Phytochemical Profiling and Proteomic Insights into the Anti-Inflammatory Effects of Jing Guan Fang in LPS-Stimulated Macrophages

Jing Guan Fang (JGF) is a traditional multi-herb formula used for hyperinflammatory conditions associated with severe viral infections; however, its protein-level effects in macrophage-mediated inflammation remain incompletely characterised. In this study, we combined ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based phytochemical profiling, functional anti-inflammatory assays, and discovery-level proteomic and secretome analyses to investigate the anti-inflammatory effects of JGF in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Phytochemical profiling revealed predominantly flavonoid- and phenolic-related annotated features, together with selected terpenoid- and alkaloid-related annotations. Functionally, JGF treatment was associated with reduced nitric oxide production and differential effects on pro-inflammatory cytokine release, with a clearer concentration-dependent reduction in interleukin-6 (IL-6) and a more variable tumour necrosis factor-α (TNF-α) response, without reducing cell viability. Label-free quantitative proteomics suggested that JGF treatment was associated with modulation of LPS-induced protein changes, including reduced abundance of inflammation-associated proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (NOS2), together with partial restoration of selected regulatory proteins such as AIMP1 and AIMP2. Secretome analysis further suggested that JGF reduced extracellular inflammatory and tissue-remodelling-related mediators, including SERPINE1/PAI-1. Exploratory pathway analysis indicated that JGF treatment was associated with changes in inflammation-related and oxidative stress-related signalling networks. Collectively, these findings provide discovery-level molecular insights into the anti-inflammatory effects of JGF in LPS-stimulated macrophages and support further targeted validation of its pharmacological activity as a candidate multi-component anti-inflammatory formula.