High glucose concentrations induce oxidative stress by inhibiting Nrf2 expression in rat Müller retinal cells in vitro

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.

Figure 1

High glucose concentrations do not affect cell viability in MC. Cells were incubated with NG (Ctr; 5 mM glucose) or HG (25 mM glucose) for different time periods (1–48 h). (a) Cell viability was determined by the MTT assay; viability was expressed as the percentage of optical density respect to cells exposed to NG (100%). Cell death was measured through LDH release and TUNEL assay. (b) The results of LDH release are expressed as units per milliliter (U/ml). (c) Representative fluorescent micrographs of TUNEL-positive cells (green) compared to total cells (DAPI, blue) in MC exposed to NG or HG; positive control (pos: plus DNAse). Data are expressed as the mean ± SEM of duplicate cultures and are representative of three independent experiments. Scale bars: 50 µm. *p < 0.05 with respect to NG.

Figure 2

High glucose concentrations increase ROS and RNS production levels in MC. Cells were incubated with NG (Ctr) or HG for different time periods (1–48 h). (a) Production of ROS in MC. In the upper part, representative fluorescent micrographs. The lower part, the percentage of relative H₂DCFDA fluorescence per µg protein. (b) Representative micrographs of DHE-positive cells (red). (c) Production of RNS in MC. The upper part, representative fluorescent micrographs. The lower part, the percentage of relative DAF-FM fluorescence per µg protein. Data are expressed as the mean ± SEM of duplicate cultures and are representative of five independent experiments. Scale bars: 50 µm. NG, normal glucose; HG, high glucose. *p < 0.05 with respect to NG, **p < 0.01 with respect to NG, ***p < 0.001 with respect to NG.

Figure 3

High glucose concentrations decrease Nrf2 and Keap1 levels in MC. Western blot analysis of Nrf2 and Keap1 expression in homogenates from MC exposed to NG (Ctr) or HG for different time periods (1–48 h). The upper part, representative western blot (a). The lower part, densitometric quantification of Nrf2 (b) and Keap1 (c) levels. The relative expression levels were normalized using actin. Values are the mean ± SEM (n = 7 per group) carried out in duplicate. NG, normal glucose; HG, high glucose. *p < 0.05 with respect to NG, ***p < 0.001 with respect to NG. Full-size blots are presented in Supplementary Fig. 3.

Figure 4

Nrf2 subcellular localization in MC. Cells were exposed to HG for the indicated time intervals. (a) Immunofluorescent localization of Nrf2 in MC. Blue marks nuclei (DAPI); Red, Nrf2-staining, and Pink, merge of blue and red indicating nuclear localization of Nrf2. (b) Nuclear and (c) Cytoplasmic Nrf2 expression in MC exposed to HG. The upper part, representative western blot of Nrf2. The lower part, quantification of the relative levels of Nrf2. The relative expression levels were normalized using actin (cytoplasmic) or H2b (histone 2b; nuclear). Values represent the mean ± SEM (n = 5 per group) carried out in duplicate. NG, normal glucose; HG, high glucose. Scale bar represents 50 µm. *p < 0.05 with respect to NG; ***p < 0.001 with respect to NG. Full-size blots are presented in Supplementary Fig. 4.

Figure 5

High glucose concentrations alter the expression of antioxidant enzymes and GSH levels. MC were exposed to HG for the indicated time intervals. mRNA levels of GCLc (a), SOD2 (b), TXN (c) and Nrf2 (f). The relative mRNA levels were normalized using ACT (actin). Values are the mean ± SEM (n = 4 per group) carried out in duplicate. (d) Glutathione (GSH) levels are expressed as nmol/mg protein. (e) Catalase activity is expressed as U/mg protein, as described in Methods. Values are the mean ± SEM (n = 5 per group) carried out in duplicate. NG, normal glucose; HG, high glucose. *p < 0.05 with respect to NG, **p < 0.01 with respect to NG, ***p < 0.001 with respect to NG.

Figure 6

GSK3-β phosphorylation. Western blot analysis of phospho-GSK3-β (Ser 9) and GSK3-β expression in homogenates from MC exposed to NG (Ctr) or HG for different time periods (1–48 h). The upper part, representative western blot. The lower part, quantification of the expression levels. The relative expression levels were normalized with total GSK3-β. Values are the mean ± SEM (n = 4 per group) carried out in duplicate. NG, normal glucose; HG, high glucose. *p < 0.05 with respect to NG, ***p < 0.001 with respect to NG. Full-size blots are presented in Supplementary Fig. 5.

Figure 7

High glucose concentrations induced the expression of inflammatory proteins in MC. Western blot analysis of NF-κB (a), IL-1β (b), and iNOS (c) expression in homogenates from MC exposed to NG (Ctr) or HG for different time periods (1–48 h). The upper part, representative western blot. The lower part, quantification of the expression levels. The relative expression levels were normalized using actin. Values are the mean ± SEM (n = 5 per group) carried out in duplicate. NG, normal glucose; HG, high glucose. *p < 0.05 with respect to NG, ***p < 0.001 with respect to NG. Full-size blots are presented in Supplementary Fig. 6.