阿司匹林对阿尔茨海默病模型鼠海马区炎性因子表达影响及机制研究

发布时间：2018-07-10 00:24

本文选题：阿尔茨海默病 + 阿司匹林　；参考：《第二军医大学》2017年硕士论文

【摘要】：阿尔茨海默病(Alzheimer's disease,AD)是一种多病因、发病机制尚不完全清楚的神经退行性疾病,以进行性认知功能下降、学习记忆能力减退、神经精神病学症状和日常行为异常等为主要临床特征,而由脑神经细胞外异常沉积的淀粉样β蛋白(Amyloid-βprotein,Aβ)和细胞内tau蛋白高度磷酸化聚集形成的神经纤维缠结(neurofibrillary tangles,NFTs)诱发的神经炎性反应、神经元凋亡、Aβ的生成与清除失衡为其主要病理学特征。AD是全球老年痴呆患者中最常见的类型,随着全球人口老龄化的加剧,其发病率也将快速增加,这将给患者、家人和社会带来沉重的经济负担。虽然对AD发病机制的研究越来越深入,但AD在当前仍然是一种不可治愈且难以阻止其进程的疾病,研发治疗AD的有效药物也成为目前面临的一个巨大挑战。大量研究发现,非甾体类抗炎药(non-steroid anti-inflammatory drugs,NSAIDs)在防治AD中具有潜在的有益作用。所以,针对AD的发病机制,如能在现有药物基础上,探索一种经济有效的防治药物将会产生巨大的社会意义和经济效益。大量研究证实中枢神经炎性反应在AD的发生和进展过程中发挥着关键作用。神经炎性反应可引起促炎性细胞因子白细胞介素1β(interleukin 1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor,TNF-α)等生成增加,而抗炎因子白细胞介素4(interleukin4,IL-4)、白细胞介素10(interleukin 10,IL-10)等不足也可能促进了神经炎性反应。IL-1β和TNF-α的神经毒性不仅可以损伤神经元和神经突触,反过来又激活NF-κB,活化的核转录因子κB(nuclear transcription factor,NF-κB)又可以诱导IL-1β、TNF-α、诱导型一氧化氮合酶(induced nitric oxide synthase,i NOS)和脂多糖(Lipopolysaccharide,LPS)等细胞因子表达,形成一个慢性神经炎性反应的恶性回路。随着神经炎性反应的持续存在,抗炎反应对促炎刺激持续缺乏,致使抗炎/促炎因子失衡,进一步促进神经炎性反应的发展。因此,通过抗炎药物抑制神经炎性反应、保护神经元,进而改善神经病理学变化可能是一种潜在的防治AD的新策略。阿司匹林为经典的NSAIDs,大量临床研究认为长期服用阿司匹林(Aspirin,Asp)的人群AD发病率低于未服用人群,阿司匹林在AD防治方面具有潜在可能性。因此,本研究通过对大鼠进行侧脑室注射淀粉样β蛋白25-35(Amyloid-βprotein 25-35,Aβ25-35)建立AD动物模型,探究阿司匹林的抗炎作用对AD模型大鼠空间学习能力及海马组织中促炎/抗炎因子IL-1β、TNF-α、IL-4和IL-10以及NF-κB、i NOS表达水平的影响,以期能为阿司匹林在防治AD中开展临床试验研究和实际应用提供部分实验室依据。研究目的中枢神经系统(central nervous system,CNS)的炎性反应和抗炎/促炎因子表达失衡被认为在AD的发生发展进程中发挥了关键作用。本课题通过Asp干预Aβ25-35诱导的AD模型鼠,研究阿司匹林对AD模型大鼠空间学习记忆能力、海马区炎性因子(IL-1β、TNF-α、IL-4和IL-10)表达水平以及抗炎/促炎因子微平衡的影响,并进一步探讨阿司匹林对AD的潜在防治作用及可能机制。研究方法40只雄性SD大鼠按照完全随机的方法分为四个实验组,10只/组。(1)对照组:自由饮用蒸馏水,喂养3周后,使用脑立体定向仪进行大鼠右侧侧脑室注射5μl的无菌生理盐水,再给予蒸馏水喂养3周;自由进食。(2)AD模型组:自由饮用蒸馏水,喂养3周后,使用脑立体定向仪进行大鼠右侧侧脑室注射5μl的Aβ25-35(10mmol/L)溶液,再给予蒸馏水喂养3周;自由进食。(3)低剂量阿司匹林干预组(1mg/ml):自由饮用蒸馏水,喂养3周后,使用脑立体定向仪进行大鼠右侧侧脑室注射5μl的Aβ25-35(10mmol/L)溶液,再给予蒸馏水喂养3周;自由进食。(4)高剂量阿司匹林干预组(2mg/ml):自由饮用蒸馏水,喂养3周后,使用脑立体定向仪进行大鼠右侧侧脑室注射5μl的Aβ25-35(10mmol/L)溶液,再给予蒸馏水喂养3周;自由进食。侧脑室注射术后继续喂养3周后,通过Morris水迷宫检测大鼠空间学习、记忆能力;采用双抗体夹心ELISA法检测海马区组织中IL-1β、TNF-α、IL-4和IL-10的表达水平;海马区组织制作病理切片,免疫组化染色观察NF-κB和i NOS表达情况,尼氏染色观察神经元凋亡情况。研究结果(1)空间学习记忆能力:与对照组比较,AD模型大鼠空间学习记忆能力显著下降(P0.001),1mg/ml Asp组大鼠与对照组差异有统计学意义(P0.05),2mg/ml Asp组大鼠与对照组比较无统计学意义(P0.05);与AD模型组比较,1mg/ml Asp组大鼠与模型组差异有统计学意义(P0.05),2mg/ml Asp组大鼠与模型组差异有统计学意义(P0.01);Asp干预组组间比较,差异无统计学意义(P0.05)。(2)海马区IL-1β表达:与对照组比较,AD模型组大鼠海马组织中IL-1β表达水平升高显著(P0.001),Asp干预组与对照组差异均无统计学意义(P0.05);与AD模型组比较,低、高剂量Asp干预组IL-1β表达水平均明显下降,差异均有统计学意义(P0.01);Asp干预组组间比较差异无统计学意义(P0.05)。(3)海马区TNF-α表达:与对照组比较,模型组大鼠海马组织中TNF-α表达水平显著升高(P0.001),1mg/ml Asp组与对照组差异有统计学意义(P0.01),2mg/ml Asp组与对照组差异无统计学意义(P0.05);Asp干预组与AD模型组比较,2mg/ml Asp组大鼠海马组织中TNF-α表达水平明显降低(P0.01),而1mg/ml Asp组与AD模型组差异无统计学意义(P0.05);Asp干预组组间比较差异无统计学意义(P0.05)。(4)海马区IL-4表达:与对照组大鼠比较,AD模型组大鼠海马组织中IL-4表达水平下降显著(P0.001),1mg/ml Asp组与对照组差异有统计学意义(P0.01),2mg/ml Asp组与对照组差异无统计学意义(P0.05);与AD模型组比较,2mg/ml Asp组大鼠海马区组织中IL-4表达水平明显升高,差异有统计学意义(P0.01),1mg/ml Asp组与AD模型组差异无统计学意义(P0.05);Asp干预组组间比较,差异无统计学意义(P0.05)。(5)海马区IL-10表达:与对照组比较,AD模型组大鼠海马区组织中IL-10表达水平下降显著(P0.01),1mg/ml Asp组与对照组差异无统计学意义(P0.05),2mg/ml Asp组与对照组差异无统计学意义(P0.05);与模型组比较,2mg/ml Asp组大鼠海马区组织中IL-10表达水平明显升高(P0.05),1mg/ml Asp组大鼠与AD模型组差异无统计学意义(P0.05);Asp干预组组间比较,差异无统计学意义(P0.05)。(6)海马区NF-κB和i NOS阳性表达:与对照组比较,AD模型组海马区组织中NF-κB和i NOS阳性表达明显显著增加,神经元缺失明显,而Asp干预组海马区组织中均有不同程度的NF-κB、i NOS阳性表达和神经元缺失,其中2mg/ml Asp干预组与对照组大致相当。(7)海马区神经元:与对照组比较,AD模型组海马区神经元缺失明显,而Asp干预组海马区均有不同程度神经元缺失,其中2mg/ml Asp干预组神经元缺失不明显,与对照组大致相当。研究结论阿司匹林干预可保护AD模型大鼠空间学习记忆能力;阿司匹林通过抑制炎性反应、NF-κB通路活化及i NOS表达,下调促炎因子IL-1β、TNF-α水平和上调抗炎因子IL-4、IL-10水平,进而调整促炎/抗炎因子失衡状态,在AD发病过程中抑制神经炎性反应发展,发挥抗炎、保护神经元和学习记忆能力的作用。
