低强度脉冲超声协同运动促进骨骼肌肥大的效果及其分子机制研究

【作者】 张静；

【导师】 唐量；

【作者基本信息】 陕西师范大学 ， 运动人体科学， 2017， 硕士

【摘要】 目的:骨骼肌是高度可塑性组织,运动训练能够引起骨骼肌的适应性改变,促使骨骼肌维度增粗、力量增加。多年来,运动科学的目标一直是以最优的方式达到最大的训练效果,然而骨骼肌疲劳损伤制约着运动员成绩的提高。因此寻找促进疲劳恢复、提高运动能力等的方法越来越受到关注。物理疗法作为康复领域的一种新兴疗法,在促进骨和肌肉疲劳损伤等恢复方面有良好的治疗效果。其中,超声波作为一种物理因子,其治疗作用在骨、韧带以及肌肉损伤等方面已有大量文献报道。但是对于超声波靶向运动系统促进运动性骨骼肌肥大的研究却较少。因此,本文将初步探讨低强度脉冲超声(Low-intensity pulsed ultrasound,LIPUS)靶向运动系统促进骨骼肌肥大的协同效果,并围绕其对骨骼肌特异性分泌因子Myostatin(MSTN)表达的影响进而调控骨骼肌重建的分子机制进行初步研究,为超声靶向运动系统改善运动能力,更好的应用于运动科学领域提供理论依据。方法:30只SD大鼠随机分成3组:正常对照组(Normal control group,NC)、跑台训练组(Treadmill Exercise group,TE)、跑台训练加低强度脉冲超声干预组(Treadmill Exercise + Low-intensity pulsed ultrasound group,TE +LIPUS),每组均为10只大鼠。正常组不进行任何操作;TE组进行坡度跑台训练,坡度是5 °,速度是28m/min,60min/d,实验周期为8 w,每周训练6d,休息1 d;LIPUS组的训练方案与运动组一样,但是每次训练结束后再施加20 min的治疗,且TE组与TE +LIPUS组的相同但不打开仪器。在实验期间,每隔3d称量一次体重每隔1w测量一次抓力。最后一次训练结束后空腹12 h,称量大鼠体重,取材,准确称量大鼠两侧腓肠肌的重量。试剂盒检测大鼠腓肠肌组织匀浆液中琥珀酸脱氢酶(Succinate dehydrogenase,SDH)和苹果酸脱氢酶(Malate dehydrogenase,MDH)的活性;形态学分析腓肠肌组织,H&E染色法分析各组大鼠肌纤维横截面积的变化;提取各组大鼠腓肠肌组织中的蛋白和RNA用于分子通路的检测。采用Western Blot 和 RT-qPCR 技术检测骨骼肌中 MSTN、ActRIIB 及 Akt、mTOR 和FoxO1的蛋白及磷酸化的表达,以及MSTN、ActRIIB及Akt、mTOR和FoxO1的基因表达。在动物实验的基础上,为进一步探究LIPUS的作用,我们还进行了细胞实验。成功培养成肌细胞C2C12细胞系,细胞种板后分为正常对照组(Normal control group,NC),低强度脉冲超声干预组(Low-intensity pulsed ultrasound group,LIPUS)。LIPUS 组每天分别进行强度为 13mW/cm2、30mW/cm2、45mW/cm2 的超声照射20 min,NC组进行同样的操作但不打开仪器。