离心运动对骨骼肌生肌调节因子的影响及针刺干预研究
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摘要
目的：通过观察离心运动及针刺干预对生肌调节因子(MRFs)与Wnt/β-catenin信号通路主要信号分子的影响,探讨MRFs变化的可能机理及其在骨骼肌损伤修复过程中的作用。
方法：实验一将SD大鼠分为安静(C)、运动(E)与运动针刺组(EA),进行坡度为-16°四周跑台运动；免疫印迹法检测MRFs蛋白表达。实验二将SD大鼠分为C、CA(安静针刺)、E与EA组,进行坡度为-16°一次性跑台运动；实时定量PCR检测Wnt1、Wnt3a mRNA表达,免疫印迹法检测β-catenin、GSK-3β蛋白磷酸化水平及MyoD、Myogenin蛋白表达。
结果：1.一、三、四周EA组MyoD、MRF4蛋白表达显著高于E组(P<0.05),一、三周EA组Myf5蛋白表达显著高于E组(P<0.05),一、二周EA组Myogenin蛋白表达显著高于E组(P<0.05),三、四周与之相反。2.CA组Wnt1 mRNA表达均低于C组,Wnt3a mRNA在12h、72h及120h显著高于C组(P<0.05)；CA组72h GSK-3β活性与120hβ-catenin磷酸化水平显著低于C组(P<0.05)；24h、48h MyoD与48h及其之后Myogenin蛋白表达显著高于C组(P<0.05)。3.EA组Wnt1、Wnt3a mRNA表达的升高程度低于E组。72h E组、EA组GSK-3β活性与β-catenin磷酸化水平均显著低于C组(P<0.05),E组β-catenin磷酸化水平显著低于EA组(P<0.05)。EA组12h与24h MyoD蛋白表达均显著高于E组与C组(P<0.05)；E组Myogenin蛋白表达在24h、72h出现两个峰值,12h、24h、120h EA组Myogenin蛋白表达显著高于E组(P<0.05)。
结论：四周离心运动能上调Myf5与Myogenin蛋白表达,下调MyoD与MRF4蛋白表达；针刺具有调控骨骼肌MRFs维持在正常水平的作用。针刺正常骨骼肌对Wnt/β-catenin信号通路有影响；一次性离心运动及运动后针刺能上调Wnt1、Wnt3a mRNA表达,降低GSK-3β活性与β-catenin磷酸化水平,针刺有上调β-catenin磷酸化水平的作用。
Objective:The aim of this study is to observe the effect of eccentric exercise and acupuncture on the myogenic regulatory factors and Wnt/β-catenin signaling, then investigate the characteristics of the MRFs and the possible mechanism as well as its effect on repairment during skeletal muscle damage.
Methods:Experiment 1:For this, rats were divided into three groups: control (C), exercise (E), exercise and acupuncture(EA). E and EA groups were submitted to four weeks of eccentric exercise on a treadmill down a 16°decline. Protein expression of MRFs were detected by western blot. Experiment 2:Rats were divided into four groups:C, control and acupuncture (CA), E and EA groups. E and EA group were submitted to a single bout of eccentric exercise on a treadmill down a 16°decline. mRNA expression of Wntl and Wnt3a were detected by RT-PCR, phosphorylation ofβ-catenin and GSK-3βas well as protein expression of MyoD and Myogenin were detected by western blot.
Results:(1) The protein expression of MyoD and MRF4 elevated significantly in EA group relative to E group in 1,3,4 weeks(P<0.05). In 1 and 3weeks, EA group Myf-5 protein expression was greater compared with E group (P<0.05);the protein expression of Myogenin elevated significantly in EA group relative to E group in 1,2 weeks(P<0.05), it was the opposite position in week 3 and 4(P<0.05). (2) CA group Wnt1 mRNA expression were lower than C group, Wnt 3a were significantly higher than C group in 12h、72h and 120h (P<0.05). GSK-3βactivity decreased significantly compared with C group in 72h (P<0.05), phosphorylation ofβ-catenin decreased significantly compared with C group in 120h (P<0.05); MyoD protein expression elevated significantly in 24h and 48h (P<0.05), Myogenin protein expression elevated significantly after 48h (P<0.05). (3) EA group wntl and wnt3a mRNA expression were lower than E group. GSK-313 activity and phosphorylation ofβ-catenin of EA group decreased significantly compared with E group in 72h (P<0.05), and phosphorylation ofβ-catenin in E group decreased significantly than EA group (P<0.05). MyoD protein expression of EA group elevated significantly in 12h and 24h compared with E and C group (P<0.05); E group Myogenin protein expression had two crest values in 24h and 72h (P<0.05); EA group Myogenin protein expression was significantly higher than E group in 12h、72h and 120h(P<0.05).
Conclusion:Four weeks eccentric exercise may up-regulate Myf-5 and Myogenin protein expression, down-regulate MyoD and MRF4 protein expression. Acupuncture can maintain MRFs in normal levels. Needling normal skeletal muscle has effect on Wnt/β-catenin signal. An eccentric exercise and needling after exercise can up-regulate Wntl and Wnt3a mRNA expression, decrease activity of GSK-3βand phosphorylation ofβ-catenin, needling has the effect on up-regulating phosphorylation ofβ-catenin.
