基于网络药理学及分子对接探讨黄芪治疗骨质疏松症的分子机制及实验验证

Discussion of molecular mechanism and experimental verification of Radix Astragali seu Hedysari in the treatment of osteoporosis based on network pharmacology and molecular docking

目的基于网络药理学和分子对接方法探讨黄芪治疗骨质疏松症的药理学作用机制,并通过体外实验进行验证。方法通过中药系统药理学数据库与分析平台获取黄芪的活性成分及药物靶点,运用GeneCard、OMIM、PharmGkb、TTD及DrugBank数据库筛选骨质疏松症的相关基因,将靶点和基因取交集,构建药物-成分-疾病-靶标调控网络,进行KEGG通路富集分析,最后进行分子对接和细胞实验验证。选取前成骨细胞系MC3T3-E1,将其分为10、20、30μmol/L组(分别加入10、20、30μmol/L异鼠李素药液)和空白组(等体积的培养液),采用CCK8法检测四组培养0、24、48、72 h的细胞增殖情况。结果共得到黄芪有效成分20个,骨质疏松症相关基因1290个,相关基因交集78个。黄芪抗骨质疏松症的核心靶标有6个,核心基因KEGG通路富集分析结果有脂质和动脉粥样硬化通路、MAPK信号通路等。分子对接结果显示,黄芪关键靶点与有效成分之间结合活性较高。选取黄芪主要有效成分异鼠李素进行细胞实验,结果显示,培养24、48、72 h的10、20、30μmol/L组细胞光密度值均高于同一时间点的空白组,20、30μmol/L组高于同一时间点的10μmol/L组,30μmol/L组高于同一时间点的20μmol/L组,差异有统计学意义(P<0.05)。结论黄芪治疗骨质疏松症的有效成分为异鼠李素,黄芪可能通过多个作用靶点参与特定的信号通路发挥调控骨代谢的作用,并可促进成骨细胞增殖。

Objective To explore the pharmacological mechanism of Radix Astragali seu Hedysari in the treatment of osteoporosis based on network pharmacology and molecular docking,and to verify by in vitro experiment.Methods The active ingredients and drug targets of Radix Astragali seu Hedysari were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.GeneCard,OMIM,PharmGkb,TTD,and DrugBank databases were used to screen the genes related to osteoporosis,and the intersection of targets and genes was established to construct the drug-compotion-disease-target regulatory network.Enrichment analysis of KEGG pathway was performed,and finally molecular docking and cell experiments were performed.Pre-osteoblast cell line MC3T3-E1 was selected and divided into 10,20,30μmol/L groups(adding 10,20,30μmol/L isorhamnosin solution),and blank group(equal volume medium).The cell proliferation of the four groups cultured for 0,24,48,72 h was detected by CCK8 method.Results A total of 20 active components,1290 osteoporosis related genes,and 78 associated gene intersections were obtained.There were six core targets of Radix Astragali seu Hedysari against osteoporosis,and the enrichment analysis of core gene KEGG pathway showed lipid and atherosclerosis pathway,MAPK signaling pathway,etc.Molecular docking results showed that the key target of Radix Astragali seu Hedysari membranaceus had higher binding activity with active components.The main active component isorhamnetin of Radix Astragali seu Hedysari was selected for cell experiment,experiment results showed that the cell optical density values of 10,20,and 30μmol/L groups at 24,48,72 h culture were higher than those of the blank group at the same time point,the 20 and 30μmol/L groups were higher than those of the 10μmol/L group at the same time point,and the 30μmol/L group was higher than those of the 20μmol/L group at the same time point,the differences were statistically significant(P<0.05).Conclusion The effective component of Radix Astragali seu Hedysari in the treatment of osteoporosis is isorhamnetin,Radix Astragali seu Hedysari may play a role in regulating bone metabolism and promoting osteoblast proliferation through multiple targets participating in specific signaling pathways.