Sidaxue ameliorates rheumatoid arthritis by inhibiting synovial neutrophil migration: Insights from an integrated multi-omics analysis.

The therapeutic outcomes for rheumatoid arthritis (RA) remain unsatisfactory. Neutrophil migration and activation in the synovium are pivotal drivers of RA pathology. Sidaxue (SX), a traditional Miao herbal formula, has shown anti-RA potential, yet its mechanism concerning neutrophil migration is unclear. This study aims to evaluate the effects of SX on neutrophil migration in RA and to uncover the novel molecular mechanisms. We integrated bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) with Mendelian randomization (MR) to pinpoint critical targets. Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) characterized SX's components, with binding affinities evaluated by molecular docking and dynamics simulations. Functional assays used SX-containing serumin vitro and a collagen-induced arthritis (CIA) model in vivo. Multi-omics analysis identified the RAC1/PAK1 pathway as central to neutrophil migration in RA. MR analysis nominated PAK1 as a potential drug target. We identified 38 major compounds in SX, which demonstrated high binding affinity to RAC1 and PAK1. SX-containing serum potently inhibited phorbol 12-myristate 13-acetate (PMA)-induced neutrophil migration, neutrophil extracellular traps (NETs) formation, and pro-inflammatory cytokine release. These effects were mediated through suppression of the RAC1/PAK1/LIMK1/Cofilin1 signaling axis, a finding corroborated by rescue experiments with the modulator Fingolimod hydrochloride (FTY). In vivo, SX alleviated joint swelling and synovitis in CIA rats, concurrently downregulating this pathway. SX ameliorates RA by inhibiting neutrophil migration and activation via the RAC1/PAK1/LIMK1/Cofilin1 pathway. This work unveils the immune-regulatory mechanism of SX, supporting its development as a natural therapeutic for RA.