运动对Sarcopenia关联的抗氧化能力及线粒体介导的细胞凋亡信号通路的影响
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摘要
常言道“人老先从腿开始”。腿部衰老的主要表现之一就是Sarcopenia现象。Sarcopenia是一种以肌肉质量和力量下降为主要特征的增龄性机能退化征。人类步入40岁以后，骨骼肌会以每年1％的速率递减，并在75岁左右表现更为突出。Sarcopenia的发生与蛋白代谢失衡、缺乏运动等密切相关，它不仅会严重影响肌肉力量与运动能力，而且还会导致老年人摔倒乃至骨折。目前世界范围内不少发达国家人口老龄化问题已经突显，由其所带来的健康成本将相当巨大。近年来的研究显示，运动能够在一定程度上促进骨骼肌正常功能的维持，进而对Sarcopenia的发生发展起到一定的延缓或抑制作用。
研究目的
本研究选用快速老化小鼠(SAMP8)作为研究对象，分别采用不同时间(8周和21周)的爬梯、跑台运动对不同月龄SAMP8小鼠进行运动干预，重点观察SAMP8小鼠骨骼肌(腓肠肌和胫骨前肌)中Sarcopenia关联的抗氧化能力以及线粒体介导的细胞凋亡信号通路的变化情况，以期在抗氧化防御以及细胞凋亡两个层面上探索Sarcopenia发生的可能归因，为运动延缓Sarcopenia的可能机制提供一些参考，并最终围绕“人老先从腿开始”这一常见现象，提出“增健需从脚下行”的应对理念。
研究方法
本研究选择不同月龄的雄性SAMP8小鼠为实验对象，利用SAMP8小鼠的快速老化特点建立衰老动物模型，选择爬梯运动和跑台运动作为运动干预方式，分别对3月龄、4月龄和6月龄SAMP8小鼠进行8周、21周和8周的运动干预。通过紫外可见分光光度法对腓肠肌与胫骨前肌中抗氧化能力相关指标(T-AOC、总SOD、CAT、MDA)进行检测，并采用实时荧光定量PCR(Quantitative Real-timePCR)法对腓肠肌与胫骨前肌中Sarcopenia关联的Caspase依赖性与Caspase非依赖性细胞凋亡信号通路中关键基因及部分凋亡调节基因进行检测。
研究结果
本研究的结果显示：
1)老年安静组SAMP8小鼠腓肠肌和胫骨前肌均已表现出Sarcopenia征状，且除21周爬梯运动组(LR组)较8周爬梯运动组(SR组)体重有所上升外，其余各组小鼠体重变化未见显著性差异；
2)随衰老进程两种骨骼肌的抗氧化能力表现出的变化不同，其中腓肠肌抗氧化能力有所下调，而胫骨前肌抗氧化能力有所上调；两种骨骼肌中凋亡调节基因均倾向于保护衰老骨骼肌，尽管如此，在氧化应激胁迫下腓肠肌和胫骨前肌凋亡可能仍成为主流趋势，并最终经Caspase-3上调来促成凋亡；
3) 8周爬梯、跑台运动对青年组SAMP8小鼠腓肠肌和胫骨前肌抗氧化能力的影响存在较大差异，说明骨骼肌中抗氧化能力对不同运动干预方式和不同肌纤维类型的适应不同；爬梯运动可能通过Caspase依赖性细胞凋亡途径弱化腓肠肌和胫骨前肌细胞凋亡的趋势，而跑台运动倾向于上调AIF而经Caspase非依赖性细胞凋亡途径促进腓肠肌和胫骨前肌趋向凋亡；
4) 8周爬梯、跑台运动均能够上调老年组SAMP8小鼠腓肠肌和胫骨前肌抗氧化酶的活性，只是对两种肌肉类型中非酶类抗氧化防御体系的影响可能存在一定差异，使得总抗氧化能力存在异向性变化，从而导致了两种运动干预后腓肠肌中氧化损伤增加，而在胫骨前肌中氧化损伤的表现存在运动方式的选择性差异。尽管如此，8周爬梯、跑台运动仍然通过抑制Caspase依赖性与非依赖性细胞凋亡途径，在很大程度上弱化了衰老骨骼肌走向凋亡的趋势；
5) 21周的爬梯、跑台运动仍然对老年组SAMP8小鼠腓肠肌和胫骨前肌中抗氧化酶活性具有上调作用，但是总抗氧化能力却依然呈现下降趋势，使得两种运动干预后胫骨前肌中氧化损伤增加，而在腓肠肌中氧化损伤则出现运动方式的选择性差异；尽管如此，跑台运动对腓肠肌和胫骨前肌以及爬梯运动对胫骨前肌仍然起到了弱化细胞凋亡趋势的作用，只是爬梯运动对老年腓肠肌却具有促进凋亡的趋势，且这种促进作用可能通过Caspase依赖性与AIF介导的Caspase非依赖性两条途径协同完成；
6) 21周爬梯、跑台运动与8周爬梯、跑台运动相比，老年组腓肠肌和胫骨前肌中抗氧化能力、细胞凋亡信号通路中的基因以及调节基因的表达未完全表现出随干预时间延长的一致性递进变化。
6．1．2结论
1) SAMP8小鼠(腓肠肌和胫骨前肌)可作为研究Sarcopenia的动物模型；
2)抗氧化酶类对不同年龄、不同肌肉类型以及不同运动方式干预的运动适应表现不同；
3)对青年组骨骼肌而言，8周爬梯运动倾向于经Caspase-3下调而弱化细胞凋亡的趋势，而8周跑台运动则可能经AIF上调而促进细胞凋亡；
4) 8周爬梯、跑台运动干预后，老年组腓肠肌和胫骨前肌均倾向于弱化细胞凋亡的趋势；
5) 21周爬梯、跑台运动干预后，老年组骨骼肌仍基本具有弱化细胞凋亡的潜能，只是爬梯运动对衰老腓肠肌凋亡的弱化作用会转而呈现为促进作用；
6) 21周爬梯、跑台运动较8周爬梯、跑台运动，老年组腓肠肌和胫骨前肌并未完全表现出随干预时间延长的一致性递进弱化凋亡作用。
