超早期推拿对骨骼肌急性钝挫伤模型大鼠肌细胞膜修复相关蛋白dysferlin表达的影响
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摘要
目的:观察超早期推拿对骨骼肌急性钝挫伤模型大鼠肌细胞膜修复相关蛋白dysferlin表达的影响,探寻dysferlin在骨骼肌钝挫伤肌细胞膜修复中的作用机制。方法:SD大鼠55只,随机分为正常对照组(n=5)、自然恢复组(n=25)、推拿组(n=25)。根据推拿治疗的天数计时,自然恢复组和推拿组分别在1d、2d、3d、5d、7d五个时间点处死,每亚组每个时间点5只,在腓肠肌标记的区域内取肌肉组织,HE观察组织的形态变化,运用Western blot检测蛋白dysferlin的表达情况,并进行统计学分析。结果:HE结果显示,与正常对照组相比,骨骼肌钝挫伤后,1d和2d肌纤维出现肿胀;3d出现炎性细胞浸润且自然恢复组比推拿组更加严重;5d炎性细胞浸润更加明显;7d推拿组炎性细胞较5d明显减少,结缔组织形成较少,7d自然恢复组有大量的结缔组织填充在肌纤维之间。WB结果显示:dysferlin在模型组中比在正常对照组中的表达明显增多;各个相应时间点推拿组较自然恢复组表达量明显增多(P<0.05),且随着时间的推移dysferlin在推拿组各亚组、自然恢复组各亚组中的表达量呈逐渐上升趋势。结论:在骨骼肌急性钝挫伤模型大鼠腓肠肌修复过程中,超早期推拿可增加膜修复蛋白dysferlin的表达,促进破裂的肌细胞膜及时修复,可能有利于损伤肌细胞的修复,从而帮助受损的骨骼肌修复。
Objective: To observe the effect of ultra-early massage on the expression of membrane repair associated protein dysferlin in rats with acute skeletal muscle injury, and demonstrate a possible mechanism how ultra-early massage protects myocytes from skeletal muscle contusion.Method: Fifty-five SD rats were randomly divided into normal control group(n=5), natural recovery group(n=25) and massage group(n=25). Rats in the natural recovery groups and the massage groups were sacrificed after treatment for 1 d, 2 d, 3 d, 5 d, 7 d. There were 5 rats in subgroups at each time point. Muscle tissue was harvested in the region of the marked muscle. Morphological changes in the skeletal muscle tissues were observed using HE stain. The expression level of dysferlin was detected by Western blot.Result: HE: Compared with the normal control group, muscle fiber blunt swelling appeared at 1 d and 2 d after skeletal muscle contusion. Inflammatory cells of the natural recovery group was more than that of the massage group at 3 d. More inflammatory cells were observed at 5 d. Inflammatory cells in 7 d massage group significantly decreased compared with that in 5 d massage group. Less extensive fibrosis was observed in the massage group than the natural recovery group at 7 d. WB: The expression level of dysferlin in the model groups wassignificantly higher than that in the normal control group. The expression of dysferlin in the massage group was significantly higher than that in the natural recovery group at each corresponding time(P<0.05). The level of dysferlin in the subgroups of the massage group and the natural recovery group presented gradual increase with the time goes on.Conclusion: In the course of acute contusion, ultra-early massage can increase the expression of dysferlin,promote the timely repair of ruptured membrane and decrease the muscle injury, which facilitate the repair of damaged skeletal muscle.
Objective: To observe the effect of ultra-early massage on the expression of membrane repair associated protein dysferlin in rats with acute skeletal muscle injury, and demonstrate a possible mechanism how ultra-early massage protects myocytes from skeletal muscle contusion.Method: Fifty-five SD rats were randomly divided into normal control group(n=5), natural recovery group(n=25) and massage group(n=25). Rats in the natural recovery groups and the massage groups were sacrificed after treatment for 1 d, 2 d, 3 d, 5 d, 7 d. There were 5 rats in subgroups at each time point. Muscle tissue was harvested in the region of the marked muscle. Morphological changes in the skeletal muscle tissues were observed using HE stain. The expression level of dysferlin was detected by Western blot.Result: HE: Compared with the normal control group, muscle fiber blunt swelling appeared at 1 d and 2 d after skeletal muscle contusion. Inflammatory cells of the natural recovery group was more than that of the massage group at 3 d. More inflammatory cells were observed at 5 d. Inflammatory cells in 7 d massage group significantly decreased compared with that in 5 d massage group. Less extensive fibrosis was observed in the massage group than the natural recovery group at 7 d. WB: The expression level of dysferlin in the model groups wassignificantly higher than that in the normal control group. The expression of dysferlin in the massage group was significantly higher than that in the natural recovery group at each corresponding time(P<0.05). The level of dysferlin in the subgroups of the massage group and the natural recovery group presented gradual increase with the time goes on.Conclusion: In the course of acute contusion, ultra-early massage can increase the expression of dysferlin,promote the timely repair of ruptured membrane and decrease the muscle injury, which facilitate the repair of damaged skeletal muscle.
