Fig. 1
IL-1 receptor blockade by IL-1Ra restored autophagy to attenuate ECM degeneration in rat OA chondrocytes. a, b Cells pretreated with IL-1β (20 ng/ml) for 36 h were treated with IL-1β (20 ng/ml)+IL-1Ra (40 ng/ml) for 36 h. The Col II (1:2000), Aggrecan (1:1000), Beclin1 (1:1000), LC3B (1:1000), and β-actin (1:40,000) protein levels were detected via western blotting (a). Col2A1, AGAN, BECN1, Map1lc3b, and GAPDH mRNA levels were detected via RT-PCR (b). c Cells pretreated with IL-1β (20 ng/ml) for 36 h were treated with IL-1β (20 ng/ml)+IL-1Ra(40 ng/ml) for 36 h, IL-1β (20 ng/ml) for 33 h followed by co-treatment with 3-MA (10 mM) for 3 h, or IL-1β (20 ng/ml)+3-MA (10 mM) for 3 h followed by co-treatment with IL-1Ra (40 ng/ml) for 33 h, respectively. The Col II (1:2000), Aggrecan (1:1000), Beclin1 (1:1000), LC3B (1:1000), P62 (1:1000), and β-actin (1:40,000) protein levels were detected via western blotting. d, e Cells pretreated with IL-1β (20 ng/ml) for 36 h were treated with IL-1β (20 ng/ml)+IL-1Ra (40 ng/ml) or IL-1β (20 ng/ml)+EBSS (as positive control) for 36 h, respectively. Representative images of immunofluorescence staining (LC3B (red), nucleus staining-DAPI (blue), original magnification × 40, d). Representative images of the transmission electron microscope (autophagic vacuoles indicated by white arrows, e). f, g Cells pretreated with IL-1β (20 ng/ml) for 36 h were treated with IL-1β (20 ng/ml)+IL-1Ra (40 ng/ml) for 36 h, followed by separation of cytoplasmic and nuclear fractions. The LC3B (1:1000), Lamin B (1:1000), and β-tubulin (1:1000) protein levels were detected via western blotting (f). Protein extracts in nucleus were subjected to immunoprecipitation with anti-LC3B antibody. The immunoprecipitates were immunoblotted with anti-acetylated-lysine antibody (1:1000) (g). h Cells pretreated with IL-1β (20 ng/ml) for 36 h were treated with IL-1β (20 ng/ml) + IL-1Ra (40 ng/ml) for 36 h. The mTOR (1:1000), p-mTOR (1:1000), ULK1 (1:1000), p-ULK1 (1:1000), Akt (1:1000), p-Akt (1:1000), NF-κB (p65, 1:1000), p-p65 (1:1000), and β-actin (1:40,000) protein levels were detected via western blotting. The values represent the means ± 95% CI of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)