基于分子对接及实验验证研究补肾健脾活血方通过Wnt信号通路防治骨质疏松症的作用机制  

Study on the Mechanism of Bushen Jianpi Huoxue Recipe(补肾健脾活血方) in Preventing and Treating Osteoporosis Through Wnt Signaling Pathway Based on Molecular Docking and Experimental Verification

作　　者：吴睿哲 吴洁 刘凯[1] 葛殊玮 伍强 曾景奇[1] 王凡[1] WU Ruizhe;WU Jie;LIU Kai;GE Shuwei;WU Qiang;ZENG Jingqi;WANG Fan(The Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha Hunan 410005,China)

机构地区：[1]湖南中医药大学第二附属医院,湖南长沙410005

出　　处：《中医药导报》2022年第8期27-31,共5页Guiding Journal of Traditional Chinese Medicine and Pharmacy

基　　金：湖南省中医药管理局科研计划(201860);湖南省中医药管理局科研计划(201862);湖南省中医骨伤临床医学研究中心(2020SK4013)。

摘　　要：目的:通过分子对接技术及细胞实验探究补肾健脾活血方对过表达Wnt信号通路抑制性蛋白Dickkopf相关蛋白1(DKK-1)及硬化蛋白(Sost)的MG63细胞中Wnt信号通路相关蛋白表达的影响,以探究补肾健脾活血方防治骨质疏松症(OP)的作用机制。方法:从TCMSP、BATMAN、Uniport数据库中筛选转化补肾健脾活血方的有效成分,GeneCards数据库中检索OP相关靶点,构建“成分-靶点-疾病”网络,使用CytoNCA插件对网络进行拓扑分析,筛选核心药物成分,使用Autodock Tools对有效成分与DKK-1及Sost进行分子对接计算;使用含药血清培养通过过表达质粒转染上述两种抑制性蛋白转染的MG63细胞,微量酶标法测定血清干预后细胞内碱性磷酸酶(ALP)活性,流式细胞仪检测细胞钙离子浓度,Western blotting法检测Wnt3a、β-catenin的表达。结果:补肾健脾活血方主要活性成分中排名前5的活性成分与DKK-1均具有较好的结合活性,有2种成分与Sost具有较好的结合活性;ALP水平方面,除空载对照组外,经含药血清干预的细胞ALP水平较空白血清干预细胞升高(P<0.01);含药血清干预的各组细胞钙离子浓度均较经空白血清干预细胞降低(P<0.05);除过表达DKK-1+Sost组外,含药血清干预的细胞中Wnt3a及β-catenin蛋白水平较空白血清干预的细胞升高,差异有统计学意义(P<0.01)。结论:补肾健脾活血方可能通过提高Wnt3a蛋白的表达量,抑制DKK-1及Sost对Wnt信号通路的抑制作用,影响成骨细胞中Wnt信号通路的表达,从而提高成骨细胞的活性及分化能力,以防治OP。Objective:To explore the effect of Bushen Jianpi Huoxue Recipe(补肾健脾活血方,BSJPHXR) on the expression of Wnt signaling pathway related proteins in in MG63 cells overexpressing the Wnt signaling pathway inhibitory protein Dickkopf related protein 1(DKK-1) and Sclerostin(Sost) through molecular docking technology and cell experiments,and to explore the mechanism of BSJPHXR in preventing and treating osteoporosis(OP).Methods:The effective components of transforming BSJPHXR were screened from TCMSP,BATMAN and Uniport databases,the relevant OP targets were retrieved from GeneCards database.The topology analysis of the network was performed using the CytoNCA plug-in,and the core drug components were screened.The molecular docking calculation of the effective components with DKK-1 and Sost was performed using Autodock Tools.MG63 cells transfected with the above two inhibitory proteins were cultured with drug containing serum and transfected with overexpression plasmids.The activity of alkaline phosphatase(ALP) in the cells after serum intervention was measured by microplate method,the concentration of calcium ions in the cells was detected by flow cytometry,and the expression of Wnt3a and β-catenin was detected by Western blotting.Results:Among the main active components of BSJPHXR,the top 5 active components have good binding activity with DKK-1,and two components have good binding activity with Sost.In terms of ALP level,except for the empty control group,the ALP level of cells treated with drug containing serum was higher than that of cells treated with blank serum(P<0.01).The calcium ion concentration of cells in each group treated with drug containing serum was lower than that of cells treated with blank serum(P<0.05).Except for the Ad-DKK-1+Ad-Sost group,the levels of Wnt3a and β-catenin protein in the cells treated with drug containing serum were significantly higher than those in the cells treated with blank serum(P<0.01).Conclusion:Bushen Jianpi Huoxue Recipe may affect the expression of Wnt signal

关 键 词：骨质疏松症 补肾健脾活血方 WNT信号通路 分子对接 Dickkopf相关蛋白1 硬化蛋白 MG63细胞 

分 类 号：R285.5[医药卫生—中药学]