引文
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[13]田源,唐成林,黄思琴,等.按摩对大鼠骨骼肌急性钝挫伤后膜修复蛋白dysferlin和annexin A1表达的影响[J].中国康复医学杂志,2016,31(8):841-846.
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[16]李晓丽,王小平.《内经》推拿疗法溯源[J].山东中医杂志,2014,(10):828-830.
[17]门志涛,张宏.中医推拿舒筋作用机制研究[J].按摩与导引,2008,24(03):10-13.
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[19]王念宏,严隽陶,孙武权,等.早期推拿对全膝关节置换患者术后股四头肌表面肌电影响的随机对照研究[J].中西医结合学报,2012,(11):1247-1253.
[20]吴志远.早期综合推拿手法对全膝关节置换术后康复及预防下肢深静脉血栓形成的影响[D].福州:福建中医药大学,2014.
[21]胡军,张喜林,严隽陶,等.Effects of massage on satellite cells of acute contusive skeletal muscles[J].针灸推拿医学(英文版),2007,5(1):6-9.
[22]吴云川.推拿对延迟性肌肉酸痛的疗效评价和机理研究[D].南京:南京中医药大学,2009.
[23]Miyake K,Mc Neil PL.Vesicle accumulation and exocytosis at sites of plasma membrane disruption[J].J Cell Biol,1995,131(6 Pt 2):1737-1745.
[24]Mc Neil PL,Vogel SS,Miyake K,et al.Patching plasma membrane disruptions with cytoplasmic membrane[J].JCell Sci,2000,113(Pt 11):1891-1902.
[25]Eddleman CS,Ballinger ML,Smyers ME,et al.Endocytotic formation of vesicles and other membranous structures induced by Ca2+and axolemmal injury[J].J Neurosci,1998,18(11):4029-4041.
[26]Lennon NJ,Kho A,Bacskai BJ,et al.Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal woundhealing[J].J Biol Chem,2003,278(50):50466-50473.
[27]Mellgren RL,Miyake K,Kramerova I,et al.Calcium-dependent plasma membrane repair requires m-or mu-calpain,but not calpain-3,the proteasome,or caspases[J].Biochim Biophys Acta,2009,1793(12):1886-1893.
[28]Lek A,Evesson FJ,Lemckert FA,et al.Calpains,cleaved mini-dysferlin C72,and L-type channels underpin calciumdependent muscle membrane repair[J].J Neurosci,2013,33(12):5085-5094.
[29]Cai C,Masumiya H,Weisleder N,et al.MG53 nucleates assembly of cell membrane repair machinery[J].Nat Cell Biol,2009,11(1):56-64.
[30]Cagliani R,Magri F,Toscano A,et al.Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies[J].Hum Mutat,2005,26(3):283.
[31]Mc Neil AK,Rescher U,Gerke V,et al.Requirement for annexin A1 in plasma membrane repair[J].J Biol Chem,2006,281(46):35202-35207.
[32]de Luna N,Gallardo E,Soriano M,et al.Absence of dysferlin alters myogenin expression and delays human muscle differentiation"in vitro"[J].J Biol Chem,2006,281(25):17092-17098.
[33]万婵娟,王玉娟.TRIM72蛋白结构及功能研究进展[J].生物技术,2015,25(01):93-97.
[2]任玉衡,史和福,敖英芳,等.优秀运动员的运动创伤流行病学调查[J].中国运动医学杂志,2000,19(4):377-386.
[3]陈世益.骨骼肌损伤后促进愈合的研究[D].上海:复旦大学,2002.
[4]张静.按摩对兔骨骼肌挫伤后修复作用的研究[D].河北:河北师范大学,2003.
[5]王荣国.电针促进家兔骨骼肌钝器伤后再生的作用和机制研究[D].北京:北京中医药大学,2012.
[6]Han R,Campbell KP.Dysferlin and muscle membrane repair[J].Curr Opin Cell Biol,2007,19(4):409-416.
[7]Bansal D,Miyake K,Vogel SS,et al.Defective membrane repair in dysferlin-deficient muscular dystrophy[J].Nature,2003,423(6936):168-172.
[8]Lek A,Evesson FJ,Sutton RB,et al.Ferlins:regulators of vesicle fusion for auditory neurotransmission,receptor trafficking and membrane repair[J].Traffic,2012,13(2):185-194.
