A novel sphingosine-1-phosphate agonist, HY-3011, protects porcine retinal pigment epithelial cells from 7-keto Cholesterol induced oxidative damage

Abstract

Purpose : The aim of the study was to investigate the protective effects of HY-3011, a sphingosine-1-phosphate (S1P) agonist, on 7-ketocholesterol (7kCh)-induced porcine retinal pigment epithelial (pRPE) cell apoptosis in vitro.

Methods : Using primary pig RPE cells as a cultured model system, an in vitro cell damage assay was developing using 7-ketocholesterol (7kCh) as the cell death signal. Cell Titer-fluor, was used as a measurement of cell viability, which detects the activity of a protease that is restricted to intact viable cells. Additionally, phalloidin staining was used to monitor the dynamic changes of pRPE cell morphology during the 7kCh insults. HY-3011 and other S1P-specific receptor subtype agonists were used to evaluate the protective effects on 7-kCh treated pRPE cells at the concentration between 10 nM to 10 µM. Pre-treated pRPE with pertussis toxin to reveal the possible intracellular signaling mechanism of HY-3011 was also investigated in this study.

Results : 7-ketocholesterol induced pRPE cell apoptosis in a time and dose-dependent manners. Cholesterol did not have any cytotoxicity while applied at the same concentration ranges. With phalloidin staining, the pRPE cells exhibited a dynamic microfilament internalization processing. Using receptor binding assay, HY-3011 as a S1P agonist can actively bind to S1P receptors with EC50=30.84 nM. At the ranges of 100 to 300 nM levels, HY-3011 exhibited notable protection against the 7-kCh-induced pRPE cell damages with an IC50= 230 nM. This protective effect was blocked by pre-treated with pertussis toxin.

Conclusions : HY-3011, a novel S1P agonist, has been demonstrated the protection of porcine RPE cells against 7kCh-induced toxicity in vitro. This action may be involved in binding on S1P receptor subtype 3 (S1P3) on RPE cells via G-protein coupled receptor (GPCR) mechanisms.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.