CN110731959A - 肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用 - Google Patents
肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用 Download PDFInfo
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Abstract
本发明涉及药物技术领域，尤其涉及肉豆蔻木酚素的新用途，包括在制备Tau蛋白寡聚化抑制剂中的应用、在制备Tau蛋白纤维化抑制剂中的应用、在制备Tau蛋白过磷酸化抑制剂中的应用、在制备预防和治疗神经退行性疾病药物中的应用。通过实验证明了肉豆蔻木酚素能够有效抑制肝素诱导的Tau蛋白纤维的形成，利用计算机分子对接，确认了肉豆蔻木酚素与Tau蛋白纤维单体的结合方式；肉豆蔻木酚素还可以降低SH‑SY5Y/Tau和HEK293/Tau细胞中Tau蛋白在丝氨酸396、404位点的磷酸化水平；肉豆蔻木酚素通过提高PP2A的活性从而促进p‑Tau蛋白的脱磷酸化作用。
Description
技术领域
本发明涉及药物技术领域，尤其涉及肉豆蔻木酚素的新用途，包括在制备Tau蛋白寡聚化抑制剂中的应用、在制备Tau蛋白纤维化抑制剂中的应用、在制备Tau蛋白过磷酸化抑制剂中的应用、在制备预防和治疗神经退行性疾病药物中的应用。
背景技术
Tau蛋白是17号染色体中MAPT基因编码的微管相关蛋白，在生理状况下维持神经正常轴突运输及微管结构稳定性，其基本功能是促进微管蛋白组装成微管，并进一步稳定已组装的微管。正常情况下，Tau蛋白上一般有两到三个位点的氨基酸进行磷酸化，当Tau蛋白发生异常过度磷酸化、基因突变或其他分子诱导后，Tau蛋白就会从微管上解聚并形成神经元纤维缠结(Neuronal fibrillary tangles，NFTs)，影响神经元的基本形态和功能，从而损害神经系统的正常功能执行。
Tau蛋白病(tauopathies)是近年来逐渐受到关注的一组与认知功能障碍密切相关的神经系统退行性疾病。将这些伴Tau蛋白异常磷酸化及其编码基因缺陷的疾病统称为Tau蛋白病，包括阿尔茨海默病(Alzheimer’s disease)、肌萎缩性侧索硬化/帕金森综合征-痴呆复征(Amyotrophic lateral sclerosis/parkinsonism-dementia complex)、嗜银颗粒性痴呆(Argyrophilic grain dementia)、皮质基底变性(Corticobasaldegeneration)、克-雅病(Creutzfeldt-Jakob disease)、拳击员痴呆(Dementiapugilistica)、伴发钙化的弥漫性神经原纤维缠结(Diffuse neurofibrillary tangleswith calcification)、唐氏综合征(Down’s syndrome)、连锁于17号染色体的伴帕金森综合征的额颞叶痴呆(Frontotemporal dementia with parkinsonism linked tochromosome17)、格-施-沙病(Gerstmann-Straussler-Scheinker disease)、哈勒沃登-施帕茨病(Hallervorden-Spatz disease)、营养不良性肌强直(Myotonic dystrophy)、C型尼曼-皮克病(Niemann-Pick disease type C)、伴发神经原纤维缠结的非关岛型运动神经元病(Non-Guamanian motor neuron disease with neurofibrillary tangles)、皮克病(Pick’s disease)、脑炎后帕金森综合征(Postencephalitic parkinsonism)、朊病毒蛋白脑淀粉样蛋白血管病(Prion protein cerebral amyloid angiopathy)、进行性皮质下神经胶质增生(Progressive subcortical gliosis)、进行性核上性麻痹(Progressivesupranuclear palsy)、亚急性硬化性全脑炎(Subacute sclerosing panencephalitis)、仅有神经纤维缠结痴呆(neurofibrillary tangle-only dementia，NFT-dementia)，以及近年提出的“全脑胶质细胞Tau蛋白病”(globular glial tauopathies，GGT)等。尽管Tau蛋白病具有多样的表型和临床特征，它们都共同存在原纤维/纤丝/纤维形式(例如，扭曲的、直链的或成对的螺旋)的不溶性的、过度磷酸化的Tau的神经原纤维缠结(NFT)。尽管上述疾病的临床症状、体征以及神经影像学改变大相径庭，但其共同特点为脑内均有异常Tau蛋白的沉积，因此在蛋白质研究层面重新进行归属，形成了“全脑胶质细胞Tau蛋白病”这一渐受瞩目的疾病群体。通过抑制Tau蛋白聚集，降低细胞内Tau蛋白及多位点磷酸化Tau蛋白的表达，将可以达到有效治疗Tau蛋白病。
肉豆蔻木酚素(Macelignan)，又称安五脂素，是从肉豆蔻科高大乔木植物种仁中提取的天然单体，分子式为C20H24O4，相对分子质量为328.40，结构式如图1所示。肉豆蔻木酚素的药理学研究发现其具有多种活性：肉豆蔻木酚素可以有效地抑制引起各种疾病的细菌和其他微生物的生长从而发挥其抗菌活性；可通过激活caspase-3诱导人早幼粒细胞性白血病细胞凋亡而发挥其抗癌作用；可显著减少细胞内炎症细胞因子的含量并降低细胞内氧化应激的水平，从而发挥抗氧化、抗炎功效。因此肉豆蔻木酚素具有多种高生物活性的特点，具有较高的开发价值。
发明内容
针对现有技术对肉豆蔻木酚素的开发利用有待进一步提高的问题，本发明提供关于肉豆蔻木酚素的新用途。
本发明的第一方面，提供肉豆蔻木酚素在制备Tau蛋白寡聚化抑制剂中的应用。
本发明的另一方面，提供肉豆蔻木酚素在制备Tau蛋白纤维化抑制剂中的应用。
本发明的另一方面，提供肉豆蔻木酚素在制备Tau蛋白过磷酸化抑制剂中的应用。
本发明的另一方面，提供肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用。
进一步地，肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用，所述的神经退行性疾病是由Tau蛋白异常聚集和/或过磷酸化引起的神经退行性疾病。
进一步地，所述的神经退行性疾病包括阿尔茨海默病、肌萎缩性侧索硬化/帕金森综合征-痴呆复征、嗜银颗粒性痴呆、皮质基底变性、克-雅病、拳击员痴呆、伴发钙化的弥漫性神经原纤维缠结、唐氏综合征、连锁于17号染色体的伴帕金森综合征的额颞叶痴呆、格-施-沙病、哈勒沃登-施帕茨病、营养不良性肌强直、C型尼曼-皮克病、伴发神经原纤维缠结的非关岛型运动神经元病、皮克病、脑炎后帕金森综合征、朊病毒蛋白脑淀粉样蛋白血管病、进行性皮质下神经胶质增生、进行性核上性麻痹、亚急性硬化性全脑炎、仅有神经纤维缠结痴呆、全脑胶质细胞Tau蛋白病。
本发明涉及化合物肉豆蔻木酚素的新用途，特别是在多种由Tau蛋白异常引起的神经退行性疾病中的应用。本发明以多种神经退行症的靶点-Tau蛋白为研究对象，通过建立体外ThT荧光检测Tau蛋白聚集的实验，发现肉豆蔻木酚素可显著降低ThT荧光值，利用透射电镜可进一步证明，在肉豆蔻木酚素的作用下，肝素钠诱导的Tau蛋白纤维数量显著减少，其纤维的完整性被破坏。同时，通过计算机仿真模拟分子对接，获取了肉豆蔻木酚素对接Tau蛋白单体的核心结构部分，辅助证明了肉豆蔻木酚素和Tau蛋白之间具有结合作用。在细胞内，肉豆蔻木酚素可显著降低SH-SY5Y/Tau和HEK293/Tau细胞Tau蛋白在Ser396、Ser404位点的磷酸化水平，并具有浓度依赖性，进一步研究发现肉豆蔻木酚素是通过提高PP2A的活性从而促进p-Tau蛋白的脱磷酸化作用。这些结果表明，肉豆蔻木酚素能够抑制Tau蛋白聚集、降低多位点的磷酸化Tau蛋白表达，可用于预防或治疗由Tau蛋白异常聚集而引发的神经退行性疾病。综上所述，肉豆蔻木酚素可用于制备预防和/或治疗与Tau蛋白异常相关的多种神经退行性疾病的药物、功能食品和保健品。
