AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES
Authors: V. Can, I. Locke, P. Grieco, S. Getting
Journal: Annals of the Rheumatic Diseases (2020)

Background: Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4]. Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear. Objectives: This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes. Methods: The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 10 6 viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M of n =4 samples repeated in triplicate. # p ≤0.05 vs control or * p ≤0.05 vs stimulus. Results: Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (# p ≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (* p ≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (* p ≤0.05). GAG content was significantly reduced by SNAP by 29% (# p ≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (* p ≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (* p ≤0.05). Conclusion: The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line. References: [1]Hunter DJ and Bierma-Zeinstra S. (2019). Lancet . 393 : 1745–59. [2]Can VC et al. (2020). Euro J Pharmacol . doi:https://doi.org/10.1016/j.ejphar.2020.172971. [3]Intekhab-Alam NY et al . (2013). Cell death &amp; disease. 4 : 1-6. [4]Getting SJ et al. (2006). Mol Pharmacol 70 : 1850-1855. [5]Kaneva MK et al. (2014). Biochem Pharmacol 92 : 336-47. [6]Greco KV et al. (2011). Biochem Pharmacol 82 : 1919-29. Disclosure of Interests: None declared