白藜芦醇对铅致AD样病变小鼠氧化应激的干预作用

发布时间：2018-04-06 00:18

本文选题：铅　切入点：白藜芦醇　出处：《郑州大学》2014年硕士论文

【摘要】：铅(Lead, Pb)是一种普遍存在的环境重金属污染物，有较强的神经毒性，并损伤认知功能，发育期的铅暴露可以诱发阿尔茨海默病(Alzheimer’s disease,AD)样病变的形成。多酚类化合物白藜芦醇(Resveratrol, Res)对神经退行性疾病和急性损伤具有保护作用，Res激活产物沉默信息调节因子1(Silent InformationRegulator1, SIRT1)具有抗氧化作用，而氧化应激是AD产生的重要机制之一，SIRT1对于氧化应激的相关调控在铅致小鼠AD样病变中可能发挥作用。目的基于铅致AD淀粉样变的实验基础，以Aβ的形成和认知效应为观察终点，采用Res进行干预研究，围绕SIRTl及其效应分子FOXO3a、PGC-lα的表达，分析铅暴露、SIRTl相关的抗氧化应激效应与神经损伤之间的可能关系，为AD的病因及发病机制研究提供理论线索。方法 1．动物模型建立：选用清洁级C57BL/6健康断乳（出生后21天）仔鼠35只作为研究对象，由本实验组进行仔鼠的繁育饲养。按体重随机分为五组：Res灌胃组、对照组、9M染铅组、9M染铅+Res灌胃组、3M染铅+Res灌胃组。以自由饮用2g/L醋酸铅溶液的方式进行铅暴露；与铅暴露三个月后进行白藜芦醇灌胃，剂量为50mg/kgBw d，，每周连续灌胃6天后，停灌1天，持续六个月。 2．行为学测试：染毒结束后，进行Morris水迷宫实验，测定小鼠空间学习记忆能力。 3．组织提取：水迷宫实验结束后两天，进行小鼠麻醉后眼球采血，收集全血及脑组织，分离出皮层，生理盐水漂洗后，称重，置于冻存管中-80℃保存。 4．实验室指标测定：用Z-5000偏振塞曼原子吸收光谱仪测定小鼠血铅；谷胱甘肽过氧化物酶(GSH-PX)测试试剂盒测定大脑皮层GSH-PX活力；还原型谷胱甘肽测试盒测定小鼠大脑皮层GSH含量；Aβ(1-40)ELISA试剂盒测定小鼠大脑皮层Aβ(1-40)含量；Western blot分析小鼠大脑皮层SIRT1、磷酸化FOXO3a（Ser253）、PGC-lα蛋白表达水平。结果 1．Morris水迷宫结果：前五天小鼠的平均逃避潜伏期随日期变化逐渐减小（F=2.799,P0.05）；9M染铅组、9M染铅+Res灌胃组的平均逃避潜伏期分别大于对照组、3M染铅+Res灌胃组（P0.05），9M染铅组的平均逃避潜伏期大于Res灌胃组（P0.05）。第6天撤去平台，9M染铅组和9M染铅+Res灌胃组的逃避潜伏期分别大于对照组、3M染铅+Res灌胃组（P0.05）。 2．小鼠血铅含量：各实验组小鼠血铅含量存在差异（F=92.21,P0.001），Res灌胃组、对照组的血铅含量分别小于9M染铅组、9M染铅+Res灌胃组、3M染铅+Res灌胃组（P0.001）；9M染铅组、9M染铅+Res灌胃组的血铅含量分别大于3M染铅+Res灌胃组（P0.001）。 3．GSH-Px活力值和GSH含量：各组小鼠大脑皮层GSH含量存在差异（F=6.177，P0.05），Res灌胃组、对照组GSH含量高于9M染铅+Res灌胃组、9M染铅组（P0.05），3M染铅+Res灌胃组GSH含量高于9M染铅组（P0.05）。各组小鼠大脑皮层GSH-Px活力值存在差异（F=10.195,P0.001），Res灌胃组GSH-Px活力值大于对照组（P0.05），9M染铅组、9M染铅+Res灌胃组、3M染铅+Res灌胃组GSH-Px活力值小于对照组（P0.05），3M染铅+Res灌胃组小鼠GSH-Px活力值大于9M染铅组（P0.05）； 4．Aβ(1-40)含量：各实验组小鼠大脑皮层Aβ(1-40)含量存在差异（F=3.285，P0.05），9M染铅组Aβ(1-40)含量分别大于Res灌胃组、对照组、3M染铅+Res灌胃组（P0.05）。 5．Western blot分析结果：各实验组小鼠大脑皮层细胞核内SIRT1蛋白表达水平存在差异（F=216.779,P0.001），对照组SIRT1水平高于其他各组（P0.05），Res灌胃组、9M染铅+Res灌胃组、3M染铅+Res灌胃组SIRT1水平低于9M染铅组（P0.05），Res灌胃组SIRT1水平高于3M染铅+Res灌胃组和9M染铅+Res灌胃组（P0.05）。各实验组小鼠大脑皮层磷酸化FOXO3a表达水平存在差异（F=18.17，P0.001），Res灌胃组磷酸化FOXO3a水平低于其他各组（P0.05），对照组磷酸化FOXO3a水平则高于其他各组（P0.05），9M染铅组磷酸化FOXO3a水平高于3M染铅+Res灌胃组（P0.05）。各实验组小鼠大脑皮层总蛋白中PGC-lα表达水平存在差异（F=58.632,P0.001），Res灌胃组PGC-lα表达水平高于对照组（P0.05），9M染铅组、9M染铅+Res灌胃组、3M染铅+Res灌胃组PGC-lα表达水平均低于对照组（P0.05），其中3M染铅+Res灌胃组PGC-lα表达水平高于9M染铅组；各实验组小鼠大脑皮层胞浆蛋白中PGC-lα表达水平也存在差异（F=67.327，P0.001），Res灌胃组小鼠PGC-lα表达水平低于对照组（P0.05），9M染铅组PGC-lα表达水平高于对照组（P0.05），9M染铅+Res灌胃组、3M染铅+Res灌胃组PGC-lα表达水平均低于9M染铅组（P0.05）且与对照组差异无统计意义（P0.05）。结论 1.发育早期铅暴露可降低小鼠大脑皮层中磷酸化FOXO3a、PGC-lα及大脑皮层细胞核内SIRT1表达水平，并增加PGC-lα在胞浆中的滞留，减少GSH-Px的酶活力以及GSH含量，降低抗氧化能力，损伤小鼠空间学习和记忆能力； 2.白藜芦醇干预可能通过SIRTI的核质转运降低小鼠大脑皮层中的磷酸化FOXO3a表达水平、升高大脑皮层PGC-lα表达水平，增进PGC-lα去乙酰化后的核内转运及转录活性，增加GSH-Px的酶活力及GSH含量，提高抗氧化能力，对铅致小鼠空间学习记忆能力损伤有保护作用。
