基于体外药效学结合网络药理学和分子对接对人参-桑椹改善骨质疏松的机制研究

目的:用网络药理学方法，探讨人参、桑椹单味药与药对的水提物、醇提物改善骨质疏松症作用的分子机制。方法:观察人参、桑椹单味药与药对的水提物、醇提物对小鼠睾丸间质细胞（TM3）增值率及睾酮分泌影响和药对的水提物及醇提物对成骨细胞（MC3T3-E1）增值率及碱性磷酸酶（ALP）活力的影响。通过中药成分数据库TCMSP、GeneCards、OMIM数据库分别收集人参、桑椹化学成分及成分与疾病相关靶点，取交集后，运用STRING数据库分析关键靶点间的蛋白相互作用，利用DAVID数据库进行生物功能和通路分析；相关结果采用Cytoscape 3.7.0进行构图和网络拓扑结构分析；并应用Autodock软件对潜在药效物质和关键靶点进行分子对接。结果:TM3给药组与模型组比较，药对各浓度细胞增殖率极显著高于单味药各浓度值，且呈浓度依赖性，其中浓度为200 μg/mL时增殖率最高，增殖率分别是97.31%、91.67%（P<0.01），睾酮分泌量分别是3.48、3.06 ng/mL。MC3T3-E1给药组与空白组比较，细胞增殖率明显升高。在药对水提物给药组中200 μg/mL浓度细胞增殖率最高，增殖率为155%；醇提物给药组中100 μg/mL时细胞增殖率最高，增殖率为142.8%；ALP分析结果显示药对水提物给药组及醇提物组均具有明显升高效果。本研究共收集到28个活性成分，69个关键靶点，GO功能富集收集到257个生物过程，34个分子功能，62个细胞组分；KEGG通路富集共收集到109条通路；分子对接结果显示，潜在药效物质与关键靶点IL6、ALB、MAPK8、CASP3对接结果能量低于0 kcal/mol。结论:本文揭示了人参-桑椹药对改善骨质疏松症可能的潜在药效物质和作用靶点，为人参-桑椹的开发和后续研究打下了基础。

Mechanism of Ginseng-Mulberry Treating Osteoporosis Based on Pharmacodynamics in Vitro Combined with Network Pharmacology and Molecular Docking

Objective: To explore the molecular mechanism of water extract and alcohol extract of ginseng and mulberry in treating osteoporosis by means of network pharmacology. Methods: The effects of water extract and alcohol extract of ginseng and mulberry on proliferation rate and testosterone secretion of mouse leydig cells (TM3), and the effects of water extract and alcohol extract of medicine pair on proliferation rate and alkaline phosphatase (ALP) activity of osteoblasts (MC3T3-E1) were observed. Collect chemical components, components and disease-related targets of ginseng and mulberry respectively through traditional Chinese medicine composition database TCMSP, GeneCards and OMIM. After the intersection of that, using STRING database to analyze protein interaction among key targets, and using DAVID database to analyze biological functions and pathways. The related results were mapped and analyzed by Cytoscape 3.7.0. Autodock software was used to perform molecular docking between potential pharmacodynamic substances and key targets. Results: Compared with the model control group, the cell proliferation rate of TM3 administration group was significantly higher than that of single drug, and it was concentration-dependent. The highest proliferation rate was found at the concentration of 200 μg/mL, which was 97.31% and 91.67% (P<0.01), and testosterone secretion was 3.48 and 3.06 ng/mL. Compared with the blank control group, the cell proliferation rate of MC3T3-E1 administration group increased significantly. The proliferation rate of cells at the concentration of 200 μg/mL was the highest in the group of water extract administration, and the proliferation rate was 155%. The highest cell proliferation rate was 142.8% at 100 μg/mL of alcohol extract. The results of ALP analysis showed that the drug had obvious increasing effect on both water extract group and alcohol extract group. In this study, 28 active components and 69 key targets were collected, and 257 biological process, 34 cell compound and 62 molecular function were collected by GO function enrichment. 109 pathways were collected from KEGG pathway enrichment. The results of molecular docking showed that the energy of potential pharmacodynamic substances docking with key targets IL6, ALB, MAPK8 and CASP3 was lower than 0 kcal/mol. Conclusion: This paper revealed the potential pharmacodynamic substances and targets of ginseng-mulberry medicine in the treatment of osteoporosis, which would lay a foundation for the development and follow-up research of ginseng-mulberry medicine.