南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (2): 171-180.doi: 10.12122/j.issn.1673-4254.2022.02.02

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龙杞方治疗糖尿病肾脏病的作用机制：基于网络药理学和大鼠验证实验

邢 磊，邢文文，郭宏敏   

  1. 南京中医药大学第一临床医学院，江苏 南京 210004；南京市高淳中医院老年病科，江苏 南京 211300
  • 出版日期:2022-02-20 发布日期:2022-03-16

Exploring the therapeutic mechanism of Longqi Fang for diabetic kidney disease based on network pharmacology and verification in rats

XING Lei, XING wenwen, GUO Hongmin   

  1. First Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing 210004, China; Department of Geriatrics, Nanjing Gaochun Hospital of Traditional Chinese Medicine, Nanjing 211300, China
  • Online:2022-02-20 Published:2022-03-16

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摘要： 目的 基于GEO数据库和网络药理学方法研究龙杞方治疗糖尿病肾脏病（DKD）的作用机制，并通过动物实验进一步验证龙杞方治疗DKD的信号通路。方法 利用GEO、TCMSP、CNKI、ChemDraw 、SwissTarget Prediction等数据库获得龙杞方和DKD靶点，VENNY获得龙杞方-DKD交集靶点，String进行蛋白互作分析，R包进行KEGG和GO富集分析，Cytoscape 3.7.2软件构建网络图。动物实验验证分析，40只健康雄性SD大鼠随机分为5组（8只/组），采用腹腔内注射链脲佐菌素（STZ）诱导的DKD大鼠模型，龙杞方低剂量组（1 g·kg-1·d-1），龙杞方中剂量组（2 g·kg-1·d-1），龙杞方高剂量组（2 g·kg-1·d-1），空白组和模型组采用等体积水灌胃；HE染色观察大鼠肾脏形态，检测肾功能情况；Western blot检测炎症指标NF-κB、p-NF-κB蛋白表达水平。结果 龙杞方共760个主要靶点，通过GEO数据库获得差异基因共1026个，通过VENNY数据库获得龙杞方-DKD交集靶点共61个，通过PPI网络互作获得的核心靶点为MYC、EGF、CASP3、VEGFA、CCL2、SPP1、VCAM1、ICAM1。GO分析龙杞方主要与核受体活性、配体激活转录因子活性等有关，KEGG发现龙杞方主要通过干预NF-κB、TNF、PI3K-AKT等炎症信号通路有关。动物实验显示龙杞方高、中、低剂量干预后DKD大鼠的肾功能指标（Scr、BUN、UALB、UACR）与模型组比较有显著改善（P<0.05）；HE染色观察出龙杞方高、中、低剂量干预DKD大鼠与模型组对照，肾小球和肾小管结构和排列改善明显，效应强度为：龙杞方高剂量组>中剂量组>低剂量组；Western blot结果显示龙杞方高、中、低剂量干预DKD大鼠，与模型组比较，可以使NF-κB、p-NF-κB表达显著下调，效应强度为：龙杞方高剂量组>中剂量组>低剂量组。结论 龙杞方通过多成分、多靶点、多信号通路治疗DKD，龙杞方可能通过抑制炎症信号通路NF-κB起到保护肾功能的作用。

关键词: 网络药理学；龙杞方；糖尿病肾脏病；动物实验；NF-κB

Abstract: Objective To study the therapeutic mechanism of Longqi Fang (LQF) for diabetic kidney disease (DKD) based on GEO database and network pharmacology. Methods LQF and DKD targets were obtained using the databases including GEO, TCMSP, CNKI, ChemDraw, and SwissTarget Prediction, and LQF-DKD intersection targets were obtained with VENNY. String was used for protein-protein interaction (PPI) analysis, and R package for KEGG and GO enrichment analysis. Cytoscape 3.7.2 software Network graphs were constructed. The results of network pharmacology analysis were verified in SD rat models of DKD by daily treatment of the rats with LQF at low (1 g/kg), medium (2 g/kg), and high (2 g/kg) doses, and kidney pathology was observed with HE staining and the changes in renal function were assessed. Western blotting was used to detect the expression levels of NF-κB and p-NF-κB proteins. Results We identified 760 main targets of LQF, and obtained 1026 differential genes using GEO database and 61 LQF-DKD intersection targets using Venny database. The core targets obtained through PPI network analysis included Myc, EGF, CASP3, VEGFA, CCL2, SPP1, VCAM1 and ICAM1. Go analysis showed that LQF affects mainly nuclear receptor activity and ligand activated transcription factor activity. KEGG analysis showed that LQF affects inflammatory signaling pathways by interfering with NF-κB, TNF, and PI3K-AKT. In rat models of DKD, treatment with LQF resulted in significant improvements of the renal functions (P<0.05) and glomerular and tubular structure and arrangement in a dose-dependent manner. Western blotting results showed that LQF dose-dependently downregulated NF-κB and p-NF-κB expressions in the rat models. Conclusion The therapeutic mechanism of LQF for DKD involves multiple components, targets and signal pathways that mediate an inhibitory effect on NF-κB signaling pathway to protect the renal function.

Key words: network pharmacology; Longqi Fang; diabetic kidney disease; animal experiments; nuclear factor-κB