted 1:3,000) and shaken at room temperature for 2 h. After the PVDF membrane was washed with TBST, the antibody-specific protein was observed in a fluorescence imager using eECL kit.

The results were expressed as the mean ± standard error (SE). Graphpad Prism 5.0 software was used for one-way analysis of variance followed by Duncan’s multiple comparisons. P < 0.05 was regarded as statistically significant and P < 0.01 was considered to indicate a statistically highly significant.

After the crude polysaccharides were separated by a DEAE-52 cellulose column, the elution curve was a single and symmetrical peak (Figure 1). Next, the eluate was collected, concentrated, dialyzed, and freeze-dried to obtain light yellow neutral polysaccharides. Then the homogeneous SLNP were obtained after being collected and frozen-drying. The HPGPC of SLNP (Figure 2) showed that this fraction had a single and symmetrical peak and the relative Mw was 3.2 × 10⁵ Da.

The IC technology was used to determine the monosaccharide composition of SLNP. As shown in Figure 3, SLNP was mainly composed of arabinose, galactose, glucose, xylose and mannose with molar ratio of 6:12:63:10:5, respectively. Galacturonic acid and glucuronic acid were not detected, which indicated that SLNP was a neutral polysaccharide (24).

Fourier transform infrared spectroscopy was used to analyze the functional group structure of the SLNP. As shown in Figure 4, the absorption peak at 3406 cm^–1 was caused by the stretching vibration of the hydrangea polysaccharide molecule -OH, the absorption peak in the range of 2780–2968 cm^–1 was caused by the stretching vibration of C-H in the polysaccharide structure (25, 26), the absorption peak at 1631 cm^–1 was the flexural vibration absorption peak of -OH, and the absorption peak near 1367 cm^–1 was the flexural vibration absorption peak of C-H (27, 28). The absorption peaks at 1154–1019 cm^–1 were the absorption peaks caused by the stretching vibration of the C-O-C structure and the C-O-H variable-angle vibration on the polysaccharide ring of S. latifolia, 1078 and 1019 cm^–1 were the characteristic absorption of the pyran ring (29). There was the characteristic absorption peak of C-H variable angle vibration of α-type glycosidic bond near 840 cm^–1, and the absorption peak near 890 cm^–1 was the characteristic absorption peak of C-H variable angle vibration of β-type glycosidic bond, indicating the existence of a pyranose ring connected by α-glycosidic bond and β-glycosidic bond (30). In addition, no absorption peak was found near 1730 cm^–1, indicating that there is no uronic acid in SLNP (31).

Nuclear magnetic resonance was used to analyze the glycosidic bond configuration of the SLNP. As shown in Figures 5A,B, in the ¹H-NMR spectrum, the chemical shift at 3.5–4.5 ppm was the proton peak of the sugar ring carbon, and there was an anomeric proton signal absorption peak in the range of 4.5–5.5 ppm. Specifically, the chemical shift of 5.0–5.5 ppm indicates that the polysaccharide is mainly in the configuration, and the weak peak at 4.5–4.6 ppm indicates that the polysaccharide structure has a small amount of β configuration. In the anomeric carbon region of the ¹³C-NMR spectrum, the two absorption peaks of 98.6 and 100.8 ppm were α-type end group carbon signals, and the absorption peaks of 102.3, 102.7, and 102.9 ppm were β-type. The end-group carbon signal, of which the absorption peak signal at 100.8 ppm was stronger, and the signal at 98.6, 102.3, 102.7, and 102.9 ppm were relatively weak, indicating that SLNP is mainly composed of five monosaccharide residues. Consistent with the results of monosaccharide composition, the main chain might consist of one α-pyranose residue, one α-type sugar residue and three β-type sugar residues to form the side chain. The chemical shifts of 70.3–78.5 ppm were assigned to the unsubstituted C₂-C₅ structure on the sugar ring.

The RAW264.7 macrophages were treated with different concentration of SLNP for 24 h to determine the cell viability by MTT assay. As shown in Figure 6, the proliferation ability of RAW264.7 cells were increased first and then decreased with the increase of SLNP concentration. The proliferation ability was significantly up-regulated after treated with SLNP (7.8125–1000 μg/mL) compared to the control group (P < 0.05). Moreover, 250 μg/mL of SLNP had the strongest ability to promote the proliferation of RAW264.7 cells and the upregulation rates were 103.84%. However, after SLNP concentration exceeded 250 μg/mL, the proliferation activity of RAW264.7 cells gradually decreased, and when the concentration was 4,000 μg/mL, SLNP strongly inhibited the proliferation activity of RAW264.7 cells with the inhibition rate of 34.54%.

