Fig. 7

Inflammatory mediators promote apoptosis via mitrochondrial oxidative stress. a Mitochondrial superoxide production was measured by MitoSox fluorescence under similar conditions (****P < 0.000 1). Bars are mean ± S. for n = 8 replicates from representative experiment. b Chondrocytes treated with IL-1β ± antimycin A (10 μmol·L⁻¹) or rotenone (100 μmol·L⁻¹) for 24 h. ROS levels in the cell were measured by DCFDA fluorescence (****P < 0.000 1). Bars represent mean ± SD for n = 8 replicates from representative experiments. c, d Chondrocytes treated with IL-1β or BP ± NAC (3 mmol·L⁻¹) for 24 h. Bax and Bcl2 gene expression normalized to actin expression. Bax and Bcl2 ratios were then determined, normalizing to untreated cells (A: Untreated vs IL-1β ***P = 0.000 5, IL-1β vs IL-1β + NAC *P = 0.041 9, B: Untreated vs BP ***P = 0.000 4, BP vs BP + NAC *P = 0.011 8). Bars are mean ± S.E.M from n = 5 independent experiments. e, f MLI surgery was performed on 12-week-old mice. Control sham surgery performed on contralateral knee. Joints were collected, embedded in paraffin and sectioned. IHC for cleaved caspase-3 and γ-H2AX was performed, with brown stain indicating positive cells. Representative images are displayed. g, h Chondrocytes were exposed to IL-1β or BP for 24 h ± rotenone (100 μmol·L⁻¹). Il6 and Mmp13 gene expression was measured by qPCR (****P < 0.000 1). Bars are mean ± SD from representative experiment. i Chondrocytes were treated with IL-1β in the presence or absence of mitoTEMPO or Rotenone doses indicated. Western blotting was performed for IκB-ζ with actin used as control. Representative immunoblot is displayed. j Chondrocytes were exposed to IL-1β ± AI-1 (40 μmol·L⁻¹) for 24 h. Il6 gene expression was measured by qPCR. Bars represent mean ± SD from n = 4 replicates. k Western blot was performed for IκB-ζ under the same conditions. Representative immunoblot is displayed