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

doi:10.3969/j.issn.1004-616x.2022.03.001

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. 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 Anqi^1,2, AI Duo^1,2, KONG Deqin¹, ZHANG Xiaodi¹, LI Wenli¹, HAI Chunxu¹, LIU Jiangzheng¹

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/Abstract

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:

R827.15

赵晨茜, 徐安琦, 艾多, 孔德钦, 张晓迪, 李文丽, 海春旭, 刘江正. 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[J]. 癌变·畸变·突变, 2022, 34(3): 161-168.

ZHAO Chenqian, XU Anqi, AI Duo, KONG Deqin, ZHANG Xiaodi, LI Wenli, HAI Chunxu, LIU Jiangzheng. Effects of Mito-TEMPO treatment on nitrogen mustard-induced cytotoxicity in BEAS-2B cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2022, 34(3): 161-168.

参考文献

[1] GRAHAM J S, SCHONEBOOM B A. Historical perspective on effects and treatment of sulfur mustard injuries[J]. Chem Biol Interact, 2013, 206(3):512-522.
[2] 崔海峰.芥子气中毒事故现场的救治[J].沈阳部队医药,2012, 25(1):77-78.
[3] 祝筱姬,朱双双,钟玉绪.芥子气肺损伤机制研究进展[J].中国药理学与毒理学杂志, 2015, 29(5):859-864.
[4] 祝筱姬,徐睿,季鹏,等.芥子气与肺损伤[J].解放军预防医学杂志, 2014, 32(1):84-86.
[5] 董训虎,陈明亮,余文珮,等.维生素D3通过抑制NLRP3炎性小体激活改善氮芥诱导角质形成细胞炎性损伤[J].第三军医大学学报, 2020, 42(3):267-272.
[6] BELLOMO A, HERBERT J, BASALY V, et al. The evaluation of acute lung injury induced by nitrogen mustard in mouse precision cut lung slices[J]. FASEB J, 2021, 35(S1):fasebj.2021.35.S1.04408.
[7] SHETTY S, KUMAR R, BHARATI S. Mito-TEMPO, a mitochondria-targeted antioxidant, prevents N-nitrosodiethylamineinduced hepatocarcinogenesis in mice[J]. Free Radic Biol Med,2019, 136:76-86.
[8] HU H T, LI M. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons[J]. Biochem Biophys Res Commun, 2016, 478(1):174-180.
[9] NI R, CAO T, XIONG S D, et al. Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy[J]. Free Radic Biol Med,2016, 90:12-23.
[10] CHEN J W, MA P W, YUAN H, et al. Mito-TEMPO attenuates oxidative stress and mitochondrial dysfunction in noiseinduced hearing loss via maintaining TFAM-mtDNA interaction and mitochondrial biogenesis[J]. Front Cell Neurosci, 2022, 16:803718.
[11] TUNCER S, AKKOCA A, CELEN M C, et al. Can MitoTEMPO protect rat sciatic nerve against ischemia-reperfusion injury?[J]. Naunyn Schmiedebergs Arch Pharmacol, 2021, 394(3):545-553.
[12] MUKEM S, THONGBUAKAEW T, KHORNCHATRI K. MitoTempo suppresses autophagic flux via the PI3K/Akt/mTOR signaling pathway in neuroblastoma SH-SY5Y cells[J]. Heliyon,2021, 7(6):e07310.
[13] BARZEGARI A, NOURI M, GUEGUEN V, et al. Mitochondriatargeted antioxidant mito-TEMPO alleviate oxidative stress induced by antimycin A in human mesenchymal stem cells[J]. J Cell Physiol, 2020, 235(7/8):5628-5636.
[14] ZHANG J Q, WANG Q, XU C O, et al. MitoTEMPO prevents oxalate induced injury in NRK-52E cells via inhibiting mitochondrial dysfunction and modulating oxidative stress[J].Oxid Med Cell Longev, 2017, 2017:7528090.
[15] 冯凯,苟渊,王文建.血清乳酸脱氢酶对儿童腺病毒肺炎严重度的预测价值[J].承德医学院学报, 2022, 39(1):22-25.
[16] 祝筱姬,朱双双,韩玮,等.芥子气经腹腔和气管致大鼠急性肺损伤细胞凋亡的变化[J].中国现代医学杂志, 2016, 26(15):11-21.
[17] SUNIL V R, VAYAS K N, CERVELLI J A, et al. Protective role of surfactant protein-D against lung injury and oxidative stress induced by nitrogen mustard[J]. Toxicol Sci, 2018, 166(1):108-122.
[18] DU K, RAMACHANDRAN A, WEEMHOFF J L, et al. Mitotempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity[J]. Arch Toxicol, 2019, 93(1):163-178.
[19] GOULD N S, WHITE C W, DAY B J. A role for mitochondrial oxidative stress in sulfur mustard analog 2-chloroethyl ethyl sulfide-induced lung cell injury and antioxidant protection[J]. J Pharmacol Exp Ther, 2009, 328(3):732-739.
[20] 贝媛媛,朱双双,张长尧,等.芥子气经腹腔和气管致大鼠急性肺损伤炎性反应的比较研究[J].重庆医学, 2016, 45(26):3608-3610, 3615.
[21] LIU Y Q, WANG Y D, DING W, et al. Mito-TEMPO alleviates renal fibrosis by reducing inflammation, mitochondrial dysfunction, and endoplasmic Reticulum stress[J]. Oxid Med Cell Longev, 2018, 2018:5828120.
[22] SHADEL G S, HORVATH T L. Mitochondrial ROS signaling in organismal homeostasis[J]. Cell, 2015, 163(3):560-569.
[23] 曾惠.线粒体在硫芥诱导大鼠脾淋巴细胞毒性中的作用机制研究[D].重庆:第三军医大学, 2004.

