巨刺“足三里”和“阿是穴”对急性骨骼肌钝挫伤大鼠的修复作用
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摘要
目的:观察巨刺"足三里"和"阿是穴"对急性骨骼肌钝挫伤大鼠的修复作用,探讨巨刺治疗骨骼肌损伤的机制。方法:雄性SD大鼠分为正常组6只、模型组24只、巨刺组24只,模型组与巨刺组再随机分为3、5、7、14d4个亚组,每个亚组6只。采用自制打击装置建立骨骼肌钝挫伤大鼠模型。巨刺组选取健侧"足三里"以及与患侧"阿是穴"相对应的健侧部位进行电针治疗,15min/次,1次/d,分别治疗3、5、7、14d。HE染色观察损伤后7、14d各组大鼠腓肠肌的形态结构及新生肌细胞横截面积;免疫荧光检测损伤后7d各组腓肠肌结蛋白(desmin)阳性表达;免疫印迹法检测损伤后3、5d腓肠肌肌细胞生成素(myoG)和7、14d腓肠肌快肌型骨骼肌肌球蛋白重链(Fast MyHC)的相对表达量。结果:正常组大鼠腓肠肌横截面的肌细胞大小均匀,排列规则,核位于细胞边缘。损伤后7d,模型组新生肌细胞较少、排列紊乱、大小不一,间质中可见胶原纤维;巨刺组新生肌细胞增多、排列较整齐、大小均一,新生肌细胞面积显著大于模型组(P<0.05)。损伤后14d,模型组仍可见排列紊乱且大小不一的新生肌细胞,间质中仍可见胶原纤维;巨刺组可见大量排列整齐且大小均一的趋于成熟的新生肌细胞,新生肌细胞面积显著大于模型组(P<0.001)。免疫荧光结果显示:在正常组中,desmin表达于肌细胞膜上。损伤后7d,模型组desmin多数表达于新生肌细胞胞质内,阳性表达的细胞小且排列紊乱、大小不一,模型组desmin表达显著高于正常组(P<0.001);巨刺组desmin多数表达于新生肌细胞膜上,接近正常组肌细胞状态,且新生肌细胞大小均一、排列整齐,巨刺组desmin表达量与模型组比较,差异无统计学意义(P>0.05)。免疫印迹结果显示:损伤后3、5d,与正常组比较,模型组腓肠肌myoG的表达量明显升高(P<0.001);与模型组比较,巨刺组腓肠肌myoG表达量明显升高(P<0.001)。损伤后7、14d,与正常组比较,模型组腓肠肌Fast MyHC的表达量明显降低(P<0.001);与模型组比较,巨刺组腓肠肌Fast MyHC的表达量明显升高(P<0.01)。结论:巨刺"足三里"和"阿是穴"可能通过正向调控急性骨骼肌钝挫伤大鼠腓肠肌myoG和Fast MyHC的表达,促进损伤骨骼肌的修复。
Objective To observe the therapeutic effect of electroacupuncture(EA)of"Zusanli"(ST36)and"Ashi"-point on the healthy side(opposing needling)on muscular injury and expression of myogenin(myoG)and fast myosin skeletal heavy chain(Fast MyHC)proteins in the gastrocnemius muscle(GM)tissues in skeletal muscle contusion rats,so as to explore its mechanism underlying improvement of skeletal muscle injury.Methods A total of 54 male SD rats were divided into normal control(n =6),model(n=24)and opposing needling(EA,n=24)groups.The latter two groups were further randomized into3,5,7 and 14 dsubgroups(n=6 per subgroup).The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device.EA(1 Hz/3 Hz,1-2 mA)was applied to ST36 and"Ashi"-point on the uninjured side of the hind-limb for 15 min every time,once a day for 3,5,7 and 14 days,respectively.The injured GM was harvested on the 3 rd,5 th,7 th and14 th day after muscular contusion.The morphological changes of the injured GM and the mean cross-sectional areas(CSAs)of the neonatal muscle cells were observed by microscope after H.E.staining.The immunoactivity of desmin protein(myogenic marker protein of myoblast cell)of GM was detected by immunofluorescence stain on the 7 th day after injury,and the expression levels of myoG(on the 3 rd and 5 th day after injury)and fast MyHC protein of GM tissues(on the 7 thand 14 th day after injury)were detected by Western blot.Results H.E.staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes,and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion,which was relatively milder in the EA group.In the EA group,the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7 th(P<0.05),14 th(P<0.001)after injury.On day 7 after muscular contusion,the desmin was found to express on the cellular membrane of GM in the normal control group,while in the model group,the desmin expressed mainly in the cellular plasma in the model group,and on the cellular membrane of neonatal myocytes in the EA group,respectively.The desmin positive myocytes showed disordered arrangement and different sizes after muscular contusion,whereas the situations of the EA group were close to those of the normal control group.Desmin expression was up-regulated in the EA group compared with the model group which was not significant difference(P>0.05).On the 3 rd and 5 th day after muscular contusion,the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group(P<0.001),and significantly up-regulated in the EA group than that in the model group(P<0.001).On the 7 th and14 th day after contusion,the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group(P<0.001),and markedly up-regulated in the EA group relevant to the model group(P<0.01).Conclusion EA of ST36 and"Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats,which may contribute to its effect in promoting the repair of skeletal muscle injury.
