背景:蒙古蒿以其在民间医学中的用途而闻名,它通常用于缓解与炎症相关的各种疾病,比如喉炎,扁桃体炎,中国西北地区的头痛和肝炎。然而,其抗炎机制尚不清楚。
目的:通过筛选蒙古曲霉的单个部分,鉴定出最具潜力的抗炎部分(AMPA)。经过网络药理分析,评价AMPA对LPS诱导的RAW264.7细胞的抗炎作用及其分子机制。
方法:选择AMPA作为最抗炎的蒙古曲霉,通过蒙古曲霉各部分对NO和COX-2的影响来测量。使用HPLC-Q-TOF-MS/MS鉴定AMPA的化学组成,使用开源数据库发现了生物活性化学物质的靶标和与炎症相关的靶标。“复合目标”网络和PPI网络是通过组合化合物和重叠目标而建立的,并通过GO和KEGG富集分析了PPI网络中的靶标。将LPS诱导的RAW26.7细胞作为炎症检查的模型。进行MTT测定以评估AMPA对LPS诱导的RAW264.7细胞的细胞毒性。用Griess法测定NO水平,用ELISA检测炎症因子。iNOS的蛋白表达水平,COX-2,MAPK,Westernblot检测NF-κB信号通路和AMPK/Nrf2相关蛋白。免疫荧光法分析了p65和Nrf2的核易位结果。
结果:共鉴定出18个具有潜在生物活性的化合物,在与640个复合预测目标和1608个炎症目标相交之后,化合物和相交的目标被用来构建“化合物-目标”和PPI网络。在AMPA中,AM6、AM7、AM11、AM8和AM1化合物在“化合物靶标”网络中是必不可少的,同时,TNF,RELA,MAPK1,NOS2,PRKAG,PTGS2目标在PPI网络中起着重要作用。基于GO和KEGG获得前10个术语和途径。细胞实验显示,在LPS诱导的RAW264.7细胞模型中,50μg/mL是无细胞毒性的AMPA的最大浓度。与LPS组相比,AMPA治疗不仅有效抑制了NO的产生,TNF-α,IL-6、PGE2、IL-1β和MCP-1在LPS诱导的RAW264.7细胞中的表达,同时下调COX-2、iNOS和p-ERK蛋白水平的表达,p-p38,p-IκB-α和p-p65抑制p65的核易位。此外,p-LKB1、p-AMPK、Nrf2和HO-1蛋白上调,促进Nrf2核易位。
结论:对于网络药理学和体外实验结果,应将AMPA视为抗炎药。在LPS刺激的RAW264.7细胞中,可以抑制MAPK通路和NF-κB通路并激活AMPK/Nrf2通路。
BACKGROUND: Artemisia mongolica is well known for its use in folk medicine, it is commonly used to alleviate a variety of diseases associated with inflammation, such as laryngitis, tonsillitis, headaches and hepatitis in northwest China. However, its anti-inflammatory mechanism is still unknown.
OBJECTIVE: The most potential anti-inflammatory part (AMPA) was identified by screening individual parts of A. Mongolica. After the network pharmacological analysis, the anti-inflammation effects and molecular mechanisms of AMPA were evaluated in RAW264.7 cells induced by LPS.
METHODS: AMPA was chosen as the most anti-inflammatory of the A. Mongolica, as measured by the effect of each part of the A. Mongolica on NO and COX-2. The chemical composition of AMPA was identified using HPLC-Q-TOF-MS/MS, and targets of bioactive chemicals and targets related to inflammation were found using open-source databases. The \"Compound-targets\" network and PPI network were established by combining compounds and overlapped targets, and targets in the PPI networks were analyzed by GO and KEGG enrichment. The RAW26.7 cells induced by LPS were used as a model of inflammation examination. MTT assay was performed to assess the cytotoxicity of AMPA on LPS-induced RAW264.7 cells. The level of NO was measured by the Griess method while the inflammatory factors were detected by ELISA. The protein expression levels of iNOS, COX-2, MAPK, NF-κB signaling pathway and AMPK/Nrf2-related proteins were determined by Western blot. The results of nuclear translocation of p65 and Nrf2 were analyzed by immunofluorescence assay.
RESULTS: A total of 18 compounds with potential bioactivity were identified, and after intersecting 640 compound-predicted targets and 1608 inflammation targets, the compounds and intersected targets were utilized to structure \"compound-target\" and PPI networks. Among AMPA, AM6, AM7, AM11, AM8 and AM1 compounds were essential in the \"compound-targets\" network, meanwhile, TNF, RELA, MAPK1, NOS2, PRKAG, and PTGS2 targets play important roles in the PPI network. The top 10 terms and pathways were obtained based on GO and KEGG. The cell experiments show that 50 μg/mL was the maximum concentration of AMPA without cytotoxicity in the LPS-induced RAW264.7 cell model. When compared with the LPS group, AMPA treatment not only effectively suppressed the generation of NO, TNF-α, IL-6, PGE2, IL-1β and MCP-1 in LPS-induced RAW264.7 cells, but also down-regulated the expression of COX-2, iNOS and the protein levels p-ERK, p-p38, p-IκB-α and p-p65, inhibited the nuclear translocation of p65. Furthermore, the expression levels of p-LKB1, p-AMPK, Nrf2 and HO-1 proteins were up-regulated and Nrf2 nuclear translocation was promoted.
CONCLUSIONS: AMPA should be considered an anti-inflammatory agent for the results of network pharmacology and in vitro, which could inhibit the MAPK pathway and NF-κB pathway and activate the AMPK/Nrf2 pathway in LPS-stimulated RAW264.7 cells.