PINK1/Parkin介导的线粒体自噬在运动性骨骼肌损伤中的作用
|
摘要
目的:探讨大负荷运动对大鼠骨骼肌线粒体自噬蛋白PINK1和Parkin表达的影响,分析PINK1/Parkin途径在运动致骨骼肌线粒体损伤中的作用。方法:48只成年雄性SpragueDaw ley大鼠,随机分为安静对照组(C,n=8)和运动组(E,n=40)。其中,E组按时相点划分为0 h、12 h、24 h、48 h、72 h等5组,每组8只。E组大鼠在跑台上进行持续性下坡跑,跑台坡度为-16°,速度为16 m/min,运动时间为90 min。各组分别于对应时间点分离比目鱼肌进行检测。使用透射电子显微镜观察骨骼肌线粒体超微结构的变化,采用ELISA方法检测各组大鼠比目鱼肌线粒体定量酶CS的含量,应用Western Blot方法检测骨骼肌PINK1和线粒体中Parkin、LC3的蛋白表达。结果:一次大负荷运动后比目鱼肌线粒体出现明显的肿胀以及在肌膜下积聚等超微结构的异常变化,且伴有大量的处于不同成熟阶段的自噬体形成,同时CS的含量明显减少,线粒体自噬蛋白PINK1、Parkin和LC3均出现一过性表达升高(P<0.05或P<0.01)。结论:一次大负荷运动后,骨骼肌线粒体结构和数量受损,有大量自噬体形成,其致骨骼肌损伤机制可能与PINK1/Parkin途径的活化及继发的过度线粒体自噬有关。
Objectives: To explore the effect of heavy load exercise on the mitophagy protein expression of PINK1 and Parkin in skeletal muscle of rats,and to analyze the role of PINK1/Parkin pathway in exercise-induced mitochondrial damage in skeletal muscles. Methods: Forty-eight male adult SD rats were randomly divided into quiet control group( C group,n = 8) and exercise group( E group,n = 40).Among them,E group was further divided into 0 h,12 h,24 h,48 h and 72 h sub-groups( n = 8) according to the time point. Rats in the E group performed a running on a treadmill down a 16° incline at 16 m/min for 90 min,and at each time point the soleus muscle was collected under anesthesia.Mitochondrial ultrastructural changes in skeletal muscle were observed by a transmission electron microscope. The content of quantitative enzyme citrate synthase( CS) was measured by ELISA; Protein expression of skeletal muscle PINK1 and mitochondrial Parkin and LC3 were detected by Western blot.Results: After heavy load exercise,the mitochondrial structure appeared the typical abnormal changes such as swelling and accumulating under cell membrane,and formed a large number of mitophagosomes which were in different stages of maturity. The content of CS was significantly decreased,and the protein expression of PINK1,Parkin and LC3 were significantly elevated( P<0.05 or P<0.01).Conclusion: After a heavy load exercise,the structure and quantity of mitochondria in skeletal muscle were impaired,and a large number of mitophagosomes were formed.The mechanism of skeletal muscle damage caused by heavy load exercise may be related to the activation of PINK1/Parkin pathway and the further initiating overactive mitophagy.
Objectives: To explore the effect of heavy load exercise on the mitophagy protein expression of PINK1 and Parkin in skeletal muscle of rats,and to analyze the role of PINK1/Parkin pathway in exercise-induced mitochondrial damage in skeletal muscles. Methods: Forty-eight male adult SD rats were randomly divided into quiet control group( C group,n = 8) and exercise group( E group,n = 40).Among them,E group was further divided into 0 h,12 h,24 h,48 h and 72 h sub-groups( n = 8) according to the time point. Rats in the E group performed a running on a treadmill down a 16° incline at 16 m/min for 90 min,and at each time point the soleus muscle was collected under anesthesia.Mitochondrial ultrastructural changes in skeletal muscle were observed by a transmission electron microscope. The content of quantitative enzyme citrate synthase( CS) was measured by ELISA; Protein expression of skeletal muscle PINK1 and mitochondrial Parkin and LC3 were detected by Western blot.Results: After heavy load exercise,the mitochondrial structure appeared the typical abnormal changes such as swelling and accumulating under cell membrane,and formed a large number of mitophagosomes which were in different stages of maturity. The content of CS was significantly decreased,and the protein expression of PINK1,Parkin and LC3 were significantly elevated( P<0.05 or P<0.01).Conclusion: After a heavy load exercise,the structure and quantity of mitochondria in skeletal muscle were impaired,and a large number of mitophagosomes were formed.The mechanism of skeletal muscle damage caused by heavy load exercise may be related to the activation of PINK1/Parkin pathway and the further initiating overactive mitophagy.
引文
[1]SANDRI M,CARRARO U,PODHORSKA-OKOLOV M,et al.Apoptosis,DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise[J].FEBS Letters,1995,373(3):291-295
[2]GREEN D R.Apoptosis:Death deceiver[J].Nature,1998,396(6712):629-630
[3]RAJ D A,BOOKER T S,BELCASTRO A N.Striated muscle calcium-stimulated cysteine protease(calpain-like)activity promotes myeloperoxidase activity with exercise[J].Pflügers Archiv,1998,435(6):804-809
[4]HO T T,WARR MR,ADELMAN E R,et al.Autophagy maintains the metabolism and function of young and old stem cells[J].Nature,2017,543(7644):205-210
[5]GARCIA-PRAT L,MARTINEZ-VICENTE M,PERDIGUERO E,et al.Autophagy maintains stemness by preventing senescence[J].Nature,2016,529(7584):37-42
[6]TAN T,ZIMMERMANN M,REICHERT A S.Controlling quality and amount of mitochondria by mitophagy:Insights into the role of ubiquitination and deubiquitination[J].Biological Chemistry,2016,397(7):637-647
[7]YOULE R J,NARENDRA D P.Mechanisms of mitophagy[J].Nature Reviews Molecular Cell Biology,2011,12(1):9-14
[8]WESTERMANN B.Mitochondrial fusion and fission in cell life and death[J].Nature Reviews Molecular Cell Biology,2010,11(12):872-884
[9]KIMI,RODRIGUEZ-ENRIQUEZ S,LEMASTERS J J.Selective degradation of mitochondria by mitophagy[J].Archives of Biochemistry and Biophysics,2007,462(2):245-253