总之，爬梯、跑台运动对Sarcopenia关联的骨骼肌抗氧化能力以及细胞凋亡信号通路的影响在青年和老年SAMP8小鼠中还是存在一定的差异。然而，不管是促进还是抑制，都是骨骼肌细胞“生存竞争”后适应性的结果。
As the saying goes,aging of human starts from the legs.Sarcopenia is one kindof characters for the aging of legs.Sarcopenia is defined as the gradual reduction ofskeletal muscle mass and strength observed with advancing age.It starts to set inaround age 40,when muscle begins to decline at a rate of about one percent per year;what's more,it deteriorates with profound repercussions after approximately 75 yearsof age.This gradual loss has been tied to protein deficiency,lack of exercise amongthe elderly.This condition strongly influences muscle strength and mobility and is afactor in the occurrence of frailty and likelihood of falls and fractures in the elderly.The world now witnesses an unprecedented trend in population aging,bearingsignificant implications for the healthcare cost problems.Recently,it is reported thatphysical activity has profound effects on the musculoskeletal system,and contributesto the maintenance of functional abilities,and prevents Sarcopenia to some extent.Objects
The present study is designed to determine the effects of ladder climbing,treadmill running exercise on Sarcopenia related anti-oxidative capacity andmitochondria mediated apoptotic signaling pathway in Gastrocnemius and Tibialisanterior muscles of Senescence Accelerated Mouse Prone/8 (SAMP8)after 8 or 21weeks of exercise training to better understand the cellular and molecular etiology dueto redox-status and apoptotic signaling pathway,and to advocate the concept of“exercise promotes life expectancy”finally.Methods
The study here selects male SAMP8 at different month's age as the researchanimal.By taking advantage of the accelerated aging character,ladder climbing andtreadmill running exercise are used to intervene the aging model after 8weeks,21weeks and 8 weeks for 3-month,4-month and 6-month age mice respectively.Ultraviolet-visible detecting method is used to detect the anti-oxidant capacity index,(T-AOC,Total SOD,CAT and MDA);quantitative Real-time PCR method is used todetect key check points and regulating points in Sarcopenia related Caspasedependent and independent apoptotic signaling pathway in Gastrocnemius andTibialis anterior muscles.
