阿尔茨海默病（AD），是一种对中老年人危害巨大的神经退行性疾病，其最常见的临床症状是记忆受损与认知障碍，主要的病理特征是在神经细胞内部Tau蛋白聚集形成的神经纤维缠结（NFTs）。Tau蛋白是一种内在无序的微管相关蛋白，它被发现可以在体内或体外环境中发生液-液相分离。其相分离由静电相互作用和疏水相互作用共同驱动，参与多种生理调控作用并受到多种因素影响。有研究表明，翻译后修饰、RNA、金属离子、分子伴侣、细胞因子和活性小分子均在Tau蛋白的液-液相分离和凝聚中起着重要的调控作用。

本实验通过原核培养、分离纯化得到Tau40蛋白，利用GSK3β催化其磷酸化，通过ThT荧光探针法筛选出木犀草素与山奈酚两种可以抑制pTau40蛋白聚集的活性小分子。在体外实验中，利用PEG诱导Tau40蛋白在拥挤环境中发生液-液相分离，使蛋白形成相对独立的液滴，通过向LLPS体系中加入活性小分子，筛选出多种对Tau40蛋白的LLPS产生抑制作用的活性小分子，其中黄芩苷与橙皮苷的抑制效果比较好，其有效作用浓度范围为5-50 μM，木犀草素、山奈酚与斑螯素也具有抑制作用。

本实验利用透射电镜法探究黄芩苷对于长时间发生LLPS的Tau40蛋白的形态转变的影响。结果表明，黄芩苷可以影响长时间孵育下液滴内聚集的Tau40蛋白的蛋白形态转变，延缓其淀粉样纤维化进程。

在细胞实验中，通过向SH-SY5Y细胞加入OA来构建以Tau蛋白过度磷酸化为特征的AD模型，利用显微镜直接观察法与CCK-8法检测筛选出的活性小分子对OA诱导的细胞凋亡与细胞形态损伤的缓解作用，发现黄芩苷在一定的浓度范围内能有效缓解OA导致的细胞毒性与细胞损伤。通过Western Blot法验证黄芩苷可以有效降低OA诱导的胞内Tau蛋白的磷酸化。

本实验还利用试剂盒探究黄芩苷发挥作用的具体机制，结果表明黄芩苷可能是通过抑制OA导致的胞内ROS水平的上升以及线粒体膜电位的下降来实现对SH-SY5Y细胞的神经保护作用，具体机制可能与氧化应激途径有关。

本实验最后还探究了黄芩苷对于Tau40寡聚体导致的细胞毒性的影响，利用CCK-8法检测发现，黄芩苷对于Tau40寡聚体导致的细胞活性下降有着缓解作用，并且在一定程度上存在浓度依赖性。综上所述，本课题的研究结果可能对AD药物开发、帮助预防或治疗AD病提供新的思路。

Alzheimer's disease (AD), a neurodegenerative disorder that poses a significant risk to middle-aged and older adults, is most commonly characterized by memory impairment and cognitive deficits, with the main pathological feature being the formation of neurofibrillary tangles (NFTs) by aggregates of Tau proteins inside neuronal cells. Tau proteins are intrinsically disordered microtubule-associated proteins, which were found to undergo fluid- liquid phase separation. Its phase separation is driven by both electrostatic and hydrophobic interactions, and it is involved in a variety of physiological regulatory roles and influenced by multiple factors. It has been shown that post-translational modifications, RNA, metal ions, molecular chaperones, cytokines, and active small molecules all play important regulatory roles in the liquid-liquid phase separation and coalescence of Tau proteins.

In this experiment, Tau40 protein was obtained by prokaryotic culture and isolation and purification, and its phosphorylation was catalyzed by GSK3β, and two active small molecules, lignans and kaempferol, which can inhibit the aggregation of pTau40 protein, were screened by the ThT fluorescent probe method. In vitro experiments, PEG was used to induce liquid-liquid phase separation of Tau40 protein in crowded environments, allowing the protein to form relatively independent droplets. By adding active small molecules to the LLPS system, various active small molecules that inhibit the LLPS of Tau40 protein were screened. Among them, baicalin and hesperidin had better inhibitory effects, with an effective concentration range of 5-50 μM, luteolin, kaempferol, and berberine also have inhibitory effects.

In this experiment, we utilized transmission electron microscopy to investigate the effect of baicalein on the morphological transformation of Tau40 protein that undergoes LLPS for a long period of time. The results showed that baicalin could affect the protein morphology transformation of Tau40 protein aggregated in droplets under prolonged incubation and retard its amyloid fibrillation process.

In cellular experiments, an AD model characterized by hyperphosphorylation of Tau protein was constructed by adding OA to SH-SY5Y cells, and the mitigating effects of the screened active small molecules on OA-induced apoptosis and cellular morphology damage were detected by using direct microscope observation and CCK-8 assay. It was found that baicalin could effectively alleviate cytotoxicity and cell damage caused by OA in a certain concentration range. It was verified by Western Blot that baicalin could effectively reduce OA-induced phosphorylation of intracellular Tau protein.

In this experiment, the kit was also used to investigate the specific mechanism of baicalin's action, and the results showed that baicalin may inhibit the increase of intracellular ROS levels and the decrease of mitochondrial membrane potential caused by OA to achieve neuroprotection in SH-SY5Y cells, and the specific mechanism may be related to the oxidative stress pathway.

Finally, the effect of baicalin on the cytotoxicity caused by Tau40 oligomers was also investigated in this experiment, and it was found that baicalin had a mitigating effect on the decrease of cellular activity caused by Tau40 oligomers by using the CCK-8 assay, and there was a concentration-dependent effect to a certain extent. In summary, the results of this study may provide new ideas for AD drug development and help prevent or treat AD disease.