Objective To observe the therapeutic effect of electroacupuncture(EA)of"Zusanli"(ST36)and"Ashi"-point on the healthy side(opposing needling)on muscular injury and expression of myogenin(myoG)and fast myosin skeletal heavy chain(Fast MyHC)proteins in the gastrocnemius muscle(GM)tissues in skeletal muscle contusion rats,so as to explore its mechanism underlying improvement of skeletal muscle injury.Methods A total of 54 male SD rats were divided into normal control(n =6),model(n=24)and opposing needling(EA,n=24)groups.The latter two groups were further randomized into3,5,7 and 14 dsubgroups(n=6 per subgroup).The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device.EA(1 Hz/3 Hz,1-2 mA)was applied to ST36 and"Ashi"-point on the uninjured side of the hind-limb for 15 min every time,once a day for 3,5,7 and 14 days,respectively.The injured GM was harvested on the 3 rd,5 th,7 th and14 th day after muscular contusion.The morphological changes of the injured GM and the mean cross-sectional areas(CSAs)of the neonatal muscle cells were observed by microscope after H.E.staining.The immunoactivity of desmin protein(myogenic marker protein of myoblast cell)of GM was detected by immunofluorescence stain on the 7 th day after injury,and the expression levels of myoG(on the 3 rd and 5 th day after injury)and fast MyHC protein of GM tissues(on the 7 thand 14 th day after injury)were detected by Western blot.Results H.E.staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes,and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion,which was relatively milder in the EA group.In the EA group,the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7 th(P<0.05),14 th(P<0.001)after injury.On day 7 after muscular contusion,the desmin was found to express on the cellular membrane of GM in the normal control group,while in the model group,the desmin expressed mainly in the cellular plasma in the model group,and on the cellular membrane of neonatal myocytes in the EA group,respectively.The desmin positive myocytes showed disordered arrangement and different sizes after muscular contusion,whereas the situations of the EA group were close to those of the normal control group.Desmin expression was up-regulated in the EA group compared with the model group which was not significant difference(P>0.05).On the 3 rd and 5 th day after muscular contusion,the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group(P<0.001),and significantly up-regulated in the EA group than that in the model group(P<0.001).On the 7 th and14 th day after contusion,the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group(P<0.001),and markedly up-regulated in the EA group relevant to the model group(P<0.01).Conclusion EA of ST36 and"Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats,which may contribute to its effect in promoting the repair of skeletal muscle injury.
引文
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[15]匡培根,吴爱华,张凤英.巨刺对穴位灌流液中乙酰胆硷含量的影响[J].针刺研究,1980,5(2):147-149.
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[18] FISCHMAN D A.The synthesis and assembly of myofibrils in embryonic muscle[J].Curr Topics Development Biol,1990(5):235-280.
[19] HORIE M,ENOMOTO M,SHIMODA M,et al.Enhancement of satellite cell differentiation and functional recovery in injured skeletal muscle by hyperbaric oxygen treatment[J].J Appl Physiol,2014,116(2):149-155.
[20] ZHU J,LI Y,LU A,et al,Follistatin improves skeletal muscle healing after injury and disease through an interaction with muscle regeneration,angiogenesis,and fibrosis[J].Am J Pathol,2011,179(2):915-930.
[21] VAITTINEN S,LUKKA R,SAHLGREN C,et al.The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle[J].J Neuropathol Exp Neurol,2001,60(6):588-597.
[22] RELAIX F,ZAMMIT P S.Satellite cells are essential for skeletal muscle regeneration:the cell on the edge returns centre stage[J].Development,2012,139(16):2845-2856.
[23] TIERNEY M T,SACCO A.Satellite cell heterogeneity in skeletal muscle homeostasis[J].Trends Cell Biol,2016,26(6):434-444.
[24]刘宇,肖卫华,罗贝贝,等.急性骨骼肌钝挫伤修复过程及MyoD、Myogenin变化的实验研究[J].中国运动医学杂志,2016,35(2):152-157.
[25]张安宁,罗雪林,黄思琴,等.PI3K/Akt/mTOR信号通路在大鼠急性骨骼肌钝挫伤修复中的作用[J].中国运动医学杂志,2018,37(7):594-600.
[26] PETTE D,STARON R S.Myosin isoforms,muscle fiber types,and transitions[J].Microsc Res Tech,2000,50(6):500-509.
[27] PEREIRA B P,HAN H C,YU Z,et al.Myosin heavy chain isoform profiles remain altered at 7 months if the lacerated medial gastrocnemius is poorly reinnervated:a study in rabbits[J].J Orthop Res,2010,28(6):732-738.