Results
The research indicates that:
(1)The Gastrocnemius and Tibialis anterior muscles of SAMP8 mice in old grouphave shown the character of Sarcopenia,and no differences of the body weight arefound among the groups except the elevation of LR group compared with SR group.
(2)The anti-oxidant capacity changes diversely in the two kinds of skeletalmuscles during the aging process,with the up-regulation and down-regulation ofGastrocnemius and Tibialis anterior muscle respectively.Most of the check points inthe apoptotic signaling pathway are inclined to prevent both muscles from furtheraging induced by apoptosis.However,Gastrocnemius and Tibialis anterior muscle arestill trending towards apoptosis under the redox status,and complete the apoptosisprocess via up-regulation of Caspase-3.
(a)The effects of eight weeks intervene of ladder climbing and treadmill runningon anti-oxidant capacity show differently in Gastrocnemius and Tibialis anteriormuscle of young group of SAMP8 mice,which reveals that different adaptation ofdifferent muscles under different exercise training.Ladder climbing exercise is likelyto weaken the potential for apoptosis via Caspase dependent apoptotic signalingpathway in both muscles,while treadmill running exercise is likely to promoteapoptosis via Caspase independent apoptotic signaling pathway according toup-regulate the expression of AIF.
(4)Eight weeks intervene of ladder climbing and treadmill running up-regulateanti-oxidase activities either in Gastrocnemius or Tibialis anterior muscle of old groupof SAMP8 mice,but may influence diversely non-enzymatic antioxidants in bothmuscles,thus altering the result of oxidative damage due to opposite changes of totalanti-oxidant capacity.Even though,eight weeks exercise training of climbing andrunning still attenuate the apoptotic trends of the aging muscles via inhibition ofCaspase dependent and independent apoptotic signaling pathway.
(5)Twenty-one weeks intervene of ladder climbing and treadmill running alsoup-regulate anti-oxidase activities either in Gastrocnemius or Tibialis anterior muscleof old group of SAMP8 mice,but the total anti-oxidant capacity still showdown-regulation,thus inducing the different results of oxidative damage for theGastrocnemius or Tibialis anterior muscle.Anyway,the effect of treadmill running onGastrocnemius or Tibialis anterior muscle and the effect of ladder climbing on Tibialisanterior muscle still show the tendency of attenuate the apoptosis potential.Unexpectedly,ladder climbing exercise training promotes apoptosis potential inGastrocnemius of old group and the promotion may be completed by the synergy ofCaspase dependent and AIF mediated Caspase independent pathways.
(6)Compared with eight weeks exercise training,twenty-one weeks exercise training have not shown further identical progressive changes with the prolongedintervene time in the index of anti-oxidant capacity,apoptotic check points andregulating points.
Conclusions
(1)Gastrocnemius and Tibialis anterior muscle of the old group of SAMP8 miceis feasible in the research of Sarcopenia.
(2)Different adaptation results exist in different anti-oxidant capacity withdifferent ages under different exercise intervene modes.
(3)In both muscles of young group,eight weeks ladder climbing is prone toattenuate apoptosis potential via down-regulation of Caspase-3,while treadmillrunning exercise training is prone to promote apoptosis via up-regulation of AIF.
(4)Eight weeks ladder climbing and treadmill running exercise training arepossibly to attenuate the apoptosis potential in Gastrocnemius and Tibialis anteriormuscle of old group.
(5)Twenty-one weeks ladder climbing and treadmill running exercise training arepossibly to attenuate the apoptosis potential in Gastrocnemius and Tibialis anteriormuscle of old group,only except the promoting effect of ladder climbing exercise onaged Gastrocnemius.
