摘要
研究蛇床子素对破骨细胞骨吸收的影响及其分子机制。采用体外分离、培养兔破骨细胞,与盖玻片及骨磨片共同培养,使用1×10?5 mol.L?1蛇床子素刺激破骨细胞,观察活体细胞并依据HE、TRAP、骨陷窝甲苯胺蓝染色鉴定破骨细胞;进行骨吸收陷窝和面积定量分析,吖啶橙染色统计凋亡细胞;real time PCR及Western blotting法检测相关基因和蛋白。与空白对照组比较,1×10?5 mol.L?1蛇床子素能够明显提高破骨细胞凋亡率并通过抑制RANKL和TRAP等相关基因及JNK1/2磷酸化水平抑制其骨吸收。结果提示,蛇床子素可以通过RANK+RANKL/TRAF6/Mkk/JNK途径刺激破骨细胞凋亡并抑制骨吸收。
This study is to investigate the effect of osthol on osteoclasts＇ activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1×10^-5 mol·L^-1 osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1~10-5 mol＇L-1 osthol. There is significant difference between control group and experiment group treated by 1×10-5 mol·L^-1 osthol. Therefore, the osthol through RANK＋RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.
出处
《药学学报》
CAS
CSCD
北大核心
2012年第2期174-179,共6页
Acta Pharmaceutica Sinica
