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两类海藻寡糖对帕金森病模型的神经保护作用机制研究
其他题名Neuroprotective effects of two kinds of algal oligosaccharides on Parkinsonian model
刘英娟
学位类型博士
导师张全斌
2018-05
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位专业海洋生物学
关键词紫菜寡糖,甘露葡萄糖醛酸寡糖,帕金森病,细胞凋亡,pc12细胞
摘要

帕金森病(Parkinson’ diseasePD)是一种由多巴胺能神经元选择性丧失所引起的神经退行性疾病。该病多发于55岁及以上的中老年人,其典型的行为学特征表现为:静止性震颤、步伐不稳、肌强直、行动缓慢或者不自主性运动等。随着中国老龄化的进展,帕金森病的发病率逐年升高。目前尚没有有效的治疗方式,临床上的治疗药物只能暂缓症状,但不能预防或减慢疾病的进展。

糖类是生物体内除蛋白质和核酸外又一类重要的生物分子,在细胞的分裂分化,生长代谢等方面发挥着重要的作用。海带多糖及紫菜多糖是从海洋植物海带及紫菜中提取的两种酸性杂多糖,已有研究证明这两种多糖具有抗氧化、抗衰老、抗肿瘤、增强免疫力及保护多巴胺能神经元免受损伤等多种生物活性,然而多糖结构复杂,对于研究其机制带来了困扰。本研究旨在通过对海带多糖及紫菜多糖进行降解,获得葡萄糖醛酸甘露糖寡糖及紫菜寡糖,并进一步探究其神经保护活性的机理。

PD模型是研究其发病机理及筛选药物的重要工具,MPTP诱导的小鼠模型在病理改变及行为学方面与PD患者有类似的特征,是目前较为理想的动物模型。6-OHDA是多巴胺能神经损毁剂,在脑内可由多巴胺代谢生成,通过生成自由基等方式损害神经元,其诱导的神经细胞损伤能较好的反应细胞受到外界刺激后的生理变化。本实验以MPTP制备PD小鼠模型,应用行为学、免疫组化、免疫印迹及电化学HPLC等技术,来探讨葡萄糖醛酸甘露寡糖及紫菜寡糖保护PD小鼠的分子生物学机制。同时,本实验以6-OHDA诱导PC12细胞损伤,以LPS诱导BV2细胞炎症,检测细胞活力、细胞凋亡及线粒体膜电位等指标,从细胞水平探讨这两类寡糖的神经保护作用。

1)对海带多糖进行酸降解,通过HPLC、核磁及质谱技术,我们获得了两个结构明确的寡糖——葡萄糖醛酸甘露二糖及四糖,即为GM1GM2。体外研究发现:GM1GM2显著地提高了PC12细胞的活力,提高细胞的抗氧化系统和线粒体膜电位水平,抑制了ROS对线粒体的损伤,从而抑制PC12细胞凋亡的发生;在体研究发现:通过腹腔连续七天注射MPTP,成功获得PD小鼠模型;经过GM1GM2处理后,显著地改善了PD小鼠的行为学障碍;提高了多巴胺的含量及酪氨酸羟化酶(TH)在纹状体黑质中的蛋白表达水平,抑制多巴胺能神经元的损伤;同时,GM1GM2可以通过调节PI3K/Akt信号通路,抑制凋亡的发生。

2)对紫菜多糖进行酸降解,获得了聚合度为2-13的紫菜寡糖混合物,其结构以3-连接的β-D-半乳糖和4-连接的α-L-半乳糖-6-硫酸基交替连接,记为OP。体外研究发现,OP显著地抑制了ROS导致的线粒体损伤,有较好的清除自由基的活力。同时,OP可以显著地抑制LPS诱导的BV2细胞活化,抑制炎症因子的产生和NO的释放,有较好的抗神经炎症的作用。在体研究发现,OP显著地改善PD小鼠的行为学障碍,抑制了多巴胺能神经元的损伤,抑制了DA能神经元的凋亡。

通过体内外研究发现,葡萄糖醛酸甘露寡糖(GM1GM2)及紫菜寡糖OP都具有较好的神经保护作用,其中GM1GM2主要抑制DA能神经元细胞的凋亡,OP还能抑制神经炎症的发生。这两类海藻寡糖作用明显,结构简单,为帕金森病的药物筛选提供了新的思路

其他摘要

Parkinson’s disease (PD) is a neurodegenerative movement disorder that is caused by a selective loss of dopaminergic neurons, which is selectively sensitive to people over 55 years old. The clinical symptoms of PD include resting tremor, bradykinesia, muscle rigidity, and postural instability. With the aging of China, the incidence of Parkinson's disease is increasing. Current PD treatments provide symptomatic relief but do not prevent or decelerate disease progression.

