Fig. 5

Resatorvid alleviates NLRP3-mediated chondrocyte pyroptosis and degeneration. A LPS was utilized as an activator of TLR4 and incubated with ATDC5 cells. Immunofluorescence detected that the expression of NLRP3 was enhanced in LPS-incubated ATDC5 cells in a dose-dependent manner, one-way ANOVA, mean ± SEM, n = 3 (*p < 0.05 vs 0 μg/ml). B, C Western blotting examining the effect of LPS stimulation on ATDC5 cells for 24 h (A). Quantitative analysis revealed that the production of TLR4, NLRP3, and degenerative mediators (ADAMTS5, MMP13 and COX-2) was increased in LPS-induced ATDC5 cells in a dose-dependent manner (B), one-way ANOVA, mean ± SEM, n = 3 (*p < 0.05, **p < 0.01 vs 0 μg/ml). D Phase-contrast and fluorescence images of ATDC5 cells stained with Hoechst (blue) and propidium iodide (PI, red, positive staining indicates lytic cell death). The pyroptotic cell bubbles (white arrows) in merged images revealed that Resatorvid treatment relieved IL-1β-irritated ATDC5 chondrocyte pyroptosis, which was reversed by NLRP3 overexpression. E, F Western blotting (E) and quantitative analysis (F) of the production of NLRP3, pyroptotic biomarkers (Caspase-1, GSDMD) and degenerative mediators (ADAMTS5, MMP13, COX-2) in ATDC5 cells treated as indicated. Treatment with Resatorvid, PDTC (NF-κB inhibitor), or MCC950 (NLRP3 inhibitor) downregulated the production of these mediators in IL-1β-induced ATDC5 cells, which was abrogated by overexpression of NLRP3, Student’s t test, mean ± SEM, n = 3 (#p < 0.05, ##p < 0.01 vs control group; *p < 0.05, **p < 0.01 vs IL-1β group, ns indicates no significant difference)