【作者】 李慧；

【导师】 张芳；

【作者基本信息】 青岛大学 ， 药理学， 2021， 硕士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是老年人最常见的以进行性认知功能障碍和行为损害为特征的神经系统疾病,虽然其病因和发病机制尚未明确,但研究表明AD最主要的病理学特征是β-淀粉样蛋白(Amyloid-βplaques,Aβ)的聚集和Tau蛋白过度磷酸化形成的神经纤维缠结(Neurofibrillary tangle,NFT)。这些病理级联反应最终都会导致患者脑内胆碱功能减退并伴有严重的神经炎症反应。胆碱能抗炎通路(Cholinergic anti-inflammatory pathway,CAP)表明,乙酰胆碱(Acetylcholine,ACh)能够通过作用于巨噬细胞表面的烟碱型乙酰胆碱受体(Nicotinic acetylcholine receptor,nAChRs),尤其是α7烟碱型乙酰胆碱受体(Alpha7nicotinic acetylcholine receptor,α7 nAChR)抑制炎症的产生。α7 nAChR是配体门控离子通道超家族的一员,是由五个α7亚基组成的同源五聚体,对钙离子具有较高的通透性。在AD患者脑内Aβ过度沉积并形成α7 nAChR-Aβ复合物,导致脑内炎症环境加重,并严重损伤α7 nAChR的生理功能。α7 nAChR的配体结合位点主要有两种,即激动剂/拮抗剂结合位点以及变构调节剂结合位点。激动剂激活受体后介导钙离子内流,但易脱敏且靶向性弱。而正向变构调节剂(Positive allosteric modulator,PAM)单独使用时不引起钙离子通道的开放,仅在内源性ACh存在的情况下增加钙离子内流,增强α7 nAChR的功能,不易脱敏且靶向性强。α7 nAChR的PAM分为I型和II型,其中I型PAM能够增强激动剂诱发的峰值电流,而不延迟脱敏,不重新激活脱敏受体;II型PAM能够延迟脱敏和重新激活脱敏受体。JWX-A0108是我们自主合成的新型α7 nAChR的I型PAM,前期实验已经证明它能够明显增加ACh对α7 nAChR的激动作用,能增加海马自主放电,且不会影响其它nAChR亚型的生理作用,具有良好的靶向性。研究目的:本实验采用APP/PS1双转基因小鼠对JWX-A0108的药效学进行评价。以确证JWX-A0108能否改善APP/PS1小鼠认知障碍,并探索其作用机制。为研发治疗AD的药物提供实验依据。研究方法:1.Morris水迷宫检测JWX-A0108对九月龄APP/PS1转基因小鼠记忆与认知功能的影响。2.尼氏染色检测JWX-A0108对九月龄APP/PS1转基因小鼠脑内海马神经元的影响。3.Western Blot和免疫荧光检测JWX-A0108对九月龄APP/PS1转基因小鼠脑内小胶质细胞的影响。4.q RT-PCR和Western Blot检测JWX-A0108对九月龄APP/PS1转基因小鼠脑内炎症因子的影响。5.Western Blot检测核因子-κB(Nuclear factor-kappa B,NF-κB)通路的激活。研究结果:1.JWX-A0108(5 mg/kg,ip)改善了APP/PS1小鼠的认知功能障碍。2.JWX-A0108(5 mg/kg,ip)改善了海马CA3区神经元细胞排列疏松以及尼氏小体表达量减少等病理损伤状态。3.JWX-A0108(5 mg/kg,ip)降低了小胶质细胞(Microglia,MG)中离子钙接头蛋白分子(Ionized calcium bindingadaptor molecule-1,Iba1)的表达。4.JWX-A0108(5 mg/kg,ip)降低了APP/PS1小鼠大脑皮层和海马中炎症因子白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)的表达。5.JWX-A0108(5 mg/kg,ip)抑制了核因子-κB p65蛋白(Nuclear factor-kappa B p65,NF-κB p65)、核因子-κBα的抑制蛋白(Inhibitor of NF-κB alpha,IκBα)的磷酸化,抑制了NF-κB通路的激活。6.JWX-A0108所发挥的作用都能被α7 nAChR特异性阻断剂甲基牛扁亭碱(Methyllycaconitine,MLA)阻断。研究结论:JWX-A0108有效地改善了APP/PS1小鼠的认知功能障碍,该作用与增强内源性ACh激动α7 nAChR的功能,抑制IκBα和p65磷酸化从而抑制了NF-κB通路的激活,降低了小鼠脑内炎症因子的表达,逆转了MG的过度激活状态,并减轻海马CA3区神经元的损伤有关。更多还原

