MFN2对骨骼肌线粒体运动适应的作用研究
发布时间：2018-09-06 12:22
【摘要】：骨骼肌为主要的运动应答器官,运动中骨骼肌对能量的需求不断地变化,线粒体在应答运动应激引起的氧耗、ATP需求增加的同时,自身形态、结构、功能都发生着积极适应变化,运动促进线粒体生物发生、提高线粒体功能的同时还促进细胞自噬、线粒体自噬等逆向适应,加速细胞新陈代谢和线粒体网络的更新。线粒体是高度动态化细胞器,不断地进行着融合、分裂,线粒体融合、分裂不但在调控线粒体形态、功能以及对凋亡信号的应答中扮演着重要的角色,还与线粒体生物发生和线粒体自噬过程紧密偶联。MFN2不但调控线粒体外膜的融合,还调控线粒体与内质网的相互作用,影响线粒体对Ca~(2+)的摄取和细胞能量代谢。MFN2还在E3泛素连接酶Parkin介导的线粒体自噬中起重要作用,MFN2缺失抑制了Parkin向功能紊乱线粒体的定位；同时,MFN2缺失还提高了PGC-1α的表达,提高线粒体生物发生。MFN2在运动调控骨骼肌线粒体生理适应中的作用尚不清楚,本研究着重从线粒体生物发生、线粒体自噬和线粒体融合、分裂三个角度探索阻断MFN2对运动训练调控骨骼肌线粒体运动适应的影响和相关分子机制。目的：本文选取MCK-Cre MFN2~(flox/flox)小鼠为研究对象,探索MFN2在骨骼肌线粒体自噬和线粒体生物发生中的作用,探索阻断MFN2对运动调控骨骼肌线粒体生理适应的影响,主要通过线粒体生物发生、线粒体自噬和线粒体融合、分裂三个角度探讨运动调控骨骼肌线粒体生理适应的机制。方法：通过Cre/Loxp技术构建条件性骨骼肌MFN2基因敲除小鼠及同窝对照小鼠,基因型分别为MCK-Cre MFN2~(flox/flox)和MFN2~(flox/flox)。1)动物水平对MFN2基因与骨骼肌线粒体自噬和线粒体生物发生的关系进行探索。实验分组：3月龄雄性MCK-Cre MFN2~(flox/flox)小鼠和同窝MFN2~(flox/flox)小鼠分别作为基因敲除组(KKnockout group, K, n=24)和正常对照组(Control group, C, n=24)。每组随机选取8只小鼠分离双侧股四头肌、腓肠肌和胫骨前肌,左侧用于RT-qPCR实验,右侧用于Western blotting实验；每组再随机选取8只小鼠,分离双侧腓肠肌和股四头肌,左侧4%多聚甲醛固定,制作石蜡切片用于HE染色和免疫组织化学指标检测,右侧2.5%戊二醛电镜固定液固定,用于电镜检测线粒体形态、自噬小体等；每组再随机选取8只小鼠,分离双侧腓肠肌用于提取线粒体,检测线粒体膜电位、柠檬酸酶(CS)活性、H_2O_2释放,部分样品用于骨骼肌氧化应激指标检测。2)动物水平对MFN2与骨骼肌线粒体运动适应的关系进行探索。实验分组：2月龄MCK-Cre MFN2~(flox/flox)小鼠和同窝MFN2~(flox/flox)小鼠随机分为正常对照组(Control group,C, n=8)、正常运动组(Exercise group, E, n=8)、基因敲除组(Knockout group,K, n=8)、基因敲除运动组(Knockout Exercise group, KE, n=8)。 E和KE组小鼠进行4周耐力跑台训练,跑速为14m/min, C和K组置于静止跑台,自由活动,最后一次运动训练结束12h处死小鼠分离双侧腓肠肌,左侧用于RT-qPCR实验,右侧用于Western blotting口氧化应激指标检测。本研究检测指标具体如下：1)代谢相关指标：体重、附睾脂肪含量及体质百分比、肝脏和心脏湿重。2)线粒体融合、分裂相关指标：RT-qPCR检测MFN2、MFN1、Drp1、FIS1、OPA1、 MFF mRNA表达；Western blotting检测MFN2、MFN1、OPA1、Drp1、MFF蛋白表达。3)细胞自噬和线粒体自噬相关指标：RT-qPCR检测Atg5、Atg12、ULK1、Parkin、PINK1. LC3、p62、BNIP3、FUNDC1、LAMP2 mRNA表达；Western blotting检测ULK1、Parkin、 PINK1、LC3、BNIP3、p62、Atg5、AMPKa蛋白表达以及AMPKa Thr~(172)和ULK1Ser~(555)磷酸化水平。4)线粒体生物发生相关指标：RT-qPCR检测PGC-1α、TFAM、NRF1、TFBIM、ATP5a. COXI、COX5b. NDUFS8 mRNA表达,mtDNA含量；Western blotting检测PGC-1α、 TFAM、NRF1、TOM20蛋白表达。5)线粒体功能指标：检测线粒体膜电位、CS活性,免疫组化COX和SDH染色。6)形态学指标：HE染色观察肌纤维形态和横截面积,电镜检测肌纤维形态、线粒体形态以及自噬小体等。7)氧化应激指标：检测离体线粒体H_2O_2释放和在体骨骼肌H_2O_2和MDA含量,T-SOD活性。结果：1)通过Cre/Loxp技术建立了骨骼肌特异性MFN2基因敲除小鼠模型,骨骼肌MFN2 mRNA和蛋白含量较比对照组均显著下降(p0.01)。2)与C组相比,K组小鼠骨骼肌中异常线粒体增加,部分线粒体出现肿胀,有的嵴折叠下降,甚至出现不规则的空泡；小鼠骨骼肌Drp1蛋白含量显著提高(p0.05)。与C组相比,K组小鼠骨骼肌线粒体膜电位显著下降(p0.05),线粒体H_2O_2释放率显著高于C组(p0.05),骨骼肌H_2O_2和MDA含量均显著高于C组(p0.05)。与C组相比,K组小鼠骨骼肌中PGC-1α、TFAM和线粒体标志物TOM20蛋白含量显著提高(p0.05)。3)与C组相比,K组小鼠骨骼肌FUNDC1、BNIP3 mRNA表达显著提高(p0.05),BNIP3、 Parkin、p62蛋白含量显著增加(p0.05), LC3-Ⅱ/Ⅰ比值显著提高(p0.05), AMPKαThr~(172)和ULK1Ser~(555)位点磷酸化水平显著下降(p0.05),肌纤维中自噬小体堆积。4)与C组相比,E组小鼠骨骼肌MFN1、MFN2、Drp1、FIS1mRNA表达显著提高(p0.05),MFN1、Drp1蛋白含量显著提高(p0.05)；与K组相比,KE组小鼠MFN1、MFN2、Drp1 FIS1mRNA表达显著提高(p0.05), MFN1、Drp1蛋白含量显著提高(p0.05)；KE组小鼠骨骼肌Drp1蛋白含量显著高于E组(p0.05)。5)与C组相比,E组小鼠骨骼肌PGC-1α、NRF1、TFAM、TFB1M、COXI、COX5b mRNA表达显著提高(P0.05或P0.01),ntDNA含量、PGC-1α、TFAM 和 TOM20蛋白含量显著提高(P0.05)；与K组相比,KE组小鼠骨骼肌PGC-1α、NRF1、TFAM、TFB1M、 COXI、COX5bmRNA表达显著提高(P0.05或P0.01), PGC-1α、TFAM和TOM20蛋白含量显著提高(P0.05)；KE组小鼠骨骼肌TFAM蛋白含量显著高于E组(P0.05)。6)与C组相比,E组小鼠骨骼肌LC3、Atg5、ULK1、BNIP3、Parkin、FUNDC1 mRNA表达显著提高(P0.05或P0.01),LC3-II/I匕值显著提高(P0.05), Parkin、PINK1、BNIP3.ULKl蛋白含量显著提高(P0.05), AMPKαThr~(172)、ULK1 Ser~(555)位点的磷酸化显著提高(P0.05)；与E组相比,KE组小鼠骨骼肌LC3、Atg5、ULK1、BNIP3、FUNDC1 mRNA表达显著提高(P0.05),LC3-Ⅱ/Ⅰ比值、p62、PINK1、ULK1蛋白含量没有显著变化,Parkin、BNIP3、AMPKαThr~(172)、ULK1 Ser~(555)位点的磷酸化显著提高(P0.05)；KE组小鼠骨骼肌BNIP3、FUNDC1 mRNA表达以及Parkin、BNIP3、p62蛋白含量显著高于E组(p0.05)。