Besides protein and nucleic acid, sugar is another important biological molecule. It plays an important role in cell division, differentiation, growth and metabolism. Laminaria japonica polysaccharides and porphyra polysaccharides are two kinds of acid heteropolysaccharide extracted from seaweed. Previous studys have shown that these two kinds of polysaccharides have many biological activities, such as antioxidant, anti-aging, anti-tumor, immunity enhancement and protection of dopamine neurons from injury. However, the complex structure of polysaccharides has caused difficulties in studying their neuroprotective mechanism. The aim of this study was to obtain glucuronomannan oligosaccharides and porphyra oligosaccharides to explore the mechanism of neuroprotective activity.

The PD model is an important tool for studying its pathogenesis and screening drugs. The mouse model induced by MPTP has similar characteristics in pathological changes and behavior. 6-OHDA is a DA neurodestructive agent, which can be metabolized by dopamine in the brain, and damage neurons by producting free radicals, reflecting the changes of cells with external stimulation. In this study, the neuroprotective mechanism of glucuronomannan oligosaccharides and porphyra oligosaccharides against MPTP was studied by behavioral, immunohistochemistry, western bloting and electrochemical HPLC. Furthermore, we also applied cell activity, apoptosis and mitochondrial membrane potential in 6-OHDA-induced PC12 cells and LPS-induced BV2 cells.

(1) We obtained the glucuronomannan oligosaccharides from Laminaria japonica through acid hydrolysis. After analysis by HPLC, NMR and MS, we obtained two oligosaccharides with the degree of polymerization of 2 and 4, named GM1 and GM2. In vitro, GM1 and GM2 significantly improved cell viability and inhibited 6-OHDA-induced apoptosis. Furthermore, GM1 and GM2 improved the mitochondrial membrane potential (MMP) and inhibited the production of ROS. In vivo, MPTP was intraperitoneally injected for 7 days to induce PD. GM1 and GM2 treatment ameliorated behavioural deficits in MPTP- induced Parkinson’ mice. GM1 and GM2 inhibited the loss of DA and tyrosine hydroxylase (TH) in stritum and substantia nigra and protected dopaminergic neurons from injury. Furthermore, GM1 and GM2 up-regulated the PI3K/Akt signalling pathway to promote neuronal survival.

(2) OP was prepared by acid hydrolysis of porphyran. The degree of polymerization of OP ranged from 2 to 13. OP was mainly composed of sulfated galactans and oligosaccharides with a linear backbone of alternating 3-linked β-D-galactose and 4-linked α-L-galactose-6-sulfate. In vitro, OP significantly restrained the overproduction of reactive oxygen species (ROS) and alleviated the MMP loss, with good free radical scavenging ability. Moreover, OP inhibited the activation of BV2 cells induced by LPS and restrained the production of cytokines and NO. In vivo, the behavioral deficits in spontaneous motor activity, latency to descend in a pole test, and suspension in a traction test were ameliorated after OP treatment. Additionally, we found that OP protected dopaminergic neurons from apoptosis.

Glucuronomannan oligosaccharides (GM1and GM2) and oligo-porphyran (OP) possessed neuroprotective effects in vivo and in vitro. GM1 and GM2 mainly inhibited DA neurons from apoptosis, and OP also inhibit nerve inflammation.These two types of algal oligosaccharides with remarkedly anti-PD acitivity and simple structure, might provide new ideas for drug screening of Parkinson's disease.

语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154524
专题实验海洋生物学重点实验室
第一作者单位中国科学院海洋研究所
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刘英娟. 两类海藻寡糖对帕金森病模型的神经保护作用机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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