急性缺氧对大弹涂鱼(Boleophthalmus pectinirostris)肝脏中缺氧应答相关基因表达、蛋白含量及酶活性的影响
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摘要
为了评估缺氧对大弹涂鱼的影响,本研究通过模拟缺氧环境(溶解氧1.5±0.11mg/L),探究急性缺氧(1h,3h和6h)对大弹涂鱼缺氧相关基因表达、蛋白含量及酶活性的影响。结果显示,6h缺氧过程中,对照组实验鱼肝脏中缺氧相关基因表达、蛋白含量和酶活性无显著性差异;缺氧处理组实验鱼肝脏中缺氧诱导因子(HIF)、葡萄糖转运体、脯氨酰羟化酶和Bcl2/腺癌E1B19KD交互蛋白3基因的表达量和蛋白含量在6h处理时间内逐渐上调,但肿瘤抑制蛋白p53基因的表达量和蛋白含量在1h时显著最高;缺氧处理组实验鱼肝脏中乳酸脱氢酶、柠檬酸合酶和丙酮酸激酶活性逐渐下调,在6h时达到最低;但肝脏中葡萄糖激酶和磷酸果糖激酶活性在3h时显著增加,随后逐渐降低,在6h时达到最低;在整个缺氧过程中,缺氧处理组实验鱼肝脏中缺氧相关基因表达、蛋白含量和酶活性显著高于对照组。本研究结果表明,HIF信号通路在缺氧应答中被激活;缺氧胁迫会抑制鱼类需氧代谢的生理过程。
To evaluate the effect of hypoxia on mudskipper, a study was carried out to simulated acute hypoxia(dissolved oxygen: 1.5±0.11 mg/L) in the laboratory and analyzed gene expressions, protein contents and enzyme activities in mudskipper exposed to various durations of acute hypoxia(1 h, 3 h and 6 h). No significant differences were found in gene expressions, protein contents and enzyme activities in control group. Fish exposed to acute hypoxia, liver m RNA expression and protein content of hypoxia-inducible factor 1, glucose transporters, prolyl-4-hydroxylase and Bcl-2/adenocarcinoma E1 B19 KD interacting protein 3 gradually increased at hour 6, on the contrary, fish exposed to hypoxia at hour 1 had the highest m RNA expression and protein content of tumour suppressor protein p53. Liver lactate dehydrogenase, citroyl synthetase and pyruvate kinase activities gradually decreased from hour 1 to hour 6, the lowest significant values were found at hour 6. However, liver glucokinase and phosphofructokinase activities abruptly increased at hour 3, and then gradually decreasing, the lowest values were found at hour 6. During the 6-hour period, gene expressions, protein contents and enzyme activities involved in hypoxic responsive of fish in the hypoxia treatment group were higher than those of fish in control group. This study indicates that the HIF signaling pathway is activated in response to hypoxia; liver enzymes are decreased, which indicated a depression in the capacity of aerobic metabolism during fish hypoxia.
To evaluate the effect of hypoxia on mudskipper, a study was carried out to simulated acute hypoxia(dissolved oxygen: 1.5±0.11 mg/L) in the laboratory and analyzed gene expressions, protein contents and enzyme activities in mudskipper exposed to various durations of acute hypoxia(1 h, 3 h and 6 h). No significant differences were found in gene expressions, protein contents and enzyme activities in control group. Fish exposed to acute hypoxia, liver m RNA expression and protein content of hypoxia-inducible factor 1, glucose transporters, prolyl-4-hydroxylase and Bcl-2/adenocarcinoma E1 B19 KD interacting protein 3 gradually increased at hour 6, on the contrary, fish exposed to hypoxia at hour 1 had the highest m RNA expression and protein content of tumour suppressor protein p53. Liver lactate dehydrogenase, citroyl synthetase and pyruvate kinase activities gradually decreased from hour 1 to hour 6, the lowest significant values were found at hour 6. However, liver glucokinase and phosphofructokinase activities abruptly increased at hour 3, and then gradually decreasing, the lowest values were found at hour 6. During the 6-hour period, gene expressions, protein contents and enzyme activities involved in hypoxic responsive of fish in the hypoxia treatment group were higher than those of fish in control group. This study indicates that the HIF signaling pathway is activated in response to hypoxia; liver enzymes are decreased, which indicated a depression in the capacity of aerobic metabolism during fish hypoxia.
