Preparation Of Lycium Barbarum Polysaccharide And Primary Study On Its Activating Mechanisms Of Macrophages

Lycium barbarum is well-known Chinese traditional medicine and also a kind of food. Lycium barbarum polysaccharides (LBPs) are its important bioactive component. Lycium barbarum polysaccharides are known to play multiple roles in pharmacological and biological functions such as immunomodulatory effect, anti-tumor function, reduction of blood pressure and blood lipid.Macrophages are important autologous immunecytes, play immunomodulatory effect, anti-infection and anti-tumor function. Activated macrophages can direct kill pathogenic microbe, eliminate apoptotic cells and mutant cells. Moreover, macrophages can secret a serials of immunologic molecules such as tumor necrosis factor(TNF-a), interferon-1(IL-1),interferon-6 (IL-6), granulocyte-macrophage colony stimulating factor(GM-CSF), nitric oxide(NO), which play noticeable roles in both innate immune defense and adaptive immune response.The objective of the present study was to investigate the molecular mechanism responsible for the activation of macrophages by LBP. Concretely this study included two parts. In the first part, water soluble polysaccharide was separated and purified bymicrofiltration and different molecular weight cut-off ultrofiltration from lycium barbarum (Zhongning, Ningxia). The polysaccharide is separated into three components (>50kDa, 30 kDa-50 kDa, and 5kDa-30kDa) according to their molecular weight. Phenol-sulfuric method was used for the determination of the content of polysaccharide. Then we used the productions of nitric oxide, TNF- a and GM-CSF as activating markers of macrophage to investigate their activating effect on macrophages.The results showed that LBP can be separated to three parts(>50kDa,30 kDa-50 kDa, and 5 kDa-30kDa) by ultrafiltration, the quantity of three parts were 2.6g, 2.0g and 3.2g respectively, the content of polysaccharide were 74.3%, 69.8% and 78.4% respectively. Griess method showed that all the three parts can induce the NO productions of macrophages, and the two later also can induce the TNF- a and GM-CSF productions of macrophages.Conclusively, the study indicated that ultrafiltration is a kind of easy, rapid and effective method, which can be used to separate and purify LBP. All of the three parts can activate macrophage in vitro.In the second part, macrophages-like line RAW264.7 cells were cultured. The cells were then stimulated with various concentrations and incubation times of LBP to study dose-dependent and time-dependent manners. The effect of LBP on the production of NO released by RAW264.7 cells was evaluated by Griess method, which was also used to evaluate the effects of NF-kB inhibitor and anti-TLR4 antibody on NO production induced by LBP. The activity of inducible nitric oxide synthase (iNOS) was determined using iNOS assay kit. Western blot was performed to detect the protein content of NF-kB in the nuclear extract.The data we obtained show that in resting macrophages, the basal levels of iNOS activity and NO production are relatively low; nevertheless, iNOS activity and NO production can be significantly induced in response to LBP stimulation. LBP (200μg/ml) significantly induced the release of NO 6h after incubation, and the amount of NO increased with time. RAW264.7 cells were treated with various doses of LBP (10, 50, 200, 500μg/ml) for 18h, we found that LBP caused marked increase iniNOS activity and NO production in a dose-dependent manner. Monoclonal antibodies directed to TLR4 blocked LBP-mediated induction of NO production. In this paper, the western blot results demonstrated that NF-kB was involved in the course of the activation induced by LBP. The protein level of NF-kB in nucleus peaks at 8h after stimulating with LBP (200μg/ml). In addition, PDTC (pyrrolidinedithiocarbamate), an inhibitor of NF-kB, whose effective concentration for NF-kB inhibition without any toxicity on cells is up to 100μmol/L, can significantly block NO production induced by LBP. To test for a possible contamination of bacterial LPS in LBP, we examined the effect of polymyxin B (PMB) on LBP-induced production of NO. The results demonstrate that the activation of macrophages by LBP was not due to the contamination of LPS in LBP.Taken together, these results suggest that LBP-mediated induction of NO production and iNOS activity in macrophages is mediated, at least in part, by NF-kB signaling pathway. This is one of the important pathways by which LBPs activate macrophage in vitro. LBP possibly activate macrophages via Toll-like receptor 4.