A potential role of chondroitin sulfate on bone in osteoarthritis: inhibition of prostaglandin E2 and matrix metalloproteinases synthesis in interleukin-1β-

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A potential role of chondroitin sulfate on bone in osteoarthritis: inhibition of prostaglandin E2 and matrix metalloproteinases synthesis in interleukin-1β- stimulated osteoblasts E. Pecchi, S. Priam, Z. Mladenovic, M. Gosset, A.-S. Saurel, L. Aguilar, F. Berenbaum, C. Jacques Osteoarthritis and Cartilage Volume 20, Issue 2, Pages 127-135 (February 2012) DOI: 10.1016/j.joca.2011.12.002 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Effect of CS on PGE2 release. Osteoblasts were treated for 7days (A) or 24h (B) in the presence or absence of CS (100μg/ml) or for 24h in the presence or absence of indomethacin (10μg/ml) (C). IL-1β (1 or 10ng/ml) was added for the latest 24h. The amount of the PGE2 release into the media (pg/ml) was measured by enzymatic immunoassay (EIA). PGE2 release are the mean with 95% CI of four independent experiments with two wells/condition, analyzed in duplicate (corrected P-values: ***P<0.001). Osteoarthritis and Cartilage 2012 20, 127-135DOI: (10.1016/j.joca.2011.12.002) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of CS on IL-1β-induced pro-inflammatory gene expression. Osteoblasts were treated for 7days in the presence or absence of CS (100μg/ml). IL-1β (1 or 10ng/ml) was added for the latest 24h. Total RNA was extracted and used in quantitative PCR to determine steady-state mRNA levels of COX-2 (A), mPGES-1 (B), and 15-PGDH (C). Standard curves for COX-2, mPGES-1, 15-PGDH and HPRT were generated by serial dilution of a cDNA mixture. The amount of COX-2, mPGES-1 and 15-PGDH mRNA was normalized against the amount of HPRT mRNA measured in the same cDNA. Values are the mean with 95% of four independent experiments with n=1/group/experiment (corrected P-values: **P=0.08 and ***P<0.001). Osteoarthritis and Cartilage 2012 20, 127-135DOI: (10.1016/j.joca.2011.12.002) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of CS on IL-1β-induced MMP-3 mRNA expression and protein release. Osteoblasts were treated for 7days in the presence or absence of CS (100μg/ml). IL-1β (1 or 10ng/ml) was added for the latest 24h. Total RNA was extracted and used in quantitative PCR to determine steady-state mRNA levels of MMP-3 (A). Media from cells incubated were assayed for analyzing MMP-3 release into the media (B). The amount of MMP-3 released into the medium (ng/ml) in response to IL-1β was measured by ELISA. Values are the mean with 95% CI of four independent experiments with n=1/group/experiment, analyzed in duplicate (corrected P-values: ***P<0.001). Osteoarthritis and Cartilage 2012 20, 127-135DOI: (10.1016/j.joca.2011.12.002) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of CS on IL-1β-induced MMP-13 mRNA expression and protein release. Osteoblasts were treated for 7days in the presence or absence of CS (100μg/ml). IL-1β (1 or 10ng/ml) was added for the latest 24h. Total RNA was extracted and used in quantitative PCR to determine steady-state mRNA levels of MMP-13 (A). Media from cells incubated were assayed for analyzing MMP-13 release into the media (B). Values are the mean with 95% CI of four independent experiments with n=1/group/experiment, analyzed in duplicate. MMP-13 protein secretion into the medium in response to IL-1β were assessed by immunoblotting. Blots in B are representative of three independent experiments. The densitometric analyses of western-blot is the mean with 95% CI of three independent experiments [corrected P-values: **P=0.076 for (IL-1 1ng/ml), **P=0.063 for (IL-1 10ng/ml), and ***P<0.001]. Osteoarthritis and Cartilage 2012 20, 127-135DOI: (10.1016/j.joca.2011.12.002) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of CS on IL-1β-induced OPG, RANKL gene expression and OPG/RANKL ratio. Osteoblasts were treated for 7days in the presence or absence of CS (100μg/ml). IL-1β (1 or 10ng/ml) was added for the latest 24h. Total RNA was extracted and used in quantitative PCR to determine steady-state mRNA levels of OPG (A), RANKL (B), and OPG/RANKL ratio (C). Values are the mean with 95% CI of three independent experiments with n=1/group/experiment (corrected P-values: ***P<0.001). Osteoarthritis and Cartilage 2012 20, 127-135DOI: (10.1016/j.joca.2011.12.002) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions