Research of the Pharmacological Mechanism of the Drug Pair Angelicae sinensis Radix-Chuanxiong Rhizoma Based on Network Pharmacology and Molecular Docking

YUAN Kexin; TAN Yi; ZHANG Chenglin; WEI Jiahui; DAI Xueshan; XU Xiaoyu; CHEN Yi

doi:10.13718/j.cnki.xdzk.2021.10.010

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This study was designed to explore the network pharmacology mechanism of the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma, and verify it with molecular docking. First, the main active components and targets in the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma were screened from literature, TCMSP and SEA databases. Next, a "component-target" network was established for key target analysis. Then, the common targets between the key targets and the FDA approved drug targets were selected to construct a "common targets-disease" network. The key targets were subjected to GO enrichment and KEGG pathway analysis. Finally, molecular docking verification was accomplished between the active ingredients and the key targets. A total of 14 active compounds were screened out with 257 targets from this drug pair. Fifty-four key targets were obtained from analyzing the "components-target" network. Thirty-four common targets between the key targets and the FDA approved drug targets were closely related to 113 diseases. The key targets were involved in 155 biological processes, 29 cell components and 66 molecular functions, and in 58 pathways, including pathways in cancer, estrogen signaling pathway and proteoglycans in cancer. In molecular docking, 6 active ingredients showed binding activity with 7 targets. The active components, targets and mechanism of the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma were revealed in this research for clinical application.

Corresponding author: CHEN Yi

Received Date: 11/06/2020

Available Online: 20/10/2021

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Abstract: This study was designed to explore the network pharmacology mechanism of the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma, and verify it with molecular docking. First, the main active components and targets in the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma were screened from literature, TCMSP and SEA databases. Next, a "component-target" network was established for key target analysis. Then, the common targets between the key targets and the FDA approved drug targets were selected to construct a "common targets-disease" network. The key targets were subjected to GO enrichment and KEGG pathway analysis. Finally, molecular docking verification was accomplished between the active ingredients and the key targets. A total of 14 active compounds were screened out with 257 targets from this drug pair. Fifty-four key targets were obtained from analyzing the "components-target" network. Thirty-four common targets between the key targets and the FDA approved drug targets were closely related to 113 diseases. The key targets were involved in 155 biological processes, 29 cell components and 66 molecular functions, and in 58 pathways, including pathways in cancer, estrogen signaling pathway and proteoglycans in cancer. In molecular docking, 6 active ingredients showed binding activity with 7 targets. The active components, targets and mechanism of the drug pair Angelicae Sinensis Radix-Chuanxiong Rhizoma were revealed in this research for clinical application.

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当归性温味甘，归肝、心、脾经，具有补血调经、活血止痛的功效；川芎性温味辛，归肝、胆、心包经，具有活血行气、祛风止痛的功效. “当归-川芎”常形成药对增强其行气活血、补血养血的作用，如四物汤、佛手散、血府逐瘀汤、补阳还五汤、温经汤和生化汤等经典名方^[1-3]，但“当归-川芎”药对的物质基础及作用机制尚未阐明. 网络药理学是融合了系统生物学、生物信息学的新兴交叉学科，近年来，被广泛应用于中药的研究中，它通过构建“药物-靶点-通路”多层次网络，系统综合地观察中药对整个疾病网络的调控作用，有利于阐明中药多成分、多靶点、多途径协同作用的机制^[4-5].

本研究通过TCMSP，SEA数据库和文献检索，收集“当归-川芎”药对活性成分及其靶点，构建“成分-靶点”网络，分析得到关键靶点，寻找其与美国食品药品监督管理局(FDA)批准药物靶点的共同靶点，利用TTD和TCMSP数据库分析与共同靶点相关的疾病，建立“共同靶点-疾病”网络. 对关键靶点进行基因功能和KEGG通路分析，将活性成分和关键靶点进行分子对接验证.

1. 材料与方法

1.1. “当归川芎”药对活性成分的筛选

通过检索TCMSP(http://lsp.nwu.edu.cn/index.php)，SEA(http://sea.bkslab.org/)数据库和文献，收集“当归-川芎”药对中当归、川芎所含成分，然后利用药代动力学参数，即口服生物利用度OB≥30%，类药性DL≥0.18及小肠上皮细胞渗透率Caco-2≥-0.40，筛选出当归、川芎的活性成分.

1.2. “当归川芎”药对“成分靶点”网络的构建和关键靶点的筛选

通过检索TCMSP，SEA数据库，获得当归、川芎活性成分的靶点，利用蛋白质数据库UniProt(http://www.uniprot.org/)的搜索功能，将靶点转换为UniProt ID导入STRING数据库，进行成分-靶点间关联性分析，运用Cytoscape 3.5.0软件构建出“当归-川芎”药对“成分-靶点”网络. 使用中间介数、接近中间度、度值3个指标，对网络进行分析，选择3个指标均大于平均值的靶点作为网络中的关键靶点.

