BMP2表达上调剂E40071体外抗骨质疏松活性研究  

Evaluation of osteogenic effects of BMP2 up-regulator E40071 in vitro

作　　者：韩小婉[1] 宫世强 李雪虹[1] 贺晓波[1] 姜威[1] 司书毅[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所国家新药(微生物)筛选实验室,北京100050

出　　处：《药学学报》2016年第3期396-402,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(30901811);国家"重大新药创制"科技重大专项资助项目(2012ZX09301002-003)

摘　　要：骨形态发生蛋白II(bone morphogenetic protein 2,BMP2)在骨发育与重建中具有重要作用,本研究利用前期已构建的BMP2表达上调剂高通量筛选模型对20 000个化合物进行了筛选,得到阳性化合物E40071[2-(4-(5-甲基-3-苯基吡唑并[1,5-a]嘧啶-7-基)哌嗪-1-基)乙-1-醇],其EC50值为2.73μmol·L-1。体外活性研究表明,E40071能上调BMP2及其下游特异性转录因子Runx2、Osx的m RNA表达,并通过促进Smad1/5/8蛋白磷酸化,上调Runx2蛋白的表达;对成骨细胞碱性磷酸酶活性具有一定的上调作用;茜素红染色结果表明,E40071能够促进成骨细胞钙化。进一步研究发现,E40071能够明显抑制RANKL诱导小鼠巨噬细胞Raw264.7分化为破骨细胞,并降低破骨细胞分化标志MMP9和NFATc1蛋白的表达水平。研究结果表明,E40071可促进成骨细胞骨形成活性并抑制破骨细胞的分化。Bone morphogenetic protein 2（BMP2） plays a key role in bone development and reestablishment. In the study, we screened up-regulators of BMP2 among 20 000 compounds through a cell-based high throughput screening model and a positive compound E40071 [2-（4-（5-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-7-yl） piperazin-1-yl）ethan-1-ol] was found as the positive hit. The EC50 value of E40071 was 2.73 μmol·L-1. In vitro, E40071 upregulated the m RNA levels of BMP2 and the downstream transcription factors, Runx2 and Osx in MC3T3-E1（subclone 14）. Protein expression of Runx2 was up-regulated by E40071 through induction of Smad1/5/8 phosphorylation. The alkaline phosphatase（ALP） activity was increased by E40071. Moreover, E40071 promoted the mineralization of MC3T3-E1（subclone 14） by Alizarin red S staining. In addition, E40071 markedly inhibited osteoclast differentiation of mice macrophage Raw264.7 induced by RANKL and reduced the expression of osteoclast differentiation markers, including MMP9 and NFATc1. The results suggest that E40071 is able to promote bone formation activity of osteoblasts and inhibit differentiation of osteoclasts.

关 键 词：2-(4-(5-甲基-3-苯基吡唑并[1 5-a]嘧啶-7-基)哌嗪-1-基)乙-1-醇 骨形态发生蛋白II 骨质疏松 成骨分化 破骨分化 

分 类 号：R965.1[医药卫生—药理学]