橄榄苦苷对破骨细胞的分化以及骨吸收功能的影响

Effect of oleuropein on osteoclast differentiation and bone resorption in vitro

目的探讨橄榄苦苷(oleuropein)对破骨细胞分化成熟以及骨吸收功能的影响及其可能机制。方法把不同浓度的橄榄苦苷(12.5、25、50μmol/L)分别加入由核因子κB受体活化因子配体(receptor activator of nuclear factor kappa B ligand,RANKL)诱导的体外破骨细胞培养体系中,用抗酒石酸酸性磷酸酶(tartrate-resist ant acid phosphatase,TRAP)染色方法观察呈TRAP阳性的多核细胞;RAW264.7细胞也用RANKL诱导,用流式细胞仪分析橄榄苦苷对破骨细胞凋亡的影响;在扫描电镜(scanning electron microscope,SEM)下观察骨吸收陷窝,并用图像分析软件统计其面积。采用Western blot法检测橄榄苦苷对破骨细胞特异性蛋白组织蛋白酶K(cathepsin K,CTSK)、激活T细胞核因子1(nuclear factor of activated T-cells 1,NFATC1)及相关信号通路蛋白表达的影响。结果 TRAP染色结果显示,橄榄苦苷能显著抑制RANKL诱导的破骨细胞的形成,且呈浓度依赖性(P<0.05);橄榄苦苷能下调破骨细胞特异性蛋白CTSK的蛋白表达水平(P<0.05);橄榄苦苷对破骨细胞的凋亡无明显影响(P>0.05);橄榄苦苷能显著减少骨吸收陷窝的面积(P<0.05);橄榄苦苷能够下调NFATC1及磷酸化p38(p-p38)的表达(P<0.05)。结论橄榄苦苷可以抑制由RANKL诱导的破骨细胞分化以及破骨细胞的骨吸收功能,可能和橄榄苦苷能够下调p38/MAPK信号通路有关。

Objective To investigate the effect of oleuropein on the differentiation and bone resorption of cultured osteoclasts and explore the possible mechanism. Methods Bone marrow macrophages from BALB/c mice were cultured in the presence of 12. 5,25,or 50 μmol/L of oleuropein in a receptor activator of nuclear factor kappa B ligand（ RANKL）-induced osteoclast culture system,and the multinucleated cells were detected with tartrate resistant acid phosphatase（ TRAP） staining. RAW264. 7 cells were induced by RANKL in the presence of oleuropein at different concentrations,and the cell apoptosis was analyzed with flow cytometry. Osteoclastic resorption pits on the bone slices were observed with scanning electron microscopy,and the areas of osteoclastic bone resorption were determined using image processing software.The expressions of the osteoclast-specific protein cathepsin K（ CTSK）,nuclear factor of activated T-cells 1（ NFATC1） and phosphorylated p38 were measured with Western blotting. Results RANKL-induced osteoclastogenesis was significantly inhibited by oleuropein in a dose-dependent manner（ P〈0. 05）. The protein expression level of CTSK in RAW264. 7 cells was down-regulated by oleuropein（ P〈0. 05）,which produced no obvious effect on apoptosis of osteoclast cells（ P〉0. 05）. Oleuropein markedly decreased the area of bone resorption pits（ P〈0. 05） and down-regulated the protein expression of NFATC1 and phosphorylated p38 in the osteoclasts. Conclusion Oleuropein inhibits RANKL-induced osteoclastogenesis and bone resorption in vitro possibly by down-regulating the p38/MAPK signaling pathway.