附图说明
图1为肉豆蔻木酚素化学结构式；
图2为Tau蛋白中分别加入硫黄素T、肝素钠、肉豆蔻木酚素后随时间变化的相对荧光强度图；
图3为肉豆蔻木酚素抑制Tau蛋白纤维化的透射电镜图；
图4为肉豆蔻木酚素与Tau蛋白分子对接的结果；
图5为肉豆蔻木酚素作用SH-SY5Y/Tau和HEK293/Tau细胞后Tau蛋白及pS396-Tau、pS404-Tau蛋白表达量的变化结果；
图6为肉豆蔻木酚素作用SH-SY5Y/Tau和HEK293/Tau细胞后PP2Aα、PP2Aα+β蛋白表达量的变化结果。
具体实施方式
为了更充分的理解本发明的技术内容，下面结合具体实施例对本发明的技术方案作进一步介绍和说明。实验中未详述的试验操作均为本领域技术人员所熟知的常规试验操作。
1.实验材料及试剂：
肉豆蔻木酚素(化学结构式如图1所示)、肝素钠、硫黄素T(ThT)购于Sigma公司；高糖DMEM培养基、F12-DMEM培养基、opti-MEM培养基、青霉素、链霉素、胎牛血清购于Gibco公司；Tau、pS396-Tau、pS404-Tau、PP2Aα、PP2Aα+β抗体购于Abcam公司；ECL发光液购于ThermoFisher Scientific公司，其他常规化学试剂均够自Sigma公司。
2.肉豆蔻木酚素抑制肝素诱导的Tau蛋白聚集
Tau蛋白在体外可以由肝素钠诱导，自聚集形成纤维，从而模拟体内Tau蛋白异常聚集产生神经纤维缠结这一病理过程。硫黄素T(ThT)能够结合在纤维的β结构上从而发射荧光，故可以通过ThT的荧光值追踪Tau蛋白的纤维化过程。用Tris-HCl缓冲液将Tau蛋白溶解至20μM并加入黑色酶标板中，再分别加入硫黄素T、肝素钠以及肉豆蔻木酚素(20μM)，利用全波长荧光酶标仪，在发射光为440nm、激发光为485nm下，连续检测25h，用相对荧光值做图。随时间变化的相对荧光强度图如图2所示，在体外，Tau蛋白在肝素钠诱导下聚集，当体系中加入肉豆蔻木酚素时，Tau蛋白的聚集作用显著被抑制。
利用透射电镜(TEM)可以直接观察Tau蛋白样品纤维化情况，可进一步验证ThT荧光实验的结果。取Tau蛋白ThT实验中样品各5μL，滴到230目铜网上，然后用滤纸吸去多余的样品，干燥后分别滴上5μL醋酸双氧铀，染色1min后吸去多余液体，干燥后放置于透射电镜(NIPPON TEKNO，JEM-1230)中，观察并拍照。结果如图3所示，对照组透射电镜下可观察到密集的Tau蛋白纤维，而肉豆蔻木酚素加入后，Tau蛋白纤维稀疏、并发生断裂，说明肉豆蔻木酚素可有效阻碍Tau蛋白纤维的形成。
3.肉豆蔻木酚素与Tau蛋白的分子对接
使用Sybyl-X 2.0软件进行分子对接，研究肉豆蔻木酚素与Tau蛋白之间的结合模式。从RCSB蛋白质数据库(http://www.rcsb.org/pdb/home/home.do)下载Tau蛋白纤维的核心结构(PDB编号为5O3L)。肉豆蔻木酚素的2D结果由ChemBioDraw Ultra 14.0软件绘制，并由ChemBio3D Ultra 14.0优化得到分子的3D结构。用Surflex-Dock程序进行对接，通过对接评分选择最佳对接方式，并由Chimera和Ligplot软件进行视觉分析。
如图4所示，肉豆蔻木酚素分子结合于Tau蛋白的疏水口袋，被Ile-360、His-362、Pro-364、Gly-367、Asn-368和Lys-369包围，疏水作用为肉豆蔻木酚素与Tau蛋白相互作用的主要作用力类型。所有这些相互作用都可以帮助肉豆蔻木酚素锚定在Tau蛋白的R3结构域中，阻断Tau蛋白的聚集，进而抑制Tau蛋白纤维的形成，从而可以有效减轻神经毒性，具有预防及治疗由Tau蛋白聚集引起的神经退行性疾病的潜能。
4.肉豆蔻木酚素降低SH-SY5Y/Tau和HEK293/Tau细胞中Tau蛋白多位点的磷酸化水平
