RA患者PBMC内MAPK途径的活化及^(99)Tc-亚甲基二膦酸盐对其的影响

The activation of mitogen-activated protein kinase pathway in peripheral blood mononuclear cells of rheumatoid arthritis patients and the regulation of ^(99)Tc-MDP on it

目的探讨类风湿关节炎(RA)患者外周血单个核细胞(PBMC)内丝裂原活化蛋白激酶 (MAPK)途径的活化状况,及99Tc-亚甲基二膦酸盐(99Tc-MDP,商品名:云克)对其的影响。方法 RA患者及正常对照各20例。Western blot检测PBMC中磷酸化MAPK(p-MAPK)的表达;不同剂量99Tc-MDP 干预PBMC,Western blot检测不同时间点p-MAPK表达的变化。另行反转录聚合酶链反应(RT-PCR)及酶联免疫吸附试验(ELISA),分别从mRNA及蛋白质水平检测该途径下游白细胞介素(IL)-1β及IL-6表达的变化。结果 RA患者PBMC中磷酸化p38(p-p38)及磷酸化JNK(p-JNK)(C-Jun氨基末端激酶)表达较正常对照显著增高,p-ERK1／2表达无明显增高;99Tc-MDP能够剂量依赖性下调PBMC中p-p38表达,以20 mg/L组最为明显(P<0．05),药物作用30 min即有明显抑制效应;99Tc-MDP对p-JNK表达无明显影响;99Tc-MDP能够剂量依赖性抑制PBMC中IL-1β及IL-6 mRNA表达(P<0．05),培养液上清中上述两种炎症介质的水平亦相应降低(P<0．05)。结论 RA患者PBMC中存在p38和JNK的明显活化,99Tc-MDP 能够抑制p38激酶磷酸化,并下调其下游炎症因子IL-1β和IL-6的表达。

Objective To explore the activation of mitogen-activated protein kinase （MAPK） pathway in peripheral blood mononuclear cells （PBMCs） of rheumatoid arthritis （RA） patients and the effect of ^99Tc-MDP on it. Methods Western blot was used for detection of phos-MAPK （p-MAPK） in freshly-isolated PBMCs from RA patients and healthy controls; PBMCs were treated with different dose of ^99Tc-MDP, after which, p-MAPK was measured by Western blot and the expression of downstream inflammatory cytokines IL-1β and IL-6 were analyzed by RT-PCR and ELISA. Results The expression of p-p38 and p-JNK was significantly higher in PBMCs from RA patients compared to healthy controls, while p-ERK1/2 was not enhanced. ^99Tc-MDP could dose-dependently reduce the expression of p-p38 （P〈0.05） and reduce the transcription and secretion of IL-1β and IL-6 from PBMCs （P〈0.05）, while it had no significant effect on the expression of p-JNK in PBMCs. Conclusion The activation of p38 and JNK is significantly enhanced in PBMCs from RA patients; and ^99Tc-MDP can significantly reduce the activation of p38 kinase and coordinately decrease the expression of its downstream cytokine IL-1β and IL-6.