低氧调控EIMD后肌细胞膜损伤MAPK机制的探索与验证
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摘要
目的:探索运动性肌损伤(EIMD)后低氧调控肌细胞膜损伤的MAPK机制,并进行验证。方法:健康雄性SD大鼠随机分为对照组、常氧24 h、48 h、72 h组和低氧24 h、48 h、72 h组。大鼠以EIMD运动模型进行间歇性下坡跑离心运动,腹腔注射伊文氏蓝荧光染色剂(EBD),取腓肠肌样本,用激光共聚焦显微镜观察EBD切片和阳性细胞率(PRC)评价膜损伤。用RT-qPCR和Westernblot测定MAPK的ERK1/2、JUN、p38和BMK1的mRNA表达、蛋白含量和磷酸化水平。再开展抑制剂实验以确认低氧调控膜损伤MAPK机制中的关键信号通路。结果:EIMD后,低氧与常氧各组均出现明显膜损伤,低氧各组阳性细胞率显著高于常氧各组和对照组(P<0.01)。低氧组ERK1、p38和BMK1通路的mRNA表达、蛋白含量和磷酸化水平显著高于常氧对应组(P<0.05),JNK信号通路蛋白含量和磷酸化水平无显著变化(P>0.05)。抑制剂实验结果表明,ERK1/2和p38抑制剂组的蛋白含量和磷酸化水平无显著变化(P>0.05)。BMK1抑制剂组24 h、48 h的磷酸化水平、阳性细胞率显著低于安慰剂对应组(P<0.001),且与对照组无显著差异(P>0.05)。结论:1)EIMD后低氧明显加剧膜损伤并造成损伤峰值前移。2)低氧调控膜损伤主要通过BMK1通路实现,抑制BMK1磷酸化能阻止低氧对膜损伤的加剧作用。BMK1/ERK5通路是低氧调控EIMD后膜损伤MAPK机制的关键途径。
Objective:To investigate and verify the effects of hypoxia on sarcolemma injury,and regulated mechanism by mitogen-activated protein kinases(MAPK) after exercise-induced muscle Damage(EIMD). Methods:The male SD rats were randomly divided into control group,normoxia(24,48 and 72 h group) and hypoxia group(24,48 and 72 h group). Rats were experienced intermittent downhill running according to EIMD model. And Rats were treated with intraperitoneal injection of Evans blue dye(EBD) after downhill running. Gastrocnemius muscle sample was isolated quickly and prepared to analyze. The positive ratio of cells(PRC) was observed by fluorescence microscope. To explore the hypoxia regulating mechanism of MAPK signal pathways in sarcolemma injury after EIMD,the m RNA,protein content and phosphylation of ERK1/2,JUN,p38 and BMK1 were tested by RT-q PCR and Western blot. Subsequently,inhibitor experiment was carried out so as to confirm the sarcolemma injury regulation mechanism of MAPK signal pathways in hypoxia.. Results:After EIMD,hypoxia and normoxia groups suffered a significant sarcolemma injury. The PRC showed a significant difference between hypoxia groups and normoxia groups(P<0.01). The m RNA,protein expression and phosphorylation of ERK1,p38 and BMK1 in hypoxia groups were significantly higher than those in normoxia groups(P< 0.05). In addition,protein expression and phosphorylation of JNK had no significant change(P>0.05). The results of inhibitor experiment showed no significant changes of phosphorylation and PRC in inhibitor group(P> 0.05). However,phosphorylation and PRC in inhibitor 24 h and 48 h BMK1 groups were significantly lower than placebo group(P<0.001). And there were no significant difference between BMK1 inhibitor groups and placebo group(P>0.05). Conclusions:1) The hypoxia exacerbates sarcolemma injury and causes the peak value shift forward after EIMD. 2) Inhibition of BMK1 phosphorylation can prevent sarcolemma injury. BMK1/ERK5 is the key signal pathway of MAPK mechanism in hypoxia regulates sarcolemma injury after EIMD.