引文
曹伏君,郭良珍,2011.大弹涂鱼(Boleophthalmus pectinirostris)窒息点及昼夜代谢规律.海洋与湖沼,42(6):760-763
Aguilar N M,Ishimatsu A,Ogawa K et al,2000.Aerial ventilatory responses of the mudskipper,Periophthalmodon schlosseri,to altered aerial and aquatic respiratory gas concentrations.Comp Biochem Physiol A Mol Integr Physiol,127(3):285-292
Baptista R B,Souza-Castro N,Almeida-Val V M F,2016.Acute hypoxia up-regulates HIF-1αand VEGF m RNA levels in Amazon hypoxia-tolerant Oscar(Astronotus ocellatus).Fish Physiol Biochem,42(5):1307-1318
Barrionuevo W R,Fernandes M N,Rocha O,2010.Aerobic and anaerobic metabolism for the zebrafish,Danio rerio,reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development.Braz J Biol,70(2):425-434
Butler P J,Jones D R,1997.Physiology of diving of birds and mammals.Physiol Rev,77(3):837-899
Cai M C,Huang Q Y,Liao W G et al,2010.Hypoxic training increases metabolic enzyme activity and composition ofα-myosin heavy chain isoform in rat ventricular myocardium.Eur J Appl Physiol,108(1):105-111
Chen N,Chen L P,Zhang J et al,2012.Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits,HIF-1α/2α/3αof the hypoxia-sensitive freshwater species,Chinese sucker.Gene,498(1):81-90
Denko N C,Fontana L A,Hudson K M et al,2003.Investigating hypoxic tumor physiology through gene expression patterns.Oncogene,22(37):5907-5914
Geng X,Feng J B,Liu S K et al,2014.Transcriptional regulation of hypoxia inducible factors alpha(HIF-α)and their inhibiting factor(FIH-1)of channel catfish(Ictalurus punctatus)under hypoxia.Comp Biochem Physiol BBiochem Mol Biol,169:38-50
Ivan M,Kondo K,Yang H F et al,2001.HIFαtargeted for VHL-mediated destruction by proline hydroxylation:implications for O2 sensing.Science,292(5516):464-468
Jing D D,Li M,Zhang Y et al,2017.Differential induction of enzymes and genes involved in oxidative stress in gill and liver tissues of mudskipper Boleophthalmus pectinirostris exposed to lead.Turk J Fish Aquat Sci,17(2):437-443
Lardon I,Eyckmans M,Vu T N et al,2013.1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish,the common carp(Cyprinus carpio).Metabolomics,9(6):1216-1227
Li Y H,Wei L,Cao J G et al,2016.Oxidative stress,DNAdamage and antioxidant enzyme activities in the pacific white shrimp(Litopenaeus vannamei)when exposed to hypoxia and reoxygenation.Chemosphere,144:234-240
Liu Z H,Lu H M,Shi H L et al,2005.PUMA overexpression induces reactive oxygen species generation and proteasome-mediated stathmin degradation in colorectal cancer cells.Cancer Res,65(5):1647-1654
Maccormack T J,Lewis J M,Almeida-Val V M F et al,2006.Carbohydrate management,anaerobic metabolism,and adenosine levels in the armoured catfish,Liposarcus pardalis(castelnau),during hypoxia.J Exp Zool A Comp Exp Biol,305A(4):363-375
Maes V,Betoulle S,Jaffal A et al,2016.Juvenile roach(Rutilus rutilus)increase their anaerobic metabolism in response to copper exposure in laboratory conditions.Ecotoxicology,25(5):900-913
Mohindra V,Tripathi R K,Singh R K et al,2013.Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits,HIF-1α,-2αand-3αin hypoxia-tolerant Indian catfish,Clarias batrachus[Linnaeus,1758].Mol Biol Rep,40(10):5805-5815
