Formononetin and Rhein from Bitong Mixture Alleviate Rheumatoid Arthritis-Related Inflammation: An Integrated WGCNA, Machine Learning and In Vitro Study

Background: Bitong Mixture (BTM) is used clinically for rheumatoid arthritis (RA), but its active components and mechanisms remain unclear. Methods: WGCNA, network pharmacology, machine learning, and ROC analysis were integrated to identify candidate targets. We used molecular docking and molecular dynamics simulations for prioritization. In vitro validation was performed in TNF-α-stimulated MH7A cells. Results: Multiple analytical approaches consistently identified JUN and PPARG. Formononetin and Rhein showed favorable binding and stable interactions. Both compounds significantly reduced IL6 and MMP9 expression and suppressed fibroblast activation. Conclusions: BTM may exert anti-inflammatory effects through a JUN- and PPARG-related regulatory framework, supported by integrated computational and experimental evidence, providing a foundation for further in vivo investigation.