β-Sitosterol protects human chondrocytes from IL-1β -induced damage by inhibiting ferroptosis

Objective To investigate the effect of β-Sitosterol on interleukin (IL)-1β-induced chondrocyte injury and the related mechanism. Methods An osteoarthritis model was constructed by treating immortalized human cartilage cells with IL-1β. The identity and purity of β-Sitosterol were confirmed by FTIR, 1 H NMR, 13 C NMR, RP-HPLC-ELSD, and UPLC-MS before cell experiments. The optimal concentrations of IL-1β and β-Sitosterol for treating cells were determined by qPCR and CCK8 assay. The cells were divided into three groups: Control group, Model group, and Model+β-Sitosterol group. Cell viability was detected by CCK8 assay. The mRNA expression of IL-18, IL-1β and tumor necrosis factor-α (TNF-α) was detected by qPCR. Collagen type II alpha 1 chain (COL2A1) and matrix metalloproteinase 13 (MMP13) expression was detected by immunofluorescence. Reactive oxygen species (ROS) and mitochondrial membrane potential were detected by flow cytometry. Malondialdehyde (MDA), Glutathione (GSH) and Fe 2+ were detected by ELISA and biochemical test. Mitochondrial structure was observed by transmission electron microscopy, and the protein expression of ferroptosis related factors was detected by Western blot. Results After induction of chondrocytes with IL-1β, the cell viability was decreased, the gene expression of inflammatory cytokines was increased, the proportion of cells with reduced ROS content and mitochondrial membrane potential were increased, MDA and Fe 2+ content were increased, and GSH content was decreased. Mitochondrial structure showed ferroptosis-like changes. The expression of COL2A1, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), total nuclear factor erythroid 2-related factor 2 (NRF2), and nuclear NRF2 was decreased. MMP13, ANO6, and p-p38/p38 levels were increased. Treatment of chondrocytes with β-Sitosterol could reverse these changes induced by IL-1β. Conclusion Spectrally confirmed, high-purity β-Sitosterol alleviated IL-1β-induced chondrocyte injury by suppressing inflammatory activation, extracellular matrix degradation, mitochondrial dysfunction, and ferroptosis-related oxidative damage, suggesting its potential as a candidate compound for osteoarthritis intervention.