As presented in Fig

As presented in Fig. phagocytosis in the cells treated with PH-EPS. Nuclear factor (NF)-B p65 was significantly translocated into the nucleus in the PH-EPS-treated cells. In addition, expression of inducible NO synthase (iNOS) and IB- degradation were enhanced in PH-EPS-treated cells. The phosphorylation levels of p38, JNK and ERK were also significantly increased in the PH-EPS-treated cells. Furthermore, IL-1 and TNF- production was markedly decreased in PH-EPS-treated cells when the mitogen-activated protein kinase (MAPK) pathways were blocked by the inhibitor Dectin-1 and by antibodies against Toll-like receptor 4 (TLR4). The present results indicated that PH-EPS from possessed the capability of activating RAW 264.7 cells via the TLR4/NF-B/MAPKs signaling pathway. and macrophages (22). To Ibotenic Acid the best of our knowledge, there are no studies related to EPSs derived from PH0016 was maintained in Hainan Provincial Key Laboratory of Tropical Medicine. Preparation of EPSs from PH0016 was performed as previously described (15). In brief, PH0016 was cultured in Potato Dextrose Agar on a rotator at 180 rpm at 28C for 10 days. Then, the concentrated supernatant of the culture was obtained using a rotary evaporator and was further processed by ethanol precipitation, dialysis and protein depletion. The crude polysaccharides were then purified through Sephadex G-75 and DEAE-Sepharose (Klamar). After being dialyzed and lyophilized, the purified PH-EPS was obtained. The PH-EPS was confirmed to be free of protein and nucleic acid by ultraviolet detection at absorbance of 260 and 280 nm, respectively. The components of PH-EPS (32.3 Ibotenic Acid kDa) included rhamnose, fucose, xylose, glucose, mannose and galactose, in which the relative molar ratio was 17.5:16.9:2.3:21.5:27.4:14.4, based on gas chromatography. Cell culture The mouse macrophage cell line RAW 264.7 was purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Ibotenic Acid Cells at the logarithmic phase were cultured with or without PH-EPS (0C800 g/ml) in DMEM supplemented with streptomycin (100 g/ml), penicillin-G (100 U/ml) and 10% FBS in a standard humidified incubator (Sanyo Electric Co., Ltd.) at 37C containing 5% CO2. MTT assay RAW 264.7 cells (4105 cells/ml) at logarithmic phase were cultured at 37C with 5% CO2 in 96-well plates overnight. They were then treated with various doses of PH-EPS (0C800 g/ml) or LPS (1 g/ml) for 24 h. Thereafter, MTT reagent (5 mg/ml, 10 l/well) was added to the well at 37C for another 4 h. Finally, the supernatant was discarded, and DMSO (150 l) was added into each well for solubilizing the formazan. The absorbance of the dissolved solutions was detected at 490 nm using a microplate reader (EXL808; BioTek Instruments, Inc.). NO production The macrophage RAW 264.7 cells at logarithmic phase were cultured at 37C with 5% CO2 for 1 day and then incubated with PH-EPS (0C600 g/ml) or LPS (1 TNFSF8 g/ml, as positive control) for a day. The NO production was detected using a commercially available Griess reagent kit (Molecular Probes; Thermo Fisher Scientific, Inc.). A sodium nitrite standard curve was established for calculating the concentration of nitrite and the 540 nm absorbance was recorded using a microplate reader. Cytokine assay RAW 264.7 macrophage cells at logarithmic phase were cultured and treated with PH-EPS (0C600 g/ml) or LPS (1 g/ml) for 1 day. The concentration of TNF- and IL-1 in the supernatants of the cell culture was detected using commercially available ELISA kits from Wuhan Boster Biological Technology, Ltd. Determination of dextran uptake by flow cytometry The dextran uptake by RAW 264.7 cells was identified as in a previous study (15). RAW 264.7 cells (5105 cells/ml in each well) were cultured with PH-EPS (0C600 g/ml) or LPS (1 g/ml) for 1 day at 37C with 5% CO2 in 6-well plates. Cells were collected, followed by suspension in 1 mg/ml FITC-labeled dextran (100 l/well). Thereafter, cells were further cultured at 37C with 5% CO2 for Ibotenic Acid 0.5 h. Then cold PBS (2 ml) mixed with 0.02% sodium azide and 1% human serum (Gibco; Thermo Fisher Scientific, Inc.) were added to each well to terminate the uptake of dextran. Finally, the RAW 264.7 cells were washed with cold PBS for three times and the level of the dextran uptake was indicated as the fluorescence intensity detected by flow cytometry (FACSCalibur; BD Biosciences) and analyzed by FlowJo software version 10 (FlowJo LLC). Immunofluorescence analysis The RAW 264.7 macrophage cells (5105 cells/ml in each well) were incubated on glass coverslips and treated with PH-EPS (200 g/ml) or LPS (1 g/ml, for positive control) for 1 h. Subsequently, the cells were rinsed with cold PB at least three times and fixed with 4% paraformaldehyde at 37C for 30C60 min. Thereafter, the cells were permeabilized with 0.2% Triton.