Abstract
Fangji Huangqi decoction (FHD), as a classic traditional Chinese medicine formula, has been clinically proven effective against rheumatoid arthritis (RA), yet its therapeutic mechanism remains unclear. This study employed network pharmacology and molecular docking methods to explore the major active components, biological targets, and signaling pathways of FHD. Subsequently, lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used as the in vitro model to validate the modulating effects of FHD on molecules/inflammatory mediators using various biomedical techniques/kits such as MTT assay, Griess reagents, flow cytometry, RT-qPCR, and immunoblotting. Network pharmacology analyses indicated a total of 20 major active components and 30 core biological targets of FHD against RA. Pathway enrichment analyses demonstrated the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the efficacy of the formula. Furthermore, experimental evidence demonstrated that FHD dose-dependently and significantly inhibited the productions of nitric oxide (NO) and reactive oxygen species; lowered the mRNA expression levels of proinflammatory mediators including iNOS, COX-2, TNF-α, ΙL-1β, and IL-6; decreased protein levels of the phosphorylated forms of p38, ERK, JNK, and NF-κB p65. Additionally, the results of molecular docking showed that tetrandrine, licochalcone A, oxonantenine, isorhamnetin, and kaempferol in FHD exerted the potent capability of binding to target molecules in the focused signaling pathway, probably being the potential effective substances for FHD. Our network pharmacology study integrated with cellular validation has elucidated that FHD exerts downregulating effects of the MAPK and NF-κB signaling pathway, ultimately leading to inhibitory effects on the productions of proinflammatory mediators in LPS-stimulated RAW264.7 cells. This work comprehensively demonstrated the effective substances, key targets, and signaling pathways involved in the anti-RA effects of the formula, and these findings provide a further understanding of the underlying mechanism of FHD in managing RA.