基于网络药理学探讨当归-牛膝防治骨关节炎的作用机制
林青1,2，王南卜3，李小云4，叶倩云1，王昊宇1，黄嘉家1，梁梓雯4，王攀攀1,2，朱晓峰1,2，杨丽4，张荣华2,4
1. 暨南大学中医学院 广州 510632；
2. 广东省中医药信息化重点实验室 广州 510632；
3. 广州中医药大学第一附属医院 广州 510405；
4. 暨南大学药学院 广州 510632
摘 要
目的 运用网络药理学、分子对接、细胞验证方法，探讨当归-牛膝防治骨关节炎（Osteoarthritis，OA）的作用机制。
方法 从 TCMSP 筛选当归-牛膝的活性成分，并用 Swiss Target Prediction 预测其作用靶点；从 GEO、GeneCards、DisGeNET、TTD 数据库收集 OA 相关靶点。取活性成分与 OA 交集靶点，借助 Cytoscape 3.8.2 的 BisoGenet 构建交集靶点的蛋白相互作用（Protein-protein interaction，PPI）网络，CytoNCA 拓扑分析获得 PPI 核心靶点，ClueGO 对 PPI 核心靶点进行 GO 和 KEGG 富集分析。运用 Autodock 4.2软件对关键活性成分与核心靶点进行分子对接。构建IL-1β诱导的软骨炎症细胞模型，RT-PCR与流式细胞仪验证预测结果。
结果 研究显示当归-牛膝活性成分与 OA 的交集靶点183个，PPI核心靶点373个；PPI核心靶点的 GO富集分析涉及有丝分裂细胞周期相变等调控，KEGG 涉及细胞周期、泛素介导的蛋白水解等信号通路；分子对接显示槲皮素、汉黄芩素、山奈酚与关键核心靶点 ESR1、RELA、MAPK1、CDKN1A、CDK2具有较好亲和力。RT-PCR显示核心药效成分可调节细胞周期信号通路靶点的mRNA表达水平，增加软骨细胞的S、G2/M期比例，提高软骨指标ACAN、COL2、SOX9的mRNA表达水平。
结论 该研究验证了当归-牛膝药对可介导细胞周期信号通路而促使炎症软骨细胞的软骨增殖与修复，其防治 OA 的综合机制可涉及调节软骨细胞周期相变、促进软骨的增殖与修复、抗炎、抗凋亡等方面的多靶点、多途径机制，对临床实践具有重要指导意义。
关键词
网络药理学 当归 牛膝 骨关节炎 分子对接 作用机制 细胞周期
主要研究及结论
本研究综合 TCMSP、TCM@Taiwan、CNKI 等数据库中当归与牛膝化合物成分数据，同时依据化合物ADME性质进行筛选，统一了活性成分的筛选条件[1]。采用 TCMSP 关联的 Drugbank 数据库和 Swiss Target Prediction来获得活性成分作用靶点，前者关联了FDA批准药物作用靶点的数据，靶点信息来源确切可信，后者通过生物活性小分子与靶点的2D和3D相似性测量计算来预测靶点，预测成功率最高可达 70%，为预测更多的当归-牛膝潜在靶点提供参考[2, 3]。此外，本次的疾病靶点主要来源于GEO数据库 OA 患者临床病理组织的基因芯片表达谱数据，可靠性强。GeneCards、DisGeNET、TTD 则作为疾病靶点的补充数据，纳入了现有研究中与 OA 相关度高的靶点，但其靶点相关性评分的筛选值还需优化与验证。
目前通过网络药理学研究发现，补肾活血中药防治 OA 的主要效应集中在抗炎、抗凋亡，涉及TNF、NF-κB、IL-17、VEGF、Hippo、AKT/ mTOR等信号通路[4, 5]，但调控有丝分裂细胞周期相变发挥抗 OA 的网络药理学机制预测并未涉及。本研究创新性地预测并验证了当归-牛膝药效成分可通过细胞周期相变调控，综合介导细胞周期信号通路相关靶点，促进炎症软骨细胞的增殖与修复，表明细胞周期信号通路亦为防治 OA 的重要机制。
综上所述，本研究对当归-牛膝药效成分的细胞周期相变调控、抗炎、抗凋亡等作用机制进行探讨，对细胞周期相变调控机制作进一步的实验验证，为后续补肾活血中药当归-牛膝干预 OA 的进一步机制研究拓展了新思路与方向，丰富了补肾活血法防治 OA 的科学内涵。
Discuss on Mechanism of Angelicae Sinensis Radix-Achyranthis Bidentatae Radix in Treating Osteoarthritis Based on Network Pharmacology
Lin Qing1,2，Wang NanBu3，Li XiaoYun4，Ye QianYun1，Wang HaoYu1，Huang JiaJia1，Liang ZiWen4，Wang PanPan1,2，Zhu XiaoFeng1,2，Yang Li4，Zhang RongHua2,4
1. School of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, China；
2. Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization, Guangzhou 510405, China；
3. The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China；
4. Pharmacy College of Jinan University, Guangzhou 510632, China
Abstract:
Objective Network pharmacology and molecular docking methods were used to discuss the mechanisms of Angelicae Sinensis Radix (AS)-Achyranthis Bidentatae (AB) Radix in treating osteoarthritis (OA).
