Immunopotentiating activities and TLR4/MAPK related molecular mechanisms research of Lycium barbarum polysaccharide

摘要

OBJECTIVE To observe the effect and molecular mechanisms of Lycium barbarum polysaccharide (LBP) and glycopeptides on T, B lymphocytes and macrophages. METHODS 3H-TdR incorporation method was used to compare the effects of LBP and glycopeptides on the proliferation lymphocytes. Peritoneal macrophages induced by sodium thioglycolate were used to compare the effects of LBP and glycopeptides. T and B lymphocytes were purified by immunomagnetic beads method. Using antibody blocking methods screening polysaccharide activity related receptors.C3H/HeJ mice were further used to observe the activity of LBP. Biolayer interference method was used to observe the binding kinetics of LBP with TLR4 in vitro.TLR4 level was tested by flow cytometry.Western blotting was used to observe the phosphorylation of p-38,SAPK/JNK and ERK.RESULTS The monosaccharide compo-sition of LBP is rhamnose, arabinose and galactose, and does not contain amino acids. The mixed lymphocyte proliferation experiment showed that LBP had more obvious effect on the proliferation of B cells,and glycosides induced T cells proliferation was more obvious.On the purify lymphocytes,it was found that LBP-induced B cells proliferation requires the involvement of macrophages. Further research found that anti-TLR4 antibody had significant inhibitory effect on LBP-induced macrophage release of TNF-α and IL-1β but not the anti-CR3 treatment.C3H/HeJ mice related results further demonstrated that TLR4 is necessary for LBP activity. Although biolayer interference showed no obvious binding ofTLR4/MD2 with LBP, flow cytometry confirmed that LBP could increase TLR4 expression. Western Blotexperiments showed that the effect of LBP on macrophage was related to its activation of p-38/MAPKpathway and inhibition of ERK/MAPK and JNK/MAPK pathways. CONCLUSION TLR4 is the activityrelated receptors of LBP. LBP cannot directly bind to TLR4/MD2 complex in vitro, but can increaseTLR4 expression and activate macrophage p- 38/MAPK signaling pathway, inhibiting ERK- MAPK andJNK-MAPK signaling pathways.