基于网络药理学探讨顾步汤治疗糖尿病足溃疡作用机制  

Discussion on Mechanism of Gubu Decoction for the Treatment of Diabetic Foot Ulcer Based on Network Pharmacology

作　　者：王继雪 杨稀瑞 马海涛[1] 代雪娜[1] 王汉杰 袁星星 周涛[1] WANG Jixue;YANG Xirui;MA Haitao;DAI Xuena;WANG Hanjie;YUAN Xingxing;ZHOU Tao(The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China;Heilongjiang University of Chinese Medicine,Harbin 150001,China)

机构地区：[1]河南中医药大学第一附属医院,河南郑州450000 [2]黑龙江省中医药科学院,黑龙江哈尔滨150001

出　　处：《中国中医药信息杂志》2023年第12期16-24,共9页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金青年基金(82205117、82205195);河南省卫生健康委员会国家中医临床研究基地科研专项课题(2022JDZX127、2022JDZX135);河南省中医药科学研究专项课题(20-21ZY1022)。

摘　　要：目的采用网络药理学方法分析顾步汤治疗糖尿病足溃疡(DFU)的可能作用靶点及相关机制,并进行细胞实验验证。方法利用TCMSP数据库查找顾步汤主要有效成分及作用靶点,通过GeneCards、OMIM数据库收集与DFU相关基因,建立疾病-靶点-化学成分网络。通过GO和KEGG通路富集分析探讨顾步汤对DFU可能的作用途径。构建NIH/3T3细胞和RAW264.7细胞共培养体系,将细胞随机分为空白组、模型组、抑制剂组及顾步汤低、中、高剂量组,除空白组外,其余各组均以脂多糖诱导RAW264.7细胞构建巨噬细胞模型,采用白细胞介素-4(20 ng/mL)诱导建立巨噬细胞M2极化模型。采用流式细胞术检测NIH/3T3细胞凋亡率及M2型巨噬细胞数量,CCK-8法检测NIH/3T3细胞增殖率,划痕实验检测NIH/3T3细胞迁移率,Western blot检测Arg-1、FIZZ1及Wnt/β-Catenin信号通路相关蛋白表达,RT-PCR检测Arg-1、FIZZ1、Wnt4a、β-Catenin mRNA表达,免疫荧光染色检测Wnt4a和β-Catenin蛋白表达。结果通过数据库检索获得顾步汤有效成分121个,主要成分对DFU的作用靶点共50个。KEGG通路富集分析得到顾步汤与DFU显著共关联的信号通路包括Wnt、PI3K-AKT、HIF-1信号通路等。细胞实验显示,顾步汤可显著降低NIH/3T3细胞凋亡率及M2型巨噬细胞数量,增加NIH/3T3细胞增殖率及迁移率,下调Arg-1、FIZZ1、Wnt4a、β-Catenin蛋白及mRNA表达,降低Wnt4a、β-Catenin免疫荧光强度。结论顾步汤能够刺激巨噬细胞M2极化,调控Wnt/β-Catenin信号通路,从而发挥治疗DFU作用。Objective To analyze the possible targets and related mechanisms of Gubu Decoction on diabetes foot ulcer(DFU)by network pharmacology method;To verify it by cell experiment.Methods TCMSP database was used to search for the main chemical components and targets of Gubu Decoction,and genes related to DFU were collected from GeneCards and OMIM databases.A disease-target-chemical component network was established.The possible pathways of action of Gubu Decoction on DFU were analyzed through GO and KEGG pathway enrichment.A co-culture system of NIH/3T3 cells and RAW264.7 cells was constructed,and the cells were randomly divided into blank group,model group,Gubu Decoction,low-,medium-,and high-dosage groups,and Wnt inhibitor group.Except for the blank group,all other groups were induced by lipopolysaccharide to construct macrophage models of RAW264.7 cells.IL-4(20 ng/mL)was used to induce the establishment of macrophage M2 polarization models.Flow cytometry was used to detect the apoptosis rate of NIH/3T3 cells and the number of M2 macrophages,respectively;CCK-8 method was used to detect the proliferation rate of NIH/3T3 cells;the migration rate of NIH/3T3 cell migration was measured by scratch test;Western blot was used to detect the expression of Arg-1,FIZZ1,and Wnt/β-Catenin signaling pathway related proteins;RT-PCR was used to detect Arg-1,FIZZ1,Wnt4a,β-Catenin gene expressions,and immunofluorescence staining was used for the detection of Wnt4a andβ-Catenin protein expressions.Results A total of 121 effective components of Gubu Decoction were retrieved through the database,and a total of 50 targets of the main components on DFU were identified.KEGG enrichment analysis revealed significant co-association between Gubu Decoction and DFU signaling pathways,including Wnt,PI3K-AKT,HIF-1,and other signaling pathways.Cell experiments showed that Gubu Decoction significantly reduced the apoptosis rate of NIH/3T3 cells and the number of M2 macrophages,up-regulated the proliferation rate and migration rate of NIH/3T3 cells,

关 键 词：网络药理学 糖尿病足溃疡 顾步汤 WNT/Β-CATENIN信号通路 巨噬细胞M2极化 

分 类 号：R259.872[医药卫生—中西医结合] R285[医药卫生—中医内科学]