藏药七十味珍珠丸改善高原低氧环境下认知功能障碍机制的研究

The cognitive impairment with Alzheimer’s disease (AD) is coming to the worldly problem with the mean life expectancy continual increasing. We have found that healthy individuals in Qinghai-Tibetan Plateau are excursions with mild cognitive impairment (MCI), which is frequently seen as a prodromal stage of AD. Current medical researches on the cognitive impairments pay a special attention on nature materials, because their traditional usages. Ratanasampil (RNSP), one of the most important Tibetan medicines with 70 components, is used to treat cerebrovascular diseases such as cerebral hemorrhage, cerebral infarction. We have previously found that RNSP improves the cognitive functions in mild-to-moderate AD patients in Qinghai-Tibetan Plateau. We have also found that RNSP improves learning and memory as well significantly decreased the β-Amyloid (Aβ)in a mouse model of AD (Tg2576) mice; however, the underling mechanisms of RNSP effects are unknown. .Microglia (MG) is the phagocytes in brain with highly branched fine processes. As constantly move their processes to eliminate unnecessary synapses by phagocytosis, MG consume much ATP to over-produce reactiveoxygenspecies(ROS) in MG, result in inducing MG-dependent neuroinflammation.We therefore rising the Microglia-aging Hypothesis(Nakanishi ＆Wu 2009). To our surprising, Aβ can only induce IL-1 release by aged MG, but not by young MG, thus further evidences our microglia-aging Hypothesis. Because MG have functional plasticity, that M1 phenotype can produce pro-inflammatory mediators, such as IL-1β,TNF-α, IL-6 to damage neuron, however, M2 phenotype can produce anti-inflammatory mediators, such as IL-10 as well as IL-4 to protectAβ-damaged neuron, therefore, we consider that change MG form M1 to M2 may give a new target for AD treatment. .In the current project, firstly,we will analyze the impacts of RNSP in brain functions of the AD patients at high altitude using functional MRI.Secondly, we will determine the effects RNSPon the cognitive impatient and the cellular mechanism in hypoxia mice models using electrical physiology technologies. Thirdly, we will analyze the effects RNSPin dynamically systemic inflammation changes in the AD patients and hypoxia mice using flow cytometer and immunohistochemical technologies. Fourthly, we will analyze the MG-dependent neuroinflammation and the neuron damages in the brain of hypoxia mice using genomics and proteomics technologies such as RT-PCR, western blot as well as ELISA. Finally, we will clarify the molecular mechanisms of RNSP effects using the cultured MG and neuron. We believe that our project will give a new idea of traditional Tibetan medicines to contribute treating the cognitive impairments with aging and the aging-related neurodegeneration diseases such as AD.

以阿尔茨海默病（AD）为主的认知功能障碍已成为全球老龄化社会面临的重大问题。研究已证实低氧加速认知功能低下，慢性低氧环境的青藏高原已成为国内外认知功能障碍研究的瞩目地区。我们多年来对传统藏药七十味珍珠丸（RNSP）的临床研究发现RNSP具有明显改善高原低氧认知功能障碍，动物实验发现RNSP可改善AD小鼠记忆功能及减少Aβ蛋白蓄积，但其作用机制尚不清楚。近年，脑内小胶质细胞依存性神经炎症参与AD的发生发展已受到关注，调控脑内小胶质细胞使其转向保护神经元的功能，成为防治AD的研究靶点。本实验将采用单细胞分离技术，培养等细胞学技术，分离小胶质细胞和神经元；流式细胞术、免疫组织化学技术，QRT-PCR等分子生物学技术，分析RNSP对低氧诱导脑内小胶质细胞活化依赖性脑内神经炎症反应和神经元损伤的调控及其途径，阐明RNSP改善高原低氧环境认知功能障碍的分子机制，为开发传统藏药防治AD提供科学依据。

阿尔茨海默病（AD）目前已成为全球老龄化社会面临的重大问题。研究已证实低氧可加速认知功能低下。我们前期发现传统藏药七十味珍珠丸RNSP具有明显改善高原低氧老年人认知功能障碍，但其作用机制尚不清楚。该项目在大量充实的前期工作基础上采用细胞学技术、流式细胞术、免疫组织化学技术，QRT-PCR等分子生物学技术，分析RNSP对低氧诱导脑内小胶质细胞活化依赖性脑内神经炎症反应和神经元损伤的调控及其途径。结果显示低氧/间歇性低氧环境下小胶质细胞（microglia，MG6）内诱导型一氧化氮合酶(iNOS)、TNF-α、IL-1β、IL- 6等促炎因子水平增加、Arginase-1，IL-10、TGF-β和降低IL-4等抗炎因子减少导致神经元的变性、死亡的发生，低氧/间歇性低氧环境下诱导神经炎症MG内p-IκBα的活性和神经细胞核内NF-κB通路相关因子p65的表达有关。通过传统藏药七十味珍珠丸(RNSP)预处理可改善缺氧作用所诱导的MG6细胞损伤提供显著的保护作用。RNAP通过氧化应激和NF-κB 活性分子通路多靶点来调控缺氧/间歇缺氧环境诱导下小胶质细胞神经炎症反应，从而改善低氧环境认知功能障碍的机制。此项研究在分子水平为开发利用传统藏药防治AD提供理论依据及调控新靶点，为防治AD提供有效科学依据。