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.