己糖激酶2通过抑制线粒体凋亡通路减少LPS引起的人肺上皮BEAS-2B细胞凋亡
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摘要
目的:探讨脂多糖(LPS)诱导人正常肺上皮BEAS-2B细胞凋亡的分子机制,并对己糖激酶2(HK2)在该效应中的作用进行分析。方法:采用不同浓度的LPS作用于BEAS-2B细胞建立损伤模型,CCK-8实验检测细胞存活率; Hoechst 33342染色及Annexin V/PI双染法分析细胞凋亡水平;通过使用线粒体凋亡通路抑制剂或者外在凋亡通路抑制剂鉴定细胞凋亡通路;在BEAS-2B细胞中转染HK2过表达质粒以验证HK2对上述效应的影响。Western blot法确认HK2过表达效果;免疫荧光实验检测HK2亚细胞定位。结果:CCK-8实验结果显示,LPS以时间和剂量依赖性方式降低BEAS-2B细胞活力; Hoechst 33342染色结果表明,给予LPS处理后的BEAS-2B细胞核出现固缩和碎裂;同时Annexin V/PI双染实验结果表明,处于凋亡状态的细胞由2. 89%增加至42. 4%,细胞凋亡率明显升高(P <0. 05)。线粒体凋亡通路执行蛋白caspase-9特异性抑制剂可显著抑制细胞凋亡,而caspase-8抑制剂却无此效应。在BEAS-2B细胞的凋亡过程中伴随着HK2的表达下调,而HK2过表达可以有效阻止以上事件的发生。结论:己糖激酶2可通过抑制线粒体凋亡通路减少LPS引起的人肺上皮细胞凋亡。
AIM: To explore the effect of hexokinase 2( HK2) on lipopolysaccharide( LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms. METHODS: BEAS-2B cells were treated with LPS to induce cell injury,and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot.The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment. RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner( P < 0. 01).The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with zLEHD-fmk,but not z-IETD-fmk,reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression,the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression. CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.
AIM: To explore the effect of hexokinase 2( HK2) on lipopolysaccharide( LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms. METHODS: BEAS-2B cells were treated with LPS to induce cell injury,and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot.The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment. RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner( P < 0. 01).The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with zLEHD-fmk,but not z-IETD-fmk,reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression,the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression. CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.
引文
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[16]Du J,Abdel-Razek O,Shi Q,et al.Surfactant protein Dattenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis,inflammation and NF-κB signaling[J].Sci Rep,2018,8(1):15393.
[17]Pei W,Wu Y,Zhang X,et al.Deletion of Apo M gene induces apoptosis in mouse kidney via mitochondrial and endoplasmic reticulum stress pathways[J].Biochem Biophys Res Commun,2018,505(3):891-897.
[18]卫冬,张晓娟,刘洁,等.Survivin抑制剂YM155对视网膜母细胞瘤Y79细胞线粒体凋亡途径的影响[J].中国病理生理杂志,2017,33(1):26-32.
[19]Neary CL,Pastorino JG.Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells[J].J Cell Physiol,2013,228(9):1943-1948.
[20]Wu R,Wyatt E,Chawla K,et al.Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production[J].EMBO Mol Med,2012,4(7):633-646.
[21]Pastorino JG,Shulga N,Hoek JB.Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis[J].J Biol Chem,2002,277(9):7610-7618.
[22]Qiu Y,Cheng X,Bai F,et al.Inhibition of NLRP9b attenuates acute lung injury through suppressing inflammation,apoptosis and oxidative stress in murine and cell models[J].Biochem Biophys Res Commun,2018,503(2):436-443.
[23]Moon JS,Hisata S,Park MA,et al.m TORC1-induced HK1-dependent glycolysis regulates NLRP3 inflammasome activation[J].Cell Rep,2015,12(1):102-115.
[24]Rowe SJ,Allen L,Ridger VC,et al.Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury[J].J Immunol,2002,169(11):6401-6407.
[2]Lee JW,Chun W,Kwon OK,et al.3,4,5-Trihydroxycinnamic acid attenuates lipopolysaccharide(LPS)-induced acute lung injury via downregulating inflammatory molecules and upregulating HO-1/AMPK activation[J].Int Immunopharmacol,2018,64:123-130.