[10]YOSHII S R,MIZUCSHIMA N.Autophagy machinery in the context of mammalian mitophagy[J].Biochimica et Biophysica Acta,2015,1853(10):2797-2801
[11]PICKRELL A M,YOULE R J.The role of PINK1,parkin,and mitochondrial fidelity in Parkinson’s disease[J].Neuron,2015,85(2):257-273
[12]PARK J,LEE S B,LEE S,et al.Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by Parkin[J].Nature,2006,441(7097):1157-1161
[13]CLARK I E,DODSON MW,JIANG C,et al.Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin[J].Nature,2006,441(7097):1162-1166
[14]YANG Y,GEHRKE S,IMAI Y,et al.Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila PINK1is rescued by Parkin[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(28):10793-10798
[15]ARMSTRONG R B,OGILVIE R W,SCHWANE J A.Eccentric exercise-induced injury to rat skeletal muscle[J].Journal of Applied Physiology:Respiratory,Environmental and Exercise Physiology,1983,54(1):80-93
[16]黄志辉,刘浩,李宪航.有氧运动对衰老大鼠心肌、肝脏细胞凋亡及线粒体膜电位的影响[J].西安体育学院学报,2007,28(1):78-80
[17]刘慧君,姜宁,赵斐,等.急性运动中骨骼肌线粒体移动相关基因表达与线粒体动力学的关系[J].天津体育学院学报,2010,25(2):118-121
[18]DAS S,MITROVSKY G,VASANTHI H R,et al.Antiaging properties of a grape-derived antioxidant are regulated by mitochondrial balance of fusion and fission leading to mitohphagy triggered by a signaling network of Sirt1-Sirt3-Foxo3-PINK1-PARKIN[J].Oxidative Medicine and Cellular Longevity,2014,2014:345105
[19]孙良,赵刚.运动性骨骼肌损伤后不同时刻大鼠肱三头肌超微结构及肌酸激酶的变化[J].沈阳体育学院学报,2015,34(3):74-78
[20]高晓娟.急性离心运动及针刺干预对骨骼肌细胞外基质的影响[D].北京:北京体育大学,2011:1-23
[21]张媛,漆正堂,郭维,等.耐力训练对高脂膳食大鼠骨骼肌线粒体脂肪氧化及PGC-1α基因表达的影响[J].天津体育学院学报,2010,25(3):193-196
[22]李世成,焦海舟.大鼠离心运动后骨骼肌微细损伤机制的研究[J].湛江师范学院学报,2006,27(3):90-95
[23]李雪,袁琼嘉,熊若虹.不同负荷运动对大鼠大脑皮质超微结构的影响[J].成都体育学院学报,2006,32(3):111-113
[24]董贵俊,吕晨曦,葛新发,等.一次和重复大强度离心运动前后大鼠骨骼肌超微结构变化[J].中国运动医学杂志,2013,32(2):142-148
[25]陈彩珍,卢健,苏有存.抗阻训练对D-半乳糖衰老模型大鼠骨骼肌线粒体膜的影响[J].西安体育学院学报,2011,28(1):83-86
[26]WATTS J A,KLINE J A,THORNTON L R,et al.Metabolic dysfunction and depletion of mitochondria in hearts of septic rats[J].Journal of Molecular and Cellular Cardiology,2004,36(1):141-150
[27]HARGREAVES I P,DUNCAN A J,WU L,et al.Inhibition of mitochondrial complexⅣleads to secondary loss complexⅡ-Ⅲactivity:Implications for the pathogenesis and treatment of mitochondrial encephalomyopathies[J].Mitochondrion,2007,7(4):284-287
[28]SANCHEZ A M,BERNARDI H,PY G,et al.Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise[J].American Journal of Physiology.Regulatory,Integrative and Comparative Physiology,2014,307(8):956-969
[29]NEEL B A,LIN Y,PESSIN J E.Skeletal muscle autophagy:A newmetabolic regulator[J].Trends in Endocrinology and Metabolism:TEM,2013,24(12):635-643
[30]TOLKOVSKY A M.Mitophagy[J].Biochimica et Biophysica Acta,2009,1793(9):1508-1515
[31]VAINSHTEIN A,TRYON LD,PAULY M,et al.Role of PGC-1αduring acute exercise-induced autophagy and mitophagy in skeletal muscle[J].American Journal of Physiology Cell Physiology,2015,308(9):710-719
