RESUMO
PURPOSE: Oxidative stress on retinal pigment epithelial (RPE) cells is thought to play a crucial role in the development and progression of age-related macular degeneration. Astaxanthin (AST) is a carotenoid that shows significant antioxidant properties. This study was designed to investigate the protective effect of AST on ARPE-19 cells against oxidative stress and the possible underlying mechanism. METHODS: ARPE-19 cells exposed to different doses of H2O2 were incubated with various concentrations of AST and cell viability subsequently detected with the (4-[3-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5- tetrazolio-1,3-benzene disulfonate]; WST-1) assay. The apoptosis rate and intracellular levels of reactive oxygen species (ROS) were measured with flow cytometry. NAD(P)H quinine oxidoreductase 1 (NQO1), hemeoxygenase-1 (HO-1), glutamate-cysteine ligase modiï¬er subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) expression were examined with real-time PCR and western blotting. The nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and the expression levels of cleaved caspase-3 and protein kinase B proteins were evaluated with western blotting. RESULTS: AST clearly reduced H2O2-induced cell viability loss, cell apoptosis, and intracellular generation of ROS. Furthermore, treatment with AST activated the Nrf2-ARE pathway by inducing Nrf2 nuclear localization. Consequently, Phase II enzymes NQO1, HO-1, GCLM, and GCLC mRNA and proteins were increased. AST inhibited expression of H2O2-induced cleaved caspase-3 protein. Activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway was involved in the protective effect of AST on the ARPE-19 cells. CONCLUSIONS: AST protected ARPE-19 cells against H2O2-induced oxidative stress via Nrf2-mediated upregulation of the expression of Phase II enzymes involving the PI3K/Akt pathway.
Assuntos
Células Epiteliais/citologia , Desintoxicação Metabólica Fase II , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , NAD(P)H Desidrogenase (Quinona) , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xantofilas/química , Xantofilas/farmacologiaRESUMO
PAX6 contributes to the development and progression of retinoblastoma (RB), but the molecular mechanism underlying the regulation of PAX6 expression is unclear. Here we found that microRNA-365b-3p (miR-365b-3p) is downregulated in human RB tissues. Ectopic expression of miR-365b-3p significantly attenuates cell growth, induces cell cycle arrest in G1 phase and cell apoptosis through inhibiting the expression of PAX6 by directly binding its 3' untranslated regions. Furthermore, overexpression of miR-365b-3p upregulates p21 and p27 but downregulates cdc2 and Cyclin D1 protein levels. Elucidating the regulatory mechanism of PAX6 by microRNAs may give new clues to the therapy against RB.
