Targeting 5-methylcytosine RNA methylation–mediated CX26/IκBα/NF-κB signaling by salvianolic acid B attenuates rheumatoid arthritis progression

Background Developing novel therapeutic targets and innovative drugs for rheumatoid arthritis (RA) remains highly challenging. Despite the involvement of 5-methylcytosine (m5C) RNA methylation in various biological processes, its specific roles in RA and potential applications in drug development remain unclear. Additionally, the role of CX26 in RA progression and its inflammatory effects on fibroblast-like synoviocytes (FLSs) have not been elucidated. Salvianolic acid B (SAB), a bioactive component derived from Salvia miltiorrhiza , possesses anti-inflammatory properties, but its potential in RA treatment has not been completely explored. Purpose This study aimed to clarify the role of m5C-methylated gene CX26 in RA-FLSs and evaluate the therapeutic potential of SAB for RA therapy. Methods Multi-omics analysis was used to determine the contribution of m5C RNA methylation to RA pathogenesis and characterize m5C-methylated genes during RA progression. Methylated RNA immunoprecipitation sequencing and RNA sequencing were employed to analyze the effect of SAB on m5C methylation of CX26 in RA-FLSs. Immunohistochemistry, quantitative real-time PCR, Western blot, Cell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine, and Transwell assays were applied to assess the effects of CX26 and SAB on RA-FLSs. The anti-arthritic activity of SAB was validated in a collagen-induced arthritis (CIA) mouse model. Results This study demonstrated a vital role of m5C RNA methylation in RA progression. CX26 was identified as an m5C-methylated gene that was selectively overexpressed in RA-FLSs. The CX26 levels in the synovium of patients with RA demonstrated significant associations with an activated immuno-inflammatory phenotype, clinical features, and treatment outcomes. Moreover, the CX26 mRNA m5C methylation, stability and expression were elevated by TNF. CX26 augmented inflammation-induced proliferation, migration, and invasion of RA-FLSs via activating the IκBα/NF-κB signaling. SAB reduced m5C methylation of CX26 mRNA and impair CX26 expression, thus abrogating CX26/IκBα/NF-κB signaling–induced pathological phenotypes of RA-FLSs. Furthermore, computational modeling and the CIA model confirmed that SAB alleviated RA progression. Conclusions This cross-validated study, based on multi-omics analysis, outlines the potential of CX26 as a highly selective target and SAB as a novel therapeutic candidate for RA treatment.