P65 Characterising the effect of novel slow-release H2S donors on pro-inflammatory enzyme activity in human cartilage cells

Abstract

Synthesis of the gaseous mediator hydrogen sulfide (H 2 S) is elevated in the joints of patients with inflammatory joint diseases such as rheumatoid arthritis and H 2 S levels in knee joint synovial fluid aspirates strongly correlate with disease activity. However, the precise role of H 2 S in inflammatory signalling is controversial and unclear since pro- and anti-inflammatory effects have been reported in diverse animal models of acute and chronic inflammation. We have hypothesised that this disparity may be explained by the manner in which cells, tissues and animals are exposed to H 2 S. For example, ‘donors’ which deliver an instantaneous bolus of H 2 S (e.g. NaSH/Na 2 S) are invariably pro-inflammatory whereas molecules which generate H 2 S in a manner comparable with endogenous H 2 S production (e.g. GYY4137) are anti-inflammatory. To further investigate the above hypothesis, we have compared the effects of GYY4137 with novel water soluble compounds synthesised by us that generate H 2 S at different rates (AP105 > AP106 > GYY4137) using primary human synoviocytes (HFLS) and articular chondrocytes (HAC) exposed to either LPS (10 μg/ml) or a cocktail of inflammatory cytokines (TNF α , IL-1 β and IFN-g; 1 ng/ml). Cells were exposed to H 2 S donors for either 1 h before or 6 h after LPS/cytokine stimulation. Isolated pro-inflammatory enzymes, tumour necrosis factor-alpha converting enzyme (TACE), COX1, COX2, iNOS and MMP-2 were also exposed to H 2 S donors and enzyme activity measured by fluorimetry and ELISA. In HFLS and HAC, GYY4137 significantly reduced LPS and cytokine-induced expression of NOS2 and COX2 (<100 μM; ANOVA, p < 0.01) and NF-kB-DNA binding activity (ELISA) as well as reduced the synthesis of PGE 2 , TNF α and NO. AP105, AP106 and GYY4137 (<200 μM) significantly (ANOVA; p < 0.01) decreased the activity of isolated TACE, NOS2, COX1 and COX2 activity in vitro (AP105 > AP106 > GYY4137). These data show that slow release H 2 S donors inhibit the expression and catalytic activity of pro-inflammatory enzymes and further highlight the anti-inflammatory and therapeutic potential of slow release H 2 S donors.