Li Rui, Lou Qi, Ji Tingting, Li Yincan, Yang Haoran, Ma Zheng, Zhu Yu, Qian Can, Yang Wulin, Wang Yijun, Luo Shengyong
Anhui Medical College (Anhui Academy of Medical Sciences), Hefei, 230061, PR China.
Department of Pharmacology, School of Basic Medicine, Anhui Medical University, Hefei, 230031, PR China.
J Ethnopharmacol. 2024 Jul 15;329:118157. doi: 10.1016/j.jep.2024.118157. Epub 2024 Apr 6.
Astragalus mongholicus Bunge (AMB) is a herb with wide application in traditional Chinese medicine, exerting a wealth of pharmacological effects. AMB has been proven to have an evident therapeutic effect on ischemic cerebrovascular diseases, including cerebral ischemia-reperfusion injury (CIRI). However, the specific mechanism underlying AMB in CIRI remains unclear.
This study aimed to investigate the potential role of AMB in CIRI through a comprehensive approach of network pharmacology and in vivo experimental research.
The intersection genes of drugs and diseases were obtained through analysis of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Gene Expression Omnibus (GEO) database. The protein-protein interaction (PPI) network was created through the string website. Meanwhile, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out using R studio, and thereafter the key genes were screened. Then, the molecular docking prediction was made between the main active ingredients and target genes, and hub genes with high binding energy were obtained. In addition, molecular dynamic (MD) simulation was used to validate the result of molecular docking. Based on the results of network pharmacology, we used animal experiments to verify the predicted hub genes. First, the rat middle cerebral artery occlusion and reperfusion (MACO/R) model was established and the effective dose of AMB in CIRI was determined by behavioral detection and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Then the target proteins corresponding to the hub genes were measured by Western blot. Moreover, the level of neuronal death was measured using hematoxylin and eosin (HE) and Nissl staining.
Based on the analysis of the TCMSP database and GEO database, a total of 62 intersection target genes of diseases and drugs were obtained. The KEGG enrichment analysis showed that the therapeutic effect of AMB on CIRI might be realized through the advanced glycation endproduct-the receptor of advanced glycation endproduct (AGE-RAGE) signaling pathway in diabetic complications, nuclear factor kappa-B (NF-κB) signaling pathway and other pathways. Molecular docking results showed that the active ingredients of AMB had good binding potential with hub genes that included Prkcb, Ikbkb, Gsk3b, Fos and Rela. Animal experiments showed that AWE (60 g/kg) could alleviate CIRI by regulating the phosphorylation of PKCβ, IKKβ, GSK3β, c-Fos and NF-κB p65 proteins.
AMB exerts multi-target and multi-pathway effects against CIRI, and the underlying mechanism may be related to anti-apoptosis, anti-inflammation, anti-oxidative stress and inhibiting calcium overload.
蒙古黄芪是一种在传统中药中广泛应用的草药，具有多种药理作用。已证明蒙古黄芪对缺血性脑血管疾病，包括脑缺血再灌注损伤（CIRI）有明显的治疗作用。然而，蒙古黄芪在CIRI中的具体机制仍不清楚。
本研究旨在通过网络药理学和体内实验研究的综合方法，探讨蒙古黄芪在CIRI中的潜在作用。
通过分析中药系统药理学（TCMSP）数据库和基因表达综合数据库（GEO）获得药物与疾病的交集基因。通过string网站构建蛋白质-蛋白质相互作用（PPI）网络。同时，使用R studio进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析，然后筛选关键基因。然后，对主要活性成分与靶基因进行分子对接预测，获得具有高结合能的枢纽基因。此外，利用分子动力学（MD）模拟验证分子对接结果。基于网络药理学结果，我们通过动物实验验证预测的枢纽基因。首先，建立大鼠大脑中动脉闭塞再灌注（MACO/R）模型，通过行为检测和2,3,5-三苯基氯化四氮唑（TTC）染色确定蒙古黄芪在CIRI中的有效剂量。然后通过蛋白质免疫印迹法检测枢纽基因对应的靶蛋白。此外，使用苏木精-伊红（HE）和尼氏染色法检测神经元死亡水平。
基于TCMSP数据库和GEO数据库的分析，共获得62个疾病与药物的交集靶基因。KEGG富集分析表明，蒙古黄芪对CIRI的治疗作用可能通过糖尿病并发症中的晚期糖基化终产物-晚期糖基化终产物受体（AGE-RAGE）信号通路、核因子κB（NF-κB）信号通路等途径实现。分子对接结果表明，蒙古黄芪的活性成分与包括Prkcb、Ikbkb、Gsk3b、Fos和Rela在内的枢纽基因具有良好的结合潜力。动物实验表明，黄芪水提取物（60 g/kg）可通过调节PKCβ、IKKβ、GSK3β、c-Fos和NF-κB p65蛋白的磷酸化来减轻CIRI。
蒙古黄芪对CIRI具有多靶点、多途径的作用，其潜在机制可能与抗凋亡、抗炎、抗氧化应激和抑制钙超载有关。
