mTOR依赖性自噬在不同模型中对心功能不全的双向调节作用

发布时间：2018-06-20 12:01

  本文选题：自噬 + 乙醇脱氢酶　； 参考：《山东大学》2016年博士论文

【摘要】：研究背景正常的心脏功能是保证人类健康与生活质量的基本条件。但心肌细胞属于永久性细胞,在分化完成后几乎不可再生。因此,维持正常的心肌细胞功能十分重要。然而,心肌细胞功能的维持面临很多挑战。基因多态性对心脏功能有先天性的影响。直接编码心脏发育相关蛋白的基因发生异常可导致心脏畸形及心脏功能严重受损。但在健康人群中,即使年龄、性别相同,体重与身高也类似的人心脏功能依然有差异,尤其是当心脏功能面临一定的应激挑战时,这种差异尤为明显。这归因于人类的基因多态性。本文将要讨论酒精代谢酶乙醇脱氢酶(Alcohol dehydrogenase, ADH)与巨噬细胞迁移抑制因子(Macrophage migration inhibitory factor, MIF)的转基因或基因敲除模型对心脏功能的影响。ADH将酒精转化为乙醛,完成酒精代谢的第一步。ADH1作为ADH的一种亚型中在酒精代谢中起最主要作用。人类编码ADH1亚结构ADH1B的基因存在基因多态性。突变型ADH1B携带者显示出超强的ADH酶活性。在东亚人群中,ADH1B的突变率为40%-90%,远高于非洲与欧洲人群。基础研究中,ADH过表达模型常用于研究酒精代谢对心脏功能的影响,但ADH蛋白本身对心脏功能的影响只有较少研究,且仍存在争议。研究ADH多态性对心脏功能的影响对东亚人群具有重要意义。MIF是T细胞分泌的介导炎症的细胞因子,在2008年发表在Nature杂志上的报道发现MIF也是代谢的调节因子,在心脏缺血状态下可激活AMPK (AMP-activated protein kniase)通路,增加心肌对糖分的摄取,使心脏有足够的能量供应,最终起到保护心肌的作用。而MIF基因也存在突变,MIF基因在启动子上的一种突变可降低MIF的表达,缺血心脏AMPK的激活随之明显减低。MIF在维持心肌稳态中起着十分重要的作用。其次,外界刺激、疾病及年龄等后天因素对心脏功能有重要影响。本文将涉及两个病理模型,一是内质网应激模型,二是衰老模型,讨论后天条件对心脏的影响。现今的环境污染、不健康的生活方式及压力等均会导致内质网应激的发生。内质网应激在多种疾病如肥胖、糖尿病、心衰、心肌缺血再灌注中扮演重要角色。内质网是细胞内的双层膜结构,它的主要功能是加工新合成的蛋白,使其正确折叠,并将折叠加工好的蛋白转运到高尔基体。同时内质网也是重要的储存钙离子的细胞器。正常状态下约有30%的蛋白非正确折叠,这些蛋白将随即被内质网中的相关机制降解。但内质网加工蛋白的过程很容易受影响,尤其当细胞面临缺氧、炎症、氧化应激等外界刺激时,使内质网处于应激状态,激活未折叠蛋白反应(Unfolded protein response, UPR),启动细胞保护程序,维持内质网的稳态。但若外界刺激持续存在,可导致UPR过度激活,发生内质网应激(Endoplasmic reticulum stress, ER stress)。衣霉素(Tunicamycin)可导致细胞内质网应激,被广泛应用于内质网应激模型的建立。衰老一直是人类难以克服的难题。衰老伴随着器官功能退化,细胞代谢水平降、低,细胞器功能减低以及相关分子表达水平和应答能力的改变。心脏衰老表现出明显的心脏收缩及舒张功能的减低、心肌能量代谢的下降以及处理钙离子能力的降低。目前的研究表明,衰老导致的心脏功能变化的分子机制包括氧化应激、线粒体功能下降等,但更详细的机制仍有待人类的探索。自噬(Autophagy)是细胞通过自我降解衰老的或损坏的蛋白及细胞器,完成细胞自我更新及物质重复利用的过程。细胞在正常状态下维持一定的自噬水平,以维持细胞活力。自噬水平过低与过高都会影响心肌稳态。自噬水平过低,心肌细胞无法完成自我更新,大量的废物堆积,代谢减慢,进而心肌收缩功能减低甚至死亡。自噬水平过高,心肌细胞正常的蛋白与细胞器被降解,心肌功能同样减低甚至死亡。自噬与多种心脏疾病息息相关,如心脏缺血再灌注、慢性心力衰竭、冠心病以及败血症引起的心衰等。mTOR (Mammalian target of rapamycin),是一种丝氨酸／苏氨酸激酶,在生物体代谢与生长过程中起重要调节作用。mTOR同时也是最重要的自噬负性调节蛋白,在自噬经典通路中起重要作用。mTOR被磷酸化而激活,磷酸化的mTOR可直接抑制自噬。mTOR受多种因子的调节,如Akt可促进mTOR的磷酸化,而AMPK可抑制mTOR的磷酸化。本研究将探讨心肌在不同的ADH或MIF基因型中,对外界刺激—内质网应激或衰老的应答及其内在机制。研究目的本文通过两部分研究(1)内质网应激对野生型和心脏特异性ADH过表达小鼠心脏结构及功能的影响,以及mTOR依赖性自噬在此过程中的作用及机制； (2)衰老对野生型和MIF敲基因小鼠心脏结构和功能的影响,以及mTOR依赖性自噬在此过程中的作用及机制,探讨外界刺激或病理状态(内质网应激、衰老)对心脏结构与功能的影响,以及ADH与MIF蛋白在此过程中的作用及内在机制。机制研究着重探讨mTOR依赖性自噬是否发挥作用及相关的上游调节机制。研究方法第一部分乙醇脱氢酶通过减轻氧化应激及自噬保护内质网应激引起的心脏收缩功能紊乱—PTEN-Akt-mTOR通路的作用1.动物模型野生型(FVB)小鼠和ADH心脏特异性过表达小鼠分别被注射衣霉素(1mg/kg,1次)或相同体积PBS,48小时后进行实验。2.心脏功能与心肌收缩功能及钙离子处理能力的检测通过多普勒超声研究心脏功能；通过Langendorff系统及Librase酶离体分离小鼠心肌细胞,在SoftEdge Myocam软件中记录心肌细胞的收缩功能,并应用fura-2/AM荧光在双向激发荧光相位倍增系统中记录心肌细胞钙离子摄取情况。3.氧化应激水平的检测应用H2DCFDA和DHE染色法分析从小鼠心脏分离的心肌细胞的氧化应激水平。4.蛋白表达水平的检测应用Western Blot分析内质网应激、自噬以及PTEN-Akt-mTOR通路相关蛋白的表达水平。5.体外实验对分离的FVB或ADH小鼠心肌细胞,应用衣霉素或(和)自噬抑制剂3-MA、Akt抑制剂AKTI及mTOR抑制剂rapamycin处理,在SoftEdge Myocam软件中记录心肌细胞的收缩功能。第二部分巨噬细胞迁移抑制因子缺陷改善衰老导致的心脏炎症,但恶化衰老导致的心脏重构和功能紊乱—自噬的作用1.实验模型(1)衰老动物模型。野生型(C57BL/6)小鼠和MIF敲除小鼠分别被正常喂养至3-4个月,为年轻组；正常喂养至24个月为衰老组。(2)注射自噬激动剂Rapamycin的动物模型。腹腔注射Rapamycin (2mg/kg/d,8周)。(3)早衰模型及MIF低表达模型。应用Doxorubicin (0.1μuM,24小时)处理H9C2细胞建立早衰模型；用MIF siRNA转染细胞建立MIF低表达模型。2.心脏功能与心肌收缩功能及钙离子处理能力的检测通过多普勒超声研究心脏功能；通过Langendorff系统及Librase酶离体分离小鼠心肌细胞,在SoftEdge Myocam软件中记录心肌细胞的收缩功能,并应用fura-2/AM荧光在双向激发荧光相位倍增系统中记录心肌细胞钙离子摄取情况。3.心肌组织结构与心脏纤维化的检测Lectin免疫染色研究心脏组织结构；Masson Trichorme染色研究心肌纤维化。