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

Abstract

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

CLC Number:

R827.15

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.

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

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