Fig. 4

Anti-arthritic effect of AT on IL-1β induced chondrocytes via activating mitochondrial autophagy through inhibiting PI3K/AKT/mTOR. A Western blot was used to analyze the proteins ATG-5, ATG-7, beclin-1, and LC-3, which are autophagy-related proteins. B Protein level of ATG-5, ATG-7, beclin-1, and LC-3 normalized to GAPDH using ImageJ software. C Real-time RT-PCR was performed to determine the gene expression of ATG-5, ATG-7, beclin-1, and LC-3. D Immunofluorescence staining for beclin-1, phalloidin was stained for cellular actin, and DAPI was stained for nuclear. Control: without IL-1β; IL-1β group: with 10 ng/mL; IL-1β + AT group: with 10 ng/mL IL-1β and 20 μM AT. E Immunofluorescence staining for mitochondria; phalloidin was stained for cellular actin, and DAPI was stained for nuclear. Control: without IL-1β; IL-1β group: with 10 ng/mL; IL-1β + AT group: with 10 ng/mL IL-1β and 5, 10 and 20 μM AT. F Western blot was used to analyze the proteins of the PI3K/AKT/mTOR signaling pathway, including PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR. G Protein level of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR normalized using ImageJ software. All experiments were performed in triplicate. Data represent the mean ± SD (n = 3), * P < 0.05, ** P < 0.01 and *** P < 0.001 versus the IL-1β group