Effects of 2-Benzoylbenzoic Acid on Viability and Nitric Oxide Production in HIG-82 Synovial Fibroblasts

Rheumatoid arthritis (RA) is characterized by persistent synovial inflammation, with synovial fibroblasts playing a key role in producing inflammatory mediators. 2-Benzoylbenzoic acid, known for its inhibitory activity against Aldo-keto reductase 1C3 (AKR1C3), was investigated for its effects on rabbit synovial fibroblasts (HIG-82) cell viability and nitric oxide (NO) production, both at rest and following stimulation with phorbol-12-myristate 13-acetate (PMA). Cells were treated with 2-benzoylbenzoic acid (3.125–300 µM) for 24, 48, or 72 hours, with or without PMA (10 nM). Reference NSAIDs, diclofenac and ibuprofen (100 µM), served as controls. The Griess reaction was used to evaluate nitrite concentrations in culture supernatants, and the MTT assay was used to assess cell viability. PMA significantly reduced cell viability over time compared to non-stimulated controls (24 h: 80.82 ± 8.70%; 48 h: 59.70 ± 12.50%; 72 h: 44.94 ± 8.40%). In non-stimulated cells, viability was generally maintained at concentrations =200 µM, whereas 300 µM reduced viability at 24 hours (67.75 ± 14.42%). In PMA-stimulated cells, viability at 72 hours was higher with 2-benzoylbenzoic acid (61.87–83.29%) than with PMA alone. Nitrite levels remained low, variable, and were not consistently decreased by the treatments, indicating that 2-benzoylbenzoic acid did not demonstrate an NO-linked anti-inflammatory effect under these conditions. Notably, despite its AKR1C3 inhibitory potential, 2-benzoylbenzoic acid did not suppress nitrite production, and no clear anti-inflammatory activity via NO modulation was observed.

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