电针对失神经大鼠腓肠肌中叉头蛋白转录因子3A/肌萎缩F-box蛋白及成肌分化抗原的影响
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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叉头蛋白转录因子3A(fork-head protein,FOXO3A)、肌萎缩F-box蛋白(muscle atrophy F-box,MAFbx)和成肌分化抗原(myogenic differentiation antigen,Myod1)蛋白及基因表达的影响,探讨电针延缓大鼠失神经肌萎缩的可能机制。方法:雄性Sprague-Dawley大鼠随机分为假手术组、模型组及电针组,每组6只。采用钳夹坐骨神经法制备大鼠失神经腓肠肌萎缩模型。电针组选取大鼠术侧"足三里""环跳"穴进行治疗,每日1次,每次10min,连续干预14d后取材。计算各组大鼠腓肠肌湿重比,HE染色测定大鼠术侧腓肠肌纤维截面积和直径,Western blot检测大鼠术侧腓肠肌中FOXO3A、MAFbx、Myod1蛋白的相对表达量,实时荧光定量PCR检测大鼠术侧腓肠肌中FOXO3A、MAFbx及Myod1mRNA的相对表达量。结果:与假手术组相比,模型组大鼠腓肠肌湿重比、肌纤维截面积和直径均显著下降(P<0.01),电针组高于模型组(P<0.01)。与假手术组相比,模型组大鼠腓肠肌中FOXO3A、MAFbx、Myod1蛋白及mRNA表达量显著升高(P<0.01);与模型组相比,电针干预后FOXO3A、MAFbx蛋白及mRNA表达量显著降低(P<0.01),Myod1蛋白及mRNA表达量显著升高(P<0.01)。结论:电针可能通过抑制FOXO3A、MAFbx的表达同时上调Myod1的表达,影响骨骼肌蛋白水解的速率和肌卫星细胞分化的水平,在延缓失神经肌萎缩的同时促进肌萎缩修复。
Objective To observe the effect of electroacupuncture(EA)on morphological changes of denervated gastrocnemius(GS)and the expression of fork-head protein(FOXO3 A),muscle atrophy F-box(MAFbx)and myogenic differentiation antigen(Myod1)in sciatic nerve injury rats,so as to reveal its mechanism underlying improvement of myoatrophy.Methods Eighteen male Sprague-Dawley rats were randomly divided into sham operation,model and EA groups(n=6 per group).The model of gastrocnemius atrophy was established by crushing the right sciatic nerve.Then,EA(2 Hz)was applied to the right"Zusanli"(ST36)and"Huantiao"(GB30)for 10 min,once a day for 14 successive days.The wet weight of the GS on both sides was weighted to calculate the wet weight ratio(the injured side/the healthy side),and the cross-sectional area(CSA)and diameter of GS fibers were measured after H.E.staining.The expressions of FOXO3 A,MAFbx and Myod1 protein and mRNA in the GS tissue were tested using Western blot and fluorescence quantitative PCR,separately.Results Following modeling,the GS wet weight ratio,CSA and fiber diameter were smaller in the model group than those in the sham group(P<0.01),and were significantly higher in the EA group than in the model group(P<0.01).H.E.staining showed that the GS fibers became smaller and the myocyte got round in the model group,while the GS fibers were bigger and the myocyte was relatively regular in morphology in the EA group.After modeling,the expression levels of FOXO3 A,MAFbx and Myod1 mRNA and protein were evidently higher in the model group(P<0.01);Moreover,after EA treatment,modeling-induced increasing of expression levels of FOXO3 Aand MAFbx mRNA and protein were revised(P<0.01),while the increased expression level of Myod1 was further up-regulated relavant to that in the model group(P<0.01).Conclusion EA of ST36 and GB30 can suppress the up-regulated expression of FOXO3 Aand MAFbx mRNA and protein and further promote the expression of Myod1 mRNA and protein in the GS tissue in rats with denervated GS atrophy,which may contribute to its function in relieving the myoatrophy,promoting the skeletal muscle protein hydrolysis and differentiation of satellite cells.
