DPP-4抑制剂对AD样神经退行性变的保护作用及其机制
发布时间：2018-08-01 14:44
【摘要】：目的:二肽基肽酶-4抑制剂对阿尔茨海默病样神经退行性变的保护作用及其机制。方法:1)SPF级10月龄雄性野生型C57BL/6小鼠和APP/PS1/Tau三重转基因(3×Tg)小鼠随机分为野生(WT)组,转基因(Tg)组,西格列汀治疗(Tg+SIT)组和沙格列汀治疗(Tg+SAX)组,每组15只。待各组小鼠适应饲养环境后,每天分别给予西格列汀2.6 mg/kg和沙格列汀3.5 mg/kg(约260μl)灌胃,WT组和Tg组每天予以等量无菌生理盐水,持续8周。监测各组小鼠体重和血糖水平;Morris水迷宫检测小鼠空间学习记忆能力;免疫组化检测小鼠脑内Aβ42沉积;改良Bielschowsky染色检测小鼠脑组织神经原纤维缠结(NFTs)的数量;Fluoro-Jade B染色和尼氏染色标记小鼠脑组织退行性变的神经元;运用微管结合实验检测Tau与微管的结合能力,western blot和免疫荧光检测小鼠脑内GLP-1及其受体GLP-1R的表达水平、Tau蛋白和神经丝(NFs)的磷酸化和糖基化修饰水平、突触蛋白水平、胰岛素受体底物-1(IRS-1)和GLP-1下游信号通路关键酶和蛋白PI3K、Akt、GSK-3β、CREB、JNK和ERK的磷酸化水平。2)取对数生长期的人神经母细胞瘤细胞SH-SY5Y分为6组:wortmannin干预组(W组,0.03μmol/L wortmannin处理12 h)、DPP-4I干预组(DPP-4I组,10μmol/L DPP-4I处理12 h)、DPP-4I与wortmannin共同干预组(DPP-4I+W组,10μmol/L DPP-4I预处理2 h,0.03μmol/L wortmannin处理12 h)、DPP-4I、wortmannin及Ex9-39共同干预组(DPP-4I+W+Ex9-39组,10μmol/L Ex9-39预处理2 h,10μmol/L DPP-4I作用2 h,最后0.03μmol/Lwortmannin处理12 h)、Ex9-39干预组(Ex9-39组,10μmol/L Ex9-39处理12h)、空白对照组(CON组,含1‰DMSO的PBS处理12 h)。MTT法检测各组细胞活性的变化,Western blot法检测各组微管相关的Tau总蛋白及其不同磷酸化位点、神经丝(NFs)以及GLP-1下游信号通路PI3K-Akt-GSK-3β中关键酶的磷酸化水平。结果:1)各组小鼠体重和血糖间差异无统计学意义。与WT小鼠相比,Tg小鼠逃避潜伏期和游泳路径长度显著延长,而穿越隐匿平台次数和目标象限游泳时间明显缩短,小鼠脑内海马和皮质区GLP-1及其受体GLP-1R的表达水平下降、淀粉样蛋白Aβ42沉积、退行性变神经元和NFTs数量增加,Tau蛋白、NFs磷酸化修饰增多而糖基化修饰减少,Tau蛋白与微管结合能力减弱,脑内突触蛋白水平下降,IRS-1和PI3K、Akt、GSK-3β、CREB及ERK磷酸化修饰水平下降,JNK磷酸化修饰水平上升;Tg小鼠经西格列汀和沙格列汀处理后上述指标明显改善。2)与CON细胞相比,经wortmannin处理的细胞活力下降,Tau蛋白在丝氨酸和苏氨酸199、202、231和396位点以及NF-H/M的磷酸化水平升高,经DPP-4抑制剂处理的细胞活力上升,上述指标的磷酸化水平下降;与经wortmannin处理的细胞相比,经DPP-4抑制剂预处理的细胞活力上升,上述指标的磷酸化水平下降。与CON细胞相比,经Ex9-39处理的细胞活力下降,Tau蛋白在丝氨酸和苏氨酸199、202、231和396位点以及NFs的磷酸化水平升高;与经DPP-4抑制剂和wortmannin共同处理的细胞相比,经Ex9-39预处理的细胞活力下降,上述指标的磷酸化水平升高。与CON细胞相比,经wortmannin处理的细胞PI3K、Akt、GSK-3β磷酸化水平下降,而经DPP-4抑制剂处理的细胞PI3K、Akt、GSK-3β磷酸化水平上升;与经wortmannin处理的细胞相比,经DPP-4抑制剂预处理的细胞PI3K、Akt、GSK-3β磷酸化水平上升。结论:二肽基肽酶-4抑制剂通过提高GLP-1水平,改善脑内GLP-1信号通路和脑内糖代谢,提高AD小鼠学习记忆能力,改善神经退行性变。
[Abstract]:Objective: the protective effect of two peptidyl peptidase -4 inhibitor on Alzheimer like neurodegeneration and its mechanism. Methods: 1) SPF grade 10 month old male wild type C57BL/6 mice and APP/PS1/Tau three weight transgenic mice (3 x Tg) mice were randomly divided into wild (WT) group, Tg group, Sig Leo Dean therapy (Tg+SIT) group and Shah Glenn Dean treatment (Tg+SAX). Groups of 15 rats in each group were treated with Sig Leo Dean 2.6 mg/kg and Shah Glenn Dean 3.5 mg/kg (about 260 L) every day after adapting to the feeding environment. Group WT and Tg were given aseptic saline for 8 weeks. The weight and blood glucose level of mice in each group were monitored, and Morris water maze was used to detect the ability of learning and memory in mice; The A beta 42 deposition in the brain of mice was detected; the number of neurogenic fibrous tangles (NFTs) in the brain tissue of mice was detected by modified Bielschowsky staining; Fluoro-Jade B staining and Nissl staining were used to mark the neurons of the degenerative brain tissue in mice; the binding ability of Tau and microtubules was detected by microtubule binding assay, and Western blot and immunofluorescence were used to detect GLP- in the brain of mice. 1 and its receptor GLP-1R expression level, phosphorylation and glycosylation level of Tau and NFs, synaptic protein level, insulin receptor substrate -1 (IRS-1) and key enzymes of the downstream signal pathway of GLP-1 and protein PI3K, Akt, GSK-3 beta, CREB, JNK, and ERK, from the logarithmic growth period of human neuroblastoma cells They were divided into 6 groups: wortmannin intervention group (group W, 0.03 mu mol/L wortmannin processing 12 h), DPP-4I intervention group (DPP-4I group, 10 micron mol/L DPP-4I processing 12 h), DPP-4I and wortmannin intervention group (10 mu 2, 0.03 micron treatment 12). Mol/L Ex9-39 pretreatment 2 h, 10 mol/L DPP-4I