Study Of Juanbi Particles On Th1/Th17 Cells Mediating Immune Suppression Effect And Mechanism

Objective:To investigate the regulating effectof Juanbi particles extracted by the ethanol on Th1/Th17 cells mediating immunosuppressive effect in vitro and thepharmacodynamics of treatment of rheumatoid arthritis(RA),to reveal its mechanism of immunosuppression and provide theoretical basis for its clinical application and new drug research.Methods:(a)Set up the mice spleen lymphocytes culture model in vitro, detecting the lymphocytic poison of Juanbi particles extracted by the ethanol and the influence of Juanbi particles extracted by the ethanol on the proliferation of T and B lymphocytes induced by Con A, anti-CD3 m Ab and LPS with the method of MTT. Purify the spleen lymphocytes of mice and set up CD4+T cells culture model in vitro,detecting the influence of Juanbi particles extracted by the ethanol on CD4+T cells cytokine production levels of IFN-?, IL-17 A,IL-6,IL-10 induced by anti-CD3 m Ab with the method of ELISA; Detecting the influence of Juanbi particles extracted by the ethanol on the phosphorylation level of P-38 and ERK of MAPK signaling pathway of the CD4+T cells induced by anti-CD3 m Ab with the method of Western blot;(b)Established bovine type II collagen-induced arthritis(collagen induced arthritis, CIA) model in DBA/1 mice, according to the clinical score was average divided into arthritis model group and Juanbi particles model group. To observed the severity of arthritis in each group of mice with the method of arthritis clinical scores and the pathological injury degree of arthritis in each group of mice with paraffin embedding slice and HE staining;Detecting the proliferation of T lymphocyte induced by CII in each group of mice with the method of MTT; Detecting the cytokine in spleen lymphocytes production levels of IFN-? and IL-17 A in each group of mice with the method of FACS.Results:(a) No cytotoxic effect on normal mice spleen lymphocytes induced by the ethanol extract of Juanbi particles in the 3-30?g/ml concentrations(P>0.05); Significant inhibition of the T and B lymphocytes proliferation induced by Con A, anti-CD3 m Ab and LPS after the ethanol extract of Juanbi particles intervention in the 3-30?g/ml concentrations(P<0.01);Significantly lower the CD4+T cells cytokine production levels of IFN-?,IL-17A(P<0.05 and P<0.01) and raise the cytokine production levels of IL-10 and lower the IL-6(P<0.05) induced by anti-CD3 m Ab after the ethanol extract of Juanbi particles intervention; Significantly inhibit the phosphorylation level of P-38 and ERK after the ethanol extract of Juanbi particles intervention in the 30?g/ml concentrations;(b) After the Juanbi particles intervention, significantly reduce the clinical scores of collagen-induced arthritis mice(P<0.05) meanwhile prevented joint synovitis, lymphocytes infiltration and injury of joint, inhibit the reaction of T lymphocyte proliferation response induced by CII(P<0.05) and cut the cytokine in spleen lymphocytes production levels of IL-17 A, however no obvious change in expression of IFN-?.Conclusion:(a)The ethanol extract of Juanbi particles can significantlyinhibit the immunoreactions of T cells, the production levels of IL-17 A, IL-6 and IFN-?,?promotethe production levels of negative regulatory factor of IL-10 and block MAPKsignaling pathways, these could be the molecular mechanism of immunosuppressive effect and the treatment of RA(rheumatoid arthritis).(b) Juanbi particles have prevention and cure function of arthritis in mice induced by collagen, lower the expression of highly pathogenic Th17, inhibit the generation of proinflammatory factor of IL-17 A, these could be one of the mechanisms.