7)与C组相比,E组小鼠T-SOD活性显著提高(p0.05),MDA显著降低(p0.05)；与K组相比,KE组小鼠T-SOD活性显著提高(p0.05)；KE组小鼠骨骼肌H2O2、MDA显著高于E组(p0.05或p0.01)。结论：1)MFN2缺失扰乱了骨骼肌线粒体融合、分裂动态平衡,引起异常形态线粒体增加,提高线粒体活性氧的产生和骨骼肌氧化应激。2)MFN2缺失提高了骨骼肌线粒体生物发生,有利于维持mtDNA含量和线粒体呼吸链功能。3)MFN2缺失抑制了骨骼肌细胞自噬和Parkin介导线粒体自噬,但代偿性提高了BNIP3依赖的线粒体自噬途径。4)耐力训练建立更高水平的骨骼肌线粒体融合、分裂,伴随着线粒体生物发生、细胞自噬和线粒体自噬的提高,提示运动训练对调控骨骼肌线粒体质量控制有积极作用,MFN2的缺失虽然抑制了运动训练诱导的骨骼肌细胞自噬和Parkin介导的线粒体自噬,但进一步提高了线粒体生物发生和BNIP3介导的线粒体自噬。5)耐力训练并不能通过激活AMPK-ULK1信号通路改善MFN2缺失小鼠骨骼肌细胞自噬水平。
[Abstract]:Skeletal muscle is the main motor response organ. The energy requirement of skeletal muscle is constantly changing during exercise. Mitochondria respond to the oxygen consumption and ATP requirement caused by exercise stress. At the same time, their morphology, structure and function have been actively adapted to changes. Exercise promotes mitochondrial biogenesis, improves mitochondrial function and promotes cells at the same time. Autophagy, mitochondrial autophagy and other adverse adaptation, accelerate cell metabolism and mitochondrial network renewal. Mitochondria are highly dynamic organelles, constantly fusing, dividing, mitochondrial fusion, division not only in the regulation of mitochondrial morphology, function and response to apoptotic signals play an important role, but also with mitochondrial organisms. MFN2 not only regulates the membrane fusion of mitochondria, but also the interaction between mitochondria and endoplasmic reticulum. MFN2 also affects the uptake of Ca ~ (2+) by mitochondria and cellular energy metabolism. MFN2 also plays an important role in E3 ubiquitin ligase Parkin-mediated mitochondrial autophagy. MFN2 deletion inhibits Parkin-directed functional turbulence. At the same time, the deletion of MFN2 also increased the expression of PGC-1a and mitochondrial biogenesis. The role of MFN2 in regulating the physiological adaptation of skeletal muscle mitochondria is still unclear. This study focused on mitochondrial biogenesis, mitochondrial autophagy and mitochondrial fusion, and mitosis to explore the effect of blocking MFN2 on exercise training regulation. Objective: To investigate the effects of MFN2 on mitochondrial autophagy and mitochondrial biogenesis in skeletal muscle of MCK-Cre MFN2 ~ (flox/flox) mice, and to explore the effects of blocking MFN2 on mitochondrial adaptation to exercise, mainly through mitochondria. Methods: The conditioned skeletal muscle MFN2 knockout mice and the homologous control mice were constructed by Cre/Loxp technique. The genotypes were MCK-Cre MFN2~ (flox/flox) and MFN2~ (flox/flox). 1) at animal level. The relationship between MFN2 gene and skeletal muscle mitochondrial autophagy and mitochondrial biogenesis was explored. The experimental groups were divided into 3-month-old male MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice as gene knockout group (KKnockout group, K, n=24) and normal control group (control group, C, n=24). The left quadriceps femoris, gastrocnemius and anterior tibial muscles were used for RT-q PCR and the right for Western blotting. Eight mice in each group were randomly selected to separate the bilateral gastrocnemius and quadriceps femoris. The left quadriceps femoris were fixed with 4% paraformaldehyde, and paraffin sections were made for HE staining and immunohistochemical detection. Fixed solution was used for electron microscopic examination of mitochondrial morphology and autophagy, and 8 mice in each group were randomly selected to isolate bilateral gastrocnemius muscle for extraction of mitochondria, detection of mitochondrial membrane potential, citrate enzyme (CS) activity, H_2O_2 release, and some samples for detection of skeletal muscle oxidative stress indicators. 