Catalpol mitigates rheumatoid arthritis by targeting neutrophil extracellular trap release

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent synovial inflammation, progressive joint damage, and systemic manifestations. Current therapies, including NSAIDs, DMARDs, and biologics, face limitations such as side effects and high costs. Neutrophil extracellular traps (NETs), driven by peptidylarginine deiminase 4 (PAD4), play a pivotal role in RA pathogenesis by promoting inflammation and cartilage degradation. Catalpol (CAT), an iridoid glycoside known for its anti-inflammatory properties, exhibits therapeutic promise through targeting NET-related mechanisms.

A collagen-induced arthritis (CIA) model was established in male DBA/1 mice. Mice were treated with CAT (30 mg/kg) or vehicle. Joint damage was assessed via micro-CT and histological staining (H&E, Safranin O, Toluidine Blue). NETs and inflammatory markers were analyzed by immunohistochemistry, Western blot, qRT-PCR, and immunofluorescence. Isolated neutrophils were stimulated with PMA in vitro to study CAT's direct effects on NETosis. Transcriptomic analysis was performed on joint tissues to identify potential targets.

CAT treatment significantly ameliorated ankle swelling, bone erosion, synovial hyperplasia, inflammatory cell infiltration, and cartilage degradation in CIA mice. It suppressed the expression of NET-associated markers (Cit-H3, MPO, NE) and pro-inflammatory cytokines (IL-1β, IL-6) in joint tissues. CAT also inhibited the expression of matrix-degrading enzymes (MMP3, MMP13) and osteoclast-related factors (MMP9, CTSK, Tracp-5b). In vitro, CAT (10 μM) effectively inhibited PMA-induced NET formation in primary neutrophils. Furthermore, CAT reduced NET-induced ROS production, mitochondrial membrane potential loss, and apoptosis in chondrocytes. Mechanistically, transcriptomic analysis suggested enrichment in neutrophil-related pathways, and Western blot confirmed that CAT inhibited PAD4 protein expression, a key regulator of NETosis.

Catalpol exerts a protective effect in the CIA model by inhibiting PAD4-mediated NET formation, thereby reducing synovial inflammation, cartilage degradation, and bone erosion. Our findings highlight NETs as a promising therapeutic target and suggest catalpol's potential as a novel agent for RA treatment.