Christy Xavier

Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation

PURPOSE Glutaredoxin 1 (Grx1) belongs to the oxidoreductase family and is a component of the endogenous antioxidant defense system. However, its physiological function remains largely unknown. In this study, we investigated whether and how Grx1 overexpression protects the retinal pigment epithelial (RPE) cells against H2O2-induced apoptosis. METHODS Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Primary human RPE cells were transfected with Grx1 small interfering RNA (siRNA) or scrambled siRNA. Cell viability was measured with the WST8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Protein kinase B (AKT) activation was examined by Western blot. Protein kinase B glutathionylation was detected by immunoprecipitation followed by immunoblotting with anti-PSSG antibody. RESULTS Glutaredoxin 1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Conversely, Grx1 gene knockdown sensitized primary human RPE cells to H2O2. Assessment of apoptosis indicated that cells transfected with the Grx1-containing plasmid were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to empty vector-transfected cells. Hydrogen peroxide-induced PSSG accumulation was also attenuated by Grx1 enrichment. Furthermore, Grx1 overexpression prevented H2O2-induced AKT glutathionylation, resulting in a sustained phospho-AKT elevation in RPE cells. CONCLUSIONS Glutaredoxin 1 can protect RPE cells against oxidative stress-induced apoptosis. The mechanism of this protection is associated with its ability to stimulate the phosphorylation of AKT by preventing AKT glutathionylation. Considering Grx1s protective abilities in RPE cells, Grx1 could be a potential pharmacological target for retinal degenerative diseases.

The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

Graphical abstract

Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1)

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.

Involvement of Nrf2 in Ocular Diseases

The human body harbors within it an intricate and delicate balance between oxidants and antioxidants. Any disruption in this checks-and-balances system can lead to harmful consequences in various organs and tissues, such as the eye. This review focuses on the effects of oxidative stress and the role of a particular antioxidant system—the Keap1-Nrf2-ARE pathway—on ocular diseases, specifically age-related macular degeneration, cataracts, diabetic retinopathy, and glaucoma. Together, they are the major causes of blindness in the world.