Mu W J,Wen H S,Li J F et al,2015.HIFs genes expression and hematology indices responses to different oxygen treatments in an ovoviviparous teleost species Sebastes schlegelii.Mar Environ Res,110:142-151
Nativ N I,Yarmush G,So A et al,2014.Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.Liver Transpl,20(8):1000-1011
Nu?ez-Hernandez D M,Felix-Portillo M,Peregrino-Uriarte A Bet al,2018.Cell cycle regulation and apoptosis mediated by P53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei.Chemosphere,190:253-259
Rahman M S,Thomas P,2007.Molecular cloning,characterization and expression of two hypoxia-inducible factor alpha subunits,HIF-1αand HIF-2α,in a hypoxia-tolerant marine teleost,Atlantic croaker(Micropogonias undulatus).Gene,396(2):273-282
Regula K M,Ens K,Kirshenbaum L A,2002.Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes.Circ Res,91(3):226-231
Schmittgen T D,Livak K J,2008.Analyzing real-time PCR data by the comparative CT method.Nat Protoc,3(6):1101-1108
Semenza G L,2000.HIF-1 and human disease:one highly involved factor.Genes Dev,14(16):1983-1991
Semenza G L,2003.Targeting HIF-1 for cancer therapy.Nat Rev Cancer,3(10):721-732
Shen W Q,Cheng K J,Bao Y Y et al,2012.Expression of GLUT-1,HIF-1α,PI3K and p-Akt in a case of ceruminous adenoma.Head Neck Oncol,4:18
Shimizu S,Eguchi Y,Kamiike W et al,1996.Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL.Cancer Res,56(9):2161-2166
Sun S M,Gu Z M,Fu H T et al,2016.Molecular cloning,characterization,and expression analysis of p53 from the oriental river prawn,Macrobrachium nipponense,in response to hypoxia.Fish Shellfish Immunol,54:68-76
Terova G,Rimoldi S,CoràS et al,2008.Acute and chronic hypoxia affects HIF-1αm RNA levels in sea bass(Dicentrarchus labrax).Aquaculture,279(1-4):150-159
Ton C,Stamatiou D,Liew C C,2003.Gene expression profile of zebrafish exposed to hypoxia during development.Physiol Genomics,13(2):97-106
Wang H J,Huang C X,Chen N et al,2015.Molecular characterization and m RNA expression of HIF-prolyl hydroxylase-2(phd2)in hypoxia-sensing pathways from Megalobrama amblycephala.Comp Biochem Physiol BBiochem Mol Biol,186:28-35
Wegener G,Krause U,2002.Different modes of activating phosphofructokinase,a key regulatory enzyme of glycolysis,in working vertebrate muscle.Biochem Soc Trans,30(2):264-270
Yang S,Yan T,Wu H et al,2017.Acute hypoxic stress:effect on blood parameters,antioxidant enzymes,and expression of HIF-1alpha and GLUT-1 genes in largemouth bass(Micropterus salmoides).Fish Shellfish Immunol,67:449-458
Yuan Z H,Liu S K,Yao J et al,2016.Expression of Bcl-2 genes in channel catfish after bacterial infection and hypoxia stress.Dev Comp Immunol,65:79-90
Zhang G S,Yin S W,Mao J Q et al,2016.Integrated analysis of Mrna-seq and mi RNA-seq in the liver of Pelteobagrus vachelli in response to hypoxia.Sci Rep,6:22907
Zhang G S,Zhao C,Wang Q T et al,2017.Identification of HIF-1 signaling pathway in Pelteobagrus vachelli using RNA-Seq:effects of acute hypoxia and reoxygenation on oxygen sensors,respiratory metabolism,and hematology indices.J Comp Physiol B,187(7):931-943
Zhu C D,Wang Z H,Yan B,2013.Strategies for hypoxia adaptation in fish species:a review.J Comp Physiol B,183(8):1005-1013
