Targeting the NOX4/NLRP inflammasome axis confers protection by isoliquiritigenin against H2O2-induced synovial inflammation

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Fibroblast-like synoviocytes (FLS) play a central role in RA pathogenesis by promoting inflammatory signaling and extracellular matrix (ECM) degradation. Although isoliquiritigenin (ISL) exhibits anti-inflammatory and antioxidant activities, its direct molecular target in RA remains unclear. Therefore, this study aimed to identify the molecular target of ISL and elucidate its underlying mechanism. To address this, integrated bioinformatics analysis, target validation (CETSA, DARTS, and molecular docking), together with mechanistic studies in H2O2-treated synovial cells and CIA rats, were performed. Bioinformatics analysis identified NADPH oxidase 4 (NOX4) as one of the overlapping candidate targets of ISL for further investigation. Target engagement of NOX4 by ISL was supported by CETSA, DARTS, and molecular docking analyses. ISL suppressed NOX4-mediated reactive oxygen species (ROS) production, thereby inhibiting inflammasome activation, pro-inflammatory cytokine production, apoptosis, and ECM degradation.ISL improved cell survival and reduced apoptosis in H2O2-treated synovial c ells while preserving ECM integrity. These protective effects were further confirmed in CIA rats, where ISL alleviated arthritis severity, oxidative stress, and cartilage destruction. Collectively, these findings identify NOX4 as a critical upstream regulator linking oxidative stress to inflammasome activation and support pharmacological targeting of the NOX4–ROS–inflammasome axis by ISL as a promising therapeutic approach for RA.