Neuroprotective effects of brimonidine on retinal ganglion cells induced by oxidative stress mitochondrial dysfunction

Objective

To explore whether brimonidine has a protective effect on retinal ganglion cells (RGCs) through improving mitochondrial function under the oxidative stress.

Methods

Mouse RGC-5 cells were cultured in DMEM medium containing low concentration of glucose (1 g/L), 10% fetal bovine serum and 100 U/ml penicillin-streptomycin solution.The cells were divided into normal control group, H₂O₂-treated group and brimonidine+ H₂O₂ group.H₂O₂ at the concentration of 800 μmol/L was added into the medium in the H₂O₂-treated group, and 1 μmol/L brimonidine was added into the medium for 2 hours prior to the addition of H₂O₂ in the brimonidine+ H₂O₂ group.The cells were sequently cultured for 24 hours.The morphology of the cell nucleus was examined by Hoechst fluorscence staining.The expressions of apoptosis-related protein in the cells were detected by Western blot assay.Mitochondrial membrane potential was assessed by JC-1 staining.

Results

The cell nuclei showed round or oval in shape with consistent size in the normal control group.The pycnosis and karyorrhexis of the cell nuclei were seen in the H₂O₂-treated group, and less abnormal nuclei were found in the brimonidine+ H₂O₂ group.The relative expression level of bcl-2 protein in the cells was 0.76±0.15, 0.50±0.13 and 0.75±0.17 in the normal control group, H₂O₂-treated group and brimonidine+ H₂O₂ group, respectively, and the expression of bcl-2 protein in the H₂O₂-treated group was significantly lower than that in the normal control group and brimonidine+ H₂O₂ group (both at P<0.05). The relative expression level of bax protein in the cells was 0.65±0.13, 0.83±0.07 and 0.70±0.10 in the normal control group, H₂O₂-treated group and brimonidine+ H₂O₂ group, respectively, and the expression of bax protein in the H₂O₂-treated group was significantly higher than that in the normal control group and brimonidine+ H₂O₂ group (both at P<0.05). A strong orange fluorescence was seen in the mitochondrial membrane of RGC-5 in the normal control group with a co-expression with the green fluorescence of cell membrane.In the H₂O₂-treated group, the orange fluorescence intensity in the cells was evidently weakened, and the number of JC-1 responsed cells was considerably increased and the orange fluorescence intensity was enhanced in the brimonidine+ H₂O₂ group.

Conclusions

Brimonidine can prevent RGCs from oxidative-stress damage by improving the mitochondrial function and therefore play a potential neuroprotective effect on optic nerve.

Neuroprotective agents/pharmacology; Cell hypoxia/drug effects; Oxidative stress; Retinal ganglion cells; Apoptosis; Mitochondria/pathology; Brimonidine; Cell line