Protective effect of myricitrin on retinal microvascular endothelial cells induced by high glucose and its regulation mechanism / 中华实验眼科杂志

ABSTRACT

Objective:To explore the effect of myricitrin on the injury of human retinal microvascular endothelial cells (HRMECs) induced by high glucose and its regulation mechanism.Methods:HRMECs were divided into normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml myricitrin groups.HRMECs transfected with pcDNA and pcDNA-circZNF292, respectively and then cultured in high-glucose medium containing 25 mmol/L D-glucose for 24 hours were assigned as pcDNA group and pcDNA-circZNF292 group.HRMECs transfected with siR-NC and siR-circZNF292, respectively and then cultured in medium containing 50.0 μg/ml myricitrin and 25 mmol/L D-glucose for 24 hours were assigned as myricitrin+ siR-NC group and myricitrin+ siR-circZNF292 group.The cell apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in cells were detected by enzyme-linked immunosorbent assay (ELISA) kits.The expression levels of circZNF292 and miR-23b-3p were detected by real-time fluorescence quantitative PCR.The targeting relationship between circZNF292 and miR-23b-3p was detected by dual-luciferase reporter assay.The relative expression levels of B-cell lymphoma-2 (bcl-2) and bcl-2-related X protein (bax) were assayed by Western blot.Results:Significant differences were found in the relative expressions of bax and bcl-2 proteins, cell apoptosis rate, MDA concentration, SOD activity, circZNF292 and miR-23b-3p among normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml, 50.0 μg/ml myricitrin groups ( F=105.707, 111.835, 74.515, 109.651, 135.020, 219.919, 116.304; all at P<0.001).With the increase of myricitrin concentration, the relative expression levels of bax protein, cell apoptosis rate, MDA concentration and miR-23b-3p in cells gradually decreased, while the relative expression levels of bcl-2 protein, SOD activity and circZNF292 increased, with statistically significant differences among groups with different concentrations of myricitrin (all at P<0.05).In the co-transfected wild-type (WT)-circZNF292 cells, the relative luciferase activity in miR-23b-3p group was 0.35±0.03, which was lower than 0.96±0.09 in microRNA-negative control group, and the difference was statistically significant ( t=11.137, P<0.001).Compared with pcDNA group, the relative expression levels of bcl-2 protein, circZNF292 and MDA concentration in cells of pcDNA-circZNF292 group were significantly increased, and the relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and SOD activity were significantly decreased (all at P<0.05).The relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and MDA concentration were reduced and relative expression levels of bcl-2 protein, circZNF292 and SOD activity were enhanced in myricitrin group and myricitrin+ siR-NC group in comparison with high glucose group and myricitrin+ siR-circZNF292 group, showing statistically significant differences (all at P<0.05). Conclusions:Myricitrin can inhibit cell apoptosis and oxidative stress by regulating the expression of circZNF292/miR-23b-3p, thereby reducing the damage of HRMECs induced by high glucose.