氧化应激与线粒体自噬在运动预适应心肌保护效应中的作用及分子机制

发布时间：2018-06-14 09:54

本文选题：运动预适应 + 心脏保护　；参考：《上海体育学院》2017年博士论文

【摘要】：研究目的:运动作为一个强度与负荷的刺激因素,极大地增加了心脏的耗氧量,导致心脏绝对或相对的缺氧。间歇大强度运动造成心脏反复短暂的绝对或相对缺血,与IP(ischemic preconditioning,IP)类似,可以诱导心脏产生内源性保护,减轻随后的急性应激造成的心脏损伤,这种运动方式称为运动预适应(exercise preconditioning,EP)。线粒体保护对心脏维持正常的生理状态至关重要,同时又与氧化应激联系紧密。最近研究表明,细胞自噬与线粒体自噬在心肌中发挥重要的线粒体保护作用,且亦与氧化应激联系紧密。本研究以氧化应激、线粒体、自噬三者为中轴线,结合EP的早、晚两个保护期,以力竭运动作为损伤性应激条件,利用自噬阻滞剂渥曼青霉素(wortmannin),并大量应用蛋白线粒体转位关系实验,深入探讨EP诱导的心脏保护中,氧化应激、线粒体、自噬三者之间作用关系,理清心脏线粒体自噬发生机制。为EP心脏保护及机制研究提供新的理论和实验依据。研究方法:200只健康雄性SD大鼠随机分为10组:C组,对照组;EE组,进行一次大强度力竭跑台运动;EEP组,一次大强度间歇的跑台运动建立EP模型,EP后0.5h取材;LEP组,EP建模后24h取材;EEP+EE组,EP后0.5h进行力竭运动;LEP+EE组,EP后24h进行力竭运动;W+EEP组,EP前0.5h腹腔注射渥曼青霉素,EP后0.5取材;W+LEP组,阻滞剂注射如W+EEP组,EP后24h取材;W+EEP+EE组,阻滞剂注射同前,EP后0.5h进行力竭运动;W+LEP+EE组,阻滞剂注射同前,EP后24h进行力竭运动。应用免疫荧光发光法检测大鼠血浆心肌肌钙蛋白I(c Tn I)水平评价心肌损伤;应用苏木素碱性复红苦味酸染色(HBFP staining)观察和评估心肌缺血缺氧程度;应用透射电镜观察心肌超微结构改变,着重观察线粒体和自噬体;应用分光光度法检测大鼠心肌丙二醛(MDA)、过氧化氢(H2O2)、锰超氧化物歧化酶(Mn SOD)和总超氧化物歧化酶活性,以及免疫印迹法检测Mn SOD含量,评价氧化应激程度;应用Phos-tag法检测电压门控阴离子通道1(VDAC1)磷酸化水平,评价线粒体渗透性转换孔道(m PTP)抑制程度;应用线粒体提取结合免疫印迹法检测,细胞色素C(Cyt-c)、自噬受体p62、线粒体自噬蛋白Parkin和Bnip3,评价上述蛋白在胞浆和线粒体的含量变化;应用免疫印迹法检测自噬关键蛋白Beclin1、Bcl-2、LC3I、LC3II水平,结合Beclin1/Bcl-2、LC3II/LC3I比值计算,评价细胞自噬水平和性质;应用组织免疫荧光双标法结合激光共聚焦技术,检测LC3对线粒体内膜蛋白COX4/1、Parkin对线粒体外膜转运酶TOM70、Bnip3对TOM20的转位水平和上述六种蛋白的荧光表达量和分布。通过上述指标深入揭示氧化应激与线粒体自噬在ep诱导心脏保护中的作用和机制。研究结果:(1)与c组相比,ee组心肌损伤标志物血浆ctni水平、hbfp染色缺血缺氧程度均有显著升高,损伤性超微结构改变明显;eep组和lep组无损伤性改变。与ee组相比,eep+ee和lep+ee组,血浆ctni水平降低、缺血缺氧和超微结构改变均有减轻。与eep+ee组相比,w+eep+ee组血浆ctni水平略有降低,但缺血缺氧未见改变且有升高趋势,线粒体肥大明显。与lep+ee组相比,w+lep+ee组血浆ctni和缺血缺氧均有升高,线粒体损伤明显。(2)与c组相比,ee组氧化应激损伤产物mda显著升高,总sod活性降低,氧化应激损伤明显,但h2o2、mnsod活性和含量无变化;eep组和lep组,mnsod、总sod活性均有升高,无mda升高和mnsod含量变化,eep组h2o2显著减低。与ee组相比,eep+ee组mda明显下降,h2o2、总sod活性明显升高;lep+ee组总sod活性升高,但eep+ee组和lep+ee组sod酶活性两数据均分别较之eep组和lep组有明显下降。与eep+ee组相比,w+eep+ee组mda和mnsod活性均明显增高。与lep+ee组相比,w+lep+ee组h2o2显著升高。(3)与c组相比,ee组和eep+ee组,抑制mptp开放的vdac1磷酸化水平显著升高。与ee组相比,lep+ee组、w+eep+ee组、w+lep+ee组vdac1磷酸化水平降低。与eep+ee组相比,w+eep+ee组vdac1磷酸化水平显著降低,线粒体cyt-c泄漏至胞浆显著增高。(4)与c组相比,ee组lc3ii和lc3ii/lc3i比值显著升高;eep组lc3ii/lc3i比值升高;eep+ee组lc3ii和beclin1含量升高提示细胞自噬增强;w+lep组lc3ii、lc3ii/lc3i比值、beclin1/bcl-2比值升高凋亡性自噬水平增高;w+eep+ee组和w+lep+ee组两种比值均升高自噬为凋亡性。与ee组相比,lep+ee组lc3ii和lc3ii/lc3i显著降低,细胞自噬水平较低;w+eep+ee组beclin1升高。(5)与c组相比,ee组lc3转位线粒体cox4/1百分比、线粒体p62降低;eep组lc3转位显著降低;w+lep组cox4/1荧光强度降低。与ee组相比,eep+ee组lc3转位程度、lc3荧光强度、胞浆p62均显著增高;lep+ee组lc3转位程度升高、lc3和cox4/1荧光强度降低;w+eep+ee组lc3转位程度降低,胞浆和线粒体p62均升高;w+lep+ee组lc3转位、线粒体p62升高,lc3、cox4/1荧光强度显著降低。与eep+ee组相比,w+eep+ee组lc3荧光强度显著降低。(6)与c组相比,ee组线粒体parkin、parkin转位tom70程度、parkin和tom70荧光强度显著降低,胞浆和线粒体bnip3、bnip3转位tom20程度、bnip3荧光强度均显著升高,tom20荧光强度无变化;eep组parkin转位、tom70和tom20荧光强度显著降低,胞浆和线粒体bnip3表达量升高;lep组bnip3转位程度、胞浆和线粒体bnip3水平显著增高;w+eep组、w+lep组分别与eep组和lep组在线粒体自噬指标上接近。与ee组相比,eep+ee组线粒体parkin和parkin转位,parkin、TOM70、Bnip3荧光强度均显著升高,胞浆和线粒体Bnip3水平和EE组接近;LEP+EE组线粒体Parkin含量、Parkin转位和TOM70荧光强度升高,Bnip3相关数据均显著下降;W+EEP+EE组Parkin相关数据均有显著升高,但Parkin转位程度显著低于EEP+EE组,胞浆Bnip3和TOM20显著升高;W+LEP+EE组Parkin转位和荧光强度升高。与EEP+EE组相比,W+EEP+EE组Parkin荧光强度显著升高,Bnip3和TOM20荧光强度显著降低。与LEP+EE组相比,W+LEP+EE组Parkin转位显著降低,Bnip3转位、Bnip3和TOM20荧光强度显著升高。研究结论:(1)一次力竭运动造成明显的心肌损伤、缺血缺氧、超微结构损伤以及氧化应激损伤。但线粒体本身损伤并不严重,也无法引起凋亡。可能通过抑制m PTP开放,和增强线粒体分裂以诱导Bnip3依赖的线粒体自噬参与有限的线粒体保护。(2)EP是无损伤的运动方式,可以诱导SOD酶活性升高,并降低H2O2水平,提供心肌适应性。在EP的早期保护时相,m PTP被抑制,H2O2诱导了修复性线粒体自噬水平升高,Parkin和Bnip3均有参与,但Bnip3可能作用更大。在EP的晚期保护时相,Parkin介导的修复性自噬占到了主导作用。(3)自噬阻滞剂wortmannin对EP诱导的线粒体保护产生负面影响,但不会引起EP本身的损伤加剧。在EP早期保护时相,被阻滞的细胞自噬引起细胞一型前凋亡表型上升,使线粒体保护丧失。在EP的晚期保护时相,反而激活了凋亡性自噬,使心肌保护丧失。
