百草枯通过激活线粒体凋亡通路诱导人肺Ⅱ型上皮样A549细胞凋亡
|
摘要
目的探讨百草枯(PQ)诱导人肺Ⅱ型上皮样A549细胞凋亡过程中线粒体凋亡通路的发生机制。方法体外培养A549细胞,对照组加入RPMI 1640培养液,实验组加入不同浓度的PQ(50、100、150和200μmol/L),2组细胞继续培养24和48 h。MTT法检测细胞活性;Hoechst 33258染色法观察细胞核的形态;流式细胞术检测细胞凋亡率以及线粒体膜电位;分光光度法检测caspase-3和caspase-9的活化程度;Western blotting检测线粒体凋亡相关蛋白Bcl-2、Bcl-x L、Bax和Bak的表达。结果 MTT结果显示,PQ对A549细胞具有显著的生长抑制作用。Hoechst染色显示,不同浓度的PQ作用于A549细胞24和48 h后,可发现细胞凋亡的特征性改变,如细胞核固缩、核碎裂、凋亡小体形成,并且细胞的凋亡程度随着PQ浓度的增大和作用时间的延长而加重。细胞凋亡率升高也证实了这一结果。线粒体膜电位出现不同程度的降低;caspase-3和caspase-9活性不同程度的增加;抗凋亡蛋白Bcl-2、Bcl-x L的表达量降低;促凋亡蛋白Bax、Bak的表达量升高。以上结果均呈时间与浓度依赖性。结论 PQ通过激活线粒体凋亡通路诱导A549细胞凋亡。
Objective To investigate the apoptosis mechanism induced by paraquat(PQ) in human typeⅡalveolar epithelial-like A549 cells. Methods A549 cells were cultured in vitro. The cells in the experimental group were exposed to various concentrations of PQ(50,100,150,and 200 μmol/L),while those in the control group were cultured in RPMI 1640 medium. After treatment for 24 and 48 h,the cell survival rate was assessed by MTT assay. Morphological changes in the nuclei were observed by Hoechst 33258 fluorescence staining. Cellular apoptosis and mitochondrial transmembrane potential were assayed by flow cytometry. The activities of caspase-3 and caspase-9 were assayed by spectrophotometry. Western blotting was used to analyze the expression of proteins in the Bcl-2 family,such as Bcl-2,Bcl-x L,Bax,and Bak. Results PQ exhibited significant anti-proliferative activity in A549 cells. PQ-treated A549 cells were subjected to Hoechst 33258 staining. The hallmarks of apoptosis were detected,and the degree of apoptosis increased. Mitochondrial membrane potential was decreased,the levels of active caspase-3 and caspase-9 increased,the expression of Bcl-2 and Bcl-x L was decreased,and the expression of Bax and Bak was increased. These effects occurred in concentration-and time-dependent manners. Conclusion PQ efficiently induced intracellular apoptosis through the mitochondrial pathway in A549 cells.
Objective To investigate the apoptosis mechanism induced by paraquat(PQ) in human typeⅡalveolar epithelial-like A549 cells. Methods A549 cells were cultured in vitro. The cells in the experimental group were exposed to various concentrations of PQ(50,100,150,and 200 μmol/L),while those in the control group were cultured in RPMI 1640 medium. After treatment for 24 and 48 h,the cell survival rate was assessed by MTT assay. Morphological changes in the nuclei were observed by Hoechst 33258 fluorescence staining. Cellular apoptosis and mitochondrial transmembrane potential were assayed by flow cytometry. The activities of caspase-3 and caspase-9 were assayed by spectrophotometry. Western blotting was used to analyze the expression of proteins in the Bcl-2 family,such as Bcl-2,Bcl-x L,Bax,and Bak. Results PQ exhibited significant anti-proliferative activity in A549 cells. PQ-treated A549 cells were subjected to Hoechst 33258 staining. The hallmarks of apoptosis were detected,and the degree of apoptosis increased. Mitochondrial membrane potential was decreased,the levels of active caspase-3 and caspase-9 increased,the expression of Bcl-2 and Bcl-x L was decreased,and the expression of Bax and Bak was increased. These effects occurred in concentration-and time-dependent manners. Conclusion PQ efficiently induced intracellular apoptosis through the mitochondrial pathway in A549 cells.
