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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 an important role in bone development and reconstruction. In this study, 20 000 compounds were screened using a high-throughput screening model of up-regulated BMP2 expression that had been constructed in the previous study. Compound E40071 was prepared in a similar manner to Example 1 except that the EC50 The value was 2.73 μmol·L-1. In vitro activity studies showed that E40071 up-regulated the mRNA expression of BMP2 and its downstream transcription factors Runx2 and Osx, and up-regulated the expression of Runx2 protein by promoting the phosphorylation of Smad1 / 5/8. The effect of E40071 on osteoblast alkaline phosphatase Activity had certain up-regulation effect; alizarin red staining results show that, E40071 can promote osteoblast calcification. Further study found that E40071 can significantly inhibit RANKL-induced mouse macrophage Raw264.7 into osteoclasts, and reduce osteoclast differentiation markers MMP9 and NFATc1 protein expression levels. The results show that, E40071 can promote osteoblast osteogenic activity and inhibit osteoclast differentiation.