[Abstract]:Research purposes: exercise, as a stimulus for intensity and load, greatly increases the oxygen consumption of the heart and leads to absolute or relative hypoxia. Intermittent large intensity exercise causes repeated absolute or relative ischemia of the heart, similar to IP (ischemic preconditioning, IP), which can induce endogenous protection in the heart and reduce the following. The heart damage caused by acute stress, which is called exercise preconditioning (EP). Mitochondrial protection is vital to the normal physiological state of the heart, and is closely associated with oxidative stress. Recent studies have shown that autophagy and mitochondrial autophagy play an important role in mitochondrial protection in the heart. This study used oxidative stress, mitochondria and autophagy three as the central axis, combined with the early and late two protective periods of EP, the exhaustive exercise as a damaging stress condition, the use of autophagic blocker walman penicillin (wortmannin), and a large amount of application of protein mitochondrial translocation experiments, to further explore the induction of EP. In the heart protection, the relationship between oxidative stress, mitochondria, autophagy and the mechanism of autophagy between three groups, clarify the mechanism of cardiac mitochondrial autophagy. Provide new theoretical and experimental basis for EP heart protection and mechanism research. Research methods: 200 healthy male SD rats were randomly divided into 10 groups: group C, group EE, a large intensity exhausted running table exercise; EEP Group, a EP model was established for a large and intermittent running platform, and 0.5h was obtained after EP; LEP group, 24h after EP modeling; EEP+EE group, EP after 0.5h carried out exhaustion movement; LEP+EE group, EP 24h carried out exhaustion movement; The blockers were injected before the same time and 0.5h was exhausted after EP; group W+LEP+EE, blockers were injected before the same time, and 24h was exhausted after EP. The level of cardiac troponin I (C Tn I) in rat plasma was evaluated by immunofluorescence. The myocardial ischemia and hypoxia was observed and evaluated by hematoxylin basic complex red bitteric acid staining (HBFP staining). The ultrastructural changes of myocardium were observed with transmission electron microscope, and mitochondria and autophagic were observed emphatically. The activity of MDA, H2O2, Mn SOD and total superoxide dismutase were detected by spectrophotometry, and the content of Mn SOD was detected by Western blot, and the degree of oxidative stress was evaluated. The level of phosphorylation of voltage gated anion channel 1 (VDAC1) was detected by Phos-tag method, and the inhibition degree of mitochondrial permeability transition channel (m PTP) was evaluated. Mitochondria extraction combined with immunoblotting, cytochrome C (Cyt-c), autophagic receptor