1.3. 共同靶点的筛选以及相关疾病分析

利用Drug Bank (https://www.drugbank.ca/)和TTD(https://db.idrblab.org/ttd/)数据库，收集FDA批准药物靶点，筛选得到关键靶点和药物靶点的共同靶点. 利用TCMSP和TTD数据库获得共同靶点相关疾病，按照医学主题词表MeSH(http://www.nlm.nih.gov/mesh/)将疾病分类，运用Cytoscape 3.5.0软件构建出“共同靶点-疾病”网络.

1.4. 关键靶点的富集分析和分子对接

将关键靶点导入DAVID数据库(https://david.ncifcrf.gov/)，进行基因功能和KEGG通路富集分析. 从Pubchem数据库(https://pubchem.ncbi.nlm.nih.gov/)中获取“当归-川芎”药对活性成分的分子结构，将其与关键靶点导入Systems Dock Web Site(http://systemsdock.unit.ois.jp/iddp/home/index)数据库进行分子对接. 对接分数为实验解离/抑制常数值(pKd/pKi)的负对数，大于4.25，表明分子与靶点具有一定的结合活性；大于5.0，表明分子与靶点结合活性较好；大于7.0，表明分子与靶点具有强烈的稳定性及活性.

3. 讨论

当归、川芎都是血分之主药，当归味甘，具有养血活血之功效，川芎味辛，是血中气药，具有活血祛瘀、行血散血的功效，两者相使配伍同用，可增强活血化瘀、养血活血之功效，因此“当归-川芎”常作为活血化瘀的药对使用，但它们配伍增效的物质基础并未阐明，我们通过数据库和文献检索获得川芎和当归14个有效成分，其中4个共有成分，分别是咖啡酸、阿魏酸、洋川芎内酯I、藁本内酯. 其中咖啡酸和阿魏酸是酚酸类成分，咖啡酸在机体内儿茶酚氧位甲基转移酶的作用下可转化成阿魏酸，而阿魏酸具有抗氧化、抗血栓、降血脂、降低心肌缺氧、抗菌、抗肿瘤和脑保护等多种药理作用^[6-7]. 洋川芎内酯I和藁本内酯是苯酞类成分，洋川芎内酯I具有保护脑、抗凝血作用，藁本内酯具有抗动脉粥样硬化、抗炎镇痛、抗老年痴呆、抗脑缺血、抗肿瘤、抗脂质过氧化、改善微循环等作用^[8-9]. 这些共有成分可能是“当归-川芎”药对配伍增效的物质基础，可以进一步通过成分敲出、敲入等技术来验证.

“当归-川芎”常常配对使用，尤其多见于一些治疗妇科疾病的名方，比如四物汤、佛手散、血府逐瘀汤、温经汤等. 通过数据库检索发现，构建和分析“当归-川芎”药对的“成分-靶点”网络，获得54个关键靶点，其中11个是川芎和当归共有靶点，分别是PTGS1，VDR，PTGS2，CDC25A，CYP19A1，NR1H4，PRKACA，AR，ESR2，ADRB2，NCOA2. 有研究发现PTGS2是FDA批准的治疗子宫内膜异位症的靶点之一^[10]，而且“当归-川芎”药对中的阿魏酸和川芎嗪能抑制PTGS2的表达^[11-14]. CYP19A1参与雌激素合成，ESR2是雌激素受体，分子对接结果中，豆甾醇、β-谷甾醇、洋川芎内酯A、藁本内酯与ESR2有较好的结合活性，也有研究发现阿魏酸和川芎嗪能抑制雌激素水平^{[4, 10]}，但是“当归-川芎”药对及其活性成分对CYP19A1和ESR2的作用还未见报道，值得研究. 其中34个关键靶点也是FDA批准药物靶点，PTGS2，ESR2可作为治疗女性生殖系统疾病及妊娠并发症的靶点，进一步KEGG通路富集分析发现，关键靶点调控多条与雌激素、孕激素相关的通路，包括Estrogen signaling pathway，Oxytocin signaling pathway，Progesterone-mediated oocyte maturation，GnRH signaling pathway，Endocrine and other factor-regulated calcium reabsorption，这可能是“当归-川芎”配对用于治疗妇科疾病的机制，值得进一步深入研究.

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Research of the Pharmacological Mechanism of the Drug Pair Angelicae sinensis Radix-Chuanxiong Rhizoma Based on Network Pharmacology and Molecular Docking

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Research of the Pharmacological Mechanism of the Drug Pair Angelicae sinensis Radix-Chuanxiong Rhizoma Based on Network Pharmacology and Molecular Docking

Corresponding author: CHEN Yi