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. 2017 Jun 12;18(1):254.
doi: 10.1186/s12891-017-1621-2.

The Mitochondrial Inhibitor Oligomycin Induces an Inflammatory Response in the Rat Knee Joint

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Free PMC article

The Mitochondrial Inhibitor Oligomycin Induces an Inflammatory Response in the Rat Knee Joint

Carlos Vaamonde-García et al. BMC Musculoskelet Disord. .
Free PMC article

Abstract

Background: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints.

Methods: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated.

Results: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue.

Conclusions: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Keywords: Cartilage; Inflammation; Mitochondria; Oxidative stress; Synovial tissue.

Figures

Fig. 1
Fig. 1
Oligomycin can induce joint swelling and inflammatory changes in the synovial tissue. a Knee joint swelling as an indicator of inflammation is estimated from joint width (as described in Methods). Values are mean ± SEM (n = 5). *P ≤ 0.005 and **P ≤ 0.001 vs contralateral vehicle-injected knees. b Representative images of synovial tissue stained with hematoxylin and eosin from each group of study. c Semi-quantitative score of pathological alterations in synovial tissue as described in Methods. Data represent mean ± SEM (n = 5 independent samples for each condition). *P ≤ 0.05 vs contralateral vehicle-injected knees. d Percentage of PMN was analyzed as described in Methods. Values are mean ± SEM (n = 5 independent synovial tissues for each condition). *P ≤ 0.05 vs contralateral vehicle-injected knees. LPS, lipopolysaccharide; OLI, oligomycin
Fig. 2
Fig. 2
Mitochondrial dysfunction increases CD68 (panel a) positive cells and IL-8 (panel b) in the synovial membrane. a Representative samples of CD68 immunohistochemistry in synovial membrane. b Synovium sections were analyzed by immunohistochemistry for IL-8. Quantitative analysis of CD68 (c) and IL-8 (d) positive cells. Values represent mean ± SEM (n = 5 independent synovial tissues). To assess non-immune non-specific binding negative control was included in each experiment. * P ≤ 0.05 vs contralateral vehicle-injected knees. LPS, lipopolysaccharide; OLI, oligomycin
Fig. 3
Fig. 3
Oligomycin increases ROS production in synovial tissue. Synovial tissue frozen sections from OLI-vehicle or OLI injected joints were incubated with a cytoplasmic (DHE; a) or mitochondrial (MitoSox; b) superoxide-sensitive fluorescent dye (red). The nuclei were counterstained with DAPI (blue). Representative images of the different color channels and their merge are shown. DHE (c) or MitoSox (d) fluorescence levels were measured and calculated as described in Methods. Values, expressed in arbitrary units (AU), represent mean ± SEM (n = 7 independent synovial sections for each condition). *P ≤ 0.05 vs OLI-vehicle knees. OLI, oligomycin; DAPI, 4′,6-diamidino-2-phenylindole; DHE, dihydroethidium; MitoSox, MitoSOX™ Red Mitochondrial Superoxide Indicator
Fig. 4
Fig. 4
Oligomycin modulates COX-IV levels in the synovial tissue. a Immunofluorescence microscopy analysis of synovial tissue sections from OLI-vehicle or OLI injected joints stained for COX-IV (red) with DAPI counterstaining (blue). Representative images are presented. b Fluorescence levels were measured and calculated as described in Methods. Values, expressed in arbitrary units (AU), represent mean ± SEM (n = 7 independent synovial sections). *P ≤ 0.05 vs OLI-vehicle knees. OLI, oligomycin; DAPI, 4′,6-diamidino-2-phenylindole; COX-IV, cytochrome c oxidase subunit IV
Fig. 5
Fig. 5
4-HNE and Nrf2 are modulated by oligomycin and Nrf2 is expressed in the synovial from OA patient. a Representative images of 4-HNE immunohistochemistry synovial membrane sections from OLI-vehicle and OLI injected joints. b Representative images of Nrf2 immunohistochemistry synovial membrane sections from OLI-vehicle and OLI injected joints. c Representative images of Nrf2 immunohistochemistry synovial membrane sections obtained from healthy and OA patients. A score positive immunostaining for 4-HNE (d) and 2 (e) in control and oligomycin rats is shown, and for healthy human donors and patients with OA in (f). Values represent mean ± SEM (n = 5 independent synovial tissues for rats; and n = 8 independent synovial tissues per each condition for human, respectively). To assess non-immune non-specific binding negative control was included in each experiment. * P ≤ 0.05 vs contralateral vehicle-injected knees, and vs healthy human synovium. OLI, oligomycin; 4-HNE, anti-4-hidroxi-2-nonenal; Nrf2, nuclear factor erythroid 2-related factor 2
Fig. 6
Fig. 6
Effects of oligomycin on histopathological and inflammatory changes in the cartilage. a Semi-quantitative score of pathological alterations in cartilage was performed as described in Methods. Data represent mean ± SEM (n = 5 independent samples). b CINC-1 mRNA expression in cartilage. Values represent mean ± SEM (n = 5 samples for each condition). c CINC-1 protein released by cartilage explants from OLI-vehicle or OLI injected joints. Values are expressed as pg CINC-1 protein released per μg of DNA (mean ± SEM, n = 6 samples for each condition). *P ≤ 0.05 vs OLI-vehicle knees. OLI, oligomycin

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