胃饥饿素对高糖下视网膜微血管内皮细胞氧化应激及铁死亡的抑制作用

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

目的研究胃饥饿素对高糖下人视网膜微血管内皮细胞(HRMEC)氧化应激及铁死亡的影响。方法将体外培养的HRMEC分为对照组、高糖组、高糖+胃饥饿素组,分别采用常规培养基、含30 mmol/L D-葡萄糖培养基、含30 mmol/L D-葡萄糖+10 nmol/L胃饥饿素培养基培养24 h。采用细胞计数试剂盒8检测细胞增生情况;采用流式细胞术检测细胞活性氧簇(ROS)水平;采用试剂盒检测细胞中氧化应激指标还原型谷胱甘肽(GSH)浓度、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性及Fe^(2+)浓度;采用透射电子显微镜观察线粒体结构;采用Western blot法检测HRMEC中铁死亡关键分子谷胱甘肽过氧化合物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)蛋白表达。结果对照组、高糖组、高糖+胃饥饿素组细胞增生率分别为(100.62±3.40)%、(63.74±4.25)%和(88.19±4.65)%,ROS荧光强度分别为15512.20±1347.53、46457.00±1072.65和22220.87±1669.20,GSH浓度分别为(68.52±7.61)、(21.45±1.57)和(55.68±5.15)μmol/L,MDA浓度分别为(0.79±0.10)、(2.47±0.27)和(1.08±0.15)μmol/L,SOD活性分别为(111.67±10.32)、(37.75±5.92)和(97.45±9.12)U/ml,Fe^(2+)浓度分别为(3.02±0.30)、(9.45±0.71)和(4.63±0.32)mmol/mgprot。各组间细胞增生率、ROS荧光强度、GSH浓度、MDA浓度、SOD活性及Fe^(2+)浓度总体比较,差异均有统计学意义(F=61.82、414.59、61.28、67.24、61.64,146.14,均P<0.001);与对照组相比,高糖组细胞增生率、GSH浓度和SOD活性均明显降低,ROS荧光强度、MDA浓度和Fe^(2+)浓度明显升高,差异均有统计学意义(均P<0.05);与高糖组相比,高糖+胃饥饿素组细胞增生率、GSH浓度和SOD活性明显升高,ROS荧光强度、MDA浓度和Fe^(2+)浓度明显降低,差异均有统计学意义(均P<0.05)。与对照组相比,高糖组细胞内线粒体铁死亡现象明显;与高糖组相比,高糖+胃饥饿素组线粒体状态明显改善。各组间细胞中GPX4、SLC7A11蛋白相对表达量总体比较,差异均有统计学意义(F=63.94、182.84,均P<0.001);与对照组相比,高糖组和高糖+胃饥饿素组细胞中GPX4、SLC7A11蛋白相对表达量均明显降低,差异均有统计学意义(均P<0.05);与高糖组相比,高糖+胃饥饿素组2种蛋白表达水平均升高,差异均有统计学意义(均P<0.01)。结论胃饥饿素可促进高糖下的HRMEC增生,抑制高糖诱导的氧化应激和铁死亡。

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 15512.20±1347.53,46457.00±1072.65 and 22220.87±1669.20,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.