Activated macrophages can secrete a series of chemokines and cytokines, which play important roles in activating adaptive immune responses and regulating other immune responses (32). ELISA was used to detect the production of NO, IL-6, TNF-α, and IFN-β. Compared with the control group, different concentrations of SLNP up-regulated significantly the levels of NO (Figure 7A), IL-6 (Figure 7B), TNF-α (Figure 7C), and IFN-β (Figure 7D) in RWA 264.7 macrophages (P < 0.01), of which 250 μg/mL of SLNP had the strongest promotion ability and the upregulation rates were 477.98, 375.45, 721.52, and 260.88%, respectively. Meanwhile, LPS could promote remarkably RWA 264.7 macrophages to produce NO, IL-6, TNF-α, and IFN-β (P < 0.01). Above results indicate that SLNP might display immune enhancing activity by inducing the production of NO and cytokines in RAW264.7 cells.

To verify whether TLR4 has participated in SLNP-induced macrophages activation, we investigated the expression of TLR4 mRNA and protein, and the effects of TLR4 antibody on the secretion of NO and cytokines induced by SLNP. The results showed that compared with the control group, the expression levels of TLR4 mRNA (Figure 8A) and protein (Figure 9B) in macrophages were significantly elevated after treatment with different doses of SLNP and 1 μg/mL of LPS (P < 0.01). And treatment with 500 μg/mL SLNP increased markedly TLR4 mRNA and protein levels by 52.33 and 96.82%, respectively, implying that TLR4 is an immune recognition receptor in which SLNP plays an immunomodulatory role. However, blocking TLR4 signaling with the specific TLR4 antibody reversed the elevation of NO and cytokines in SLNP-induced RAW264.7 macrophages. As shown in Figure 10, the levels of NO (Figure 10A), IL-6 (Figure 10B), TNF-α (Figure 10C), and IFN-β (Figure 10D) were remarkably increased in macrophages RAW264.7 treated with 250 μg/mL of SLNP or LPS compared with the control group (P < 0.01). However, the TLR4 antibody decreased significantly the levels of NO (Figure 10A), IL-6 (Figure 10B), TNF-α (Figure 10C), and IFN-β (Figure 10D) by 68.87, 68.45, 75.38, and 47.25% compared with 250 μg/mL of SLNP group. The similar trends were found in RAW264.7 cells induced by 1 μg/mL of LPS. In short, these results illustrate that TLR4 participates in SLNP-induced macrophage activation.

In order to explore whether the SLNP-induced up-regulation of TLR4 caused the activation of MAPK signaling pathway, the cells were treated with different concentrations of SLNP, and the mRNA levels of TRAF6, JNK, ERK, and p38 were measured by qPCR. Meanwhile, to further clarify the mechanism by which SLNP activates the MyD88-dependent pathway, the total levels of TRAF6 and phosphorylation levels of JNK, ERK and p38 were measured by Western Blot (Figure 9A).

As shown in Figure 8, different concentrations of SLNP significantly up-regulated the mRNA levels of TRAF6 (Figure 8B), JNK (Figure 8C), ERK (Figure 8D), and p38 (Figure 8E) compared with the control group (P < 0.05). Treatment with 250 μg/mL of SLNP elevated remarkably these indexes by 72.70, 86.54, 82.77, and 56.98%, respectively. The similar trends were found in the protein levels of TRAF6 (Figure 9C), JNK (Figures 9D,E), ERK (Figures 9F,G) and p38 (Figures 9H,I). Compared with the control group, the relative protein levels of TRAF6 (Figure 9C), JNK (Figures 9D,E), ERK (Figures 9E,G) and p38 (Figures 9H,I) in RAW264.7 cells were significantly up-regulated in 250 μg/mL of SLNP group, with the up-regulation rate of 115.13, 147.66, 131.21, and 214.01%. These results suggest that SLNP exerts the immunomodulatory effects by activating MyD88-dependent pathway and the downstream MAPK signaling pathway.

TLR4 initiates MyD88-independent signaling pathways which increase the expression of IRF3 resulting in the expression of IFN-β (33). As shown in Figure 8F, after being incubated with SLNP, the expression of IRF3 mRNA in RAW264.7 cells was increased significantly compared with the control group. The expression of IRF3 protein also showed an increasing trend after treated with SLNP (Figure 9J). Treatment with 500 μg/mL of SLNP increased remarkably the levels of IRF3 mRNA and protein by 28.13 and 18.00% in the RAW264.7 cells, respectively. These results indicate that SLNP activates the MyD88-independent signaling pathway.