Aguilar N M,Ishimatsu A,Ogawa K et al,2000.Aerial ventilatory responses of the mudskipper,Periophthalmodon schlosseri,to altered aerial and aquatic respiratory gas concentrations.Comp Biochem Physiol A Mol Integr Physiol,127(3):285-292
Baptista R B,Souza-Castro N,Almeida-Val V M F,2016.Acute hypoxia up-regulates HIF-1αand VEGF m RNA levels in Amazon hypoxia-tolerant Oscar(Astronotus ocellatus).Fish Physiol Biochem,42(5):1307-1318
Barrionuevo W R,Fernandes M N,Rocha O,2010.Aerobic and anaerobic metabolism for the zebrafish,Danio rerio,reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development.Braz J Biol,70(2):425-434
Butler P J,Jones D R,1997.Physiology of diving of birds and mammals.Physiol Rev,77(3):837-899
Cai M C,Huang Q Y,Liao W G et al,2010.Hypoxic training increases metabolic enzyme activity and composition ofα-myosin heavy chain isoform in rat ventricular myocardium.Eur J Appl Physiol,108(1):105-111
Chen N,Chen L P,Zhang J et al,2012.Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits,HIF-1α/2α/3αof the hypoxia-sensitive freshwater species,Chinese sucker.Gene,498(1):81-90
Denko N C,Fontana L A,Hudson K M et al,2003.Investigating hypoxic tumor physiology through gene expression patterns.Oncogene,22(37):5907-5914
Geng X,Feng J B,Liu S K et al,2014.Transcriptional regulation of hypoxia inducible factors alpha(HIF-α)and their inhibiting factor(FIH-1)of channel catfish(Ictalurus punctatus)under hypoxia.Comp Biochem Physiol BBiochem Mol Biol,169:38-50
Ivan M,Kondo K,Yang H F et al,2001.HIFαtargeted for VHL-mediated destruction by proline hydroxylation:implications for O2 sensing.Science,292(5516):464-468
Jing D D,Li M,Zhang Y et al,2017.Differential induction of enzymes and genes involved in oxidative stress in gill and liver tissues of mudskipper Boleophthalmus pectinirostris exposed to lead.Turk J Fish Aquat Sci,17(2):437-443
Lardon I,Eyckmans M,Vu T N et al,2013.1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish,the common carp(Cyprinus carpio).Metabolomics,9(6):1216-1227
Li Y H,Wei L,Cao J G et al,2016.Oxidative stress,DNAdamage and antioxidant enzyme activities in the pacific white shrimp(Litopenaeus vannamei)when exposed to hypoxia and reoxygenation.Chemosphere,144:234-240
Liu Z H,Lu H M,Shi H L et al,2005.PUMA overexpression induces reactive oxygen species generation and proteasome-mediated stathmin degradation in colorectal cancer cells.Cancer Res,65(5):1647-1654
Maccormack T J,Lewis J M,Almeida-Val V M F et al,2006.Carbohydrate management,anaerobic metabolism,and adenosine levels in the armoured catfish,Liposarcus pardalis(castelnau),during hypoxia.J Exp Zool A Comp Exp Biol,305A(4):363-375
Maes V,Betoulle S,Jaffal A et al,2016.Juvenile roach(Rutilus rutilus)increase their anaerobic metabolism in response to copper exposure in laboratory conditions.Ecotoxicology,25(5):900-913
Mohindra V,Tripathi R K,Singh R K et al,2013.Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits,HIF-1α,-2αand-3αin hypoxia-tolerant Indian catfish,Clarias batrachus[Linnaeus,1758].Mol Biol Rep,40(10):5805-5815