将SH-SY5Y/Tau、HEK293/Tau细胞接种于培养板中，细胞贴壁后弃上清，加入含肉豆蔻木酚素的无血清培养基处理24h。利用细胞裂解液提取细胞总蛋白，通过Western Blot法检测细胞内磷酸化Tau蛋白的表达量。
如图5所示，利用Western Blot法检测发现，肉豆蔻木酚素可显著性降低SH-SY5Y/Tau和HEK293/Tau细胞Tau蛋白在丝氨酸396、404位点的磷酸化水平，且具有浓度依赖性。
5.肉豆蔻木酚素提高了SH-SY5Y/Tau和HEK293/Tau细胞中PP2Aα、PP2Aα+β蛋白的表达
将SH-SY5Y/Tau、HEK293/Tau细胞接种于培养板中，细胞贴壁后弃上清，加入含肉豆蔻木酚素的无血清培养基处理24h。利用细胞裂解液提取细胞总蛋白，通过Western Blot法检测细胞内PP2Aα、PP2Aα+β蛋白的表达量。
如图6所示，利用Western Blot法检测发现，肉豆蔻木酚素可显著性提高SH-SY5Y/Tau和HEK293/Tau细胞PP2Aα、PP2Aα+β蛋白的表达。
由上述实验结果可知，肉豆蔻木酚素可有效抑制肝素诱导的Tau蛋白聚集，有效阻碍Tau蛋白纤维的形成。利用计算机仿真模拟分子对接，确认了肉豆蔻木酚素可结合于Tau蛋白单体的核心结构部分，辅助证明了肉豆蔻木酚素和Tau蛋白之间具有的结合作用。此外，肉豆蔻木酚素可通过增强SH-SY5Y/Tau和HEK293/Tau细胞内PP2Aα、PP2Aα+β的表达从而抑制Tau蛋白在丝氨酸396、404位点的磷酸化水平。即由上述实验可得出以下结论：
第一：肉豆蔻木酚素体外能够有效抑制Tau蛋白聚集，进而抑制神经纤维缠结的形成。
第二：通过了计算机仿真模拟分子对接，获取了肉豆蔻木酚素对接Tau蛋白单体的核心结构部分，辅助证明了肉豆蔻木酚素和Tau蛋白之间具有结合作用及可能的结合方式。
第三：肉豆蔻木酚素能够提高SH-SY5Y/Tau和HEK293/Tau细胞内PP2Aα、PP2Aα+β的表达，从而降低细胞内Tau蛋白在丝氨酸396、404位点的磷酸化水平。
综上所述，肉豆蔻木酚素能够抑制Tau蛋白聚集，降低细胞内Tau蛋白在多位点的磷酸化水平，可用于预防或治疗由Tau蛋白异常聚集、多磷酸化而引发的神经退行性疾病。
以上所述仅以实施例来进一步说明本发明的技术内容，以便于读者更容易理解，但不代表本发明的实施方式仅限于此，任何依本发明所做的技术延伸或再创造，均受本发明的保护。
Claims (6)
1.肉豆蔻木酚素在制备Tau蛋白寡聚化抑制剂中的应用。
2.肉豆蔻木酚素在制备Tau蛋白纤维化抑制剂中的应用。
3.肉豆蔻木酚素在制备Tau蛋白过磷酸化抑制剂中的应用。
4.肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用。
5.权利要求4所述的应用，其特征在于：所述的神经退行性疾病是由Tau蛋白异常聚集和/或过磷酸化引起的神经退行性疾病。
6.权利要求5所述的应用，其特征在于：所述的神经退行性疾病包括阿尔茨海默病、肌萎缩性侧索硬化/帕金森综合征-痴呆复征、嗜银颗粒性痴呆、皮质基底变性、克-雅病、拳击员痴呆、伴发钙化的弥漫性神经原纤维缠结、唐氏综合征、连锁于17号染色体的伴帕金森综合征的额颞叶痴呆、格-施-沙病、哈勒沃登-施帕茨病、营养不良性肌强直、C型尼曼-皮克病、伴发神经原纤维缠结的非关岛型运动神经元病、皮克病、脑炎后帕金森综合征、朊病毒蛋白脑淀粉样蛋白血管病、进行性皮质下神经胶质增生、进行性核上性麻痹、亚急性硬化性全脑炎、仅有神经纤维缠结痴呆、全脑胶质细胞Tau蛋白病。
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| WO2021103119A1 (zh) * | 2019-11-26 | 2021-06-03 | 深圳大学 | 肉豆蔻木酚素在制备预防和治疗神经退行性疾病药物中的应用 |
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