2) Animal level of MFN2 and skeletal muscle mitochondria mitochondria. The experimental group: 2-month-old MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice were randomly divided into normal control group (control group, C, n = 8), normal exercise group (Exercise group, E, n = 8), gene knockout group (Knockout group, K, n = 8), gene knockout exercise group (Knockout Exercise group, KE, n = 8). 8) The mice in E and KE groups were trained on the treadmill for 4 weeks at a running speed of 14 m/min. The mice in C and K groups were placed on the stationary treadmill to move freely. At the end of the last exercise training, the mice were sacrificed to separate bilateral gastrocnemius muscles. The left side was used for RT-qPCR test, and the right side was used for the detection of oxidative stress in Western blotting mouth. Relevant indicators: body weight, epididymal fat content and body mass percentage, liver and heart wet weight. 2) Mitochondrial fusion, mitosis related indicators: RT-qPCR detection of MFN2, MFN1, Drp1, FIS1, OPA1, MFF mRNA expression; Western blotting detection of MFN2, MFN1, OPA1, Drp1, MFF protein expression. 3) cell autophagy and mitochondrial autophagy related indicators: RT-qPCR detection of Atg5, Drp1, MFF protein expression. Atg12, ULK12, ULK1, Parkin, PINK1.LC3, p62, BNIP3, BNIP3, FUNDC1, FUNDC1, LAMP2 mRNAexpression; Western blotdetection ULK1, Parkin, PINK1, LC3, BNIP3, p62, Atg5, AMPKa protein expression and AMPKa Thr ~ (172) and ULK1 Ser ~ (555) phosphophosphorylation levels. 4) Mitochondriabiogenerelated indicators: RT-qPCR detection of PGC-1alpha, TFAM, TFAM, NRF1, NRF1, TFBIM, TFBIM, LCP5a, COCOCOP5a. COCOCOA. COCOCOA. COXFS8.5, COX 8.RN Mitochondrial function index: Mitochondrial membrane potential, CS activity, immunohistochemical COX and SDH staining. 6) Morphological index: HE staining to observe muscle fiber morphology and cross-sectional area, electron microscopy to detect muscle fiber morphology, mitochondrial morphology and autophagy. 7) Oxidative stress index: The release of H_2O_2 from mitochondria in vitro, the content of H_2O_2 and MDA in skeletal muscle in vivo, and the activity of T-SOD were detected. Results: 1) The skeletal muscle-specific MFN2 knockout mice model was established by Cre/Loxp technique. The content of MFN2 mRNA and protein in skeletal muscle of K group was significantly lower than that of control group (p0.01). The content of Drp1 protein in skeletal muscle of mice was significantly increased (p0.05). Compared with group C, the mitochondrial membrane potential of skeletal muscle of mice in group K was significantly decreased (p0.05), the release rate of H_2O_2 in mitochondria was significantly higher than that of group C (p0.05). Compared with group C, the contents of PGC-1a, TFAM and mitochondrial marker TOM20 protein in