(6)Compared with eight weeks exercise training,twenty-one weeks exercisetraining have not shown further identical progressive changes with the prolongedintervene time in the index of anti-oxidant capacity,apoptotic check points andregulating points.
Taken all together,the effects of ladder climbing and treadmill running exercisetraining on Sarcopenia related anti-oxidant capacity and apoptotic signaling pathwayare different to some extent in the muscles of young group and old group of SAMP8mice.Nevertheless,either promoting or attenuating effect is the fate of adaptation ofskeletal muscles after competition.
研究目的
本研究选用快速老化小鼠(SAMP8)作为研究对象，分别采用不同时间(8周和21周)的爬梯、跑台运动对不同月龄SAMP8小鼠进行运动干预，重点观察SAMP8小鼠骨骼肌(腓肠肌和胫骨前肌)中Sarcopenia关联的抗氧化能力以及线粒体介导的细胞凋亡信号通路的变化情况，以期在抗氧化防御以及细胞凋亡两个层面上探索Sarcopenia发生的可能归因，为运动延缓Sarcopenia的可能机制提供一些参考，并最终围绕“人老先从腿开始”这一常见现象，提出“增健需从脚下行”的应对理念。
研究方法
本研究选择不同月龄的雄性SAMP8小鼠为实验对象，利用SAMP8小鼠的快速老化特点建立衰老动物模型，选择爬梯运动和跑台运动作为运动干预方式，分别对3月龄、4月龄和6月龄SAMP8小鼠进行8周、21周和8周的运动干预。通过紫外可见分光光度法对腓肠肌与胫骨前肌中抗氧化能力相关指标(T-AOC、总SOD、CAT、MDA)进行检测，并采用实时荧光定量PCR(Quantitative Real-timePCR)法对腓肠肌与胫骨前肌中Sarcopenia关联的Caspase依赖性与Caspase非依赖性细胞凋亡信号通路中关键基因及部分凋亡调节基因进行检测。
研究结果
本研究的结果显示：
1)老年安静组SAMP8小鼠腓肠肌和胫骨前肌均已表现出Sarcopenia征状，且除21周爬梯运动组(LR组)较8周爬梯运动组(SR组)体重有所上升外，其余各组小鼠体重变化未见显著性差异；
2)随衰老进程两种骨骼肌的抗氧化能力表现出的变化不同，其中腓肠肌抗氧化能力有所下调，而胫骨前肌抗氧化能力有所上调；两种骨骼肌中凋亡调节基因均倾向于保护衰老骨骼肌，尽管如此，在氧化应激胁迫下腓肠肌和胫骨前肌凋亡可能仍成为主流趋势，并最终经Caspase-3上调来促成凋亡；
3) 8周爬梯、跑台运动对青年组SAMP8小鼠腓肠肌和胫骨前肌抗氧化能力的影响存在较大差异，说明骨骼肌中抗氧化能力对不同运动干预方式和不同肌纤维类型的适应不同；爬梯运动可能通过Caspase依赖性细胞凋亡途径弱化腓肠肌和胫骨前肌细胞凋亡的趋势，而跑台运动倾向于上调AIF而经Caspase非依赖性细胞凋亡途径促进腓肠肌和胫骨前肌趋向凋亡；
4) 8周爬梯、跑台运动均能够上调老年组SAMP8小鼠腓肠肌和胫骨前肌抗氧化酶的活性，只是对两种肌肉类型中非酶类抗氧化防御体系的影响可能存在一定差异，使得总抗氧化能力存在异向性变化，从而导致了两种运动干预后腓肠肌中氧化损伤增加，而在胫骨前肌中氧化损伤的表现存在运动方式的选择性差异。尽管如此，8周爬梯、跑台运动仍然通过抑制Caspase依赖性与非依赖性细胞凋亡途径，在很大程度上弱化了衰老骨骼肌走向凋亡的趋势；
5) 21周的爬梯、跑台运动仍然对老年组SAMP8小鼠腓肠肌和胫骨前肌中抗氧化酶活性具有上调作用，但是总抗氧化能力却依然呈现下降趋势，使得两种运动干预后胫骨前肌中氧化损伤增加，而在腓肠肌中氧化损伤则出现运动方式的选择性差异；尽管如此，跑台运动对腓肠肌和胫骨前肌以及爬梯运动对胫骨前肌仍然起到了弱化细胞凋亡趋势的作用，只是爬梯运动对老年腓肠肌却具有促进凋亡的趋势，且这种促进作用可能通过Caspase依赖性与AIF介导的Caspase非依赖性两条途径协同完成；
6) 21周爬梯、跑台运动与8周爬梯、跑台运动相比，老年组腓肠肌和胫骨前肌中抗氧化能力、细胞凋亡信号通路中的基因以及调节基因的表达未完全表现出随干预时间延长的一致性递进变化。
6．1．2结论
1) SAMP8小鼠(腓肠肌和胫骨前肌)可作为研究Sarcopenia的动物模型；
2)抗氧化酶类对不同年龄、不同肌肉类型以及不同运动方式干预的运动适应表现不同；
3)对青年组骨骼肌而言，8周爬梯运动倾向于经Caspase-3下调而弱化细胞凋亡的趋势，而8周跑台运动则可能经AIF上调而促进细胞凋亡；
4) 8周爬梯、跑台运动干预后，老年组腓肠肌和胫骨前肌均倾向于弱化细胞凋亡的趋势；
5) 21周爬梯、跑台运动干预后，老年组骨骼肌仍基本具有弱化细胞凋亡的潜能，只是爬梯运动对衰老腓肠肌凋亡的弱化作用会转而呈现为促进作用；
