Abstract: BACKGROUND: Studies have shown that irisin can delay joint degeneration by modulating the metabolic homeostasis of chondrocytes and inhibiting inflammatory responses and oxidative stress.
OBJECTIVE: To further explore the mechanism by which irisin exerts therapeutic effects on osteoarthritis. 
METHODS: (1) Bioinformatics analysis: Gene expression data from cartilage tissues of patients with osteoarthritis and healthy controls were obtained from the GSE51588 and GSE207881 datasets. Gene set variation analysis assessed the activation of programmed cell death in osteoarthritis. Differential expression analysis identified differentially expressed genes between osteoarthritis and control samples, followed by pathway enrichment analysis. Ferroptosis-related differentially expressed genes were further identified, with those having an area under the receiver operating characteristic curve greater than 0.9 designated as core genes. (2) Cell experiment. Human articular chondrocytes were divided into four groups: control (chondrocytes), model (inflammatory models were established in chondrocytes induced by tumor necrosis factor α), model+Erastin (a ferroptosis inducer), and model+Erastin+irisin. ELISA measured glutathione, malondialdehyde and reactive oxygen species levels, while JC-1 assays assessed mitochondrial membrane potential. RT-qPCR quantified mRNA levels of core and ferroptosis-related genes, and western blot analyzed the expression of core genes, ferroptosis-related proteins, ERK pathway components, and apoptotic proteins. 
RESULTS AND CONCLUSION: (1) Gene set variation analysis indicated significant ferroptosis activation in osteoarthritis. (2) Differential expression analysis highlighted significant enrichment of differentially expressed genes in the ERK signaling pathway. Sixteen ferroptosis-related differentially expressed genes including HMOX1, G6PD, and ALOX5, were identified, all with the area under the curve values above 0.9. (3) In the human articular chondrocytes model+Erastin group, glutathione levels and mitochondrial membrane potential decreased significantly, while malondialdehyde and reactive oxygen species levels increased. The expression of HMOX1, G6PD, ALOX5, glutathione peroxidase 4, and SLC7A11 was significantly downregulated, while p-ERK and Bax levels rose, and Bcl2 decreased (all P < 0.05). Irisin treatment significantly improved ferroptosis-related markers (all P < 0.05). To conclude, HMOX1, G6PD, and ALOX5 may be potential therapeutic targets of osteoarthritis. Irisin offers protective effects in osteoarthritis by modulating ferroptosis-related genes and pathways.

Key words: osteoarthritis, irisin, ferroptosis, therapeutic target, core gene, bioinformatics analysis, signaling pathway