【Abstract】 Alzheimer’s disease(AD)is the most common of the neurodegenerative diseases among the aging population,characterized by progressive memory impairment,significant cognitive deficits and irreversible changes in personality and behavior.Although the specific molecular and cellular mechanisms responsible for the etiology and pathogenesis of AD have not been defined,research has revealed that the main pathological characteristics of AD are amyloid-β(Aβ)aggregation and the hyperphosphorylation of tau protein,which eventually develop into senile plaque and neurofibrillary tangles.Together with inflammation and oxidative stress,these pathological cascades contribute to the decline of cholinergic function in the brain.Cholinergic anti-inflammatory pathway(CAP)indicates that acetylcholine(ACh)can inhibit inflammation by acting on nicotinic acetylcholine receptors(nAChRs)on the surface of macrophages,especially α7 nAChR.This receptor belongs to the super family of pentameric ligand-gated ion channels.It is a homopentamer composed of five α7subunits and has high permeability to calcium ions.The α7 nAChR-Aβ complex in the brain leads to the aggravation of inflammatory environment and serious damage α7nAChR.There are two main ligand binding sites of α7 nAChR,namely agonist/antagonist binding sites and allosteric regulatory sites.Activation of receptor by agonists induces calcium influx,with fast desensitization and off-target effects.Positive allosteric modulator(PAM)of α7 nAChR with low intrinsic activity,only increased calcium influx in the presence of endogenous ACh,and it is not easy to desensitize and with specific targeting.The α7 nAChR PAMs have been classified into two categories: type I and type II.Type I PAMs increase peak current in the presence of an orthosteric agonist without affecting receptor desensitization.Type II PAMs,on the other hand,significantly reduce the fast desensitization of α7 nAChRs.JWX-A0108 is a new type I PAM of α7 nAChR developed and synthesized by our laboratory independently.Previous experiments have proved that JWX-A0108 can significantly increase the excitatory effect of ACh on α7 nAChR,increase the spontaneous discharge of hippocampus,it can specifically target α7 nAChR and the physiological effects of other nAChR subtypes were not affected.Objective: In this study,APP/PS1 double transgenic mice were used to evaluate the pharmacodynamics of JWX-A0108.To confirm whether JWX-A0108 can improve cognitive impairment in APP/PS1 mice and explore its mechanism.To provide experimental basis for the research and development of drugs for the treatment of AD.Methods:1.Morris water maze was used to detect the effect of JWX-A0108 on the cognitive and memory function of nine months old APP/PS1 transgenic mice.2.Nissl staining was used to detect the effect of JWX-A0108 on hippocampal neurons in nine months old APP/PS1 transgenic mice.3.Western Blot and immunofluorescence were used to detect the effect of JWX-A0108 on microglia in nine months old APP/PS1 transgenic mice.4.q RT-PCR and Western Blot were used to detect the effects of JWX-A0108 on inflammatory factors in the brain of nine months old APP/PS1 transgenic mice.5.Western Blot was used to detect the activation of NF-κB pathway.Results:1.JWX-A0108(5 mg/kg,ip)improved the cognitive impairment of APP/PS1 mice.2.JWX-A0108(5 mg/kg,ip)improved the pathological damage of hippocampal CA3 area,such as loose cell arrangement and decreased Nissl body expression.3.JWX-A0108(5 mg/kg,ip)decreased the expression of Iba1 in microglia.4.JWX-A0108(5 mg/kg,ip)decreased the expression of IL-1β,TNF-α and IL-6 in cerebral cortex and hippocampus of APP/PS1 mice.5.JWX-A0108(5 mg/kg,ip)inhibited the phosphorylation of NF-κB p65,IκBα and the activation of NF-κB,the effects of JWX-A0108 were blocked by MLA,a specific α7nAChR blocker.Conclusions:JWX-A0108 can significantly improve the learning and cognitive function of APP/PS1 mice by enhancing the activation of endogenous ACh on α7 nAChR,thus inhibiting the over-activation of the NF-κB pathway in microglia and reducing the inflammatory responses.更多还原