基于网络药理学与动物实验探究人参—黄连—三七药串治疗糖尿病肾病的作用机制

Amechanistic study on the Ginseng-Coptis chinensis-Radix notoginseng Herbal combination for diabetic nephropathy based on network pharmacology and animal experiments

目的 应用网络药理学的研究方法筛选人参—黄连—三七药串治疗糖尿病肾病的作用靶点及相关信号通路,通过动物实验明确其疗效与作用机制。方法 运用Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)数据库检索获取人参—黄连—三七的主要化学成分以及相关作用靶点;使用DisGeNET筛选胰岛素抵抗与糖尿病肾病的相关靶标基因;通过Venn软件筛选出药物与疾病的共同作用靶点;构建出疾病—靶点—成分—药物网络;使用STRING数据库完成蛋白质—蛋白质相互作用网络(protein-protein interaction networks, PPI)的构建;通过基因本体(gene ontology, GO)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路富集分析对有效作用靶点进行分析。以db/db小鼠作为动物模型,于给药第4、第8周后留取各组小鼠8小时尿液,检测各组小鼠尿微量白蛋白排泄水平;采用蛋白免疫印迹法法对网络药理学的研究预测的关键通路及靶点进行进一步的实验验证。结果 本研究共筛选得到人参—黄连—三七药串的人参皂苷Rh2、人参皂苷F2、槲皮素、小檗碱等39种有效成分;获得人参—黄连—三七药串治疗糖尿病肾病潜在靶点18个,其中PTEN、CCL2、IL6、TNF、HIF1A等11个与其他靶点相互作用较强,可能在网络中起到关键作用;通过GO分析和KEGG分析分别获得2595个条目和107条信号通路,其中包括磷脂酰肌醇-3-激酶丝氨酸/苏氨酸特异性蛋白激酶(phosphatidylinositol-3-kinase/protein kinase B,PI3K/Akt)信号通路、晚期糖基化终末产物—晚期糖基化终末产物受体信号通路、低氧诱导因子-1信号通路、细胞迁移的正调控、细胞因子活性、细胞因子受体结合、血小板α颗粒等。动物实验显示,在给药第4周及第8周后,中药组小鼠尿微量白蛋白排泄水平均较模型组下降(P<0.05)。对于PI3K/Akt信号通路的相关蛋白检测结果表明,中药组小鼠肾脏PI3K、p-Akt表达水平较模型组明显下降(P<0.05),磷酸酶张力蛋白同源物(phosphatase and tensin homolog deleted on chromosome ten, PTEN)水平较模型组升高(P<0.05)。结论 人参—黄连—三七药串能够有效改善db/db小鼠糖尿病肾病引起的蛋白尿,其作用机制可能与激活PTEN抑制PI3K/Akt信号通路有关。

Objective To screen and clarify the therapeutic effect and mechanism of the Ginseng-Coptis chinensis-Radix notoginseng herbal combination in the treatment of diabetic nephropathy by network pharmacology and animal experiments.Methods By using the TCMSP database of traditional Chinese medicine system pharmacology analysis platform,the main chemical components and related target genes of Ginseng-Coptis chinensis-Radix notoginseng herbal combination were retrieved.The target genes related to insulin resistance and diabetic nephropathy were selected by using DisGeNET.The common target genes between drugs and diseases were screened out by using Venn software.The disease-target-component-drug network and protein-protein interaction network(PPI)was constructed by STRING database.The effective target genes were analyzed by using gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The db/db mice model was used.The urine samples were collected from each group of mice at week 4 and week 8 after drug administration,and the level of urinary microalbumin excretion was detected in each group of mice.The key pathways and targets predicted by network pharmacology were further experimentally verified using western-blot method.Results This study identified 39 effective compounds from the Ginseng-Coptis chinensis-Radix notoginseng herbal combination,including ginsenoside Rh2,ginsenoside F2,quercetin and berberine.A total of 18 potential targets of Ginseng-Coptis chinensis-Radix notoginseng herbal combination in the treatment of diabetic nephropathy were identified,among which PTEN,CCL2,IL6,TNF,and HIF1A,had strong interaction with other targets and may play a key role in the network.Through GO analysis and KEGG analysis,2595 entries and 107 signaling pathways were obtained,including PI3K/Akt signaling pathway,AGE-RAGE signaling pathway,HIF-1 signaling pathway,positive regulation of cell migration,activity of cytokines,binding of cytokine receptors and platelet alpha granules.Animal experiments showed that the Ginseng-Coptis chinensis-Radix notoginseng herbal combination could effectively reduced the urinary microalbumin excretion level of db/db mice at week 4 and week 8 after drug administration(P<0.05).Western-blot results showed that the PI3K and p-Akt expression levels in the herbal group were significantly lower than those in the model group(P<0.05),while the PTEN level was significantly higher(P<0.05)than that of the model group.Conclusion Ginseng-Coptis chinensis-Radix notoginseng herbal combination can protect against proteinuria caused by diabetic nephropathy in db/db mice.Its mechanism of action may be related to activating PTEN to inhibit the PI3K/Akt signaling pathway.