Effects Of 3-O-Me-Galactan From Cantharelles Cibarius On The Activation Of Macrophage

Cantharelles cibarius is a commonly edible fungus,which is not only nutritious,but also has therapeutic effects.Polysaccharides,as the main component in chanterelles,has good biological activity,such as anti-oxidation,anti-tumor,promoting the proliferation of spleen cells and immunomodulation.At present,there are many studies on its activity,but few studies on its active mechanism.In this paper,the mechanism of activation of macrophages by polysaccharide from C.cibarius was studied.The relationship between molecular weight and immunoregulatory mechanism in macrophages,and immunosuppressive effect of macrophage on the growth of colon cacncer cells were discussed.The specific contents are as follows:The total polysaccharides of C.cibarius are prepared by water extraction and ethanol precipitation.The activity study showed that WCCP could activate macrophages to release NO,TNF-? and IL-6.In order to study the active components of WCCP that can activate macrophages,ion exchange chromatorgraphy and gel chromatography were used to obtain a neutral averageWCCP-N-b and a acid average WCCP-A-b,the molecular weights was 18 kDa and 10 kDa,respectively.Analysis of its monosaccharide composition,WCCP-N-b consists mainly of Gal and Me-Gal,WCCP-A-b is mainly composed of Glc and Man and GlcA.The preliminary results of the laboratory showed that the structure of WCCP-N-b was linear 3-O-Me-Galactan,and WCCP-A-b was composed of ?-1,6-D-Glc as backbone with ?-1,4-D-Man side chains.The activity of WCCP-N-b and WCCP-A-b activated macrophages were analyzed.The results showed that both of these fractions could activate macrophages,but WCCP-N-b had a better effect on the activation of macrophages.Therefore,we choose to conduct an in-depth study of the WCCP-N-b fraction.WCCP-N-b can promote macrophage phahocytosis and product NO and cytokines.The mechanism of WCCP-N-b activation of macrophages was studied by WB,ELISA,immunofluorescence and knockdown.The results showed that WCCP-N-b activates MAPK and NF-?B signaling pathway in macrophages via binding to the TLR2 receptor on the surface of macrophages.The activation of signaling pathway allows NF-?B to enter the nucleus,thereby activating macrophages and promoting the expression of NO,TNF-? and IL-6.WCCP-N-b not only activates the RAW264.7 mouse macrophage cell line,but also activates primary peritoneal macrophages isolated from the abdominal cavity of Balb/c mice,primary bone marrow isolated from the bone marrow of Balb/c mice,and THP-1 human macrophage cell lines,this result proves that WCCP-N-b can activate a variety of macrophages.In order to further study the relationship between molecular weight and the activation of macrophages,four molecular weight fractions of WCCP-N-b prepared by partial acid hydrolysis,named WCCP-N-b1~4(16.1 kDa,11.2 kDa,5 kDa,3.5 kDa),respectively.The results of monosaccharide composition revealed that the composition of WCCP-N-b1~4 monosaccharide is basically the same and is the same as the parent fraction WCCP-N-b,which indicated that partial acid hydrolysis only changed the molecular weight and did not change the composition of these polysaccharides.Immunological activity analyses showed that the macrophage activation effects of these degraded polysaccharide fragments were significantly weaker than that of WCCP-N-b.The lower the molecular weight,the weaker the macrophage activation effect.Furthermore,we noted that when the molecular weight was decreased to 3.5 kDa,the polysaccharide fragment could not induce the secretion of NO,TNF-? and IL-6.Therefore,we speculated that molecular weight of WCCP-N-b plays a crucial role inactivation of macrophages.In order to study the relationship between the activated macrophages of WCCPN-b and the inhibition of the growth of colon cancer cells,MTT,flow cytometry and WB were used to analyz the relationship.The results showed that the supernatant of WCCP-N-b activated RAW264.7 cells could inhibit the proliferation of HCT116 and HT29 cells,and promote the caspase-dependent apoptosis of HCT116 and HT29 cells.In addition,the supernatant of WCCP-N-b activated BMDMs and peritoneal macrophage can inhibit the proliferation of the HCT116 cells.When MAPK,NF-?B signaling pathway and TLR2 receptors were suppressed,their inhibitory effect on the proliferation of HCT116 cells was weakened.These results suggest that WCCP-N-b can indirectly inhibit the growth of colon cancer cells by activating macrophages and releasing immunoactive substances.In summary,the linear 3-O-Me-Galactan(WCCP-N-b)obtained from the C.cibarius can activated MAPK and NF-?B signaling pathway of macrophage via TLR2 receptor.Molecular weight is the key to affect its activity.In addition,WCCP-N-b can indirectly inhibit the growth of colon cancer cells by activating macrophages.These studies enrich the data of polysaccharide immunomodulation and antitumor research,and provide methodological reference and theoretical basis for the study of the relationship between immunomodulatory activity and molecular weight of polysaccharides.It also provides theoretical support for the development of C.cibarius as functional food.