4.小鼠糖代谢与心肌ATP水平的检测IPGTT实验测定葡萄糖代谢,分析小鼠的葡萄糖利用能力。应用氯仿-甲醇法用分光光度计(激发光波长350nm,发射光波长485nm)测心脏组织匀浆的ATP水平。5.蛋白表达水平的检测应用Western Blot分析衰老、自噬、AMPK-mTOR通路及炎症相关蛋白的表达水平。研究结果第一部分乙醇脱氢酶通过减轻氧化应激及自噬保护内质网应激引起的心脏收缩功能紊乱—PTEN-Akt-mTOR通路的作用1.ADH缓解了内质网应激引起的心脏功能下降。在衣霉素作用下,野生型小鼠的心脏功能明显下降,而ADH过表达小鼠的心脏功能明显优于野生型小鼠。给予衣霉素注射后,野生型小鼠心肌摄取钙离子的能力明显下降,而ADH过表达小鼠钙离子处理能力得到改善。2.ADH降低了内质网应激导致的氧化应激。3.ADH缓解了衣霉素导致的内质网应激及自噬水平的升高,改善了PTEN-Akt-mTOR通路蛋白表达的下降。衣霉素的应用导致野生型小鼠内质网应激相关蛋白BIP和GADD153及自噬相关蛋白LC3BI、Atg5、Atg7、P62的表达上升,而ADH过表达小鼠中,这些蛋白的上升程度均明显低于野生型小鼠。衣霉素的应用在野生型小鼠中同样导致了PTEN、Akt、mTOR磷酸化水平的下降,而在ADH过表达小鼠中,这些分子通路蛋白的下降同样得到了改善。4.ADH通过Akt-mTOR通路抑制自噬,保护内质网应激引起的心肌功能紊乱。在体外研究中,衣霉素的应用使离体心肌细胞收缩功能明显下降,而在ADH过表心肌细胞中,或给予自噬抑制剂3-MA后,衣霉素导致的细胞收缩功能减低明显得到缓解。但在ADH过表达心肌中加入Akt抑制剂AktI或mTOR抑制剂Rapamycin,ADH的抗衣霉素作用不复存在。第二部分巨噬细胞迁移抑制因子缺陷改善衰老导致的心脏炎症,但恶化衰老导致的心脏重构和功能紊乱—自噬的作用1.MIF缺陷恶化了衰老导致的糖利用功能减低及心脏功能下降。IPGTT实验中,在给小鼠注射葡萄糖后的15分钟和60分钟时间点, 衰老小鼠的血糖水平明显低于年轻小鼠,而衰老的MIF敲除小鼠降低更为明显。衰老小鼠的心脏功能与心肌收缩功能明显减低,而在MIF敲除小鼠中心脏功能进一步降低。2.MIF缺陷进一步恶化了衰老导致的心肌肥大与纤维化。3.衰老心肌AMPK-mTOR通路受抑制,自噬水平下降,这在MIF缺陷的衰老小鼠中更加明显。衰老心肌的自噬标志蛋白LC2BII表达下降,P62上升,表明自噬水平下降,废物堆积增加；AMPK磷酸化水平的下降和mTOR磷酸化水平的增高表明AMPK-mTOR通路收到抑制。MIF缺陷进一步加重了这些变化。4. Doxorubicin导致H9C2细胞早衰标志物SA-β-Gal的表达增加,应用MIFsiRNA加重了早衰水平,恢复MIF表达后早衰水平得到缓解。5.自噬激动剂Rapamycin缓解了MIF缺陷对衰老心脏功能的恶化。6.MIF缺陷缓解了衰老导致的炎症水平升高,但通过Rapamycin激动自噬对炎症并无明显影响。研究结论我们的研究首次表明ADH自身对内质网应激导致心功能损伤的保护作用,并进一步说明其机制是通过缓解内质网应激导致的氧化应激水平,以及降低PTEN-Akt-mTOR通路介导的自噬水平起到心肌保护作用。我们的研究同样首次表明了MIF缺陷进一步恶化衰老导致的心脏功能下降。衰老导致自噬水平的降低,从而使心脏能量代谢及废物更新受阻,心脏功能下降。MIF介导的AMPK-mTOR通路可恢复自噬水平。因此MIF的缺失导致自噬水平进一步降低,心脏功能也进一步恶化。在这两个研究中,mTOR依赖性自噬起到了相反的作用。在ADH-内质网应激模型中,内质网应激导致了mTOR依赖性自噬的过度激活,从而细胞死亡增加,心脏功能受损；而在MIF-衰老模型中,衰老导致mTOR依赖性自噬的下降,使心肌代谢及供能下降,心脏功能受损。因此,为达到心肌保护的目的,应当使自噬维持在适当的水平。mTOR是调节自噬的重要靶点,针对mTOR调节的药物有很大的潜在临床应用价值。
[Abstract]:Normal cardiac function is the basic condition for ensuring human health and quality of life. However, myocardial cells belong to permanent cells and are almost nonrenewable after the differentiation is completed. Therefore, it is important to maintain normal cardiac function. However, the maintenance of cardiac function is facing many challenges. Genetic abnormalities that directly encode cardiac development related proteins can lead to cardiac abnormalities and severe cardiac dysfunction. But in healthy people, people who are of the same age, sex, body weight and height are still different in heart function, especially in the face of a certain stress challenge. This is particularly evident. This is due to human genetic polymorphisms. This article will discuss the effects of Alcohol dehydrogenase (ADH) and macrophage migration inhibitory factor (Macrophage migration inhibitory factor, MIF) on the cardiac function of the transgenic or gene knockout model..ADH will convert alcohol into acetaldehyde and complete alcohol. .ADH1, the first step in sperm metabolism, plays the most important role in alcohol metabolism as a subtype of ADH. The gene polymorphisms of human encoded ADH1 substructure ADH1B are gene polymorphisms. Mutant ADH1B carriers show super strong ADH activity. In East Asia, the mutation rate of ADH1B is 40%-90%, far higher than that in African and European populations. The ADH overexpression