Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells / 中华眼底病杂志

ABSTRACT

Objective:To investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:An animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H 2DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. Results:In vivo experiment: compared with WT group, the expression level of STING ( t=73.248) and the relative expression amount of mRNA ( t=67.385) in the retina of DM group mice increased significantly ( P<0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased ( F=84.352, P<0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly ( F=35.251, P<0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly ( F=26.374, P<0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group ( F=41.362, P<0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference ( F=68.741, P<0.01). Conclusion:Inhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.