Targeted screening of phenylpropanoids from Dendropanax proteus for rheumatoid arthritis therapy and mechanistic insights

Rheumatoid arthritis (RA) is a chronic inflammatory disorder. Toll-like receptor 4 (TLR4) and cyclooxygenase-2 (COX-2) are key mediators in its pathogenesis. Despite their crucial role, few studies have examined inhibitors targeting the TLR4–COX-2 axis. In this study, a combination of affinity ultrafiltration (AUF), surface plasmon resonance (SPR), serum pharmacochemistry, UHPLC-MS/MS, network pharmacology, cellular thermal shift assay (CETSA), isothermal dose–response (ITDR), and drug affinity–responsive target stability (DARTS) techniques were employed to explore TLR4–COX-2 inhibitors from Dendropanax proteus root extract (DPR). As a result, 10 phenylpropanoids were successfully identified from DPR. These constituents exhibited COX-2 inhibitory activity, with IC₅₀ values ranging from 5.2 to 36.2 μM. Molecular docking and molecular dynamics simulations confirmed their strong binding affinity to COX-2. In TNF-α–stimulated MH7A cells, these constituents significantly decreased COX-2 and PGE2 protein expression at concentrations of 2.5 and 5 μM. DPR modulates TLR4-mediated inflammatory signaling pathway against RA through network pharmacology, which was confirmed by CETSA, ITDR, and DARTS experiments. These phenylpropanoids significantly reduced TLR4 protein expression in TNF-α–stimulated MH7A cells at concentrations of 2.5 and 5 μM. Collectively, phenylpropanoids are the major anti-RA constituents of DPR, exerting their effects by inhibition of the TLR4-COX-2 signaling axis. While these findings are limited to in vitro and in silico models, they provide a strong scientific foundation for future in vivo efficacy studies and structural optimization of these phenylpropanoid leads.