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Updated: Oct 5, 2025
Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells
Published on: May 13, 2022
High glucose concentrations induce oxidative stress by inhibiting Nrf2 expression in rat Müller retinal cells in
Jesús Silvestre Albert-Garay1, Juan Rafael Riesgo-Escovar2, Rocío Salceda3
1Departamento de Neurodesarrollo y Fisiología, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510, CDMX, Mexico. silvestre.alb@gmail.com.
Abstract:
Diabetic retinopathy (DR) is a complication of diabetes. Several studies have implicated oxidative stress as a fundamental factor in the progression of the disease. The nuclear factor erythroid-2-related factor 2 (Nrf2) is one of the main regulators of redox homeostasis. Glia Müller cells (MC) maintain the structural and functional stability of the retina. The objective of this study was to evaluate the effect of high glucose concentrations on reactive oxygen species (ROS) production and Nrf2 expression levels in rat MC. MC were incubated with normal (NG; 5 mM) or high glucose (HG; 25 mM) for different times. Incubation with HG increased ROS levels from 12 to 48 h but did not affect cell viability. However, exposure to 3 h of HG caused a transient decrease Nrf2 levels. At that time, we also observed a decrease in the mRNA expression of Nrf2 target genes, glutathione levels, and catalase activity, all of which increased significantly beyond initial levels after 48 h of incubation. HG exposure leads to an increase in the p65 subunit of nuclear factor-κB (NF-kB) levels, and its target genes. These results suggest that high glucose concentrations lead to alteration of the redox regulatory capacity of Nrf2 mediated by NF-kB regulation.
Insights
High glucose levels increase oxidative stress and alter Nrf2 regulation in Müller cells, impacting diabetic retinopathy progression. This suggests NF-kB activation plays a role in Nrf2 dysfunction.
Area of Science:
- Ophthalmology
- Cell Biology
- Metabolic Disorders
Background:
- Diabetic retinopathy (DR) is a diabetes complication linked to oxidative stress.
- Nuclear factor erythroid-2-related factor 2 (Nrf2) regulates redox homeostasis.
- Müller cells (MC) are crucial for retinal stability.
Purpose of the Study:
- To investigate the impact of high glucose on reactive oxygen species (ROS) and Nrf2 in rat MC.
- To explore the role of nuclear factor-kappa B (NF-kB) in high glucose-induced cellular changes.
Main Methods:
- Rat MC were exposed to normal (5 mM) or high glucose (25 mM) for varying durations.
- Assessed cell viability, ROS production, Nrf2 expression, and Nrf2 target gene mRNA levels.
- Measured glutathione levels, catalase activity, and NF-kB p65 subunit levels.
Main Results:
- High glucose increased ROS levels from 12 to 48 hours without affecting cell viability.
- A transient decrease in Nrf2 levels and its target gene expression was observed after 3 hours of high glucose exposure.
- High glucose elevated NF-kB p65 levels and its target genes, while glutathione and catalase activity initially decreased then increased.
Conclusions:
- High glucose concentrations disrupt MC redox balance by transiently downregulating Nrf2.
- NF-kB activation appears to mediate the high glucose-induced alterations in Nrf2 regulatory capacity.
- These findings highlight potential therapeutic targets for mitigating diabetic retinopathy-associated oxidative stress.