[Abstract]:Lead (Lead, Pb) is a ubiquitous environmental heavy metal pollutants, toxicity, and cognitive impairment, developmental lead exposure can induce Alzheimer's disease (Alzheimer 's disease, AD) formed like lesions. The polyphenolic compound resveratrol (Resveratrol, Res) has a protective effect on nerve degenerative diseases and acute injury, activation of Res product of silent information regulator 1 (Silent, InformationRegulator1, SIRT1) with antioxidant effects, and oxidative stress is one of the important mechanisms of AD, SIRT1 in regulation of oxidative stress may play a role in lead induced mouse AD like lesions.
objective
The experimental basis of lead induced AD amyloidosis based on form and cognitive effects of A beta to observe the end point, the intervention study was conducted by Res, around SIRTl and its effector molecule FOXO3a, expression of PGC-l, analysis of lead exposure, the possible relationship between SIRTl anti oxidative stress effects associated with nerve injury, to provide theoretical clues for the study the etiology and pathogenesis of AD.
Method
1. animal model: choose healthy C57BL/6 weaning (postnatal day 21) 35 mice as the research object, the experimental group of rats. Breeding breeding were randomly divided into five groups: Res group, control group, 9M exposure group, 9M lead +Res group 3M, lead +Res gavage group. By drinking 2g/L lead acetate solution of lead exposure; and lead exposure three months after intragastric administration of resveratrol, the dose of 50mg/kgBw D, a weekly intragastric administration for 6 days, stopping perfusion for 1 days, lasted for six months.
2. behavior test: after the end of the drug, the Morris water maze test was carried out to determine the learning and memory ability of the mice.
3. tissue extraction: two days after the water maze test, the blood samples were collected from the eyeballs after anesthesia, and the whole blood and brain tissues were collected. The cortex was isolated. After rinsing, the saline was weighed and stored in the cryopreservation tube at -80 C.
Determination of 4. laboratory index: Z-5000 Polarized Zeeman atomic absorption spectrometer for determination of lead in mice; glutathione peroxidase (GSH-PX) test kit for determination of GSH-PX activity in the cerebral cortex; Determination of GSH content in cerebral cortex glutathione test kit; A beta (1-40) ELISA kit mouse cerebral cortex A beta (1-40) content; Western blot analysis of mouse cerebral cortex SIRT1, phosphorylation of FOXO3a (Ser253), the expression level of PGC-l protein.
