Comparison Of Structural Characteristics And Immunoregulatory Activities Of Four Natural Polysaccharides

Polysaccharides are composed of more than ten monosaccharides and exist in animals,plants,microorgnisams and algas,which have various bioactivities,such as antitumor,antiviral,antioxidant and immunostimulatory activities.They can activate immune cells including macrophages,T cells,B cells,NK cells and dendritic cells(DC),and promote the production of cytokines,chemokines,and complements to improve the immunity.DC are professional antigen-presenting cells that capture antigens and present them to na?ve T cells to induce antigen-specific immune responses.In our previous study,Pleurotus ferulae polysaccharides(PFPS)and Glycyrrhiza uralensis polysaccharides(GUPS)were purified and these two polysaccharides promoted the maturation of murine bone marrow-derived DC and,as adjuvant,improved the antitumor efficacy of DC-based vaccine.To further explore the relationship of structure and activity of polysaccharides,the crude polysaccharides of wheat(Triticum aestivum l.)and rice(Oryza sativa l.)were prepared by water extraction and ethanol precipitation,and their polysaccharides with higher polysaccharide content and immunostimulatory activity were purified by column chromatography of DEAE-52 Cellulose.The structural characteristics of polysaccharides were analyzed by scanning electron microscope,Fourier transform infrared(FT-IR)and nuclear magnetic resonance(NMR)spectroscopy,and compared with GUPS and PFPS.The immunostimulatory activities of these four polysaccharides were further compared.Murine bone marrow-derived DC were treated with these four polysaccharides.Flow cytometry and ELISA were used to detect the expression of surface molecules and the production of cytokines of DC,respectively.Mixed lymphocyte reaction(MLR)was used to measure the ability of DCs to stimulate the proliferation of allogeneic T cells.Inhibitor pretreatment and Western blot were used to investigate the mechanism of DC maturation.The in vivo immunostimulatory activity of the four polysaccharides was evaluated in mouse model.(1)Purification of wheat and rice polysaccharides.The crude polysaccharides of wheat and rice were obtained by water extraction and ethanol precipitation,and their polysaccharide contents were 35%(35%±0.009)and 25%(25%±0.011),respectively,then purified by column chromatography of DEAE-52 Cellulose eluted with gradient Na Cl solutions.Polysaccharide components eluted with 0.2 M Na Cl contained 81%(81%±0.048)and 79%(79%±0.037)of polysaccharide contents,respectively,and showed stronger activity on DC maturation.They were named as TAPS and OSPS.(2)Comparison of the structural characteristics of GUPS,PFPS,TAPS and OSPS.PFPS with 1600 k Da molecular weight was mainly composed of glucose.GUPS with 29 k Da molecular weight mainly consisted of glucose and arabinose.TAPS with 98 k Da molecular weight was mainly made up of glucose and glucuronic acid.OSPS with 309.9 k Da molecular weight mainly contained arabinose and xylose.The FT-IR spectra showed that the main glycoside bonds and functional groups of the four polysaccharides were similar except that OSPS contained N-H signal.The NMR spectra showed that all the four polysaccharides contained the structure ofα-andβ-glycosides.GUPS and PFPS containedα-L-Ara but TAPS and OSPS did not.(3)Comparison of the effects of GUPS,PFPS,TAPS and OSPS on DC maturation and function and TLR4 signaling pathway.Flow cytometry results showed that GUPS and PFPS induced higher expression levels of surface molecules(CD40,CD80,MHC-II)and cytokines(IL-12p40 and TNF-α)of DCs and reduced the capacity of antigen uptake for FITC-dextran compared with TAPS and OSPS.In MLR,GUPS and PFPS significantly enhanced DC capacity to stimulate the proliferation of CD8~+T cells,while TAPS and OSPS had no that effect.After pretreatment with TLR4inhibitor TAK-242,all the four polysaccharides promoted DC maturation through TLR4.Western blot showed that all the four polysaccharides increased the phosphorylation of p38,ERK and IKKα/β.The phosphorylation level of gups and PFPS is the strongest.(4)Comparison of the effects of GUPS,PFPS,TAPS and OSPS on the immunity of mice.The four polysaccharides did not affect the body weight of mice,in which GUPS and PFPS significantly increased the spleen and thymus indexes.The four polysaccharides significantly up-regulated the levels of IL-12p40 in serum but did not change the levels of TNF-α.The percentages of T cells and B cells in spleen were significantly increased by PFPS,GUPS and TAPS,and the activation of CD8~+and CD4~+T cells were enhanced by PFPS and GUPS.Taken together,the molecule weight,monosaccharide composition and glycoside are different in the four polysaccharides.The stimulative activities of GUPS and PFPS were higher than those of TAPS and OSPS.GUPS and PFPS could promote DC function.All of the four polysaccharides could promote DC maturation through TLR4signaling pathway,but the downstream signaling pathway activated was different.The immune responses of GUPS and PFPS in vivo were higher than those of TAPS and OSPS.The immunoenhancement activities of the four polysaccharides were PFPS=GUPS>TAPS>OSPS,indicating that PFPS and GUPS could be potential Immunoenhancers or adjuvants.