Autophagy is activated in compression-induced cell degeneration and is mediated by reactive oxygen species in nucleus pulposus cells exposed to compression

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Autophagy is activated in compression-induced cell degeneration and is mediated by reactive oxygen species in nucleus pulposus cells exposed to compression K.-G. Ma, Z.-W. Shao, S.-H. Yang, J. Wang, B.-C. Wang, L.-M. Xiong, Q. Wu, S.-F. Chen Osteoarthritis and Cartilage Volume 21, Issue 12, Pages 2030-2038 (December 2013) DOI: 10.1016/j.joca.2013.10.002 Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Effects of compression on cell morphology. The effects of compression on rat NP cell morphology as assessed using an inverted microscope. Rat NP cells were subjected to 1 MPa compression for 0 (a), 12 (b), 24 (c), 36 (d) and 48 h (e), and displayed a time-dependent increase in cell death. The injured cells were characterized by cell shrinkage, and detachment from the plates when rat NP cells were exposed to compression for 48 h. (scale bar = 20 μm). Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 The cell viability of rat NP cells exposed to compression was determined by CCK-8 at 0, 12, 24, 36 and 48 h. The autophagy inhibitor 3-MA (5 mM) was applied to observe the effect of autophagy on the cell viability under stress conditions, and the compression treatment groups without 3-MA were selected as the control. Data were expressed as the means ± 95% confidence intervals. Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Ultrastructural observations of autophagic vesicles deposited in rat NP cells. The effects of 1 MPa compression on rat NP cell morphology were assessed by TEM. Untreated controls (a, b) displayed normal cell morphology. Double arrowheads indicated normal endoplasmic reticula. Compression-treated cells (c–e, 36 h exposure, f–h 48 h exposure) demonstrated special cell morphology as indicated by the double-membraned autophagosomes marked by arrowheads. The expansive endoplasmic reticula are indicated by double arrowheads and the autophagosomes by single arrowheads (e). Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The conversion of LC3B-I to LC3B-II in rat NP cells treated with compression. (A) 3-MA (5 mM) and CQ (25 μM) were applied to observe effects of autophagy inhibitors on compression-induced autophagy at 24 h. (B) A quantitative analysis of western blotting was shown by LC3B-II/GAPDH ratios. The data are expressed as the means ± 95% confidence intervals. (*P < 0.05, ***P < 0.001 vs the control group cells, ANOVA/LSD). Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 MDC staining of rat NP cells treated with 1 MPa compression. (A) Representative fluorescent images of MDC staining of rat NP cells treated with compression for 0 (a), 12 (b), 24 (c), 36 (d) and 48 h (e) (scale bar = 20 μm). Arrows point to cells stained positive for MDC; (B) Representative graphs of cell autophagy obtained by flow cytometry analysis after MDC staining. (C) Statistical analysis of the positive cell number of rat NP cells treated with compression and 3-MA (5 mM) for 0, 12, 24, 36 and 48 h. The values are expressed as the means ± 95% confidence intervals from three independent experiments. The P value of the 12 h group was 0.2835 vs control, while the P values were less than 0.01 vs control at 24, 36 and 48 h. Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 The effects of the autophagy inhibitor 3-MA (5 mM) on the compression-induced apoptosis in rat NP cells. (A) Representative graphs of cell apoptosis obtained by flow cytometry analysis after Annexin-V/PI dual staining. Statistical analysis of the apoptotic rate (B) and cell survival rate (C) of rat NP cells exposed to compression for 0, 12, 24, 36 and 48 h. The values are expressed as the means ± 95% confidence intervals from three independent experiments. Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 The level of intracellular ROS was measured by flow cytometry using an oxidation-sensitive fluorescent probe, DCFH-DA, which is oxidized to DCF in the presence of ROS. The histogram for statistical analysis shows the ROS levels in rat NP cells treated with compression, NAC (5 mM) and 3-MA (5 mM) for 0, 12, 24, 36 and 48 h. Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 The effects of the ROS scavenger NAC (5 mM) and the autophagy inhibitor 3-MA (5 mM) on the compression-induced autophagy in rat NP cells. (A) Representative western blotting graphs for LC3B, beclin-1 and GAPDH in rat NP cells subjected to compression for 0, 12, 24, 36 and 48 h. (B) Statistical analysis of the western blotting of LC3B-II- and GAPDH in these five groups. (C) Statistical analysis of the western blotting of beclin-1 and GAPDH in these five groups. Osteoarthritis and Cartilage 2013 21, 2030-2038DOI: (10.1016/j.joca.2013.10.002) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions