Annatto prevents retinal degeneration induced by endoplasmic reticulum stress in vitro and in vivo.
METHODS
Annatto (Bixa orellana) seeds have been used as a colorant in butter and in a variety of other foods. In this study, we investigated the amelioration of retinal damage by an acetone extract of annatto (A-ext.), bixin (a main component of annatto), and four bixin derivatives (Bx-1, Bx-2, Bx-3, and Bx-4) that we have synthesized.


RESULTS
We used cultured retinal ganglion cells (RGC-5) to examine in vitro effects of A-ext. on stress pathways, focusing on intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, caspase-3 activation, and cell membrane damage. In vivo retinal damage in mice following intravitreous injection of tunicamycin was evaluated by counting the cell numbers in the ganglion cell layer (GCL) and measuring the thickness of outer nuclear layer (ONL). A-ext., bixin, and Bx-1 treatment inhibited both tunicamycin- and H₂O₂-induced cell death. Bixin derivatives also inhibited tunicamycin-induced cell death. Treatment with A-ext., bixin, and Bx-1 reduced tunicamycin-induced caspase-3 activity and inhibited the inversion of phosphatidylserine, an early apoptotic event without antioxidant effect or reduction of ER stress itself. A-ext., bixin, and Bx-1 significantly inhibited the tunicamycin-induced loss of cells from the GCL, and these materials also suppressed the tunicamycin-induced thinning of ONL.


CONCLUSIONS
A-ext., its main component bixin, and bixin derivatives may therefore be useful for preventive and therapeutic treatment of retinal-related diseases.
Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan.
Kazuhiro
K
Tsuruma
Hiroki
H
Shimazaki
Ken-Ichi
K
Nakashima
Mika
M
Yamauchi
Sou
S
Sugitani
Masamitsu
M
Shimazawa
Munekazu
M
Iinuma
Hideaki
H
Hara
D000818:Animals; D031212:Bixaceae; D002338:Carotenoids / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D059865:Endoplasmic Reticulum Stress; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010718:Phosphatidylserines / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology*; D012162:Retinal Degeneration / Q000209:etiology / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000187:drug effects; D014415:Tunicamycin / Q000009:adverse effects
D010718:Phosphatidylserines; D010936:Plant Extracts; D014415:Tunicamycin; D002338:Carotenoids; C054041:annatto; C004588:bixin; D006861:Hydrogen Peroxide; D053148:Caspase 3