Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis Z. Zainal,

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Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis Z. Zainal, A.J. Longman, S. Hurst, K. Duggan, B. Caterson, C.E. Hughes, J.L. Harwood Osteoarthritis and Cartilage Volume 17, Issue 7, Pages 896-905 (July 2009) DOI: 10.1016/j.joca.2008.12.009 Copyright © 2009 Terms and Conditions

Fig. 1 Uptake of α-linolenate into chondrocyte cultures. GLC traces are shown with control and IL-1α treated cultures and increasing amounts of α-linolenate added. See Materials and methods for procedures. Incubations with added linolenate were for 8 h. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 2 Morphology of chondrocytes in culture. Pictures were taken by light microscopy of control cells (A), cells treated with IL-1α (B), cells pre-incubated with 10μg/ml EPA followed by IL-1α treatment (C) and cells pre-incubated with 30μg/ml EPA followed by IL-1α treatment (D). Time points represent 0, 24, 48 and 72h treatment, respectively. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 3 Chondrocyte metabolism as measured by lactate production. Lactate release into serum-free culture medium with or without IL-1 was measured after the experimental period of 96h. Cultures were either pre-treated with or without fatty acids or BSA solution alone (as labelled in A–D). Means±S.D. are shown (n=3). Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 4 Variable effects of different PUFAs on mRNA levels for the proteinase ADAMTS-4. For procedures see Materials and methods. (A) shows RT-PCRs for ADAMTS-4 whose expression is induced by IL-1α and reduced by n-3 PUFAs. Pre-exposure to fatty acids or BSA alone (controls) was for 8h and incubation with or without 10ng/ml IL-1α was for 4 days. The histogram shows data from scans of gels from three separate experiments with means±S.D. given. Individual lanes show (L to R): controls (C), controls +30μg/ml fatty acid (FA), +IL-1α, +IL-1α and 2.5μg/ml FA, +IL-α and 5μg/ml FA, +IL-1α and 10μg/ml FA, +20μg/ml FA, +IL-1α and 30μg/ml FA. * indicate significantly different from +IL-1α alone (P<0.05) by Student's t test. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 5 Effects of exogenous PUFAs on mRNA levels for the proteinase ADAMTS-5. For procedures see Materials and methods. The results for three experiments (means±S.D.) are shown with EPA, DHA or ALA being compared. See legend to Fig. 4 for details. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 6 Effects of exogenous fatty acids on mRNA levels for the MMP-3 and MMP-13. (A) shows results for MMP-3 and (B) data for MMP-13. Exposure to EPA or DHA was for 8h and incubations with or without 10ng/ml IL-1α were for 4 days. Abbreviations and further details as for Fig. 4. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 7 n-3 PUFAs reduce mRNA levels for the inducible COX-2 but not for COX-1 in chondrocyte cultures. (A) shows results for COX-1 and (B) data for COX-2. Exposure to fatty acids was for 8h and incubations with or without 10ng/ml IL-1α were for 4 days. Abbreviations and further details as for Fig. 4. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions

Fig. 8 n-3 PUFAs reduce mRNA levels for inflammatory cytokines in chondrocyte cultures. RT-PCRs are shown for IL-1α (A), IL-1β (B) and TNF-α (C). Exposure to fatty acids was for 8h and incubations with 10ng/ml IL-1α for 4 days. The histograms show means±S.D. (n=3). Abbreviations as for Fig. 4 and IL-α, IL-1α; IL-1β; TNF-α. Individual conditions are as for Fig. 4 with results for EPA, DHA and ALA shown. Osteoarthritis and Cartilage 2009 17, 896-905DOI: (10.1016/j.joca.2008.12.009) Copyright © 2009 Terms and Conditions