地乌三萜皂苷W_1对破骨细胞分化和骨吸收功能的影响  

Effect of triterpene saponin W_1 from rhizome of Anemone Flaccid on osteoclasts differentiation and bone resoroption

作　　者：杨悦[1] 孔祥英[1] 万红叶[1] 苏晓慧[1] 林娜[1] 

机构地区：[1]中国中医科学院中药研究所中药理论与本草文献研究中心,北京100700

出　　处：《环球中医药》2014年第3期191-196,共6页Global Traditional Chinese Medicine

基　　金：国家科技重大专项(2011ZX09101-006-03);北京市自然科学基金(7112096);中国中医科学院基本科研业务费自主选题(ZZ070836)

摘　　要：目的探讨地乌三萜皂苷类成分W1对体外破骨细胞分化及骨吸收功能的影响。方法3个不同浓度(0.0625,0.125,0.25μg/ml)的W1与核因子-κB受体活化因子配体(receptor activator of nuclear factor kB ligand,RANKL)诱导的小鼠单核/巨噬细胞RAW 264.7共孵育7天,抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色观察TRAP阳性多核细胞的形成,甲苯胺蓝染色及扫描电镜观察骨吸收陷窝的形成,图像分析计算骨吸收陷窝面积百分比;同3个浓度的W1与RANKL诱导的RAW264.7细胞共孵育24小时后收集细胞上清液,放射免疫法检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的含量;甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测W1对RAW264.7细胞毒性影响。结果 RANKL组诱导RAW 264.7细胞形成多量TRAP阳性多核细胞,形成明显的骨吸收陷窝,并诱导TNF-α在RAW264.7细胞上清中异常高表达;与RANKL组相比,3个浓度的W1均能显著减少TRAP阳性细胞数(P<0.01),显著减少骨吸收陷窝面积(P<0.01,P<0.001,P<0.001),明显降低TNF-α的表达量(P<0.05,P<0.01,P<0.01),且未观察到对RAW264.7的细胞毒性。结论 W1对由RANKL诱导的体外破骨细胞分化和骨吸收功能有一定的负调节作用。Objective To observe the effect of W1 from rhizome of Anemone Flaccid on osteoclasts formation and bone resoroption in vitro. Methods RAW264. 7 cells induced by RANKL were cultured with three variable concentration（0. 0625,0. 125,0.25 μg · ml^-1 ）of Wl in vitro for 7 days. The influence of W1 on osteoclasts differentiation was assessed by osteoclast-like number and tartrate resistant acid phos- phatase （TRAP） activity; resorption lacunae were stained by toluidine blue and were visualized by Philips XL30 scanning electron microscope. The number of TRAP ＋ cells and the percentage of resorption pits were counted. RAW264. 7 ceils induced by RANKL were incubated with three variable concentration of W1 in vitro for 24 hours, the level of TNF-α in cell culture supernatant was examined by Iodine [ ^125I ] Tunmor Necrosis Factor Radioimnmnoassay kit; RAW264. 7 cell viability was assayed by MTT. Results RANKL could induce RAW264.7 ceils to form TRAP positive multinucleated cells and form bone resorption pits on bone. RANKL could induce abnormally high expression of TNF-α in RAW264.7 cells. W1 （0. 0625,0. 125,0.25 μg · ml^-1 ） could decrease the number of TRAP ＋ cells （ P 〈 0.01 ） as well as the area of bone resorption pits （P 〈 0.01, P 〈 0. 001, P 〈 0. 001 ） , and showed no cytotoxicity to RAW264.7 ; W1 （0. 0625-0.25 μg · ml^-1 ） could suppresse the expression of TNF-α（ P 〈 0.05, P 〈 0.01 , P 〈 0.01 ）. Conclusion RANKL induced osteoclastogenesis in these monocytic cells, and W1 inhibited RANKL-induced tumor necrosis factor-co production and osteoclast differentiation and bone re- sorption pit formation.

关 键 词：地乌三萜皂苷W1 小鼠单核 巨噬细胞RAW264 7 肿瘤坏死因子Α 骨吸收 

分 类 号：R285.5[医药卫生—中药学]