Study On The Immune Pharmacological Effects And Intracellular Signal Transduction Mechanisms Of The Two Polysaccharides XG And MP-A Acting On Toll-like Receptor 4 (TLR4)

Objectives:Polysaccharides are a kind of important macromolecule substances, with many aspects of pharmacological activity, such as immune regulation, antioxidant,anti-tumor, hypolipidemic,antiviral, hypotensive, hypoglycemic, enhancing bone marrow hematopoietic function and so on. Polysaccharides have good pharmacological activity, and almost no toxicity , which attract more and more domestic and foreign biochemists and pharmacists to study.BBG and LNT-S, two ?-glucans which isolated respectively from S. cerevisiae and Lentinus edodes, have been reported to inhibit LPS-induced inflammatory cytokines NO, TNF-a, IL-1 and so on, and this inhibition is achieved by suppressing the phosphorylation of ERK1/2 and JNK1/2 through mitogen-activated protein kinase(MAPK) pathway. It is still unknown whether other a or ?-glucans besides LNT-S and BBG have the same bioactivity of inhibiting proinflammatory mediators through MAPK signaling pathways. In other words, does this structure-effectiveness relationship be generic? To solve these problems need to select more D-glucans to verify.Our laboratory has done a lot of work on the activity of these polysaccharides.The subject is based on the preliminary work of our laboratory, mainly studied on the immunoregulation effects and related molecular mechanisms of the two kinds polysaccharides of which D-glucans as the main chain. One is xanthan gum (XG)which derived from Xanthomonas camperstris bacterial extracellular heteropolysaccharide, the other is an a-D-glucan purified from the mussels (Mytilus coruscus) and named MP-A. Their main chain was both contained D-glucans. Our previous study indicated that injection of XG could inhibit the continuous lesions on the cartilage and delayed the progression of osteoarthritis (OA) and MP-A had lipid-lowering and anti- -inflammatory effect according to the animal experiments.Furthmore, they both have no significant side effects. Their good safety and pefect effectiveness indicates the excellent development prospects.The biological activity of polysaccharides is closely related to their primary structure, advanced structure and physicochemical properties. Although XG and MP-A are different in source, their main chains also contain D-glucan structures which are similar to LNT-S and BBG. XG is a heteropolysaccharide containing?-D-glucan backbone structure, And MP-A is the homoglycan containing a-D-glucan.Whether they have similar biological activity and molecular mechanisms as LNT-S and BBG is still unknown. Furthermore, there is a lack of relevant data on the activity of these two polysaccharides.However, the immunological pharmacological activity of these two polysaccharides is still lack of relevant research data.In this paper, the immunological pharmacological activities of these two polysaccharides were investigated. We attempts to find their extracellular target receptors and their possible molecular mechanism and investigate the in vitro immunomodulatory activity of XG through studying its effect on RAW264.7 macrophages, especially on LPS-induced macrophages. We also try to study the possible binding receptors of XG and intracellular signaling mechanism.Another aim is to investigate the effect of MP-A on the activation of THP-1 cells in vitro such as the effect of MP-A on the proliferation of human THP-1 macrophages, the phagocytosis the possible receptors, and its intracellular signaling mechanisms.Finally,the similarities and differences of the immunoregulatory activity of the two D-glucan polysaccharides XG and MP-A were summarized, and more experimental data were provided for the study of the structure-activity relationship of the active polysaccharides.Research ideas and methods:1 Study on the pharmacological effects of xanthan gum (XG) in vitroFirst, we used RAW264.7 cells as an in vitro experimental model and LPS-induced RAW264.7 cells as an inflammatory model. CCK-8 kit was used to detect XG proliferation or toxicity. The effect of XG on RAW264.7 cell activation was evaluated as follows: The effect of XG on the morphology of mouse RAW264.7 cells was observed by Scanning electron microscopy (SEM); the phagocytosis of macrophages were detected by FITC-dextran assay with Flow cytometry; the level of NO produced by RAW264.7 cells was determined with Griess regent method; the secretions of IL-6, IL-1? and TNF-a cytokines were assayed with ELISA kits; and the mRNA levels of iNOS, COX-2, IL-6, IL-1? and