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

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。

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

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.