低氧调控离心运动后大鼠骨骼肌Dystrophin的表达
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摘要
目的观察急性离心运动后低氧暴露对大鼠腓肠肌细胞膜通透性及膜骨架蛋白dystrophin的影响,探讨骨骼肌细胞膜损伤的作用机制,为高住低训提供理论依据。方法 70只雄性SD大鼠分为安静对照组、运动后常氧恢复24 h、48 h、72 h组和运动后低氧暴露24 h、48 h、72 h组。运动后低氧暴露组在离心运动后于常压低氧环境中进行恢复。采用免疫组化、Western blot、qRT-PCR等方法检测大鼠腓肠肌细胞膜完整性和dystrophin蛋白及基因表达。结果 (1)离心运动后低氧暴露,大鼠腓肠肌阳性细胞率在各时间点与常氧恢复组比较,呈现显著性上升、下降再上升的趋势;(2)急性离心运动后,dystrophin蛋白含量在各组中未出现显著性变化;(3)常氧恢复组和低氧暴露组dystrophin mRNA表达水平均显著性低于安静对照组。结论 (1)离心运动后急性低氧暴露可加剧骨骼肌细胞膜的损伤,随着低氧暴露时间的延长,机体虽然产生了短暂适应,但其恢复速率仍较常氧下恢复低;(2)膜骨架蛋白dystrophin在运动后无论是常氧恢复组还是低氧暴露组,并未发生显著性变化,且其基因表达与蛋白变化在时间上存在一定滞后,提示除mRNA转录调节之外,可能存在一种转录外调节机制;(3)"高住低训"的训练方案并不利于运动后骨骼肌的快速恢复。
Objective The purpose of this study was firstly to observe the effects of hypoxia on rat gastrocnemius membrane permeabilityand Dystrophin after acute eccentric exercise. The results would explore the mechanism of the injury of skeletal muscle cells and provide an important reference for the HiLo.Methods 70 male Sprague-Dawley rats were randomly divided into 7 groups including a normoxic control group(C),a post-exercise 24 h、48 h、72 h normoxic recovery groups,and post-exercise 24 h、48 h、72 h hypoxic exposed groups.All hypoxic groupsrecovered in Nnormobaric Hypoxiaaftereccentric exercise. Both studies were used immunohistochemistry、Western blot and qRT-PCR to measure themembrane permeability、Dystrophin level and Dystrophin mRNA in each group. Results(1)The ratio of EBD-positive cell were significantly higher, lowerand higher in hypoxic exposed groupscomparing with normoxic recovery groupsat 24 h, 48 h and 72 h respectively.(2)The expression of dystrophin protein was not statistically different after acute eccentric exercise in all groups.(3)Compared withnormoxic control group,the expression of dystrophin mRNA was significantly decreased in normoxic recovery groupsand hypoxia exposed groups. Conclusions(1)After eccentric exercise, the acute hypoxic exposure can aggravate the damage of sarcolemma. With the extension of hypoxic exposure time, the body adapted to the situation for a short time butits recovery rate was still lower than normoxic recovery.(2)There was no significant change in dystrophin either in normoxic recovery groups or hypoxic exposed groups after exercise. And there is a time lag between dystrophin mRNA and protein expression, suggesting that there may be an extra transcriptional regulation mechanism except for mRNA.(3)HiLo is not conducive to the recovery rate of skeletal muscle after exercise.
Objective The purpose of this study was firstly to observe the effects of hypoxia on rat gastrocnemius membrane permeabilityand Dystrophin after acute eccentric exercise. The results would explore the mechanism of the injury of skeletal muscle cells and provide an important reference for the HiLo.Methods 70 male Sprague-Dawley rats were randomly divided into 7 groups including a normoxic control group(C),a post-exercise 24 h、48 h、72 h normoxic recovery groups,and post-exercise 24 h、48 h、72 h hypoxic exposed groups.All hypoxic groupsrecovered in Nnormobaric Hypoxiaaftereccentric exercise. Both studies were used immunohistochemistry、Western blot and qRT-PCR to measure themembrane permeability、Dystrophin level and Dystrophin mRNA in each group. Results(1)The ratio of EBD-positive cell were significantly higher, lowerand higher in hypoxic exposed groupscomparing with normoxic recovery groupsat 24 h, 48 h and 72 h respectively.(2)The expression of dystrophin protein was not statistically different after acute eccentric exercise in all groups.(3)Compared withnormoxic control group,the expression of dystrophin mRNA was significantly decreased in normoxic recovery groupsand hypoxia exposed groups. Conclusions(1)After eccentric exercise, the acute hypoxic exposure can aggravate the damage of sarcolemma. With the extension of hypoxic exposure time, the body adapted to the situation for a short time butits recovery rate was still lower than normoxic recovery.(2)There was no significant change in dystrophin either in normoxic recovery groups or hypoxic exposed groups after exercise. And there is a time lag between dystrophin mRNA and protein expression, suggesting that there may be an extra transcriptional regulation mechanism except for mRNA.(3)HiLo is not conducive to the recovery rate of skeletal muscle after exercise.