6) 21周爬梯、跑台运动较8周爬梯、跑台运动，老年组腓肠肌和胫骨前肌并未完全表现出随干预时间延长的一致性递进弱化凋亡作用。
总之，爬梯、跑台运动对Sarcopenia关联的骨骼肌抗氧化能力以及细胞凋亡信号通路的影响在青年和老年SAMP8小鼠中还是存在一定的差异。然而，不管是促进还是抑制，都是骨骼肌细胞“生存竞争”后适应性的结果。
As the saying goes,aging of human starts from the legs.Sarcopenia is one kindof characters for the aging of legs.Sarcopenia is defined as the gradual reduction ofskeletal muscle mass and strength observed with advancing age.It starts to set inaround age 40,when muscle begins to decline at a rate of about one percent per year;what's more,it deteriorates with profound repercussions after approximately 75 yearsof age.This gradual loss has been tied to protein deficiency,lack of exercise amongthe elderly.This condition strongly influences muscle strength and mobility and is afactor in the occurrence of frailty and likelihood of falls and fractures in the elderly.The world now witnesses an unprecedented trend in population aging,bearingsignificant implications for the healthcare cost problems.Recently,it is reported thatphysical activity has profound effects on the musculoskeletal system,and contributesto the maintenance of functional abilities,and prevents Sarcopenia to some extent.Objects
The present study is designed to determine the effects of ladder climbing,treadmill running exercise on Sarcopenia related anti-oxidative capacity andmitochondria mediated apoptotic signaling pathway in Gastrocnemius and Tibialisanterior muscles of Senescence Accelerated Mouse Prone/8 (SAMP8)after 8 or 21weeks of exercise training to better understand the cellular and molecular etiology dueto redox-status and apoptotic signaling pathway,and to advocate the concept of“exercise promotes life expectancy”finally.Methods
The study here selects male SAMP8 at different month's age as the researchanimal.By taking advantage of the accelerated aging character,ladder climbing andtreadmill running exercise are used to intervene the aging model after 8weeks,21weeks and 8 weeks for 3-month,4-month and 6-month age mice respectively.Ultraviolet-visible detecting method is used to detect the anti-oxidant capacity index,(T-AOC,Total SOD,CAT and MDA);quantitative Real-time PCR method is used todetect key check points and regulating points in Sarcopenia related Caspasedependent and independent apoptotic signaling pathway in Gastrocnemius andTibialis anterior muscles.
