Multi-Targeted Therapeutic Mechanisms of Huangqi Guizhi Wuwu Decoction Against Rheumatoid Arthritis: An Integrated Approach Combining Serum Pharmacochemistry, Network Pharmacology, Metabolomics, and Experimental Validation

<b>Background</b>: The pathogenesis of rheumatoid arthritis (RA) is closely related to multiple disorders in the immune and metabolic systems, which indicates that a multi-target therapy strategy may have advantages over traditional single-target therapy. Huangqi Guizhi Wuwu Decoction (HGWD), as a classic traditional Chinese medicine formula that has been used to treat RA in clinic, is a potential source of multi-target natural medicine. However, its active components and mechanism of action still need further research. <b>Methods</b>: This study combined serum pharmacochemistry, non-targeted metabonomics, network pharmacology, and experimental verification and comprehensively analyzed the therapeutic mechanism and pharmacodynamic basis of HGWD. <b>Results</b>: Through HPLC-Q-TOF-MS/MS, a total of 99 chemical components were identified. Among them, 25 prototype compounds were absorbed into the systemic circulation. The study of network pharmacology indicates that these compounds are concentrated in TNF, IL-17, and MAPK signaling pathways. In collagen-induced arthritis rats, HGWD can effectively alleviate joint inflammation, inhibit the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-17), block the activation of the MAPK pathway, and restore 13 abnormal metabolic markers related to lipid and amino acid metabolism. In addition, the researchers identified and verified the combination of four active components (calycosin, paeoniflorin, 6-gingerol, and formononetin) in vitro, and its anti-inflammatory and anti-migration activities were equivalent to or stronger than those of the complete extract. Pharmacokinetic analysis also confirmed that these components were fully exposed in vivo. <b>Conclusions</b>: These findings reveal the mechanism of multi-component therapy of HGWD and identify the potential bioactive components, which can be used to develop multi-target therapeutic drugs for RA based on natural products.