CN105748461A - 苏木精抑制β-淀粉样蛋白聚集及其相关毒性的应用 - Google Patents
苏木精抑制β-淀粉样蛋白聚集及其相关毒性的应用 Download PDFInfo
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Abstract
本发明涉及苏木精抑制β?淀粉样蛋白聚集及其相关毒性的应用;通过多种实验手段证明,苏木精与β?淀粉样蛋白摩尔浓度比在0.5~1:1时,能够最有效地抑制了Aβ42纤维的生成;延缓Aβ42二级结构向β?折叠结构转化,降低Aβ42在溶液中形成的β?折叠的含量;并引导了Aβ42的不同聚集路径,改变了Aβ42的纤维形貌;最终大幅降低了Aβ42聚集过程中所产生的细胞毒性作用,是抑制Aβ42聚集的最佳实施方案。苏木精与β?淀粉样蛋白的摩尔浓度比在0.5~1:1条件下,对β?淀粉样蛋白聚集及其相关毒性具备最佳抑制效果;提供了用于药物和保健品开发方面的用途和用于阿尔兹海默症的药物的原料药应用。
Description
技术领域
本发明涉及苏木精与β-淀粉样蛋白摩尔浓度比在0.5~1:1时,对β-淀粉样蛋白聚集及其相关细胞毒性的有效抑制作用,及其潜在的药物和保健品开发方面的用途。
背景技术
阿尔兹海默症(Alzheimer’s Disease,AD)是最为常见的老年痴呆症,其病症表现为认知功能障碍、记忆能力衰退、情绪不稳定等症状。大量研究表明AD发病过程中伴随着β-淀粉样蛋白(amyloid β-protein,Aβ)的聚集过程,在患者脑内神经元细胞中产生纤维状斑块。因此,可以认为Aβ聚集成纤维与AD发病之间存在着紧密的联系。
现有研究表明Aβ单体在水溶液条件下呈现无规则卷曲结构,且被证明无毒性。而当Aβ自组装成为包含了cross-β-折叠结构的纤维后能够引发神经细胞的细胞凋亡。“成核模型”是现在公认的Aβ聚集模型。在该模型的描述中,Aβ单体首先通过构象转换形成富含β-折叠的二级结构,Aβ分子之间通过β-折叠结构上的氢键、盐桥、疏水性相互作用等作用方式聚集在一起,形成富含β-折叠片层结构的寡聚体。多个寡聚体之间相互聚集,形成聚集核,而其它寡聚体或单体继续向聚集核上聚集形成丝状的原纤维,并进一步聚集形成成熟的纤维,最终形成淀粉样斑块。其中形成的可溶性Aβ寡聚体和原纤维是最具神经毒性的。因此阻止Aβ单体向纤维聚集体转化成为了开发AD治疗药物的主要手段。
目前为止,人们发现了很多不同种类的Aβ聚集抑制剂,主要包括有机小分子、多肽、抗体和纳米颗粒几大类。而由于具备易穿过血脑屏障、低的生物毒性和高的体内稳定性等优势,有机小分子药物成为了人们研究Aβ聚集抑制剂的热点。近年来人们发现很多优秀的天然有机小分子如EGCG、姜黄素和白藜芦醇等都对Aβ的聚集和随之产生的细胞毒性有着很好的抑制效果。
苏木精是由苏木属植物的心材中提取得到,是一种常用的细胞染色剂和生物医学诊断剂,同时它也是一种现有的食用染色剂。苏木精为多酚羟基结构,带有两个苯环,自身常含有3分子的结晶水。它易溶于热水和乙醇,在水溶液中特别是碱性溶液中易被空气中的氧气氧化为红棕色的氧化苏木精。目前研究发现苏木精具备一定的药物价值,并被广泛地运用在一些特定的肿瘤和糖尿病治疗过程中。它还具备了产量大、成本低、毒性低等优点,可以成为一种有潜力的小分子药物。
发明内容
本发明的目的是发现更多的抑制β-淀粉样蛋白聚集的小分子药物,在此提供一种新的小分子抑制剂苏木精,以及其抑制β-淀粉样蛋白聚集和相关细胞毒性方面的应用。
本发明的技术方案概述如下:
苏木精抑制β-淀粉样蛋白聚集及其相关毒性的应用。
苏木精与β-淀粉样蛋白摩尔浓度比在0.5~1:1。
苏木精与β‐淀粉样蛋白摩尔浓度比在0.5~1:1,用于药物和保健品开发方面的用途。
苏木精用于阿尔兹海默症的药物的原料药。
本发明的优点:
多种实验手段证明,苏木精与β-淀粉样蛋白摩尔浓度比在0.5~1:1时,能够最有效地抑制了Aβ42纤维的生成;延缓Aβ42二级结构向β-折叠结构转化,降低Aβ42在溶液中形成的β-折叠的含量;并引导了Aβ42的不同聚集路径,改变了Aβ42的纤维形貌;最终大幅降低了Aβ42聚集过程中所产生的细胞毒性作用,是抑制Aβ42聚集的最佳实施方案。苏木精与β-淀粉样蛋白的摩尔浓度比在0.5~1:1条件下,对β-淀粉样蛋白聚集及其相关毒性具备最佳抑制效果。
