Inhibitory effects of ghrelin on oxidative stress and ferroptosis in retinal microvascular endothelial cells under high glucose

Objective  To investigate the effects of ghrelin on oxidative stress and ferroptosis in retinal microvascular endothelial cells (HRMEC) under high glucose conditions.

Methods  HRMEC were divided into control group, high glucose group, high glucose+ ghrelin group and cultured with conventional medium, 30 mmol/L D-glucose medium, and 30 mmol/L D-glucose+ 10 nmol/L ghrelin medium in vitro for 24 hours accordingly.The cell proliferation was identified by cell counting kit-8 assay.The reactive oxygen species (ROS) levels were detected by flow cytometry.The oxidative stress indexes glutathione (GSH) concentration, malondialdehyde (MDA) superoxide dismutase (SOD) activity and Fe ²⁺ concentration were detected by the corresponding kit.The mitochondrial structure was observed by transmission electron microscopy.The expression levels of glutathione peroxidase 4 (GPX4) and recombinant solute carrier family 7 member 11 (SLC7A11) proteins were detected by Western blot.

Results  The cell proliferation rates of control group, high glucose group and high glucose+ ghrelin group were (100.62±3.40)%, (63.74±4.25)% and (88.19±4.65)%, respectively.The ROS fluorescence intensity of control group, high glucose group and high glucose+ ghrelin group was 15 512.20±1 347.53, 46 457.00±1 072.65 and 22 220.87±1 669.20, GSH concentration was (68.52±7.61), (21.45±1.57) and (55.68±5.15)μmol/L, MDA concentration was (0.79±0.10), (2.47±0.27) and (1.08±0.15)μmol/L, SOD activity was (111.67±10.32), (37.75±5.92) and (97.45±9.12)U/ml, Fe ²⁺ concentration was (3.02±0.30), (9.45±0.71) and (4.63±0.32)mmol/mgprot, respectively.There were statistically significant differences in cell proliferation rate, ROS fluorescence intensity, GSH, MDA, and Fe ²⁺ concentrations and SOD activity among the three groups ( F=61.82, 414.59, 61.28, 67.24, 61.64, 146.14; all at P<0.001).Compared with the control group, the cell proliferation rate, GSH concentration and SOD activity were reduced, ROS fluorescence intensity, MDA and Fe ²⁺ concentrations were increased in the high glucose group, with statistically significant differences (all at P<0.05).Compared with the high glucose group, the cell proliferation rate, GSH concentration and SOD activity were significantly increased, ROS fluorescence intensity, MDA and Fe ²⁺ concentrations were decreased in the high glucose+ ghrelin group, with statistically significant differences (all at P<0.05).Compared with the control group, the ferroptosis of mitochondria in high glucose group was obvious.Compared with the high glucose group, the mitochondrial status of the high glucose+ ghrelin group was significantly improved.There were significantly differences in the relative expression levels of GPX4 and SLC7A11 proteins in cells among the three groups ( F=63.94, 182.84; both at P<0.001).Compared with the control group, the relative expression levels of GPX4 and SLC7A11 proteins were significantly decreased in the high glucose group and high glucose+ ghrelin group (all at P<0.05).Compared with the high glucose group, the relative expression levels of the two proteins in the high glucose+ ghrelin group were increased, with statistically significant differences (both at P<0.01).

Conclusions  Ghrelin can promote proliferation of HRMEC under high glucose conditions, and inhibit high glucose-induced oxidative stress and ferroptosis.

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