木犀草素对骨缺损成骨修复的作用及机制研究

Study on the effects and mechanism of luteolin on osteogenic repair of bone defects

目的探讨木犀草素对骨缺损成骨修复的作用及机制。方法通过网络药理学方法筛选木犀草素治疗骨缺损的作用靶点及潜在通路,利用Hub基因筛选排名前2位的靶点进行分子对接验证,以结合能作为评判标准。对大鼠骨髓间充质干细胞(BMSC)及脐静脉内皮细胞(RUVEC)进行体外实验,采用碱性磷酸酶染色、茜素红S染色、体外血管形成实验进行表型验证,采用Western blot法检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B1(Akt1)蛋白的表达情况,以验证木犀草素对BMSC成骨分化及RUVEC体外血管形成的作用机制。结果网络药理学结果显示,木犀草素对骨缺损的血管形成和骨修复作用主要与Akt1、SRC蛋白、雌激素受体1、表皮生长因子受体、环加氧酶2、基质金属蛋白酶9靶点有关,且与PI3K/Akt信号通路关系密切。分子对接结果显示,木犀草素与Akt1、SRC蛋白结合稳定。体外实验结果表明,木犀草素能显著增高BMSC内碱性磷酸酶的表达水平和活性,增加钙盐沉积和钙化结节的数量,促进BMSC的钙化;与木犀草素0μmol/L组比较,木犀草素1、10μmol/L组的RUVEC血管形成能力显著增强、血管长度显著增加,PI3K、Akt1的蛋白表达水平显著提高(P<0.05或P<0.01),且浓度越高效果越好。结论木犀草素可能通过激活PI3K/Akt信号通路,促进PI3K、Akt1蛋白的表达来发挥增强骨折端的血管形成及骨修复的功能。

OBJECTIVE To investigate the effects and mechanism of luteolin on osteogenic repair of bone defects.METHODS The targets and potential pathways of luteolin in the treatment of bone defects were screened by network pharmacology method,and then the top 2 targets were selected by Hub gene screening for molecular docking verification,with binding energy as the evaluation standard.In vitro experiments were conducted on rat bone mesenchymal stem cells(BMSC)and rat umbilical vein endothelial cells(RUVEC).Phenotypic validation was performed using alkaline phosphatase staining,alizarin red S staining,and in vitro angiogenesis experiments.Western blot assay was used to detect the protein expressions of phosphatidylinositol 3 kinase(PI3K)and protein kinase 1(Akt1),so as to validate the mechanism of luteolin on osteogenic differentiation of BMSC and angiogenesis of RUVEC in vitro.RESULTS The results of network pharmacology showed that the effects of luteolin on vascular formation and bone repair in bone defects were mainly related to Akt1,SRC,estrogen receptor 1,epidermal growth factor receptor,cyclooxygenase 2,matrix metalloproteinase 9 targets,and were closely related to PI3K-Akt signaling pathway.The results of molecular docking showed that luteolin binding to Akt1 and SRC proteins was stable.The results of in vitro experiments showed that luteolin could significantly improve the expressions and activities of alkaline phosphatase in BMSC,increased the number of calcium salt deposits and calcified nodules,and promoted calcification of BMSC.Compared with luteolin 0μmol/L group,the angiogenesis ability of RUVEC was enhanced significantly in luteolin 1,10μmol/L groups,the length of blood vessels and the protein expressions of PI3K and Akt1 were significantly increased(P<0.05 or P<0.01);the higher the concentration,the better the effect.CONCLUSIONS Luteolin may play a role in promoting angiogenesis and bone repair at the fracture site by activating PI3K/Akt signaling pathway and promoting the protein expressions of PI3K and Akt1.