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

doi:10.3969/j.issn.1004-616x.2020.02.003

摘要/Abstract

摘要： 目的： 探讨1，25-二羟基维生素D₃[1，25-（OH）₂D₃]对细颗粒物（PM_2.5）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+PM_2.5染毒组、PM_2.5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+PM_2.5染毒组用0.1%乙醇溶剂处理24 h后更换为含乙醇的PM_2.5（200 μg/mL）染毒液继续处理24 h，PM_2.5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PM_2.5（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）-Glo^TM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： PM_2.5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PM_2.5染毒的细胞存活率降低至75.8%。与对照相比，PM_2.5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PM_2.5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，PM_2.5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PM_2.5染毒组细胞内Nrf-2和HO-1蛋白的表达水平。结论： PM_2.5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PM_2.5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PM_2.5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 PM_2.5 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 + PM_2.5-treated group,and PM_2.5+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 PM_2.5 (200 μg/mL) and ethanol (0.1%) or PM_2.5 (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-Glo^TM assay kit,and Western blot,respectively. RESULTS: In the PM_2.5-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 PM_2.5-treated HBE cells was further reduced to 75.8%. After PM_2.5 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 PM_2.5-exposed group were significantly improved (P < 0.05),together with decreased MDA concentrations and increased GSH/GSSG ratios. Protein analyses reveal that PM_2.5 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 PM_2.5-treated HBE cells. CONCLUSIONS:PM_2.5 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 PM_2.5-induced oxidative stress,probably through regulation of Nrf-2/HO-1 pathway via VDR. Furthermore,1,25-(OH)₂D₃ exposure attenuated PM_2.5-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 PM_2.5-induced cell apoptosis which was promoted by 1,25-(OH)₂D₃.

Key words: PM_2.5, 1,25-(OH)₂D₃, oxidative stress, Nrf-2, HO-1

中图分类号:

R114

孙娇娇, 车碧众, 罗秋林, 翟兵中, 信丽丽. 1,25-二羟基维生素D₃对PM_2.5所致HBE细胞氧化损伤的保护作用[J]. 癌变·畸变·突变, 2020, 32(2): 92-97.

SUN Jiaojiao, CHE Bizhong, LUO Qiulin, ZHAI Bingzhong, XIN Lili. 1,25-(OH)₂D₃ attenuates PM_2.5-induced oxidative stress in HBE cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2020, 32(2): 92-97.

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1,25-二羟基维生素D₃对PM_2.5所致HBE细胞氧化损伤的保护作用

1,25-(OH)₂D₃ attenuates PM_2.5-induced oxidative stress in HBE cells

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