action 2 h, final 0.03 mol/Lwortmannin treatment 12 h), Ex9-39 intervention group (Ex9-39 group, 10 mu mol/L Ex9-39), the blank control group (1 per thousand, 12) The phosphorylation level of the same phosphorylation site, the nerve filament (NFs) and the key enzyme in the downstream signal pathway of GLP-1. Results: 1) there was no significant difference between the weight and blood sugar of the mice in each group. Compared with the WT mice, the escape latency and the swimming path length of the Tg mice were significantly prolonged, while the number of hidden platforms and the target quadrant were swimming in the Tg mice. The expression level of GLP-1 and its receptor GLP-1R in the hippocampus and cortex of mice decreased significantly, the deposition of amyloid A beta 42, the number of degenerative neurons and NFTs, the increase of Tau protein, NFs phosphorylation and glycosylation modification, the weakening of Tau protein and microtubule binding capacity, the decrease of synapse protein levels in the brain, IRS-1 and PI3K, Akt, GSK-3 beta, CREB and ERK phosphorylation modification level decreased, JNK phosphorylation modification level increased; Tg mice improved.2 after treatment with Western glipetine and Shah Glenn Dean. Compared with CON cells, wortmannin treated cell vitality decreased, Tau protein at serine and threonine 199202231 and 396 loci and NF-H/M phosphorylated water. The activity of the cells treated by DPP-4 increased and the phosphorylation level of the above index decreased. Compared with the cells treated with wortmannin, the activity of cells pretreated by the DPP-4 inhibitor increased and the phosphorylation level of the above index decreased. Compared with the CON cells, the cell vitality of the Ex9-39 treated cells decreased, and the Tau protein was in serine and SSU. The phosphorylation level of the 199202231 and 396 sites of ammonia and NFs increased. Compared with the cells treated with DPP-4 inhibitors and wortmannin, the activity of the cells pretreated by Ex9-39 decreased and the level of phosphorylation of the above indexes increased. The level of PI3K, Akt, GSK-3 beta phosphorylation of wortmannin treated cells decreased compared with CON cells, and the DPP-4 was reduced by DPP-4. The level of phosphorylation of PI3K, Akt, GSK-3 beta in the cells treated by inhibitors increased; compared with wortmannin treated cells, PI3K, Akt, and GSK-3 beta phosphorylation levels of cells treated by DPP-4 inhibitor increased. Conclusion: two peptidyl peptidase -4 inhibitors improve AD mice learning by improving GLP-1 level, improving the GLP-1 signaling pathway and brain glucose metabolism in the brain. Memory ability improves neurodegenerative changes.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
本文编号：2157910
[Abstract]:Objective: the protective effect of two peptidyl peptidase -4 inhibitor on Alzheimer like neurodegeneration and its mechanism. Methods: 1) SPF grade 10 month old male wild type C57BL/6 mice and APP/PS1/Tau three weight transgenic mice (3 x Tg) mice were randomly divided into wild (WT) group, Tg group, Sig Leo Dean therapy (Tg+SIT) group and Shah Glenn Dean treatment (Tg+SAX). Groups of 15 rats in each group were treated with Sig Leo Dean 2.6 mg/kg and Shah Glenn Dean 3.5 mg/kg (about 260 L) every day after adapting to the feeding environment. Group WT and Tg were given aseptic saline for 8 weeks. The weight and blood glucose level of mice in each group were monitored, and Morris water maze was used to detect the ability of learning and memory in mice; The A beta 42 deposition in the brain of mice was detected; the number of neurogenic fibrous tangles (NFTs) in the brain tissue of mice was detected by modified Bielschowsky staining; Fluoro-Jade B