蠲痹颗粒醇提物调控MAPK信号转导抑制T细胞活化效应机制的研究

Mechanism of Juanbi Granules Ethanol Extract Suppressing the Activation of T Cells through the MAPK Signaling Transduction Pathway

目的探讨蠲痹颗粒醇提物(JBKL)在丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号转导通路中抑制T细胞活化的效应机制,揭示扶阳学派应用扶阳理论治疗类风湿关节炎(rheumatoid arthritis,RA)的现代免疫学机制。方法噻唑蓝(MTT)比色法检测JBKL对伴刀豆球蛋白(Con A)、脂多糖(LPS)诱导T、B脾淋巴细胞增殖活性及细胞毒性的影响;建立体外抗CD3单克隆抗体(Anti-CD3 mAb)诱导的T细胞活化模型,MTT检测JBKL对T细胞体外活化增殖的影响,酶联免疫吸附法(ELISA)测定干扰素-γ(IFN-γ)、白细胞介素-17A(IL-17A)、IL-10和IL-6在细胞上清中的含量;蛋白质印迹法(Western blot)检测体外纯化CD4~+T细胞中ERK和p38蛋白的磷酸化水平。结果JBKL在浓度30μg/mL时显著抑制Con A、Anti-CD3 mAb和LPS诱导的T细胞和B细胞增殖功能;JBKL在浓度(3~30)μg/mL时对正常脾淋巴细胞无明显的细胞毒性作用,明显下调IFN-γ、IL-17A和促炎因子IL-6的含量,而促进IL-10的分泌;JBKL在浓度100μg/mL时抑制MAPK信号通路,可降低纯化CD4~+T细胞磷酸化ERK和p38蛋白的表达。结论JBKL阻碍T细胞介导的免疫反应,减少MAPK信号通路磷酸化ERK和p38蛋白表达,可能是其治疗RA的重要分子机制之一。

Objective To investigate the potential mechanism of the ethanol extract of Juanbi Granules(JBKL)on T cell activation via the mitogen-activated protein kinase(MAPK)signaling transduction pathway,and to reveal the mechanism of immunosuppression on the application of Yang-aided theory in rheumatoid arthritis(RA)treatment.Methods T cells and B cells proliferation assay were induced by concanavalin A(Con A)and lipopolysaccharide(LPS),respectively.Splenocyte proliferation and cytotoxicity were detected by MTT assay.The T cells were further stimulated in vitro with Anti-CD3 mAb to establish Anti-CD3 mAb-induced T cells activation;ELISA assay was used to measure the supernatant concentrations of interferon-γ(IFN-γ),interleukin(IL)-17A,IL-10 and IL-6.The expression of MAPK signaling pathway related proteins(p-ERK and p-p38)in purified CD4T cells were detected by Western blot assay.Results Compared with the vehicle control group,JBKL(30μg/mL)significantly inhibited the proliferation of T cells induced by Con A and Anti-CD3 mAb,and the proliferation of B cells responded to LPS.Further,JBKL groups had no remarkable cytotoxicity in normal mouse spleen lymphocytes.The levels of IFN-γL-17A,IL-6 content were obviously decreased in the JBKL(3~30μg/mL)group and the IL-10 content was up-regulated.The phosphorylation levels of ERK and p38 proteins were lower in the JBKL(100μg/mL)group than those in the vehicle group.Conclusion JBKL can significantly restrain the activation and proliferation of CD4T cells,which may be related to the inhibition of p-ERK and p-p38 MAPK signaling pathways.This may be an important molecular mechanism contributes to the effect of JBKL in rheumatoid arthritis treatment.