Objective:To investigate and verify the effects of hypoxia on sarcolemma injury,and regulated mechanism by mitogen-activated protein kinases(MAPK) after exercise-induced muscle Damage(EIMD). Methods:The male SD rats were randomly divided into control group,normoxia(24,48 and 72 h group) and hypoxia group(24,48 and 72 h group). Rats were experienced intermittent downhill running according to EIMD model. And Rats were treated with intraperitoneal injection of Evans blue dye(EBD) after downhill running. Gastrocnemius muscle sample was isolated quickly and prepared to analyze. The positive ratio of cells(PRC) was observed by fluorescence microscope. To explore the hypoxia regulating mechanism of MAPK signal pathways in sarcolemma injury after EIMD,the m RNA,protein content and phosphylation of ERK1/2,JUN,p38 and BMK1 were tested by RT-q PCR and Western blot. Subsequently,inhibitor experiment was carried out so as to confirm the sarcolemma injury regulation mechanism of MAPK signal pathways in hypoxia.. Results:After EIMD,hypoxia and normoxia groups suffered a significant sarcolemma injury. The PRC showed a significant difference between hypoxia groups and normoxia groups(P<0.01). The m RNA,protein expression and phosphorylation of ERK1,p38 and BMK1 in hypoxia groups were significantly higher than those in normoxia groups(P< 0.05). In addition,protein expression and phosphorylation of JNK had no significant change(P>0.05). The results of inhibitor experiment showed no significant changes of phosphorylation and PRC in inhibitor group(P> 0.05). However,phosphorylation and PRC in inhibitor 24 h and 48 h BMK1 groups were significantly lower than placebo group(P<0.001). And there were no significant difference between BMK1 inhibitor groups and placebo group(P>0.05). Conclusions:1) The hypoxia exacerbates sarcolemma injury and causes the peak value shift forward after EIMD. 2) Inhibition of BMK1 phosphorylation can prevent sarcolemma injury. BMK1/ERK5 is the key signal pathway of MAPK mechanism in hypoxia regulates sarcolemma injury after EIMD.
引文
[1]曹师承,孙黎光,赵刚,等.运动干预对大鼠骨骼肌JNK磷酸化与表达的影响[J].中国应用生理学杂志,2009,25(1):103-106.
[2]曹师承,孙黎光,赵刚,等.耐力运动对大鼠骨骼肌ERK1/2活性的影响[J].中国应用生理学杂志,2007,23(3):351-354.
[3]孙超,刘景生,彭雪贤.信号转导与人类疾病[M].北京:化学工业出版社,2006:84-87.
[4]王今越,丁树哲,王小虹,等.p38、NF-κB、IL-6在长期大强度运动诱导大鼠骨骼肌发生中的作用[J].体育科学,2010,30(2):75-82.
[5]王金发.细胞生物学[M].北京:科学出版社,2003:213-218.
[6]王洋,佟钧,张忍发,等.MAPK信号通路在雄激素和运动对大鼠骨骼肌蛋白合成中的作用[J].昆明医科大学学报,2016,37(1):60-64.
[7]吴迎,曾凡星,李奕,等.长期不同负荷运动对心肌MAPK信号通路的影响[J].中国体育科技,2013,49(6):94-99.
[8]翟帅,陈佩林.运动心脏重塑过程中mi RNA-350/JNK信号通路的动态变化[J].体育科学,2014,34(5):9-14.
[9]张雪,丁树哲.耐力训练对SD大鼠氧化应激及MAPKERK1/2的影响[J].体育科学,2010,30(5):49-55.
[10]ARMSTRONG R B,OGILVIE R W,SCHWANE J A.Eccentric exercise-induced injury to rat skeletal muscle[J].J Appl Physiol Respir Environ Exerc Physiol,1983,54(1):80-93.
[11]BOPPART M D,ARONSON D,GIBSON L,et al.Eccentric exercise markedly increases c-Jun NH(2)-terminal kinase activity in human skeletal muscle[J].J Appl Physiol(1985),1999,87(5):1668-1673.