[32]樊申元,靳二辉.耐力训练对帕金森模型小鼠中脑线粒体自噬相关基因表达的影响[J].中国康复医学杂志,2015,30(5):437-442
[33]崔迪,邱守涛,王海燕,等.耐力运动对营养性肥胖小鼠骨骼肌细胞自噬及线粒体自噬的影响[J].体育科学,2014,34(12):63-71
[34]崔迪,贺杰,孙婧瑜,等.耐力训练与p53抑制剂PFT-α对小鼠骨骼肌线粒体自噬相关基因表达的影响[J].天津体育学院学报,2013,28(5):422-426
[35]KIMY,PARK J,KIMS,et al.PINK1 controls mitochondrial localization of Parkin through direct phosphorylation[J].Biochemical and Biophysical Research Communications,2008,377(3):975-980
[36]赵晓琴.Omi在离心运动诱导骨骼肌细胞凋亡及针刺干预中的作用[D].北京:北京体育大学,2014:1-16
[37]BIRAL D,JAKUBIEC-PUKA A,CIECHOMSKA I,et al.Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension[J].Acta Neuropathologica,2000,100(6):618-626
[38]KIME H,SOHN S,KWON H J,et al.Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells[J].Cancer Research,2007,67(13):6314-6324
[39]CHAKRABARTI L,ENG J,IVANOV N,et al.Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuronal death[J].Molecular Brain,2009,2:24
[2]GREEN D R.Apoptosis:Death deceiver[J].Nature,1998,396(6712):629-630
[3]RAJ D A,BOOKER T S,BELCASTRO A N.Striated muscle calcium-stimulated cysteine protease(calpain-like)activity promotes myeloperoxidase activity with exercise[J].Pflügers Archiv,1998,435(6):804-809
[4]HO T T,WARR MR,ADELMAN E R,et al.Autophagy maintains the metabolism and function of young and old stem cells[J].Nature,2017,543(7644):205-210
[5]GARCIA-PRAT L,MARTINEZ-VICENTE M,PERDIGUERO E,et al.Autophagy maintains stemness by preventing senescence[J].Nature,2016,529(7584):37-42
[6]TAN T,ZIMMERMANN M,REICHERT A S.Controlling quality and amount of mitochondria by mitophagy:Insights into the role of ubiquitination and deubiquitination[J].Biological Chemistry,2016,397(7):637-647
[7]YOULE R J,NARENDRA D P.Mechanisms of mitophagy[J].Nature Reviews Molecular Cell Biology,2011,12(1):9-14
[8]WESTERMANN B.Mitochondrial fusion and fission in cell life and death[J].Nature Reviews Molecular Cell Biology,2010,11(12):872-884
[9]KIMI,RODRIGUEZ-ENRIQUEZ S,LEMASTERS J J.Selective degradation of mitochondria by mitophagy[J].Archives of Biochemistry and Biophysics,2007,462(2):245-253
[10]YOSHII S R,MIZUCSHIMA N.Autophagy machinery in the context of mammalian mitophagy[J].Biochimica et Biophysica Acta,2015,1853(10):2797-2801
[11]PICKRELL A M,YOULE R J.The role of PINK1,parkin,and mitochondrial fidelity in Parkinson’s disease[J].Neuron,2015,85(2):257-273
[12]PARK J,LEE S B,LEE S,et al.Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by Parkin[J].Nature,2006,441(7097):1157-1161
[13]CLARK I E,DODSON MW,JIANG C,et al.Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin[J].Nature,2006,441(7097):1162-1166
[14]YANG Y,GEHRKE S,IMAI Y,et al.Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila PINK1is rescued by Parkin[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(28):10793-10798
[15]ARMSTRONG R B,OGILVIE R W,SCHWANE J A.Eccentric exercise-induced injury to rat skeletal muscle[J].Journal of Applied Physiology:Respiratory,Environmental and Exercise Physiology,1983,54(1):80-93
