Regulation On Function Of Dendritic Cells By Selenium-Hericium Erinaceus Polysaccharide

Objective:The objective of this study is to compare the effect on the immune function of DCs by HEP and sHEP.To reveal the selenylation modification may enhance the immune activity of HEP and select the best sHEP and investigate the effects of polysaccharides on signaling pathway.Methods:1.Hericium erinaceus polysaccharide(HEP)as the research object which was extracted by water decoction and ethanol precipitation,purified through column chromatography of DEAE-52 and SephadexG-100,and selenizingly modified by HNO3-Na2SeO3 method according to orthogonal design.Finally,sHEP was obtained.2.The establishment of the dendritic cell model:Bone marrow was harvested from the tibia and femur of 6?8weeks' ICR mice.Bone marrow cells were cultured in complete medium with GM-CSF and IL-4 at 37 °C and 5%C02 incubator.Using microscope and flow cytometry instrument to identify the maturity of the cells.3.Detecting the maximum safe concentration of HEP and sHEP.In order to select three kinds of selenium polysaccharide,so we investigated the effects of HEP or sHEP on proliferation of T-cell stimulated by DCs in the unified security concentration.Comparison with the three selenium polysaccharide and HEP in the next phase of the test.To verify whether the selenylation modification could enhance the immune-enhancing activity of non-modified polysaccharide thus we compared HEP and sHEP to improve the ability of antigen-presenting and the expression levels of IFN-?,IL-12,MHC II and CD86 on DCs.We chose the best selenium polysaccharide from the three kinds of selenium polysaccharide for next experiment.4.The best selenium polysaccharide and HEP were used to stimulate the DCs at the same time,in order to analyze the associated protein levels of MAPK and NF-kB signaling pathway.Results:1.The extraction rate of crude polysaccharide was 7.01%,its carbohydrate content was 42.05%.After purification,its carbohydrate content was increased to 81.3%.HEP was selenizingly modified by HNO3-Na2SeO3 method to get nine sHEP.2.The dendritic cell model was successfully established:With the increase of incubation time,the typical morphological characters were observed in the cells.When culturing for 7 days,CD11c+ yieled 88.2%,CD86 yielded 87.2%and MHC ?yielded 46.1%.3.According to the changes of DCs precursors proliferation of each polysaccharide,we set safe concentration is 12.5 p.g/mL.Examined HEP and sHEP on the effect of DCs on stimulating T lymphocyte proliferation in the unified security concentration,we found sHEPi,sHEP2 and sHEP8 had better effect,then chose them.Comparison with the three selenium polysaccharide and polysaccharide in the next phase of the test.(1)sHEPi,SHEP2,sHEP8 and HEP could decreased the uptake dextran and enhance antigen presenting ability of DCs-The effects of SHEP2 was superior to other groups.(2)Compared with DCs treated with sHEP(sHEPi,SHEP2,sHEP8)and DCs treated with HEP on production of cytokines,we found HEP2 and sHEP8 increased the production of IFN-y better than HEP and sHEPi.sHEP raised the production of IL-12 better than HEP.(3)Compared with DCs treated with sHEP(sHEP1,SHEP2,sHEP8)and DCs treated with HEP on the expression levels of CD86 and MHC ? molecule.These results demonstrated that HEP and sHEP(sHEP1,sHEP2,sHEP8)both could increase the expression level of CD86 significantly.In the expression level of MHC ?,sHEP2 possessed the best efficacy.These results indicated that selenylation modification could significantly enhance the immune-enhancing activity of HEP,induce the maturation of the phenotype and functions of DCs.In the three selenium polysaccharide,sHEP2 possessed the best activity.4.Investigated the effect of DCs treated with sHEP2 and DCs treated with HEP on the MAPK and NF-kB signaling pathway,we found that HEP and sHEP2 both could activate the MAPK and NF-kB signaling pathways.Especially,sHEP2 increased the phosphorylation of p38,ERK and JNK,promoted IkB?/? degradation in the cytoplasm and increased nuclear translocation of NF-KB subunits p50 and p65 better than HEP.Conclusion:selenylation modification could significantly enhance the immune-enhancing activity of HEP and strengthen the immune ability of DCs.HEP and sHEP2 induced the maturation of the phenotype and functions of DCs might through activate the MAPK and NF-kB signaling pathways.