低氧预适应对小鼠骨骼肌Nrf2和线粒体呼吸链复合物蛋白表达及运动能力的影响
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摘要
目的:通过对小鼠进行低氧预适应,探究其对骨骼肌Nrf2及线粒体呼吸链复合物蛋白表达和运动能力的影响。方法:健康8周龄C57BL/6J小鼠(WT)共40只,随机分为4组:未低氧预适应安静组(NC)、低氧预适应安静组(HC)、未低氧预适应低氧运动组(NE)和低氧预适应低氧运动组(HE),每组10只。对低氧预适应组小鼠进行48 h持续低氧暴露,氧浓度为11. 2%,未低氧预适应组小鼠饲养于常氧下。低氧预适应组低氧暴露结束后,对运动组小鼠进行运动能力测试。运动后即刻取小鼠腿部胫骨前肌检测Nrf2及线粒体呼吸链复合物的蛋白表达,实时荧光定量PCR检测NRF1、TFAM的基因表达。结果:1)与未低氧预适应组相比,低氧预适应组Nrf2蛋白表达均显著增加(P<0. 05)。2)安静状态下,低氧预适应小鼠TFAM mRNA表达和线粒体呼吸链复合物CⅠ～CⅤ蛋白表达呈下降趋势。3)一次性低氧力竭运动后,低氧预适应小鼠NRF1和TFAM mRNA和线粒体呼吸链复合物CⅠ、CⅢ、CⅣ、CⅤ蛋白表达呈增加趋势,CⅡ蛋白表达显著下降(P<0. 05)。4)经过低氧预适应的小鼠在低氧环境中的运动能力显著增加,体现为力竭时小鼠跑动距离显著增加和达到力竭时所用时长显著增加。结论:48 h的11. 2%氧浓度的低氧预适应,可显著增加低氧运动后小鼠骨骼肌Nrf2和线粒体呼吸链复合物CⅣ和CⅤ蛋白表达,这可能是低氧预适应提高小鼠运动能力的原因之一。
Objective: This study tried to explore the influence of hypoxic preconditioning on the exercise capacity and the expression of mitochondrial respiratory chain complex protein expression in mice. Methods: Forty healthy 8-week-old C57 BL/6 J mice( WT) were randomly divided into four groups( n = 10) : normal control( NC),hypoxic preconditioning control( HC),normal exercise( NE) and hypoxic preconditioning and exercise( HE) groups.Mice in the HC and HE groups were exposed to hypoxic for 48 h at oxygen concentration of 11. 2%,while those in the NC and NE groups were kept under normoxic. After the hypoxia preconditioning,the exercise capacity of mice in the exercise groups was tested. The anterior tibial muscles of mice were taken immediately after exercise,the protein expression of mitochondrial respiratory chain complex was detected,and the gene expressions of NRF1,TFAM were detected by real-time fluorescence quantitative PCR. Results: 1) Compared with NC and NE groups,the Nrf2 protein expression increased in HC and HE groups( P < 0. 05). 2) The mRNA expression of TFAM and the protein expression of mitochondrial respiratory chain complex CⅠ-CⅤ in HC mice showed a downward trend.3) After one-time exhaustive exercise under hypoxic environment,the expression of NRF1 and TFAM and mitochondrial respiratory chain complex CⅠ,CⅢ,CⅣ and CⅤ in HE increased,while the expression of CⅡ protein decreased significantly( P < 0. 05). 4) The exercise capacity of hypoxic preconditioned mice in hypoxic environment was significantly increased,which was reflected in the significant increase of running distance and the significant increase of time spent in exhaustion. Conclusions: Hypoxic preconditioning with 11. 2% oxygen concentration at 48 hours significantly increased the expression of Nrf 2 and mitochondrial respiratory chain complex CⅣ and CⅤprotein in skeletal muscle of mice after hypoxic exercise,which may be one of the reasons for hypoxic preconditioning to improve the exercise capacity of mice.
