目的研究秦皮乙素对叔丁基过氧化氢(t-BHP)诱导的人视网膜色素上皮细胞系ARPE-19细胞氧化损伤的影响及其机制。
方法将传代ARPE-19细胞贴壁培养后分为空白对照组、模型对照组、20 μmol/L秦皮乙素组、40 μmol/L秦皮乙素组、80 μmol/L秦皮乙素组和100 μmol/L秦皮乙素组，空白对照组细胞采用正常培养液培养，模型对照组使用900 μmol/L t-BHP单独作用细胞4 h，后4个组分别使用900 μmol/L t-BHP＋不同浓度的秦皮乙素共同作用细胞4 h。采用MTS法检测细胞存活率；采用荧光染色分析法检测细胞内活性氧簇(ROS)活性；采用试剂盒检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性，以及丙二醛(MDA)含量。
结果空白对照组、模型对照组、20 μmol/L秦皮乙素组、40 μmol/L秦皮乙素组、80 μmol/L秦皮乙素组和100 μmol/L秦皮乙素组细胞存活率分别为(100.00±1.58)%、(49.19±1.06)%、(76.82±3.48)%、(103.90±1.60)%、(111.70±3.36)%和(113.40±3.08)%，组间总体比较差异有统计学意义( F＝95.44， P<0.01)，其中与空白对照组比较，模型对照组细胞存活率明显降低，差异有统计学意义( P<0.01)；与模型对照组比较，各浓度秦皮乙素组细胞存活率明显升高，差异均有统计学意义(均 P<0.01)。各组ROS荧光强度相对值、MDA水平、SOD活力值、CAT活力值、GSH-Px活力值总体比较差异均有统计学意义( F＝575.20、40.61、1 802.00、41.62、38.31，均 P<0.01)，其中与模型对照组比较，各浓度秦皮乙素组ROS荧光强度相对值、MDA水平明显降低，SOD活性、CAT活性和GSH-Px活性明显升高，差异均有统计学意义(均 P<0.01)。
结论秦皮乙素通过上调细胞内抗氧化酶类活性或抗氧化蛋白的表达对氧化损伤的ARPE-19细胞起到保护作用。
ObjectiveTo study the protective effect and the mechanism of esculetin on oxidative-stressed human retinal pigment epithelial cells (ARPE-19) induced by tert-butyl hydroperoxide (t-BHP).
MethodsThe ARPE-19 cells were divided into blank control group, model control group, 20 μmol/L esculetin group, 40 μmol/L esculetin group, 80 μmol/L esculetin group and 100 μmol/L esculetin group.The cells in the blank control group were normally cultured.The cells in the model control group were treated with 900 μmol/L t-BHP for 4 hours.The rest four groups were treated with 900 μmol/L t-BHP+ different molar concentrations of esculetin respectively for 4 hours.The cell viability of the each group was detected by MTS method.The activity of reactive oxygen species (ROS) was detected by fluorescence staining, and the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) as well as the levels of malondialdehyde (MDA) of the cells from each group were measured with each corresponding assay kit, respectively.
ResultsThe relative viabilities of the cells in the blank control group, model control group, 20 μmol/L esculetin group, 40 μmol/L esculetin group, 80 μmol/L esculetin group and 100 μmol/L esculetin group were (100.00±1.58)%, (49.19±1.06)%, (76.82±3.48)%, (103.90±1.60)%, (111.70±3.36)% and (113.40±3.08)%, respectively.There was a significant difference among the groups ( F=95.44, P<0.01). Compared with the blank control group, the viability of the cells in the model control group was decreased significantly ( P<0.01). Compared with the model control group, the cell viabilities in different concentrations of esculetin groups were increased significantly (all at P<0.01). There were significant differences between the groups in the relative value of ROS fluorescence intensity, MDA level, SOD activity, CAT activity and GSH-Px activity ( F=575.20, 40.61, 1 802.00, 41.62, 38.31; all at P<0.01). Compared with the model control group, the levels of ROS and MDA were decreased significantly, while the activities of SOD, CAT and GSH-Px were increased significantly in different concentrations of esculetin-treated groups (all at P<0.01).
ConclusionsEsculetin can protect the oxidative damaged ARPE-19 cells by up-regulating the expression of antioxidant enzymes or antioxidant proteins.
张英俊,白鸽,何向东,等. 秦皮乙素对氧化损伤ARPE-19细胞的保护作用及其机制[J]. 中华实验眼科杂志,2020,38(12)：1025-1031.
DOI:10.3760/cma.j.cn115989-20200224-00101版权归中华医学会所有。
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