[12]BOPPART M D,ASP S,WOJTASZEWSKI J F,et al.Marathon running transiently increases c-Jun NH2-terminal kinase and p38activities in human skeletal muscle[J].J Physiol,2000,526(Pt 3):663-669.
[13]BROWN J L,ROSA-CALDWELL M E,LEE D E,et al.PGC-1alpha4 gene expression is suppressed by the IL-6-MEKERK 1/2 MAPK signalling axis and altered by resistance exercise,obesity and muscle injury[J].Acta Physiol(Oxf),2017,220(2):275-288.
[14]CHEN T,MOORE T M,EBBERT M T,et al.Liver kinase B1inhibits the expression of inflammation-related genes postcontraction in skeletal muscle[J].J Appl Physiol(1985),2016,120(8):876-888.
[15]GE Y,PAN S,GUAN D,et al.Micro RNA-350 induces pathological heart hypertrophy by repressing both p38 and JNK pathways[J].Biochim Biophys Acta,2013,1832(1):1-10.
[16]GONZALEZ A,HOFFMAN J R,TOWNSEND J R,et al.MAPK signaling following high volume and high intensity resistance exercise protocols in trained men[J].Med Sci Sports Exerc,2016,48(5 Suppl 1):17.
[17]GONZALEZ A M,HOFFMAN J R,TOWNSEND J R,et al.Intramuscular MAPK signaling following high volume and high intensity resistance exercise protocols in trained men[J].Eur J Appl Physiol,2016,116(9):1663-1670.
[18]HAMER P W,MCGEACHIE J M,DAVIES M J,et al.Evans Blue Dye as an in vivo marker of myofibre damage:Optimising parameters for detecting initial myofibre membrane permeability[J].J Anat,2002,200(Pt 1):69-79.
[19]HE Y,JIAN C X,ZHANG H Y,et al.Hypoxia enhances periodontal ligament stem cell proliferation via the MAPK signaling pathway[J].Genet Mol Res,2016,15(4):1-10.
[20]KATO Y,TAPPING R I,HUANG S,et al.Bmk1/Erk5 is required for cell proliferation induced by epidermal growth factor[J].Nat,1998,395(6703):713-716.
[21]KIM J S,LEE Y H,CHANG Y U,et al.PPARgamma regulates inflammatory reaction by inhibiting the MAPK/NF-kappa B pathway in C2C12 skeletal muscle cells[J].J Physiol Biochem,2017,73(1):49-57.
[22]LEE J S,BRUCE C R,SPURRELL B E,et al.Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38mitogen-activated protein kinase pathways in rat soleus muscle[J].Clin Exp Pharmacol Physiol,2002,29(8):655-660.
[23]LI C,VU K,HAZELGROVE K,et al.Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn’s disease and smooth muscle hypertrophy[J].Am J Physiol Gastr L,2015,309(11):G888-899.
[24]LIEBER R L,THORNELL L E,FRIDEN J.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction[J].J Appl Physiol,(1985).1996,80(1):278-284.
[25]LUO F,SHI J,SHI Q,et al.Mitogen-activated protein kinases and hypoxic/Ischemic nephropathy[J].Cell Physiol Biochem,2016,39(3):1051-1067.
[26]MCNEIL P L,KHAKEE R.Disruptions of muscle fiber plasma membranes.Role in exercise-induced damage[J].Am J Pathol,1992,140(5):1097-1109.
[27]NICOLL J X,FRY A C,GALPIN A J,et al.Changes in resting mitogen-activated protein kinases following resistance exercise overreaching and overtraining[J].Eur J Appl Physiol,2016,116(11-12):2401-2413.
[28]NOSAKA K,CLARKSON P M.Muscle damage following repeated bouts of high force eccentric exercise[J].Med Sci Sports Exerc,1995,27(9):1263-1269.
[29]QIU J,ZHENG Y,HU J,et al.Biomechanical regulation of vascular smooth muscle cell functions:From in vitro to in vivo understanding[J].J R Soc Interface,2014,11(90):2013-2052.
