Screening the effective metabolites of Mosla soochowensis Matsuda on LPS-induced inflammation in RAW264.7 cells by integrated LC-triple TOF-MS/MS and network pharmacology

Background Mosla soochowensis Matsuda (MSM) is a traditional Chinese herbal medicine empirically used for treating inflammatory conditions such as tonsillitis and the common cold. Previous studies focused solely on volatile metabolites and rudimentary antibacterial activity, but there is still a lack of detailed research on its non-volatile metabolites and their potential anti-inflammatory mechanisms. This lack of comprehensive phytochemical and mechanistic insight limits the scientific validation and modern application of MSM. In this study, the non-volatile metabolites of MSM were analyzed by using ultrafast liquid chromatography combined with triple quadrupole time-of-flight tandem mass spectrometry (LC-triple TOF-MS/MS). Potential anti- inflammatory targets were predicted via network pharmacology and molecular docking, and verified by in vitro experiments on LPS-induced RAW264.7 cells (ELISA for cytokines, RT-qPCR for gene expression). A total of 84 metabolites were identified; potential anti-inflammatory targets were predicted, with HSP90AA1, AKT1 and STAT3 emerging as central mediators. Molecular docking simulations supported strong binding interactions between core metabolites (moslosooflavone, mosloflavone, pachypodol) and these targets. Experiments in vitro using LPS-induced RAW264.7 macrophages demonstrated that the herb extract and key metabolites significantly suppressed the secretion of pro-inflammatory cytokines TNF- α and IL-6, and upregulated the gene expression of HSP90AA1, AKT1 and STAT3. This study could characterize MSM’s non-volatile metabolites for the first time, and preliminarily reveal a novel molecular mechanism underlying its anti-inflammatory effects, aligning with its traditional use. This multi-modal strategy bridges traditional knowledge with contemporary pharmacology, offering a scientific foundation for quality control and future development of MSM as a potential anti-inflammatory agent.