β-淀粉样蛋白对叉头蛋白O3a和突触后致密蛋白95的影响  

Eff ects ofβ-amyloid on the Expression of Forkhead Protein O3a and Postsynaptic Density Protein 95

作　　者：牛波[1] 王欣怡 徐江平[1] 汪海涛[1] NIU Bo;WANG Xin-yi;XU Jiang-ping;WANG Hai-tao(Department of Neuropharmacology,School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学药学院神经药理学科,广东广州510515

出　　处：《中山大学学报（医学科学版）》2021年第5期676-685,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81301099)。

摘　　要：【目的】为了研究β-淀粉样蛋白(Aβ)产生神经毒性的机制,本研究探讨了Aβ对凋亡相关核转录因子叉头蛋白O3a(FoxO3a)和突触后致密蛋白95(PSD95)的作用及可能的机制。【方法】采用梯度浓度(5、10、20μmol/L)寡聚化的Aβ25-35处理PC12细胞和神经元24 h,采用Westernblot法检测FoxO3a和PSD95的变化。免疫荧光法检测PC12细胞中PSD95表达量的变化和FoxO3a在细胞内的定位。在Aβ25-35海马注射的大鼠和APP/PS1转基因鼠中,考察脑组织中PSD95和FoxO3a的变化。采用WesternBlot法检测体外环境和在体环境中,Aβ对FoxO3a和蛋白激酶B(AKT)磷酸化的影响。【结果】与正常对照组相比,20μmol/L的Aβ25-35处理组PSD95蛋白水平下调至(45.09±1.61)%(F=7.487,P=0.0540)。与之对应,20μmol/L的Aβ25-35上调FoxO3a的蛋白水平为对照组的(228.7±20.44)%(F=17.48,P=0.0210)。在原代培养的神经元中,获得了相似的结果。另外,免疫荧光的结果显示Aβ25-35可以促进FoxO3a进入细胞核。大鼠海马注射Aβ25-35后,水迷宫实验中目标区域的平均停留时间为(24.35±1.29)s(F=2.843,P=0.098),平均穿越次数为(2.53±0.49)次(F=3.459,P=0.0149),与对照组相比都显著降低。RT-PCR结果显示Aβ25-35组大鼠海马组织中PSD95的mRNA水平下降为对照组的(58.40±8.28)%(F=1.193,P=0.0101),同时FoxO3a的mRNA的表达量上调(140.90±7.45)%(F=2.378,P=0.0496)。在7月龄的APP/PS1转基因小鼠的大脑组织中,PSD95的mRNA和蛋白水平分别下调为野生型小鼠的(60.89±1.53)%(F=20.05,P=0.0088)和(59.63±13.55)%(F=8.496,P=0.0445),而FoxO3a的mRNA和蛋白水平则分别上调为对照组的(172.4±4.87)%(F=2.351,P=0.0004)和(235.00±39.03)%(F=2.754,P=0.0320)。20μmol/L的Aβ25-35处理PC12细胞不同时间后(5、10、20和40 min),FoxO3a和AKT的磷酸化水平随着时间增长而降低,APP/PS1转基因小鼠脑组织中的AKT和FoxO3a的磷酸化水平与对照组相比也显著下降至(65.75±3.51)%(F=6.362,P=0.0236)和(46.62±9.64)%(F【Objective】To understand the mechanism of β-amyloid(Aβ)-induced neurotoxicity,this study aimed to investigate the effects and possible pathways of Aβ on apoptosis-related nuclear transcription forkhead protein O3a(FoxO3a)and postsynaptic density protein 95(PSD 95).【Methods】PC 12 cells and neurons were treated with gradient con centrations(5,10,20μmol/L)of Aβ25-35 for 24 h,and the alterations of FoxO 3 a and PSD 95 were detected by RT PCR and Western blot.Immunofluorescence assay was used to detect the level of PSD 95 in PC 12 cells and the subcellular localization of FoxO3a in the cells.Then in rats injected with Aβ25-35 and APP/PS 1 transgenic mice,the changes of PSD 95 and FoxO3a in brain tissue were investigated.Western Blot was used to detect the effects of Aβon the phosphorylation of FoxO3a and protein kinase B(AKT)in vitro and in vivo.【Results】Compared with cells in the normal control group,the protein levels of PSD 95 in the cells treated with Aβ25-35 at the dosage of 20μmol/L were down-regulated to(45.09±1.61)%(P=0.0540).Correspondingly,the protein levels of FoxO3a were increased to(228.70±20.44)%(F=17.48,P=0.0210)when the cells were treated with 20μmol/L of Aβ25-35.In the primary cultured neurons,similar results were obtained.In addition,the results of immunofluorescence showed that Aβ25-35 promoted the nuclear translocation of FoxO 3 a.The residence time of Aβ25-35-injected group was(24.35±1.29)s(F=2.843,P=0.098)and the number of crossings was 2.53±0.49(F=3.459,P=0.0149)of rats in the water maze test.There was significant difference between the CTL group and Aβ25-35-injected group(P<0.05).The RT-PCR assay showed that the mRNA level of PSD 95 in the hippocampus of rats treated with Aβ25-35 was decreased to(58.40±8.28)%(F=1.193,P=0.0101)of that in the CTL group,and the mRNA expression of FoxO3a was increased to(140.90±7.45)%(F=2.378,P=0.0496).In the brain tissue of 7-month-old APP/PS 1 transgenic mice,the mRNA and protein levels of PSD 95 were down-regulated to(60.89±1.53

关 键 词：Β-淀粉样蛋白 叉头框蛋白O3a 突触后致密蛋白95 蛋白激酶B 老年性痴呆 

分 类 号：R741.02[医药卫生—神经病学与精神病学]