model is often used to study the effect of alcohol metabolism on cardiac function, but the effect of ADH protein itself on cardiac function is only less and still controversial. The study of the effect of ADH polymorphism on the heart function for East Asian population.MIF is the cytokine that mediates inflammation in T cells, published in Natu in 2008. The report in the re magazine found that MIF is also a regulatory factor in metabolism, which activates the AMPK (AMP-activated protein kniase) pathway in the ischemic state of the heart, increases the uptake of sugar in the myocardium, makes the heart sufficient energy supply, and ultimately protects the myocardium. The MIF gene also has a mutation and a mutation of the MIF gene in the promoter. It can reduce the expression of MIF, and the activation of AMPK in the ischemic heart significantly reduces the role of.MIF in the maintenance of myocardial homeostasis. Secondly, external stimuli, disease and age have important effects on cardiac function. This article will involve two pathological models, one is the endoplasmic reticulum stress model, the two is the aging model, and the postnatal days are discussed. The effects on the heart. Current environmental pollution, unhealthy lifestyles and stress can lead to endoplasmic reticulum stress. Endoplasmic reticulum stress plays an important role in a variety of diseases such as obesity, diabetes, heart failure, and myocardial ischemia reperfusion. The endoplasmic reticulum is a double layer membrane structure within the cell and its main function is to process newly synthesized eggs. White, folding it correctly, and transferring the processed protein to the Golgi apparatus. And the endoplasmic reticulum is also an important organelle for storing calcium ions. About 30% of the proteins are not properly folded in normal state, and these proteins will be degraded in the endoplasmic reticulum, but the process of protein processing in the endoplasmic reticulum is easily affected. When the cells are exposed to external stimuli such as hypoxia, inflammation, and oxidative stress, the endoplasmic reticulum is in a stress state, activating the unfolded protein reaction (Unfolded protein response, UPR), initiating the cell protection program and maintaining the homeostasis homeostasis. But if the external stimulus persists, it can lead to excessive activation of UPR and the occurrence of endoplasmic reticulum stress (Endoplasmic reticulu). M stress, ER stress). Ycomycin (Tunicamycin) can lead to endoplasmic reticulum stress and is widely used in the establishment of endoplasmic reticulum stress model. Senescence has been a difficult problem to be overcome by human beings. Aging is accompanied by degradation of organ function, decrease of cell metabolism level, low cell power, and modification of related molecular expression level and response ability. Cardiac senescence shows a significant reduction in cardiac contractile and diastolic function, a decline in cardiac energy metabolism and a decrease in the ability to deal with calcium ions. Current research shows that molecular mechanisms of cardiac function