引文
[1]GIL HW,HONG JR,JANG SH,et al.Diagnostic and therapeutic approach for acute paraquat intoxication[J].Korean Med Sci,2014,29(11):1441-1449.DOI:10.3346/jkms.2014.29.11.1441.
[2]张宝兰,姚朗,欧艺.1991-2008年我国百草枯中毒文献分析[J].中国急救医学,2010,30(2):139-141.DIO:10.3969/j.issn.1002-1949.2010.02.013.
[3]DINIS-OLIVEIRA RJ,DUARTE JA,S NCHEZ-NAVARRO A,et al.Paraquat poisonings:mechanisms of lung toxicity,clinical features and treatment[J].Crit Rev Toxicol,2008,38(1):13-71.
[4]VENKATESAN N.Pulmonary protective effects of curcumin against paraquat toxicity[J].Life Sci,2000,66(2):PL21-PL28.
[5]TOMITA M,OKUYAMA T,KATSUYAMA H,et al.Mouse model of paraquat-poisoned lungs and its gene expression profile[J].Toxicology,2007,231(2/3):200-209.DOI:10.1016/j.tox.2006.12.005.
[6]MITSOPOULOS P,SUNTRES ZE.Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat[J].Chem Biol Interact,2010,188(3):427-436.DOI:10.1016/j.cbi.2010.09.022.
[7]HIRAI K,WITSCHI H,C T MG.Mitochondrial injury of pulmonary alveolar epithelial cells in acute paraquat intoxication[J].Exp Mol Pathol,1985,43(2):242-252.
[8]HE Y,ZOU L,ZHOU Y,et al.Adiponectin ameliorates the apoptotic effects of paraquat on alveolar typeⅡcells via improvements in mitochondrial function[J].Mol Med Rep,2016,14(1):746-752.DOI:10.3892/mmr.2016.5328.
[9]UHAL BD.Epithelial apoptosis in the initiation of lung fibrosis[J].Eur Respir J Suppl,2003,44:7s-9s.DOI:10.1183/09031936.03.00000303.
[10]KUWANO K,HAGIMOTO N,NAKANISHI Y.The role of apoptosis in pulmonary fibrosis[J].Histol Histopathol,2004,19(3):867-881.DOI:10.14670/HH-19.867.
[11]KIM SJ,CHERESH P,JABLONSKI RP,et al.The role of mitochondrial DNA in mediating alveolar epithelial cell apoptosis and pulmonary fibrosis[J].Int J Mol Sci,2015,16(9):21486-21519.DOI:10.3390/ijms160921486.
[12]LIU S,LIU K,SUN Q,et al.Consumption of hydrogen water reduces paraquat-induced acute lung injury in rats[J].J Biomed Biotechnol,2011,2011:305086.DOI:10.1155/2011/305086.
[13]PARK HK,KIM SJ,KWON DY,et al.Protective effect of quercetin against paraquat-induced lung injury in rats[J].Life Sci,2010,87(5/6):181-186.DOI:10.1016/j.lfs.2010.06.011.
[14]王晔.Bcl-2/Bax在百草枯中毒鼠肺组织中的表达及三七总皂苷的保护作用[J].中国急救医学,2012,32(8):714-717.DOI:10.3969/j.issn.1002-1949.2012.08.011.
[15]赖登攀,夏金明,王建峰,等.百草枯中毒大鼠肺组织线粒体中电压依赖性阴离子通路及caspase-3、8、9的变化[J].中华劳动卫生职业病杂志,2015,33(5):363-365.DOI:10.3760/cma.j.issn.1001-9391.2015.05.014.
[16]FADEEL B,ORRENIUS S.Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease[J].J Intern Med,2005,258(6):479-517.DOI:10.1111/j.1365-2796.2005.01570.x.
[17]MARTINOU JC,YOULE RJ.Mitochondria in apoptosis:Bcl-2 family members and mitochondrial dynamics[J].Dev Cell,2011,21(1):92-101.DOI:10.1016/j.devcel.2011.06.017.
[18]PENG W,WU JG,JIANG YB,et al.Antitumor activity of 4-O-(2″-O-acetyl-6″-O-p-coumaroyl-β-D-glucopyranosyl)-p-coumaric acid against lung cancers via mitochondrial-mediated apoptosis[J].Chem Biol Inter,2015,233:8-13.DOI:10.1016/j.cbi.2015.03.014.