[30]RIZO-ROCA D,SANTOS-ALVES E,RIOS-KRISTJANSSON J G,et al.Intermittent hypoxia increases mitochondrial dynamics and biogenesis after eccentric exercise-induced muscle damage in trained rats[J].Med Sci Sports Exerc,2016,48(5 Suppl 1):899-900.
[31]ROBERTS O L,HOLMES K,MULLER J,et al.ERK5 and the regulation of endothelial cell function[J].Biochem Soc Trans,2009,37(Pt 6):1254-1259.
[32]ROVIDA E,DI MAIRA G,TUSA I,et al.The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma[J].Gut,2015,64(9):1454-1465.
[33]STAUBER W T,KNACK K K,MILLER G R,et al.Fibrosis and intercellular collagen connections from four weeks of muscle strains[J].Muscle Nerve,1996,19(4):423-430.
[34]SUTTON K M,HAYAT S,CHAU N M,et al.Selective inhibition of MEK1/2 reveals a differential requirement for ERK1/2signalling in the regulation of HIF-1 in response to hypoxia and IGF-1[J].Oncogene,2007,26(27):3920-3929.
[35]VAN GINNEKEN M M,DE GRAAF-ROELFSEMA E,KEIZER H A,et al.Effect of exercise on activation of the p38 mitogen-activated protein kinase pathway,c-Jun NH2 terminal kinase,and heat shock protein 27 in equine skeletal muscle[J].Am J Vet Res,2006,67(5):837-844.
[36]WAGNER E F,NEBREDA A R.Signal integration by JNK and p38 MAPK pathways in cancer development[J].Nat Rev Cancer,2009,9(8):537-549.
[37]WIDEGREN U,RYDER J W,ZIERATH J R.Mitogen-activated protein kinase signal transduction in skeletal muscle:Effects of exercise and muscle contraction[J].Acta Physiol Scand,2001,172(3):227-238.
[38]WILLIAMSON D,GALLAGHER P,HARBER M,et al.Mitogen-activated protein kinase(MAPK)pathway activation:Effects of age and acute exercise on human skeletal muscle[J].J Physiol,2003,547(Pt 3):977-987.
[39]XU Y M,LI J P,WANG R Y.Changes of dystrophin and desmin in rat gastrocnemius under micro-damage induced by hypoxia[J].Acta Physiol Sin,2010,62(4):339-348.
[40]YANAGISAWA O,SAKUMA J,KAWAKAMI Y,et al.Effect of exercise-induced muscle damage on muscle hardness evaluated by ultrasound real-time tissue elastography[J].Springerplus,2015,4:308.
[41]ZBINDEN-FONCEA H,RAYMACKERS J M,DELDICQUE L,et al.TLR2 and TLR4 activate p38 MAPK and JNK during endurance exercise in skeletal muscle[J].Med Sci Sports Exerc,2012,44(8):1463-1472.
(1)此通路在文献中有BMK1/ERK5、BMK1和ERK5 3种表述方式,为简练表达并与ERK1/2通路区别,本文采用BMK1表述。但在引用文献内容时,尊重并保留原文献中BMK1/ERK5或ERK5的表述。
(1)EBD注射时间点为运动后即刻。
[2]曹师承,孙黎光,赵刚,等.耐力运动对大鼠骨骼肌ERK1/2活性的影响[J].中国应用生理学杂志,2007,23(3):351-354.
[3]孙超,刘景生,彭雪贤.信号转导与人类疾病[M].北京:化学工业出版社,2006:84-87.
[4]王今越,丁树哲,王小虹,等.p38、NF-κB、IL-6在长期大强度运动诱导大鼠骨骼肌发生中的作用[J].体育科学,2010,30(2):75-82.
[5]王金发.细胞生物学[M].北京:科学出版社,2003:213-218.
[6]王洋,佟钧,张忍发,等.MAPK信号通路在雄激素和运动对大鼠骨骼肌蛋白合成中的作用[J].昆明医科大学学报,2016,37(1):60-64.
[7]吴迎,曾凡星,李奕,等.长期不同负荷运动对心肌MAPK信号通路的影响[J].中国体育科技,2013,49(6):94-99.