Results
The research indicates that:
(1)The Gastrocnemius and Tibialis anterior muscles of SAMP8 mice in old grouphave shown the character of Sarcopenia,and no differences of the body weight arefound among the groups except the elevation of LR group compared with SR group.
(2)The anti-oxidant capacity changes diversely in the two kinds of skeletalmuscles during the aging process,with the up-regulation and down-regulation ofGastrocnemius and Tibialis anterior muscle respectively.Most of the check points inthe apoptotic signaling pathway are inclined to prevent both muscles from furtheraging induced by apoptosis.However,Gastrocnemius and Tibialis anterior muscle arestill trending towards apoptosis under the redox status,and complete the apoptosisprocess via up-regulation of Caspase-3.
(a)The effects of eight weeks intervene of ladder climbing and treadmill runningon anti-oxidant capacity show differently in Gastrocnemius and Tibialis anteriormuscle of young group of SAMP8 mice,which reveals that different adaptation ofdifferent muscles under different exercise training.Ladder climbing exercise is likelyto weaken the potential for apoptosis via Caspase dependent apoptotic signalingpathway in both muscles,while treadmill running exercise is likely to promoteapoptosis via Caspase independent apoptotic signaling pathway according toup-regulate the expression of AIF.
(4)Eight weeks intervene of ladder climbing and treadmill running up-regulateanti-oxidase activities either in Gastrocnemius or Tibialis anterior muscle of old groupof SAMP8 mice,but may influence diversely non-enzymatic antioxidants in bothmuscles,thus altering the result of oxidative damage due to opposite changes of totalanti-oxidant capacity.Even though,eight weeks exercise training of climbing andrunning still attenuate the apoptotic trends of the aging muscles via inhibition ofCaspase dependent and independent apoptotic signaling pathway.
(5)Twenty-one weeks intervene of ladder climbing and treadmill running alsoup-regulate anti-oxidase activities either in Gastrocnemius or Tibialis anterior muscleof old group of SAMP8 mice,but the total anti-oxidant capacity still showdown-regulation,thus inducing the different results of oxidative damage for theGastrocnemius or Tibialis anterior muscle.Anyway,the effect of treadmill running onGastrocnemius or Tibialis anterior muscle and the effect of ladder climbing on Tibialisanterior muscle still show the tendency of attenuate the apoptosis potential.Unexpectedly,ladder climbing exercise training promotes apoptosis potential inGastrocnemius of old group and the promotion may be completed by the synergy ofCaspase dependent and AIF mediated Caspase independent pathways.
(6)Compared with eight weeks exercise training,twenty-one weeks exercise training have not shown further identical progressive changes with the prolongedintervene time in the index of anti-oxidant capacity,apoptotic check points andregulating points.
Conclusions
(1)Gastrocnemius and Tibialis anterior muscle of the old group of SAMP8 miceis feasible in the research of Sarcopenia.
(2)Different adaptation results exist in different anti-oxidant capacity withdifferent ages under different exercise intervene modes.
(3)In both muscles of young group,eight weeks ladder climbing is prone toattenuate apoptosis potential via down-regulation of Caspase-3,while treadmillrunning exercise training is prone to promote apoptosis via up-regulation of AIF.
(4)Eight weeks ladder climbing and treadmill running exercise training arepossibly to attenuate the apoptosis potential in Gastrocnemius and Tibialis anteriormuscle of old group.
(5)Twenty-one weeks ladder climbing and treadmill running exercise training arepossibly to attenuate the apoptosis potential in Gastrocnemius and Tibialis anteriormuscle of old group,only except the promoting effect of ladder climbing exercise onaged Gastrocnemius.
(6)Compared with eight weeks exercise training,twenty-one weeks exercisetraining have not shown further identical progressive changes with the prolongedintervene time in the index of anti-oxidant capacity,apoptotic check points andregulating points.
Taken all together,the effects of ladder climbing and treadmill running exercisetraining on Sarcopenia related anti-oxidant capacity and apoptotic signaling pathwayare different to some extent in the muscles of young group and old group of SAMP8mice.Nevertheless,either promoting or attenuating effect is the fate of adaptation ofskeletal muscles after competition.
引文
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