Suppression of cAMP/PKA/CREB signaling ameliorates retinal injury in diabetic retinopathy

Abstract

The blood-retinal barrier (BRB), homeostasis, neuronal integrity, and metabolic processes are all directly influenced by Müller cells, the most important retinal glial cells. We isolated primary Müller cells from Sprague-Dawley (SD) neonatal rats and treated them with glucose at varying doses. CCK-8 was used to quantify cellular viability, and a TUNEL assay was performed to detect cell apoptosis. ELISA, immunofluorescence, and western blotting were used to assess cAMP/PKA/CREB signaling, Kir4.1, AQP4, GFAP, and VEGF levels, respectively. H&E staining was used to examine histopathological alterations in diabetic retinopathy (DR)-affected retinal tissue in rats. As glucose concentration increases, gliosis of Müller cells became apparent, as evidenced by a decline in cell activity, an increase in apoptosis, downregulation of Kir4.1 level, and overexpression of GFAP, AQP4, and VEGF. Treatments with low, intermediate, and high glucose levels led to aberrant activation of cAMP/PKA/CREB signaling. Interestingly, blocking cAMP and PKA reduced high glucose-induced Müller cell damage and gliosis by a significant amount. Further in vivo results suggested that cAMP or PKA inhibition significantly improved edema, bleeding, and retinal disorders. Our findings showed that high glucose exacerbated Müller cell damage and gliosis via a mechanism involving cAMP/PKA/CREB signaling.

Keywords: Müller cells; cAMP/PKA/CREB signaling; diabetic retinopathy.

FIGURE 1

Effects of different concentrations of glucose on Müller cells. The primary Müller cells were isolated from SD rats and cultured in a medium with various glucose concentrations (control, 5 mmol/L; LG, 12.5 mmol/L; MG, 25 mmol/L; HG, 50 mmol/L) for 48 h. (A) The images show morphological changes in Müller cells as observed under an inverted microscope. The arrow represents the cells with morphological change. (B) The cell viability of Müller cells detected using CCK‐8. (C) Cell apoptosis in Müller cells by TUNEL detection. All data were analyzed in three independent replicates. The data were presented as the mean ± SD of three individual experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

FIGURE 2

Different concentrations of glucose induce gliosis of Müller cells. The primary Müller cells were stimulated with various concentrations of glucose (control, 5 mmol/L; LG, 12.5 mmol/L; MG, 25 mmol/L; HG, 50 mmol/L) for 48 h. (A) The expressions of AQP4, GFAP, and Kir4.1 as determined by the IF assay. (B) Western blot showing the protein levels of VEGF, AQP4, GFAP, and Kir4.1. The results were calculated using the data of three independent replicates and are denoted as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

FIGURE 3

Role of glucose concentrations in cAMP/PKA/CREB signaling pathway. The primary Müller cells were exposed to various concentrations of glucose (control, 5 mmol/L; LG, 12.5 mmol/L; MG, 25 mmol/L; HG, 50 mmol/L) for 48 h. (A, B) Assessment of levels of cAMP and PKA by ELISA. (C) Western blot showing protein levels of p‐CREB and CREB. The data of triplicate is expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

FIGURE 4

Inhibition of cAMP or PKA reverses the damage of Müller cells induced by HG. The primary Müller cells were pretreated with SQ22536 (100 μmol/L, cAMP inhibitor) or H‐89 (10 μmol/L, PKA inhibitor), respectively, and further stimulated with HG (50 mmol/L) for 48 h. (A, B) Assessment of the inhibition efficiency of cAMP or PKA inhibitor using ELISA. (C) Images showing morphological changes in Müller cells as observed under an inverted microscope. The arrow represents the cells with morphological change. (D) The change in Müller cell viability as detected by the CCK‐8 assay. (E) TUNEL staining to determine Müller cell apoptosis. The data of triplicate is expressed as mean ± SD. ***p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the HG group.

FIGURE 5

Inhibition of cAMP or PKA restrains HG‐induced Müller cell gliosis. Müller cells pretreated with cAMP or PKA inhibitor were stimulated with HG for 48 h. (A) IF staining to evaluate the levels of AQP4, GFAP, and Kir4.1. (B) Western blot of VEGF, AQP4, GFAP Kir4.1, p‐CREB, and CREB. The data of triplicate is expressed as mean ± SD. ***p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the HG group.

FIGURE 6

cAMP/PKA/CREB signaling is activated in retinal tissues of DR rats. SD rat was given an intraperitoneal injection of 60 mg/kg streptozotocin to establish a DR animal model, and then 10 μL SQ22536 (10 μg) or H‐89 (8 nmol) was administered intravenously once a day for 1 week, respectively. (A, B) ELISA of cAMP and PKA in rat retinal tissues in each group. (C) Western blot of VEGF, AQP4, GFAP Kir4.1, p‐CREB, and CREB. The data were expressed as mean ± SD from five rats per group. ***p < 0.001 vs. the control group; ^## p < 0.01, ^### p < 0.001 vs. the DR group.

FIGURE 7

Inhibition of cAMP or PKA alleviates retinal injury in DR rats. SD rat was given an intraperitoneal injection of 60 mg/kg streptozotocin to establish a DR animal model, and then 10 μL SQ22536 (0.05 mg/kg) or H‐89 (0.02 mg/kg) was administered intravenously once a day for 1 week, respectively. (A) H&E staining showed the pathological features in early stage of DR. The arrow represents the location of injury. (B) IF assay for AQP4 and GFAP. The experimental data were collected from five rats per group.