基于网络药理学及实验验证探究环丙沙星加重主动脉夹层的潜在致病机制

Potential pathogenic mechanism of ciprofloxacin in regulating aortic dissection based on network pharmacology and experimental verification

目的 通过网络药理学及分子对接技术,筛选环丙沙星调控主动脉夹层发生发展的关键靶点基因,并通过动物及细胞实验进一步探究其潜在致病机制。方法 通过TargetPrediction、PharmMapper、GeneCards、CTD数据库分别筛选环丙沙星及主动脉夹层的相关靶点,二者取交集后得到环丙沙星调控主动脉夹层的潜在靶点;使用基因与蛋白质相互作用检索数据库(STRING)构建蛋白质相互作用(PPI)网络分析潜在靶点间的关联。通过对潜在靶点进行京都基因与基因组百科全书(KEGG)通路富集分析和基因本体(GO)功能富集分析,构建“药物–靶点–通路–疾病”网络。利用Cytoscape软件及蛋白质复合物聚类算法(MCODE)等插件进一步筛选出关键靶点,通过AutoDock vina和PyMol软件把环丙沙星化学结构与筛选出的关键靶点进行分子对接及可视化呈现。最后结合动物模型构建、苏木精–伊红(HE)染色法、细胞增殖和毒性检测实验、体内外实时荧光定量反应(RT-qPCR)实验及蛋白质免疫印迹(Western blotting),检测小鼠主动脉组织及人主动脉平滑肌细胞(HASMCs)关键靶点基因表达。结果 本研究筛选出515个环丙沙星相关靶点,9 004个主动脉夹层相关靶点,并得到412个环丙沙星介导主动脉夹层的潜在靶点。KEGG通路富集分析结果显示,主要富集于血脂与动脉粥样硬化、白细胞介素(IL)-17信号通路、肿瘤坏死因子(TNF)信号通路等。GO功能富集分析结果显示,细胞定位(CC)主要富集于膜筏、膜微区等;分子功能(MF)主要富集于受体结合、异生物跨膜转运蛋白活性等;生物学过程(BP)主要富集于对脂多糖的反应、对细菌来源分子的反应等。STRING及Cytoscape分析构建PPI网络图及关键子模块,获得了15个关键靶点,分别为B淋巴细胞瘤-2(Bcl-2)、转录因子AP-1(JUN)、CD44、胱天蛋白酶3(CASP3)、Toll样受体4(TLR4)、γ干扰素(IFNG)、肿瘤蛋白P53(TP53)、蛋白激酶B1(Akt1)、白蛋白(ALB)、基质金属蛋白酶9(MMP9)、白细胞介素(IL)-6、IL-10、IL-1β、甘油醛-3-磷酸脱氢酶(GAPDH)、肿瘤坏死因子(TNF)。分子对接结果显示,环丙沙星与MMP9、ALB、GAPDH、Akt1、TP53、CASP3、IL-1β的对接模式较好。体内实验表明,相较于模型组,模型+环丙沙星组在成膜率和死亡率上均增加,管壁弹性结构破坏也更加严重;促凋亡基因及促炎基因的m RNA表达水平及蛋白表达水平均增加。体外实验发现,相较于对照组,不同浓度环丙沙星刺激HASMCs细胞后,MMP9、IL-6、CASP3、JUN、IL-1β、TP53和TLR4表达水平明显上调,Akt1表达水平明显下调。结论 环丙沙星是介导主动脉夹层发生发展的重要因素之一,可能通过刺激炎症因子表达及VASMCs凋亡发挥调控作用。

Objective To screen the key target genes of ciprofloxacin in regulation of the occurrence and development of aortic dissection through network pharmacology and molecular docking technology,and further explore its potential pathogenic mechanism through animal and cell experiments.Methods TO screen the related targets of ciprofloxacin and aortic dissection were from TargetPrediction,PharmMapper,GeneCards,and CTD databases respectively,and the potential targets of ciprofloxacin regulating aortic dissection were obtained after the intersection of the related targets.PPI network was constructed using the search tool for STRING database to analyze the association between potential targets.The potential targets were analyzed by KEGG pathway enrichment and GO function enrichment analysis,and the“drug-target-pathway-disease”network was constructed.Cytoscape software and MCODE and other plug-ins were used to further screen out the key targets.The chemical structure of ciprofloxacin and the key targets were docked and visualized by AutoDock vina and PyMol software.Combined with animal model construction,HE staining,cell proliferation,and toxicity detection experiments,RT-Qpcr experiments,and Western blotting were used to detect the key target gene expression in mouse aortic tissues and human aortic smooth muscle cells(HASMCs).Results In this study,515 Ciprofloxacin-related targets,and 9004 aortic dissection-related targets were screened,resulting in 412 potential ciprofloxacin-regulated aortic dissection targets.KEGG pathway enrichment analysis showed that they were mainly enriched in lipid and atherosclerosis,IL-17 signaling pathway,TNF signaling pathway,etc.GO functional enrichment analysis showed that CC was mainly enriched in membrane raft and membrane microdomain.MF was mainly enriched in cytokine receptor binding and xenobiotic transmembrane transporter activity.BP were mainly enriched in the response to lipopolysaccharide,the response to molecule bacterial origin,etc.STRING and Cytoscape analysis were used to construct the PPI network and key sub-modules,and 15 key targets were obtained.They were Bcl-2,JUN,CD44,CASP3,TLR4,IFNG,TP53,Akt1,ALB,MMP9,IL-6,IL10,IL-1B,GAPDH,and TNF.Molecular docking results showed that ciprofloxacin had a good docking mode with MMP9,ALB,GAPDH,Akt1,TP53,CASP3,and IL1β.In vivo experiments showed that compared with the model group,the model+ciprofloxacin model group had increased film formation rate and mortality rate,and more serious damage to the elastic structure of the vessel wall.The mRNA and protein expression levels of pro-apoptotic and pro-inflammatory genes were increased.In vitro experiments showed that compared with the control group,the expression levels of MMP9,IL-6,CASP3,JUN,IL-1β,TP53,and TLR4 were significantly up-regulated,and the expression level of Akt1 was significantly down-regulated in HASMCs cell stimulated with different concentrations of ciprofloxacin.Conclusion Ciprofloxacin is one of the important factors mediating the occurrence and development of aortic dissection,which may play a regulatory role by stimulating the expression of inflammatory factors and VASMCs cell apoptosis.