The mechanisms of N-Acetylcysteine protect against cobalt chloride induced 661W Cone Photoreceptor cell toxicity

Abstract

Purpose : Proliferative diabetic retinopathy (PDR) is the leading cause of blindness due to the apoptotic death of the photoreceptors among working age adults. Photoreceptors are the most common and metabolically demanding cells in the retina thus oxygen is vital for retinal function. Hypoxia induced metabolic stress leads to photoreceptor atrophy and retinopathy. However, the protection of hypoxia-induced cytotoxicity in cone photoreceptor cells has not been investigated extensively. The aim of the present study was to further characterize the biochemical changes in cultured 661W photoreceptor cells during hypoxic exposure and to investigate the protective mechanisms of N-acetyl cysteine (NAC) against hypoxia-induced cytotoxicity.

Methods : 661W were cultured at 37°C in DMEM with FBS. Cobalt (II) Chloride hexahydrate (CoCl₂, 400 mM) was used to induce hypoxia. NAC was dissolved in the culture medium. For protection assay, cells were pretreated with 3-4mM NAC for 2 hours followed by addition of 400-500µm CoCl₂ for 24 hours. Cell viability was determined by MTT assay. Morphological changes of the cells were observed under phase-contrast microscope and levels of protein expression was measured by Western blot.

Results : Treatment with CoCl₂ significantly reduced cell viability, and induced apoptotic protein expression, including PARP cleavage, and caspase-3 activation. Inhibition of cell proliferations and disruption of cell morphology, were significantly reversed by NAC treatment. In addition, CoCl₂ treatment led to protein ubiquitination, activation of autophagy and ERK pathways in cells. Autophagy inhibitor 3-MA eliminated NAC protection against hypoxia induced damage. Interestingly, NAC was found to strengthen CoCl₂ induced Hif1a expression and protein ubiquitination without changing its protective effect on CoCl₂ induced cell apoptosis and damage.

Conclusions : Hypoxia induced HIF-1a expression, significant apoptosis, and protein ubiquitination in 661W cells. Moreover, hypoxia also activated autophagy and upregulated ERK signaling.The HIF-1a expression and protein ubiquitination strengthened by combination treatment with NAC and CoCl₂ remains to be determined. The overall, NAC dramatically reversed hypoxia-induced cytotoxicity in 661W cells. Therefore, treatment with NAC may be a potential strategy to treat hypoxia-induced degeneration of cone photoreceptor cells.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.