p62, mitochondria autophagic egg white Parkin and Bnip3 were used to evaluate the content of these proteins in cytoplasm and mitochondria. The key proteins of autophagy Beclin1, Bcl-2, LC3I, LC3II were detected by Western blot, and the level and properties of autophagy were evaluated by the ratio of Beclin1/Bcl-2 and LC3II/LC3I. The tissue immunofluorescence double labeling method combined with laser confocal technique was used to detect the endometrial protein COX4/1 of LC3 and Parkin to the mitochondrial membrane transporter TOM70. Bnip3's transposition level of TOM20 and the fluorescence expression and distribution of the above six proteins. Through the above indicators, the role and mechanism of oxidative stress and mitochondrial autophagy in EP induced cardiac protection were revealed. (1) compared with the C group, the level of plasma cTnI in the myocardial damage markers in EE group and the degree of ischemic anoxia by HBFP staining were significantly increased. In the group eep and the group LEP, the level of plasma cTnI decreased and the ischemic anoxia and ultrastructural changes were reduced. Compared with the group of eep+ee, the level of cTnI decreased slightly in the group of w+eep+ee, but in the group of w+eep+ee, the blood oxygen deficiency was not changed and increased, and the mitochondrial fertilizer Daming was found. Compared with the lep+ee group, the plasma cTnI and ischemic anoxia in the w+lep+ee group were increased and the mitochondrial damage was obvious. (2) compared with the C group, the oxidative stress damage product MDA was significantly increased in the EE group, the total SOD activity was reduced, the oxidative stress was obviously damaged, but the activity and content of MnSOD were not changed, and the EEP and LEP groups, MnSOD, no higher activity increased. With the change of MnSOD content, H2O2 in group eep decreased significantly. Compared with group EE, MDA decreased significantly in group eep+ee, H2O2, total SOD activity increased obviously, and the total SOD activity of lep+ee group increased, but the two data of eep+ee group and lep+ee group decreased significantly. Compared with group lep+ee, H2O2 was significantly higher in group w+lep+ee. (3) the phosphorylation level of Vdac1 in group EE and eep+ee group was significantly higher than that in group EE and eep+ee group. Compared with group EE, the phosphorylation level of lep+ee, w+eep+ee, and w+lep+ee groups decreased. (4) the ratio of lc3ii to lc3ii/lc3i in group EE was significantly higher than that in group C, and the ratio of lc3ii/lc3i in eep group increased. The increase of lc3ii and Beclin1 content in eep+ee group suggested that the cell autophagy increased, w+lep lc3ii, lc3ii/lc3i ratio, increase of apoptotic autophagy increased, and the ratio of two kinds of autophagy increased in both group and group. Compared with group EE, lc3ii and lc3ii/lc3i decreased significantly in group lep+ee, the level of autophagy was lower and Beclin1 in w+eep+ee group increased. (5) compared with the C group, the percentage of cox4/1 in the LC3 transposition of EE group was lower than that in the C group. Fluorescence intensity, cytoplasmic p62 