RESUMO
Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.
Assuntos
Antioxidantes/farmacologia , Benzofuranos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Glutarredoxinas/genética , Peróxido de Hidrogênio/antagonistas & inibidores , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feto , Regulação da Expressão Gênica , Glutarredoxinas/antagonistas & inibidores , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Estresse Oxidativo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
CONTEXT: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. OBJECTIVE: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. MATERIALS AND METHODS: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. RESULTS: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. DISCUSSION: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. CONCLUSION: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.