FIGURE 3.

Erastin initiated inflammation responses and ECM degradation in chondrocytes that could be alleviated by Ferrostatin-1. (A,B) Cell viability determined by CCK-8 assay and toluidine blue staining. (C,D) The protein expression levels of collagen Ⅱ, MMP13, ACSL4, LOX15, lpcat3, P53, and SLC7A11 GPX4 when treated by erastin (5 μM) were detected by Western blot, and band density ratios of collagen Ⅱ, MMP13, ACSL4, LOX15, lpcat3, P53, and SLC7A11 GPX4 to GAPDH in the Western blots were quantified by densitometry (n = 3). (E,F) The protein expression levels of collagen Ⅱ, MMP13, ACSL4, LOX15, lpcat3, P53, and SLC7A11 GPX4 when treated by Erastin (5 μM) with fer-1 (1 μM) or equal volume of DMSO were detected by Western blot, and band density ratios of collagen Ⅱ, MMP13, ACSL4, LOX15, lpcat3, P53, and SLC7A11 GPX4 to GAPDH in the Western blots were quantified by densitometry (n = 3). (G) Intracellular ROS level detected by DCFH-DA fluorescent probe (scale bar: 200 µm). (H) Intracellular lipid-ROS level detected by C11 BODIPY fluorescent probe (scale bar: 100 µm). Red, reduced form of C11-BODIPY; green, oxidized form of C11-BODIPY. (I) The intracellular level of MDA and Fe²⁺ was determined using the MDA assay kit and iron assay kit (n = 3). (J) The collagen Ⅱ, MMP13, and ACSL4 expression levels in the cartilage samples were measured using immunohistochemistry staining. Dotted arrows indicate positive cells for MMP13 and ACSL4 and positive staining of collagen Ⅱ (scale bar: 100 µm). (K) Quantification of MMP13- and ACSL4-positive cells and collagen Ⅱ–positive staining in vivo. *p < 0.05 versus control or the sham group, **p < 0.01 versus control or the sham group, ***p < 0.001 versus control or the sham group, ****p < 0.0001 versus control or the sham group, #p < 0.05 versus IL-1β–treated group, ##p < 0.01 versus IL-1β–treated group, and ###p < 0.001 versus IL-1β–treated group. Error bars represent SD.