引文
[1] FORNASIER-SANTOS C, MILLET G P, WOORONS X. Repeated-sprint training in hypoxia induced by voluntary hypoventilation improves running repeated-sprint ability in rugby players[J]. Eur J Sport Sci, 2018: 1-9.
[2] KASAI N, KOJIMA C, SUMI D, et al. Impact of 5 days of sprint training in hypoxia on performance and muscle energy substances[J]. Int J Sports Med, 2017, 38(13): 983-991.
[3] 李洁, 王世超. 高住高练低训不同时程大鼠骨骼肌线粒体呼吸链功能的变化[J]. 中国运动医学杂志, 2016, 35(1): 32-35.
[4] BO H, LI L, DUAN F Q, et al.Exercise training in hypoxia prevents hypoxia induced mitochondrial DNA oxidative damage in skeletal muscle[J]. Acta Physiologica Sinica, 2014, 66(5): 597-604.
[5] 徐玉明,李俊平,王瑞元. 低氧训练诱导大鼠腓肠肌抗肌营养不良蛋白和代谢酶变化及延长高速力竭运动时间[J]. 生理学报, 2012, 64(1): 455–462.
[6] XU Y M, CAO J M, LI J P, et al. Analyses of exercise-induced muscle damage-specific microRNA expression and molecular target of sarcolemmal damage in rats[J]. Sheng Li Xue Bao, 2017, 69(3): 276-284.
[7] GAWOR M, PROSZYNSKI T J. The molecular cross talk of the dystrophin-glycoprotein complex[J]. Ann N Y Acad Sci, 2018, 1412(1): 62-72.
[8] 徐玉明, 曹建民, 李俊平, 等. 大鼠运动性肌损伤特征micro RNA表达及其膜损伤调控靶点的预测[J]. 生理学报, 2017, 69(3): 276-284.
[9] 徐玉明. 低氧、低氧运动对骨骼肌Dystrophin、Desmin的影响[D]. 北京: 北京体育大学, 2007.
[10] HAMER P W, MCGEACHIE J M, DAVIESM 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(1): 69-79.
[11] 徐玉明,李俊平,王瑞元. 低氧性微损伤时大鼠腓肠肌抗肌营养不良蛋白和结蛋白的变化 [J]. 生理学报, 2010, 62(4): 339-348.
[12] WEBSTER C, SILBERSTEIN L, HAYSA P, et al. Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy[J]. Cell, 1988, 52(4): 503-513.
[13] WANG R, URSO M L, ZAMBRASKIE J, et al. Adenosine A(3) receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury[J]. Am J Physiol Regul Integr Comp Physiol, 2010, 299(1): 259-267.
[14] 邓文骞, 王璐, 王玉. 低氧预处理对骨骼肌抗氧化能力及运动性肌肉损伤的影响[J]. 现代预防医学, 2014, 41(18): 3369-3372.
[15] MURPHY R M, VERBURG E, LAMB G D. Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle[J]. J Physiol, 2006, 576(Pt 2): 595-612.
[16] 赵蕾, 胡超平, 王艺. Duchenne型肌营养不良症患儿肌膜抗肌萎缩蛋白-糖蛋白复合物表达研究[J]. 中国现代神经疾病杂志, 2015, 15(6): 448-452.
[17] DE LUCA A, NICO B, ROLLAND J F, et al. Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle[J]. Neurobiol Dis, 2008, 32(2): 243-253.
