基于网络药理学探讨臭灵丹抗炎的作用机制  

Anti-inflammatory mechanism of Laggerae Herba based on network pharmacology

作　　者：周永芝 舒腾云 宋玉莹 郑立雄 李海舟[1] ZHOU Yong-zhi;SHU Teng-yun;SONG Yu-ying;ZHENG Li-xiong;LI Hai-zhou(Kunming University of Science and Technology,Faculty of Life Science and Technology,Kunming 650500,China;National Engineering Research Center for Modernization of Traditional Chinese Medicine,Weihe Pharmaceutical Co.,Ltd.,Yuxi 653101,China)

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]国家中药现代化工程研究中心西南分中心,云南维和药业股份有限公司,玉溪653101

出　　处：《天然产物研究与开发》2024年第8期1420-1431,共12页Natural Product Research and Development

基　　金：国家自然科学基金(181360643);昆明理工大学药食同源学科方向支持项目(331814078324)。

摘　　要：基于LC-MS和网络药理学对臭灵丹乙酸乙酯部位(ethyl acetate fraction of Laggerae Herba,ELH)进行成分和靶点通路分析,并通过LPS(lipopolysaccharides,LPS)诱导RAW 264.7炎症模型探讨ELH抗炎的作用机制。通过Griess实验,比较臭灵丹不同萃取部位的抗炎活性;利用LC-MS对ELH的化学成分进行分析;基于网络药理学预测和筛选ELH发挥抗炎的核心成分,靶基因和主要通路,并用分子对接和蛋白免疫印迹(Western blot)对靶点和通路进行验证。Griess实验结果表明,ELH可显著抑制LPS诱导的RAW 264.7细胞释放NO,抑制率达103.07%,是臭灵丹发挥抗炎作用的活性部位;LC-MS分析得到ELH中23个化学成分,包括21个倍半萜,1个黄酮,1个有机酸体;基于4个数据库,获得66个共同靶点;蛋白互作网络(protein-protein interaction,PPI)分析发现获得白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF)等degree值大于40的6个核心靶点;GO、KEGG富集分析发现,核心靶点可能通过PI3K-Akt、IL-17、NF-κB和TNF等信号通路发挥抗炎作用;分子对接结果显示23个化学成分与核心靶点具有良好的结合能力;Western blot实验结果显示ELH处理后Akt的磷酸化水平和NF-κB表达显著下降(P<0.05)。综上,ELH能抑制AKT磷酸化,在一定程度上抑制LPS诱导的RAW 264.7细胞中NF-κB信号通路的激活,从而发挥抗炎作用。This study aims to analyze the components and target pathways of the ethyl acetate fraction of Laggerae Herba(ELH)based on LC-MS and network pharmacology,exploring its anti-inflammatory mechanisms in vitro using an lipopolysaccharides(LPS)-induced RAW 264.7 inflammatory model.The anti-inflammatory activities of various extracts of Laggerae Herba were assessed using the Griess assay.Additionally,LC-MS was utilized to analyze the chemical components of ELH.Network pharmacology aided in predicting and screening the core components,target genes,and principal pathways that mediate anti-inflammatory effects,which were then confirmed through molecular docking and Western blot analysis.Results from the Griess assay demonstrated that ELH significantly inhibited NO release in LPS-induced RAW 264.7 cells,achieving an inhibition rate of 103.07%and identifying it as the active fraction.LC-MS analysis revealed 23 chemical components in ELH,including 21 sesquiterpenes,one flavonoid,and one organic acid.Using four different databases,66 common targets were identified.Protein-protein interaction(PPI)analysis pinpointed six core targets,including interleukin-6(IL-6)and tumor necrosis factor(TNF),each with a degree value exceeding 40.GO and KEGG enrichment analyses indicated that these core targets could modulate anti-inflammatory effects through the PI3K-Akt,IL-17,NF-κB,and TNF signaling pathways.Molecular docking confirmed the strong binding affinity of the 23 chemical components to the core targets.Western blot results indicated significant reductions in Akt phosphorylation and NF-κB expression post-ELH treatment(P<0.05).In conclusion,ELH can inhibit Akt phosphorylation and,to a certain extent,suppress the activation of the NF-κB signaling pathway induced by LPS in RAW 264.7 cells,thereby exerting potent anti-inflammatory effects.

关 键 词：臭灵丹 网络药理学 炎症 分子对接 LC-MS分析 

分 类 号：R285.5[医药卫生—中药学]