MnCl2致人肺细胞线粒体功能的损伤

MnCl2-induced functional damage of mitochondria in human lung cells in vitro

目的探讨MnCl2导致的人肺细胞线粒体功能损伤，初步研究核呼吸因子1（NRF-1）在Mn导致线粒体功能损伤中的表达改变。方法用不同浓度的MnCl2分别处理人支气管上皮细胞16HBE和人肺腺癌细胞A549，四甲基偶氮唑蓝（MTT）法检测细胞存活和线粒体酶活性，荧光分光光度法检测线粒体膜通透性转运孔（PTP）的开放程度和线粒体膜电位（△ψm）的变化；流式细胞仪分析细胞凋亡；Western blot法检测NRF-1及线粒体转录因子（mtTFA）的蛋白表达量变化。结果MnCl2可导致16HBE和A549细胞存活下降，其半数抑制浓度（IC50）分别为1．91mmol／L和1．98mmol／L。MnCl2可抑制细胞线粒体总酶活性，且存在浓度效应关系，其中1．00mmol／L MnCl2对16HBE细胞和A549细胞的线粒体总酶活性抑制率最高，分别为（52．8±5．4）％和（50．6±2．2）％。线粒体PTP开放程度随着MnCl2作用浓度的增加而增强，且同一浓度MnCl2作用时间增加，PTP开放程度增强。当PTP开放程度增大时，细胞△ψm降低，与对照组相比，0．25、0．50、1．00mmol／L MnCl2处理16HBE细胞后，△ψm分别下降了（7．9±3．0）％、（26．2±2．2）％和（27．8±4．1）％；0．25、0．50、1．00mmol／L MnCl2处理A549后，AtOm分别下降了（4．7±1．0）％、（14．9±2．4）％和（27．5±1．2）％。MnCl2可导致细胞凋亡率增加，当1．00mmol／L MnCl，处理16HBE和A549细胞48h后，细胞凋亡率分别为（12．3±1．9）％和（6．0±0．4）％。Western blot结果显示，MnCl2可导致16HBE和A549细胞中NRF-1和mtTFA蛋白的表达水平降低，与对照组差异有统计学意义（P〈0．05）。结论MnCl2可诱导16HBE和A549细胞线粒体功能损伤，NRF-1蛋白表达降低，NRF-1可能参与Mn引起的线粒体功能损伤的过程。

Objective To explore the effect of MnCl2 on the mitochondrial function of human lung cells, and to study the changes of protein expression level of nuclear respiratory factor-1 （NRF-1） in mitochondrial dysfunction induced by MnCl2. Methods The effects of MnCl2 on cell survival rate were assessed by the reductions of tetrazolium dye （MTT） in cultured cell lines 16HBE and A549 cells. All testedl6HBE and A549 cells were incubated with different concentrations of MnCl2. The permeability transition pore （PTP） of mitochondria, mitochondrial membrane potential and the inhibition rate of mitochondrial enzymes as indicators of mitochondrial damage were measured by fluorescent spectrometry and MTT assay, respectively. Apoptosis was determined by flow cytometry. Protein levels of NRF-1 and mtTFA were measured by Western blot assay. Results MnCl2 decreased the survival rate of the two cell lines. The IC50 of 16HBE and A549 cells were 1.91 mmoL/L and 1.98 mmol/L, respectively. MnCl2 caused a concentration-dependent decrease of mitochondrial enzymes and the inhibition rate of mitochondrial enzymes of the two cell lines induced by 1.00 mmol/L MnCl2 were （52. 8 ±5.4）% and （50.6 ±2.2）%, respectively. The PTP opening increased in MnCl2-treated cells in a dose- and time-dependent manner. Compared with the control group, mitochondrial membrane potential in the two cell lines was decreased by MnCl2, by （7.9±3.0）%, （26.2±2.2）% and （27.8±4. 1）% in the 16HBE cells, and （4.7± 1.0） %, （ 14.9 ± 2.4） % and （27.5 ± 1.2） % in the A549 cells. Increased apoptosis rates of the two cell lines were induced by 1.00 mmol/L MnCl2, （ 12.3 ± 1.9） % and （6.0 ± 0.4） %, respectively. The results of Western blot assay revealed that the protein levels of NRF-1 and mtTFA were decreased in manganesetreated cells in a dose-dependent manner, with a significant difference compared with that of the control cells （P 〈 0.05 ）. Conclusion MnCl2 induces mitochondrial dysfunction in 16HBE and A549 cells, and decreases the expression level of nuclear respiratory factor-1 （ NRF-1 ）, indicating that NRF-1 may play an important role in mitochondrial dysfunction.