[Abstract]:Alzheimer's disease (AD) is a multiple cause of neurodegenerative disease whose pathogenesis is not completely clear. The main clinical features are progressive cognitive impairment, impairment of learning and memory, neuropsychiatry symptoms and daily behavior abnormalities, and amyloid beta protein (amyloid beta protein), which is abnormally deposited outside the nerve cells of the brain. Amyloid- beta protein, A beta, and highly phosphorylated intracellular tau protein aggregation (neurofibrillary tangles, NFTs) induced neuroinflammatory response, neuronal apoptosis, the formation and clearance of A beta, the main pathological feature of A beta, as the most common type of age idiot in the world, with the aging of the global population. The increasing incidence of the disease will also increase rapidly, which will bring a heavy financial burden to patients, family and society. Although the research on the pathogenesis of AD is becoming more and more deep, AD is still a disease that is not curable and difficult to prevent its process. The development of effective drugs for the treatment of AD has also become a huge challenge. Quantitative studies have found that non-steroid anti-inflammatory drugs (NSAIDs) has a potential beneficial effect in the prevention and control of AD. Therefore, in view of the pathogenesis of AD, the exploration of an economic and effective control drug on the basis of existing drugs will produce great social and economic benefits. A large number of studies have confirmed the center. The neuroinflammatory response plays a key role in the development and progression of AD. The neuroinflammatory response can cause the increase of pro-inflammatory cytokines interleukin 1 beta (interleukin 1 beta, IL-1 beta), tumor necrosis factor alpha (tumor necrosis factor, TNF- alpha), and the anti inflammatory factor interleukin 4 (interleukin4, IL-4), and interleukin 10 (I). Nterleukin 10, IL-10) and other deficiencies may also promote the neuroinflammatory response to the neurotoxicity of.IL-1 beta and TNF- alpha, which not only damage neurons and synapses, but also activate NF- kappa B, and the activated nuclear factor kappa B (nuclear transcription factor, NF- kappa B) can also induce the beta, alpha, inducible nitric oxide synthase (inducible nitric oxide synthase). The expression of cytokines such as oxide synthase, I NOS) and lipopolysaccharide (Lipopolysaccharide, LPS) forms a malignant loop of chronic inflammatory response. With the persistent neuroinflammatory response, the anti-inflammatory response to proinflammatory stimuli is continuously lacking, causing anti inflammatory / proinflammatory factors to balance and further promoting the development of neuroinflammatory response. Anti inflammatory drugs to inhibit neuroinflammatory response, protect neurons, and then improve neuropathological changes may be a potential new strategy for the prevention and control of AD. Aspirin is a classic NSAIDs. A large number of clinical studies believe that the incidence of AD in people who have long taken aspirin (Aspirin, Asp) is lower than that of non taking people and aspirin in the prevention and treatment of AD. Therefore, in this study, the rat model of AD was established by injecting amyloid beta protein 25-35 (Amyloid- beta protein 25-35, A beta 25-35) into the lateral ventricle of the rat, to explore the spatial learning ability of aspirin in AD model rats and the proinflammatory / anti-inflammatory factor IL-1 beta, TNF- a, IL-4 and IL-10, and NF- kappa B in the hippocampus. The