Alpha Lipoic Acid Protects Retinal Pigment Epithelial Cells Against Oxidative Stress Via Induction Of Heme Oxygenase-1Expression Via PI3K/Akt And Nrf2Pathways

ObjectiveTo observe the effect and molecular mechanism of alpha lipopic acid (ALA) to theexogenous H2O2on production of Heme oxygenase (HO)-1in human retinalpigment epithelium cellsMethodsHuman retinal pigment epithelium cells ARPE-19was cultured in vitro and wasdivided into6groups according to different experimental purposes:(1) Control group(0.1%medium);(2) H2O2group (ARPE-19cells were added by12.5mmol/L H2O2for30min;(3) ALA intervention group (Cells were treated with10~30μmol/L ALA for4~24h after H2O2administration;(4) PI3K inhibitor group (Cells were incubated with25μmol/L LY294002for30min after ALA treatment);(5) siRNA group (Cells weretransfected with specific siRNA for Nrf2or HO-1, and then treated with H2O2orALA);(6) CoPP or ZnPP group (10μmol/L CoPP or ZnPP was added afterALAtreatment). Expression of HO-1mRNA and protein were detected by RT-PCRand Western blot, respective. Apoptosis of ARPE-19cells were analyzed by flowcytometry. Production of reactive oxygen species (ROS) was detected by fluorescentprobe. Phosphorylation of Akt and nuclear translocation of Nrf2were detected byWestern blot. Results1. RT-PCR results showed the expression levels of HO-1mRNA and protein werelow in the control group.12.5μmol/L H2O2only slightly increased theexpression level of HO-1. However, After incubated with ALA, HO-1expression were upregulated as the concentration of ALA increased;2. H2O2treatment for30min could slightly induce Akt phosphorylation. After10~30μmol/L ALA treatment for4h, the phosphorylated Akt significantlyincreased;3. There were no Nrf2translocation in control group.30μmol/L ALA significantlyincreased the nuclear translocation of Nrf2, and this could be inhibit by25μmol/L PI3K inhibitor LY294002;4. Silencing of Nrf2by specific siRNA significantly inhibited HO-1expression;5. H2O2could induce ARPE-19cells apoptosis and ROS production. After30μmol/L ALA treatment, the apoptosis rate and ROS significantly decreased.However, an HO-1inhibitor ZnPP, or HO-1siRNA, could reverse theinhibitory effect of ALA-decreased apoptosis and ROS production, while theHO-1agonist CoPP had a synergy effect on ALA to decrease the apoptosis rateand ROS production.ConclusionAlpha lipoic acid protects retinal pigment epithelial cells against oxidativestress via induction of heme oxygenase-1expression via PI3K/Akt and Nrf2pathways...