Immunomodulation Activity And Mechanism Of Polysaccharide From Plantago Asiatic L.Seeds

Polysaccharide is the most abundant biological polymer in the nature,mading up of monosaccharide,polysaccharide was also one of the basic materials to maintain the normal lives of the body with the biological function of energy storaging,structural supporting,defensing and antigen qualiting.The the alkali-extractable polysaccharides from Plantago named PLP were isolated and purified by ethanol precipitation,dialysis,anion-exchange and gel filtration chromatography.To further elucidate the role of PLP as a biological response modifier,the immunomoduating activities and mechanism of PLP was explored in current study.The results showed that PLP selectively stimulated B cells to proliferate in a dose-dependent manner.By contrast,significant proliferation can not be detected in PLP-treated T cells at all concentrations tested,and PLP stimulated macrophage cells RAW264.7 to phagocytosis and the discharge of NO.We next focused our study on the binding characteristics of PLP to macrophage cells,especially the involvement of TLR signaling in PLP-mediated macrophage cell responses.The binding assay indicated that PLP was able to interact with macrophage cells directly.The immunomodulating activities of PLP were then nicely correlated with TLR2 and TLR4,since defunctionalization of TLR2 and TLR4 by antibody blocking using anti-TLR2 and anti-TLR4 impaired the macrophage-cell proliferative to PLP.Taken together,the findings demonstrated that TLR2 and TLR4,were responsible for the immunostimulating property of PLP in macrophage cells.We further performed affinity-precipitation assay to confirm the interactions of PLP with TLR2 or TLR4.To start with,PLP was conjugated to EAHSepharose 4B,a kind of agarose beads containing active amino groups,to prepare PLP-coupled affinity beads.The PLP-Sepharose 4B beads were then utilized to precipitate PLP-bound ingredients from macrophage cell membrane extracts,in which both TLR2 and TLR4 were detected by Western blot analysis.These findings,together with those obtained from antibody blocking assay and TLR-knockout mice,provided convincing evidence that TLR2 and TLR4 were the functional receptors for PLP.To evaluate the consequential downstream events upon PLP binding to TLR2 and TLR4 in macrophage cells,the responses of MAPK signaling,which have been well-characterized as the downstream signal transducers for TLR2 and TLR4,was investigated.We found that PLP increased the phosphorylation of three independent MAPK members,including p38 MAPK,ERK and JNK,in a dose-dependent manner without affecting the total expression of p38,ERK or JNK significantly.Furthermore,specific inhibitors of p38,ERK and JNK could attenuate the ability of PLP to induce macrophage cell proliferation in varying degrees.According to their inhibition ratios,ERK has the strongest ability in the induction of macrophage cells prolifieration.