[19]ORRENIUS S.Mitochondrial regulation of apoptotic cell death[J].Toxicol Lett,2004,149(1/3):19-23.
[2]张宝兰,姚朗,欧艺.1991-2008年我国百草枯中毒文献分析[J].中国急救医学,2010,30(2):139-141.DIO:10.3969/j.issn.1002-1949.2010.02.013.
[3]DINIS-OLIVEIRA RJ,DUARTE JA,S NCHEZ-NAVARRO A,et al.Paraquat poisonings:mechanisms of lung toxicity,clinical features and treatment[J].Crit Rev Toxicol,2008,38(1):13-71.
[4]VENKATESAN N.Pulmonary protective effects of curcumin against paraquat toxicity[J].Life Sci,2000,66(2):PL21-PL28.
[5]TOMITA M,OKUYAMA T,KATSUYAMA H,et al.Mouse model of paraquat-poisoned lungs and its gene expression profile[J].Toxicology,2007,231(2/3):200-209.DOI:10.1016/j.tox.2006.12.005.
[6]MITSOPOULOS P,SUNTRES ZE.Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat[J].Chem Biol Interact,2010,188(3):427-436.DOI:10.1016/j.cbi.2010.09.022.
[7]HIRAI K,WITSCHI H,C T MG.Mitochondrial injury of pulmonary alveolar epithelial cells in acute paraquat intoxication[J].Exp Mol Pathol,1985,43(2):242-252.
[8]HE Y,ZOU L,ZHOU Y,et al.Adiponectin ameliorates the apoptotic effects of paraquat on alveolar typeⅡcells via improvements in mitochondrial function[J].Mol Med Rep,2016,14(1):746-752.DOI:10.3892/mmr.2016.5328.
[9]UHAL BD.Epithelial apoptosis in the initiation of lung fibrosis[J].Eur Respir J Suppl,2003,44:7s-9s.DOI:10.1183/09031936.03.00000303.
[10]KUWANO K,HAGIMOTO N,NAKANISHI Y.The role of apoptosis in pulmonary fibrosis[J].Histol Histopathol,2004,19(3):867-881.DOI:10.14670/HH-19.867.
[11]KIM SJ,CHERESH P,JABLONSKI RP,et al.The role of mitochondrial DNA in mediating alveolar epithelial cell apoptosis and pulmonary fibrosis[J].Int J Mol Sci,2015,16(9):21486-21519.DOI:10.3390/ijms160921486.
[12]LIU S,LIU K,SUN Q,et al.Consumption of hydrogen water reduces paraquat-induced acute lung injury in rats[J].J Biomed Biotechnol,2011,2011:305086.DOI:10.1155/2011/305086.
[13]PARK HK,KIM SJ,KWON DY,et al.Protective effect of quercetin against paraquat-induced lung injury in rats[J].Life Sci,2010,87(5/6):181-186.DOI:10.1016/j.lfs.2010.06.011.
[14]王晔.Bcl-2/Bax在百草枯中毒鼠肺组织中的表达及三七总皂苷的保护作用[J].中国急救医学,2012,32(8):714-717.DOI:10.3969/j.issn.1002-1949.2012.08.011.
[15]赖登攀,夏金明,王建峰,等.百草枯中毒大鼠肺组织线粒体中电压依赖性阴离子通路及caspase-3、8、9的变化[J].中华劳动卫生职业病杂志,2015,33(5):363-365.DOI:10.3760/cma.j.issn.1001-9391.2015.05.014.
[16]FADEEL B,ORRENIUS S.Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease[J].J Intern Med,2005,258(6):479-517.DOI:10.1111/j.1365-2796.2005.01570.x.
[17]MARTINOU JC,YOULE RJ.Mitochondria in apoptosis:Bcl-2 family members and mitochondrial dynamics[J].Dev Cell,2011,21(1):92-101.DOI:10.1016/j.devcel.2011.06.017.
[18]PENG W,WU JG,JIANG YB,et al.Antitumor activity of 4-O-(2″-O-acetyl-6″-O-p-coumaroyl-β-D-glucopyranosyl)-p-coumaric acid against lung cancers via mitochondrial-mediated apoptosis[J].Chem Biol Inter,2015,233:8-13.DOI:10.1016/j.cbi.2015.03.014.
[19]ORRENIUS S.Mitochondrial regulation of apoptotic cell death[J].Toxicol Lett,2004,149(1/3):19-23.