Exploring the Mechanism of Angelica Sinensis-Radix Paeoniae Alba in the Treatment of Angiogenesis Disorder at the Maternal Fetal Interface of Recurrent Spontaneous Abortion Based on Network Pharmacology and Animal Experiments
1. Department of Gynecology, Ningbo Hospital of Traditional Chinese Medicine Affiliated to Zhejiang Chinese Medicine University, Ningbo 315010, China; 2. College of Traditional Chinese Medicine, Zhejiang Pharmaceutical Vocational University, Ningbo 315100, China
摘要目的 利用网络药理学及动物实验探讨当归-白芍药对治疗复发性流产(recurrent spontaneous abortion,RSA)母胎界面血管生成障碍的作用机制,为中药的临床推广提供依据。方法 利用中药系统药理学数据库与分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP)数据库和中药分子机制的生物信息学分析工具(bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine,BATMAN-TCM)数据库收集并筛选当归-白芍药对的活性物质成分和作用靶点。在人类基因数据库(GeneCards)与在线人类孟德尔遗传(online Mendelian inheritance in man,OMIM)数据库中筛选RSA以及血管生成相关靶点。利用韦恩图(Venn)在线制图网站获得当归-白芍、RSA和血管生成的共同靶点,分别利用基因/蛋白相互作用检索搜查工具(search tool for the retrieval of interacting genes/proteins,STRING)数据库和Cytoscape对共同靶点进行蛋白互作网络分析(Protein-protein interaction,PPI)分析,构建药物、活性和共同靶点的作用网络。并且,对共同靶点进行富集分析。以雌性CBA/J小鼠配雄性DBA/2小鼠建立复发性流产模型,分别给予不同浓度当归-白芍水煎剂或黄体酮干预,以雌性CBA/J小鼠配雄性BALB/C小鼠建立正常妊娠模型,给予等体积生理盐水。所有小鼠均连续灌胃15 d后处死,用苏木素-伊红法观察各组小鼠蜕膜组织形态和血管数量;免疫组织化学法和免疫印迹法检测各组小鼠蜕膜组织缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF1α)/血管内皮生长因子(vascular endothelial growth factor, VEGF)/血管内皮生长因子受体2(vascular endothelial growth factor receptor 2, VEGFR2)蛋白的分布和表达含量。结果 通过网络药理学分析获得当归-白芍药对潜在活性成分27个,对应1 084个靶点。在GeneCards数据库与OMIM数据库中共获得496个RSA相关蛋白靶点以及150个血管生成相关靶点。Venn图得到当归-白芍、RSA、血管生成障碍的交集靶点28个。PPI可视化共同靶点间的相互关系,并得到10个关键基因(hub基因)。28个共同靶点富集于486个基因本体(Gene Ontology, GO)条目(P<0.05)和51条京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路(P<0.05)。动物模型验证结果表明,与模型组相比,当归-白芍低、中、高剂量组及黄体酮组能够显著改善蜕膜组织中血管生成,显著提高蜕膜组织中HIF-1α、VEGF和VEGFR2的水平(P<0.01)。结论 当归-白芍药对改善RSA母胎血管生成具有多组分、多靶点和多通路的优势,其中当归-白芍药对可能通过作用于HIF-1α/VEGF/VEGFR2通路基因的表达改善RSA母胎界面的血管生成,达到治疗功效。
Abstract：OBJECTIVE To explore the mechanism of Angelica sinensis-Radix Paeoniae Alba in the treatment of angiogenesis disorder at the maternal fetal interface of recurrent spontaneous abortion (RSA) by using network pharmacology and animal experiments, so as to provide basis for the clinical promotion of traditional Chinese medicine. METHODS Traditional Chinese Medicine systems pharmacology database and analysis platform (TCMSP) database and a bioinformatics analysis tool for molecular mechanism of Traditional Chinese Medicine (BATMAN-TCM) database were used to collect and screen the active components and action targets of Angelica sinensis and Radix Paeoniae Alba. RSA and angiogenesis-related genes were screened in GeneCards database and OMIM database. The common targets of Angelica sinensis-Radix Paeoniae Alba, RSA and angiogenesis were obtained by Venn online mapping website. The protein-protein interaction (PPI) network analysis of the common targets was analyzed and visualized by STRING database and Cytoscape, respectively. And then the network of Chinese herbs, active compounds and common targets was constructed. Moreover, enrichment analysis of GO and KEGG on common targets were conducted. RSA model was established by mating female CBA/J mice with male DBA/2 mice, and RSA mice were intervened with different concentrations of Angelica sinensis-Radix Paeoniae Alba decoction or progesterone. Normal pregnancy model was established by mating female CBA/J mice with male BALB/c mice, and the mice were intervened with equal volume of normal saline. All mice were sacrificed after 15 days of continuous gavage. The morphology of decidua and the number of blood vessels were observed by hematoxylin eosin staining. Distribution and expression of HIF-1 α/VEGF/VEGFR2 in decidua of mice in each group were detected by immunohistochemistry and Western blotting. RESULTS Through network pharmacological analysis, 27 potential active components in Angelica sinensis-Radix Paeoniae Alba were obtained, targeting 1 084 core targets. A total of 496 RSA-related protein targets and 150 angiogenesis-related targets were obtained in Genecards database and OMIM database. Twenty-eight overlapped targets were obtained by Venn diagram. PPI network was utilized to visualize the relationship between overlapped targets and 10 hub genes were obtained. These 28 overlapped targets were enriched in 486 GO entries (P<0.05) and 51 KEGG pathways (P<0.05). The results of animal model validated that compared with the model group, the low, medium and high dose of Angelica sinensis-Radix Paeoniae Alba, as well as progesterone, could significantly improve angiogenesis in decidual tissue and increased the levels of HIF-1α, VEGF and VEGFR2 (P<0.01). CONCLUSION Angelica sinensis-Radix Paeoniae Alba has the advantages of multi-component, multi-target and multi-pathway in improving angiogenesis in maternal fetal interface of RSA. The drug pair of Angelica sinensis-Radix Paeoniae Alba may act on the expression of HIF-1α/VEGF/VEGFR2 pathway, thus improving angiogenesis at the maternal fetal interface of RSA and achieving therapeutic effect.
蒋婴, 钱诚, 张蔚苓, 常淑华, 钱旭武. 基于网络药理学和动物实验探究当归-白芍药对治疗复发性流产母胎界面血管生成障碍的机制[J]. 中国药学杂志, 2023, 58(2): 139-150.
JIANG Ying, QIAN Cheng, ZHANG Wei-ling, CHANG Shu-hua, QIAN Xu-wu. Exploring the Mechanism of Angelica Sinensis-Radix Paeoniae Alba in the Treatment of Angiogenesis Disorder at the Maternal Fetal Interface of Recurrent Spontaneous Abortion Based on Network Pharmacology and Animal Experiments. Chinese Pharmaceutical Journal, 2023, 58(2): 139-150.
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