断藤益母汤对破骨细胞活性及Wnt信号通路的影响研究

Effect of Duanteng Yimu Decoction (DTYMT) on Osteoclast activity and Wnt Signaling Pathway

目的：探讨断藤益母汤（DTYMT）对破骨细胞活性及Wnt信号通路的影响。方法：通过诱导RAW264. 7巨噬细胞建立体外破骨细胞培养体系;设空白组、模型组、断藤益母汤400、600、800、1000μg/m L浓度组、甲氨蝶呤2μg/m L组,然后通过CCK-8法检测细胞生存率,蛋白免疫印迹法检测Wnt信号通路关键蛋白表达。结果：TRAP染色试验显示,与模型组相比,DTYMT 800、1000μg/m L浓度组融合细胞数目显著减少,具有统计学意义（P 〈0. 01）。CCK-8结果显示,与模型组比较,DTYMD 600、800、1000μg/m L浓度组和甲氨蝶呤2μg/m L组生存率明显下降（P 〈0. 05,P 〈0. 01）。免疫蛋白印迹结果显示,与模型组相比,DTYMT 800、1000μg/m L、甲氨蝶呤2μg/m L组中Wnt3a、β-catenin蛋白表达量显著上升（P 〈0. 05,P 〈0. 01）,DKK1蛋白表达水平显著下降（P 〈0. 05,P 〈0. 01）。结论：断藤益母汤可能是通过影响Wnt信号通路,即上调Wnt3a、β-catenin蛋白表达、下调DDK1蛋白表达以及抑制破骨细胞的分化和增殖而起到减缓RA的骨破坏作用。

Objective： To investigate the effect of DTYMT on Wnt signaling pathway and MMP-9 in osteoclasts.Methods： In vitro osteoclast culture system was established by inducing RAW264. 7 macrophages. Blank group,model group,methotrexate group of 2 μg/m L and DTYMD groups of 400,600,800,1000 μg/m L were set up. Cell viability was then measured by CCK-8 and the expression of key protein in Wnt signaling pathway was detected by Western blotting. Results：The TRAP staining showed that the number of fusion cells was reduced in the DTYMT 800 μg/m L and 1000μg/m L concentrations compared to the model group（P〈0. 01）. CCK-8 results showed that DTYMD 600,800,1000μg/m L concentration groups and methotrexate 2 μg/m L group significantly reduced OC survival rate compared with model group（ P〈0. 05,P〈0. 01）. The expressions of Wnt3 a and β-catenin protein in DTYMT 800,1000 μg/m L and methotrexate 2 μg/m L groups were significantly increased compared with that of the model group（ P〈0. 05,P〈0. 01）,and the expression of DKK1 protein was significantly decreased（ P 〈0. 05,P〈0. 01）. Conclusion： DTYMT may play a role in mitigating the bone destruction of RA by affecting Wnt signaling pathway,that is up-regulation of Wnt3 a,β-catenin protein expressions,down-regulation of DDK1 protein expression and inhibition of osteoclast differentiation and proliferation.