雷公藤氯内酯醇通过改善突触可塑性、调节Aβ代谢、抑制神经炎症反应减轻5XFAD转基因小鼠的认知损害
发布时间：2019-01-09 06:49
【摘要】：【目的】β淀粉样蛋白（β-amyloid，Aβ）代谢异常是阿尔茨海默病（Alzheimer’sdisease，，AD）发病机制的核心环节。Aβ的生成和清除失平衡导致其在脑内异常增多并聚集，触发了一系列病理生理级联反应，包括突触功能障碍、胶质细胞活化诱发的炎症反应等，最终导致神经元损伤并引起痴呆。本研究以5XFAD转基因小鼠为动物模型，观察雷公藤氯内酯醇（tripchlorolide，T_4）对该转基因小鼠认知功能的影响，并分别从突触可塑性、Aβ代谢和神经炎症的角度较为深入的探讨T_4作用的可能分子机制。 【方法】SPF级5月龄雄性5XFAD转基因小鼠和野生型小鼠，各自分别随机分为溶媒对照组（0.9%NaCl）、T_4处理组（5μg/kg，25μg/kg），每组8～11只，腹腔注射给药，隔日1次，共60天。 1、采用Y迷宫和Morris水迷宫两种行为学实验，检测动物学习记忆功能。 2、电镜下观察海马CA1区突触超微结构；Western blot法检测海马和皮层突触可塑性相关蛋白synaptophsin、 PSD-95、 p-NMDAR1/NMDAR1、p-CaMKIIα/CaMKIIα、p-CREB/CREB和PI3K/Akt/mTOR信号通路中pAkt/Akt、pmTOR/mTOR的表达。 3、通过免疫组化、ELISA和Western blot检测脑内Aβ及其生成和降解的关键酶（包括β分泌酶、α分泌酶、NEP和IDE）的表达。 4、通过免疫组化观察脑内小胶质细胞和星形胶质细胞的活化情况，通过Western blot检测脑内GFAP的表达，利用real-time PCR检测海马炎症介质的mRNA水平。 【结果】 1、T_4可改善5XFAD转基因小鼠视空间学习记忆障碍。 2、T_4可改善5XFAD转基因小鼠海马突触超微结构异常，增加脑内突触可塑性相关蛋白的表达，同时增加pAkt/Akt、pmTOR/mTOR的表达。 3、T_4能够减少5XFAD转基因小鼠脑内Aβ沉积，降低BACE1及其酶切产物sAPP-β、CTF-β的表达，增加NEP的表达，但对APP、CTF-α、ADAM10、ADAM17、IDE的表达无影响。 4、T_4能够抑制5XFAD转基因小鼠脑内小胶质细胞活化，下调炎症介质IL-1β、TNF-α、IL-6、TGF-β、CCL2、NOS2的mRNA水平，但对星形胶质细胞活化无明显影响。 【结论】5XFAD转基因小鼠存在视空间学习记忆障碍、突触功能损害、Aβ沉积和神经炎症反应。T_4可能通过活化PI3K-Akt-mTOR通路促进突触可塑性相关蛋白的合成；抑制BACE1活性，增加NEP表达，减少Aβ生成并促进其降解清除；抑制小胶质细胞活化所致的神经炎症反应，从而改善该转基因小鼠的认知功能障碍。因此，T_4有望成为AD治疗有前途的候选药物。
[Abstract]:[objective] abnormal metabolism of 尾 -amyloid protein (尾 -amyloid 尾) is the key link in the pathogenesis of Alzheimer's disease (Alzheimer'sdisease,AD). It triggers a series of pathophysiological cascade reactions, including synaptic dysfunction and inflammation induced by the activation of glial cells, leading to neuronal damage and dementia. The effects of tripterygium wilfordii chloride lactone (tripchlorolide,T_4) on the cognitive function of 5XFAD transgenic mice were studied, and the synaptic plasticity was observed. The possible molecular mechanism of T4 was discussed in detail from the perspective of A 尾 metabolism and neuroinflammation. [methods] SPF grade 5-month-old male 5XFAD transgenic mice and wild-type mice were randomly divided into two groups: solvent control group (0.9%NaCl), T4 treated group (5 渭 g / kg ~ 25 渭 g/kg), each group (n = 8). Once every other day for 60 days. 1. Two behavioral experiments, Y maze and Morris water maze, were used to detect the learning and memory function of animals. 2. Ultrastructure of synapses in hippocampal CA1 area was observed under electron microscope. The expression of pAkt/Akt,pmTOR/mTOR in hippocampal and cortical synaptic plasticity associated protein (synaptophsin, PSD-95, p-NMDAR1 / p-CaMKII 伪 / CaMKII 伪, p-CREB/CREB and PI3K/Akt/mTOR signaling pathway) was detected by Western blot method. 3The expression of A 尾 and its key enzymes (including 尾 -secretase, 伪 -secretase, NEP and IDE) in brain were detected by immunohistochemistry, ELISA and Western blot. (4) the activation of microglia and astrocytes in brain was observed by immunohistochemistry, the expression of GFAP in brain was detected by Western blot, and the mRNA level of inflammatory mediators in hippocampus was detected by real-time PCR. [results] 1. Tip4 could improve the visual spatial learning and memory impairment of 5XFAD transgenic mice. 2TX _ 4 could improve the synaptic ultrastructure abnormality, increase the expression of synaptic plasticity related protein and increase the expression of pAkt/Akt,pmTOR/mTOR in hippocampal synaptic ultrastructure of 5XFAD transgenic mice. 3T _ 4 could reduce A 尾 deposition in brain of 5XFAD transgenic mice, decrease the expression of sAPP- 尾 and CTF- 尾, and increase the expression of NEP, but had no effect on the expression of APP,CTF- 伪 and ADAM10,ADAM17,IDE. 4T _ 4 could inhibit the activation of microglia in the brain of 5XFAD transgenic mice and down-regulate the mRNA levels of inflammatory mediators IL-1 尾, TNF- 伪, IL-6,TGF- 尾 and CCL2,NOS2, but had no effect on the activation of astrocytes. [conclusion] 5XFAD transgenic mice have visual spatial learning and memory impairment, synaptic dysfunction, A 尾 deposition and neuroinflammatory response. Tap4 may promote the synthesis of synaptic plasticity related proteins by activating PI3K-Akt-mTOR pathway. Inhibition of BACE1 activity, increase of NEP expression, reduction of A 尾 production and promotion of its degradation and clearance, inhibition of neuroinflammation induced by activation of microglia, and improvement of cognitive impairment in transgenic mice. Therefore, T-d-4 is expected to be a promising drug candidate for AD therapy.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16