changes, including oxidative stress and decrease in mitochondrial function, are still needed to be explored by human beings. Autophagy is the process of cell self regeneration and reutilization of cells by self degradation of aging or damaged proteins and organelles. Cells maintain a certain level of autophagy to maintain cell viability under normal conditions. The low and high level of autophagy will affect the homeostasis of the myocardium. The level of autophagy is too low and the cardiac myocytes are not. Complete self renewal, accumulation of large amounts of waste, slow metabolism, and reduced myocardial contractile function or even death. High autophagy, normal protein and organelles of cardiac myocytes are degraded, cardiac function is also reduced or even death. Autophagy is closely related to a variety of heart diseases, such as cardiac ischemia reperfusion, chronic heart failure, coronary heart disease .mTOR (Mammalian target of rapamycin), which is caused by septicemia, is a serine / threonine kinase, which plays an important regulatory role in the metabolism and growth of organisms..mTOR is also the most important autophagic negative regulatory protein. It plays an important role in the classical pathway of autophagy, which is activated by phosphorylation and phosphorylation of mTOR. Autophagic.MTOR can be directly inhibited by a variety of factors, such as Akt can promote the phosphorylation of mTOR, and AMPK can inhibit the phosphorylation of mTOR. This study will explore the response of myocardium to the response to external stimulation of endoplasmic reticulum stress or senescence in different ADH or MIF genotypes and its intrinsic mechanism. The purpose of this study was to study the endoplasmic reticulum in two parts (1) endoplasmic reticulum should be studied. The effects and mechanisms of mTOR dependent autophagy on the heart structure and function of the wild type and heart specific ADH overexpressed mice; (2) the effect of senescence on the heart structure and function of wild type and MIF knockout mice, as well as the role and mechanism of mTOR dependent autophagy in this process, and to explore the external stimuli or the external stimuli. The effect of the pathological state (endoplasmic reticulum stress, aging) on the structure and function of the heart, and the role of ADH and MIF protein in this process and its intrinsic mechanism. The mechanism study focuses on the role of mTOR dependent autophagy and the related upstream regulation mechanism. Cardiac contractile dysfunction induced by endoplasmic reticulum stress - the effect of PTEN-Akt-mTOR pathway 1. animal model wild type (FVB) mice and ADH heart specific overexpressed mice were injected with mycophencin (1mg/kg, 1 times) or the same volume PBS respectively. After 48 hours, the test of.2. heart function and myocardial contractile function and calcium ion processing ability were examined. The cardiac function was measured by Doppler ultrasound. The systolic function of cardiac myocytes was recorded in SoftEdge Myocam software through the isolation of Langendorff system and Librase enzyme in vitro, and the fura-2/AM fluorescence was used to record the level of.3. oxidative stress in the myocardial cell calcium uptake in the bi-directional excitation fluorescence phase multiplier system. Detection and application of H2DCFDA and DHE staining methods to detect the level of oxidative stress level.4. protein expression from cardiac isolated cardiomyocytes in mice by Western Blot analysis of endoplasmic reticulum stress, autophagy and the expression level of PTEN-Akt-mTOR pathway related proteins in.5. in vitro experiments on isolated FVB or ADH murine cardiomyocytes, using mycophenycin Or (and) (and) autophagic inhibitor 3-MA, Akt inhibitor AKTI and mTOR inhibitor rapamycin treatment, recording cardiac contractile function in SoftEdge Myocam software. Second partial macrophage migration inhibitory factor defect improves aging induced heart inflammation, but deteriorates senescence caused by cardiac remodeling and dysfunction - the role of autophagy in the 1. experimental model Type (1) aging animal model. Wild type (C57BL/6) mice and MIF knockout mice were fed to 3-4 months, respectively, for young group; normal feeding to 24 months as senescence group. (2) animal model of autophagic agonist Rapamycin injection. Intraperitoneal injection of Rapamycin (2mg/kg/d, 8 weeks). (3) premature failure model and MIF low expression model. Doxorubicin (0.1). UM, 24 hours) treatment of H9C2 cells to establish premature aging model; MIF siRNA transfected cells to establish MIF low expression model.2. cardiac function and myocardial contractile function and calcium ion processing ability detection by Doppler ultrasound study of cardiac function; Langendorff system and Librase enzyme isolation of mouse cardiomyocytes in SoftEdge Myocam soft The systolic function of cardiac myocytes was recorded in the part, and fura-2/AM fluorescence was used to record cardiac tissue structure of myocardial cells and cardiac fibrosis in the bi-directional excitation fluorescence phase multiplier system. The structure of cardiac tissue and cardiac fibrosis were detected by Lectin immunostaining. Masson Trichorme staining was used to study the glucose metabolism in.4. mice of myocardial fibrosis. Glucose metabolism was measured by IPGTT test with myocardial ATP level, and the glucose utilization ability of mice was analyzed. The detection of ATP level of ATP level of cardiac tissue homogenate by chloroform methanol spectrophotometer (excitation light wavelength 350nm, emission wavelength 485nm) should be detected by Western Blot to analyze senescence, autophagy, AMPK-mTOR pathway and The expression level of inflammation related proteins. Part 1 of the study, ethanol dehydrogenase alleviated the decline in cardiac function induced by endoplasmic reticulum stress by reducing oxidative stress and protecting endoplasmic reticulum stress dysfunction caused by endoplasmic reticulum stress and the effect of PTEN-Akt-mTOR pathway on cardiac dysfunction induced by endoplasmic reticulum stress. In the action of ycomycin, heart work of wild type mice The heart function of ADH overexpressed mice was obviously better than that of wild type mice. After injection of ycomycin, the ability of calcium ion uptake in the myocardium of wild type mice decreased significantly, while the ability of calcium ion treatment in ADH overexpressed mice was improved by.2.ADH and the oxidative stress caused by endoplasmic reticulum stress was reduced by.3.ADH. The increase of endoplasmic reticulum stress and autophagy increased the decrease in the expression of PTEN-Akt-mTOR pathway protein. The application of mycophencin led to the increase in the expression of BIP and GADD153 and autophagy related protein LC3BI, Atg5, Atg7 and P62 in the endoplasmic reticulum of wild type mice, and the increase of these proteins in ADH overexpressed mice was significantly lower than that in the wild mice. The application of ycomycin in the wild type mice also led to a decrease in the phosphorylation level of PTEN, Akt, and mTOR in the wild mice, and in the ADH overexpressed mice, the decrease of these molecular pathway proteins also improved the inhibition of autophagy through the Akt-mTOR pathway and the protection of the myocardial dysfunction induced by the endoplasmic reticulum. In vitro studies, in vitro The application of mycophentin reduced the contractile function of isolated cardiomyocytes, and the decrease of contractile function caused by mycophenin was obviously alleviated in the ADH overexpressed cardiomyocytes or after the autophagy inhibitor 3-MA was given, but the effect of the Akt inhibitor AktI or mTOR inhibitor Rapamycin in the ADH overexpressed myocardium was no longer in existence. The second part of the macrophage migration inhibitory factor deficiency improves the heart inflammation caused by aging, but the cardiac remodeling and dysfunction caused by the deterioration of aging - the role of autophagy 1.MIF deficiency worsens the aging induced impaired glucose utilization and the decline of the heart function in the.IPGTT experiment, when the glucose injection is given to mice for 15 and 60 minutes The blood sugar level of aging mice was significantly lower than that of young mice, while the aging MIF knockout mice decreased more obviously. The cardiac function and myocardial contractile function of aging mice decreased significantly, while in MIF knockout mice, the heart function further reduced the.2.MIF deficiency and further worsened the myocardial hypertrophy induced by aging and the fibrosis of the.3. senescence heart. The AMPK-mTOR pathway was inhibited and autophagy decreased, which was more obvious in the aging mice with MIF defects. The expression of autophagic marker protein LC2BII in the aging myocardium decreased, P62 increased, indicating that the level of autophagy decreased and the accumulation of waste increased; the decline of AMPK phosphorylation level and the increase of mTOR phosphorylation level indicated that the AMPK-mTOR pathway received the inhibition of.MIF. Defects further aggravate these changes..4. Doxorubicin leads to increased expression of SA- beta -Gal in H9C2 cell premature senescence, MIFsiRNA plus
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.6
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本文编号：2044187