基于网络药理学探究王琦院士治疗精液不液化核心组方的作用机制及实验验证

Exploring the Mechanism of Academician Wang Qi′s Core Prescription Against Semen Non-liquefaction Based on Network Pharmacology and Experimental Validation

目的:利用网络药理学和分子对接技术探究核心组方治疗精液不液化的作用机制。方法:利用中药系统药理学数据库与分析平台(TCMSP)和Uniprot数据库对王琦院士的核心组方药物进行成分和靶点的处理收集,再借助Gene Cards数据库、在线人类孟德尔遗传数据库(OMIM)对疾病靶点进行收集,与药物靶点取“药物-疾病”交集,获得核心靶点,使用String数据库绘制核心靶点蛋白质-蛋白质相互作用(PPI)网络。运用R语言平台进行基因本体(GO)和京都基因和基因组百科全书(KEGG)信号通路富集分析,构建核心组方“成分-靶点-疾病”通路图。对关键成分与核心靶点进行分子对接,最后通过造模和药物对大鼠进行干预后,利用酶联免疫吸附试验(ELISA)检测各组大鼠前列腺中细胞因子水平,实时荧光定量-聚合酶链反应(RT-PCR)法检测IL-17mRNA的表达量,对核心作用靶点及信号通路进行动物实验验证。结果:核心组方中有活性成分29个,对应靶点194个,与疾病靶点交集靶点24个,关键成分有槲皮素、山柰酚、β-谷甾醇等,核心靶点涉及肿瘤坏死因子(TNF)、纤溶酶原激活物抑制因子(SERPINE1)、白细胞介素6(IL-6)等。主要通过糖尿病并发症AGE-RAGE信号通路及IL-17炎症通路等发挥药效。核心组方中的槲皮素、β-谷甾醇等核心靶点之间具有较强的结合活性。核心组方能显著降低模型组大鼠前列腺组织中TNF、SERPINE1、IL-6、IL-1β的水平及IL-17 mRNA的表达量(P<0.01)。结论:王琦院士所拟核心组方中的关键成分槲皮素、β-谷甾醇等可能通过作用于TNF、SERPINE1等靶点调节多条通路,可能通过抑制炎症反应、抗氧化和调节纤维蛋白等作用治疗精液不液化。

Objective:To explore the mechanism of the core prescription in the treatment of semen non-liquefaction using network pharmacology and molecular docking.Methods:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and UniProt were used to collect the components and targets of Academician Wang Qi′s core prescription.The disease targets were collected by GeneCards and Online Mendelian Inheritance in Man(OMIM),and then intersected with the drug targets to obtain the core targets.String was used to draw the protein-protein interaction(PPI)network of the core targets.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed using R language platform to construct the“component-target-disease”map of the core prescription,and the key components were docked with the core targets.Finally,after modeling and drug intervention on rats,enzyme-linked immunosorbent assay(ELISA)was conducted to detect the cytokine level in the prostate of rats in each group,and real-time polymerase chain reaction(RT-PCR)was conducted to detect the expression of IL-17 mRNA,followed by animal experimental validation on core targets and signaling pathways.Results:There were 29 active components in the core prescription,acting on 194 targets,and 24 key components intersected with disease targets,including quercetin,kaempferol andβ-sitosterol.The core targets included tumor necrosis factor(TNF),serine protease inhibitor clade E member 1(SERPINE1),and interleukin-6(IL-6).KEGG enrichment analysis revealed that the core prescription produced effects mainly through AGE-RAGE signaling pathway in diabetic complications and IL-17 signaling pathway.Molecular docking showed that quercetin andβ-sitosterol had strong binding activity with the core targets.Additionally,the core prescription reduced the content of TNF,SERPINE1,IL-6,and IL-1βand the expression of IL-17 mRNA in prostate tissue of rats in model group(P<0.01).Conclusion:The key components of Academician Wang Qi′s core prescription such as quercetin andβ-sitosterol may regulate multiple pathways by acting on targets such as TNF and SERPINE1,and treat semen non-liquefaction via inhibiting inflammatory reaction,resisting oxidation,and regulating fibrin.