Objective To observe the effect of electroacupuncture(EA)on morphological changes of denervated gastrocnemius(GS)and the expression of fork-head protein(FOXO3 A),muscle atrophy F-box(MAFbx)and myogenic differentiation antigen(Myod1)in sciatic nerve injury rats,so as to reveal its mechanism underlying improvement of myoatrophy.Methods Eighteen male Sprague-Dawley rats were randomly divided into sham operation,model and EA groups(n=6 per group).The model of gastrocnemius atrophy was established by crushing the right sciatic nerve.Then,EA(2 Hz)was applied to the right"Zusanli"(ST36)and"Huantiao"(GB30)for 10 min,once a day for 14 successive days.The wet weight of the GS on both sides was weighted to calculate the wet weight ratio(the injured side/the healthy side),and the cross-sectional area(CSA)and diameter of GS fibers were measured after H.E.staining.The expressions of FOXO3 A,MAFbx and Myod1 protein and mRNA in the GS tissue were tested using Western blot and fluorescence quantitative PCR,separately.Results Following modeling,the GS wet weight ratio,CSA and fiber diameter were smaller in the model group than those in the sham group(P<0.01),and were significantly higher in the EA group than in the model group(P<0.01).H.E.staining showed that the GS fibers became smaller and the myocyte got round in the model group,while the GS fibers were bigger and the myocyte was relatively regular in morphology in the EA group.After modeling,the expression levels of FOXO3 A,MAFbx and Myod1 mRNA and protein were evidently higher in the model group(P<0.01);Moreover,after EA treatment,modeling-induced increasing of expression levels of FOXO3 Aand MAFbx mRNA and protein were revised(P<0.01),while the increased expression level of Myod1 was further up-regulated relavant to that in the model group(P<0.01).Conclusion EA of ST36 and GB30 can suppress the up-regulated expression of FOXO3 Aand MAFbx mRNA and protein and further promote the expression of Myod1 mRNA and protein in the GS tissue in rats with denervated GS atrophy,which may contribute to its function in relieving the myoatrophy,promoting the skeletal muscle protein hydrolysis and differentiation of satellite cells.
引文
[1]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.
[2]吴佳佳,徐建光,马书杰.失神经骨骼肌萎缩的机制及康复治疗[J].安徽医药,2017,21(11):1949-1953.
[3]陶星,马铁明.针刺治疗周围神经损伤及再生修复相关机制探讨[J].针刺研究,2016,41(1):90-93.
[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]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.
[6]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.
[7]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:316.
[8]刘宪彤,陶星,马铁明,等.电针“环跳”穴不同组织对坐骨神经损伤大鼠脊髓JNK、c-jun磷酸化表达的影响[J].针刺研究,2015,40(5):373-377.
[9]吴珍元,黄英如,冼华,等.电针对失神经骨骼肌萎缩及纤维化的影响[J].中国康复医学杂志,2016,31(2):177-182.
[10]VENTADOUR S,ATTAIX D.Mechanisms of skeletal muscle atrophy[J].Curr Opin Rheumatol,2006,18(6):631-635.
[11]高睿琦,唐成林,曹净,等.电针对去神经支配骨骼肌萎缩大鼠PI3K/Akt/mTOR信号通路的影响[J].中国康复医学杂志,2018,33(1):15-21.
[12]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.
[13]BODINE S C,BAEHR L M.Skeletal muscle atrophy and the E3ubiquitin ligases MuRF1and MAFbx/atrogin-1[J].Am JPhysiol Endocrinol Metab,2014,307(6):e469-e484.
[14]ROWAN S L,RYGIEL K,PURVES-SMITH F M,et al.Denervation causes fiber atrophy and myosin heavy chain coexpression in senescent skeletal muscle[J].PLoS One,2012,7(1):e29082.
[15]LGER B,CARTONI R,PRAZ M,et al.Akt signaling through GSK-3Bbeta,mTOR and Foxo1is involved in human skeletal muscle hypertrophy and atrophy[J].J Physiol Lond,2006,576(Pt3):923-933.
[16]GOMES M D,LECKER S H,JAGOE R T,et al.Atrogin-1,a muscle-specific F-box protein highly expressed during muscle atrophy[J].Proc Natl Acad Sci USA,2001,98(25):14440-14445.
[17]ZHENG B,OHKAWA S,LI H Y,et al.FOXO3amediates signaling crosstalk that coordinates ubiquitin and atrogin-1/MAFbx expression during glucocorticoid-induced skeletal muscle atrophy[J].FASEB J,2010,24(8):2660-2669.
[18]MILAN G,ROMANELLO V,PESCATORE F,et al.Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy[J].Nat Commun,2015,6:6670.
[19]CHEN B L,WU Q,QIONG Z J,et al.Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway[J].Acta Biochim Biophys Sin(Shanghai),2016,48(9):827-832.
[20]裴艳宏,刘坤祥.黄芪丹参联合应用可延缓大鼠失神经骨骼肌萎缩[J].解剖学报,2014,45(2):278-282.
[21]CERLETTI M,JURGA S,WITCZAK C A,et al.Highly efficient,functional engraftment of skeletal muscle stem cells in dystrophic muscles[J].Cell,2008,134(1):37-47.
[22]KOTTLORS M,KIRSCHNER J.Elevated satellite cell number in Duchenne muscular dystrophy[J].Cell Tissue Res,2010,340(3):541-548.
[23]HUANG Q K,QIAO H Y,FU M H,et al.MiR-206attenuates denervation-induced skeletal muscle atrophy in rats through regulation of satellite cell differentiation via TGF-β1,smad3,and HDAC4signaling[J].Med Sci Monit,2016,22:1161-1170.
