电针干预对失神经肌萎缩大鼠肌卫星细胞分化及肌纤维类型转化的影响
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摘要
目的:观察电针干预对失神经肌萎缩大鼠腓肠肌中配对盒转录因子7(Pax7)、成肌分化抗原(Myod1)、肌细胞生成素(Myog)、肌球蛋白重链-Ⅱa(Myh2)、肌球蛋白重链-Ⅱx(Myh1)及肌球蛋白重链-Ⅰ(Myh7)表达的影响,探讨电针延缓失神经肌萎缩发展的可能机制。方法:将66只雄性SD大鼠随机分为假手术组24只、模型组24只和电针组18只。采用慢性坐骨神经压迫损伤制备大鼠右后肢失神经肌萎缩模型。造模1周后电针组取大鼠术侧"足三里""环跳"穴予电针治疗,每次10min,每日1次,每周6次,分别连续干预1、2、4周。称重计算各组大鼠的腓肠肌湿重比,HE染色测定腓肠肌纤维截面积及直径;实时荧光定量PCR检测造模第3周各组大鼠腓肠肌中Pax7、Myod1、Myog、Myh2、Myh1和Myh7mRNA的相对表达量。结果:与假手术组相比,造模1周后各时间点模型组大鼠腓肠肌湿重比显著降低(P<0.05)。模型组和电针组腓肠肌湿重比差异无统计学意义(P>0.05)。与假手术组相比,各时间点模型组大鼠术侧腓肠肌纤维截面积及直径显著减小(P<0.05);与模型组比较,各时间点电针组大鼠术侧腓肠肌纤维截面积及直径显著升高(P<0.05)。造模3周时,与假手术组相比,模型组大鼠术侧腓肠肌中Myod1和Myog mRNA表达量明显升高(P<0.01),而Myh2、Myh1和Myh7mRNA表达量明显降低(P<0.05,P<0.01);与模型组比较,电针干预则能有效上调Myod1、Myog和Myh7mRNA表达量(P<0.05,P<0.01);3组间Pax7mRNA表达量差异无统计学意义(P>0.05)。结论:电针干预大鼠"足三里""环跳"穴可能通过调控Myod1、Myog、Myh7mRNA的表达,影响肌卫星细胞的成肌分化水平及肌纤维类型的转换,延缓腓肠肌失神经肌萎缩的发展。
Objective To explore the effect of electroacupuncture(EA)on amyotrophia and expression of paired box7(Pax7),myogenic differentiation antigen-1(Myod1),myogenin(Myog),myosin heavy chain-Ⅱa(Myh2),myosin heavy chain-Ⅱx(Myh1)and myosin heavy chain-Ⅰ(Myh7)of denervated gastrocnemius in rats with chronic constriction injury(CCI)of the right sciatic nerve,so as to explore its mechanisms underlying postponing development of amyotrophia.Methods Sixty-six SD adult male rats were randomly divided into sham operation(sham)group(n=24),model group(n=24)and EA group(n=18).The denervated muscle(gastrocnemius)atrophy model was established by CCI of the right sciatic nerve.EA(2 Hz,1.0 mA)was applied to the right"Zusanli"(ST36)and"Huantiao"(GB30)for 10 min,once a day,six times a week and for 1,2 and 4 weeks,respectively.After complete dissection of bilateral gastrocnemius muscles,their wet weight levels were measured and the ratio of wet weight(=that of the operation side/that of the non-operation side)was calculated,and the cross-sectional area(CSA)and diameter of the gastronemius were detected after fixation in 4%paraformaldehyde,sectioning,and H.E.staining.The expression levels of Pax7,Myod1,Myog,Myh2,Myh1 and Myh7 mRNAs in the gastrocnemius tissue after 3 weeks of modeling were detected with quantitative real time-PCR(qPCR).Results After 1 week of modeling,the ratios of wet weight of gastrocnemius and the CSA and fiber diameter at the 2 nd,3 rd and 5 th week were significantly smaller in the model group than in the sham group(P<0.05).The expression levels of Myod1 and Myog mRNAs were significantly up-regulated(P<0.01),and those of Myh2,Myh1 and Myh7 considerably down-regulated in the model group compared with the sham group(P<0.05,P<0.01).No significant changes were found in the expression levels of Pax7 mRNA after modeling and EA intervention(P>0.05).Following EA intervention,the CSA and diameterof the gastronemius were obviously increased(P<0.05),and the expression levels of Myod1,Myog and Myh7 further markedly or remarkably up-regulated in comparison with the model group(P<0.05,P<0.01).No significant changes were found in the ratio of wet weight of gastrocnemius at the 3 time-points,and the expression levels of Myh2 mRNA and Myh1 mRNA in the EA group relevant to the model group after 3 weeks of modeling(P>0.05).Conclusion EA treatment may delay gastrocnemius atrophy in CCI rats,which is possibly associated with its effects in up-regulating the expression of Myod1,Myog and Myh7 mRNAs to control the differentiation of the satellite cells and the muscle fiber type transformation.
Objective To explore the effect of electroacupuncture(EA)on amyotrophia and expression of paired box7(Pax7),myogenic differentiation antigen-1(Myod1),myogenin(Myog),myosin heavy chain-Ⅱa(Myh2),myosin heavy chain-Ⅱx(Myh1)and myosin heavy chain-Ⅰ(Myh7)of denervated gastrocnemius in rats with chronic constriction injury(CCI)of the right sciatic nerve,so as to explore its mechanisms underlying postponing development of amyotrophia.Methods Sixty-six SD adult male rats were randomly divided into sham operation(sham)group(n=24),model group(n=24)and EA group(n=18).The denervated muscle(gastrocnemius)atrophy model was established by CCI of the right sciatic nerve.EA(2 Hz,1.0 mA)was applied to the right"Zusanli"(ST36)and"Huantiao"(GB30)for 10 min,once a day,six times a week and for 1,2 and 4 weeks,respectively.After complete dissection of bilateral gastrocnemius muscles,their wet weight levels were measured and the ratio of wet weight(=that of the operation side/that of the non-operation side)was calculated,and the cross-sectional area(CSA)and diameter of the gastronemius were detected after fixation in 4%paraformaldehyde,sectioning,and H.E.staining.The expression levels of Pax7,Myod1,Myog,Myh2,Myh1 and Myh7 mRNAs in the gastrocnemius tissue after 3 weeks of modeling were detected with quantitative real time-PCR(qPCR).Results After 1 week of modeling,the ratios of wet weight of gastrocnemius and the CSA and fiber diameter at the 2 nd,3 rd and 5 th week were significantly smaller in the model group than in the sham group(P<0.05).The expression levels of Myod1 and Myog mRNAs were significantly up-regulated(P<0.01),and those of Myh2,Myh1 and Myh7 considerably down-regulated in the model group compared with the sham group(P<0.05,P<0.01).No significant changes were found in the expression levels of Pax7 mRNA after modeling and EA intervention(P>0.05).Following EA intervention,the CSA and diameterof the gastronemius were obviously increased(P<0.05),and the expression levels of Myod1,Myog and Myh7 further markedly or remarkably up-regulated in comparison with the model group(P<0.05,P<0.01).No significant changes were found in the ratio of wet weight of gastrocnemius at the 3 time-points,and the expression levels of Myh2 mRNA and Myh1 mRNA in the EA group relevant to the model group after 3 weeks of modeling(P>0.05).Conclusion EA treatment may delay gastrocnemius atrophy in CCI rats,which is possibly associated with its effects in up-regulating the expression of Myod1,Myog and Myh7 mRNAs to control the differentiation of the satellite cells and the muscle fiber type transformation.
引文
[1]侯志儒,梁炳生,梁勇,等.经皮电刺激大鼠骨骼肌失神经肌萎缩时成肌调节因子基因的表达[J].中国组织工程研究与临床康复,2009,13(33):6496-6499.
[2]庄伊洢,陈玄,叶笑然,等.宽波电脉冲电针足三里防治下肢肌萎缩研究[J].上海针灸杂志,2016,35(6):742-744.
[3]高睿琦,唐成林,曹净,等.电针对失神经骨骼肌萎缩大鼠胰岛素样生长因子1、肌肉生长抑制素及肌卫星细胞增殖的影响[J].中国康复理论与实践,2016,22(11):1259-1263.
[4]高睿琦,唐成林,黄思琴,等.电针对失坐骨神经大鼠腓肠肌细胞凋亡及相关蛋白的影响[J].针刺研究,2017,42(4):302-307.
[5]车光昇,郭源秩,宋光熠.神经源性肌萎缩病理动物模型的制备[J].辽宁医学杂志,2014,28(5):301.
[6]张毅,唐成林,田源,等.电针联合饮食调控对非酒精性脂肪性肝病大鼠过氧化物酶体增殖物激活受体α和肝型脂肪酸结合蛋白的影响[J].针刺研究,2015,40(5):345-351.
[7]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:316.
[8]MORESI V,WILLIAMS A H,MEADOWS E,et al.Myogenin and classⅡHDACs control neurogenic muscle atrophy by inducing E3ubiquitin ligases[J].Cell,2010,143(1):35-45.
