Mechanism of the DDX3X/NLRP3/GSDMD Signaling Axis in Pyroptosis and Inflammatory Response in Osteoarthritis Chondrocytes

Osteoarthritis (OA) is a chronic disorder involving pain and functional disturbance due to its impact on joint tissues. This study investigated the mechanism of the DEAD-box helicase 3 X-linked (DDX3X)/NOD-like receptor protein 3 (NLRP3)/gasdermin D (GSDMD) pathway in pyroptosis and inflammatory responses in OA chondrocytes. Mouse chondrocyte ATDC5 cells were cultured in vitro and induced for chondrogenic differentiation, followed by H₂O₂ treatment to simulate OA. Cells were transfected for 24 h with siRNA-DDX3X, pcDNA3.1-NLRP3, pcDNA3.1-GSDMD, and corresponding negative controls. Cell viability was evaluated by CCK-8. Reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β, and IL-18 levels in the supernatant, DDX3X, GSDMD, and NLRP3 mRNA and protein levels, and pyroptosis-related protein (Collagen II, Aggrecan, Cleave-Caspase-1, full-length GSDMD [GSDMD-FL], GSDMD-N-terminal domain [GSDMD-NT], apoptosis-associated spot-like protein [ASC]) levels were assessed by ELISA, RT-qPCR, and western blot. The NLRP3-DDX3X interaction was predicted and validated by the STRING database and Co-IP assay. H₂O₂-treated ATDC5 showed decreased cell viability, and Collagen II and Aggrecan levels, increased GSDMD-FL cleavage, supernatant ROS, IL-6, TNF-α, IL-1β, and IL-18 levels, DDX3X mRNA and protein expression, Cleave-Caspase-1, GSDMD-NT, NLRP3, and ASC levels, and an enhanced NLRP3-DDX3X interaction. However, DDX3X knockdown partially reversed the above phenomenon in H₂O₂-treated ATDC5, indicating that suppressing the DDX3X/GSDMD axis attenuated OA chondrocyte pyroptosis and inflammatory response in vitro. NLRP3 or GSDMD overexpression partly counteracted DDX3X knockdown-improved OA chondrocyte pyroptosis and inflammation. Inhibiting the DDX3X/NLRP3/GSDMD axis alleviated chondrocyte pyroptosis and inflammatory response in OA in vitro, thus ameliorating chondrocyte injury.