effect of S expression level is expected to provide some laboratory basis for the clinical trials and practical applications of aspirin in the prevention and control of AD. The inflammatory response of the central nervous system (CNS) and the imbalance of the expression of anti-inflammatory / proinflammatory factors are considered to play a key role in the development of AD. The AD model rats induced by A beta 25-35 were used to study the spatial learning and memory ability of aspirin on AD model rats, the expression level of inflammatory factors (IL-1 beta, TNF- a, IL-4 and IL-10) in the hippocampus and the effect of the micro balance of anti-inflammatory and proinflammatory factors in the hippocampus, and further explore the potential preventive and therapeutic effects of aspirin on AD and its possible mechanism. 40 male SD rats were divided into four experimental groups according to the complete random method, 10 / group. (1) the control group: free drinking distilled water. After feeding for 3 weeks, the right side ventricle of the rat was injected with the sterile saline of 5 mu in the right lateral ventricle of the rat and fed with distilled water for 3 weeks. (2) the free drinking of distilled water and feeding for 3 weeks (2) AD model group. After that, the right lateral ventricle of the rat was injected with 5 L A beta 25-35 (10mmol/L) solution, and then fed with distilled water for 3 weeks, and free feeding. (3) low dose aspirin intervention group (1mg/ml): free drinking distilled water, and after feeding for 3 weeks, the right lateral ventricle of rats was injected with 5 L A beta 25-35 (10mmol/L) by injection of brain stereotaxic apparatus. The liquid was given to distilled water for 3 weeks; free feeding. (4) high dose aspirin intervention group (2mg/ml): free drinking distilled water. After feeding for 3 weeks, the right lateral ventricle of the rat was injected with 5 u l A beta 25-35 (10mmol/L), and then fed with distilled water for 3 weeks. Morris water maze was used to detect the spatial learning and memory ability of rats, and the expression level of IL-1 beta, TNF- a, IL-4 and IL-10 in hippocampus tissue was detected by double antibody sandwich ELISA; the expression of NF- kappa B and I NOS was observed by immunohistochemical staining in hippocampus tissue, and neuronal apoptosis was observed by immunohistochemical staining, and the results of Nissl staining were observed. (1) space Learning and memory ability: compared with the control group, the spatial learning and memory ability of the AD model rats decreased significantly (P0.001). The difference between the 1mg/ml Asp group and the control group was statistically significant (P0.05), and there was no significant difference between the 2mg/ml Asp group and the control group (P0.05), and the difference between the 1mg/ml Asp group and the model group was statistically significant compared with the AD model group. (P0.05) the difference between the 2mg/ml Asp group and the model group was statistically significant (P0.01), and the difference between the Asp intervention group was not statistically significant (P0.05). (2) the expression of IL-1 beta in the hippocampus: the increase of IL-1 beta expression in the hippocampus of the AD model group was significantly higher than that in the control group (P0.001), and there was no significant difference between the Asp intervention group and the control group (P0.0). 