ABSTRACT: Background: Chondrocyte homeostasis is vital for maintaining the extracellular matrix (ECM) and overall cartilage health. In osteoarthritis (OA), oxidative stress, often caused by redox imbalances, can disrupt chondrocyte homeostasis, leading to cartilage degradation. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a key mediator of oxidative stress and contributes to chondrocyte apoptosis and ECM degradation. Previous studies have explored individual protein responses to oxidative stress; however, a comprehensive proteomic analysis in chondrocytes has not been conducted. In this study, we aimed to assess the global proteomic alterations in chondrocytes exposed to H2O2 using a shotgun proteomics approach, which enables the detection of a broader spectrum of proteomic changes. Methods: Chondrocytes were treated with H2O2 for 1, 4, and 16 h followed by protein extraction and processing, including denaturation, alkylation, and trypsin digestion. The peptides were then acidified, desalted, dried, and resuspended for LC-MS/MS. Proteomics data were analyzed using MaxQuant software to identify and quantify proteins. Secretome analysis was performed to examine protein secretion changes under oxidative stress. The statistical significance of all proteomics and secretome data was assessed using a two-tailed Student’s t-test in the Perseus software. Complementary methods, including quantitative PCR, western blotting, and immunofluorescence, were employed to validate the proteomic analysis. Results: Our findings revealed that oxidative stress primarily affected the endoplasmic reticulum (ER), causing notable alterations in the expression of ER-associated proteins, redox-responsive enzymes, chaperones, and sialyltransferases. These changes increased intracellular accumulation of ECM proteins and decreased secretion into the extracellular environment, indicating impaired protein trafficking and secretion. Additionally, immune-related pathways were activated in the long term, with a short-term upregulation of inflammatory markers, such as interleukin (IL)-6) and IL-18, although the levels of matrix metalloproteinases (MMPs) remained stable, indicating a complex inflammatory response and stress responses that disrupt ECM homeostasis. Conclusions: Our study demonstrates a detailed proteomic view of the stress response of H2O2-treated chondrocytes, highlighting the significant changes in ER function, cytoskeletal remodeling, protein secretion, and immune responses. These changes suggest that oxidative stress impacts ECM balance and contributes to OA progression, offering new insights into the molecular mechanisms underlying oxidative stress in OA.
Hellen Valerio 
