Resveratrol prevents hypoxia-induced retinal ganglion cell death related with ErbB2

Abstract

Aim: To confirm the changes in proteins related with hypoxia-induced retinal cell death and to assess the effects of resveratrol (Res).

Methods: The therapeutic effect of Res was verified using an ischemic/reperfusion (I/R) model in vivo and a hypoxia modelin retinal ganglion cells (RGCs) in vitro. Death of RGCs were confirmed by TUNEL assay. Protein expression was confirmed by Western blotting and immunohistochemistry. In addition, flow cytometric analysis was used to confirm the response in the cell unit to obtain more accurate data.

Results: ErbB2 expression and apoptosis in the ganglion cell layer (GCL) increased after I/R injury. Treatment of Res rescued I/R-induced ganglion cell death, downregulated apoptosis and ErbB2 protein expression in the retina. In subsequent in vitro models, Res affects apoptosis by regulating the phosphorylation and expression of mouse double minute 2 homolog (MDM2), along with those of ErbB2. These results suggest that Res reverses GCL-specific apoptosis via downregulation of ErbB2 in ischemic injury.

Conclusion: In light of Res favorable properties, it should be evaluated in the treatment of RGC death and related retinal disease characterized by ErbB2 and MDM2 expression. Therefore, Res is appropriate therapeutic agent for treating ischemic injury-related eye diseases by targeting the expression of ErbB2 and MDM2.

Keywords: ErbB2; hypoxia; ischemia/reperfusion injury; resveratrol; retinal ganglion cell.

Figure 1. Schematic diagrams of the resveratrol (Res) treatment and retinal I/R injury

A: Schematic diagram for in vivo; B: Schematic diagram for in vitro.

Figure 2. Effects of Res on I/R injury-induced TUNEL-positive cells in the GCL

A: TUNEL and API staining of retinal cross-sections by group; B: Number of TUNEL-positive cells in the GCL (white arrows) were counted using Image J software (top of the GCL-bottom of the INL). Data are represented as the means±SEM and were analyzed using one-way ANOVA analysis of variance with Dunnett's post-test (n=3-4 for each group). Scale bar: 50 µm. ^aP<0.001 vs control, ^bP<0.001 vs I/R.

Figure 3. Effect of Res on I/R injury-induced ErbB2 expression in the retina

A: Retinal ErbB2 expression was detected by immunohistochemistry (hematoxylin counterstain). Scale bar represents 50 µm; B: ErbB2 protein expression decided using Image J software and displayed as the change value of each group relative to the control by an arbitrary unit. Data are indicated as the means±SEM and were tested using one-way analysis of variance with Dunnett's post-test (n=3 for each group). ^aP<0.05 vs control, ^bP<0.01 vs I/R.

Figure 4. Effect of Res on expression of cell death-related upstream regulator

A: RGC-5 cells were incubated in 1% O₂ hypoxic condition (hypoxia), pretreated with Res (10 µmol/L) for 2h and then co-treated with 1% O₂ and Res (10 µmol/L) for 6h (hypoxia+Res) or treated with Res (10 µmol/L) for 8h (Res). Western blots showing the protein expression of ErbB2, p-MDM2, and MDM2 in RGC-5 cells. B: ErbB2 protein expression levels. C: The p-MDM2/MDM2 protein expression ratio performed using Image J software and displayed as the change value of each group relative to the control (CTL) by an arbitrary unit. Data are indicated as the means±SEM and were analyzed using one-way analysis of variance with Dunnett's post-test (n=3). ^aP<0.05, ^bP<0.01 vs control, ^cP<0.05 vs hypoxia.

Figure 5. Effects of Res on CoCl₂-induced ROS production, cell cycle arrest and CoCl₂-induced apoptosis

A: Cells were pretreated with Res (10 µmol/L) for 2h and then co-treated with CoCl₂ (200 µmol/L) and Res for 24h. ROS production measured by carboxy-H₂DCFDA staining. B: Cell cycle arrest measured by propidium iodide staining. C: Apoptosis measured by annexin V-FITC/PI staining. Data are represented as the means±SEM and were analyzed by one-way analysis of variance with Dunnett's post-test (n=4-6 for each group). ^aP<0.001 vs control, ^bP<0.001 vs CoCl₂.