Figure 6.

AMPK/FoxO3a signaling is involved in ASIC1a-mediated articular chondrocytes autophagy. (A) rat articular chondrocytes were treated with extracellular acid (pH 6.0) for the indicated time periods. Cells were then lysed to determine the expression level of phosphorylated AMPKα (Thr172) by Western blot analysis; (B) pretreatment with ASIC1a-specific blocker, PcTx1 and calcium chelating agent, BAPTA-AM for 1 h, articular chondrocytes were stimulated with extracellular acid (pH 6.0) for 30 min. The level of p-AMPK/AMPK was determined by Western blotting; (C) rat articular chondrocytes were transfected with siRNA specific for AMPKα or scrambled control and then treated with extracellular acid (pH 6.0). After 30 min incubation, cells were then lysed to determine the expression level of FoxO3a by Western blot analysis; (D) rat articular chondrocytes transfected with siRNA for AMPKα or scrambled control were treated with extracellular acid. The levels of Beclin1, LC3B-II protein expression were determined by Western blot followed by densitometry analysis; (E,F) rat articular chondrocytes were pretreated with Compound C (10 µM) followed by treatment with extracellular acid (pH 6.0) for 30 min. The protein expression levels of total and nuclear FoxO3a were examined by Western blotting; (G,H) rat articular chondrocytes were incubated with or without 10 μM Compound C for 1 h and then stimulated with extracellular acid (pH 6.0) for 3 h. The mRNA and protein expression of Beclin1, LC3B-II were determined by qRT-PCR and Western blotting; (I) articular chondrocytes were transfected with EGFP-LC3 plasmid and then treated as for (H) followed by examining with fluorescence microscopy. Scale bar = 20 μm. GFP-LC3 puncta were quantified for each experiment, with at least 30 cells counted in each experiment. Data were presented as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus pH 6.0 group.