Targeting mitochondrial fission-autophagy axis in rheumatoid arthritis: leflunomide promote apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and joint destruction, driven by aberrantly activated fibroblast-like synoviocytes (RA-FLS). Mitochondrial dysfunction, particularly excessive mitochondrial fission, contributes to RA-FLS activation and apoptosis resistance, yet the impact of disease-modifying antirheumatic drugs (DMARDs) on mitochondrial dynamics remains unclear. Here, we examined the correlation between mitochondrial dynamics proteins and RA disease activity and investigated the effects of leflunomide and methotrexate (MTX) on mitochondrial dynamics, autophagy, and apoptosis in RA-FLS and collagen-induced arthritis (CIA) mice. Mitochondrial dynamics proteins in synovial fluid correlated more strongly with disease activity than those in peripheral blood, and were partially normalized in RA patients receiving leflunomide or MTX. Both leflunomide and MTX attenuated TNF-induced mitochondrial fragmentation and decreased mitochondrial membrane potential in RA-FLS. Notably, leflunomide, but not MTX inhibited DRP1 phosphorylation at Ser616, increased reactive oxygen species, and induced apoptosis via the BCL-2/BAX/caspase-3 pathway. Additionally, leflunomide influenced autophagy by promoting LC3B II/I and enhancing p62 expression. In CIA mice, leflunomide reduced the expression and phosphorylation of DRP1 on synovium, increased the expression of OPA1, and alleviated joint inflammation and destruction. These findings identify mitochondrial dynamics as a therapeutic target in RA and suggest that leflunomide promotes apoptosis of RA-FLS by modulating the mitochondrial fission-autophagy axis.