TNF-? were determined with fluorescence quantitative (FQ)-PCR technology. Furthermore, the expression of iNOS and COX-2 protein in RAW264.7 cells was also investigated by Western blot and the binding of XG to recombinant TLR4 was analyzed by SPR technique.2 Study on intracellular signal transduction mechanisms of XG through TLR4 in RAW264.7 cellsThe cytokine signaling mechanism of LPS-activated macrophages was studied by using mouse RAW264.7 cell line. The effects of XG on the ultrastructure of RAW264.7 cells were observed by transmission electron microscopy (TEM). Griess regent method was used to determine the level of NO produced in RAW264.7 cells with anti-TLR4 antibody pretreatment.The effect of XG on the expression ofmitogen-activated protein kinase (MAPKs) in RAW264.7 cells was detected by Western blot.And immunofluorescence were used to detect the effect of NF-?B on nuclear translocation. SPR technique was used to analyze the binding of XG to TLR4 and other commonly known receptors as dectin-1 and TLR2.3 Study on pharmacological effects and intracellular signal transduction mechanisms ofMP-A through TLR4 in THP-1 cellsWe used THP-1 as an in vitro experimental model and LPS-induced THP-1 macrophages as an inflammatory model. The CCK-8 kit was used to detect MP-A proliferation or toxicity. The main test methods included: FITC-dextran assay was used to analyze the effect of MP-A on phagocytosis of macrophages by flow cytometry; Griess reagent was used to determine the effect of MP-A on the level of NO produced;The effect of MP-A on secretion of TNF-a and PGE2 in THP-1 cells was detected by ELISA kit.The effects of expression of iNOS, COX-2 protein ,MAPKs and NF-?B pathway-related protein of MP-A in LPS-induced THP-1 cells were detected by Western blot.The effect of MP-A on the nuclear transport of NF-?B was detected by immunofluorescence. Finally, the binding of MP-A to recombinant TLR4, dectin-1 and TLR2 was analyzed by SPR technique.Results:The results show that they both have immunomodulatory activity, and can reduce the expression of inflammatory factors in the activation of macrophages. We investigated the affinity of two polysaccharides with the known receptor Dectin-1,TLR4 and TLR2 by using Surface PlasmonResonance (SPR) technology, and confirmed that TLR4 is the receptor of these two polysaccharides.1. In vitro experiments showed that both XG and MP-A had immunomodulatory activity, which could down-regulate the expression of inflammatory protein NO, TNF-a, interleukin (IL) or prostaglandin 2 (PGE2) induced by LPS in macrophages. XG and MP-A have enhanced FITC-labeled dextran internalization, showing a certain degree of immunogenicity. This Suggested that XG and MP-A may have two-way immunomodulatory effects ,indicating that inflammatory factors and phagocytosis may be caused by different mechanisms.2. It was confirmed that both XG and MP-A could be identified by extracellular TLR4 receptors. XG and MP-A could suppress the phosphorylation of mitogen-activated protein kinase (MAPK) and the activation of nuclear transcription factor(NF-?B) and nuclear translocation in activated macrophage cells to complete the intracellular signal transmission and play a pharmacological role.3. XG and MP-A could down-regulate activated macrophage inflammatory factors, and inhibit MAPK and NF-KB-related proteins in TLR4 pathway,showing strong anti-inflammatory activity The inhibition can provide a potential therapeutic regimen for the treatment of inflammatory diseases. We suppose that XG and MP-A can compete with LPS to bind TLR4 protein,which inhibits the phosphorylation of ERK and JNK protein in TLR4 downstream signal pathway. This is consistat the D-glucan structure may be a necessary group for its biological activity.Novelties and Conclusions:1. In this paper, we firstly investigated the immunoregulatory activity of XG and MP-A in vitro, and studied their effects on macrophage inflammatory factor expression, phagocytosis and intracellular expression.2. The affinity of XG and MP-A to common receptors as dectin-1, TLR4 and TLR2 were studied by SPR technique for the first time. TLR4 was first confirmed as the cell surface receptors of XG and MP-A in macrophage cells.3. Intracellular signaling pathway of XG and MP-A were firstly examined through TLR4 receptor. It was further confirmed that the two D-glucans were regulated by phosphorylationand of MAPKs and NF-?B activation and nuclear translocation to complete intracellular signaling and play anti-inflammatory effect, providing new insights for XG and MP-A for thetreatment of inflammatory diseases.