| The effects of T cell immune abnormalities play an important role in many autoimmune diseases, including the dowm-regulation of T cells subsets and abnormal activation of T cells. The abnormal activation of T cells have a variety of abnormal behavior, such as the imbalance of cytokine secretion and continue to increase their reactive T cells, increases T cell apoptosis, etc. The study to research of the abnormal activation of T cells can help us to explore new pathogenesis of autoimmune diseases and find new drugs.Immunoglobulin D (IgD) including membrane IgD (mIgD) and secreted IgD (sIgD), which coexpressed on T and B lymphocyte and innate immune cells, and plays an important role in autoimmune disease development. In addition, the IgD receptor (IgD-R) expressed in T cells can be combined with sIgD or mIgD, activate the downstream signal transduction. Research shows that through the inhibition of protein tyrosine kinase pathway can block IgD the IgD-R by IgD induced, which involved including P56Lck, Fyn, ZAP-70 and PLC-y, etc. According to the results of early reserches showed us that the elevateion of abnormal sIgD in rheumatoid arthritis(RA), and IgD can significantly promote T cells activation and inflammatory cytokine secretion. Therefore, excessive expression of IgD and IgD-R in the activation of T cells may be the key links of autoimmune disease, explore IgD-R targeted drugs in the activation of T cells can provide reasonable treatment of autoimmune diseases and new ideas about the new drug research and development.Paeoniflorin-6’-O-benzene sulfonate (CP-25) was from the chemistry constructs modifications of Paeoniflorin (Pae). CP-25 has higher lipid solubility and bioavailability than Pae. Our previous studies have shown that the CP-25 has a therapeutic effect in collagen induced arthritis (CIA), its therapeutic effect related to the regulation of T and B lymphocytes. Thus we assume that the whether CP-25 can suppress the abnormal activation of T cells induced by IgD? and whether inhibit the abnormal activation of T cells is related to the IgD-R and downstream signaling pathways?To observe the effect of CP-25 though IgD-R and downstream signaling pathways on the activation T cells, we choose human PBMC, to examined the effect of CP-25 on proliferation in T cells, the effect of CP-25 on the expression of T cells subsets and IgD-R in T cells subsets, variation of inflammatory cells secreted production, and IgD-R or P56Lck in T cells.Objective:To observe the effect of CP-25 though IgD-R and downstream signaling pathways on the activation T cells, we examined the effect of CP-25 on proliferation in T cells, the effect of CP-25 on the expression of T cells subsets and IgD-R in T cells subsets, variation of inflammatory cells secreted production, and IgD-R or P56Lck in T cells.Methods:PBMCs were freshly isolated from blood from health patients, CD4+ T cells were purified by Ficoll-Hypaque through incubation of PBMCs with CD4+ antibody-conjugated microbeads. PBMC and T cells proliferation were analyzed by CCK-8. Analysis of the expression of IgD-R in T cells subsets by flow-cytometry. Simultaneous quantification of 10 cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-10, IL-8, IL-4, INF-γ, IL-13, and MCP-1)was performed on a Microarray scanner. Measurement of inflammatory factors was performed according to the manufacturer’s instructions. Expressions of p56 Lck and IgD-R were analyzed by western blot.Results:1 Effect of CP-25 on the activation of T lymphocytesAfter 24 h of culture, IgD (3 μg/ml) increased the expression of activated T cells (CD4+/CD154+, CD4+/CD69+), but had no impact in immature T cells (CD4+/CD62L+) and T helper cells (CD3+/CD4+). CP-25(10-7,10-6, 10-5mol/L) decreased the expression of activated T cells (CD4+/CD154+,CD4+/CD69+), also had no significant effect on the expression of immature T cells and T helper cells. CP-25 had no impact in immature T cells、 T helper cells and activated T cells.IgD (3μg/ml) increased the expression of IgD-R in activated T cells (CD4+/CD154+, CD4+/CD62L+), T helper cells (CD3+/CD4+), and total T cells (CD3+), but had no impact in immature T cells (CD4+/CD62L+). CP-25 (10-7,10-6, 10-5mol/L) decreased the expression of IgD-R in activated T cells (CD4+/CD154+,CD4+/CD62L+), T helper cells (CD3+/CD4+), and total T cells (CD3+), also had no significant effect on the expression of IgD-R in immature T cells.Compared with the control group, IgD significantly increased concentrations of IL-a、IL-1b、IL-6、IL-8、IL-10、TNF-α TNF-γ and MCP-1, had no impact on IL-4 and IL-13. Supernatants from cells treated with CP-25(10"5, 10-6,10-7mol/L) exhibited significantly group decreased concentrations of IL-a、 IL-1b、 IL-6、 IL-8、 TNF-α、 TNF-γ, and MCP-1, increased IL-4, and IL-10, and had no impact on IL-13. As expected, IgD can significantly enhanced the proliferation of T cells, compared with control group. After 24h, CP-25 (10-7,10-6,10-5mol/L) significantly decreased the proliferation of T cells by IgD induced in vitro.2 Effect of CP-25 on the expression of IgD-R and potential signaling pathway on the activation of T lymphocytesWestern blot analyses of the expression of IgD-R in healthy human T cells. IgD (1,3, lOμg/ml) significantly increased the expression of IgD-R compared with PHA group. CP-25 (10-7,10-6,10-5mol/L) significantly decreased the expression of T cells by IgD induced. Western blot analyses of the expression of p56Lck in healthy human T cells. IgD (3μg/ml) significantly increased the expression of p56Lck. CP-25 (10-7,10-6, 10"5mol/L) significantly decreased the expression of p56Lck by IgD induced. Meanwhile, IgD and CP-25 had no impact on the expression of Lck (not phosphorylation).Conclusions:1. CP-25 mediated inflammatory immune may through inhibiting the activation of T lymphocytes and decreasing variation of inflammatory cells secreted production.2. CP-25 inhibited the activation of T lymphocytes may though inhibiting the phosphorylation of p56Lck, dowm-regulating the expression of IgD-R, and decreasing the over expression of IgD combined with IgD-R. |
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