方法：实验一将SD大鼠分为安静(C)、运动(E)与运动针刺组(EA),进行坡度为-16°四周跑台运动；免疫印迹法检测MRFs蛋白表达。实验二将SD大鼠分为C、CA(安静针刺)、E与EA组,进行坡度为-16°一次性跑台运动；实时定量PCR检测Wnt1、Wnt3a mRNA表达,免疫印迹法检测β-catenin、GSK-3β蛋白磷酸化水平及MyoD、Myogenin蛋白表达。
结果：1.一、三、四周EA组MyoD、MRF4蛋白表达显著高于E组(P<0.05),一、三周EA组Myf5蛋白表达显著高于E组(P<0.05),一、二周EA组Myogenin蛋白表达显著高于E组(P<0.05),三、四周与之相反。2.CA组Wnt1 mRNA表达均低于C组,Wnt3a mRNA在12h、72h及120h显著高于C组(P<0.05)；CA组72h GSK-3β活性与120hβ-catenin磷酸化水平显著低于C组(P<0.05)；24h、48h MyoD与48h及其之后Myogenin蛋白表达显著高于C组(P<0.05)。3.EA组Wnt1、Wnt3a mRNA表达的升高程度低于E组。72h E组、EA组GSK-3β活性与β-catenin磷酸化水平均显著低于C组(P<0.05),E组β-catenin磷酸化水平显著低于EA组(P<0.05)。EA组12h与24h MyoD蛋白表达均显著高于E组与C组(P<0.05)；E组Myogenin蛋白表达在24h、72h出现两个峰值,12h、24h、120h EA组Myogenin蛋白表达显著高于E组(P<0.05)。
结论：四周离心运动能上调Myf5与Myogenin蛋白表达,下调MyoD与MRF4蛋白表达；针刺具有调控骨骼肌MRFs维持在正常水平的作用。针刺正常骨骼肌对Wnt/β-catenin信号通路有影响；一次性离心运动及运动后针刺能上调Wnt1、Wnt3a mRNA表达,降低GSK-3β活性与β-catenin磷酸化水平,针刺有上调β-catenin磷酸化水平的作用。
Objective:The aim of this study is to observe the effect of eccentric exercise and acupuncture on the myogenic regulatory factors and Wnt/β-catenin signaling, then investigate the characteristics of the MRFs and the possible mechanism as well as its effect on repairment during skeletal muscle damage.
Methods:Experiment 1:For this, rats were divided into three groups: control (C), exercise (E), exercise and acupuncture(EA). E and EA groups were submitted to four weeks of eccentric exercise on a treadmill down a 16°decline. Protein expression of MRFs were detected by western blot. Experiment 2:Rats were divided into four groups:C, control and acupuncture (CA), E and EA groups. E and EA group were submitted to a single bout of eccentric exercise on a treadmill down a 16°decline. mRNA expression of Wntl and Wnt3a were detected by RT-PCR, phosphorylation ofβ-catenin and GSK-3βas well as protein expression of MyoD and Myogenin were detected by western blot.
Results:(1) The protein expression of MyoD and MRF4 elevated significantly in EA group relative to E group in 1,3,4 weeks(P<0.05). In 1 and 3weeks, EA group Myf-5 protein expression was greater compared with E group (P<0.05);the protein expression of Myogenin elevated significantly in EA group relative to E group in 1,2 weeks(P<0.05), it was the opposite position in week 3 and 4(P<0.05). (2) CA group Wnt1 mRNA expression were lower than C group, Wnt 3a were significantly higher than C group in 12h、72h and 120h (P<0.05). GSK-3βactivity decreased significantly compared with C group in 72h (P<0.05), phosphorylation ofβ-catenin decreased significantly compared with C group in 120h (P<0.05); MyoD protein expression elevated significantly in 24h and 48h (P<0.05), Myogenin protein expression elevated significantly after 48h (P<0.05). (3) EA group wntl and wnt3a mRNA expression were lower than E group. GSK-313 activity and phosphorylation ofβ-catenin of EA group decreased significantly compared with E group in 72h (P<0.05), and phosphorylation ofβ-catenin in E group decreased significantly than EA group (P<0.05). MyoD protein expression of EA group elevated significantly in 12h and 24h compared with E and C group (P<0.05); E group Myogenin protein expression had two crest values in 24h and 72h (P<0.05); EA group Myogenin protein expression was significantly higher than E group in 12h、72h and 120h(P<0.05).
Conclusion:Four weeks eccentric exercise may up-regulate Myf-5 and Myogenin protein expression, down-regulate MyoD and MRF4 protein expression. Acupuncture can maintain MRFs in normal levels. Needling normal skeletal muscle has effect on Wnt/β-catenin signal. An eccentric exercise and needling after exercise can up-regulate Wntl and Wnt3a mRNA expression, decrease activity of GSK-3βand phosphorylation ofβ-catenin, needling has the effect on up-regulating phosphorylation ofβ-catenin.
引文
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