Immunomodulatory Activity Of A ?-glucan From Amillariella Mellea On RAW264.7

Fungi,a medicinal and functional resource,contain a variety of active ingredients.Polysaccharides are one of the major active ingredients.Amillariella Mellea genus Basidiomycetes,Agaricales,Armillaria,is a traditional medicinal and edible fungus.Macrophages play a unique role in the immune system,not only related to the innate immune response,but also have the function of antigen processing and presentation.Therefore,macrophages become target cells for many anticancer drugs and immunomodulatory drugs.A homogeneous polysaccharide fraction AAMP-A70 was isolated from the fruit body of Armillaria mellea by alkali extraction,ion exchange column chromatography and fractional alcohol precipitation.The molecular weight was 5.6 KDa using TSK detection analysis.The results of monosaccharide composition analysis showed that AAMP-A70 consists of Glc(81.0%)and Gal(14.9%)and also contains trace amounts of Man.Further infrared spectroscopy analysis and methylation analysis showed that the ?-D-Glc pyranose ring was present in AAMP-A70.This result was verified in the subsequent NMR analysis.We concluded that the structure of AAMP-A70 is dominated by ?-D-(1?6)-glucan,and O-3 in Glc? is replaced with a single ?-Glc? or very small ?-D-(1?6)-Gal? side chain structure.Further study on the immunoregulatory effect of AAMP-A70 on mouse macrophage RAW264.7 and its mechanism revealed that AAMP-A70 did not affect RAW264.7 cell proliferation,but produced NO,ROS,and secretion after AAMP-A70 treatment.The inflammatory cytokines TNF-?,IL-6 and IL-1?,macrophage phagocytosis capacity were significantly increased.To investigate its mechanism of activating macrophages,we investigated the effect of AAMP-A70 on the NF?B and MAPK classical signaling pathways.The results showed that AAMP-A70 can degrade after activation of I?B?,and NF?B phosphorylates to the nucleus,which promotes the transcription and expression of cytokines.In order to further verify this conclusion,we used the inhibitor of NF?B to pretreat cells to block the signaling pathway and found that macrophage activity was significantly inhibited.At the same time,AAMP-A70 phosphorylates p38,ERK,and JNK in Raw264.7 cells.When the cells were pretreated with the corresponding inhibitors(SB203580,U0126,and SP600125),the phosphorylation of p38,ERK,and JNK was significantly inhibited,and the macrophage activity was also significantly inhibited.These results indicate that AAMP-A70 activates macrophages through NF?B and MAPK signaling pathways.Polysaccharides cannot cross the cell membrane as biological macromolecules.To determine the membrane receptors of AAMP-A70,we used specific blocking antibodies to block the pattern recognition receptor Dectin1,TLR-2,and TLR-4 receptors associated with polysaccharides,and then stimulated RAW264.7 cells with AAMP-A70..The results showed that the activity of RAW264.7 macrophages was significantly reduced after the TLR-2 receptor was blocked.The above results indicate that the TLR-2 receptor may be a pattern recognition receptor for AAMP-A70.In summary,we extracted and purified the polysaccharide fraction AAMP-A70 with a molecular weight of 5.6 KDa from the fruit body of Amillariella mellea.?-D-(1?6)-linked.Glc? is the main chain and has a side chain structure consisting of a single ?-Glc? residue or a small amount of ?-D-(1?6)-linked Gal? at the O-3 position of Glc?.AAMP-A70 activated the NF?B and MAPK signaling pathways through TLR-2 receptor.AAMP-A70 has the potential as a supplement to enhance immunity.