[16]黄志辉,刘浩,李宪航.有氧运动对衰老大鼠心肌、肝脏细胞凋亡及线粒体膜电位的影响[J].西安体育学院学报,2007,28(1):78-80
[17]刘慧君,姜宁,赵斐,等.急性运动中骨骼肌线粒体移动相关基因表达与线粒体动力学的关系[J].天津体育学院学报,2010,25(2):118-121
[18]DAS S,MITROVSKY G,VASANTHI H R,et al.Antiaging properties of a grape-derived antioxidant are regulated by mitochondrial balance of fusion and fission leading to mitohphagy triggered by a signaling network of Sirt1-Sirt3-Foxo3-PINK1-PARKIN[J].Oxidative Medicine and Cellular Longevity,2014,2014:345105
[19]孙良,赵刚.运动性骨骼肌损伤后不同时刻大鼠肱三头肌超微结构及肌酸激酶的变化[J].沈阳体育学院学报,2015,34(3):74-78
[20]高晓娟.急性离心运动及针刺干预对骨骼肌细胞外基质的影响[D].北京:北京体育大学,2011:1-23
[21]张媛,漆正堂,郭维,等.耐力训练对高脂膳食大鼠骨骼肌线粒体脂肪氧化及PGC-1α基因表达的影响[J].天津体育学院学报,2010,25(3):193-196
[22]李世成,焦海舟.大鼠离心运动后骨骼肌微细损伤机制的研究[J].湛江师范学院学报,2006,27(3):90-95
[23]李雪,袁琼嘉,熊若虹.不同负荷运动对大鼠大脑皮质超微结构的影响[J].成都体育学院学报,2006,32(3):111-113
[24]董贵俊,吕晨曦,葛新发,等.一次和重复大强度离心运动前后大鼠骨骼肌超微结构变化[J].中国运动医学杂志,2013,32(2):142-148
[25]陈彩珍,卢健,苏有存.抗阻训练对D-半乳糖衰老模型大鼠骨骼肌线粒体膜的影响[J].西安体育学院学报,2011,28(1):83-86
[26]WATTS J A,KLINE J A,THORNTON L R,et al.Metabolic dysfunction and depletion of mitochondria in hearts of septic rats[J].Journal of Molecular and Cellular Cardiology,2004,36(1):141-150
[27]HARGREAVES I P,DUNCAN A J,WU L,et al.Inhibition of mitochondrial complexⅣleads to secondary loss complexⅡ-Ⅲactivity:Implications for the pathogenesis and treatment of mitochondrial encephalomyopathies[J].Mitochondrion,2007,7(4):284-287
[28]SANCHEZ A M,BERNARDI H,PY G,et al.Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise[J].American Journal of Physiology.Regulatory,Integrative and Comparative Physiology,2014,307(8):956-969
[29]NEEL B A,LIN Y,PESSIN J E.Skeletal muscle autophagy:A newmetabolic regulator[J].Trends in Endocrinology and Metabolism:TEM,2013,24(12):635-643
[30]TOLKOVSKY A M.Mitophagy[J].Biochimica et Biophysica Acta,2009,1793(9):1508-1515
[31]VAINSHTEIN A,TRYON LD,PAULY M,et al.Role of PGC-1αduring acute exercise-induced autophagy and mitophagy in skeletal muscle[J].American Journal of Physiology Cell Physiology,2015,308(9):710-719
[32]樊申元,靳二辉.耐力训练对帕金森模型小鼠中脑线粒体自噬相关基因表达的影响[J].中国康复医学杂志,2015,30(5):437-442
[33]崔迪,邱守涛,王海燕,等.耐力运动对营养性肥胖小鼠骨骼肌细胞自噬及线粒体自噬的影响[J].体育科学,2014,34(12):63-71
[34]崔迪,贺杰,孙婧瑜,等.耐力训练与p53抑制剂PFT-α对小鼠骨骼肌线粒体自噬相关基因表达的影响[J].天津体育学院学报,2013,28(5):422-426
[35]KIMY,PARK J,KIMS,et al.PINK1 controls mitochondrial localization of Parkin through direct phosphorylation[J].Biochemical and Biophysical Research Communications,2008,377(3):975-980
[36]赵晓琴.Omi在离心运动诱导骨骼肌细胞凋亡及针刺干预中的作用[D].北京:北京体育大学,2014:1-16
[37]BIRAL D,JAKUBIEC-PUKA A,CIECHOMSKA I,et al.Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension[J].Acta Neuropathologica,2000,100(6):618-626
[38]KIME H,SOHN S,KWON H J,et al.Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells[J].Cancer Research,2007,67(13):6314-6324
[39]CHAKRABARTI L,ENG J,IVANOV N,et al.Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuronal death[J].Molecular Brain,2009,2:24
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |