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Updated: Apr 13, 2026
Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness
Published on: March 18, 2022
ALOX5 knockdown alleviates cartilage damage in osteoarthritis by inhibiting the NF-κB signaling pathway
Gang Cheng1, Kaiwen Chen2, Zhigang Wang2
1Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu Province 215004, China; Department of Orthopedics, The No. 2 People's Hospital of Wuhu City, Anhui Province 241000, China.
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.
Insights
Arachidonate 5-lipoxygenase (ALOX5) is upregulated in osteoarthritis (OA). Inhibiting ALOX5 alleviates OA by reducing ferroptosis and extracellular matrix degradation via the NF-κB pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Pathology
Background:
- Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage breakdown.
- The molecular mechanisms underlying OA pathogenesis remain incompletely understood, necessitating the identification of novel therapeutic targets.
Purpose of the Study:
- To investigate the role and mechanisms of arachidonate 5-lipoxygenase (ALOX5) in OA.
- To explore ALOX5 as a potential therapeutic target for OA treatment.
Main Methods:
- Transcriptome sequencing and differential gene expression analysis in OA cartilage.
- Immunohistochemistry, Western blot, and database analysis to confirm ALOX5 expression.
- In vitro (IL-1β) and in vivo (DMM) OA models to assess ALOX5 function in ferroptosis and extracellular matrix (ECM) degradation.
- KEGG-GSEA and Western blot to analyze downstream pathways, including NF-κB signaling.
Main Results:
- ALOX5 expression is significantly increased in OA cartilage and chondrocytes.
- ALOX5 knockdown reduces pro-inflammatory cytokines, ferroptosis markers (ROS, Fe2+), and ECM degradation factors (MMP3, MMP13, ADAMTS5).
- ALOX5 inhibition upregulates ferroptosis inhibitors (GPX4, SLC7A11), restores collagen II, alleviates OA in rats, and suppresses NF-κB pathway activation.
Conclusions:
- ALOX5 is a key mediator in OA progression.
- Inactivating the NF-κB pathway through ALOX5 inhibition mitigates ferroptosis and ECM degradation, offering a potential therapeutic strategy for OA.