staining and Nissl staining were used to mark the neurons of the degenerative brain tissue in mice; the binding ability of Tau and microtubules was detected by microtubule binding assay, and Western blot and immunofluorescence were used to detect GLP- in the brain of mice. 1 and its receptor GLP-1R expression level, phosphorylation and glycosylation level of Tau and NFs, synaptic protein level, insulin receptor substrate -1 (IRS-1) and key enzymes of the downstream signal pathway of GLP-1 and protein PI3K, Akt, GSK-3 beta, CREB, JNK, and ERK, from the logarithmic growth period of human neuroblastoma cells They were divided into 6 groups: wortmannin intervention group (group W, 0.03 mu mol/L wortmannin processing 12 h), DPP-4I intervention group (DPP-4I group, 10 micron mol/L DPP-4I processing 12 h), DPP-4I and wortmannin intervention group (10 mu 2, 0.03 micron treatment 12). Mol/L Ex9-39 pretreatment 2 h, 10 mol/L DPP-4I action 2 h, final 0.03 mol/Lwortmannin treatment 12 h), Ex9-39 intervention group (Ex9-39 group, 10 mu mol/L Ex9-39), the blank control group (1 per thousand, 12) The phosphorylation level of the same phosphorylation site, the nerve filament (NFs) and the key enzyme in the downstream signal pathway of GLP-1. Results: 1) there was no significant difference between the weight and blood sugar of the mice in each group. Compared with the WT mice, the escape latency and the swimming path length of the Tg mice were significantly prolonged, while the number of hidden platforms and the target quadrant were swimming in the Tg mice. The expression level of GLP-1 and its receptor GLP-1R in the hippocampus and cortex of mice decreased significantly, the deposition of amyloid A beta 42, the number of degenerative neurons and NFTs, the increase of Tau protein, NFs phosphorylation and glycosylation modification, the weakening of Tau protein and microtubule binding capacity, the decrease of synapse protein levels in the brain, IRS-1 and PI3K, Akt, GSK-3 beta, CREB and ERK phosphorylation modification level decreased, JNK phosphorylation modification level increased; Tg mice improved.2 after treatment with Western glipetine and Shah Glenn Dean. Compared with CON cells, wortmannin treated cell vitality decreased, Tau protein at serine and threonine 199202231 and 396 loci and NF-H/M phosphorylated water. The activity of the cells treated by DPP-4 increased and the phosphorylation level of the above index decreased. Compared with the cells treated with wortmannin, the activity of cells pretreated by the DPP-4 inhibitor increased and the phosphorylation level of the above index decreased. Compared with the CON cells, the cell vitality of the Ex9-39 treated cells decreased, and the Tau protein was in serine and SSU. The phosphorylation level of the 199202231 and 396 sites of ammonia and NFs increased. Compared with the cells treated with DPP-4 inhibitors and wortmannin, the activity of the cells pretreated by Ex9-39 decreased and the level of phosphorylation of the above indexes increased. The level of PI3K, Akt, GSK-3 beta phosphorylation of wortmannin treated cells decreased compared with CON cells, and the DPP-4 was reduced by DPP-4. The level of phosphorylation of PI3K, Akt, GSK-3 beta in the cells treated by inhibitors increased; compared with wortmannin treated cells, PI3K, Akt, and GSK-3 beta phosphorylation levels of cells treated by DPP-4 inhibitor increased. Conclusion: two peptidyl peptidase -4 inhibitors improve AD mice learning by improving GLP-1 level, improving the GLP-1 signaling pathway and brain glucose metabolism in the brain. Memory ability improves neurodegenerative changes.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
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