Objective: This study tried to explore the influence of hypoxic preconditioning on the exercise capacity and the expression of mitochondrial respiratory chain complex protein expression in mice. Methods: Forty healthy 8-week-old C57 BL/6 J mice( WT) were randomly divided into four groups( n = 10) : normal control( NC),hypoxic preconditioning control( HC),normal exercise( NE) and hypoxic preconditioning and exercise( HE) groups.Mice in the HC and HE groups were exposed to hypoxic for 48 h at oxygen concentration of 11. 2%,while those in the NC and NE groups were kept under normoxic. After the hypoxia preconditioning,the exercise capacity of mice in the exercise groups was tested. The anterior tibial muscles of mice were taken immediately after exercise,the protein expression of mitochondrial respiratory chain complex was detected,and the gene expressions of NRF1,TFAM were detected by real-time fluorescence quantitative PCR. Results: 1) Compared with NC and NE groups,the Nrf2 protein expression increased in HC and HE groups( P < 0. 05). 2) The mRNA expression of TFAM and the protein expression of mitochondrial respiratory chain complex CⅠ-CⅤ in HC mice showed a downward trend.3) After one-time exhaustive exercise under hypoxic environment,the expression of NRF1 and TFAM and mitochondrial respiratory chain complex CⅠ,CⅢ,CⅣ and CⅤ in HE increased,while the expression of CⅡ protein decreased significantly( P < 0. 05). 4) The exercise capacity of hypoxic preconditioned mice in hypoxic environment was significantly increased,which was reflected in the significant increase of running distance and the significant increase of time spent in exhaustion. Conclusions: Hypoxic preconditioning with 11. 2% oxygen concentration at 48 hours significantly increased the expression of Nrf 2 and mitochondrial respiratory chain complex CⅣ and CⅤprotein in skeletal muscle of mice after hypoxic exercise,which may be one of the reasons for hypoxic preconditioning to improve the exercise capacity of mice.
引文
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[5] MOTOMURA A,SHIMIZU M,KATO A,et al.Remote ischemic preconditioning protects human neural stem cells from oxidative stress[J].Apoptosis,2017,22(11):1353-1361.
[6] BEIDLEMAN BA,MUZA SI,FULCO CS,et al.Intermittent altitude exposures reduce acute mountain sickness at 4300 m[J]. Clin Sci(Lond),2004,106(3):321.
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[10] MILLER CJ,GOUNDER SS,KANNAN S,et al.Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle[J].Biochim Biophys Acta,2012,1822(6):1038-1050.
[11] STROM J,XU B,TIAN X,et al. Nrf2 protects mitochondrial decay by oxidative stress[J]. FASEB J,2016,30(1):66.
[12] WU KC,CUI JY,KLAASSEN CD. Beneficial role of Nrf2 in regulating NADPH generation and consumption[J]. Toxicol Sci,2011,123:590-600.
[13] CRILLY MJ,TRYON LD,ERLICH AT,et al. The role of Nrf2 in skeletal muscle contractile and mitochondrial function[J]. J Appl Physiol,2016,121:730-740.
[14] BURCH N,ARNOLD AS,ITEM F,et al. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle[J]. PLoS One,2010,5(6):e10970.
[15] ZHANG YK,WU KC,KLAASSEN CD. Genetic activation of Nrf2 protects against fasting-induced oxidative stress in livers of mice[J]. PLoS One,2013,8:e59122.
[16] HOLMSTROM KM,BAIRD L,ZHANG Y,et al.Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration[J]. Biol Open,2013,2:761-770.
[17] PIANTADOSI CA,CARRAWAY MS,BABIKER A,et al. Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1[J]. Circ Res,2008,103:1232-1240.
[18] KOLAMUNNE RT,DIAS IH,VERNALLIS AB,et al. Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts:the roles of reactive oxygen and nitrogen species[J].Redox Bio,2013,1:418-426.
[19]周珊珊.Nrf2与MT的协同作用在保护慢性间歇性低氧所致心脏损伤中的作用[D].长春:吉林大学,2015.
[20] HERZIG RP,SCACCO S,SCARPULLA RC. Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome[J]. J Biol Chem,2000,275(17):13134-13141.