[18] INGBER D E. Tensegrity: the architectural basis of cellular mechanotransduction[J]. Annu Rev Physiol, 1997, 59(3): 575-599.
[19] NIELSEN S, SCHEELE C, YFANTIC, et al. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle[J]. J Physiol, 2010, 588(Pt 20): 4029-4037.
[20] WANG L, ZHOU L, JIANG P, et al. Loss of miR-29 in myoblasts contributes to dystrophic muscle pathogenesis[J]. Mol Ther, 2012, 20(6): 1222-1233.
[2] KASAI N, KOJIMA C, SUMI D, et al. Impact of 5 days of sprint training in hypoxia on performance and muscle energy substances[J]. Int J Sports Med, 2017, 38(13): 983-991.
[3] 李洁, 王世超. 高住高练低训不同时程大鼠骨骼肌线粒体呼吸链功能的变化[J]. 中国运动医学杂志, 2016, 35(1): 32-35.
[4] BO H, LI L, DUAN F Q, et al.Exercise training in hypoxia prevents hypoxia induced mitochondrial DNA oxidative damage in skeletal muscle[J]. Acta Physiologica Sinica, 2014, 66(5): 597-604.
[5] 徐玉明,李俊平,王瑞元. 低氧训练诱导大鼠腓肠肌抗肌营养不良蛋白和代谢酶变化及延长高速力竭运动时间[J]. 生理学报, 2012, 64(1): 455–462.
[6] XU Y M, CAO J M, LI J P, et al. Analyses of exercise-induced muscle damage-specific microRNA expression and molecular target of sarcolemmal damage in rats[J]. Sheng Li Xue Bao, 2017, 69(3): 276-284.
[7] GAWOR M, PROSZYNSKI T J. The molecular cross talk of the dystrophin-glycoprotein complex[J]. Ann N Y Acad Sci, 2018, 1412(1): 62-72.
[8] 徐玉明, 曹建民, 李俊平, 等. 大鼠运动性肌损伤特征micro RNA表达及其膜损伤调控靶点的预测[J]. 生理学报, 2017, 69(3): 276-284.
[9] 徐玉明. 低氧、低氧运动对骨骼肌Dystrophin、Desmin的影响[D]. 北京: 北京体育大学, 2007.
[10] HAMER P W, MCGEACHIE J M, DAVIESM 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(1): 69-79.
[11] 徐玉明,李俊平,王瑞元. 低氧性微损伤时大鼠腓肠肌抗肌营养不良蛋白和结蛋白的变化 [J]. 生理学报, 2010, 62(4): 339-348.
[12] WEBSTER C, SILBERSTEIN L, HAYSA P, et al. Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy[J]. Cell, 1988, 52(4): 503-513.
[13] WANG R, URSO M L, ZAMBRASKIE J, et al. Adenosine A(3) receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury[J]. Am J Physiol Regul Integr Comp Physiol, 2010, 299(1): 259-267.
[14] 邓文骞, 王璐, 王玉. 低氧预处理对骨骼肌抗氧化能力及运动性肌肉损伤的影响[J]. 现代预防医学, 2014, 41(18): 3369-3372.
[15] MURPHY R M, VERBURG E, LAMB G D. Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle[J]. J Physiol, 2006, 576(Pt 2): 595-612.
[16] 赵蕾, 胡超平, 王艺. Duchenne型肌营养不良症患儿肌膜抗肌萎缩蛋白-糖蛋白复合物表达研究[J]. 中国现代神经疾病杂志, 2015, 15(6): 448-452.
[17] DE LUCA A, NICO B, ROLLAND J F, et al. Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle[J]. Neurobiol Dis, 2008, 32(2): 243-253.
[18] INGBER D E. Tensegrity: the architectural basis of cellular mechanotransduction[J]. Annu Rev Physiol, 1997, 59(3): 575-599.
[19] NIELSEN S, SCHEELE C, YFANTIC, et al. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle[J]. J Physiol, 2010, 588(Pt 20): 4029-4037.
[20] WANG L, ZHOU L, JIANG P, et al. Loss of miR-29 in myoblasts contributes to dystrophic muscle pathogenesis[J]. Mol Ther, 2012, 20(6): 1222-1233.
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