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. 2015 Sep:86:57-64.
doi: 10.1016/j.freeradbiomed.2015.05.008. Epub 2015 May 19.

Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions

John A Collins  1 , Robert J Moots  2 , Peter D Clegg  1 , Peter I Milner  3
Affiliations

Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions

John A Collins et al. Free Radic Biol Med. 2015 Sep.

Abstract

Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in <1, 2, 5, and 21% O2 at pH 7.2 or 6.2 in the absence or presence of the proinflammatory cytokine, interleukin-1β (10ng/ml).In addition, chondrocytes were cultured with resveratrol (10µM) or N-acetylcysteine (NAC) (2mM).Cell viability, glycosaminoglycan (GAG) release, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), GSH:GSSG ratio, and SOD1 and SOD2 protein expression were measured. Very low levels of oxygen (<1%), acidosis (pH 6.2), and exposure to IL-1β led to reductions in cell viability, increased GAG release, alterations in ΔΨm and ROS levels, and reduced GSH:GSSG ratio. In addition, SOD1 and SOD2 protein expressions were reduced. Both resveratrol and NAC partially restored ΔΨm and ROS levels and prevented GAG release and cell loss and normalized SOD1 and SOD2 protein expression. In particular NAC was highly effective at restoring the GSH:GSSG ratio.These results show that the antioxidants resveratrol and N-acetylcysteine can counteract the redox imbalance in articular chondrocytes induced by low oxygen and acidic conditions.

Keywords: Acidosis; Antioxidants; Cartilage; Oxygen; Redox balance.

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Figures

Fig. 1
Fig. 1
Effect of oxygen tension, pH, and IL-1β on glyosaminoglycan (GAG) release from equine articular chondrocytes in the absence or presence of resveratrol or N-acetylcysteine. Equine articular chondrocytes were cultured in 3D alginate beads for 48 h in <1, 2, 5, or 21% O2 at pH 7.2 (A), pH 6.2 (B), pH 7.2 plus 10 ng/ml IL-1β (C) or pH 6.2 plus 10 ng/ml IL-1β (D) in the absence or presence of N-acetylcysteine (2 mM) or resveratrol (10 µM). GAG release was measured in media using the dimethylmethylene blue (DMMB) assay. Bar charts represent mean±SEM, n=3. *P<0.05 versus control (time=0, 5%O2, pH 7.2).
Fig. 2
Fig. 2
Effect of oxygen tension, pH, and IL-1β on mitochondrial membrane potential (ΔΨm) in equine articular chondrocytes in the absence or presence of resveratrol or N-acetylcysteine. Equine articular chondrocytes were cultured in 3D alginate beads for 48 h in <1, 2, 5, or 21% O2 at pH 7.2 (A), pH 6.2 (B), pH 7.2 plus 10 ng/ml IL-1β (C) or pH 6.2 plus 10 ng/ml IL-1β (D) in the absence or presence of N-acetylcysteine (2 mM) or resveratrol (10 µM). ΔΨm was measured using the fluorescent probe JC-1. Bar charts represent mean±SEM, n=3. *P<0.05;†P<0.01 versus control (time=0, 5%O2, pH 7.2) or between groups where shown.
Fig. 3
Fig. 3
Effect of oxygen tension, pH, and IL-1β on reactive oxygen species levels in equine articular chondrocytes in the absence or presence of resveratrol or N-acetylcysteine. Equine articular chondrocytes were cultured in 3D alginate beads for 48 h in <1, 2, 5, or 21% O2 at pH 7.2 (A), pH 6.2 (B), pH 7.2 plus 10 ng/ml IL-1β (C) or pH 6.2 plus 10 ng/ml IL-1β (D) in the absence or presence of N-acetylcysteine (2 mM) or resveratrol (10 µM). ROS levels were measured using the fluorescent probe DCF-DA. Bar charts represent mean±SEM, n=3. *P<0.05; †P<0.01 versus control (time=0, 5%O2, pH 7.2) or between groups where shown.
Fig. 4
Fig. 4
Effect of oxygen tension, pH, and IL-1β on GSH:GSSG ratio in equine articular chondrocytes in the absence or presence of resveratrol or N-acetylcysteine. Equine articular chondrocytes were cultured in 3Dalginate beads for 48 h in <1, 2, 5, or 21% O2 at pH 7.2 (A), pH 6.2 (B), pH 7.2 plus 10 ng/ml IL-1β (C) or pH 6.2 plus 10 ng/ml IL-1β (D) in the absence or presence of N-acetylcysteine (2 mM) or resveratrol (10 µM). GSH:GSSG ratio was calculated using the GSH/GSSG-Glo assay. Bar charts represent mean±SEM, n=3. *P<0.05; †P<0.01 versus control (time=0, 5%O2, pH 7.2) or between groups where shown.
Fig. 5
Fig. 5
Representative Western blots showing the effect of oxygen tension, pH, and IL-1β on protein expression of superoxide dismutase 1 (Cu/ZnSOD, SOD1), superoxide dismutase 2 (MnSOD, SOD2), and α-tubulin from equine articular chondrocytes. Equine articular chondrocytes were cultured in 3D alginate beads for 48 h in <1, 2, 5, or 21% O2 at pH 7.2 or pH6.2 in the presence or absence of 10 ng/ml IL-1β (A), in the presence of resveratrol (10 µM) (B) or N-acetylcysteine (2 mM) (C).
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