[21]宋银娟,廖轶,赵德明,等.线粒体转录因子A的调节和功能[J].动物医学进展,2017,38(11):112-116.
[22] LAN L,UUO M,AI Y,et al. Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM[J]. Biosci Rep, 2017, 37(3):BSR20170319.
[23] VIRBASIUS JV,SCARPULLA RC. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors:a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis[J]. Proc Natl Acad Sci USA,1994,91(4):1309-1313.
[24] DHAR SS, ONGWIJITWAT S, WONG-RILEY MTT. Nuclear respiratory factor 1 regulates all ten nuclear-encoded subunits of cytochrome coxidase in neurons[J]. J Biol Chem,2008,283(6):3120-3129.
[25] DHAR SS,LIANG HL,WONG-RILEY MT. Transcriptional coupling of synaptic transmission and energy metabolism:role of nuclear respiratory factor 1in coregulating neuronal nitric oxide synthase and cytochrome coxidas genes in neurons[J]. Biochim Biophys Acta,2009,1793(10):1604-1613.
[26] RUSTIN P,CHRETIEN D,BOURGERON T,et al. Bio-chemical and molecular investigations in respiratory chain deficiencies[J]. Clin Chim Acta,1994,228:35-51.
[27]张前锋,徐晓阳,李捷,等.不同训练方案对大鼠股直肌线粒体呼吸链复合体酶活性的影响[J].体育科学,2014,34(10):67-71.
[28] ALI SS,HSl AO M,ZHAO HW,et al. Hypoxia adaptation involves mitochondrial metabolic depression and decreased ROS leakage[J]. PLoS One,2012,7(5):c36801.
[29] FFRGUSON M,MOCKETT RJ,SHEN Y,et al.Age-associated decline in mitochondrial respiration and electron transport in Drosophila mclanogaster[J]. Biol Chem,2005,390(2):501-511.
[30] HORSTMAN DH,WEISKOPF RB,JACKSON RE.Work capacity during 3-week sojourn at 4300 m:effects of relative polycythemia[J]. J Appl Physiol,1980,35:385-390.
[31] FULCO CS,KAMBIS KW. Carbohydrate supplementation improves time-trial cycle performance during energy deficit at 4300 m altitude[J]. J Appl Physiol,2005,99:867-876.
[32] MUZA SR. Military applications of hypoxic training for high-altitude operations[J]. Medicine&Science in Sports&Exercise,2007,39(9):1625-1631.
[2] DIRNAGL U,BECKER K,MEISEL A. Preconditioning and tolerance against cerebral ischaemia:from experimental strategies to clinical use[J]. Lancet Neurol,2009,8(4):398-412.
[3] SPRICK JD,MALLET RT. Ischemic and hypoxic conditioning:potential for protection of vital organs.[J]. Exp Physiol,2018,9(5):1-40.
[4] MANUKHINA EB,MASHINA S,SMIRIN BV,et al.Role of nitric oxide in adaptation to hypoxia and adaptive defense[J]. Physiol Res,2000,49(1):89-97.
[5] MOTOMURA A,SHIMIZU M,KATO A,et al.Remote ischemic preconditioning protects human neural stem cells from oxidative stress[J].Apoptosis,2017,22(11):1353-1361.
[6] BEIDLEMAN BA,MUZA SI,FULCO CS,et al.Intermittent altitude exposures reduce acute mountain sickness at 4300 m[J]. Clin Sci(Lond),2004,106(3):321.
[7]高钮棋.高原军事医学[M].重庆:重庆出版社,2005.
[8] ROACH RC,GREENE ER,SCHOENE RB,et al.Arterial oxygen saturation for prediction of acute mountain sickness[J]. Aviat Space Environ Med,1998,69(12):1182-1185.
[9] HAYAT A,HUSSAIN MM,AZIZ S,et al. Hyperventilatory capacity:a predictor of altitude sickness[J]. J Awb Med Coll Abbottabad,2006,18(2):17-20.
[10] MILLER CJ,GOUNDER SS,KANNAN S,et al.Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle[J].Biochim Biophys Acta,2012,1822(6):1038-1050.