increased significantly, LC3 transposition in lep+ee group increased, LC3 and cox4/1 fluorescence intensity decreased, LC3 transposition in w+eep+ee group decreased, cytoplasm and mitochondrial p62 increased, w+lep+ee group LC3 transposition, mitochondria p62 increased, LC3, fluorescence intensity decreased significantly (6 Compared with the C group, the mitochondrial Parkin, parkin transposition tom70, parkin and tom70 fluorescence intensity decreased significantly, the cytoplasm and mitochondrial BNIP3, the BNIP3 transposition Tom20 degree, BNIP3 fluorescence intensity increased significantly, and the Tom20 fluorescence intensity was not changed. The level of BNIP3 transposition and the level of cytoplasm and mitochondrial BNIP3 increased significantly in group LEP, and in group w+eep, group w+lep was close to the mitochondrial autophagy index in group eep and LEP group. Compared with group EE, the mitochondrial parkin and parkin transposition of eep+ee group was significantly higher than that of EE group, and the level of cytoplasm and mitochondria was close to that of the group. Mitochondrial Parkin content, Parkin transposition and TOM70 fluorescence intensity increased, and Bnip3 related data were significantly decreased, and Parkin related data in W+EEP+EE group were significantly increased, but the degree of Parkin transposition was significantly lower than that in EEP+EE group, and Bnip3 and TOM20 increased significantly in the cytoplasm; W+LEP+EE group Parkin transposition and fluorescence intensity increased. The fluorescence intensity of Bnip3 and TOM20 decreased significantly. Compared with the LEP+EE group, the Parkin transposition of the W+LEP+EE group decreased significantly, the Bnip3 transposition, and the fluorescence intensity of Bnip3 and TOM20 increased significantly. (1) the first exhaustive exercise resulted in obvious myocardial injury, ischemic oxygen deficiency, ultrastructural damage and oxidative stress injury. Its own damage is not serious, nor can it cause apoptosis. It may be possible by inhibiting m PTP opening and enhancing mitochondrial division to induce Bnip3 dependent mitochondrial autophagy to participate in limited mitochondrial protection. (2) EP is a noninvasive mode of exercise that can induce the increase of SOD enzyme activity and reduce the level of H2O2 and provide myocardial adaptability. In the early protection of EP Phase, m PTP was inhibited, H2O2 induced elevated mitochondrial autophagy, Parkin and Bnip3 were involved, but Bnip3 may play a greater role. In the late protection phase of EP, Parkin mediated repair autophagy dominated the role. (3) autophagic blocker wortmannin has a negative effect on EP induced mitochondrial protection, but does not cause EP Ben. The damage to the body is aggravated. In the early protection of EP, the blocked cell autophagy caused the apoptotic phenotype of the cell to rise, causing the loss of mitochondrial protection. In the late EP protection phase, the apoptotic autophagy was activated and the myocardial protection was lost.
【学位授予单位】：上海体育学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：G804.2

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