线粒体自噬参与大鼠骨骼肌钝挫伤能量代偿的理论研究

发布时间：2018-02-28 22:36

本文关键词： 骨骼肌钝挫伤线粒体自噬骨骼肌超微结构缺氧 AMPKα2 HIF-1α BNIP3 NIX 活性氧线粒体电子传递链酶复合体 ATP合成酶　出处：《上海体育学院》2016年博士论文　论文类型：学位论文

【摘要】：研究目的:骨骼肌钝挫伤发生后,受损骨骼肌除了发生经典的血管反应、炎症反应外,其内部还会形成缺氧环境,这种环境形成后可能引起线粒体功能降低,导致ATP产生不足,最终势必会使损伤的恢复时程延长,功能恢复不完全,甚至丧失正常解剖结构,预后较差。现有研究已证实,线粒体自噬是维持线粒体质量的重要力量,也是机体对代谢改变做出的一种重要的代偿性反应,该过程可及时清除损伤、功能障碍的线粒体,从而减少ROS的生成,而过多的ROS会直接损害健康线粒体,那么及时清除破损线粒体就可以防止损伤范围扩大。根据上述依据提出假设:1.损伤后骨骼肌超微结构发生改变:线粒体形态改变、完整性缺失,可见到线粒体自噬体的存在;2.钝挫伤发生后在骨骼肌恢复过程中ROS水平增高,线粒体功能下降;3.在观察骨骼肌钝挫伤恢复时程内,线粒体自噬被诱导且在损伤早期最为显著,而线粒体功能下降也发生在这一时期。本研究用重物砸伤动物骨骼肌制作动物模型,模拟运动中的骨骼肌钝挫伤,使用透射电镜观察线粒体自噬体及数量、检测受损组织中ROS生成量、对比线粒体呼吸链酶复合体活性及线粒体自噬相关指标的时程性变化等,通过这些指标的变化来观测和评价钝挫伤发生后线粒体自噬、线粒体功能的改变,从一个崭新视角来观察、解释骨骼肌钝挫伤发生、恢复的可能机制,并推测该过程中诱导线粒体自噬发生可能因素,希望可以为今后骨骼肌钝挫伤的临床治疗、康复、预防提供更多的理论支持。研究方法:随机选取64只雄性Wistar大鼠,分为8组:正常对照组以及钝挫伤后12h、2d、5d、7d、10d、15d、30d取材组(h:小时;d:天),每组8只。除对照组外其他各组用重物砸伤造模后按各组所示时间点取材,检测腓肠肌AMPKα2、HIF-1α、NIX、BNIP3蛋白、m RNA表达的变化;用透射电镜观察钝挫伤后骨骼肌超微结构的变化,观察是否存在自噬体及其自噬体数量的时程性改变;检测ROS、线粒体跨膜电位(ΔΨm)、电子传递链酶复合体CⅠ-Ⅳ、ATP合成酶等体现线粒体功能指标的时程性改变。通过检测损伤发生后上述各指标的变化情况,分析该过程中线粒体功能的改变,以及是否在该过程中发生了线粒体自噬,并推断线粒体自噬发生机制、可能产生的代偿后果;线粒体功能是否被损害,推测钝挫伤损伤线粒体功能降低的可能机制及可能产生的后果。研究结果:1透射电镜下观察骨骼肌超微结构在骨骼肌钝挫伤发生后时程性改变:从损伤发生直至伤后30天,均能观察到被双层膜包裹、半包裹的线粒体,伤后10天内更易观察到线粒体自噬体。2代谢相关蛋白AMPKα2、缺氧相关因子HIF-1α在损伤初期都有显著的升高(p0.05),随着时间推移,二者表达从伤后5-7d就基本回落到正常水平,直至伤后30d。就二者m RNA表现来看,只有HIF-1α蛋白表达与其m RNA表达不一致。3线粒体自噬相关蛋白BNIP3、NIX表达水平在伤后12h-5d升高明显(p0.05)HIF-1α表达与二者表现基本接近。此外,在二者水平增高组,电镜下可观察到较多数量被溶酶体包裹的线粒体。4钝挫伤后ΔΨm呈现明显的时程性改变:在损伤早期(12h、2d、5d)ΔΨm显著下降(p0.05),该指标改变同BNIP3、NIX表达增高所在时间段基本吻合。5损伤后12h-5d各组ROS与对照组相比均有升高,且差异显著(p0.05)。6损伤后12h-5d,线粒体电子传递链酶复合体C1、CⅢ活性升高,与对照组相比差异显著(p0.05),这也同ROS水平的升高发生在相同时间段,而另外两个酶复合体活性并未发生显著变化。ATP合成酶的活性在损伤早期下降明显(p0.05),从伤后7d开始逐渐回升至正常水平并保持到伤后30d。结论:1高浓度的ROS可直接攻击线粒体呼吸链造成线粒体损伤,而产生内源性ROS的部位又恰好是在线粒体电子传递链上。ROS升高的阶段与CⅠ、CⅢ活性升高的时间段基本一致,这说明线粒体电子传递链通过影响CⅠ、CⅢ活性直接对缺氧产生反应,CⅠ、CⅢ又是ROS产生的主要部位,故该阶段受损线粒体产生大量ROS,而体内的ROS清除系统无法及时清除过多的ROS。2代谢调节关键因子AMPKα2、缺氧相关因子HIF-1α均在线粒体功能变化期间出现高表达,该阶段更易观察到线粒体自噬体,这说明二者可能从对合成分解代谢的调整和线粒体质量控制方面做出代偿性调节。3线粒体自噬相关蛋白BNIP3、NIX在线粒体功能下降期间出现明显的高表达,同时透射电镜观察结果显示该阶段受损骨骼肌也存在许多被溶酶体全包裹或半包裹的线粒体。这说明在损伤后线粒体功能下降期间发生了线粒体自噬,且参与了机体的调整过程,通过对受损线粒体的清除,达到减少ROS的产生的目的,防止损伤范围扩大。虽然过度的线粒体自噬会引起细胞的凋亡,但就本实验来看,伤侧肢体功能是逐渐恢复的,且并未出现骨骼肌的坏死,说明线粒体自噬程度并未过度,作用是积极的。4正常线粒体跨膜电位(ΔΨm)的维持,是线粒体能否维持功能的关键,该指标的变化也是发生线粒体自噬和细胞凋亡的重要条件。在钝挫伤骨骼肌恢复过程中,ΔΨm表现出明显的时程性改变。在损伤早期(12h、2d、5d)ΔΨm显著下降,该结果结合BNIP3、NIX表达及电镜观察结果来推测,损伤后线粒体自噬被诱导,同时对比线粒体功能下降时间段推测线粒体自噬参与到损伤后的代偿机制中。
[Abstract]:Objective: To study skeletal muscle contusion, damaged skeletal muscle vascular reaction in addition to the classic, inflammatory reaction, but also the formation of its internal anoxic environment, this environment may cause the formation of reduced mitochondrial function, leading to ATP problems, will eventually be restored to the prolonged injury, incomplete functional recovery, and even loss the normal anatomic structure, poor prognosis. Existing studies have confirmed that mitochondrial autophagy is an important force to maintain mitochondrial quality, an important compensatory response is the body to make metabolic changes, the process can be timely removal of damage, mitochondrial dysfunction, to reduce the production of ROS, and too much ROS will directly harm the health of mitochondria then, the timely removal of damaged mitochondria can prevent the damage scope. According to the above basis for assumptions: 1. after the injury of skeletal muscle ultrastructure change: Mitochondria Body shape, lack of integrity, visible