脂联素调节C2C12细胞氧化应激诱导的线粒体自噬和凋亡

发布时间：2018-06-24 20:03

本文选题：脂联素 + 线粒体自噬　；参考：《天津医科大学》2017年硕士论文

【摘要】：背景与目的脂联素,也被称为apM1,Acrp30,GBP28和adipoQ,主要由脂肪组织产生的循环激素。许多药理研究表明,该蛋白具有抗糖尿病、抗动脉粥样硬化和抗炎作用。虽然已经有一些研究证明这种蛋白质有抗氧化活性,但是在骨骼肌中具体的调节机制还不是很清楚。本研究的目的是探讨脂联素对小鼠骨骼肌细胞C2C12氧化损伤的保护作用及分子机制。方法本研究通过过氧化氢(H2O2)氧化应激的经典模型来模拟氧化损伤。脂联素预处理骨骼肌细胞C2C12之后,用过氧化氢处理来模拟氧化损伤。通过检测细胞活力来确定过氧化氢处理的浓度和时间以及脂联素的适宜浓度。通过检测活性氧来分析脂联素对细胞氧化应激水平的影响。通过Annexin V/PI双染色法检测脂联素对细胞凋亡的影响。蛋白印记检测脂联素对抗氧化物质的影响。通过检测线粒体膜电位的变化,证明脂联素对线粒体的保护作用。通过线粒体通透性转运孔抑制剂米酵菌酸(BA)来探讨脂联素对线粒体膜电位改变的调控机制。利用共聚焦显微镜检测脂联素对氧化损伤引起的自噬流的影响,线粒体自噬的影响,线粒体和溶酶体的共定位情况的影响。通过加入自噬抑制剂氯喹(CQ)进一步验证脂联素对线粒体自噬改变的影响。通过线粒体DNA拷贝数检测脂联素对脂联素对线粒体DNA的影响。通过蛋白印迹和实时定量聚合酶链反应探讨脂联素对线粒体自噬和凋亡影响的分子机制。结果通过细胞活力检测发现,脂联素浓度为1~30μg/mL时对骨骼肌细胞没有任何毒性,浓度为30μg/m L时对细胞活力有显著升高作用。过氧化氢诱导的氧化损伤对细胞活力的抑制作用具有时间和剂量依赖性,脂联素预处理能够显著提高过氧化氢诱导的细胞活力抑制作用。过氧化氢处理之后,提高骨骼肌细胞活性氧(ROS)水平,脂联素预处理之后能够显著抑制活性氧的水平,与此同时,脂联素预处理之后显著抑制了过氧化氢诱导的细胞凋亡。脂联素能够增强抗氧化物质Nrf2和HO-1的表达。过氧化氢处理之后显著降低线粒体膜电位,脂联素预处理显著抑制线粒体膜电位的下降。线粒体通透性转运孔抑制剂米酵菌酸预处理能够提高过氧化氢引起的细胞活力下降。过氧化氢处理之后,自噬小体和自噬溶酶体都增多,脂联素预处理之后显著降低自噬小体和自噬溶酶体的数量,抑制过氧化氢诱导的线粒体自噬,同时也抑制溶酶体和线粒体的共定位。过氧化氢处理之后,线粒体自噬指示剂线粒体内膜蛋白TIM23逐渐降低,LC3-II逐渐增加,脂联素预处理之后TIM23降解减少,而对LC3-I向LC3-II转换没有影响。脂联素预处理能够抑制过氧化氢诱导的Pink1和Parkin的增加。加入自噬抑制剂氯喹之后,能够显著逆转过氧化氢诱导的TIM23降解,而对脂联素预处理的TIM23水平无明显影响。脂联素抑制过氧化氢诱导的线粒体DNA的降解。脂联素抑制过氧化氢处理之后,凋亡相关Bax/Bcl-2比率升高,脂联素抑制Bax的转录水平,明显降低Bax/Bcl-2的比率,抑制细胞凋亡相关蛋白的转录水平。结论这些结果表明,脂联素对小鼠骨骼肌细胞氧化损伤具有细胞保护作用,脂联素缓解过氧化氢诱导的生长抑制,增加细胞抗氧化能力,清除过氧化氢诱导的活性氧的能力。脂联素抑制过氧化氢诱导的自噬流增加,抑制Pink1、Parkin的增加,抑制线粒体自噬,部分抑制溶酶体和线粒体的共定位。此外,脂联素抑制过氧化氢诱导的细胞凋亡,抑制Bax的转录水平和蛋白水平及凋亡相关蛋白的转录水平。这些结果表明,脂联素在氧化应激诱导的线粒体自噬过程中具有一定的调节作用,抑制细胞凋亡。由于其抗氧化损伤的作用,可以考虑将其作为一个骨骼肌氧化损伤有关疾病的防治药物。
[Abstract]:Background and objective adiponectin, also known as apM1, Acrp30, GBP28, and adipoQ, mainly produced by the adipose tissue. Many pharmacological studies have shown that the protein has anti diabetic, anti atherosclerotic and anti-inflammatory effects. Although some studies have shown that the protein has antioxidant activity, it is specific in skeletal muscle. The purpose of this study is to explore the protective effect and molecular mechanism of adiponectin on C2C12 oxidative damage in skeletal muscle cells in mice. Methods this study simulated oxidative damage through the classical model of oxidative stress of hydrogen peroxide (H2O2). After adiponectin pretreated skeletal muscle cells C2C12, hydrogen peroxide treatment was used to simulate the oxidative damage. Oxidative damage. The concentration and time of hydrogen peroxide treatment and the appropriate concentration of adiponectin were determined by detecting cell viability. The effects of adiponectin on the level of oxidative stress were analyzed by detecting reactive oxygen species. The effect of adiponectin on apoptosis was detected by Annexin V/PI double staining. Protein imprint detection of adiponectin against oxidation The effects of substance. By detecting the changes in mitochondrial membrane potential, the protective effect of adiponectin on mitochondria was demonstrated. The regulation mechanism of adiponectin to mitochondrial membrane potential change was explored by BA. The effect of adiponectin on autophagic flow induced by oxidative damage was detected by confocal microscopy. The effects of mitochondrial autophagy, the co localization of mitochondria and lysosomes. The effect of adiponectin on mitochondrial autophagy by adding autophagic inhibitor chloroquine (CQ). The effects of adiponectin on the mitochondrial DNA were detected by the mitochondrial DNA copy number. The molecular mechanism of the effect of adiponectin on autophagy and apoptosis should be explored. Results by cell viability detection, it is found that the concentration of adiponectin is 1~30 mu g/mL without any toxicity to the skeletal muscle cells, and the cell viability is significantly elevated when the concentration is 30 u g/m L. The inhibition effect of oxidative damage induced by hydrogen peroxide on cell viability In time and dose dependence, adiponectin preconditioning could significantly increase the inhibitory effect of hydrogen peroxide induced cell activity. After hydrogen peroxide treatment, the activity oxygen (ROS) level of skeletal muscle cells was enhanced. Adiponectin pretreatment could significantly inhibit the level of reactive oxygen species, and the preconditioning of adiponectin inhibited hydrogen peroxide significantly after adiponectin pretreatment. Induced apoptosis. Adiponectin can enhance the expression of antioxidant Nrf2 and HO-1. After hydrogen peroxide treatment, the mitochondrial membrane potential is significantly reduced. Adiponectin preconditioning significantly inhibits the decrease of mitochondrial membrane potential. Mitochondrial permeability translocation inhibitor michcolic acid preconditioning can increase the activity of hydrogen peroxide induced cell viability. After hydrogen peroxide treatment, autophagosome and autophagosome are increased. Adiponectin pretreatment significantly reduces the number of autophagosomes and autophagosomes, inhibits the mitochondrial autophagy induced by hydrogen peroxide, and inhibits the co localization of lysosomes and mitochondria. After hydrogen peroxide treatment, mitochondrial autophagy indicator mitochondria are in the mitochondria. The membrane protein TIM23 gradually decreased, LC3-II increased gradually, and the degradation of TIM23 decreased after adiponectin pretreatment, but had no effect on LC3-I conversion to LC3-II. The adiponectin pretreatment could inhibit the increase of Pink1 and Parkin induced by hydrogen peroxide. After adding the autophagic inhibitor chloroquine, the degradation of TIM23 induced by hydrogen peroxide could be markedly reversed, and the lipoprotein association was found. Adiponectin inhibited the degradation of mitochondrial DNA induced by H2O2. Adiponectin inhibited the degradation of mitochondrial DNA induced by hydrogen peroxide. After the inhibition of hydrogen peroxide treatment, adiponectin inhibited the apoptosis related Bax/Bcl-2 ratio, inhibited the transcriptional level of Bax, significantly reduced the ratio of Bax/Bcl-2 and inhibited the transcription of apoptosis related proteins. The results show that adiponectin has a protective effect on the oxidative damage of skeletal muscle cells in mice. Adiponectin relieves the growth inhibition induced by hydrogen peroxide, increases the antioxidant capacity of the cells, and clears the ability of hydrogen peroxide induced reactive oxygen species. Adiponectin inhibits the increase of the autophagic flow induced by hydrogen peroxide, inhibits the increase of Pink1, Parkin, and inhibits the grain size. Autophagy partially inhibits the co localization of lysosomes and mitochondria. In addition, adiponectin inhibits the apoptosis induced by hydrogen peroxide and inhibits the transcriptional level and protein level of Bax and the transcriptional level of apoptosis related proteins. These results suggest that adiponectin has a certain regulatory role in the process of autophagy induced by oxidative stress. Apoptosis can be considered as a preventive and therapeutic agent for oxidative damage related to skeletal muscle because of its antioxidant effects.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R363

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