Abstract
Purpose :
(a) To assess the pharmacological action of different concentrations of LPS on the expression of CBS in porcine ICB explants, and (b) to investigate the effect of a H2S-releasing compound on LPS-induced changes in CBS expression.
Methods :
Freshly isolated porcine ICB explants were cut into quadrants and cultured in RPMI 1640 medium containing 1% penicillin-streptomycin. Explants were then exposed to different concentrations of LPS (10 ng/ml – 200 ng/ml) for 20 h. The H2S-producing compound, S-allyl cysteine, (SAC, 1 nM – 1 µM) was added to the media 30 min before the end of the incubation period. CBS enzyme expression was determined by immunoblotting. Briefly, 50 µg aliquots of protein extract from ICB were loaded on a 4-20% bis-tris SDS-PAGE gel, run, and transferred onto a nitrocellulose membrane. After probing with primary antibodies: anti-CBS (1:1,000), and anti-GAPDH (1:2,000) and secondary antibody, bands were visualized using Bio-Rad ChemiDoc imaging system.
Results :
In the concentration range of 10 - 200 ng/ml, LPS elicited a decrease in the expression of CBS with a maximum response observed at 25 ng/ml. For instance, LPS (25 ng/ml) caused a 43.7 ± 20.7 % (n = 3) decrease in CBS expression when compared to the 200 ng/ml concentration (23.5 ± 15.5 %, n = 3). In the presence of SAC (1 nM – 1 µM), there was a blockade of the LPS (25 ng/ml)-induced decrease in CBS expression. Indeed, SAC (300 nM) completely reversed the LPS (25 ng/ml)-induced reduction of CBS expression.
Conclusions :
We conclude that CBS expression was decreased during inflammation induced by LPS in the porcine anterior uvea, a response that was reversed by the administration of a H2S-releasing compound.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.