skeletal muscle of mice in group K were significantly increased (p0.05). Compared with group C, the expressions of FUNDC1 and BNIP3 mRNA in skeletal muscle of mice in group K were significantly increased (p0.05), the contents of BNIP3, Parkin and p62 protein were significantly increased (p0.05), and the ratio of LC3-II/I was significantly increased (p0.05). Compared with group C, the expression of MFN1, MFN2, Drp1, FIS1 mRNA in skeletal muscle of mice in group E was significantly increased (p0.05), and the expression of MFN1, MFN2, and P1 FIS1 mRNA in skeletal muscle of mice in group K was significantly increased (p0.05). Compared with group C, the expression of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in group E increased significantly (P 0.05 or P 0.01), and the content of ntDNA, PGC-1a, TFAM and TOM20 protein in group K increased significantly (P 0.05). Compared with KE group, the expressions of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in KE group were significantly increased (P 0.05 or P 0.01), and the contents of PGC-1a, TFAM and TOM20 protein were significantly increased (P 0.05); the contents of TFAM protein in skeletal muscle of mice in KE group were significantly higher than those in E group (P 0.05). LC3-II/I dagger value was significantly increased (P 0.05), Parkin, PINK1, BNIP 3.ULKl protein content was significantly increased (P 0.05), AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) phosphorylation was significantly increased (P 0.05), compared with E group, the expression of LC3, Atg5, ULK1, BNIP 3, FUNDC 1 mRNA in skeletal muscle of KE group was significantly increased (P 0.05), LC3-II/I ratio, p62, p62, ULK1 Ser ~ (555). PINK1, ULK1 protein content did not change significantly, Parkin, BNIP3, AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) site phosphorylation significantly increased (P 0.05); KE group mice skeletal muscle BNIP3, FUNDC1 mRNA expression and Parkin, BNIP3, p62 protein content significantly higher than E group (p 0.05). 7 compared with C group, E group mice T-SOD activity significantly increased (p 0.05), MDA significantly decreased (p 0.05). Compared with K group, the activity of T-SOD in KE group was significantly increased (p0.05); H2O2 and MDA in skeletal muscle of KE group were significantly higher than those of E group (p0.05 or p0.01). Conclusion: 1) MFN2 deficiency disturbed mitochondrial fusion, mitotic homeostasis, increased abnormal mitochondrial morphology, increased production of reactive oxygen species and oxidative stress in skeletal muscle. Loss of MFN2 inhibits autophagy of skeletal muscle cells and mitochondrial autophagy mediated by Parkin, but compensates for BNIP3-dependent mitochondrial autophagy pathway. 4) Endurance training establishes a higher level of mitochondrial fusion and division in skeletal muscle. With the development of mitochondrial biogenesis, autophagy and mitochondrial autophagy increased, suggesting that exercise training plays an active role in regulating the quality control of skeletal muscle mitochondria. Although the deletion of MFN2 inhibits the autophagy of skeletal muscle cells induced by exercise training and the autophagy of mitochondria mediated by Parkin, it further improves mitochondrial biogenesis and BNIP3. Mediated mitochondrial autophagy.5) Endurance training can not improve the autophagy level of skeletal muscle cells in MFN2-deficient mice by activating AMPK-ULK1 signaling pathway.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：G804.2