[8]翟帅,陈佩林.运动心脏重塑过程中mi RNA-350/JNK信号通路的动态变化[J].体育科学,2014,34(5):9-14.
[9]张雪,丁树哲.耐力训练对SD大鼠氧化应激及MAPKERK1/2的影响[J].体育科学,2010,30(5):49-55.
[10]ARMSTRONG R B,OGILVIE R W,SCHWANE J A.Eccentric exercise-induced injury to rat skeletal muscle[J].J Appl Physiol Respir Environ Exerc Physiol,1983,54(1):80-93.
[11]BOPPART M D,ARONSON D,GIBSON L,et al.Eccentric exercise markedly increases c-Jun NH(2)-terminal kinase activity in human skeletal muscle[J].J Appl Physiol(1985),1999,87(5):1668-1673.
[12]BOPPART M D,ASP S,WOJTASZEWSKI J F,et al.Marathon running transiently increases c-Jun NH2-terminal kinase and p38activities in human skeletal muscle[J].J Physiol,2000,526(Pt 3):663-669.
[13]BROWN J L,ROSA-CALDWELL M E,LEE D E,et al.PGC-1alpha4 gene expression is suppressed by the IL-6-MEKERK 1/2 MAPK signalling axis and altered by resistance exercise,obesity and muscle injury[J].Acta Physiol(Oxf),2017,220(2):275-288.
[14]CHEN T,MOORE T M,EBBERT M T,et al.Liver kinase B1inhibits the expression of inflammation-related genes postcontraction in skeletal muscle[J].J Appl Physiol(1985),2016,120(8):876-888.
[15]GE Y,PAN S,GUAN D,et al.Micro RNA-350 induces pathological heart hypertrophy by repressing both p38 and JNK pathways[J].Biochim Biophys Acta,2013,1832(1):1-10.
[16]GONZALEZ A,HOFFMAN J R,TOWNSEND J R,et al.MAPK signaling following high volume and high intensity resistance exercise protocols in trained men[J].Med Sci Sports Exerc,2016,48(5 Suppl 1):17.
[17]GONZALEZ A M,HOFFMAN J R,TOWNSEND J R,et al.Intramuscular MAPK signaling following high volume and high intensity resistance exercise protocols in trained men[J].Eur J Appl Physiol,2016,116(9):1663-1670.
[18]HAMER P W,MCGEACHIE J M,DAVIES M J,et al.Evans Blue Dye as an in vivo marker of myofibre damage:Optimising parameters for detecting initial myofibre membrane permeability[J].J Anat,2002,200(Pt 1):69-79.
[19]HE Y,JIAN C X,ZHANG H Y,et al.Hypoxia enhances periodontal ligament stem cell proliferation via the MAPK signaling pathway[J].Genet Mol Res,2016,15(4):1-10.
[20]KATO Y,TAPPING R I,HUANG S,et al.Bmk1/Erk5 is required for cell proliferation induced by epidermal growth factor[J].Nat,1998,395(6703):713-716.
[21]KIM J S,LEE Y H,CHANG Y U,et al.PPARgamma regulates inflammatory reaction by inhibiting the MAPK/NF-kappa B pathway in C2C12 skeletal muscle cells[J].J Physiol Biochem,2017,73(1):49-57.
[22]LEE J S,BRUCE C R,SPURRELL B E,et al.Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38mitogen-activated protein kinase pathways in rat soleus muscle[J].Clin Exp Pharmacol Physiol,2002,29(8):655-660.
[23]LI C,VU K,HAZELGROVE K,et al.Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn’s disease and smooth muscle hypertrophy[J].Am J Physiol Gastr L,2015,309(11):G888-899.
[24]LIEBER R L,THORNELL L E,FRIDEN J.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction[J].J Appl Physiol,(1985).1996,80(1):278-284.
[25]LUO F,SHI J,SHI Q,et al.Mitogen-activated protein kinases and hypoxic/Ischemic nephropathy[J].Cell Physiol Biochem,2016,39(3):1051-1067.
