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Chinese Journal of Alzheimer's Disease and Related Disorders

Abbreviation (ISO4): Chinese Journal of Alzheimer's Disease and Related Disorders      Editor in chief: Jun WANG

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Chinese Journal of Alzheimer's Disease and Related Disorders >

2020 , Vol. 3 >Issue 1: 56 - 60

DOI: https://doi.org/10.3969/j.issn.2096-5516.2020.01.014

Effects of AcSDKP on apoptosis induced by β-amyloid protein

  • ZHAI Wanying , 1, 2 ,
  • QIAN Yihua , 1 ,
  • MA Kaige 3 ,
  • CHANG Kewei 1 ,
  • ZONG Hangfan 1 ,
  • YANG Weina 1 ,
  • HAN Hua 1
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  • 1 Department of Human Anatomy and Histology-Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center
  • 2 Department of Dermatology, Plastic Surgery Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College
  • 3 Institute of Neurobiology

Received date: 2019-11-16

  Revised date: 2019-12-06

  Online published: 2020-03-25

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Abstract

Objective: To establish Alzheimer’s disease (AD)-like cellular model induced by Oligomeric Amyloid-beta1-42 (oAβ1-42) and explore the effects of acetyl-Ser-Asp-Lys-Pro (AcSDKP) on apoptosis induced by β-amyloid protein. Methods: SH-SY5Y cells were treated with oAβ1-42 and AcSDKP. The viability of SH-SY5Y cells was examined, the expression of BCL2 and BAX protein, the release of cytochrome C (Cyt c) and the signal molecular NF-κB were assayed by Western blot The neuroprotection of AcSDKP was explored on the cells. Results: SH-SY5Y cells were treated with oAβ1-42 for 24 hours, MTT assay showed that the toxicity of oAβ1-42 to SH-SY5Y cells was concentration-dependent, more than 2.5 μmol oAβ1-42 could significantly decrease the viability of cells, while viability was decreased by about 50% after treatment with 5 μmol of oAβ1-42 for 24 h. Western blot showed that the level of BAX in AcSDKP group was significantly lower than that in Aβ treatment group, and the level of BCL2 in AcSDKP treatment group was significantly higher than that in Aβ treatment group. The results also showed that AcSDKP significantly decreased the release of Cytc. At the same time, it was observed that oAβ1-42 promoted the expression of NF-κB, while AcSDKP could significantly inhibit the expression of NF-κB. Conclusion: AcSDKP could inhibit apoptosis induced by oAβ1-42, which may be due to the inhibition of NF-κB signaling pathway.

Key words： Alzheimer’s disease; Oligomeric Amyloid-beta; AcSDKP; Aoptosis; NF-κB

Cite this article

ZHAI Wanying , QIAN Yihua , MA Kaige , CHANG Kewei , ZONG Hangfan , YANG Weina , HAN Hua . Effects of AcSDKP on apoptosis induced by β-amyloid protein[J]. Chinese Journal of Alzheimer's Disease and Related Disorders, 2020 , 3(1) : 56 -60 . DOI: 10.3969/j.issn.2096-5516.2020.01.014

阿尔茨海默病（Alzheimer’s disease,AD）是老年人群常见神经变性疾病之一。AD典型的病理学改变为大量淀粉样蛋白在脑内沉积,神经元内神经原纤维缠结形成,神经元丢失。同时较多报道提出AD病人脑内有明显的神经炎症反应表现[1⇓-3]。关于AD发病机制有多种学说,如淀粉样蛋白级联假说、tau蛋白假说、基因遗传假说、神经炎症假说等。由于AD发病的复杂性,故有关AD的有效防治一直是研究的焦点。目前关于AD治疗的研究主要围绕在它的病理改变上。而Aβ的沉积是导致AD的关键因素,Aβ神经毒性可以继发导致神经炎症等多种病理性损伤,最终引起细胞凋亡、突触功能变化,学习记忆障碍。而目前有关AD有效防治尚无有效措施,属于亟待解决的重要问题。为了探索针对AD神经炎症这一关键组织病理学变化,本课题组选取炎症免疫调节分子胸腺素β4（thymosin β4,Tβ4）,探索其是否有防治AD的作用及其可能机制。
Tβ4在许多生理和病理活动中起重要作用,研究证明Tβ4不仅参与细胞骨架平衡、炎症反应等多种生理功能,还能促进创伤愈合、角膜损伤修复及心肌修复,并且它可显著抑制细胞凋亡,起到保护内皮细胞、间充质干细胞、心肌细胞等的作用[4⇓-6]。有关Tβ4对神经系统疾病的防治研究鲜见。而N-乙酰-丝-天冬-赖-脯氨酸（AcSDKP）是Tβ4 N端的氨基酸序列,即第四位脯氨酸残基与第五位天冬氨酸残基之间肽键的蛋白酶切产物。在人体内广泛存在且生物功能多样化。研究报道表明四肽AcSDKP可以促进血管发生、抗炎症等多种功效[7,8]。鉴此本课题组测AcSDKP可能对Aβ引起的神经元损伤具有保护作用,以达到防治AD的作用。