5): compared with the AD model group, the expression level of IL-1 beta in the low and high dose Asp intervention group was significantly decreased, the difference was statistically significant (P0.01), and there was no statistical difference between the Asp intervention group (P0.05). (3) the expression of TNF- a in the hippocampus: compared with the control group, the expression of TNF- a in the hippocampus of the model rats was significantly increased (P0.001), 1mg/ml Asp. There was significant difference between the group and the control group (P0.01), but there was no significant difference between the 2mg/ml Asp group and the control group (P0.05), and the expression level of TNF- alpha in the hippocampus of 2mg/ml Asp group was significantly lower than that of the AD model group (P0.01), but there was no significant difference between the 1mg/ml Asp group and the model group. There was no statistical significance (P0.05). (4) the expression of IL-4 in the hippocampus: compared with the control group, the level of IL-4 expression in the hippocampus of the AD model rats decreased significantly (P0.001), and the difference between the 1mg/ml Asp group and the control group was statistically significant (P0.01), and the 2mg/ml Asp group was different from the control group (P0.05); compared with the AD model group, the group was larger than the AD model group. The expression level of IL-4 in the rat hippocampus was significantly increased (P0.01), and there was no significant difference between the 1mg/ml Asp group and the AD model group (P0.05), and there was no statistical difference between the Asp intervention group and the Asp intervention group (P0.05). (5) the IL-10 expression in the hippocampus: the IL-10 expression level in the hippocampus of the AD model group was lower than that in the control group. There was no significant difference between the 1mg/ml Asp group and the control group (P0.05), but there was no significant difference between the 2mg/ml Asp group and the control group (P0.05). Compared with the model group, the IL-10 expression level of the hippocampus tissue in the group 2mg/ml Asp group was significantly higher (P0.05). There was no statistical difference between the rats of the 2mg/ml Asp group and the P0.01 group. There was no significant difference between the groups (P0.05). (6) the positive expression of NF- kappa B and I NOS in the hippocampus: compared with the control group, the positive expression of NF- kappa B and I NOS increased significantly in the hippocampus tissue of the AD model group, and the neuron deletion was obvious. 2mg/ml Asp intervention group was roughly equivalent to the control group. (7) hippocampal neurons: compared with the control group, the neuron loss in the hippocampus of the AD model group was obvious, while the hippocampus of the Asp intervention group had different degrees of neuron loss, and the neuron deletion in the 2mg/ml Asp intervention group was not obvious, which was roughly equivalent to the control group. Protecting AD model rats' spatial learning and memory ability; aspirin can inhibit inflammatory response, activation of NF- kappa B pathway and I NOS expression, down-regulation of proinflammatory factor IL-1 beta, TNF- a level and up regulation of IL-4, IL-10 level, and then regulate the state of inflammation and anti-inflammatory factors, inhibit the development of neuroinflammatory reaction in the course of AD hair disease, play anti-inflammatory and protect it. The function of protecting neurons and learning and memory.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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