| Abstract | Purpose: Normal 0 false false false EN-GB X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} Osteoarthritis (OA) is estimated to affect more than 15% of the worldwide population and at present this degenerative joint disease requires novel treatment options. A role for the melanocortin peptides exerting anti-inflammatory effects has been shown, although the receptor subtype involved is unclear. This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human MC1 receptor agonist BMS-470539 dihydrochloride, selective human MC3 receptor agonist PG-992 and the human MC1/3 receptor agonist [DTrp8]-γ-MSH on S-Nitroso-N-acetyl-DL-penicillamine activated chondrocytes. Methods: The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106 viable cells/ml into 96-well plates. Micromass cultures were stimulated with S-Nitroso-N-acetyl-DL-penicillamine (1.0 mM) and then treated with the melanocortin peptides BMS-470539 dihydrochloride (10.0 μg/ml), PG-992 (10.0 μg/ml) and [DTrp8]-γ-MSH (10.0 μg/ml) for 6h. Gene expression for hMC1, hMC3, ADAMTS-4 and ADAMTS-5 determined by RT-PCR/Western Blot. Cell viability was determined by Neutral Red assay, pro-inflammatory mediator release (IL-6 and IL-8) and tissue destructive matrix metalloproteinase (MMP-1 and MMP-13) determined by ELISA. Data are expressed as Mean ±SD of n=4 samples repeated in triplicate. #p≤0.05 vs control or *p≤0.05 vs stimulus. Results: RT-PCR showed hMC1 and hMC3 receptor expression. Cell viability analysis showed SNAP stimulation caused a maximal cell death of 21% (#p≤0.05), BMS-470539 dihydrochloride, PG-992 and [DTrp8]-γ-MSH inhibited cell death by 19%, 126% and 126% respectively (*p≤0.05). SNAP stimulation caused a significant increase in IL-6, IL-8, MMP-1 and MMP-13 protein release, which was inhibited by the melanocortin peptides. BMS-470539 dihydrochloride inhibited IL-6, IL-8, MMP-1 and MMP-13 release by 3%, 14%, 19% and 4% respectively (*p≤0.05). PG-992 inhibited IL-6, IL-8, MMP-1 and MMP-13 release by 50%, 44%, 69% and 80% respectively (*p≤0.05). [DTrp8]-γ-MSH inhibited IL-6, IL-8, MMP-1 and MMP-13 release by 36%, 39%, 45% and 28% respectively (*p≤0.05). SNAP also caused a significant increase in ADAMTS-4 and ADAMTS-5 expression, which was reduced by BMS-470539 dihydrochloride, PG-992 and [DTrp8]-γ-MSH by 1.8-fold, 2.2-fold and 2.2-fold respectively (*p≤0.05). Conclusions: The selective hMC3 receptor agonist PG-992 exhibited both chondroprotection and modulation of the inflammatory pathways following SNAP activation better than the selective hMC1 receptor agonist BMS-470539 dihydrochloride, which suggests that targeting the human melanocortin receptor 3 sub-type for the development of treatment options for OA would be a possible avenue to explore. |
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