[11] STROM J,XU B,TIAN X,et al. Nrf2 protects mitochondrial decay by oxidative stress[J]. FASEB J,2016,30(1):66.
[12] WU KC,CUI JY,KLAASSEN CD. Beneficial role of Nrf2 in regulating NADPH generation and consumption[J]. Toxicol Sci,2011,123:590-600.
[13] CRILLY MJ,TRYON LD,ERLICH AT,et al. The role of Nrf2 in skeletal muscle contractile and mitochondrial function[J]. J Appl Physiol,2016,121:730-740.
[14] BURCH N,ARNOLD AS,ITEM F,et al. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle[J]. PLoS One,2010,5(6):e10970.
[15] ZHANG YK,WU KC,KLAASSEN CD. Genetic activation of Nrf2 protects against fasting-induced oxidative stress in livers of mice[J]. PLoS One,2013,8:e59122.
[16] HOLMSTROM KM,BAIRD L,ZHANG Y,et al.Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration[J]. Biol Open,2013,2:761-770.
[17] PIANTADOSI CA,CARRAWAY MS,BABIKER A,et al. Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1[J]. Circ Res,2008,103:1232-1240.
[18] KOLAMUNNE RT,DIAS IH,VERNALLIS AB,et al. Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts:the roles of reactive oxygen and nitrogen species[J].Redox Bio,2013,1:418-426.
[19]周珊珊.Nrf2与MT的协同作用在保护慢性间歇性低氧所致心脏损伤中的作用[D].长春:吉林大学,2015.
[20] HERZIG RP,SCACCO S,SCARPULLA RC. Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome[J]. J Biol Chem,2000,275(17):13134-13141.
[21]宋银娟,廖轶,赵德明,等.线粒体转录因子A的调节和功能[J].动物医学进展,2017,38(11):112-116.
[22] LAN L,UUO M,AI Y,et al. Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM[J]. Biosci Rep, 2017, 37(3):BSR20170319.
[23] VIRBASIUS JV,SCARPULLA RC. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors:a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis[J]. Proc Natl Acad Sci USA,1994,91(4):1309-1313.
[24] DHAR SS, ONGWIJITWAT S, WONG-RILEY MTT. Nuclear respiratory factor 1 regulates all ten nuclear-encoded subunits of cytochrome coxidase in neurons[J]. J Biol Chem,2008,283(6):3120-3129.
[25] DHAR SS,LIANG HL,WONG-RILEY MT. Transcriptional coupling of synaptic transmission and energy metabolism:role of nuclear respiratory factor 1in coregulating neuronal nitric oxide synthase and cytochrome coxidas genes in neurons[J]. Biochim Biophys Acta,2009,1793(10):1604-1613.
[26] RUSTIN P,CHRETIEN D,BOURGERON T,et al. Bio-chemical and molecular investigations in respiratory chain deficiencies[J]. Clin Chim Acta,1994,228:35-51.
[27]张前锋,徐晓阳,李捷,等.不同训练方案对大鼠股直肌线粒体呼吸链复合体酶活性的影响[J].体育科学,2014,34(10):67-71.
[28] ALI SS,HSl AO M,ZHAO HW,et al. Hypoxia adaptation involves mitochondrial metabolic depression and decreased ROS leakage[J]. PLoS One,2012,7(5):c36801.
[29] FFRGUSON M,MOCKETT RJ,SHEN Y,et al.Age-associated decline in mitochondrial respiration and electron transport in Drosophila mclanogaster[J]. Biol Chem,2005,390(2):501-511.
[30] HORSTMAN DH,WEISKOPF RB,JACKSON RE.Work capacity during 3-week sojourn at 4300 m:effects of relative polycythemia[J]. J Appl Physiol,1980,35:385-390.
[31] FULCO CS,KAMBIS KW. Carbohydrate supplementation improves time-trial cycle performance during energy deficit at 4300 m altitude[J]. J Appl Physiol,2005,99:867-876.
[32] MUZA SR. Military applications of hypoxic training for high-altitude operations[J]. Medicine&Science in Sports&Exercise,2007,39(9):1625-1631.
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