Integrated single-cell and experimental validation links effector chondrocyte-associated oxidative stress to EIF6-related p47phox-NOX2 signaling in osteoarthritis.

Background: Oxidative stress is closely involved in osteoarthritis (OA), but the cell-type-specific oxidative stress landscape and related regulatory molecules in human cartilage remain incompletely defined. This study aimed to identify effector chondrocyte (EC)-associated oxidative stress features in OA and to prioritize candidate molecules linked to p47phox-NOX2-related ROS responses. Methods: Public human cartilage single-cell RNA sequencing datasets were integrated to construct an OA cartilage atlas. Oxidative stress activity was evaluated using multiple scoring methods, and high oxidative stress (HOS) cells were defined according to the top 10% of integrated oxidative stress scores. Cell-state and communication features were examined using CytoTRACE and CellChat. High-dimensional weighted gene co-expression network analysis, machine learning, protein-protein interaction network analysis, summary-data-based Mendelian randomization, and molecular docking were combined to prioritize candidate genes. EIF6 and YWHAB were examined in an inflammatory EC-like model, and EIF6-associated p47phox/TGF-β responses were further assessed in DMM-induced OA mouse tissues. Results: Single-cell analysis identified an EC population enriched in OA cartilage. ECs showed relatively high oxidative stress activity and represented the largest component of HOS cells. The proportion of ECs among HOS cells increased in OA samples, and HOS cells were concentrated near the EC-enriched region in the UMAP space. HOS/EC-enriched populations showed broad communication with other chondrocyte populations and were associated with inflammation, stress-related signaling, and extracellular matrix remodeling pathways. Integrated computational analyses prioritized EIF6 and YWHAB as candidate molecules associated with EC-related oxidative stress. In inflammatory EC-like cells, IL-1β and TNF-α increased EIF6 and YWHAB expression. EIF6 or YWHAB knockdown attenuated IL-1β-induced p47phox and NOX2 expression and reduced ROS-related fluorescence, with EIF6 knockdown showing a stronger effect. Co-immunoprecipitation suggested altered YWHAB-p47phox interaction under inflammatory stimulation. In DMM-induced OA tissues, EIF6 knockdown reduced p47phox upregulation, whereas EIF6 overexpression increased TGF-β staining, which was partly attenuated by p47phox knockdown. Conclusion: This study identifies EC-associated oxidative stress features in OA cartilage and prioritizes EIF6/YWHAB-associated p47phox-NOX2 signaling as a candidate axis linked to inflammatory ROS-related responses and tissue-level OA responses.