Effects of cyclooxygenase and soluble epoxide hydrolase inhibitors on apoptosis of cultured primary equine chondrocytes

Background: Apoptosis is an important mechanism underlying chondrocyte loss in osteoarthritis that could be affected by modulation of lipid signaling via inhibition of cyclooxygenases (COX) and soluble epoxide hydrolase (sEH). Objective: To determine the impact of inhibiting COX and sEH alone or in combination on apoptosis of equine chondrocytes. Methods: Cultured primary equine chondrocytes were subjected to serum deprivation or incubation with 1 μg/ml tunicamycin for 24 h to induce apoptosis via caspase activation and endoplasmic reticulum (ER) stress, respectively. Cells were treated with the non-selective COX inhibitor phenylbutazone, the COX-2 selective inhibitor firocoxib and the sEH inhibitor t-TUCB alone or in combination. The inhibitors were used at half-maximal (IC₅₀), 80% of maximal (IC₈₀) and 10-fold the 80% inhibitory concentration (10xIC₈₀) for the equine enzymes. Apoptosis was quantified via ELISA technique. Data were analyzed with unpaired two-tailed t-test or one-way ANOVA followed by Bonferroni's post-hoc while correcting for multiple comparisons via statistical hypothesis testing. P < 0.05 was considered significant. Results: In the caspase model, 10xIC₈₀ t-TUCB significantly decreased whereas 10xIC₈₀ phenylbutazone significantly enhanced apoptosis. Apoptosis enhancement by phenylbutazone was significantly attenuated by concurrent 10xIC₈₀ t-TUCB. The remaining treatments and concentrations had no effect on apoptosis development. In the ER stress model, IC₅₀ and IC₈₀ phenylbutazone and firocoxib significantly enhanced apoptosis, which was fully prevented by concurrent 10xIC₈₀ t-TUCB. Main limitations: In vitro findings that will need to be verified in vivo. Conclusions: Chondrocyte apoptosis caused by ER stress can be enhanced by COX inhibition but prevented by concurrent inhibition of sEH.