[9]谢辉.在骨骼肌损伤修复过程中按摩对自由基相关酶和成肌调节因子的影响研究[D].重庆:重庆医科大学,2014.
[10]谢辉,唐成林,陈晓琳,等.按摩通过改善VEGF活性及血供促进兔骨骼肌损伤修复[J].中国老年学杂志,2014,12(24):6984-6986.
[11]杨辉,常青,唐成林,等.跑台运动训练与按摩联合作用对大鼠骨骼肌急性损伤修复过程中炎症的发展及肌卫星细胞增殖的影响[J].体育科学,2015,35(03):51-58.
[12]谢辉,唐成林,陈晓琳,等.按摩促进骨骼肌损伤后肌纤维再生机理的研究[J].重庆医科大学学报,2015,40(02):171-175.
[13]田源,唐成林,黄思琴,等.按摩对大鼠骨骼肌急性钝挫伤后膜修复蛋白dysferlin和annexin A1表达的影响[J].中国康复医学杂志,2016,31(8):841-846.
[14]Markert CD,Merrick MA,Kirby TE,et al.Nonthermal ultrasound and exercise in skeletal muscle regeneration[J].Arch Phys Med Rehabil,2005,86(7):1304-1310.
[15]Shu B,Yang Z,Li X,et al.Effect of different intensity pulsed ultrasound on the restoration of rat skeletal muscle contusion[J].Cell Biochem Biophys,2012,62(2):329-336.
[16]李晓丽,王小平.《内经》推拿疗法溯源[J].山东中医杂志,2014,(10):828-830.
[17]门志涛,张宏.中医推拿舒筋作用机制研究[J].按摩与导引,2008,24(03):10-13.
[18]李俊华.针刺对大鼠骨骼肌挫伤后组织修复的影响[D].北京:北京体育大学,2011.
[19]王念宏,严隽陶,孙武权,等.早期推拿对全膝关节置换患者术后股四头肌表面肌电影响的随机对照研究[J].中西医结合学报,2012,(11):1247-1253.
[20]吴志远.早期综合推拿手法对全膝关节置换术后康复及预防下肢深静脉血栓形成的影响[D].福州:福建中医药大学,2014.
[21]胡军,张喜林,严隽陶,等.Effects of massage on satellite cells of acute contusive skeletal muscles[J].针灸推拿医学(英文版),2007,5(1):6-9.
[22]吴云川.推拿对延迟性肌肉酸痛的疗效评价和机理研究[D].南京:南京中医药大学,2009.
[23]Miyake K,Mc Neil PL.Vesicle accumulation and exocytosis at sites of plasma membrane disruption[J].J Cell Biol,1995,131(6 Pt 2):1737-1745.
[24]Mc Neil PL,Vogel SS,Miyake K,et al.Patching plasma membrane disruptions with cytoplasmic membrane[J].JCell Sci,2000,113(Pt 11):1891-1902.
[25]Eddleman CS,Ballinger ML,Smyers ME,et al.Endocytotic formation of vesicles and other membranous structures induced by Ca2+and axolemmal injury[J].J Neurosci,1998,18(11):4029-4041.
[26]Lennon NJ,Kho A,Bacskai BJ,et al.Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal woundhealing[J].J Biol Chem,2003,278(50):50466-50473.
[27]Mellgren RL,Miyake K,Kramerova I,et al.Calcium-dependent plasma membrane repair requires m-or mu-calpain,but not calpain-3,the proteasome,or caspases[J].Biochim Biophys Acta,2009,1793(12):1886-1893.
[28]Lek A,Evesson FJ,Lemckert FA,et al.Calpains,cleaved mini-dysferlin C72,and L-type channels underpin calciumdependent muscle membrane repair[J].J Neurosci,2013,33(12):5085-5094.
[29]Cai C,Masumiya H,Weisleder N,et al.MG53 nucleates assembly of cell membrane repair machinery[J].Nat Cell Biol,2009,11(1):56-64.
[30]Cagliani R,Magri F,Toscano A,et al.Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies[J].Hum Mutat,2005,26(3):283.
[31]Mc Neil AK,Rescher U,Gerke V,et al.Requirement for annexin A1 in plasma membrane repair[J].J Biol Chem,2006,281(46):35202-35207.
[32]de Luna N,Gallardo E,Soriano M,et al.Absence of dysferlin alters myogenin expression and delays human muscle differentiation"in vitro"[J].J Biol Chem,2006,281(25):17092-17098.
[33]万婵娟,王玉娟.TRIM72蛋白结构及功能研究进展[J].生物技术,2015,25(01):93-97.