基于网络药理学和体外实验研究血必净注射液治疗脓毒症相关ARDS的作用机制

Mechanism of Xuebijing Injection in treatment of sepsis-associated ARDS based on network pharmacology and in vitro experiment

使用网络药理学和体外实验的方法探讨血必净注射液在脓毒症相关急性呼吸窘迫综合征(脓毒症相关ARDS)中的作用及其分子机制。通过TCMSP数据库对血必净注射液活性成分进行筛选与靶点预测。在GeneCards、DisGeNet、OMIM、TTD数据库中进行脓毒症相关ARDS疾病靶点的检索及筛选。运用微生信平台将血必净注射液中主要活性成分的靶点与脓毒症相关ARDS的靶点进行映射取交集靶点,制作韦恩图。利用Cytoscape 3.9.1构建“药物-活性成分-交集靶点-疾病”网络图。将交集靶点输入到STRING数据库分析获得蛋白-蛋白互作(PPI)网络,然后导入Cytoscape 3.9.1软件进行可视化处理。使用DAVID 6.8对交集靶点进行基因本体(GO)及京都基因与基因组百科全书(KEGG)富集分析,然后使用微生信平台进行富集分析可视化。选取前20个KEGG信号通路导入Cytoscape 3.9.1软件中构建KEGG关系网络图。最后,使用分子对接与体外细胞实验对分析结果进行验证。在上述数据库中筛选得到血必净注射液115种活性成分、217个靶点,360个脓毒症相关ARDS疾病靶点,63个药物-疾病交集靶点。交集靶点中的核心靶点包括白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白蛋白(ALB)、丝氨酸/苏氨酸蛋白激酶1(AKT1)、血管内皮生长因子A(VEGFA)等。GO富集分析确定了453个条目,其中包括361个生物学过程(BP)、33个细胞成分(CC)和59个分子功能条目(MF),主要涉及细胞对脂多糖的反应、细胞凋亡过程的负调控、脂多糖介导的信号通路、RNA聚合酶Ⅱ启动子转录的正向调控、对缺氧的反应、炎症应答等。KEGG富集分析得到85条通路,剔除疾病、广义通路后,筛选出HIF-1信号通路、TNF信号通路、NF-κB信号通路、Toll样受体信号通路、NOD样受体信号通路等。分子对接表明血必净注射液主要活性成分与核心靶点具有良好的结合活性。体外实验证实,血必净注射液可以下调HIF-1、TNF、NF-κB、Toll样受体、NOD样受体信号通路,抑制细胞凋亡与ROS的生成,降低细胞TNF-α、IL-1β、IL-6的表达。因此,血必净注射液能够通过作用于HIF-1、TNF、NF-κB、Toll样受体、NOD样受体信号通路,调控凋亡、炎症、氧化应激等,治疗脓毒症相关ARDS。

The aim of this study was to investigate the effect and molecular mechanism of Xuebijing Injection in the treatment of sepsis-associated acute respiratory distress syndrome(ARDS)based on network pharmacology and in vitro experiment.The active components of Xuebijing Injection were screened and the targets were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The targets of sepsis-associated ARDS were searched against GeneCards,DisGeNet,OMIM,and TTD.Weishengxin platform was used to map the targets of the main active components in Xuebijing Injection and the targets of sepsis-associated ARDS,and Venn diagram was established to identify the common targets.Cytoscape 3.9.1 was used to build the"drug-active components-common targets-disease"network.The common targets were imported into STRING for the building of the protein-protein interaction(PPI)network,which was then imported into Cytoscape 3.9.1 for visualization.DAVID 6.8 was used for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KECG)enrichment of the common targets,and then Weishengxin platform was used for visualization of the enrichment results.The top 20 KECG signaling pathways were selected and imported into Cytoscape 3.9.1 to establish the KEGG network.Finally,molecular docking and in vitro cell experiment were performed to verify the prediction results.A total of 115 active components and 217 targets of Xuebijing Injection and 360 targets of sepsis-associated ARDS were obtained,among which 63 common targets were shared by Xuebijing Injection and the disease.The core targets included interleukin-1 beta(IL-1β),IL-6,albumin(ALB),serine/threonine-protein kinase(AKT1),and vascular endothelial growth factor A(VEGFA).A total of 453 GO terms were annotated,including 361 terms of biological processes(BP),33 terms of cellular components(CC),and 59 terms of molecular functions(MF).The terms mainly involved cellular response to lipopolysaccharide,negative regulation of apoptotic process,lipopolysaccharide-mediated signaling pathway,positive regulation of transcription from RNA polymerase I promoter,response to hypoxia,and inflammatory response.The KECG enrichment revealed 85 pathways.After diseases and generalized pathways were eliminated,hypoxia-inducible factor-1(HIF-1),tumor necrosis factor(TNF),nuclear factor-kappa B(NF-kB),Tll-like receptor,and NOD-like receptor signaling pathways were screened out.Molecular docking showed that the main active components of Xuebijing Injection had good binding activity with the core targets.The in vitro experiment confirmed that Xuebijing Injection suppressed the HIF-1,TNF,NF-B,Tll-like receptor,and NOD-like receptor signaling pathways,inhibited cell apoptosis and reactive oxygen species generation,and down-regulated the expression of TNF-α,IL-1β,and IL-6 in cells.In conclusion,Xuebijing Injection can regulate apoptosis and response to inflammation and oxidative stress by acting on HIF-1,TNF,NF-kB,Tolllike receptor,and NOD-like receptor signaling pathways to treat sepsis-associated ARDS.