摘要
目的:探讨木犀草素对H2O2诱导的视网膜色素上皮(RPE)细胞氧化损伤的保护作用及其机制。方法:将ARPE-19细胞分为对照组、H2O2组、不同剂量木犀草素组和Nrf2抑制剂组,除对照组外均采用100μmol/L H2O2制备RPE氧化损伤模型。采用四甲基偶氮唑盐(MTT)法检测细胞活力并确定木犀草素处理浓度,采用流式细胞仪检测细胞凋亡率及活性氧(ROS)含量,采用试剂盒法检测细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)含量,采用Western blot检测细胞Caspase-3、多聚腺苷酸二磷酸核糖聚合酶(PARP)、B细胞淋巴瘤-2(Bcl-2)、核因子E2相关因子2(Nrf2)及血红素氧化酶-1(HO-1)蛋白表达水平。结果:100μmol/L木犀草素对ARPE-19具有毒性作用,因此选择25μmol/L和50μmol/L木犀草素进行后续实验。与H2O2组比较,25μmol/L和50μmol/L木犀草素组细胞活力明显升高,凋亡率降低,ROS和MDA含量明显减少,SOD活性明显升高,Caspase-3和PARP蛋白表达水平明显降低,Bcl-2、Nrf2及HO-1蛋白表达水平明显升高(P<0.05)。与50μmol/L木犀草素组比较,Nrf2抑制剂组细胞活力明显降低,凋亡率明显升高,ROS和MDA含量明显升高[(654.96±26.99)vs(446.52±29.42),(3.89±0.29)nmol/mL vs(2.06±0.19)nmol/mL],SOD活性明显降低[(13.83±1.49)U/mL vs(22.69±1.83)U/mL],Caspase-3和PARP蛋白表达水平明显升高,Bcl-2、Nrf2及HO-1蛋白表达水平明显降低(P<0.05)。结论:木犀草素能够改善H2O2诱导的RPE细胞氧化损伤,其作用机制与激活Nrf2/HO-1通路有关。
AIM:To investigate the protective effect and mechanism of luteolin on H2O2-induced oxidative damage of retinal pigment epithelium(RPE)cells.METHODS:ARPE-19 cells were divided into the control group,H2O2 group,different doses of luteolin groups and Nrf2 inhibitor group,and the oxidative damage model of RPE was prepared by 100μmol/L H2O2,except for the control group.Cell activity was detected by Methyl thiazolyl tetrazolium(MTT)assay and proper experimental concentration of luteolin was determined.The cell morphology and activity was observed in each group.Cell apoptosis rate and reactive oxygen species(ROS)were detected by flow cytometry,malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by kit method,and the expression of caspase-3,poly adeno-sine diphosphate ribose polymerase(PARP),B cell lymphoma-2(Bcl-2),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)proteins were detected by Western blot.RESULTS:100μmol/L luteolin has toxic effects on ARPE-19 cells,so 25μmol/L and 50μmol/L luteolin were selected for subsequent experiments.The cell activity,SOD activity and the protein expression levels of Bcl-2,Nrf2,HO-1 in 25μmol/L and 50μmol/L luteolin groups were significantly higher than the H2O2 group(P<0.05).The apoptosis rate,ROS,MDA content and the protein expression levels of Caspase-3 and PARP in 25μmol/L and 50μmol/L luteolin groups were significantly lower than the H2O2 group(P<0.05).The cell activity,SOD activity[(13.83±1.49)U/mL vs(22.69±1.83)U/mL]and the protein expression levels of Bcl-2,Nrf2 and HO-1 protein expression in the Nrf2 inhibitor group were significantly lower than the 50μmol/L luteolin group(P<0.05).The apoptosis rate,ROS,MDA content[(654.96±26.99)vs(446.52±29.42),(3.89±0.29)nmol/mL vs(2.06±0.19)nmol/mL]and the protein expression levels of Caspase-3 and PARP in the Nrf2 inhibitor group were significantly higher than the 50μmol/L luteolin group(P<0.05).CONCLUSION:Luteolin can improve the oxidative damage of RPE cells induced by H2O2,and its mechanism may be related to the activation of the Nrf2/HO-1 pathway.
作者
洪萌
洪道先
石荣先
魏聪
赵晓丽
李权达
Meng Hong;Dao-Xian Hong;Rong-Xian Shi;Cong Wei;Xiao-Li Zhao;Quan-Da Li(Department of Ophthalmology,the First Affiliated Hospital of Henan University,Kaifeng 475001,Henan Province,China;Department of Anesthesiology,the First Affiliated Hospital of Henan University,Kaifeng 475001,Henan Province,China)
出处
《国际眼科杂志》
CAS
北大核心
2021年第1期21-26,共6页
International Eye Science
基金
开封市科技发展计划项目(No.1903065)。
