Title : IL-1beta -induced oxidative stress and ferroptosis in osteoarthritis-derived chondrocytes: targeting ferroptosis inhibition through genetic therapy
Abstract:
Problem Statement: Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage breakdown and chronic inflammation. Interleukin-1 beta (IL-1β), a key pro-inflammatory cytokine in OA, disrupts iron metabolism, leading to excess intracellular iron and reactive oxygen species (ROS) generation. This imbalance may contribute to ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation. The role of ferroptosis in OA remains underexplored, particularly in relation to the enzyme ACSL4, which facilitates lipid peroxidation.
Objective: This study aimed to (1) establish an in vitro OA model using IL-1β to induce ferroptosis in chondrocytes and (2) investigate the effect of ACSL4 gene silencing via CRISPR-Cas9 to inhibit ferroptosis.
Methods: Primary human chondrocytes derived from OA patients were treated with IL-1β at concentrations ranging from 2 to 14 ng/ml for 24, 48, and 72 hours. Cell viability was assessed using the AlamarBlue assay, and morphological changes were evaluated microscopically. A CRISPR-Cas9 construct with sgRNA targeting ACSL4 was developed and transfected into chondrocytes. Transfection efficiency was analyzed by flow cytometry using green fluorescent protein (GFP) markers, and successful gene targeting was confirmed through Sanger sequencing.
Results: IL-1β treatment induced a dose- and time-dependent reduction in cell viability, along with noticeable morphological changes, confirming oxidative stress-induced damage. No significant difference was observed in cytotoxicity between OA and non-OA chondrocytes at 48 hours, supporting the use of OA-derived chondrocytes for modeling. CRISPR-mediated ACSL4 silencing was successful, as confirmed by both fluorescence intensity shifts in flow cytometry and sequencing.
Conclusion: This study successfully established an in vitro OA model and demonstrated the potential of targeting ferroptosis via ACSL4 silencing. The findings support further exploration of genetic therapies aimed at modulating ferroptosis as a novel approach for OA treatment.