to mitochondrial autophagy; 2. in skeletal muscle contusion after recovery of ROS levels in the process of increased mitochondrial dysfunction; 3. in the recovery of skeletal muscle contusion duration, mitochondrial autophagy was induced and the injury was the most significant, but also a decline in mitochondrial function during this period. The study of animal models with bruise animal skeletal muscle, simulate the movement in skeletal muscle contusion, and the number of autophagosomes were observed using transmission electron microscopy, ROS content detection of damaged tissue, comparing related indicators of mitochondrial respiratory chain enzyme complex activity and mitochondrial autophagy duration changes. The changes in these indicators to monitor and evaluate the contusion after the occurrence of mitochondrial autophagy, mitochondrial function changes, from a new perspective, to explain the occurrence of skeletal muscle contusion, recovery The possible mechanism of the complex, and that induced mitochondrial autophagy in this process may occur, the hope can for clinical treatment in skeletal muscle contusion rehabilitation, prevention to provide more theoretical support. Methods: 64 randomly selected male Wistar rats were divided into 8 groups: normal control group and 12h after contusion 2D, 5D, 7d, 10d, 15d, 30d, material group (h: hours; d: days), 8 rats in each group. Except the control group the other groups with bruise after modeling were shown by time were detected in gastrocnemius AMPK alpha 2, alpha HIF-1, NIX, BNIP3 protein, RNA m the change of expression; transmission electron microscope was used to observe the ultrastructure of skeletal muscle after contusion, observe whether there is any history of the number of autophagosomes and autophagy change detection; ROS, mitochondrial membrane potential (delta m), electron transport chain complexes C I - IV, ATP synthase reflects the history of mitochondrial function index changes. The change of damage detection after the occurrence of the above indicators, analysis of mitochondrial function in the process of change, and whether the occurrence of mitochondrial autophagy in this process, and infer the mechanism of mitochondrial autophagy, the possible consequences of compensation; mitochondrial function has been damaged, speculated that the mechanism of functional damage of mitochondria and may reduce blunt contusion the consequences of the results: 1. Transmission electron microscopy was used to observe the ultrastructure of skeletal muscle in the process of changing the occurrence of skeletal muscle contusion: from the injury until 30 days after injury, were observed by double membrane wrapped mitochondria injury within 10 days after it is easier to study mitochondrial autophagy.2 metabolism related protein AMPK alpha 2, alpha HIF-1 hypoxia related factors are significantly increased in the early stage of injury (P0.05), with the passage of time, the expression of two 5-7d after injury from basic back to normal level, until the 30D. after injury two m RNA performance, only HIF-1 expression and expression of M RNA with.3 mitochondrial autophagy related protein BNIP3, NIX expression level at 12h-5d after injury was significantly increased (P0.05) HIF-1 expression and two basic approaches. In addition, in the two group. The level of observation under electron microscopy the number was wrapped in