Mu W J,Wen H S,Li J F et al,2015.HIFs genes expression and hematology indices responses to different oxygen treatments in an ovoviviparous teleost species Sebastes schlegelii.Mar Environ Res,110:142-151
Nativ N I,Yarmush G,So A et al,2014.Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.Liver Transpl,20(8):1000-1011
Nu?ez-Hernandez D M,Felix-Portillo M,Peregrino-Uriarte A Bet al,2018.Cell cycle regulation and apoptosis mediated by P53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei.Chemosphere,190:253-259
Rahman M S,Thomas P,2007.Molecular cloning,characterization and expression of two hypoxia-inducible factor alpha subunits,HIF-1αand HIF-2α,in a hypoxia-tolerant marine teleost,Atlantic croaker(Micropogonias undulatus).Gene,396(2):273-282
Regula K M,Ens K,Kirshenbaum L A,2002.Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes.Circ Res,91(3):226-231
Schmittgen T D,Livak K J,2008.Analyzing real-time PCR data by the comparative CT method.Nat Protoc,3(6):1101-1108
Semenza G L,2000.HIF-1 and human disease:one highly involved factor.Genes Dev,14(16):1983-1991
Semenza G L,2003.Targeting HIF-1 for cancer therapy.Nat Rev Cancer,3(10):721-732
Shen W Q,Cheng K J,Bao Y Y et al,2012.Expression of GLUT-1,HIF-1α,PI3K and p-Akt in a case of ceruminous adenoma.Head Neck Oncol,4:18
Shimizu S,Eguchi Y,Kamiike W et al,1996.Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL.Cancer Res,56(9):2161-2166
Sun S M,Gu Z M,Fu H T et al,2016.Molecular cloning,characterization,and expression analysis of p53 from the oriental river prawn,Macrobrachium nipponense,in response to hypoxia.Fish Shellfish Immunol,54:68-76
Terova G,Rimoldi S,CoràS et al,2008.Acute and chronic hypoxia affects HIF-1αm RNA levels in sea bass(Dicentrarchus labrax).Aquaculture,279(1-4):150-159
Ton C,Stamatiou D,Liew C C,2003.Gene expression profile of zebrafish exposed to hypoxia during development.Physiol Genomics,13(2):97-106
Wang H J,Huang C X,Chen N et al,2015.Molecular characterization and m RNA expression of HIF-prolyl hydroxylase-2(phd2)in hypoxia-sensing pathways from Megalobrama amblycephala.Comp Biochem Physiol BBiochem Mol Biol,186:28-35
Wegener G,Krause U,2002.Different modes of activating phosphofructokinase,a key regulatory enzyme of glycolysis,in working vertebrate muscle.Biochem Soc Trans,30(2):264-270
Yang S,Yan T,Wu H et al,2017.Acute hypoxic stress:effect on blood parameters,antioxidant enzymes,and expression of HIF-1alpha and GLUT-1 genes in largemouth bass(Micropterus salmoides).Fish Shellfish Immunol,67:449-458
Yuan Z H,Liu S K,Yao J et al,2016.Expression of Bcl-2 genes in channel catfish after bacterial infection and hypoxia stress.Dev Comp Immunol,65:79-90
Zhang G S,Yin S W,Mao J Q et al,2016.Integrated analysis of Mrna-seq and mi RNA-seq in the liver of Pelteobagrus vachelli in response to hypoxia.Sci Rep,6:22907
Zhang G S,Zhao C,Wang Q T et al,2017.Identification of HIF-1 signaling pathway in Pelteobagrus vachelli using RNA-Seq:effects of acute hypoxia and reoxygenation on oxygen sensors,respiratory metabolism,and hematology indices.J Comp Physiol B,187(7):931-943
Zhu C D,Wang Z H,Yan B,2013.Strategies for hypoxia adaptation in fish species:a review.J Comp Physiol B,183(8):1005-1013