Protective effect of myricitrin on retinal microvascular endothelial cells induced by high glucose and its regulation mechanism

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.

Myricitrin; Apoptosis; Oxidative stress; Human retinal microvascular endothelial cells; CircZNF292; MiR-23b-3p