lysosomal mitochondrial.4 after contusion was only m presents the history of change in the early injury (12h, 2D, 5d) was significantly decreased while m (P0.05), the index change with BNIP3, the increased expression of NIX where the time is consistent with the.5 after injury in 12h-5d group and control ROS compared group were increased, and the difference was significant (P0.05).6 12h-5d after injury, the mitochondrial electron transport chain complexes C1, increased the activity of C III, compared with the control group had significant difference (P0.05), this also with high levels of ROS occurred at the same time, while the other two enzyme complex activity Did not change significantly.ATP synthetase activity decreased significantly in the early injury (P0.05), from 7d after injury began to rise gradually to the normal level and kept to 30D. after injury. Conclusion: high concentration of 1 ROS can directly attack the mitochondrial respiratory chain caused by the injury of mitochondria, and the endogenous ROS of the site is just in the mitochondrial electron transfer stage and C chain of.ROS increased 1 time, increased the activity of C III is basically the same, indicating that the mitochondrial electron transport chain by C I, C III activity directly respond to hypoxia, C I, C III is the main part of ROS, the stage of damaged mitochondria produce large amounts of ROS. The in vivo ROS scavenging system can not be timely cleared many key factors regulating the metabolism of ROS.2 AMPK alpha 2, high expression of hypoxia related factor HIF-1 alpha were changes in mitochondrial function during this stage, it is easier to study mitochondrial autophagy, This shows that the two may make compensatory adjustments and from the mitochondrial quality control synthesis metabolism regulation of.3 mitochondrial autophagy related protein BNIP3, NIX decreased during high expression was evident in mitochondrial function, and transmission electron microscopy results showed that the stage of injured skeletal muscle also has many lysosomal coated or semi wrapped mitochondria. This shows that in the injury of mitochondrial dysfunction occurred during mitochondrial autophagy is involved in the adjustment process, and through the body, for the removal of damaged mitochondria, to reduce the production of ROS to prevent the damage scope. Although excessive apoptosis mitochondrial autophagy leads to cell, but in this experiment, the limb function injury side is gradually recovered, and did not appear in skeletal muscle necrosis, indicating mitochondrial autophagy degree are not excessive, the positive effects of.4 normal mitochondrial transmembrane Potential (a ~ m) maintenance, is the key to maintain mitochondrial function, an important condition for change in the index is mitochondrial autophagy and apoptosis in skeletal muscle contusion in the recovery process, while m showed a history of significant changes. In the early injury (12h, 2D, 5d). While m decreased significantly, the results combined with BNIP3, the expression of NIX and electron microscope results suggest that mitochondrial autophagy was induced after injury, compared with a decline in mitochondrial function time that mitochondrial autophagy involved in the compensatory mechanism of injury.

【学位授予单位】：上海体育学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R873

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