LIPUS刺激3 d,每天镜下观察并拍照,CCK-8检测细胞增殖情况,H&E染色分析细胞分化情况。结果:1.通过8 w的动物训练,各组(NC组、TE组、TE +LIPUS组)的体重、抓力均有显著性的变化(P<0.05)。体重结果显示,三组大鼠体重与之前比较均有所增加,其中NC组增幅最大。与NC组相比,TE组的体重减轻但无显著性差异,TE + LIPUS组体重明显减少(P<0.05)。抓力测试结果显示,与NC组的相比,TE组和TE + LIPUS组的抓力均有极显著性的提高(P<0.01,P<0.01),TE+ LIPUS组与TE组相比较抓力也有所增加,且具有显著性差异(P<0.05)。2.腓肠肌的湿重结果显示,与NC组相比,腓肠肌湿重在TE组(P<0.05)和TE + LIPUS组(P<0.01)均有明显的增加,并且TE + LIPUS组和TE组相比差异显著(P<0.05)。3.生化指标显示,与NC组相比,TE组和TE + LIPUS组的SDH活性均高于NC组,具有极显著性差异(P<0.01,P<0.01);与TE组相比,LIPUS组不具有显著性差异。与NC组相比,TE组和TE + LIPUS组的MDH活性显著性的增加(P<0.05,P<0.01),并且TE + LIPUS组与TE组相比MDH的活性有显著的增加(P<0.05)。4.形态学指标显示,与NC组相比,TE组和TE + LIPUS组肌纤维的平均横截面积均有极显著性的增加(P<0.01,P<0.01),且TE + LIPUS组与TE组相比肌纤维横截面积的平均值增加并且有差异极显著(P<0.01)。5.蛋白和基因表达结果显示,LIPUS明显上调了 Akt,mTOR蛋白及其磷酸化的表达,明显下调了 MSTN,ActRIIB蛋白表达及其FoxO1的蛋白及其磷酸化的表达;基因检测结果与蛋白相同。6.细胞实验结果显示,CCK-8检测细胞增殖情况,与NC相比,LIPUS各组虽有所增加,但无统计学意义;其中当强度为30 mW/cm2时,LIPUS组比其他组呈现出更高的趋势,但仍无统计学差异。通过H&E染色分析细胞分化结果,在强度是30 mW/cm2,LIPUS组细胞核的融合指数增多(P<0.05)。结论:1.动物实验结果表明,LIPUS刺激能够促进运动诱导性骨骼肌肥大,通过增加肌肉的力量、质量、肌纤维的横截面积;可能通过调节三羧酸循环中关键酶的活性,提高有氧代谢的速率,使骨骼肌代谢能力增强;可能通过抑制腓肠肌MSTN及其受体的蛋白和基因表达,抑制肌肉萎缩,平衡与骨骼肌代谢息息相关的Akt及其主要下游信号分子,使mTOR的表达量增加,加速蛋白质合成,使FoxO1的表达量降低,减慢蛋白质降解,从而促进运动性骨骼肌肥大。2.细胞实验结果表明,LIPUS刺激虽在细胞增殖上作用不明显,但在细胞分化上起到了一定的促进作用,说明在没有运动训练的基础上LIPUS在骨骼肌肥大上也发挥着有利的影响。3.LIPUS作为一种新型的理疗方法,有望成为一种促进健身爱好者、运动员和一些肌萎缩的病人等的肌肉质量增加的辅助疗法,但对其普遍的应用于运动科学和健康科学领域仍需进行深入的研究。更多还原

