干扰素基因刺激蛋白抑制剂改善视网膜血管内皮细胞白细胞粘附及糖酵解水平

Interferon gene stimulating protein inhibitor improves leukocyte adhesion and glycolysis of retinal vascular endothelial cells

目的观察氧化应激条件下干扰素基因刺激蛋白(STING)抑制剂对人视网膜微血管内皮细胞(hRMEC)的影响。方法实验研究。体内动物实验:将雄性健康C57BL/6J小鼠48只随机分为野生型小鼠组(WT组)、糖尿病(DM)组,每组各24只。DM组小鼠经链脲佐菌素诱导建立DM模型。建模成功后,DM组再分为DM+二甲基亚砜(DMSO)组、DM+C176组,每组各12只小鼠。DM+DMSO组小鼠按50 mg/kg的剂量腹腔注射DMSO;DM+C176组小鼠按50 mg/kg的剂量腹腔注射STING抑制剂C176共750 nmol。建模后4周,采用免疫组织化学染色、蛋白质免疫印迹法、实时荧光定量聚合酶链反应检测WT组、DM组小鼠视网膜STING表达情况;白细胞粘附实验检测WT组、DM+DMSO组、DM+C176组小鼠体内白细胞粘附于hRMEC数量。体外细胞实验:将hRMEC随机分为常规培养细胞组(N组)、DMSO组(加入DMSO干预)、C176组(加入C176干预)。各组加入150μg/ml糖基化终末产物诱导细胞,体外白细胞粘附实验联合4',6-二脒基-2-苯基吲哚染色检测白细胞粘附于hRMEC数量;流式细胞仪对粘附的白细胞进行定量分析;H2DCFDA/活性氧(ROS)荧光探针检测细胞中ROS表达情况;Seahorse XFe96细胞能量代谢分析仪测定细胞内糖酵解代谢水平。两组间比较采用t检验;三组间比较采用单因素方差分析。结果体内动物实验:与WT组比较,DM组小鼠视网膜中STING表达水平(t=73.248)及mRNA(t=67.385)、蛋白(t=69.371)相对表达量升高,差异均有统计学意义(P<0.05)。与WT组小鼠视网膜血管中白细胞粘附数量比较,DM+DMSO组显著增加,DM+C176组显著降低,差异有统计学意义(F=84.352,P<0.01)。体外细胞实验:与N组、DMSO组比较,C176组hRMEC上白细胞粘附数量降低,差异有统计学意义(F=35.251,P<0.01);与N组比较,DMSO组、C176组hRMEC上外周血白细胞粘附数量降低,差异有统计学意义(F=26.374,P<0.01);C176组hRMEC内ROS水平较N组、C176组降低,差异有统计学意义(F=41.362,P<0.01);与N组、DMSO组比较,C176组hRMEC的糖酵解水平显著降低,差异有统计学意义(F=68.741,P<0.01)。结论抑制白细胞粘附、ROS生成、下调糖酵解水平可抑制STING表达,从而改善视网膜血管内皮细胞功能。

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 C176750 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 150μg/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;H2DCFDA/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.