Inhibiting Arachidonate-5-lipoxygenase expression ameliorates osteoarthritis progression by suppressing ferroptosis via the JAK2/STAT3 signaling pathway.

Abstract

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease. Recent studies have shown that ferroptosis plays a critical role in OA. Arachidonate-5-lipoxygenase (ALOX5), a pivotal enzyme regulating arachidonic acid metabolism, is involved in the synthesis of the pro-inflammatory leukotrienes. However, its role in ferroptosis and OA has not been elucidated. METHODS: Measuring the expression of ALOX5 and ferroptosis-related markers in human cartilage and synovial fluid. Constructed small interfering RNA or plasmids were used to knockdown or overexpress Alox5 to explore its role in chondrocyte. Transcription factors regulating Alox5 were predicted through database analysis, while RNA sequencing revealed signaling pathways modulated by Alox5. Subsequently, the role of ALOX5 in destabilization of medial meniscus (DMM) OA model was investigated by intra-articular injection of knockdown (AAV-ShAlox5) or overexpression (AAV-OEAlox5) adeno-associated virus. Finally, the therapeutic efficacy of ALOX5 inhibitor Zileuton, was verified in vivo and in vitro. RESULTS: We observed upregulated ALOX5 expression in human cartilage and Alox5 knockdown mitigated IL-1β- and FAC-induced chondrocyte damage by suppressing ferroptosis. Through database predictive analysis and experimental verification, ALOX5 was modulated by the JNK-p53 signaling axis. Through RNA-seq analysis, it was found that knockdown of Alox5 inhibited the activation of the JAK2-STAT3 signaling pathway, thereby delaying the process of ferroptosis and slowing down the wear of articular cartilage. In the mouse DMM model, intra-articular injection AAV-ShAlox5 delayed DMM-induced cartilage degeneration, whereas AAV-OEAlox5 exacerbated cartilage damage. Similarly, treatment with Zileuton alleviated ferroptosis and chondrocyte injury,demonstrating a protective effect in vivo and in vitro. CONCLUSIONS: In summary, our study demonstrated knockdown of Alox5 inhibited the JAK2/STAT3 signaling, thereby suppressing ferroptosis and alleviating cartilage damage. Intra-articular injection of the ALOX5 inhibitor Zileuton or AAV-ShAlox5 in the knee joint alleviated cartilage damage in the mouse DMM model. Mechanistically, our study reveals that the JNK-p53-ALOX5 signaling axis alleviates OA by regulating ferroptosis in chondrocytes.