青藤碱合用乌头原碱对CD4^+ T细胞RANKL表达和破骨细胞生成的影响

Effects of sinomenine and aconine on the expression of RANKL in CD4^+ T cells and the osteoclast formation

目的:研究青藤碱和乌头原碱单独或配伍使用对活化的T淋巴细胞RANKL表达和两药合用对破骨细胞生成的影响。方法:采用anti-CD3激活小鼠CD4^+T细胞,用流式细胞仪检测青藤碱、乌头原碱对小鼠辅助性T细胞RANKL表达的影响;同时用TRAP染色法观察两药合用对RANKL诱导破骨细胞生成的影响。结果:0.25~1mmol/L青藤碱能够剂量依赖性地抑制小鼠CD4^+T细胞RANKL表达;0.25、0.5mmol/L乌头原碱也能显著地降低CD4^+T细胞RANKL表达,而另一毒性较大的活性成分苯甲酰乌头原碱只在高剂量0.5mmol/L时才能下调RANKL表达。乌头原碱与低浓度青藤碱合用后能使CD4^+T细胞RANKL表达进一步降低,而苯甲酰乌头原碱与低浓度青藤碱合用后对RANKL表达无影响。研究发现,青藤碱与乌头原碱合用后对RANKL诱导的RAW264.7细胞分化成破骨细胞的抑制作用较两药单独使用明显增强。结论:青风藤和附子活性成分均可能通过下调活化的T细胞RANKL表达间接抑制RANKL诱导的破骨细胞生成和骨吸收,改善类风湿性关节炎引起的骨破坏;附子久煎后的无毒活性成分乌头原碱与青藤碱配伍使用后对T细胞RANKL表达和RANKL相关破骨细胞生成的作用均优于单用,提示中医临床使用附子时久煎不仅能够减毒,还可能更有利于配伍其它药物治疗类风湿性关节炎骨破坏。

Objective： It is to investigate the single and combining effects of sinomenine and aconine on RANKL （ receptor activator of nuclear fac- tor-KB ligand） expression in activated splenic lymphocytes and the osteoclast formation. Methods： Murine CD4 ~T cells were activated by anti-CD3, then the effects of main active components sinomenine of Caulis sinomeniior and nontoxic active ingredient aconine of Radix Aconiti Lateralis Preparata, were analyzed on the expression of RANKL in mouse T helper ceils by flow cytometry; TRAP staining was used to ob- serve the synergistic effects of two drugs on RANKL induced osteoclast formation. Results： The expression of RANKL on anti-CD3-stimulated CIM＇T cells were inhibited by sinomenine （1,0.5, 0.25mmol/L）, aconine （0.5, 0.25mmol/L）, benzoylaconine （0.5mmol/L） signifi- candy （ P 〈0. 001 ）. Synergistic effects were present when aconine （0.5, O. 25mmol/L） was respectively used together with 0.25mmol/L of sinomenine, and there is no significant difference when benzoylaconine （0.5, 0.25mmol/L） was respectively combined with 0.25mmol/L of sinomenine （ P 〉 0.05 ）. Further study found that the combining effects of sinomenine and aconine on the inhibition of RANKL induced osteo- clast formation in RAW264.7 cells, was significantly greater than the effects of two drugs used alone. Conclusion ： The main active ingredi- ents of Caulis sinomenii and Radix Aconiti Lateralis Preparata can both decrease the expression of RANKL in activated CD4 ~ T cells, which may indirectly contribute to the inhibitory functions of these drugs on the osteoclast formation and bone resorption followed by the alleviation the pain of bone destruction resulted by RA. The co-administration of aconine and sinomenine can play a better role on decreasing the expres- sion of RANKL of T cells and inhibiting the RANKL induced formation of osteoclast. This suggested that decocting of Radix Aconiti Lateralis Preparata for a long time could not only reduce the drug toxicity but also be benefit for the treatment of bone destruction of RA.