本文编号：2405269
[Abstract]:[objective] abnormal metabolism of 尾 -amyloid protein (尾 -amyloid 尾) is the key link in the pathogenesis of Alzheimer's disease (Alzheimer'sdisease,AD). It triggers a series of pathophysiological cascade reactions, including synaptic dysfunction and inflammation induced by the activation of glial cells, leading to neuronal damage and dementia. The effects of tripterygium wilfordii chloride lactone (tripchlorolide,T_4) on the cognitive function of 5XFAD transgenic mice were studied, and the synaptic plasticity was observed. The possible molecular mechanism of T4 was discussed in detail from the perspective of A 尾 metabolism and neuroinflammation. [methods] SPF grade 5-month-old male 5XFAD transgenic mice and wild-type mice were randomly divided into two groups: solvent control group (0.9%NaCl), T4 treated group (5 渭 g / kg ~ 25 渭 g/kg), each group (n = 8). Once every other day for 60 days. 1. Two behavioral experiments, Y maze and Morris water maze, were used to detect the learning and memory function of animals. 2. Ultrastructure of synapses in hippocampal CA1 area was observed under electron microscope. The expression of pAkt/Akt,pmTOR/mTOR in hippocampal and cortical synaptic plasticity associated protein (synaptophsin, PSD-95, p-NMDAR1 / p-CaMKII 伪 / CaMKII 伪, p-CREB/CREB and PI3K/Akt/mTOR signaling pathway) was detected by Western blot method. 3The expression of A 尾 and its key enzymes (including 尾 -secretase, 伪 -secretase, NEP and IDE) in brain were detected by immunohistochemistry, ELISA and Western blot. (4) the activation of microglia and astrocytes in brain was observed by immunohistochemistry, the expression of GFAP in brain was detected by Western blot, and the mRNA level of inflammatory mediators in hippocampus was detected by real-time PCR. [results] 1. Tip4 could improve the visual spatial learning and memory impairment of 5XFAD transgenic mice. 2TX _ 4 could improve the synaptic ultrastructure abnormality, increase the expression of synaptic plasticity related protein and increase the expression of pAkt/Akt,pmTOR/mTOR in hippocampal synaptic ultrastructure of 5XFAD transgenic mice. 3T _ 4 could reduce A 尾 deposition in brain of 5XFAD transgenic mice, decrease the expression of sAPP- 尾 and CTF- 尾, and increase the expression of NEP, but had no effect on the expression of APP,CTF- 伪 and ADAM10,ADAM17,IDE. 4T _ 4 could inhibit the activation of microglia in the brain of 5XFAD transgenic mice and down-regulate the mRNA levels of inflammatory mediators IL-1 尾, TNF- 伪, IL-6,TGF- 尾 and CCL2,NOS2, but had no effect on the activation of astrocytes. [conclusion] 5XFAD transgenic mice have visual spatial learning and memory impairment, synaptic dysfunction, A 尾 deposition and neuroinflammatory response. Tap4 may promote the synthesis of synaptic plasticity related proteins by activating PI3K-Akt-mTOR pathway. Inhibition of BACE1 activity, increase of NEP expression, reduction of A 尾 production and promotion of its degradation and clearance, inhibition of neuroinflammation induced by activation of microglia, and improvement of cognitive impairment in transgenic mice. Therefore, T-d-4 is expected to be a promising drug candidate for AD therapy.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16
【参考文献】
相关期刊论文 前2条
1 胡小令,吕诚,万卫,张宪郁;雷公藤多甙对Alzheimer病大鼠学习记忆和小胶质细胞的影响[J];中国老年学杂志;2005年02期
2 吕诚;胡小令;万斌;杨宝林;;雷公藤内酯醇对阿尔茨海默病模型大鼠学习记忆和海马核因子-κB表达的影响[J];中国老年学杂志;2009年17期
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