| Agaricus bisporus,a common edible fungi,is rich in protein,fat,polysaccharides and other components.It has a variety of biological activities,such as immune regulation,anti-oxidation,anti-tumor,etc.Many reports had indicated that Agaricus bisporus polysaccharides played a crucial role in immunoregulatory effect,and the immunostimulation mechanism was necessary to study further.In the present study,a variety of chromatographic techniques were used for preparation of Agaricus bisporus polysaccharides.Its structure was identified by various analytical methods,and its immunostimulation mechanism was assayed on murine macrophage RAW264.7 cells.The proliferation,NO production,COX expression,inflammatory cytokines mRNA expression and related proteins expression on RAW264.7 cells were analyzed to explore the regulation mechanism immune of Agaricus bisporus polysaccharide.The main findings of this paper were as follows:?1?Agaricus bisporus polysaccharide was extracted by microwave assisted method,and was purified with macroporous resin,ion exchange resin,and molecular sieve chromatography.The polysaccharide content was determined by phenol-sulfuric acid method to be 99.2%?W/W,calculated as glucose?.The molecular weight of the polysaccharides was 822 K Da,which was determined by exclusion chromatography with the dextran standards.?2?Monosaccharide composition analysis was determined by HPLC using precolumn derivatization.The results revealed that the purified polysaccharide contains mannose,glucose,galactose and xylose.Infrared spectra showed that the polysaccharides had the characteristic absorption peaks of polysaccharides,and the characteristic absorption peaks of 1035.82 cm-1 and 886.57 cm-1 showed that the main type of this polysaccharides were ?-pyranose configuration.The analysis by NMR spectroscopy?1H-NMR?showed that the polysaccharide was mostly composed of ?-pyranose configuration and had characteristic absorption peaks of monosaccharide residues containing mannose,glucose,galactose and xylose,which was consistent with the results of infrared spectrum analysis and monosaccharide composition analysis.?3?The effect of Agaricus bisporus polysaccharides on the proliferation of RAW264.7 cells was detected by MTT.The results showed that the low concentration(1 ?g·mL-1)of Agaricus bisporus polysaccharides can significantly promote the proliferation of RAW264.7 cells,and the high concentration(10 ?g·mL-1)of Agaricus bisporus polysaccharides can significantly inhibit the proliferation of RAW264.7 cells.In general,with the increasing concentration,the effect of Agaricus bisporus polysaccharide on the proliferation of RAW264.7 cells showed a trend of gradual change from the promotion to inhibition?4?The results of Griess and Western blot test showed that Agaricus bisporus polysaccharide could significantly promote the release of NO and the expression of stress response protein?COX-2 and iNOS?in RAW264.7 cells,which indicated that the polysaccharide has a certain immunomodulatory effect.RT-PCR analysis showed that Agaricus bisporus polysaccharide can significantly promote the mRNA expression of IL-6,IL-1?,TNF-? and COX-2 in a dose-dependent manner on RAW264.7 cells?relative to ?-actin?,and further illustrated the immunomodulatory effects of Agaricus bisporus polysaccharide.In addition,Agaricus bisporus polysaccharides can significantly promote the phosphorylation of I?B-? and ERK protein,which indicated that they can activate I?B/NF-?B pathway and ERK/MAPK pathway on RAW264.7 cells.In conclusion,Agaricus bisporus polysaccharide can regulate cellular immune function though activativing the I?B/NF-?B pathway and ERK/MAPK pathway.?5?The results of Western blot testshowed that Agaricus bisporus polysaccharide could induce the apoptosis through the activation of Caspase-3 in a dose-dependent manner on RAW264.7 cells.Agaricus bisporus polysaccharide can also cause the phosphorylation of JNK protein,thus activating the JNK/MAPK signaling pathway.It can also inhibited Bcl-2 protein expression and promote Bim protein expression to induce RAW264.7 cell apoptosis,and the change of Bim protein is mainly induced by activating the JNK/MAPK signaling pathway.These results suggest that Agaricus bisporus polysaccharide can induce apoptosis of RAW264.7 cells by inducing the activation of JNK/MAPK signaling pathway,and it is mainly through the mediating effects of the Bcl-2 and Bim protein.In summary,Agaricus bisporus polysaccharide can induce the immune regulation and apoptosis on RAW264.7 cells.Moreover,with the increase of the concentration of Agaricus bisporus polysaccharide,the immune regulatory function was enhanced,and the apoptosis was also enhanced.These results suggest that the apoptosis induced by Agaricus bisporus polysaccharide can stimulate the immune response of RAW264.7 cells,and further illustrate the mechanism of the immune regulation of Agaricus bisporus polysaccharide on RAW264.7 cells. |
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