Polyunsaturated Fatty Acid-Phloroglucinol conjugates protect RPE and Neural Retina against All-trans-Retinal-induced Damages

Abstract

Purpose: We previouly showed that phloroglucinol (PG), a monomer of phloritannins with a wide range of clinical applications, has a dual action as anti-carbonyl stress from all-trans-retinal (atRAL) and anti-oxidant in retinal pigment epithelium (RPE). A major disadvantage of such a compound for the treatment of retinal disorder is its low bioavailibility. We develop strategy to improve the lipophilicity and enhance reactivity with atRAL relies on synthesis of PUFA-O-alkylated-PG conjuguates. The aim of this study was to assess their protective effect in cultured RPE and neural retina (NR) against atRAL-induced cell damages.

Methods: PG conjugates was synthetized as described (Crauste C., EurJOC, 2014). ARPE19 cell culture was treated with different concentrations of PG conjugates for 1h and exposed to 25 µM atRAL for 4h in serum-free medium. Similar co-treatments were applied to primary cultures of rat RPE and mouse NR. Cell viability was determined by MTT assay. The type of cell death was determined by flow cytometric using AnnexinV-FITC and propidium iodide staining. Mitochondrial respiratory chain complex function was analyzed by oxygraphy and enzymatic activities. Citrate synthase activity and respiratory chain complex protein quantification assessed the mitochondrial mass.

Results: Cell viability of ARPE19 was significantly increased by introduction of docosahexanoic acid (DHA) and ispopropyl to the polyphenol structure compare to the initial PG in the presence of atRAL (66.8 ± 8.6 %, 41.6 ± 9.2 %, and 29.1 ± 5.4 %, respectively). Monoisopropyl-phloroglucinol-DHA (PG-OIP-DHA) from 10 to 80 µM dose-dependently increased survival of both ARPE19, RPE and NR primary cultures. In contrast, PG-OIP had no effect and DHA decreased ARPE19 survival. PG-OIP-DHA almost fully rescued ARPE19 cells from atRAL-induced late apoptosis and necrosis. Linoleic acid (LA) substitution had similar effect than DHA on cell viability, linolenic (ALA) and ecosapentanoic (EPA) acids showed intermediate efficacy and saturated C22:0 had almost no efficacy. Both PG-OIP-DHA and PG-OIP-LA rescue atRAL-induced damage on mitochondrial function.

Conclusions: The protective effect of the PUFA-O-alkylated-PG conjugates was demonstrated in cultured RPE and neural retina challenged with a toxic dose of atRAL. Both PUFA and isopropyl were confirmed to be essential for the activity of these derivatives.