附图说明
图1:实施例1中不同浓度苏木精与Aβ42共同培养物随着时间变化的ThT荧光动力学图。
图2:实施例2中摩尔浓度比为1:1的苏木精与Aβ42共培养24h后培养物的透射电镜图。
图3:实施例3中摩尔浓度比为1:1的苏木精与Aβ42共培养不同时间后培养物的圆二色谱图。
图4:实施例4中不同浓度苏木精与Aβ42共培养24h后培养物对SH-SY5Y的细胞存活率影响图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
我们通过多种物理化学生物的检测手段证实苏木精可以有效抑制Aβ42聚集,并能降低Aβ42聚集过程中所产生的细胞毒性。
苏木精结构式:
实施例1:不同浓度苏木精与Aβ42共同培养物随着时间变化的ThT荧光动力学变化。
首先将存储于-80℃冰箱的Aβ42置于4℃冰箱下,待其温度升至4℃后,取出Aβ42粉末以1mg/mL的浓度溶于六氟异丙醇溶液中。将此溶液置于4℃冰箱中静置2h待Aβ完全溶解后,在冰浴中超声2min以打散Aβ42的聚集体。接着将溶液于4℃离心机中16000g离心20min,取75%的上清液。最后在-80℃下冻实,并冷冻干燥24h,得到与处理好的絮状Aβ42,并置于-20℃下保存。
称取3.1mg的Aβ42,溶于2.5mL 20mM的NaOH溶液中,使Aβ42终浓度为275μM,冰浴中超声2min使之完全溶解。用含27.5μM的ThT磷酸盐缓冲液(PBS,phosphate buffer saline)稀释10倍(其中磷酸盐浓度为100mM,NaCl浓度为10mM),最终得到浓度为25μM的Aβ42溶液。
将0.24mg的苏木精(含3分子结晶水)溶于5mL的含27.5μM的ThT的PBS缓冲液中,获得浓度为137.5μM的苏木精母液,然后按梯度稀释为2.75μM、5.5μM、13.75μM和27.5μM浓度的苏木精溶液。取浓度为275μM的Aβ42溶液分别与上述浓度的苏木精溶液以体积比1:10混合,得到苏木精终浓度分别为2.5μM、5μM、12.5μM和25μM的2.5μM的Aβ42溶液。即4种溶液中Aβ42与苏木精的摩尔浓度比分别为1:1、1:0.5、1:0.2和1:0.1。
将上述溶液取200μL滴入96孔板中,每组浓度做3组平行,通过酶标仪进行连续测量。酶标仪测定的激发波长为440nm,发射波长为480nm,激发光带宽为9nm,发射光带宽为20nm,每隔15分钟测量一次,每次测量前摇晃混匀5s,温度设定为37℃,培养24h。将480nm处的荧光强度对时间作图。结果如图1所示。
由图1可以看到,加入苏木精的Aβ42ThT荧光强度显著降低,并随着Aβ与苏木精的浓度比的上升而下降,这说明了苏木精能够有效抑制Aβ聚集过程中β-折叠片层结构的形成。当苏木精与蛋白的摩尔浓度比达到0.5:1至1:1时荧光强度下降到最低,具备最佳抑制效果。
实施例2:摩尔浓度比为1:1的苏木精与Aβ42共培养24h后培养物的纤维形貌差异。
按照与实施例1相同的方法处理Aβ42单体,并配制成终浓度为25μM的Aβ42溶液(PBS溶液中不含ThT),其中Aβ42与苏木精的摩尔浓度比为1:1。将上述溶液于37℃,200rpm条件下进行培养。
在培养了24h后,取100μL的Aβ42培养液,超声10min,将5-10μL溶液滴加在300目的碳膜铜网上,自然干燥。待铜网上的溶液将干未干时,滴加1%的磷钨酸溶液(pH 6.5)染色30s后吸掉多余液体,自然干燥。然后用透射电镜(JEM100CXII)进行观察,检测电压为100kV,选取放大后标尺为100nm的图像进行观察,结果如图2所示。
由图2可以看到,未加入苏木精的Aβ42聚集成了交错的长纤维形态,而加入了苏木精的Aβ42聚集体形貌有着很大的区别。视野中并没有出现长纤维,而是仅有无规则的颗粒状Aβ42聚集体。这说明了苏木精改变了Aβ42的聚集途径,改变了Aβ42聚集体的形貌,阻止了纤维化的进程。
实施例3:摩尔浓度比为1:1的苏木精与Aβ42共培养不同时间后培养物的二级结构变化。