，
本文编号：2226335
[Abstract]:Skeletal muscle is the main motor response organ. The energy requirement of skeletal muscle is constantly changing during exercise. Mitochondria respond to the oxygen consumption and ATP requirement caused by exercise stress. At the same time, their morphology, structure and function have been actively adapted to changes. Exercise promotes mitochondrial biogenesis, improves mitochondrial function and promotes cells at the same time. Autophagy, mitochondrial autophagy and other adverse adaptation, accelerate cell metabolism and mitochondrial network renewal. Mitochondria are highly dynamic organelles, constantly fusing, dividing, mitochondrial fusion, division not only in the regulation of mitochondrial morphology, function and response to apoptotic signals play an important role, but also with mitochondrial organisms. MFN2 not only regulates the membrane fusion of mitochondria, but also the interaction between mitochondria and endoplasmic reticulum. MFN2 also affects the uptake of Ca ~ (2+) by mitochondria and cellular energy metabolism. MFN2 also plays an important role in E3 ubiquitin ligase Parkin-mediated mitochondrial autophagy. MFN2 deletion inhibits Parkin-directed functional turbulence. At the same time, the deletion of MFN2 also increased the expression of PGC-1a and mitochondrial biogenesis. The role of MFN2 in regulating the physiological adaptation of skeletal muscle mitochondria is still unclear. This study focused on mitochondrial biogenesis, mitochondrial autophagy and mitochondrial fusion, and mitosis to explore the effect of blocking MFN2 on exercise training regulation. Objective: To investigate the effects of MFN2 on mitochondrial autophagy and mitochondrial biogenesis in skeletal muscle of MCK-Cre MFN2 ~ (flox/flox) mice, and to explore the effects of blocking MFN2 on mitochondrial adaptation to exercise, mainly through mitochondria. Methods: The conditioned skeletal muscle MFN2 knockout mice and the homologous control mice were constructed by Cre/Loxp technique. The genotypes were MCK-Cre MFN2~ (flox/flox) and MFN2~ (flox/flox). 1) at animal level. The relationship between MFN2 gene and skeletal muscle mitochondrial autophagy and mitochondrial biogenesis was explored. The experimental groups were divided into 3-month-old male MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice as gene knockout group (KKnockout group, K, n=24) and normal control group (control group, C, n=24). The left quadriceps femoris, gastrocnemius and anterior tibial muscles were used for RT-q PCR and the right for Western blotting. Eight mice in each group were randomly selected to separate the bilateral gastrocnemius and quadriceps femoris. The left quadriceps femoris were fixed with 4% paraformaldehyde, and paraffin sections were made for HE staining and immunohistochemical detection. Fixed solution was used for electron microscopic examination of mitochondrial morphology and autophagy, and 8 mice in each group were randomly selected to isolate bilateral gastrocnemius muscle for extraction of mitochondria, detection of mitochondrial membrane potential, citrate enzyme (CS) activity, H_2O_2 release, and some samples for detection of skeletal muscle oxidative stress indicators. 