Abstract

Background Anoikis is programmed cell death triggered by loss of cell–extracellular matrix adhesion and is essential for synovial homeostasis. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) can acquire anoikis resistance, promoting synovial hyperplasia and inflammation. We tested whether inadequate responses to methotrexate (MTX) relate to persistent anoikis resistance and sought compounds that may restore anoikis sensitivity. Methods RA synovial transcriptomic datasets were analyzed to quantify anoikis-related gene (ARG) signatures before and after MTX therapy. Anoikis-related differentially expressed genes were used for network analysis and compound screening using the Connectivity Map (CMap). Triptolide was prioritized, assessed by molecular docking, and validated in detachment-induced anoikis assays in RA-FLS and in a collagen-induced arthritis (CIA) rat model. Results ARG signatures remained dysregulated after MTX treatment, consistent with persistent anoikis resistance. Protein–protein interaction analysis identified CDKN1A, PARP1, MAPK1, and CASP8 as hub genes. CMap ranked triptolide as the top candidate, and docking supported stable interactions with these targets. In vitro, triptolide increased detachment-induced apoptosis in RA-FLS versus MTX alone and regulated ARG expression, with corresponding protein changes confirmed by western blotting. In vivo, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) showed increased synovial apoptosis in triptolide-treated CIA rats. Conclusion MTX does not fully reverse anoikis resistance in RA synovium, whereas triptolide enhances anoikis-associated responses and modulates key hubs, supporting anoikis dysregulation as a therapeutic target and triptolide as a potential adjunct strategy for MTX-insufficient RA.