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

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	蔡娇, 孔德钦, 龙子, 彭洁, 刘江正, 刘瑞, 海春旭. IRAK4在对乙酰氨基酚诱导的急性肝细胞损伤中的作用及其机制[J]. 癌变·畸变·突变, 2022, 34(4): 255-261.
[2]	张月, 王耀一, 武雪亮, 张志生, 杨修明, 姜洋, 乔志飞, 梁晚平, 薛军. ¹²⁵I粒子联合AZD1152对三阴性乳腺癌细胞增殖和凋亡的影响[J]. 癌变·畸变·突变, 2022, 34(4): 295-299,306.
[3]	段惠娟, 孙照刚, 郝卫东, 魏雪涛, 褚洪迁. 4种化学物质对HaCaT细胞氧化应激相关基因表达的影响[J]. 癌变·畸变·突变, 2022, 34(3): 192-199,205.
[4]	于婧, 冯丹丹, 潘文森. 桑黄素在脂多糖诱导的急性肺损伤中的相关机制[J]. 癌变·畸变·突变, 2022, 34(3): 213-218.
[5]	张琪, 刘宇峻, 庄喜兵, 乔田奎. 附子提取物对肺癌A549细胞增殖抑制及放射增敏作用的研究[J]. 癌变·畸变·突变, 2022, 34(3): 227-232.
[6]	涂永梅, 彭洁, 龙子, 孔德钦, 陈宇豪, 覃梓瀚, 刘瑞, 李文丽, 于卫华. PPAR-γ在巨噬细胞炎症调控中的作用及机制的研究进展[J]. 癌变·畸变·突变, 2022, 34(2): 158-161.
[7]	陈东亚, 陆罗定, 陈耿, 张颖, 俞萍, 吕中明, 卞倩. 丹参酮ⅡA磺酸钠对环境细颗粒物诱导的大鼠肺部损伤的保护作用[J]. 癌变·畸变·突变, 2022, 34(1): 25-29,34.
[8]	艾多, 徐安琦, 孔德钦, 刘颖, 于卫华, 王钊, 彭洁, 刘瑞, 张晓迪, 海春旭, 李文丽, 王欣, 刘江正. 催化性抗氧化剂AEOL-10150对氮芥诱导小鼠急性肝损伤的保护作用[J]. 癌变·畸变·突变, 2022, 34(1): 40-46,61.
[9]	尹亚萍, 王宇, 田雨阳, 马焜, 李百祥. 褪黑素在帕金森病治疗中的研究进展[J]. 癌变·畸变·突变, 2022, 34(1): 75-78,81.
[10]	曹家豪, 彭越, 段佳琪, 刘桥松, 刘启玲, 秦绪军. 酒精性脂肪肝的线粒体作用机制研究进展[J]. 癌变·畸变·突变, 2021, 33(6): 475-481.
[11]	李柏茹, 秦双建, 关岚, 李闰冰, 蔡颖, 蒲桔宁, 曾明, 肖芳, 徐新云. PM_2.5对HK-2细胞氧化损伤和凋亡的影响[J]. 癌变·畸变·突变, 2021, 33(5): 360-364.
[12]	李宏伟, 孔德钦, 于卫华, 吴昊, 王钊, 刘瑞, 海春旭, 王欣, 刘江正, 李文丽. 齐墩果酸对酒精诱导的大鼠胃壁氧化损伤的保护作用[J]. 癌变·畸变·突变, 2021, 33(5): 365-369.
[13]	于卫华, 孔德钦, 龙子, 刘瑞, 李文丽, 海春旭. 抗氧化悖论真的成立吗？[J]. 癌变·畸变·突变, 2021, 33(5): 388-392.
[14]	黄晓华, 牛永东, 石刚刚. 核受体FXR激活对宫颈癌CaSki细胞增殖的抑制效应及其作用机制[J]. 癌变·畸变·突变, 2021, 33(4): 302-306.
[15]	王熙凯, 孟庆贺, 高燕鲁. miR-223-3p通过调控ECT2诱导非小细胞肺癌细胞凋亡的实验研究[J]. 癌变·畸变·突变, 2021, 33(3): 187-192.