水飞蓟宾对阿尔茨海默病动物模型的保护作用及机制研究

发布时间：2018-04-08 17:18

本文选题：水飞蓟宾　切入点：阿尔茨海默病　出处：《沈阳药科大学》2017年博士论文

【摘要】：目的:阿尔茨海默病(Alzheimer's disease,AD)是一种以进行性认知功能障碍和记忆损伤为特征的神经退行性疾病,是最为普遍的一种老年痴呆症。水飞蓟宾作为菊科植物水飞蓟中的黄酮木质素类药物,其在临床上主要被用作保肝药物。研究表明水飞蓟宾表现出良好的神经保护功能,但其神经保护作用机制尚不明确。因此,本研究分别在LPS、Aβ25-35及Aβ1-42所致AD模型中考察水飞蓟宾对模型大鼠学习记忆损伤的改善作用及作用机制。本研究为水飞蓟宾在皮肤、肝及胰腺等器官中药理作用机制研究提供理论基础。方法:实验通过侧脑室注射LPS、海马CA1区注射Aβ25-35及Aβ1-42建立三个AD动物模型,口服灌胃给予不同剂量的水飞蓟宾(25,50 and 100mg/kg)、白黎芦醇(30mg/kg)、多奈哌齐(1mg/kg)或对照溶剂。通过Y迷宫、Morris水迷宫、新物体辨别实验及高架十字迷宫等行为学方法测定水飞蓟宾对模型大鼠学习记忆障碍的影响;使用尼氏染色及Fluoro-JadeB(FJB)染色方法考察大鼠海马区域形态学改变;采用ELISA测定促炎性细胞因子IL-1β、TNFα及抗炎性细胞因子IL-4的水平;Western blot方法测定炎症、自噬和雌激素受体(estrogen receβtors,ERs)及相关通路蛋白表达。结果:本研究首先采用LPS作为工具药物建立神经炎症模型,研究水飞蓟宾的神经保护作用。研究发现水飞蓟宾对侧脑室注射LPS诱导的大鼠学习记忆损伤具有显著的改善作用。与LPS注射大鼠比较,给予水飞蓟宾显著降低Morris水迷宫中大鼠的逃避潜伏期,并显著提高Y迷宫中大鼠的自发交替反应率。水飞蓟宾通过抑制海马内促炎性因子IL-1β的表达、促进抗炎性细胞因子IL-4的表达抑制LPS诱导的神经炎症,并活化海马内ROS-BDNF-TrkB通路发挥神经保护作用。在LPS模型研究基础上,建立Aβ25-35诱导的AD模型。研究发现水飞蓟宾抑制海马内MDA水平促进GSH的水平,抑制Aβ25-35诱导的氧化应激。水飞蓟宾抑制海马内炎性介质NF-κB、COX-2及iNOS抑制炎症水平,并上调海马内自噬水平和BDNF-TrkB通路对抗神经损伤。Aβ1-42具有朊病毒样作用诱导Aβ斑块病理变化,我们通过行为学Morris 水迷宫、Y迷宫及高架十字迷宫测定显示Aβ1-42较Aβ25-35发挥更强毒性作用。水飞蓟宾通过下调自噬水平,抑制炎症反应以及上调BDNF-TrkB通路对Aβ1-42诱导的神经损伤发挥保护作用。基于水飞蓟宾具有雌激素样作用,而且雌激素受体在神经细胞中有较高表达,这提示我们水飞蓟宾很可能是通过调节雌激素受体以及相关信号传导而发挥神经保护作用。在Aβ1-42损伤动物中,水飞蓟宾下调ERs水平以及其介导的PI3K-Akt、MAPKs通路发挥神经保护作用结论:水飞蓟宾对LPS、Aβ25_35及Aβ1-42诱导的学习记忆障碍具有保护作用,这种保护作用可能是通过抑制海马内炎症水平、氧化应激,调节自噬以及ERs及其相关PI3K-Akt、MAPKs通路实现的。本研究为水飞蓟宾作为神经保护药物的开发,为水飞蓟宾在皮肤、肝及胰腺等器官中药理作用的研究以及针对氧化应激-自噬-炎症-雌激素受体作为靶点开发药物提供了有力的理论支持。
[Abstract]:Objective: Alzheimer's disease (Alzheimer's disease AD) is a kind of cognitive dysfunction and memory impairment characterized by neurodegenerative disease, is one of the most common dementia. Silybin as flavonoids wood Compositae in Silybum marianum hormone drugs, the clinical is mostly used as hepatoprotective drug. The research showed that silibinin showed neuroprotective function, but its neuroprotective mechanism is still not clear. Therefore, the research on LPS, to improve the learning effect and the mechanism of memory impairment of A A beta 25-35 and beta 1-42 induced AD model of silybin on the model rats. Study of silybin in skin, provide a theoretical basis for the study on pharmacological mechanism of liver and pancreas and other organs. Methods: the experiment by intracerebroventricular injection of LPS, hippocampus CA1 injection of A A beta 25-35 and beta 1-42 build three AD animal model, oral Intragastric administration of different doses of silymarin (25,50 and 100mg/kg), resveratrol (30mg/kg), donepezil (1mg/kg) or solvent controls by Y maze and Morris water maze, determination of effects of silibinin on rat model of learning and memory impairment method of object recognition test and elevated plus maze behavior; using Nissl staining and Fluoro-JadeB staining (FJB) method to study the morphological changes of hippocampus in rats; Determination of proinflammatory cytokines by ELISA IL-1 beta, TNF