摘要
目的:探讨1,25-二羟基维生素D3[1,25-(OH)2D3]对细颗粒物(PM2.5)致人支气管上皮细胞(HBE)氧化损伤的保护作用。方法:根据处理因素不同将细胞分为4组,溶剂(乙醇)对照组、1,25-(OH)2D3干预组、乙醇+PM2.5染毒组、PM2.5染毒+1,25-(OH)2D3干预组。溶剂对照组细胞用0.1%乙醇处理48 h,1,25-(OH)2D3干预组用1×10^-9mol/L 1,25-(OH)2D3处理48 h,乙醇+PM2.5染毒组用0.1%乙醇溶剂处理24 h后更换为含乙醇的PM2.5(200μg/m L)染毒液继续处理24 h,PM2.5染毒+1,25-(OH)2D3干预组用1×10^-9mol/L 1,25-(OH)2D3预处理24 h后更换为含1,25-(OH)2D3的PM2.5(200μg/m L)染毒液继续处理24 h。染毒结束后,用CCK-8试剂盒测定细胞存活率、丙二醛(MDA)和还原型谷胱甘肽(GSH)/氧化型谷胱甘肽(GSSG)-GloTM试剂盒分别测定MDA浓度和GSH/GSSG比值,Western blot实验测定维生素D受体(VDR)、转录因子NF-E2相关因子2(Nrf-2)与血红素加氧酶-1(HO-1)蛋白的表达水平。结果:PM2.5染毒处理后,HBE细胞的存活率降至80.8%,1,25-(OH)2D3预处理24 h后再进行PM2.5染毒的细胞存活率降低至75.8%。与对照相比,PM2.5染毒后,HBE细胞MDA浓度显著增加(P<0.05),而GSH/GSSG的比值却明显降低(P<0.01)。1,25-(OH)2D3处理48 h后可显著改善PM2.5染毒细胞的抗氧化水平(P<0.05),主要表现为MDA浓度的降低和GSH/GSSG比值的增加。蛋白分析结果发现,PM2.5可诱导细胞Nrf-2和HO-1蛋白表达的增加。1,25-(OH)2D3干预48 h后可上调HBE细胞内VDR水平,并可增加PM2.5染毒组细胞内Nrf-2和HO-1蛋白的表达水平。结论:PM2.5可诱导HBE细胞氧化损伤,主要表现为脂质过氧化水平升高、GSH/GSSG比值下降和抗氧化蛋白Nrf-2与HO-1表达水平的增加。在PM2.5所致细胞氧化应激效应中,1,25-(OH)2D3可起到一定的保护作用,这可能与VDR及Nrf-2/HO-1信号通路有关。而1,25-(OH)2D3所致细胞存活率的降低可能与其诱导细胞周期阻滞及促进PM2.5所诱导损伤细胞的凋亡有关。
OBJECTIVE:The aim of this study was to investigate whether 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]would inhibit oxidative damage which was induced by PM2.5in human bronchial epithelial cells(HBE).METHODS:HBE cells were divided into four groups:vehicle(ethanol,0.1%)control,1,25-(OH)2D3-treated group,ethanol+PM2.5-treated group,and PM2.5+1,25-(OH)2D3-treated group.HBE cells were pretreated with ethanol(0.1%)or 1×10^-9mol/L of 1,25-(OH)2D3for 24 h.Afterwards,the cell culture medium was replaced with medium containing PM2.5(200μg/m L)and ethanol(0.1%)or PM2.5(200μg/m L)and 1×10^-9mol/L 1,25-(OH)2D3.After another 24 hours of incubation of the cultures,cells were harvested for determination of cell viability,MDA concentrations,ratios of reduced glutathione(GSH)to oxidized glutathione(GSSG),and expression levels of transcription factor NF-E2-related factor 2(Nrf-2)and heme oxygenase-1(HO-1)protein.These expressions were detected using CCK-8 assay kit,cell MDA assay kit,GSH/GSSG-GloTMassay kit,and Western blot,respectively.RESULTS:In the PM2.5-treated groups,the cell viabilities were obviously decreased to 80.8%as compared with the control.After 48-h treatment of 1,25-(OH)2D3,the viability of PM2.5-treated HBE cells was further reduced to 75.8%.After PM2.5exposure,MDA concentrations were significantly increased(P<0.05),and the ratios of GSH/GSSG were found to be decreased significantly(P<0.01)as compared with the control.After 48 h-treatment with 1,25-(OH)2D3,the anti-oxidative levels in the PM2.5-exposed group were significantly improved(P<0.05),together with decreased MDA concentrations and increased GSH/GSSG ratios.Protein analyses reveal that PM2.5could increase levels of Nrf-2 and HO-1proteins in HBE cells.The 48 h-intervention of 1,25-(OH)2D3could up-regulate VDR level and slightly increased levels of Nrf-2 and HO-1 in PM2.5-treated HBE cells.CONCLUSIONS:PM2.5exposure induced oxidative damage in HBE cells,together with elevated lipid peroxidation,decreased GSH/GSSG ratios,and increased levels of antioxidant proteins including Nrf-2 and HO-1 proteins.However,1,25-(OH)2D3exposure attenuated PM2.5-induced oxidative stress,probably through regulation of Nrf-2/HO-1 pathway via VDR.Furthermore,1,25-(OH)2D3exposure attenuated PM2.5-induced oxidative stress,probably related to VDR and Nrf-2/HO-1 signal pathways.Finally,1,25-(OH)2D3exposure decreased cell viability,probably attributed to cell cycle arrest,and PM2.5-induced cell apoptosis which was promoted by 1,25-(OH)2D3.
作者
孙娇娇
车碧众
罗秋林
翟兵中
信丽丽
SUN Jiaojiao;CHE Bizhong;LUO Qiulin;ZHAI Bingzhong;XIN Lili(School of Public Health,Medical College of Soochow University,Suzhou 215123,Jiangsu,China)
出处
《癌变．畸变．突变》
CAS
2020年第2期92-97,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
环境与健康教育部重点实验室开放课题基金(2018GWFJJ02)。
