Posters
Presentation Type
Poster
Discipline Track
Biomedical Science
Abstract Type
Research/Clinical
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 inl 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 5% CO2 at 37 ̊C in DMEM with 10% FBS, 1% a penicillin/streptomycin. Cobalt (II) Chloride hexahydrate (CoCl2) was used to induce hypoxia. NAC was dissolved in H20 and diluted in the culture medium. For protection assay, pretreated cells with either 3 or 4mM NAC for 2 hours followed by addition of either 400 or 500μm CoCl2 for 24 hours. Cell viability was determined by MTT assay in a 96 well plate. Morphological changes of the cells were observed and photographed under phase-contrast microscope and the levels of protein expression was measured by Western blot analysis. Statistical analysis was undertaken using independent two-tailed Students t-test using SPSS Statistics software.
Results: Treatment with CoCl2 significantly inhibited 661W cell proliferation, reduced cell viability, and induced apoptotic protein expression, including PARP cleavage, and caspase 3 activation. These effects, including 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 MAPK/ERK pathways in the 661W cells. Autophagy inhibitor 3-Methyladenine (3-MA) eliminated NAC protection against hypoxia induced cell 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 hypoxia-inducible factor-1a (HIF-1a) expression, significant apoptosis, and protein ubiquitination in 661W cone photoreceptors. Moreover, hypoxia also activated autophagy and upregulated MAPK/ERK signaling. Our preliminary study demonstrated that the possible role of MAPK/ERK and autophagy activation at the early stages may have increased the tolerance to hypoxia for a short period. 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 photoreceptor cells. Therefore, treatment with NAC may be a potential strategy to treat hypoxia-induced degeneration of cone photoreceptor cells.
Academic/Professional Position
Graduate Student
Academic/Professional Position (Other)
UTRGV SOM
Recommended Citation
Guerra, Lili; Cheng, Benxu; and Tsin, Andrew, "The mechanisms of N-Acetylcysteine protect against cobalt chloride induced 661W Cone Photoreceptor cell toxicity." (2024). Research Symposium. 70.
https://scholarworks.utrgv.edu/somrs/2023/posters/70
Included in
The mechanisms of N-Acetylcysteine protect against cobalt chloride induced 661W Cone Photoreceptor cell toxicity.
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 inl 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 5% CO2 at 37 ̊C in DMEM with 10% FBS, 1% a penicillin/streptomycin. Cobalt (II) Chloride hexahydrate (CoCl2) was used to induce hypoxia. NAC was dissolved in H20 and diluted in the culture medium. For protection assay, pretreated cells with either 3 or 4mM NAC for 2 hours followed by addition of either 400 or 500μm CoCl2 for 24 hours. Cell viability was determined by MTT assay in a 96 well plate. Morphological changes of the cells were observed and photographed under phase-contrast microscope and the levels of protein expression was measured by Western blot analysis. Statistical analysis was undertaken using independent two-tailed Students t-test using SPSS Statistics software.
Results: Treatment with CoCl2 significantly inhibited 661W cell proliferation, reduced cell viability, and induced apoptotic protein expression, including PARP cleavage, and caspase 3 activation. These effects, including 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 MAPK/ERK pathways in the 661W cells. Autophagy inhibitor 3-Methyladenine (3-MA) eliminated NAC protection against hypoxia induced cell 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 hypoxia-inducible factor-1a (HIF-1a) expression, significant apoptosis, and protein ubiquitination in 661W cone photoreceptors. Moreover, hypoxia also activated autophagy and upregulated MAPK/ERK signaling. Our preliminary study demonstrated that the possible role of MAPK/ERK and autophagy activation at the early stages may have increased the tolerance to hypoxia for a short period. 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 photoreceptor cells. Therefore, treatment with NAC may be a potential strategy to treat hypoxia-induced degeneration of cone photoreceptor cells.