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目的：基于网络药理学、分子对接及细胞实验验证探讨高粱泡叶醇提物（AELR）抗炎的疗效及作用机制。方法：采用超高效液相色谱-高分辨四极杆轨道阱质谱（UHPLC-Q-Exactive HF/MS）技术分析AELR的化学成分，通过SwissADME平台筛选AELR化学成分，再用SwissTargetPrediction数据库检索潜在靶点；OMIM、GeneCards、TTD数据库检索疾病靶点；Venny2.1平台得到“成分-疾病”交集靶点，并构建蛋白质-蛋白质相互作用（protein-protein interaction,PPI）网络；利用DAVID数据库对交集靶点进行GO功能及KEGG信号通路富集分析；用AutoDock等软件对关键活性成分和核心靶点进行分子对接验证；最后，采用LPS诱导RAW264.7细胞构建体外炎症模型，通过Griess、ELISA以及流式细胞仪法检测细胞上清液相关炎症因子水平，RT-PCR、Western blot技术检测细胞相关蛋白mRNA和信号通路蛋白表达。结果：UHPLC-Q-Exactive HF/MS共鉴定出56种化合物；网络药理学筛选得到21种AELR活性成分及419个靶点，炎症疾病靶点1 923个，交集靶点187个，其中IL-1B、AKT1、STAT3、EGFR、PTGS2为核心靶点，与石吊兰素、木犀草素、异泽兰黄素、苜蓿素、棕矢车菊素活性成分分子对接结果良好；GO和KEGG分析发现，AELR抗炎可能通过PI3K/AKT、MAPK、NF-κB信号通路转导。体外细胞实验表明，与模型组比较，AELR能显著降低细胞上清液中的NO、IL-6、TNF-α以及细胞内ROS水平含量（P<0.01）；显著降低细胞COX-2、IL-6、TNF-α、IL-1βmRNA表达（P<0.01）,COX-2(P<0.01)蛋白表达下调，p-p65/p65、p-IκBα/IκBα、p-JNK/JNK、p-ERK/ERK、p-Akt/Akt蛋白表达比值均降低（P<0.01）。结论：AELR具有抗炎作用，其抗炎作用机制可能是通过多成分和多靶点来抑制NF-κB、MAPK及Akt信号通路的蛋白表达。
Abstract：Objective: To explore the anti-inflammatory efficacy and mechanism of action of Rubus lambertianus leaf ethanol extract(AELR) based on network pharmacology,molecular docking and cellular experimental validation. Methods: The chemical composition of AELR was analyzed by ultra-high performance liquid chromatography coupled with high-resolution quadrupole orbit trap mass spectrometry(UHPLC-Q-Exactive HF/MS).The AELR chemistry was screened by SwissADME platform and then the SwissTargetPrediction database was used to retrieve potential targets;OMIM,GeneCards,and TTD databases were used to search for disease targets;Venny2.1 platform was used to obtain“component-disease”intersection targets and construct protein-protein interaction(PPI) networks;GO function and KEGG signaling pathway enrichment analysis of intersecting targets were conducted using the DAVID database;Molecular docking validation of key active ingredients and core targets was performed using software such as AutoDock;Finally,an in vitro inflammation model was constructed using LPS-induced RAW264.7 cells.The levels of cell supernatant-associated inflammatory factors were detected by Griess,ELISA,and flow cytometry,and cell-associated protein mRNA and signaling pathway protein expression were detected by RT-PCR and Western blot techniques.Results: A total of 56 compounds were identified by UHPLC-Q-Exactive HF/MS;21 AELR active ingredients and 419 targets were obtained by network pharmacological screening,with 1 923 inflammatory disease targets and 187 intersecting targets,among which IL-1B,AKT1,STAT3,EGFR,PTGS2 were the core targets,and the molecular docking results with the active ingredients of stigmasterol,lignoceroside,isoxanthin,brown cornflower,cloverberry Xanthin,brown cornflower,alfalfa active ingredient molecular docking results were good;GO and KEGG analyses revealed that AELR anti-inflammatory may be transduced through PI3K/AKT,MAPK,and NF-κB signaling pathways.In vitro cellular experiments showed that AELR significantly reduced NO,IL-6,TNF-αand intracellular ROS levels in cell supernatants compared with the model group(P<0.01),significantly reduced cellular COX-2、IL-6、TNF-α、IL-1β mRNA expression reduction(P<0.01),and significantly down-regulated p-p65/p65、p-IκBα/IκBα、p-JNK/JNK、p-ERK/ERK、p-Akt/Akt protein expression(P<0.01). Conclusion: AELR has anti-inflammatory effects,and its anti-inflammatory mechanism of action may be inhibition of the protein expression of NF-κB,MAPK and Akt signaling pathways through multiple components and targets.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20241106.001
中图分类号:R285.5
引用信息:
[1]田孟斌,吴静澜,李旭东,等.基于网络药理学、分子对接和细胞实验探讨高粱泡叶醇提物抗炎的作用机制[J].海南医科大学学报,2025,31(12):938-953.DOI:10.13210/j.cnki.jhmu.20241106.001.
基金信息:
贵州省中医药管理局中医药、民族医药科学技术课题（QZYY-2022-011）; 贵州中医药大学药用高分子材料研究中心（贵中医党办发[2019]70号）~~
2024-11-06
2024-11-06
2024-11-06