Cannabinoid receptor 1 blockade protects human retinal pigment epithelial cells from oxidative injury

Abstract

Background: Because oxidative stress is assumed to be a key mechanism in the pathological process of age-related macular degeneration (AMD), increasing numbers of studies have focused on discovering new pathways and treatments for reducing oxidative damage. Our work investigates the potential role of the cannabinoid receptor 1 (CB1) in oxidative stress of primary human retinal pigment epithelial (RPE) cells, a cellular model of AMD.

Methods: Primary human RPE cells were cultured and exposed to hydrogen peroxide for 24 h to induce oxidative damage. The expression of and changes in the CB1 receptor were determined with western blot assay and confocal imaging. The CB1 receptor in the RPE cells was inhibited with small interfering RNA (siRNA) or rimonabant (SR141716). Cell viability, apoptosis, and reactive oxygen species production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulforhodamine B assay, annexin V and propidium iodide staining, and the dichlorofluorescein fluorescence assay, respectively. Intracellular superoxide dismutase activity was assayed with a commercially available assay kit. Phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) protein expression and activation of signaling molecules were assessed with western blot analysis.

Results: We showed that human RPE cells express the CB1 receptor. In addition, oxidative stress upregulates the expression of the CB1 receptor. Deleting the CB1 receptor or treating with the CB1 receptor antagonist rimonabant (SR141716) rescued RPE cells from hydrogen peroxide-induced oxidative damage. Rimonabant pretreatment effectively reduced the apoptosis of RPE cells, inhibited the generation of intracellular reactive oxygen species and elevated the activity of superoxide dismutase. In addition, rimonabant significantly strengthened the oxidative stress-induced activation of the PI3K/Akt signaling pathway.

Conclusions: The results demonstrate the expression and regulation of CB1 receptors in human RPE cells. Inhibiting the CB1 receptor may be an effective therapeutic strategy for AMD by downregulating oxidative stress signaling and facilitating PI3K/Akt activation.

Figure 1

Expression of and changes in the CB₁ receptor in human retinal pigment epithelial (RPE) cells. A: In the western blot analysis of CB₁ receptor protein expression changes in primary RPE cells, CB₁ receptor protein level was significantly increased by H₂O₂ incubation in a dose-dependent manner. B: CB₁ receptor protein localized to the cytoplasm and cellular membrane as demonstrated with immunofluorescence staining (Bar=20 μm). Quantitative analysis of the fluorescent levels is indicated in the right panel. *p<0.05 versus control, the sample number is n=5 per group and we performed t test here. C: A representative photograph of primary cultured RPE cells seeded for 24 h.

Figure 2

Downregulation of the CB₁ receptor protected RPE cells from H₂O₂-induced damage. A: Primary human RPE cells were transfected with CB₁ receptor siRNA for 24 h. CB₁ receptor mRNA levels were detected using real time RT–PCR. B: Primary human RPE cells were transfected with CB₁ receptor siRNA for 48 h, and CB₁ receptor protein levels were detected by western blot assay. C: After the si-NC or si-CB₁ siRNA-treated RPE cells received different concentrations of H₂O₂ for 24 h, cell viability was examined with the MTT assay. ***p<0.001, si-NC versus si-CB₁ siRNA-treated cells. D: After the si-NC or si-CB₁ siRNA-treated RPE cells received different concentrations of H₂O₂ for 24 h, cell viability was examined with the SRB assay. *p<0.05,***p<0.001, si-NC versus si-CB₁ siRNA-treated cells. The statistical test of C and D are two way ANOVA, n=4 per group.

Figure 3

Rimonabant attenuates H₂O₂-induced cytotoxicity and apoptosis. A: Inhibition of the H₂O₂-induced decrease in RPE cell viability by rimonabant. RPE cells were pretreated with rimonabant (0 to 5 μM) for 15 min before being exposed to H₂O₂ (200 μM) for 24 h, and cell viability was measured with the MTT assay. Values are the percentage of control (no H₂O₂, no rimonabant). *p<0.05 versus H₂O₂. B: RPE cells were pretreated with 1 μM ACEA for 15 min in the presence or absence of rimonabant (1 μM) before being exposed to H₂O₂ (200 μM) for 24 h. *p<0.05 versus H₂O₂. **p<0.01 versus rimonabant without ACEA. C: Flow cytometric analysis of cell death with DMSO, H₂O₂ (200 μM), rimonabant (1 μM), and rimonabant (1 μM) + H₂O₂ (200 μM). Cells were treated with different media as indicated for 24 h. Summary of the results showing a significant increase in apoptosis in RPE cells maintained in H₂O₂ (200 μM) compared with those maintained in vehicle. When the cells were incubated with rimonabant (1 μM), H₂O₂-induced apoptosis was significantly reduced. Treatment of RPE cells with rimonabant (1 μM) alone did not alter cell death. *p<0.05 versus vehicle H₂O₂ (n=4). The statistical test of A and B are one way ANOVA, n=4 per group. In the C, the test is two way ANOVA, n=4 per group.

Figure 4

Rimonabant inhibited the H₂O₂-induced increase in intracellular ROS and activated the H₂O₂-induced decrease in intracellular superoxide dismutase (SOD) in RPE cells. A: RPE cells were pretreated with rimonabant (0 to 1 μM) for 15 min before being exposed to H₂O₂ (200 μM) for 24 h. Intracellular ROS was measured with the DCF-DA assay. *p<0.05 versus H₂O₂. B: SOD activity was assayed with a commercially available assay kit. *p<0.05 versus control (no H₂O₂ no rimonabant). #p<0.05 versus H₂O₂. The statistical test of A is one way ANOVA, n=4 per group. In the B, the test is two way ANOVA, n=4 per group.

Figure 5

Rimonabant modulates phosphorylation of the PI3K/Akt signaling pathway. A: Representative western blot analysis shows that rimonabant (1 μM) enhanced the H₂O₂-induced activation of p-PI3K/Akt in RPE cells. RPE cells were pretreated with rimonabant (0.1, 1 μM, 15 min) and then exposed to H₂O₂ (200 μM, 24 h). B: Statistical analysis of the results indicated a 2.4-fold increase. C: RPE cells were pretreated with or without LY294002 (LY, 10 μM) or wortmannin (WMN, 10 μM) for 15 min in the presence or absence of rimonabant (1 μM) before being exposed to H₂O₂ (200 μM) for 24 h (***p<0.001 versus rimonabant + H₂O₂). The statistical test of C is one way ANOVA, n=4 per group.

Figure 6

The mechanism chart of CB₁ receptor blockade protects human RPE cells from H₂O₂-induced damage.