[26]MCNEIL P L,KHAKEE R.Disruptions of muscle fiber plasma membranes.Role in exercise-induced damage[J].Am J Pathol,1992,140(5):1097-1109.
[27]NICOLL J X,FRY A C,GALPIN A J,et al.Changes in resting mitogen-activated protein kinases following resistance exercise overreaching and overtraining[J].Eur J Appl Physiol,2016,116(11-12):2401-2413.
[28]NOSAKA K,CLARKSON P M.Muscle damage following repeated bouts of high force eccentric exercise[J].Med Sci Sports Exerc,1995,27(9):1263-1269.
[29]QIU J,ZHENG Y,HU J,et al.Biomechanical regulation of vascular smooth muscle cell functions:From in vitro to in vivo understanding[J].J R Soc Interface,2014,11(90):2013-2052.
[30]RIZO-ROCA D,SANTOS-ALVES E,RIOS-KRISTJANSSON J G,et al.Intermittent hypoxia increases mitochondrial dynamics and biogenesis after eccentric exercise-induced muscle damage in trained rats[J].Med Sci Sports Exerc,2016,48(5 Suppl 1):899-900.
[31]ROBERTS O L,HOLMES K,MULLER J,et al.ERK5 and the regulation of endothelial cell function[J].Biochem Soc Trans,2009,37(Pt 6):1254-1259.
[32]ROVIDA E,DI MAIRA G,TUSA I,et al.The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma[J].Gut,2015,64(9):1454-1465.
[33]STAUBER W T,KNACK K K,MILLER G R,et al.Fibrosis and intercellular collagen connections from four weeks of muscle strains[J].Muscle Nerve,1996,19(4):423-430.
[34]SUTTON K M,HAYAT S,CHAU N M,et al.Selective inhibition of MEK1/2 reveals a differential requirement for ERK1/2signalling in the regulation of HIF-1 in response to hypoxia and IGF-1[J].Oncogene,2007,26(27):3920-3929.
[35]VAN GINNEKEN M M,DE GRAAF-ROELFSEMA E,KEIZER H A,et al.Effect of exercise on activation of the p38 mitogen-activated protein kinase pathway,c-Jun NH2 terminal kinase,and heat shock protein 27 in equine skeletal muscle[J].Am J Vet Res,2006,67(5):837-844.
[36]WAGNER E F,NEBREDA A R.Signal integration by JNK and p38 MAPK pathways in cancer development[J].Nat Rev Cancer,2009,9(8):537-549.
[37]WIDEGREN U,RYDER J W,ZIERATH J R.Mitogen-activated protein kinase signal transduction in skeletal muscle:Effects of exercise and muscle contraction[J].Acta Physiol Scand,2001,172(3):227-238.
[38]WILLIAMSON D,GALLAGHER P,HARBER M,et al.Mitogen-activated protein kinase(MAPK)pathway activation:Effects of age and acute exercise on human skeletal muscle[J].J Physiol,2003,547(Pt 3):977-987.
[39]XU Y M,LI J P,WANG R Y.Changes of dystrophin and desmin in rat gastrocnemius under micro-damage induced by hypoxia[J].Acta Physiol Sin,2010,62(4):339-348.
[40]YANAGISAWA O,SAKUMA J,KAWAKAMI Y,et al.Effect of exercise-induced muscle damage on muscle hardness evaluated by ultrasound real-time tissue elastography[J].Springerplus,2015,4:308.
[41]ZBINDEN-FONCEA H,RAYMACKERS J M,DELDICQUE L,et al.TLR2 and TLR4 activate p38 MAPK and JNK during endurance exercise in skeletal muscle[J].Med Sci Sports Exerc,2012,44(8):1463-1472.
(1)此通路在文献中有BMK1/ERK5、BMK1和ERK5 3种表述方式,为简练表达并与ERK1/2通路区别,本文采用BMK1表述。但在引用文献内容时,尊重并保留原文献中BMK1/ERK5或ERK5的表述。
(1)EBD注射时间点为运动后即刻。