基于网络药理学和分子对接探讨木犀草素抗急性肺损伤的分子机制

Molecular mechanism of luteolin against acute lung injury based on network pharmacology and molecular docking

目的通过网络药理学及分子对接技术探讨木犀草素抗急性肺损伤的作用机制,并进行实验验证。方法利用PubChem和SwissTargetPrediction数据库预测木犀草素作用的相关靶点;通过GeneCards数据库收集急性肺损伤相关靶点;以Venny 2.1绘制韦恩图,并得到药物与疾病的共同靶点;应用String在线平台建立蛋白互作网络(PPI),通过Cytoscape 3.8.2软件筛选出核心靶点;利用DAVID数据库将共同靶点进行GO功能富集分析和KEGG通路富集分析,将其结果可视化;最后,通过Auto Dock软件进行分子对接,并运用Pymol对分子结果进行可视化。构建小鼠急性肺损伤模型,采用苏木精伊红(HE)染色检测组织病理学,蛋白免疫印迹(Western blot)检测肺组织中相关蛋白。结果经筛选得到85个共同靶点,其中,核心靶点有AKT1、EGFR、SRC、MMP9、ESR1、PTGS2等。GO富集分析获得生物过程265条,主要包括信号转导、蛋白磷酸化、凋亡过程的负调控等过程;细胞组成48条,主要包括质膜、细胞溶质、细胞质等;分子功能107条,主要包括ATP结合、蛋白丝氨酸/苏氨酸/酪氨酸激酶活性、蛋白激酶活性等。KEGG通路富集分析共获得92条信号通路,主要作用于PI3K-AKT信号通路、ErbB信号通路、VEGF信号通路等。分子对接结果显示木犀草素与核心靶点AKT1、EGFR、SRC、MMP9、ESR1、PTGS2、MMP2、GSK3B、KDR、PARP1均具有良好的对接活性,其中ERS1、GSK3B、MMP2结合能低于-5.0 kal·mol^(-1),与木犀草素之间的亲和力更强。肺组织病理学结果显示,木犀草素在LPS诱导的小鼠急性肺损伤模型中,能够抑制炎症浸润,有较强的抗炎作用。Western blot实验显示,木犀草素可能通过抑制AKT的磷酸化缓解炎症反应。结论木犀草素可通过多靶点、多途径机制发挥抗急性肺损伤的作用,其中可能与抑制AKT的磷酸化密切相关。

Aim To explore the molecular mechanism of luteolin against acute lung injury by network pharmacology and molecular docking technology,and to conduct experimental verification.Methods The related targets of luteolin were predicted by PubChem and Swiss Target Prediction databases.Acute lung injury-related targets were collected through the GeneCards database.Venny 2.1 was used to draw the Venn diagram,and the common targets of drug and disease were obtained.The protein interaction network(PPI)was established by String online platform,and the core targets were screened by Cytoscape 3.8.2 software.The functional enrichment analysis of Gene Ontology(GO)and pathway enrichment analysis of Kyoto Encyclopedia of Gene and Genome(KEGG)were performed on the common targets using the DAVID database,and the results were visualized.Finally,molecular docking was performed by Auto Dock software,and the molecular results were visualized by Pymol.The mouse acute lung injury model was constructed.HE staining was used to detect histopathology,and Western blot was used to detect lung tissue related proteins.Results After screening,85 common targets were obtained.Among them,the core targets were AKT1,EGFR,SRC,MMP9,ESR1,PTGS2,etc.GO enrichment analysis obtained 265 biological processes,including signal transduction,protein phosphorylation,and negative regulation of apoptosis.There were 48 cells,mainly including plasma membrane,cell solute,cytoplasm,etc.There are 107 molecular functions,mainly including ATP binding,protein serine/threonine/tyrosine kinase activity,protein kinase activity and so on.A total of 92 signaling pathway were obtained by KEGG pathway enrichment analysis,which mainly acted on PI3K-AKT signaling pathway,ErbB signaling pathway,VEGF signaling pathway,etc.Molecular docking results showed that luteolin had good docking activity with core targets AKT1,EGFR,SRC,MMP9,ESR1,PTGS2,MMP2,GSK3 B,KDR and PARP1.The binding energy of ERS1,GSK3B and MMP2 was lower than-5.0 kal·mol^(-1),and the affinity with luteolin was stronger.The pathological results of lung tissue showed that luteolin could inhibit inflammatory infiltration and had a strong anti-inflammatory effect in LPS-induced acute lung injury model in mice.Western blot experiments showed that luteolin might alleviate the inflammatory response by inhibiting the phosphorylation of AKT.Conclusions Luteolin can play an anti-acute lung injury role through multi-target and multi-channel mechanisms,which may be closely related to the inhibition of AKT phosphorylation.