按照与实施例1相同的方法处理Aβ42分子,并配制含25μM的Aβ42培养液(PBS溶液中不含ThT),其中Aβ42终浓度为25μM,将上述溶液置于37℃,200rpm条件下进行培养。在培养分别进行到0、2、4和24h时刻,取培养液300μL加入光程为1mm的CD检测池中进行检测,波长扫描范围为195-260nm,带宽2nm,扫描速度为100nm/min,实验结果为三次扫描平均值,结果如图3所示。
从图3A中可以看到,反应开始时(0h),是否添加苏木精的Aβ42溶液均在204nm处呈现一个特征的负峰,这是典型的α-螺旋无规则结构。随着培养进行至2h-4h,单独培养的Aβ42在217nm处开始出现负峰,说明开始出现了β-折叠结构;而混合有苏木精的样品中负峰依旧停留在204nm处,说明了苏木精的加入延缓了Aβ42中β-折叠结构的生成。经过24h培养后,两种样品在204nm处的负峰完全消失,而在217nm出现β-折叠典型负峰,这说明α-螺旋完全转变为β-折叠结构。有所不同的是,当加入苏木精后,培养24h后217nm负峰值显著高于单独培养的Aβ42,而在200nm处的正锋值低于单独培养的Aβ42。这说明苏木精的加入延缓了Aβ42单体二级结构向β-折叠结构的转化,最终降低了Aβ42中β-折叠结构含量。
实施例4:不同浓度苏木精与Aβ42共培养24h后培养物对SH-SY5Y的细胞存活率的影响。
细胞毒性实验中使用的细胞为人神经母细胞瘤细胞株(SH-SY5Y)。培养基为DMEM/F-12培养基,含20%FBS,2mmol/L谷氨酰胺,100U/mL双抗。培养环境为5%CO2,37℃恒温培养。按照与实施例1相同的方法处理Aβ42,并配制含不同浓度(12.5、25、50、125和250μM)的苏木精的Aβ42溶液,溶液中Aβ42的终浓度为250μmol/L。
首先将细胞按每孔90μL培养液,5000个细胞的密度加入96孔板中。培养24h后,加入10μL老化后的Aβ42溶液样品,使每个孔中Aβ42的终浓度为25μM,苏木精浓度分别为1.25、2.5、5、12.5和25μM。继续培养48h后移除培养基,将细胞使用pH=7.4的PBS溶液清洗并更换为无血清培养基。然后在每孔中加入10μL MTT溶液(浓度为6mg/mL),继续培养4h。检测时,将96孔板以1500rpm的速度离心10min,离心后移除96孔板中溶液,每孔加入100μL的DMSO摇床震荡10min。待紫色晶体完全溶解后,使用酶标仪在570nm下测定吸光度,计算细胞存活率。以不含细胞的样品作为空白组(0%);不含Aβ和抑制剂,仅含有细胞的样品作为对照组(100%),计算加药组的细胞存活率。
结果如图4所示,加入单独培养后的Aβ42,细胞的存活率下降至67%,表明单独培养的Aβ42对SH-SY5Y细胞具有很大的毒性作用。而当将Aβ42混合苏木精共同培养后,细胞存活率显著提高。在一定浓度范围内细胞的存活率随着苏木精浓度的升高而提高,当加入的苏木精与Aβ42的摩尔浓度比达到0.5:1时,细胞的存活率上升到最高值(92%)。因此,可以说明在一定浓度比范围内苏木精能够有效地抑制Aβ42聚集所产生的细胞毒性作用。此外在该浓度比范围内,苏木精对Aβ42聚集引起的对PC12细胞株(鼠嗜铬细胞瘤细胞株)细胞毒性具备同样有效的抑制效果。
本发明提出了小分子抑制剂苏木精与β-淀粉样蛋白摩尔浓度比在0.5~1:1时能够有效抑制β-淀粉样蛋白聚集,降低Aβ42聚集相关的细胞毒性,具备潜在的药物和保健品开发方面的用途。已通过多项实施例对其作用于Aβ42的聚集、形貌、构象转化和细胞毒性抑制的效果进行了描述,相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合,来实现本发明技术。特别需要指出的是,所有相类似的替代和改动对本领域技术人员来说是显而易见的,他们都被视为包括在本发明精神、范围和内容中。
Claims (4)
1.苏木精抑制β-淀粉样蛋白聚集及其相关毒性的应用。
2.苏木精与β-淀粉样蛋白摩尔浓度比在0.5~1:1。
3.苏木精与β‐淀粉样蛋白摩尔浓度比在0.5~1:1时,用于药物和保健品开发方面的用途。
4.苏木精用于阿尔兹海默症的药物的原料药。
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