黄芪甲苷对人视网膜色素上皮细胞线粒体氧化损伤的调控作用

Protective Effects of Astragaloside IV on Mitochondrial Oxidative Damage in Human Retinal Pigment Epithelial Cells

目的探究黄芪甲苷对H_(2)O_(2)诱导人视网膜上皮细胞(Retinal pigment epithelium,RPE)线粒体氧化损伤的调控作用。方法建立H_(2)O_(2)诱导人RPEARPE-19和hTERT RPE-1氧化损伤模型,并加入不同剂量黄芪甲苷,用CCK-8法检测ARPE-19和hTERT RPE-1细胞增殖活性,用荧光探针标记法检测ARPE-19细胞活性氧(Reactive oxygen,ROS)和脂质过氧化水平,用化学显色法检测ARPE-19细胞内SOD、LDH和MDA水平,用JC-1标记法检测ARPE-19细胞线粒体膜电势,用Calcein AM荧光染色法检测ARPE-19线粒体通透性,用蛋白质免疫印迹实验检测ARPE-19细胞Nrf2表达及磷酸化水平。结果与对照组比较,H_(2)O_(2)模型组ARPE-19和hTERT RPE-1细胞增殖活性明显下降(P<0.05),细胞内ROS积累增加(P<0.05),脂质过氧化水平升高(P<0.05),SOD和LDH表达水平下降(P<0.05),MDA水平升高(P<0.05),线粒体膜电势下降(P<0.05),通透性增加(P<0.05),Nrf2表达水平升高(P<0.05),磷酸化水平增加(P<0.05)。与H_(2)O_(2)模型组比较,低剂量、中剂量和高剂量黄芪甲苷处理的ARPE-19细胞增殖活性明显升高(P<0.05),中剂量和高剂量黄芪甲苷处理的hTERT RPE-1细胞增殖活性明显升高(P<0.05)。ARPE-19细胞内ROS和脂质过氧化水平下降(均P<0.05),SOD和LDH表达水平升高(P<0.05),MDA水平下降(P<0.05),线粒体膜电势升高(P<0.05),通透性降低(P<0.05),Nrf2表达无明显变化(P>0.05),磷酸化水平增加(P<0.05)。结论黄芪甲苷缓解H_(2)O_(2)诱导的人RPE氧化应激和线粒体功能损伤。

Objective This study investigates the regulatory effects of astragaloside IV on H_(2)O_(2)-induced mitochondrial oxidative damage in human retinal pigment epithelial cells.Methods The ARPE-19 and hTERT RPE-1 cell model with H_(2)O_(2)-induced oxidative damage was established,and various doses of astragaloside IV were added.Cell proliferation activity was assessed using the CCK-8 method.Cellular reactive oxygen species(ROS)and lipid peroxidation levels were measured using fluorescent probes.The intracellular levels of superoxide dismutase(SOD),lactate dehydrogenase(LDH),and malondialdehyde(MDA)were determined using chemical staining methods.Mitochondrial membrane potential was assessed with JC-1 staining,and mitochondrial permeability was examined using Calcein AM fluorescent staining.Western blot was used to detect the expression and phosphorylation of Nrf2 in ARPE-19 cells.Results Compared to the control group,the H_(2)O_(2)model group showed significantly decreased proliferation activity of ARPE-19 and hTERT RPE-1 cells(P<0.05),increased intracellular ROS accumulation(P<0.05),elevated lipid peroxidation levels(P<0.05),decreased SOD and LDH expression levels(P<0.05),increased MDA level(P<0.05),decreased mitochondrial membrane potential(P<0.05),increased permeability(P<0.05),elevated expression level of Nrf2(P<0.05),and increased phosphorylation level(P<0.05).Compared to the H_(2)O_(2)model group,the low-dose,medium-dose,and high-dose astragaloside IV groups exhibited greatly increased proliferation activity of ARPE-19 cells(P<0.05),and the medium-dose,and high-dose astragaloside IV groups showed markedly increased proliferation activity of hTERT RPE-1 cells(P<0.05).Additionally,ARPE-19 cells treated with astragaloside IV showed decreased intracellular ROS and lipid peroxidation levels(P<0.05),increased SOD and LDH expression levels(P<0.05),decreased MDA level(P<0.05),increased mitochondrial membrane potential(P<0.05),decreased permeability(P<0.05),increased phosphorylation level(P<0.05),and no significant change in the expression of Nrf2(P>0.05).Conclusion Astragaloside IV can alleviate oxidative stress and mitochondrial function damage induced by H_(2)O_(2)in human retinal pigment epithelial cells.