Result
The results of the 1.Morris water maze: the average escape latency of five days before the date with mice gradually decreased (F=2.799, P0.05); 9M lead group, the average escape latency of 9M lead +Res gavage group were higher than the control group, 3M lead +Res gavage group (P0.05), the average escape latency of 9M in lead exposed group more than Res group (P0.05). Sixth days removed platform, 9M lead group and 9M lead +Res group the escape latency were higher than the control group, 3M lead +Res gavage group (P0.05).
2. mouse blood lead content: there are differences in each experimental group of mice blood lead levels (F=92.21, P0.001), Res group, blood lead content in control group were less than 9M in lead group, 9M lead +Res gavage group, 3M lead +Res gavage group (P0.001); 9M blood lead group. The lead content of 9M lead +Res gavage group were higher than 3M lead +Res gavage group (P0.001).
3.GSH-Px activity and GSH content: there were differences in GSH content in cerebral cortex of each group (F=6.177, P0.05), Res in gavage group, GSH content in control group was higher than 9M, lead and +Res gavage group, 9M lead exposure group (P0.05), and the level of lead in group B was higher than that in group B (group B).
There were differences in mouse cerebral cortex GSH-Px activity value (F=10.195, P0.001), Res group GSH-Px activity was higher than that of the control group (P0.05), 9M lead group, 9M lead +Res gavage group, 3M lead +Res gavage group GSH-Px activity value is less than the control group (P0.05), 3M +Res was exposed to lead mice gastric GSH-Px activity value greater than 9M in lead group (P0.05);
4.A beta (1-40) content: there was a difference in the content of A beta (1-40) in the cerebral cortex of each experimental group (F=3.285, P0.05), and the content of A beta (1-40) in 9M lead staining group was higher than that in Res gavage group, while in control group, 3M was exposed to lead and +Res in gavage group (P0.05).
5.Western blot analysis: the experimental group of mice cerebral cortex nucleus SIRT1 protein expression level differences (F=216.779, P0.001), the control group SIRT1 was higher than other groups (P0.05), Res group, 9M lead +Res group, 3M lead +Res group SIRT1 level was lower than that of 9M in lead group (P0.05, Res) with SIRT1 level in gastric lead exposed group was higher than that in 3M +Res group and 9M lead +Res gavage group (P0.05).
Differences in the level of expression of each experimental group in cerebral cortex of phosphorylated FOXO3a (F=18.17, P0.001), Res gavage group phosphorylated FOXO3a levels lower than the other groups (P0.05), the control group the level of phosphorylated FOXO3a was higher than other groups (P0.05), 9M lead group FOXO3a phosphorylation level was higher than that of 3M lead +Res gavage group (P0.05).
Differences in the level of PGC-l expression in each experimental group total protein in mouse cerebral cortex (F=58.632, P0.001), Res group PGC-l expression level was higher than that of the control group (P0.05), 9M lead group, 9M lead +Res gavage group, 3M lead +Res group PGC-l expression levels were lower than the control group (P0.05), of which 3M lead +Res group PGC-l expression level was higher than that of 9M in lead group; the experimental group cells of cerebral cortex in mice plasma protein PGC-l expression level differences (F=67.327, P0.001), Res mice fed the PGC-l expression level was lower than that of the control group (P0.05), 9M lead group PGC-l expression level was higher than that of the control group (P0.05), 9M lead +Res gavage group, 3M lead +Res group PGC-l expression levels were lower than that of 9M in lead group (P0.05) and the control group showed no statistical significance (P0.05).
conclusion
1. early developmental lead exposure could decrease the phosphorylation of FOXO3a in mouse cerebral cortex, the expression level of SIRT1 alpha and PGC-l in cerebral cortex of the nucleus, and increased PGC-l alpha stranded in the cytoplasm, decrease of GSH-Px activity and GSH content, decreased antioxidant capacity, impaired spatial learning and memory in mice;
2. resveratrol intervention may reduce the expression level of phosphorylated FOXO3a in the cerebral cortex of mice through nucleocytoplasmic transport of SIRTI, increased cortical PGC-l expression level, to enhance PGC-l alpha nuclear translocation and transcriptional activity after acetylation, increase enzyme activity and GSH content of GSH-Px, improve the antioxidant ability of mice induced by lead the ability of spatial learning and memory has a protective effect on injury.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5

【参考文献】