Effect Of Different Molecular Weights Of Lycium Barbarum Polysaccharides On The Maturation Of Dendritic Cells

Objective:Lycium barbarum polysaccharides(LBP),the important component of Lycium barbarum L.,have various biological activities,such as immunomodulation,anti-oxidation,anti-tumor,anti-diabetes and hepatoprotection.Dendritic cells(DCs)are most potent antigen presenting cells(APCs)and link the innate and adaptive immune responses.It has been reported that polysaccharides from Lycium barbarum L.could induced the maturation of DCs.But the different molecular weights of LBP remain to be further researched for their effects on the maturation of DCs.In this study,water extraction and alcohol precipitation was used to extract LBP and four different sizes of membrane permeability were used to obtain five different molecular weight components,such as LBP1,LBP2,LBP3,LBP4 and LBP5.Granulocyte-macrophage colony stimulating factor(GM-CSF)and intreleukin-4(IL-4)were used to culture the immature dendritic cells(iDCs).LBP2,LBP3,LBP4 and LBP5,dissolved easily in water,were incubated with iDCs for 24 hours and the nitrix oxide(NO),IL-6,IL-10,monocyte chemotactic protein-1(MCP-1),interferon-gamma(IFN-y)and tumor necrosis factor(TNF)were detected in the supernatant.Preliminary screening of iDCs has good activity of polysaccharide component.Then the active components of LBP were screened and continued to go on further expriments in vivo and in vitro.The active components of LBP were incubated with iDCs for 24h.The cell morphology,phagocytosis,cytokine production and the effect of stimulating the same allogenic lymphocyte proliferation were observed to confirm the effect of inducing the maturation of DCs.The expression of nuclear transcription factor p65(NF-?B p65)were detected to explore the molecular mechanism of the active components to induce the maturation of DCs.Methods:1.The mouse bone marrow cells were cultured by RPMI-1640 medium supplemented with 10%FBS and with GM-CSF and IL-4.Cells were cultured at 37? in a incubator for 8 days,loosely-adherent and non-adherent cells were harvested.The ratio of CD11c+DCs was detected.2.LBP and iDCs were incubated for 24h,the nitric oxide(NO)was detected by Griess reagent.3.The cytokines of IL-6,IL-10,MCP-1,IFN-? and TNF were detected by cytometric bead assay(CBA).4.The DCs were observed under a microscope.5.The expression of CD11c,MHC-?,CD80 and CD86 was detected by flow cytometry.6.MTT assay was used to detect the effect of LBP3 treated DCs on the proliferation of allogenic splenic lymphocytes.7.The effect of LBP on the phagocytosis of DCs was detected by flow cytometry.8.The effect of LBP on the expression of NF-?B p65 in DCs was detecte by Western blot.9.The effect of LBP on the maturation of DCs in the immature mice spleen and mesenteric lymph nodes was detected by flow cytometry.Results:1.The results showed that the bone marrow cells were cultured with 10ng/mL GM-CSF and lng/mL IL-4 for 8 days.The ratio of CDllc+DCs was 82.1%.2.The results showed that LBP3,LBP4,and LBP5 promoted the production of NO in a dose-depengdent manner.At the same dose,LBP3 was more effective on stimulating DCs.3.The results showed that LBP3 and LBP5 promoted the production of IL-6,IL-10,MCP-1,IFN-y and TNF while LBP4 promoted the production of IL-6,MCP-1 and TNF.LBP3 was more effective on stimulating DCs than LBP4 and LBP5.4.After the stimulation of LBP3,DCs had more wrinkles and branches on the cell surface.5.The results showed that LBP3 increased the ratio of CD 11 c+MHC-?+,CDllc+CD80+,CD11c+CD86+ DCs and the mean fluorescence intensity of CD11c,MHC-II,CD80 and CD86.6.After the stimulation of LBP3,DCs induced the proliferation of allogeneic splenic lymphocytes.7.Both phagocytic rate and phagocytic index of DCs in LBP3 groups were lower than those in the control group.8.LBP3 increased the expression of NF-?B p65 in DCs' nucleus.9.The results in vivo showed that LBP3 increased the ratio of CD11c+MHC-?+ and CD11c+CD86+ DCs in mouse spleen.Conclusion:Different molecular weights of LBP were different in inducing the maturation of DCs.The results of NO and cytokines showed that LBP3,LBP4 and LBP5 had the effect on the maturation of DCs while LBP3 was the most effective component.The results in vitro showed that LBP3 induced the maturation of DCs though NF-?B signaling pathway.The results in vivo showed that LBP3 induced the maturation of mouse splenic DCs.The results revealed that LBP3might had anti-tumor effect.