(R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage

Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4302-10. doi: 10.1167/iovs.04-1098.


Purpose: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / pharmacology*
  • Apoptosis
  • Cell Survival
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Cytoprotection / drug effects
  • Fluorescent Dyes
  • Glutamate-Cysteine Ligase / genetics
  • Glutathione / metabolism
  • Glutathione Disulfide / metabolism
  • Humans
  • Oxidative Stress*
  • Pigment Epithelium of Eye / cytology
  • Pigment Epithelium of Eye / drug effects*
  • Pigment Epithelium of Eye / metabolism
  • RNA, Messenger / metabolism
  • Reactive Oxygen Species / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Thioctic Acid / pharmacology*
  • Up-Regulation
  • tert-Butylhydroperoxide / toxicity


  • Antioxidants
  • Fluorescent Dyes
  • RNA, Messenger
  • Reactive Oxygen Species
  • Thioctic Acid
  • tert-Butylhydroperoxide
  • Glutamate-Cysteine Ligase
  • Glutathione
  • Glutathione Disulfide