Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β
1 Musculoskeletal Research Group, School of Veterinary Medicine and Science, Faculty of Medicine and Health Sciences, The University of Nottingham, Sutton Bonington Campus, Sutton Bonington LE12 5RD, United Kingdom
2 Bruker UK Limited, Coventry CV4 9GH, United Kingdom
3 WALTHAM® Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire LE14 4RT, United Kingdom
4 Proteomics Laboratory, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom
5 The D-BOARD European Consortium for Biomarker Discovery, The University of Nottingham, University Park, Nottingham NG7 2RD, UK
6 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Nottingham University Hospitals, Nottingham NG7 2UH, United Kingdom
7 Arthritis Research UK Pain Centre, The University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom
8 Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, The University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom
9 Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Center (KFMRC), King AbdulAziz University, Jeddah 21589, Kingdom of Saudi Arabia
10 Schools of Life Sciences and Pharmacy, University of Bradford, Richmond Road, Bradford BD7 1DP, United Kingdom
11 Faculty of Medicine and Health Sciences, The University of Nottingham, Sutton Bonington Campus, Sutton Bonington LE12 5RD, United Kingdom
Arthritis Research & Therapy 2013, 15:R223 doi:10.1186/ar4424Published: 30 December 2013
Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.
Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).
Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.
Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.