ALOX5 knockdown alleviates cartilage damage in osteoarthritis by inhibiting the NF-κB signaling pathway.

PURPOSE: This study aims to elucidate the role and underlying mechanisms of arachidonate 5-lipoxygenase (ALOX5) in osteoarthritis (OA) to identify novel therapeutic targets for this disease.

METHODS

Five samples of normal cartilage tissue and five samples of OA cartilage tissue were collected, followed by transcriptome sequencing was performed to analyze differentially expressed genes (DEGs) in OA. Immunohistochemistry (IHC), western blot, and analysis of the GEO database were used to verify ALOX5 expression in both humans and rat OA cartilage. IL-1β and destabilization of the medial meniscus (DMM) were employed to establish invitro and in vivo OA models, respectively, to assess the role of ALOX5 in Fe2+ accumulation, ROS generation, inflammatory cytokines release, ferroptosis, and extracellular matrix (ECM) degradation. Additionally, KEGG-GSEA was conducted to identify key downstream pathways regulated by ALOX5. Furthermore, western blot was used to assess the effect of ALOX5 on the NF-κB pathway, and the NF-κB activator betulinic acid (BA) was administered to chondrocytes to validate the regulatory role of ALOX5 in this pathway.

RESULTS

ALOX5 expression was significantly upregulated in OA cartilage tissues and in IL-1β-induced chondrocytes. Knockdown of ALOX5 attenuated the production of pro-inflammatory cytokines, including IL-6 and TNF-α; reduced the acculation of ferroptosis-associated markers such as ROS and Fe2+; and suppressed the ezpression of key ECM degradation-related factors, including MMP3, MMP13, and ADAMTS5. Conversely, ALOX5 silencing led to a significant upregulation of ferroptosis inhibitors GPX4 and SLC7A11, as well as trstored expression of collagen II, the major cartilage component. In OA rats, intra-articular injection of AAV-shALOX5 significantly alleviated cartilage damage. Moreover, ALOX5 knockdown significantly reduced the expression ratios of p-p65/p65 and p-IKKβ/IKKβ. Notably, treatment with BA reversed the protective effects conferred by ALOX5 knockdown, restoring NF-κB pathway activation and subsequently promoting ferroptosis and ECM degradation in chondrocytes.

CONCLUSION

ALOX5 is upregulated in OA. Knockdown of ALOX5 alleviates OA progression by inactivating the NF-κB signaling pathway, thereby inhibiting ferroptosis and ECM degradation in chondrocytes.