基于网络药理学和实验验证探讨三七总皂苷治疗糖尿病肾病的作用机制研究

Mechanism of Panax notoginseng saponins in treating diabetic kidney disease based on network pharmacology and experimental verification

基于网络药理学、分子对接、动物实验,探究三七总皂苷(Panax notoginseng saponins,PNS)对糖尿病肾病(diabetic kidney disease,DKD)的治疗作用及其机制。基于网络药理学筛选潜在作用靶点,利用STRING数据库构建蛋白-蛋白互作网络,将筛选的核心靶点进行基因本体论(Gene Ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路的富集分析,并构建“成分-靶点-通路”的可视化网络,预测PNS防治DKD的潜在作用机制。从PNS中筛选出5个有效成分,其治疗DKD的核心靶点有AKT1、STAT3、ESR1、HSP90AA1、MTOR等;通过KEGG分析发现PNS治疗DKD相关的通路有癌症通路、化学致癌-受体激活、PI3K-AKT信号通路等;通过GO分析发现PNS治疗DKD主要涉及的生物过程有蛋白质结合、同种蛋白质结合、酶结合以及ATP结合等生物过程。将40只雄性6周龄db/db小鼠随机分为模型组、达格列净组、PNS低剂量组、PNS高剂量组,每组10只,将10只同周龄db/m小鼠作为空白组。干预组灌胃给药干预8周,空白组和模型组每日给予等量蒸馏水灌胃,每天1次,监测小鼠体质量和空腹血糖,并检测肾脏指数、尿微量白蛋白、肌酐、尿微量白蛋白/肌酐比值及尿素含量。采用苏木精-伊红(HE)染色、过碘酸雪夫(PAS)染色、马松(Masson)染色观察PNS对db/db小鼠的肾脏保护作用。结果发现PNS可显著降低db/db小鼠的空腹血糖水平,改善其肾脏损伤情况。Western blot验证实验显示PNS干预组可降低p-AKT1、p-STAT3的蛋白表达水平及p-AKT1/AKT1、p-STAT3/STAT3比值。此外,PNS高剂量组可下调PIK3CA的蛋白表达水平。综上,三七总皂苷可能是通过PI3K-AKT信号通路抑制STAT3靶点蛋白发挥良好的糖尿病肾脏保护作用。

This study aims to investigate the therapeutic effect and mechanism of Panax notoginseng saponins(PNS)on diabetic kidney disease(DKD)based on network pharmacology,molecular docking,animal experiments.Network pharmacology was employed to screen the potential targets,and STRING was employed to build the protein-protein interaction network.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were carried out for the core targets screened out,and a″components-targets-pathways″visualization network was constructed to predict the potential mechanism of PNS in treating DKD.Five active ingredients were screened from PNS,the core targets of which for treating DKD were AKT1,STAT3,ESR1,HSP90AA1,MTOR,et al.The KEGG enrichment analysis showed that the pathways related to PNS for treating DKD included the pathway in cancer,chemical carcinogenesis-receptor activation,and PI3K-AKT signaling pathway.GO analysis revealed that protein binding,homologous protein binding,enzyme binding,and ATP binding were the main biological processes involved in the treatment of DKD with PNS.Male 6-week-old db/db mice were randomized into model,dapagliflozin,and low-dose and high-dose PNS groups,with 10 mice in each group.Ten 6-week-old db/m mice were used as the control group.Mice were administrated with corresponding drugs or distilled water(control and model groups)by gavage once a day for 8 weeks.The body weight,fasting blood glucose,kidney index,microalbuminuria,creatinine,microalbuminuria/creatinine ratio,and urea nitrogen content in the urine of mice were determined.Hematoxylin-eosin(HE)staining,periodic acid-Schiff(PAS)staining,and Masson staining were performed to observe the protective effect of PNS on the renal tissues in db/db mice.The results showed that PNS could significantly reduce the fasting blood glucose level and improve the renal damage in db/db mice.Western blot results showed that PNS down-regulated the protein levels of p-AKT1 and p-STAT3 and decreased the p-AKT1/AKT1 and p-STAT3/STAT3 ratios.In addition,high-dose PNS down-regulated the protein level of PIK3CA.In conclusion,PNS may exert the kidney-protecting effects in DKD by inhibiting STAT3 via the PI3K-AKT signaling pathway.