2) Animal level of MFN2 and skeletal muscle mitochondria mitochondria. The experimental group: 2-month-old MCK-Cre MFN2~ (flox/flox) mice and the same litter MFN2~ (flox/flox) mice were randomly divided into normal control group (control group, C, n = 8), normal exercise group (Exercise group, E, n = 8), gene knockout group (Knockout group, K, n = 8), gene knockout exercise group (Knockout Exercise group, KE, n = 8). 8) The mice in E and KE groups were trained on the treadmill for 4 weeks at a running speed of 14 m/min. The mice in C and K groups were placed on the stationary treadmill to move freely. At the end of the last exercise training, the mice were sacrificed to separate bilateral gastrocnemius muscles. The left side was used for RT-qPCR test, and the right side was used for the detection of oxidative stress in Western blotting mouth. Relevant indicators: body weight, epididymal fat content and body mass percentage, liver and heart wet weight. 2) Mitochondrial fusion, mitosis related indicators: RT-qPCR detection of MFN2, MFN1, Drp1, FIS1, OPA1, MFF mRNA expression; Western blotting detection of MFN2, MFN1, OPA1, Drp1, MFF protein expression. 3) cell autophagy and mitochondrial autophagy related indicators: RT-qPCR detection of Atg5, Drp1, MFF protein expression. Atg12, ULK12, ULK1, Parkin, PINK1.LC3, p62, BNIP3, BNIP3, FUNDC1, FUNDC1, LAMP2 mRNAexpression; Western blotdetection ULK1, Parkin, PINK1, LC3, BNIP3, p62, Atg5, AMPKa protein expression and AMPKa Thr ~ (172) and ULK1 Ser ~ (555) phosphophosphorylation levels. 4) Mitochondriabiogenerelated indicators: RT-qPCR detection of PGC-1alpha, TFAM, TFAM, NRF1, NRF1, TFBIM, TFBIM, LCP5a, COCOCOP5a. COCOCOA. COCOCOA. COXFS8.5, COX 8.RN Mitochondrial function index: Mitochondrial membrane potential, CS activity, immunohistochemical COX and SDH staining. 6) Morphological index: HE staining to observe muscle fiber morphology and cross-sectional area, electron microscopy to detect muscle fiber morphology, mitochondrial morphology and autophagy. 7) Oxidative stress index: The release of H_2O_2 from mitochondria in vitro, the content of H_2O_2 and MDA in skeletal muscle in vivo, and the activity of T-SOD were detected. Results: 1) The skeletal muscle-specific MFN2 knockout mice model was established by Cre/Loxp technique. The content of MFN2 mRNA and protein in skeletal muscle of K group was significantly lower than that of control group (p0.01). The content of Drp1 protein in skeletal muscle of mice was significantly increased (p0.05). Compared with group C, the mitochondrial membrane potential of skeletal muscle of mice in group K was significantly decreased (p0.05), the release rate of H_2O_2 in mitochondria was significantly higher than that of group C (p0.05). Compared with group C, the contents of PGC-1a, TFAM and mitochondrial marker TOM20 protein in skeletal muscle of mice in group K were significantly increased (p0.05). Compared with group C, the expressions of FUNDC1 and BNIP3 mRNA in skeletal muscle of mice in group K were significantly increased (p0.05), the contents of BNIP3, Parkin and p62 protein were significantly increased (p0.05), and