[9]NAKAGAWA K,TAMAKI H,HAYAO H,et al.Electrical stimulation of denervated rat skeletal muscle retards capillary and muscle loss in early stages of disuse atrophy[J].Biomed Res Int,2017,2017:5695217.
[10]银欢,赵建文.建立大鼠失神经支配骨骼肌萎缩模型适宜方法的研究[J].沈阳医学院学报,2008,13(2):81-82.
[11]秦秋.独取阳明治疗痿症100例临床观察[J].中医临床研究,2011,3(9):49.
[12]王昕,赵明亮,吉长福,等.针刺“足三里”穴对脾虚证大鼠血清中睾酮和雌二醇水平的影响[J].针刺研究,2011,36(4):268-271.
[13]勇入琳,曲怡,李欣欣,等.电针“足三里”对脾气虚大鼠空肠组织胃生长激素释放激素/环磷酸腺苷/蛋白激酶A表达的影响[J].针刺研究,2016,41(6):497-501.
[14]刘玉丽,李野,任路,等.深刺“环跳”穴对大鼠损伤坐骨神经的修复作用[J].针刺研究,2014,39(2):93-99.
[15]MAURO A.Satellite cell of skeletal muscle fibers[J].J Cell Biol,1961,9(2):493-495.
[16]PALLAFACCHINA G,BLAAUW B,SCHIAFFINO S.Role of satellite cells in muscle growth and maintenance of muscle mass[J].Nutr Metab Cardiovasc Dis,2012,23:S12-S18.
[17]YIN H,PRICE F,RUDNICKI M A.Satellite cells and the muscle stem cell niche[J].Physiol Rev,2013,93(1):23-67.
[18]SCHIAFFINO S,DYAR K A,CICILIOT S,et al.Mechanisms regulating skeletal muscle growth and atrophy[J].FEBS J,2013,280(17):4294-4314.
[19]KUANG S,RUDNICKI M A.The emerging biology of satellite cells and their therapeutic potential[J].Trends Mol Med,2008,14(2):82-91.
[20]MOTOHASHI N,ASAKURA A.Molecular regulation of muscle satellite cell self-renewal[J].J Stem Cell Res Ther,2012,1(Suppl 11):e002.
[21]TIAN Z L,JIANG S K,ZHANG M,et al.Detection of satellite cells during skeletal muscle wound healing in rats:time-dependent expressions of Pax7and Myod in relation to wound age[J].Int J Legal Med,2016,130(1):163-172.
[22]ZANOU N,GAILLY P.Skeletal muscle hypertrophy and regeneration:interplay between the myogenic regulatory factors(MRFs)and insulin-like growth factors(IGFs)pathways[J].Cell Mol Life Sci,2013,70(21):4117-4130.
[23]SU Z,HU L,CHENG J,et al.Acupuncture plus low-frequency electrical stimulation(Acu-LFES)attenuates denervation-induced muscle atrophy[J].J Appl Physiol,2016,120(4):426-436.
[24]廖华,王齐,徐达传,等.失神经萎缩肌组织中卫星细胞池的耗竭与Pax7的相关性研究[J].中国修复重建外科杂志,2009,23(1):92-96.
[25]郭秋平,文超越,王文龙,等.肌纤维类型转化的分子信号通路及其营养调控进展[J].动物营养学报,2017,29(6):1836-1842.
[26]SCHIAFFINO S,REGGIANI C.Fiber types in mammalian skeletal muscles[J].Physiol Rev,2011,91(4):1447-1531.
[27]LI J,CHAN M C,YU Y,et al.MiR-29bcontributes to multiple types of muscle atrophy[J].Nat Commun,2017(8):15201.
[28]VERDIJK L B,KOOPMAN R,SCHAART G,et al.Satellite cell content is specifically reduced in typeⅡskeletal muscle fibers in the elderly[J].Am J Physiol Endocrinol Metab,2007,292(1):E151-E157.
[2]庄伊洢,陈玄,叶笑然,等.宽波电脉冲电针足三里防治下肢肌萎缩研究[J].上海针灸杂志,2016,35(6):742-744.
[3]高睿琦,唐成林,曹净,等.电针对失神经骨骼肌萎缩大鼠胰岛素样生长因子1、肌肉生长抑制素及肌卫星细胞增殖的影响[J].中国康复理论与实践,2016,22(11):1259-1263.
[4]高睿琦,唐成林,黄思琴,等.电针对失坐骨神经大鼠腓肠肌细胞凋亡及相关蛋白的影响[J].针刺研究,2017,42(4):302-307.
