N-acetyl-L-cysteine ethyl ester (NACET) induces the transcription factor NRF2 in the retina and prevents its aging and diabetic retinopathy. [IV]

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are leading causes of visual impairment in older people, with oxidative stress playing a central role in the development of these diseases. In fact, the cells of the retina are particularly susceptible to oxidative damage due to high metabolic activity and exposure to light. Glutathione (GSH), a key intracellular antioxidant, is essential for retinal protection but it becomes limited during aging and in diabetes patients. Cysteine (Cys), the rate-limiting precursor for GSH synthesis, can be supplemented by derivatives such as N-acetylcysteine ethyl ester (NACET), which has better bioavailability and cellular uptake compared to the widely used N-acetylcysteine (NAC). NACET effectively increases intracellular Cys and GSH levels, improves the viability of retinal pigment epithelium (RPE) cells under oxidative stress and increases in vivo GSH levels after oral administration. In this study, we show that NACET strongly stimulates NRF2 expression and activity in RPE cells, with a distinctive transcriptomic response. Using RNA interference, mass spectrometry and KEAP1 mutagenesis, we identify direct cysteinylation of sensor residues Cys226 and Cys613 on KEAP1 as the molecular mechanism underlying NRF2 activation after NACET treatment. Furthermore, in vivo administration of NACET induces NRF2 activity and increases GSH content in the retina, mitigating oxidative damage in aging and diabetic mouse models. These results position NACET as a promising therapeutic candidate for oxidative stress-related retinal diseases such as AMD and DR by targeting the KEAP1–NRF2–GSH axis. Overall design: Akita mice were used to test the effect of NACET administration as compared with that obtained in C57BL/6J mice (WT), used as normoglycemic control. Only male mice were involved in the study as female Akita mice are reported to develop a milder and less consistent diabetic phenotype (Al-Awar et al., 2016). All the procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the EU Directive (2010/63/EU), and the Italian guidelines for animal care (DL 26/14; Italian Ministry of Health, decree number 606/2024-PR). Akita mice were obtained from The Jackson Laboratories (Bar Harbor, ME, USA). At 4 weeks of age, WT and Akita mice were randomly assigned to either vehicle- or NACET-treatment group. NACET was dissolved in drinking water at the concentration of 0.33 mg/mL to obtain a daily oral intake of 50 mg/Kg. Mice received the oral administration of vehicle (water) or NACET everyday up to the 24th week of age. During the treatment period, mice underwent a longitudinal assessment of blood glucose level using a OneTouch Ultra glucometer (LifeScan Inc., Milpitas, CA, USA).