【Abstract】 Purpose:Skeletal muscle is a highly adaptable tissue,exercise training can cause adaptive changes of skeletal muscles,increase the skeletal muscle dimension and strength.These years,the goal of sports science has always been to achieve maximum training by the best possible way,however,the fatigue damage of skeletal muscles restricts the improvement of athletes’ performance.Therefore,more and more attention has been paid to find ways to promote fatigue recovery and improve exercise ability.Physical therapy was as an emerging therapy in the field of rehabilitation,with a good therapeutic effect on bone and muscle fatigue recovery.Thereinto,ultrasound as a kind of acoustic therapy,has a large number of literature report its therapeutical effect on bone,ligament and muscle damage.Howere,there is little research on the application of ultrasound on exercise system to promote the exercise-induced skeletal muscle hypertrophy.Therefore,this paper will preliminary discussion the low intensity pulsed ultrasound(LIPUS)target sports system improved skeletal muscle hypertrophy of synergistic effect,and the influence of skeletal muscle specific secrete factors secreted Myostatin(MSTN)to regulate the molecular mechanisms of skeletal muscle reconstruction.In order to expore ultrasound targeted sports system to improve sports ability and be providing a theoretical basis about being applied better in the field of sports science.Methods:Thirty SD rats were randomly divided into three groups:normal control group(NC),treadmill exercise group(TE),treadmill exercise and low-intensity pulsed ultrasound group(TE + LIPUS),10 rats in each group.NC without any operation,TE was carried out slope treadmill training,slope is 5 °,speed is 28 m/min,60 min/d,experimental period for eight weeks,training 6 d a week,only one day rest.LIPUS group training methods were similarly to TE,but every time after training to exert treatment for 20 min and TE group was treated similarly to the TE+LIPUS group,expect that the power was not turn on.During the experiment,weight was measured once every 3 d and grip test was measured once a week.After the last training,empty stomach for 12 h,weighing the rat body weight,draw materials,accurate weighing on both sides of gastrocnemius on rats.The activity of Succinate dehydrogenase(SDH)and Malate dehydrogenase(MDH)level of gastrocnemius was analyzed by standard colorimetric using commercial kits;morphometric analysis gastrocnemius that HE stains analyze the change of the average areas of the myofibers in different groups;extract gastrocnemius of protein and RNA in different group test the molecular pathways.Western Blotting was used to detect skeletal muscle the expression of protein and phosphorylation using MSTN、ActRIIB、Akt、mTOR、FoxO1,and RT-qPCR was used to detect the expression of gene using MSTN ActRIIB、Akt、mTOR、FoxO1.On the basis of animal experiments,to further explore the effect of LIPUS,we also conducted cell experiments.Successful culture myoblast C2C12 cell line,cells were randomly divided into two groups:normal control group(NC),low-intensity pulsed ultrasound group(LIPUS).And LIPUS groups were accepted different intensity stimulate with 13 mW/cm2、30 mW/cm2、45 mW/cm2,20 min/d,NC group was treated similarly to the LIPUS group,expect that the power was not turn on.LIPUS stimulated three days,every day the microscopic observation and take photos,CCK-8 was used to test cell proliferation,H&E staining analysis cell differentiation.Results:1.Through 8 weeks of animal experiments,each group(NC、TE、TE+LIPUS)of weight and grip both have significantly different(P<0.05).The result of weight showed,compared with before,three groups of rat weight had increased and among them the largest rise was NC group.Compare with NC group,TE group had increased but no significantly different,TE+LIPUS showed a significant decrease(P<0.05).The result of grip strength showed,both TE and TE+LIPUS group significantly enhanced the grip strength of rats compared with the NC group(P<0.01,P<0.01).Especially,the grip strength in the TE+LIPUS group was significantly higher than the TE group(P<0.05).2.The results of the gastrocnemius wet weight showed,the gastrocnemius wet weight in both TE group(P<0.05)and TE+LIPUS group(P<0.01)were significantly increase compared with the NC group,and there was a significant difference between TE group and TE+LIPUS group(P<0.05).3.The results of the biochemical analysis showed,the SDH activity in both the TE and TE+LIPUS group were significantly higher than that in the NC group(P<0.01,P<0.01);but there was no significant difference between the TE and TE+LIPUS group.Compared with the NC group,both the TE and TE+LIPUS treatment correlated with an increase in the activity of MDH(P<0.05,P<0.01),and the activity of MDH in the TE+LIPUS group was significantly higher compared with the TE group(P<0.05).4.The results of the morphometric analysis showed,compared with the NC group,both TE and TE+LIPUS group presented increased average surface of muscle fibers(P<0.01,P<0.01).Furthermore,compared with the TE group,TE+LIPUS further significantly increased the cross-sectional area of muscle fibers(P<0.01).5.The results of the expression of protein and gene showed,LIPUS treatment significantly up regulated the expression of Akt,mTOR,p-Akt,p-mTOR,and significantly down regulated the expression of MSTN,ActRIIB,FoxO1 and its phosphorylation;genetic testing results with the protein had consistency.6.The result of the cell experiment showed,CCK-8 was used to detect cell proliferation,compared with the NC group,LIPUS stimulate different group but all no significant difference;among them,Lhe intensity was 30mw/cm2 showed a higher trend than others but also no significant difference.Through HE stains to analysis the cell differentiation,when the intensity was 30mw/cm2 that LIPUS group of nucleus fusion index had increased(P<0.05).Conclusion:1.The result of animal experiment showed that LIPUS can promote exercise-induced muscle hypertrophy,through increasing muscle strength,mass,muscle fiber cross-sectional area,regulating the activity of key enzymes in the Krebs cycle,improving aerobic metabolism rate,to make the skeletal muscle energy metabolism ability enhancement,inhibiting gastrocnemius the expression of MSTN protein and gene and its receptors to inhibit muscle atrophy,balancing Akt and its main downstream signaling molecules which is closely related to metabolism of skeletal muscle,making the expression of mTOR increased,accelerating protein synthesis,reduce the expression of FoxO1,leading to protein degradation,further promotng exercise-induced skeletal muscle hypertrophy.2.The result of myoblast experiment showed that LIPUS stimulation cell proliferation was not obvious,but playing an important role in cell differentiation.It is showed that in the absence basis of training.LIPUS also played a beneficial effect on skeletal muscle hypertrophy.3.LIPUS as a new kind of physical therapy method,was expected to become promotion of the fitness fanatics,athletes and some patients of muscle atrophy to increase skeletal muscle mass of adjuvant therapy,but for the widespread application in sports science and health science remained to be further explored.更多还原