Structural Characterization,immunomodulatory Activities And Underlying Mechanism Of Hericium Erinaceus Polysaccharide

Hericium erinaceus(H.erinaceus)also known as Lion's Mane Mushroom,Houtou,Monkey's Mushroom,Bear's Head and Yamabushitake(in Japanese),belongs to the class Basidiomycetes,subclass Holobasidiomycetidae,order Hericiales and family Hericiaceae.H.erinaceus contains various bioactive compounds,such as hericenones,erinacines,glycoprotein,polysaccharides and alkaloids,etc.Among these compounds,polysaccharides are considered to be the major active ingredient in H.erinaceus,which possess various pharmacological activities such as antioxidant property,anti-chronic atrophic gastritis activity,etc.To date,although more and more polysaccharides have been isolated and purified from H.erinaceus,little information is available about their immunomodulatory activities in vivo and in vitro and the underlying mechanism.In the present study,a systemactic study was carried out focused on the purification,structural characterization,chain conformation,immunomodulatory effects in vitro and in vivo for the polysaccharide from the fruiting bodies of H.erinaceus.Meanwhile,the immune mechanism of HEP-W was also studied and elucidated using murine macrophage RAW 264.7 cells.The main research results are listed as follows:(1)Crude polysaccharide was obtained at a yield of(2.56±0.14)%(w/w)by water extraction,alcohol precipitation and Sevage deproteinization.The fraction,HEP-W was obtained after purification by a DEAE-Sepharose fast flow column chromatography and a Sephadex G-100 column chromatography.Structural characterization revealed that HEP-W had an average molecular weight of 1.59×10~4 Da and was composed of rhamnose,fucose,mannose,glucose and galactose at a molar ratio of 0.98:1.59:0.89:5.60:7.06.The main glycosidic linkage types of HEP-W consisted of(1?)?-D-Glc,(1?3,6)-?-D-Glc,(1?2,6)-?-D-Gal,T-?-Gal,(1?3,4)-?-D-Man,(1?3)-?-Rha,and(1?2)-?-L-Fuc by FT-IR,periodate oxidation-Smith degradation and NMR analysis.Thermogravimetry-differential scanning calorimetry(TG-DSC)was applied to analyze the stability of HEP-W.The results showed that HEP-W had good thermal stability with the glass transition temperature at 77.2? and thermally decomposed temperature at 320.9?,respectively.(2)Based on the atomic force microscopy(AFM)images,HEP-W was found to appear as flexible chain conformation in the solution,the height and diameter of the single chain ranged from 0.12 to 1.5 nm and 250 to 615 nm,respectively.X-ray diffraction and Congo-red analysis showed that HEP-W was amorphous structure and non-three-helix conformation,respectively.The viscosity of HEP-W solution was typical pseudoplastic fluid,and the viscosities increased with the concentration increasing.(3)The CE-TAL test excluded endotoxin contamination for the immune activation of HEP-W.HEP-W could significantly improve the cell immune function through the proliferation of T and B lymphocytes in normal mice.Meanwhile,compared with the negative control group,HEP-W could significantly promote the secretion of IL-2,IL-4 and IFN-?(p<0.05)in a dose-dependent manner.(4)Murine macrophage RAW 264.7 cells were used as the cell model to evaluate the immunity of HEP-W in vitro.The bioactivity tests showed that HEP-W(at 100~1000?g/mL)could significantly promote the pinocytic and phagocytic capacity and increase the NO,IL-6and TNF-?secretion by activating corresponding mRNA expression in macrophages(p<0.05).(5)Toll-like receptor 2(TLR 2)and mannose receptor(MR)were found to act as synergistic PRRs to co-regulate the immunomodulatory response to HEP-W in RAW 264.7cells.Meanwhile,MyD88/IRAK-1/TRAF-6,PI3K/AKT and MAPKs signaling pathway were all involved in the process of HEP-W-mediated activation in macrophages.(6)Animal experiments indicated that HEP-W had significantly improvement effects on the cyclophosphamide(Cy)-induced immunosuppression in mice:HEP-W could significantly increase the counts of peripheral white blood cells and bone marrow nucleated cells of the Cy-induced mice(p<0.05).Furthermore,HEP-W could increase T and B splenic lymphocyte proliferation induced by ConA and LPS,as well as the splenic nature killer cell activity and the level of interleukin-2 in serum(p<0.05).Finally,HEP-W could also significantly increase the activities of SOD and GSH,and decrease the MDA level in Cy-treated mice.