alpha and anti-inflammatory cytokine IL-4 level; Western blot method for the determination of inflammation, autophagy and estrogen receptor (estrogen rece beta tors, ERs) expression and related signaling pathways protein. Results: in this study we used LPS as a tool for drug establishment of inflammatory model nerve, nerve protective effect of silybin. The study found that silibinin on learning and memory impairment induced by intracerebroventricular injection of LPS rats Has a significant role in the improvement. Compared with the LPS injected rats given silymarin significantly decreased in Morris water maze rats escape latency, and increase the reaction rate of spontaneous alternation of Y rats in the maze. Silybin by inhibiting the expression of proinflammatory cytokines in the hippocampus of IL-1 beta expression in inflammation, nerve the anti-inflammatory cytokine IL-4 inhibit LPS induced activation in the hippocampus, and ROS-BDNF-TrkB pathway play a neuroprotective effect. On the basis of LPS model, the establishment of A beta 25-35 induced AD model. The study found that silibinin inhibits the MDA level in the hippocampus and promote the level of GSH, oxidative stress inhibits A beta 25-35 induced by silymarin. Inhibition of inflammatory mediators in the hippocampus. NF- COX-2 and iNOS K B, inhibit the inflammatory level, and increased in the hippocampus of autophagy and BDNF-TrkB pathway against nerve injury with A.A beta 1-42 beta patch pathological changes induced by prion like effect, I The behavior of Morris water maze and Y maze and elevated plus maze test showed that A beta 1-42 beta 25-35 A is stronger. The toxicity of silybin through down-regulation of autophagy, inhibiting the inflammatory reaction and up regulation of BDNF-TrkB pathway play a protective effect on nerve injury induced by A beta 1-42. Silybin based has estrogen like effect. And estrogen receptors in nerve cells have high expression, this suggests that silibinin is likely to play a neuroprotective role by regulating estrogen receptor and signal transduction. In A beta 1-42 damage animal, silybin lowered the level of ERs and PI3K-Akt mediated MAPKs pathway play a neuroprotective role. Conclusion: milk silibinin on LPS, learning and memory of A beta 25_35 and A beta 1-42 induced a protective effect of the protective effect may be through inhibition of the inflammatory level in the hippocampus, oxidative stress, regulating Section of autophagy and ERs and its related PI3K-Akt, MAPKs pathway. The research of silybin as neuroprotective drug development, as silybin in skin, pharmacological studies of liver and pancreas and other organs as well as for oxidative stress and autophagy - inflammation - estrogen receptor as a target for drug development provides a strong support the theory.

【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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