基于网络药理学及动物实验研究温肾宣痹汤抗骨质疏松的机制  

Mechanism of Wen Shen Xuan Bi Tang in treatment of osteoporosis based on network pharmacology and animal experimental verification

作　　者：王海平 袁兆丰 夏天卫 张超[1] 沈计荣[1] WANG Hai-ping;YUAN Zhao-feng;XIA Tian-wei;ZHANG Chao;SHEN Ji-rong(The First Clinical Medicine College of Nanjing University of Chinese Medicine/Dept of Orthopedics,Jiangsu Province Hospital of Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210029,China;Dept of Orthopedics,Dafeng Hospital of Traditional Chinese Medicine,Yancheng 224100,China)

机构地区：[1]南京中医药大学附属医院/江苏省中医院骨伤科,江苏南京210029 [2]盐城市大丰中医院骨伤科,江苏盐城224100

出　　处：《中国药理学通报》2024年第2期344-351,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82274552)。

摘　　要：目的应用网络药理学、分子对接及体内实验技术,预测以及验证温肾宣痹汤(wenshen xuanbi tang,WSXBT)抗骨质疏松(osteoporosis,OP)的作用机制。方法利用网络药理学筛选WSXBT治疗OP的关键成分及核心靶点,Metascape数据库对核心靶点进行基因本体论(gene ontology,GO)功能分析和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析,AutoDockTools 1.5.7软件进行分子对接模拟关键活性成分与核心靶点的结合活性。切除大鼠双侧卵巢建立OP大鼠模型,研究WSXBT对OP大鼠的药效及验证相关靶点通路。酶联免疫吸附(ELISA)法检测血清中OPG、PINP、RANKL水平,生物力学测试仪测定大鼠股骨生物力学,Micro-CT检测股骨骨密度,进而通过Western blot法检测PI3K和Akt的蛋白表达水平。结果筛选出WSXBT活性成分156个,涉及229个潜在作用靶点,筛选核心靶点23个,共涉及145条信号通路;分子对接结果显示,槲皮素、异补骨脂黄酮、β-谷甾醇等关键成分与PIK3R1、AKT1核心靶点均拥有良好的结合能力。体内实验结果表明,WSXBT可明显升高OP大鼠骨密度,改善OP大鼠骨组织微结构,提高大鼠股骨生物力学,增加大鼠PINP表达及OPG/RANKL比值,Western blot结果显示,WSXBT可明显升高p-PI3K/PI3K、p-Akt/Akt比值。结论WSXBT可能通过PI3K/Akt信号通路及升高OPG/RANKL比值改善绝经后OP大鼠骨密度。Aim To predict and validate the mechanism of wenshen xuanbi tang(WSXBT)in treatment of osteoporosis(OP)based on network pharmacology,molecular docking techniques and in vivo experimental techniques.Methods Network pharmacology was used to screen the key ingredients and core targets of WSXBT for the treatment of osteoporosis.Metascape database was used for gene ontology(GO)biological process enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG)signaling pathway enrichment analysis of core targets.AutoDockTools 1.5.7 software was applied in molecular docking to simulate the binding activity of key active ingredients to core targets.To study the efficacy of WSXBT on rats with osteoporosis and to verify the related targets and pathways,rat models of osteoporosis were established by excising the bilateral ovaries of rats.Enzyme-linked immunosorbent assay(ELISA)was used to detect the serum OPG,PINP and RANKL content.Biomechanical tester was applied to test the biomechanics of rat femurs.Micro-CT was applied to detect the femoral bone density.Then,Western blot was employed to measure the protein expression levels of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt).Results A total of 156 active ingredients of WSXBT were screened,involving 229 potential targets,23 core targets and 145 signaling pathways.The molecular docking results showed that five key ingredients,including quercetin,kaempferol,naringenin,isobavachin and licochalcone a,possessed good binding ability to the core targets of PIK3R1 and AKT1.The results of in vivo experiments showed that WSXBT could significantly increase bone density,improve bone tissue microstructure,enhance femur biomechanics and increase PINP expression and OPG/RANKL ratio in rats with osteoporosis.Results of WB showed that WSXBT significantly increased p-PI3K/PI3K and p-Akt/Akt ratios.Conclusions WSXBT could improve bone mineral density in postmenopausal osteoporotic rats through PI3K/Akt signaling pathway and increasing OPG/RANKL ratio.

关 键 词：网络药理学 分子对接 温肾宣痹汤 骨质疏松 p-PI3K/PI3K 

分 类 号：R-332[医药卫生] R289.5R319R336R681