| Tumor necrosis factor-a (TNF-a) is a pleiotropic cytokine, which play an important role in many physiological and pathological processes of inflammation, cellular immunity, tumor immunity, etc. TNF-a promotes a derangement in the immune regulation and directly involves in the pathogenesis of inflammatory autoimmune diseases, such as rheumatoid arthritis (RA) and so on. TNF-a exerts its biological functions by interaction with two members of the TNF receptor (TNFR), TNFR1 and TNFR2. TNF-a binding to various TNF receptors lead to different signaling pathways, and thus play a different role in physiological and pathological effects. TNF-a recruits downstream signaling molecules through bind to its receptor, and final activation of NF-κB signaling pathway, which induces immune and inflammatory response. Dendritic cells (DCs) were a population of highly specialized antigen-presenting cells (APCs) recognized for their crucial role as regulators of innate and adaptive immunity. DCs have a dual role:immunogenicity and tolerability. The immature dendritic cells (iDCs) show the characteristics of immune tolerance, the mature dendritic cells (mDCs) promote immune function. The mature dendritic cells express highly CD80, CD86, CD40, MHC II and CD83, and secret proinflammatory cytokines such as IL-12, IL-6 and IL-1β. Activation of mDCs can through the release of TNF-a, MMPs, IL-6 and soluble IL-6 receptor-induced cartilage collagen degradation of RA. TNF-a can promote maturation of DCs by reducing the ability to uptake antigens, and enhancing antigen-presenting capacity of mDCs. However, does TNF-a regulate the function of bone marrow-derived DCs in mice through TRADD-TRAF2-NF-kB signaling pathway mediated by TNFR1? It has been unclear.Paeoniflorin-6’-O-benzene (namely CP-25) is a new active monomers drug of Pae monomer derivative. The studies in our lab showed that CP-25 relieved significantly inflammation progress in mice with collagen induced arthritis (CIA) in vivo, which related to regulating the functions of T and B lymphocytes. However, whether CP-25 regulates the phenotype and function of bone marrow-derived DCs stimulated by TNF-alpha in mice, and whether CP-25 regulates DCs function through TNF-alpha-TNFR1-TRADD-TRAF2-NF-KB signaling pathway? It has been unclear.Aim:To investigate the effect of TNF-a on the phenotype and function of bone marrow-derived dendritic cells in mice and its mechanisms. To investigate the regulatory effect of CP-25 on the phenotype and function of bone marrow-derived dendritic cell in mice under the stimulation of TNF-a and its mechanism, which reveal the mechanism of TNF-a involved in the regulation of bone marrow-derived DCs in mice function through receptors and the effect of CP-25, and provide an improtant experimental evidence.Methods:Bone marrow-derived DCs in mice were research subjects. The expressions of CD83 on bone marrow-derived DCs in mice stimulated by TNF-a under the different concentrations and different time were analyzed, and then select suboptimal conditions as follow experimental conditions. The changes of the expression of CD80, CD86, CD40, MHC-Ⅱ and CD83 and the ability of antigen uptake on bone marrow-derived DCs in mice stimulated by TNF-α were observed. The proliferation of T cells co-cultured with bone marrow-derived DCs in mice stimulated by TNF-α was observed. The changes of the expression of TNFR1, TNFR2, TRADD, TRAF2, NF-κB p65 in bone marrow-derived DCs in mice stimulated by TNF-α, and the effects of CP-25 on the above changes were also observed at the same time. The expressions of CD80, CD86, CD40, MHC-Ⅱ and CD83 on bone marrow-derived DCs in mice and the ability of antigen uptake were analyzed by flow cytometry. T cells proliferation was determined by the 3-(4,5-2 dimethylthiazal-2yl) 2,5-diphenyltetrazoliumbromide (MTT) assay. The expression of TNFRland TNFR2 in bone marrow-derived DCs in mice was analyzed by flow cytometry, which use the mean fluorescence intensity as the level of expression. The expressions of TNFR1, TRADD, TRAF2 and NF-κB p65 in bone marrow-derived DCs in mice were determined by Western blot.Results:1. TNF-α promotes the maturation of DCs, characterized by up-regulation the expression of CD80, CD86, CD40, MHC-II and CD83, inhibition of antigen uptake ability, promotion the proliferation of T cellsBone marrow-derived DCs in mice were cultured with TNF-α (20ng/mL) for 24h. The flow cytometry results show that TNF-α was enhanced significantly the expression of CD80, CD86, CD40, MHC-II and CD83 on bone marrow-derived DCs in mice, and inhibition antigen uptake capacity of bone marrow-derived DCs in mice. The MTT results show that TNF-α-treated bone marrow-derived DCs in mice can promote T cell proliferation.2. TNF-α up-regulated the expression of mouse bone marrow-derived DCs phenotype and enhance DCs function, which may related to regulateTNFR1-TRADD-TRAF2-NF-κB signaling pathwayBone marrow-derived DCs in mice were cultured with TNF-a (20ng/mL) for 24h. The flow cytometry results show that TNF-a was increased the expression of TNFR1, but there was no significant effect on the expression of TNFR2. The western blot results show that TNF-a could up-regulate the expression of TNFR1, TRADD, TRAF2 and NF-κB p65.3. CP-25 could down-regulate the expression of CD80, CD86, CD40, MHC-II and CD83 on bone marrow-derived DCs, increased the ability of antigen uptake of DCs, inhibition the proliferation of T cells stimulated by TNF-a.After bone marrow-derived DCs in mice were cultured with TNF-a (20ng/mL) for 24h, then treatment of CP-25 (10-9,10-8,10-7,10-6,10-5mol/L). The flow cytometry results show that CP-25 (10-7,10-6,10-5mol/L) could significantly decrease the CD80, CD86, CD40, MHC-II and CD83 expression on TNF-a-induced bone marrow-derived DCs in mice, suppressed the proliferation of T cell, and increased the ability of antigen uptake.4. CP-25 recovered the abnormal function of bone marrow-derived DCs in mice may by regulating the TNFR1-TRADD-TRAF2-NF-κB signaling pathwayAfter bone marrow-derived DCs in mice were cultured with TNF-a (20ng/mL) for 24h, then treatment of CP-25 (10-9,10-8,10-7,10-6,10-5mol/L). The flow cytometry results show that CP-25 (10-7,10-6,10-5mol/L) could significantly down-regulate the expression of TNFR1, but there was no significant effect on the expression of TNFR2. The western blot results show that CP-25 (10-7,10-6,10-5mol/L) could decrease the expression of TNFR1, TRADD, TRAF2 and NF-κB p65.Conclusion:1. TNF-a could up-regulate the function of bone marrow-derived DCs in mice by promoting expression of costimulatory molecules, inhibiting antigen uptake;2. TNF-a up-regulates the function of bone marrow-derived DCs in mice, which may be by activing TNFR1-TRADD-TRAF2-NF-κB signaling pathway;3. CP-25 could inhibite the maturation of bone marrow-derived DCs stimulated by TNF-a in mice with down-regulated the expression of costimulatory molecules, recovered the ability of antigen uptake;4. CP-25 could inhibit the function of bone marrow-derived DCs stimulated by TNF-ain mice, which may be relative to regulating TRADD-TRAF2-NF-κB signaling pathway mediated by TNF-α/TNFRl. |
PDF Full Text Request