1 材料与方法

1.1 方法

本实验采用的是人神经母细胞瘤细胞系即SH-SY5Y细胞系。

1.1.2 主要试剂

Aβ1-42 （北京博奥森生物工程公司）,DMEM高糖培养基（Invitrogen 公司）,胰蛋白酶、DMSO、多聚赖氨酸、MTT（美国Sigma公司）,兔抗BAX抗体、兔抗Cytochrome c抗体、兔抗NF-κB抗体（美国Proteintech公司）,鼠抗GAPDH抗体（美国Santa Cruz公司）,HRP 标记的抗兔二抗、HRP标记的抗鼠二抗,SuperSignal化学发光试剂盒（美国Pierce公司）,兔抗BCL2抗体（CST公司）,AcSDKP（西安华广生物工程有限公司惠赠）。

1.2 方法

1.2.1 Aβ1-42寡聚体的制备

在超净工作台内,用六氟异丙醇溶解Aβ1-42,充分溶解后4 ℃放置72h,制备Aβ寡聚体,-80 ℃冷冻1 h,入低温冷冻干燥机到-40 ℃,真空、干燥6 h,然后-80 ℃保存待用。

1.2.2 人神经母细胞瘤SH-SY5Y细胞的体外培养

SH-SY5Y细胞培养在含有10%胎牛血清的DMEM培养基中,置于37 ℃、5% CO2浓度的培养箱中培养。

1.2.3 MTT 法

接种于96孔板的SH-SY5Y细胞,按照相应分组,加入oAβ1-42孵育后,进行MTT检测;每组至少3孔细胞,并预留3个空白组（只加入培养基）。每孔加入噻唑蓝（5 mg∕ml）20 μl置于37 ℃、5%CO2培养箱孵育4 h,吸弃每孔的培养液后,每孔加入150 μl DMSO,振荡器震荡直至紫色结晶完全溶解,用酶联免疫检测仪测定OD值,波长为492 nm,校正参数为630,重复检测3次。

1.2.4 Western blot检测BCL2、BAX、Cytochrome c、NF-κB的蛋白表达

分别提取各组实验细胞的总蛋白,根据BCA试剂盒检测蛋白浓度,确定上样量。经过SDS-PVDF凝胶电泳后,4 ℃过夜孵育一抗BCL2（1:500）、BAX（1:1000）、Cytochrome c (1:1000)、NF-κB（1:500）、GAPDH（1:1000）,室温孵育二抗1.5 h。通过ECL显影、化学成像后,半定量检测BCL2、BAX、Cytochrome c、NF-κB蛋白表达。

1.3 统计学分析

采用SPSS13.0进行统计分析,所有数据均采用平均值±标准差表示。对于不同组的数据,通过单因素方差分析（One-way ANOVA）进行分析,首先进行方差检验,如果方差检验显示为方差齐性,用LSD（Least Significant Difference）检验,方差不齐时采用Tamhane’s T2进行统计分析,P<0.05表示差异有统计学意义。

2 结果

2.1 Aβ1-42对SH-SY5Y细胞活力影响的量效分析

在时间一定的条件下,Aβ的浓度是决定其引起的是生理效应还是毒理效应的关键因素。本课题组用MTT法检测不同浓度oAβ1-42对SH-SY5Y作用24 h,引起细胞活力的变化。图1显示,dayudegnyu≥2.5 μmol oAβ1-42,明显降低细胞存活率（P<0.01）,而5 μmol oAβ1-42处理细胞24 h,细胞的活力呈现明显降低约50%。
图1 MTT检测不同浓度oAβ1-42对SH-SY5Y细胞活力影响(*: P<0.01 vs control, n=3)

