(R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage.
OBJECTIVE
To determine whether (R)-alpha-lipoic acid (LA) protects cultured human fetal retinal pigment epithelial (hfRPE) cells against oxidative injury and identify the pathways that may mediate protection.


METHODS
Cultured hfRPE cells were pretreated with various concentrations of LA for 14 to 16 hours followed by treatment with a chemical oxidant, tert-butylhydroperoxide (t-BuOOH; 0.8 mM, 3 hours). Reactive oxygen species (ROS) production and cell viability were measured using H(2)DCF and MTT assays, respectively. RPE cells were evaluated with fluorescent dyes (SYTOX Orange and SYTO Green; Molecular Probes, Eugene, OR), which differentiate between live and dead cells. Apoptosis was visualized by using the TUNEL assay. Changes in mitochondrial membrane potential were detected by JC-1 dye. Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by HPLC. Regulation of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme of GSH production, was assayed by RT-PCR.


RESULTS
Pretreatment of hfRPE cells with LA, 0.2 mM and 0.5 mM, significantly reduced the levels of t-BuOOH-induced intracellular ROS, by 23% and 49%, respectively. LA (0.5 mM) prevented oxidant-induced cell death and apoptosis and also increased the viability of oxidant-treated hfRPE cells from 38% to 90% of control. LA upregulated the mRNA expression of GCL, and was protective against t-BuOOH-induced decreases in both mitochondrial membrane potential and intracellular levels of GSH and GSH/GSSG.


CONCLUSIONS
The present study suggests that the protective effect of LA involves multiple pathways and that LA could be effective against age-associated increase in oxidative stress and mitochondrial dysfunction in RPE cells.
Voloboueva
Ludmila A
LA
Biophysics Graduate Group, University of California, Berkeley, USA.
Liu
Jiankang
J
Suh
Jung H
JH
Ames
Bruce N
BN
Miller
Sheldon S
SS
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D019610:Cytoprotection / Q000187:drug effects; D005456:Fluorescent Dyes; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008063:Thioctic Acid / Q000494:pharmacology*; D015854:Up-Regulation; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D000975:Antioxidants; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D008063:Thioctic Acid; D020122:tert-Butylhydroperoxide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D019803:Glutathione Disulfide