[28] HURME T,KALIMO H.Activation of myogenic precursor cells after muscle injury[J].Med Sci Sports Exerc,1992,24(2):197-205.
[2] BEINER J M,JOKL P.Muscle contusion injuries:current treatment options[J].J Am Acad Orthop Surg,2001,9(4):227-237.
[3] EKSTRAND J,HAGGLUND M,WALDEN M.Epidemiology of muscle injuries in professional football(soccer)[J].Am J Sports Med,2011,39(6):1226-1232.
[4]袁海洲,唐成林,田源,等.电针对大鼠急性骨骼肌损伤修复中生长相关蛋白和聚集蛋白表达的影响[J].中国康复医学杂志,2016,31(8):857-861.
[5]余飞,吕威,黄怡然,等.针刺对骨骼肌钝挫伤模型大鼠肝细胞生长因子表达的影响[J].针刺研究,2015,40(1):50-55.
[6]季传婷,徐平,陈新旺,等.电针对糖尿病小鼠骨骼肌磷酸化蛋白激酶B、天冬氨酸特异性半胱氨酸蛋白酶-3蛋白表达的影响[J].针刺研究,2012,37(2):104-107.
[7]肖慧玲,郑禹,王娜,等.巨刺法的临床应用概况[J].现代中西医结合杂志,2012,21(2):220-222.
[8]万应强.巨刺法为主治疗四肢软组织损伤251例疗效观察[J].贵阳中医学院学报,1993,15(1):32-33.
[9]刘林,李凤仙,张秀莲.电针巨刺法治疗四肢软组织损伤600例疗效观察[J].中国针灸,1994,14(S1):345.
[10] SAMBASIVAN R,COMAI G,LE ROUX I,et al.Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4[J].Dev Biol,2013,381(1):241-255.
[11] DALBIS A,COUTEAUX R,JANMOT C,et al.Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals.myosin isoform analysis[J].Eur J Biochem,1988,174(1):103-110.
[12] MARKERT C D,MERRICK M A,KIRBY T E,et al.Nonthermal ultrasound and exercise in skeletal muscle regeneration[J].Arch Phys Med Rehabil,2005,86(7):1304-1310.
[13]蔡晓刚.巨刺阿是穴为主治疗肩周炎临床观察[J].针刺研究,2001,26(3):184.
[14]刘光亭,肖永俭.电针巨刺对中风偏瘫患者甲皱微循环的影响[J].针刺研究,1990,15(1):40-43.
[15]匡培根,吴爱华,张凤英.巨刺对穴位灌流液中乙酰胆硷含量的影响[J].针刺研究,1980,5(2):147-149.
[16]王和鸣.中医骨伤科学[M].2版,北京:中国中医药出版社,2007.
[17]解秸萍.巨刺法神经解剖学机制探讨[J].上海针灸杂志,1997,16(2):28-29.
[18] FISCHMAN D A.The synthesis and assembly of myofibrils in embryonic muscle[J].Curr Topics Development Biol,1990(5):235-280.
[19] HORIE M,ENOMOTO M,SHIMODA M,et al.Enhancement of satellite cell differentiation and functional recovery in injured skeletal muscle by hyperbaric oxygen treatment[J].J Appl Physiol,2014,116(2):149-155.
[20] ZHU J,LI Y,LU A,et al,Follistatin improves skeletal muscle healing after injury and disease through an interaction with muscle regeneration,angiogenesis,and fibrosis[J].Am J Pathol,2011,179(2):915-930.
[21] VAITTINEN S,LUKKA R,SAHLGREN C,et al.The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle[J].J Neuropathol Exp Neurol,2001,60(6):588-597.
[22] RELAIX F,ZAMMIT P S.Satellite cells are essential for skeletal muscle regeneration:the cell on the edge returns centre stage[J].Development,2012,139(16):2845-2856.
[23] TIERNEY M T,SACCO A.Satellite cell heterogeneity in skeletal muscle homeostasis[J].Trends Cell Biol,2016,26(6):434-444.
[24]刘宇,肖卫华,罗贝贝,等.急性骨骼肌钝挫伤修复过程及MyoD、Myogenin变化的实验研究[J].中国运动医学杂志,2016,35(2):152-157.
[25]张安宁,罗雪林,黄思琴,等.PI3K/Akt/mTOR信号通路在大鼠急性骨骼肌钝挫伤修复中的作用[J].中国运动医学杂志,2018,37(7):594-600.
[26] PETTE D,STARON R S.Myosin isoforms,muscle fiber types,and transitions[J].Microsc Res Tech,2000,50(6):500-509.
[27] PEREIRA B P,HAN H C,YU Z,et al.Myosin heavy chain isoform profiles remain altered at 7 months if the lacerated medial gastrocnemius is poorly reinnervated:a study in rabbits[J].J Orthop Res,2010,28(6):732-738.
[28] HURME T,KALIMO H.Activation of myogenic precursor cells after muscle injury[J].Med Sci Sports Exerc,1992,24(2):197-205.