[5]车光昇,郭源秩,宋光熠.神经源性肌萎缩病理动物模型的制备[J].辽宁医学杂志,2014,28(5):301.
[6]张毅,唐成林,田源,等.电针联合饮食调控对非酒精性脂肪性肝病大鼠过氧化物酶体增殖物激活受体α和肝型脂肪酸结合蛋白的影响[J].针刺研究,2015,40(5):345-351.
[7]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:316.
[8]MORESI V,WILLIAMS A H,MEADOWS E,et al.Myogenin and classⅡHDACs control neurogenic muscle atrophy by inducing E3ubiquitin ligases[J].Cell,2010,143(1):35-45.
[9]NAKAGAWA K,TAMAKI H,HAYAO H,et al.Electrical stimulation of denervated rat skeletal muscle retards capillary and muscle loss in early stages of disuse atrophy[J].Biomed Res Int,2017,2017:5695217.
[10]银欢,赵建文.建立大鼠失神经支配骨骼肌萎缩模型适宜方法的研究[J].沈阳医学院学报,2008,13(2):81-82.
[11]秦秋.独取阳明治疗痿症100例临床观察[J].中医临床研究,2011,3(9):49.
[12]王昕,赵明亮,吉长福,等.针刺“足三里”穴对脾虚证大鼠血清中睾酮和雌二醇水平的影响[J].针刺研究,2011,36(4):268-271.
[13]勇入琳,曲怡,李欣欣,等.电针“足三里”对脾气虚大鼠空肠组织胃生长激素释放激素/环磷酸腺苷/蛋白激酶A表达的影响[J].针刺研究,2016,41(6):497-501.
[14]刘玉丽,李野,任路,等.深刺“环跳”穴对大鼠损伤坐骨神经的修复作用[J].针刺研究,2014,39(2):93-99.
[15]MAURO A.Satellite cell of skeletal muscle fibers[J].J Cell Biol,1961,9(2):493-495.
[16]PALLAFACCHINA G,BLAAUW B,SCHIAFFINO S.Role of satellite cells in muscle growth and maintenance of muscle mass[J].Nutr Metab Cardiovasc Dis,2012,23:S12-S18.
[17]YIN H,PRICE F,RUDNICKI M A.Satellite cells and the muscle stem cell niche[J].Physiol Rev,2013,93(1):23-67.
[18]SCHIAFFINO S,DYAR K A,CICILIOT S,et al.Mechanisms regulating skeletal muscle growth and atrophy[J].FEBS J,2013,280(17):4294-4314.
[19]KUANG S,RUDNICKI M A.The emerging biology of satellite cells and their therapeutic potential[J].Trends Mol Med,2008,14(2):82-91.
[20]MOTOHASHI N,ASAKURA A.Molecular regulation of muscle satellite cell self-renewal[J].J Stem Cell Res Ther,2012,1(Suppl 11):e002.
[21]TIAN Z L,JIANG S K,ZHANG M,et al.Detection of satellite cells during skeletal muscle wound healing in rats:time-dependent expressions of Pax7and Myod in relation to wound age[J].Int J Legal Med,2016,130(1):163-172.
[22]ZANOU N,GAILLY P.Skeletal muscle hypertrophy and regeneration:interplay between the myogenic regulatory factors(MRFs)and insulin-like growth factors(IGFs)pathways[J].Cell Mol Life Sci,2013,70(21):4117-4130.
[23]SU Z,HU L,CHENG J,et al.Acupuncture plus low-frequency electrical stimulation(Acu-LFES)attenuates denervation-induced muscle atrophy[J].J Appl Physiol,2016,120(4):426-436.
[24]廖华,王齐,徐达传,等.失神经萎缩肌组织中卫星细胞池的耗竭与Pax7的相关性研究[J].中国修复重建外科杂志,2009,23(1):92-96.
[25]郭秋平,文超越,王文龙,等.肌纤维类型转化的分子信号通路及其营养调控进展[J].动物营养学报,2017,29(6):1836-1842.
[26]SCHIAFFINO S,REGGIANI C.Fiber types in mammalian skeletal muscles[J].Physiol Rev,2011,91(4):1447-1531.
[27]LI J,CHAN M C,YU Y,et al.MiR-29bcontributes to multiple types of muscle atrophy[J].Nat Commun,2017(8):15201.
[28]VERDIJK L B,KOOPMAN R,SCHAART G,et al.Satellite cell content is specifically reduced in typeⅡskeletal muscle fibers in the elderly[J].Am J Physiol Endocrinol Metab,2007,292(1):E151-E157.