the ratio of LC3-II/I was significantly increased (p0.05). Compared with group C, the expression of MFN1, MFN2, Drp1, FIS1 mRNA in skeletal muscle of mice in group E was significantly increased (p0.05), and the expression of MFN1, MFN2, and P1 FIS1 mRNA in skeletal muscle of mice in group K was significantly increased (p0.05). Compared with group C, the expression of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in group E increased significantly (P 0.05 or P 0.01), and the content of ntDNA, PGC-1a, TFAM and TOM20 protein in group K increased significantly (P 0.05). Compared with KE group, the expressions of PGC-1a, NRF1, TFAM, TFB1M, COXI, COX5b mRNA in skeletal muscle of mice in KE group were significantly increased (P 0.05 or P 0.01), and the contents of PGC-1a, TFAM and TOM20 protein were significantly increased (P 0.05); the contents of TFAM protein in skeletal muscle of mice in KE group were significantly higher than those in E group (P 0.05). LC3-II/I dagger value was significantly increased (P 0.05), Parkin, PINK1, BNIP 3.ULKl protein content was significantly increased (P 0.05), AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) phosphorylation was significantly increased (P 0.05), compared with E group, the expression of LC3, Atg5, ULK1, BNIP 3, FUNDC 1 mRNA in skeletal muscle of KE group was significantly increased (P 0.05), LC3-II/I ratio, p62, p62, ULK1 Ser ~ (555). PINK1, ULK1 protein content did not change significantly, Parkin, BNIP3, AMPK alpha Thr ~ (172), ULK1 Ser ~ (555) site phosphorylation significantly increased (P 0.05); KE group mice skeletal muscle BNIP3, FUNDC1 mRNA expression and Parkin, BNIP3, p62 protein content significantly higher than E group (p 0.05). 7 compared with C group, E group mice T-SOD activity significantly increased (p 0.05), MDA significantly decreased (p 0.05). Compared with K group, the activity of T-SOD in KE group was significantly increased (p0.05); H2O2 and MDA in skeletal muscle of KE group were significantly higher than those of E group (p0.05 or p0.01). Conclusion: 1) MFN2 deficiency disturbed mitochondrial fusion, mitotic homeostasis, increased abnormal mitochondrial morphology, increased production of reactive oxygen species and oxidative stress in skeletal muscle. Loss of MFN2 inhibits autophagy of skeletal muscle cells and mitochondrial autophagy mediated by Parkin, but compensates for BNIP3-dependent mitochondrial autophagy pathway. 4) Endurance training establishes a higher level of mitochondrial fusion and division in skeletal muscle. With the development of mitochondrial biogenesis, autophagy and mitochondrial autophagy increased, suggesting that exercise training plays an active role in regulating the quality control of skeletal muscle mitochondria. Although the deletion of MFN2 inhibits the autophagy of skeletal muscle cells induced by exercise training and the autophagy of mitochondria mediated by Parkin, it further improves mitochondrial biogenesis and BNIP3. Mediated mitochondrial autophagy.5) Endurance training can not improve the autophagy level of skeletal muscle cells in MFN2-deficient mice by activating AMPK-ULK1 signaling pathway.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：G804.2
，
本文编号：2226335
本文链接：https://www.wllwen.com/shoufeilunwen/sklbs/2226335.html