[3]Gupta S,Sankar J,Lodha R,et al.Comparison of prevalence and outcomes of pediatric acute respiratory distress syndrome using Pediatric Acute Lung Injury Consensus Conference criteria and Berlin definition[J].Front Pediatr,2018,6:93.
[4]Tai W,Zhou Z,Zheng B,et al.Inhibitory effect of circulating fibrocytes on injury repair in acute lung injury/acute respiratory distress syndrome mice model[J].J Cell Biochem,2018,119(10):7982-7990.
[5]Thompson BT,Chambers RC,Liu KD.Acute respiratory distress syndrome[J].N Engl J Med,2017,377(19):1904-1905.
[6]Hara MR,Agrawal N,Kim SF,et al.S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding[J].Nat Cell Biol,2005,7(7):665-674.
[7]Palsson-McDermott EM,Curtis AM,Goel G,et al.Pyruvate kinase M2 regulates Hif-1αactivity and IL-1βinduction and is a critical determinant of the warburg effect in LPS-activated macrophages[J].Cell Metab,2015,21(1):65-80.
[8]Boukouris AE,Zervopoulos SD,Michelakis ED.Metabolic enzymes moonlighting in the nucleus:metabolic regulation of gene transcription[J].Trends Biochem Sci,2016,41(8):712-730.
[9]Roberts DJ,Miyamoto S.Hexokinase II integrates energy metabolism and cellular protection:Akting on mitochondria and TORCing to autophagy[J].Cell Death Differ,2015,22(2):248-257.
[10]Wolf A,Agnihotri S,Micallef J,et al.Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme[J].J Exp Med,2011,208(2):313-326.
[11]Crane RK,Sosa A.The association of hexokinase with particulate fractions of brain and other tissue homogenates[J].J Biol Chem,1953,203(1):273-292.
[12]Ahmad A,Ahmad S,Schneider BK,et al.Elevated expression of hexokinase II protects human lung epitheliallike A549 cells against oxidative injury[J].Am J Physiol Lung Cell Mol Physiol,2002,283(3):L573-L584.
[13]Yao M,Wang X,Tang Y,et al.Dicer mediating the expression of mi R-143 and mi R-155 regulates hexokinase IIassociated cellular response to hypoxia[J].Am J Physiol Lung Cell Mol Physiol,2014,307(11):L829-L837.
[14]Liu X,Shao K,Sun T.SIRT1 regulates the human alveolar epithelial A549 cell apoptosis induced by Pseudomonas aeruginosa lipopolysaccharide[J].Cell Physiol Biochem,2013,31(1):92-101.
[15]蓝琳友,洪溪屏,蔡元晖.MicroRNA-132转染对脂多糖诱导的肺泡巨噬细胞炎症反应的作用[J].中国病理生理杂志,2014,30(12):2190-2194.
[16]Du J,Abdel-Razek O,Shi Q,et al.Surfactant protein Dattenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis,inflammation and NF-κB signaling[J].Sci Rep,2018,8(1):15393.
[17]Pei W,Wu Y,Zhang X,et al.Deletion of Apo M gene induces apoptosis in mouse kidney via mitochondrial and endoplasmic reticulum stress pathways[J].Biochem Biophys Res Commun,2018,505(3):891-897.
[18]卫冬,张晓娟,刘洁,等.Survivin抑制剂YM155对视网膜母细胞瘤Y79细胞线粒体凋亡途径的影响[J].中国病理生理杂志,2017,33(1):26-32.
[19]Neary CL,Pastorino JG.Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells[J].J Cell Physiol,2013,228(9):1943-1948.
[20]Wu R,Wyatt E,Chawla K,et al.Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production[J].EMBO Mol Med,2012,4(7):633-646.
[21]Pastorino JG,Shulga N,Hoek JB.Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis[J].J Biol Chem,2002,277(9):7610-7618.
[22]Qiu Y,Cheng X,Bai F,et al.Inhibition of NLRP9b attenuates acute lung injury through suppressing inflammation,apoptosis and oxidative stress in murine and cell models[J].Biochem Biophys Res Commun,2018,503(2):436-443.
[23]Moon JS,Hisata S,Park MA,et al.m TORC1-induced HK1-dependent glycolysis regulates NLRP3 inflammasome activation[J].Cell Rep,2015,12(1):102-115.
[24]Rowe SJ,Allen L,Ridger VC,et al.Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury[J].J Immunol,2002,169(11):6401-6407.
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