[24]HYATT J P,ROY R R,BALDWIN K M,et al.Nerve activity-independent regulation of skeletal muscle atrophy:role of MyoD and myogenin in satellite cells and myonuclei[J].Am J Physiol Cell Physiol,2003,285(5):C1161-C1173.
[25]TINTIGNAC L A,LAGIRAND J,BATONNET S,et al.Degradation of MyoD mediated by the SCF(MAFbx)ubiquitin ligase[J].J Biol Chem,2005,280(4):2847-2856.
[26]LAGIRAND-CANTALOUBE J,CORNILLE K,CSIBI A,et al.Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo[J].PLoSOne,2009,4(3):e4973.
[2]吴佳佳,徐建光,马书杰.失神经骨骼肌萎缩的机制及康复治疗[J].安徽医药,2017,21(11):1949-1953.
[3]陶星,马铁明.针刺治疗周围神经损伤及再生修复相关机制探讨[J].针刺研究,2016,41(1):90-93.
[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]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.
[6]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.
[7]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:316.
[8]刘宪彤,陶星,马铁明,等.电针“环跳”穴不同组织对坐骨神经损伤大鼠脊髓JNK、c-jun磷酸化表达的影响[J].针刺研究,2015,40(5):373-377.
[9]吴珍元,黄英如,冼华,等.电针对失神经骨骼肌萎缩及纤维化的影响[J].中国康复医学杂志,2016,31(2):177-182.
[10]VENTADOUR S,ATTAIX D.Mechanisms of skeletal muscle atrophy[J].Curr Opin Rheumatol,2006,18(6):631-635.
[11]高睿琦,唐成林,曹净,等.电针对去神经支配骨骼肌萎缩大鼠PI3K/Akt/mTOR信号通路的影响[J].中国康复医学杂志,2018,33(1):15-21.
[12]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.
[13]BODINE S C,BAEHR L M.Skeletal muscle atrophy and the E3ubiquitin ligases MuRF1and MAFbx/atrogin-1[J].Am JPhysiol Endocrinol Metab,2014,307(6):e469-e484.
[14]ROWAN S L,RYGIEL K,PURVES-SMITH F M,et al.Denervation causes fiber atrophy and myosin heavy chain coexpression in senescent skeletal muscle[J].PLoS One,2012,7(1):e29082.
[15]LGER B,CARTONI R,PRAZ M,et al.Akt signaling through GSK-3Bbeta,mTOR and Foxo1is involved in human skeletal muscle hypertrophy and atrophy[J].J Physiol Lond,2006,576(Pt3):923-933.
[16]GOMES M D,LECKER S H,JAGOE R T,et al.Atrogin-1,a muscle-specific F-box protein highly expressed during muscle atrophy[J].Proc Natl Acad Sci USA,2001,98(25):14440-14445.
[17]ZHENG B,OHKAWA S,LI H Y,et al.FOXO3amediates signaling crosstalk that coordinates ubiquitin and atrogin-1/MAFbx expression during glucocorticoid-induced skeletal muscle atrophy[J].FASEB J,2010,24(8):2660-2669.
[18]MILAN G,ROMANELLO V,PESCATORE F,et al.Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy[J].Nat Commun,2015,6:6670.
[19]CHEN B L,WU Q,QIONG Z J,et al.Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway[J].Acta Biochim Biophys Sin(Shanghai),2016,48(9):827-832.
[20]裴艳宏,刘坤祥.黄芪丹参联合应用可延缓大鼠失神经骨骼肌萎缩[J].解剖学报,2014,45(2):278-282.
[21]CERLETTI M,JURGA S,WITCZAK C A,et al.Highly efficient,functional engraftment of skeletal muscle stem cells in dystrophic muscles[J].Cell,2008,134(1):37-47.
[22]KOTTLORS M,KIRSCHNER J.Elevated satellite cell number in Duchenne muscular dystrophy[J].Cell Tissue Res,2010,340(3):541-548.
[23]HUANG Q K,QIAO H Y,FU M H,et al.MiR-206attenuates denervation-induced skeletal muscle atrophy in rats through regulation of satellite cell differentiation via TGF-β1,smad3,and HDAC4signaling[J].Med Sci Monit,2016,22:1161-1170.
[24]HYATT J P,ROY R R,BALDWIN K M,et al.Nerve activity-independent regulation of skeletal muscle atrophy:role of MyoD and myogenin in satellite cells and myonuclei[J].Am J Physiol Cell Physiol,2003,285(5):C1161-C1173.
[25]TINTIGNAC L A,LAGIRAND J,BATONNET S,et al.Degradation of MyoD mediated by the SCF(MAFbx)ubiquitin ligase[J].J Biol Chem,2005,280(4):2847-2856.
[26]LAGIRAND-CANTALOUBE J,CORNILLE K,CSIBI A,et al.Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo[J].PLoSOne,2009,4(3):e4973.