2.2 AcSDKP对BCL2和BAX蛋白表达的影响

实验分为3组：①对照组;②Aβ1-42组;③AcSDKP+Aβ1-42组。首先向AcSDKP+Aβ1-42组SH-SY5Y细胞加入160nmol AcSDKP共孵育24 h,然后分别用5 μmol oAβ1-42与Aβ1-42组和AcSDKP+Aβ1-42组细胞共孵育6 h,而对照组加入等体积的培养基共孵育6 h。收集细胞,提取总蛋白,采用Western blot方法检测BCL2和BAX蛋白表达。
结果显示,在Aβ处理组,BAX的表达水平显著高于生理盐水组（如图2A,P<0.05）;AcSDKP+Aβ1-42处理组,BAX的水平显著低于Aβ处理组（如图2A,P<0.05）。并且在AcSDKP+Aβ1-42处理组,BCL2的水平较Aβ处理组和生理盐水处理组都要高（如图2B,P<0.05）,提示AcSDKP可以下调oAβ1-42所诱导的凋亡反应。
图2 oAβ1-42及AcSDKP对BAX及BCL2表达的影响（oAβ1-42:5μmol; AcSDKP:160nmol;*：P<0.05 vs control; #：P<0.05 vs Aβ1-42, n=3）

2.3 AcSDKP对Cytc释放和NF-κB蛋白表达的影响

研究已表明BCL2可与BAX形成异源二聚体,降低线粒体膜通透性,从而起到抑制凋亡的作用;而BAX可以打开线粒体膜通道,增加Cyt c的释放,起到促进凋亡的作用。我们进行了Cyt c的检测。结果表明,在oAβ1-42处理组,Cyt c的表达显著高于生理盐水组（如图3A,P<0.05）。与oAβ1-42处理组和生理盐水组相比,AcSDKP+Aβ1-42处理组Cyt c的释放显著减少。随后进行了可能的机制探究,假设AcSDKP是通过抑制NF-κB的表达来实现抗凋亡的作用。实验结果显示,在oAβ1-42处理组,NF-κB表达水平显著高于生理盐水组（如图3B,P<0.05）,而在AcSDKP+Aβ1-42处理组,加入AcSDKP后NF-κB表达降低。
图3 oAβ1-42及AcSDKP对Cyt c及NF-κB表达的影响（Aβ:5μmol; AcSDKP:160nmol;*：P<0.05 vs saline; #：P<0.05 vs Aβ1-42, n=3）

3 讨论

AD是一种以进行性认知功能减退为特征的中枢神经变性疾病。目前AD的发病机制仍不明确,发病机制上主要以β淀粉样级联瀑布假说占据中心事件,即AD病人Aβ在脑内大量沉积,级联神经毒性作用导致一系列病理学改变,其中神经炎症反应越来越多引起研究者的重视。出现神经炎症时白介素1β(IL-1β),白介素6（IL-6）和TNF-α等分泌增多,损伤神经元,导致神经元凋亡[9-10]。因此,减轻神经炎症是探索有效防治AD重要的靶点和思路。
Tβ4是一种炎症免疫调节分子,它在许多生理和病理活动中起重要作用。早些时期研究表明Tβ4和球形肌动蛋白作用,参与细胞骨架微管的重塑,保护损伤后心肌和多种器官前体细胞的迁移和组织结构、机能的再建[11]。同时小分子的Tβ4能够通过细胞的自分泌、旁分泌机制分泌到细胞间,在分泌过程中的再加工形成氨基端乙酰化的AcSDKP、核心区15肽、7肽,羧基端短肽多种生物机能肽,具有促进血管生成的特性,保护组织免受损伤,并促进组织再生[12]。
Tβ4/AcSDKP被誉为器官损伤的修复因子。AD是一种慢性神经变性疾病,以Aβ毒性作用引起的细胞凋亡、神经元大量丢失、炎症反应等为主要病理特征。而Tβ4的主要作用是修复损伤、抗细胞凋亡、抗炎症反应、促进再生等[13-14]。本实验结果提示AcSDKP可能具有降低Cyt c释放、增加抑凋亡蛋白的表达,以对抗细胞凋亡,起到对细胞保护作用。
本次研究结果还提示AcSDKP可能是通过调节核转录因子NF-κB的表达来起到对抗神经细胞凋亡的作用。近些年来关于核转录因子的研究越来越多[15-16],但是它在AD中对抗凋亡的作用还有待于阐明。NF-κB发挥作用复杂,不是简单的如本研究所提到的表达地变化,NF-κB结合DNA形成的同源和/或异源二聚体与靶基因上10 bp特定的序列（-κB 位点）结合调节基因转录,不同的NF-κB二聚体在选择结合序列时可能略有差异,这是NF-κB通过不同的二聚体的形式对不同基因的表达进行精细调节的一种方式;其次NF-κB信号通路激活引起系列信号转导反应出现NF-κB会进入细胞核,与有NF-κB结合位点的基因结合,启动转录进程。因此NF-κB发挥作用还涉及其磷酸化位点与水平、进核等复杂分子机制[17]。下一步本课题组将在此实验基础上完善课题设计,进一步探索AcSDKP保护神经元的可能机制。

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