癌变·畸变·突变 ›› 2022, Vol. 34 ›› Issue (3): 161-168.doi: 10.3969/j.issn.1004-616x.2022.03.001

• 论著 •    下一篇

线粒体靶向抗氧化剂Mito-TEMPO对氮芥诱导BEAS-2B细胞损伤的影响

赵晨茜1, 徐安琦1,2, 艾多1,2, 孔德钦1, 张晓迪1, 李文丽1, 海春旭1, 刘江正1   

  1. 1. 空军军医大学军事预防医学系军事毒理学与防化医学教研室, 陕西省自由基生物学与医学重点实验室, 教育部特殊作业环境危害评估与防治重点实验室, 陕西 西安 710032;
    2. 空军军医大学基础医学院学员二大队, 陕西 西安 710032
  • 收稿日期:2022-03-24 修回日期:2022-05-13 发布日期:2022-06-10
  • 通讯作者: 刘江正
  • 作者简介:赵晨茜,E-mail:517026523@qq.com
  • 基金资助:
    国家自然科学基金青年基金(31900892)

Effects of Mito-TEMPO treatment on nitrogen mustard-induced cytotoxicity in BEAS-2B cells

ZHAO Chenqian1, XU Anqi1,2, AI Duo1,2, KONG Deqin1, ZHANG Xiaodi1, LI Wenli1, HAI Chunxu1, LIU Jiangzheng1   

  1. 1. Department of Military Toxicology and Chemical Defense Medicine, School of Military Preventive Medicine, Air Force Medical University, Key Laboratory of Free Radical Biology and Medicine of Shaanxi Province, Key Laboratory of Environmental Hazard Assessment and Prevention of Special Operations of Ministry of Education, Xi'an 710032;
    2. The Second Brigade of Basic Medical College, Air Force Medical University, Xi'an 710032, Shaanxi, China
  • Received:2022-03-24 Revised:2022-05-13 Published:2022-06-10

摘要: 目的: 探讨线粒体靶向抗氧化剂Mito-TEMPO对氮芥(HN2)诱导人支气管上皮细胞系BEAS-2B的影响。方法: BEAS-2B细胞分为正常对照组、Mito-TEMPO对照组、HN2染毒组和Mito-TEMPO干预组,其中正常对照组和Mito-TEMPO对照组分别给予含DMSO(体积分数为0.1%)和Mito-TEMPO(100μmol/L)的无血清培养基处理26 h,HN2染毒组给予含DMSO的无血清培养基预处理2 h,然后给予HN2(8μmol/L)染毒24 h,Mito-TEMPO干预组给予Mito-TEMPO(100μmol/L)预处理2 h,然后HN2(8μmol/L)和Mito-TEMPO(100μmol/L)共处理24 h。分别采用CCK-8法检测细胞活性,速率法检测细胞培养基上清乳酸脱氢酶(LDH)活性,倒置显微镜观察细胞形态,Annexin V/PI探针法流式细胞术检测细胞凋亡,JC-1探针法检测线粒体膜电位,紫外分光光度法检测细胞匀浆ATP含量,MitoSOX、DCFH-DA、DHE探针法分别检测细胞内线粒体ROS、H2O2及总ROS水平,实时荧光定量PCR检测炎症因子TNF-α、IL-6的mRNA表达水平。结果: 与HN2染毒组相比,Mito-TEMPO干预组细胞活性降低了约42.6%(P<0.05),细胞上清LDH水平显著增加(P<0.05),同时伴有异常形态细胞增多,Mito-TEMPO干预组细胞线粒体ROS水平降低了53.6%(P<0.05),细胞内H2O2水平及总ROS水平分别升高了171%和43.4%(均为P<0.05)。Mito-TEMPO干预对HN2染毒导致的细胞凋亡比例、线粒体膜电位、ATP含量、TNF-α、IL-6的mRNA表达水平等指标改变均无显著影响(P>0.05)。结论: 100μmol/L的Mito-TEMPO干预在HN2染毒BEAS-2B细胞模型中促进了细胞损伤,其机制可能与提高细胞内H2O2水平及总ROS水平有关。

关键词: 线粒体靶向抗氧化剂, Mito-TEMPO, 氮芥, 肺损伤, 凋亡, 氧化应激, 炎症

Abstract: OBJECTIVE: To investigate effects of mitochondrial-targeted antioxidant Mito-TEMPO on cytotoxicity which was induced by nitrogen mustard(HN2) in the human bronchial epithelial cell line BEAS-2B cells. METHODS: BEAS-2B cells were divided into four groups:normal control,Mito-TEMPO control,HN2exposure and Mito-TEMPO intervention groups. The two control groups were treated with serum-free medium containing DMSO(0.1%) or Mito-TEMPO(100 μmol/L) for 26 h,respectively. The HN2 exposure group was pretreated with DMSO-containing serum-free medium for 2 h,and then with HN2(8 μmol/L) for 24 h. The Mito-TEMPO intervention group was pretreated with Mito-TEMPO(100 μmol/L) for 2 h,and then co-treated with HN2(8 μmol/L) and Mito-TEMPO(100 μmol/L) for 24 h. Cell viability was detected by CCK-8 method,LDH activity in cell culture medium supernatant was detected by rate method,cell morphology was observed by inverted microscope,cell apoptosis was detected by Annexin V/PI probe method using flow cytometry,and mitochondrial membrane potential was detected by JC-1 probe. UV spectrophotometry were used to detect thecontent of ATP in cell homogenate. MitoSOX,DCFH-DA and DHE probe were used to detect the level ofmitochondrial ROS(reactive oxygen species), intracellular H2O2and intracellular total ROS respectively.RT-PCR was used to detect the mRNA expression levels of inflammatory factor TNFα and IL-6. RESULTS: Compared with the nitrogen mustard exposure group,the cell viability of the Mito-TEMPO intervention groupdecreased by about 42.6%(P<0.05),and the LDH level in the cell supernatant increased by about 73.5%(P<0.05), accompanied by increase of abnormal cell morphology. Compared with the HN2 exposure group, theMito-TEMPO intervention group showed the level of mitochondrial ROS decreased by 53.6%(P<0.05),and thelevel of intracellular H2O2and total ROS increased by 171%(P<0.05) and 43.4%(P<0.05) respectively.Mito-TEMPO intervention had no significant effects on changes of apoptosis ratio, mitochondrial membranepotential, ATP content, TNF-α and IL-6 mRNA expression levels caused by HN2 exposure(P>0.05). CONCLUSION: 100 μmol/L Mito-TEMPO intervention significantly promoted cell damage in the HN2exposure BEAS-2B cell model, and the mechanisms might be related to increase of intracellular H2O2andtotal ROS levels.

Key words: mitochondrial-targeted antioxidants, Mito-TEMPO, nitrogen mustard, apoptosis, oxidative stress, inflammation

中图分类号: