癌变·畸变·突变 ›› 2020, Vol. 32 ›› Issue (2): 92-97.doi: 10.3969/j.issn.1004-616x.2020.02.003

• 论著 • 上一篇    下一篇

1,25-二羟基维生素D3对PM2.5所致HBE细胞氧化损伤的保护作用

孙娇娇, 车碧众, 罗秋林, 翟兵中, 信丽丽   

  1. 苏州大学医学部公共卫生学院, 江苏 苏州 215123
  • 收稿日期:2019-05-16 修回日期:2019-12-05 出版日期:2020-03-30 发布日期:2020-04-10
  • 通讯作者: 信丽丽,E-mail:llxin@suda.edu.cn E-mail:llxin@suda.edu.cn
  • 作者简介:孙娇娇,E-mail:20184247014@stu.suda.edu.cn。
  • 基金资助:
    环境与健康教育部重点实验室开放课题基金(2018GWFJJ02)

1,25-(OH)2D3 attenuates PM2.5-induced oxidative stress in HBE cells

SUN Jiaojiao, CHE Bizhong, LUO Qiulin, ZHAI Bingzhong, XIN Lili   

  1. School of Public Health, Medical College of Soochow University, Suzhou 215123, Jiangsu, China
  • Received:2019-05-16 Revised:2019-12-05 Online:2020-03-30 Published:2020-04-10

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1×10-9 mol/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(200 μg/mL)染毒液继续处理24 h,PM2.5染毒+1,25-(OH)2D3干预组用1×10-9 mol/L 1,25-(OH)2D3预处理24 h后更换为含1,25-(OH)2D3的PM2.5(200 μg/mL)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。

关键词: 细颗粒物, 1,25-二羟基维生素D3, 氧化应激, 转录因子NF-E2相关因子, 血红素加氧酶-1

Abstract: 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 PM2.5 in 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 1×10-9 mol/L of 1,25-(OH)2D3 for 24 h. Afterwards,the cell culture medium was replaced with medium containing PM2.5 (200 μg/mL) and ethanol (0.1%) or PM2.5 (200 μg/mL) and 1×10-9 mol/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-GloTM assay 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 PM2.5 exposure,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 PM2.5 could increase levels of Nrf-2 and HO-1 proteins in HBE cells. The 48 h-intervention of 1,25-(OH)2D3 could up-regulate VDR level and slightly increased levels of Nrf-2 and HO-1 in PM2.5-treated HBE cells. CONCLUSIONS:PM2.5 exposure 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)2D3 exposure attenuated PM2.5-induced oxidative stress,probably through regulation of Nrf-2/HO-1 pathway via VDR. Furthermore,1,25-(OH)2D3 exposure attenuated PM2.5-induced oxidative stress,probably related to VDR and Nrf-2/HO-1 signal pathways. Finally,1,25-(OH)2D3 exposure decreased cell viability,probably attributed to cell cycle arrest,and PM2.5-induced cell apoptosis which was promoted by 1,25-(OH)2D3.

Key words: PM2.5, 1,25-(OH)2D3, oxidative stress, Nrf-2, HO-1

中图分类号: