海藻糖抑制短暂性缺血诱导的蛋白质聚集的机制研究

发布时间：2018-05-19 21:23

本文选题：海藻糖 + 蛋白质聚集　；参考：《吉林大学》2017年博士论文

【摘要】：海藻糖抑制短暂性缺血诱导的蛋白质聚集的机制研究目的:探讨海藻糖对缺血性脑损害引起的蛋白质聚集的影响,并探索其机制。方法:建立大鼠的短暂性全脑缺血模型以及SH-SY5Y的氧糖剥夺模型。应用LDH释放测定和台盼蓝测定、细胞内活性氧测定、Morris水迷宫行为学评估、苏木精及伊红染色和组织学检查、免疫组织化学分析、蛋白酶体活性测定、蛋白印迹分析和印迹密度分析探索海藻糖对缺血性损害引起的蛋白质聚集的影响及其机制。结果:(1)苏木精-伊红染色观察发现:与假手术组相比,缺血组中海马CA1区神经元在缺血后再灌注72小时后,其形态学表现为细胞质粉染、固定核呈多边形。用3.0%浓度的海藻糖提前48小时预处理可显著抑制短暂性脑缺血引起的海马CA1区神经元的形态学改变。缺血前使用3.0%浓度的海藻糖预处理后,缺血/再灌注后72小时时存活神经元的百分比从5.52±2.31%增加到62.56±8.17%。水迷宫实验中典型的游泳轨迹表明海藻糖预处理可以使缺血大鼠能以更为合适的方法搜索水中隐藏平台,缩短寻找到水中隐藏平台的潜伏期、减少在QII内的游泳轨迹;与假手术组相比,缺血组大鼠在QII中停留时间更短暂,但是应用海藻糖预处理显著延长了大鼠在目标象限中的停留时间。(2)蛋白印迹分析显示,在3.0%海藻糖+缺血组,再灌注24小时时泛素标记蛋白质聚集体的增加明显减弱。免疫组织化学染色显示:在再灌注24小时时,经3.0%浓度的海藻糖预处理的海马ca1区神经元中泛素标记的蛋白质聚集体明显受到抑制。与缺血组相比,经3.0%浓度的海藻糖预处理后在缺血/再灌注12小时、24小时和48小时时,海马ca1区神经元内蛋白酶体活性明显得到逆转,其活性分别从47.56%±4.89%上升至71.88%±7.12%,53.25%±4.21%上升至73.35%±6.96%和46.88%±3.79%上升至75.28%±7.86%。(3)乳酸脱氢酶释放测定显示:在复氧6小时时,提前48小时应用海藻糖预处理(0.5或5.0mmol/l)可以显著抑制氧糖剥夺诱导的sh-sy5y细胞死亡。蛋白印迹分析显示:在复氧2小时时,经5.0mmol/l浓度的海藻糖预处理后的sh-sy5y样本,其逆转了氧糖剥夺诱导的lc3ii、beclin-1水平的增加及自噬p62选择性底物的减少。我们发现使用自噬抑制剂3ma以2mmol/l浓度预处理1小时后,它不仅抑制了氧糖剥夺诱导的lc3ii,beclin-1和p62蛋白水平的变化,而且在复氧6小时时减少了细胞的死亡。(4)5.0mmol/l浓度的海藻糖预处理可以显著抑制mg-132诱导的蛋白酶体活性下降、乳酸脱氢酶释放增加和泛素标记蛋白质聚集物的形成。(5)荧光显微镜下的图像显示用0.5mmol/l或5.0mmol/l的海藻糖提前48h预处理可以显著抑制氧糖剥夺24小时并复氧2小时时人sh-sy5y细胞中绿色荧光的增加。荧光密度的统计分析证明0.5mmol/l和5.0mmol/l浓度的海藻糖均可以有效的抑制由氧糖剥夺引起的细胞内氧化应激的增加。(6)蛋白印迹分析和印迹密度分析显示,在复氧2小时时,经5.0mmol/L浓度的海藻糖预处理的人SH-Y5Y细胞样本中,氧糖剥夺诱导的PERK,磷酸化PERK,磷酸化eIF2α,IRE-1和GRP78的表达上调明显受抑。这意味着海藻糖预处理抑制了人SH-SY5Y细胞样本中氧糖剥夺诱导的内质网应激。结论:在本研究中,我们证明海藻糖通过抑制氧化应激和内质网应激来保护细胞内蛋白酶体活性,从而实现其对缺血性损伤引起的蛋白质聚集的抑制作用。蛋白酶体在调节缺血性损伤引起的蛋白质聚集中起到关键性作用;海藻糖是一种有效的神经细胞保护剂,可以抑制缺血性神经元死亡。
[Abstract]:Study on the mechanism of trehalose inhibiting protein aggregation induced by transient ischemia: To explore the effect of trehalose on protein aggregation induced by ischemic brain damage and explore its mechanism. Methods: establishing rat transient global cerebral ischemia model and SH-SY5Y oxygen deprivation model. LDH release assay and trypan blue assay, intracellular Active oxygen assay, Morris water maze behavior assessment, hematoxylin and eosin staining and histological examination, immunohistochemical analysis, proteasome activity assay, Western blot analysis and imprinting density analysis to explore the effect and mechanism of trehalose on protein aggregation induced by ischemic damage. Results: (1) observation of hematoxylin eosin staining Compared with the sham operation group, the hippocampal CA1 area neurons in the ischemic group were stained with cytoplasm and polygonal after 72 hours of reperfusion after ischemia, and the preconditioning with 3% concentration of Trehalose before 48 hours preconditioning could significantly inhibit the morphological changes of the hippocampal neurons in the hippocampal CA1 region. Before ischemia, 3 were used. After the concentration of trehalose preconditioning, the percentage of surviving neurons at 72 hours after ischemia / reperfusion increased from 5.52 + 2.31% to 62.56 + 8.17%. in the water maze experiment, which showed that trehalose preconditioning could make ischemic rats search the hidden platform in water more appropriately and shorten the hidden platform in the water. Incubation period, reducing the swimming track in QII; compared with the sham group, the retention time of the rats in the ischemic group was shorter in the QII, but the application of trehalose preconditioning significantly prolonged the residence time of the rats in the target quadrant. (2) Western blot analysis showed that in the 3% trehalose + ischemia group and 24 hours of reperfusion, the ubiquitin labeled protein aggregates The increase in the immuno histochemical staining showed that the ubiquitin labeled protein aggregates in the hippocampal CA1 region neurons pretreated by 3% concentration of trehalose were significantly inhibited at 24 hours of reperfusion. Compared with the ischemic group, 3% concentration of trehalose was pretreated at 12 hours, 24 hours and 48 hours after ischemia / reperfusion. The activity of proteasome in the hippocampal CA1 region was obviously reversed, and its activity increased from 47.56% + 4.89% to 71.88% + 7.12% respectively. 53.25% + 4.21% increased to 73.35% + 6.96% and 46.88% + 3.79% to 75.28% + 7.86%. (3). L) could significantly inhibit the SH-SY5Y cell death induced by oxygen glucose deprivation. Western blot analysis showed that the SH-SY5Y sample pretreated by 5.0mmol/l concentration of trehalose at 2 hours reversed the lc3ii of oxygen deprivation induced, the increase of beclin-1 level and the decrease of autophagic p62 selective substrate. We found the use of autophagy inhibitor 3M. When a was pretreated with 2mmol/l concentration for 1 hours, it not only inhibited the changes in lc3ii, beclin-1 and p62 protein levels induced by oxygen glucose deprivation, but also reduced the cell death at the time of reoxygenation 6. (4) trehalose preconditioning in the concentration of 5.0mmol/l could significantly inhibit the decline of the activity of the white enzyme body induced by MG-132, the release of lactate dehydrogenase and the general release of lactate dehydrogenase. The formation of protein tagged protein aggregates. (5) images under the fluorescence microscope show that 0.5mmol/l or 5.0mmol/l trehalose preconditioning can significantly inhibit the increase of green fluorescence in human SH-SY5Y cells when oxygen glucose deprivation is 24 hours and reoxygenation for 2 hours. The statistical analysis of fluorescence density shows the trehalose concentration of 0.5mmol/l and 5.0mmol/l. The increase in intracellular oxidative stress caused by oxygen glucose deprivation can be effectively suppressed. (6) Western blot analysis and imprinting density analysis showed that the expression of oxygen glucose deprivation induced PERK, phosphorylated PERK, phosphorylated eIF2 a, IRE-1 and GRP78 was up-regulated in the samples of human SH-Y5Y cells pretreated by 5.0mmol/L concentration of trehalose at 2 hours of reoxygenation. This means that trehalose preconditioning inhibits endoplasmic reticulum stress induced by oxygen glucose deprivation in human SH-SY5Y cell samples. Conclusion: in this study, we have demonstrated that trehalose protects the activity of proteasome by inhibiting oxidative stress and endoplasmic reticulum stress to achieve protein aggregation caused by ischemic damage. The proteasome plays a key role in regulating the aggregation of proteins caused by ischemic injury; trehalose is an effective neuroprotective agent that inhibits the death of ischemic neurons.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.31

【相似文献】