Methods The active components and potential targets of AS-AB were screened and predicted from TCMSP and Swiss Target Prediction. OA related targets were also collected from GEO、GeneCards、DisGeNET、TTD databases. Secondly, BisoGenet was used to construct PPI network of the intersection targets, obtaining the core targets of PPI network through topological analysis with CytoNCA. The core targets of PPI were analyzed by GO and KEGG with ClueGO. Molecular docking verification were also carried out by Autodock 4.2 software. Cartilage inflammatory cell model was constructed with IL-1β, and RTPCR and flow cytometry were used to verify the predictions.
Results The intersection targets of the active components of AS-AB and OA were 183, and the core targets of PPI were 373. The core targets' GO analysis involved the regulation of mitotic cell cycle phase transition and so on, and KEGG analysis involved the signaling pathway of cell cycle,ubiquitin mediated proteolysis etc. The critical active components contained quercetin, wogonin and kaempferol, showed great binding activities with ESR1, RELA, MAPK1, CDKN1A, and CDK2. RT-PCR showed that core ingredients regulate the mRNA expression level of intersection targets of cell cycle signaling pathway and cartilage index with ACAN、COL2、SOX9, increasing the ratio of S and G2/M phases of cells.
Conclusions This study analyzed that ASAB can promote cartilage proliferation and repair of inflammatory cartilage cells via cell cycle signaling pathways, and its comprehensive mechanism for preventing OA may involve regulating chondrocyte cycle phase transition, promoting cartilage proliferation, repair, anti-inflammatory, anti-apoptotic and regeneration, which revealing its potential multitarget and multi-path mechanism, lying a foundation for clinical practice of its combination in treating OA.
Keywords
Network pharmacology, Angelicae Sinensis Radix, Achyranthis Bidentatae Radix, Osteoarthritis, Molecular docking, Mechanisms of action, Cell Cycle
注释
1 Liu J L, Pei M J, Zheng C L, et al. A systems-pharmacology analysis of herbal medicines used in health improvement treatment: Predicting potential new drugs and targets. Evidence-Based Complementary and Alternative Medicine, 2013, 2013: 938764.
2 Wishart D S, Feunang Y D, Guo A C, et al. DrugBank 5.0: A major update to the DrugBank database for 2018. Nucleic Acids Research,2017, 46(D1): D1074-D1082.
3 Daina A, Michielin O, Zoete V. SwissTargetPrediction: Updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Research, 2019, 47(W1): W357-W364.
4 葛海雅, 鄢来军, 张燕, 等 . 基于网络药理学方法分析当归-川芎药对治疗骨关节炎的潜在靶点和机制. 中医正骨, 2020, 32(9): 1-8.
5 黄泽灵, 何俊君, 施珊妮, 等 . 基于网络药理学分析牛膝-桑寄生药对治疗骨关节炎的作用机制 . 中国组织工程研究, 2020, 24(29): 4599-4604
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