基于mTOR-TFEB研究花色苷改善阿尔茨海默病认知障碍的自噬调控机制

Alzheimer’s disease (AD) is ranking No.4 in the cause for death of the older adults. Unfortunately, there is still no effective ways to prevent/or cure this disease. The previous findings of our group revealed that supplementation of the anthocyanins extracts from blueberry may postphone the process of cognitive dysfunction of AD. Futhermore, cyanidin 3-O-glucoside (Cy-3-G)downregulated the expression of autophagy-related genes including LC3 and Becline-1. In the present project, we will demonstrate the hypothesis that anthocyanins supplementation might improve the cognitive dysfunction of AD by inhibiting autophagy of hippocampus mediated by mTOR-TFEB and promoting the clearance of Aβ. First, the Aβdamaged hippocampus neuron will be used to screen the new active ingredients from Cy-3-G and protocatechuic acid (PA); and explore how the inhibition of autophagy in hippocampus via mTOR-TFEB could work as the related underlying mechanism. Second, AD transgenetic mice will be used to compare the cognition-enhancing effects of the anthocyanins extracts from blueberry, and Cy-3G or PA. Finally, the autophagy-regulating mechanism will be developed to confirm the findings from in vitro experiment combined with the intervention study in RFP-EGFP-LC3 transgenetic mice and AD transgenetic mice. These results will not only help finding the new mechanism of improving the cognitive dysfunction of AD by anthocyanins but also help developing the foods rich in anthocyanin. Finally, it will contribute to maintain the brain health in the elderly and realize health aging in China.

阿尔茨海默病（AD）为我国老年人第4位死因，迄今仍缺乏有效防治手段。前期研究发现，蓝莓花色苷提取物延缓AD认知障碍进程，矢车菊-3-O-葡萄糖苷（Cy-3-G）明显下调Aβ25-35孵育海马神经元自噬标志蛋白LC3和Becline-1表达。为验证“花色苷通过mTOR激活TFEB，抑制海马自噬，减少Aβ沉积与清除障碍，改善AD认知障碍”的科学假设，本项目拟首先通过Aβ损伤海马神经细胞模型，筛选花色苷活性成分，探明Cy-3G/原儿茶酸通过mTOR激活TFEB、抑制细胞自噬的机制；进而利用AD转基因小鼠模型，比较蓝莓花色苷提取物、Cy-3G/原儿茶酸的认知干预效应，并结合RFP-EGFP-LC3转基因小鼠干预实验，验证细胞试验的发现。研究结果不仅对发现花色苷改善AD认知功能障碍的新机制具有重要学术价值，还有利于富含花色苷食物的开发利用，有利于维护老年人脑功能、促进健康老龄化。

阿尔茨海默病（AD）严重威胁老年人脑健康，其机制尚未阐明且缺乏有效防治手段。本项目主要完成了以下四部分研究内容：①蓝莓提取物、矢车菊-3-O-葡萄糖苷（Cy3G）改善APP/PS1双转基因小鼠认知障碍的作用及自噬调控机制；②蓝莓提取物早期干预改善APP/PS1子代小鼠认知障碍的作用及自噬调控机制；③Cy3G对Aβ25-35致SH-SY5Y细胞损伤的保护作用及自噬调控机制；④原儿茶酸（PCA）对Aβ25-35致海马神经细胞损伤的保护作用及自噬调控机制研究。研究发现：①蓝莓提取物干预能有效改善APP/PS1小鼠及其子代学习记忆功能障碍，发挥保护神经元的功能，其机制与促进细胞自噬有关；②Cy3G具有减少Aβ25-35诱导的细胞内活性氧升高、促进细胞自噬相关蛋白表达从而保护神经细胞的作用；③蓝莓提取物及花色苷的主要代谢物PCA同样具有保护原代海马神经元、促进细胞自噬的作用，并且通过加入自噬诱导剂和自噬抑制剂，证明了PCA能通过促进自噬相关蛋白表达，增强溶酶体活性从而诱导自噬体和溶酶体融合，加速细胞自噬进程。结果提示：①蓝莓提取物、Cy3G及PCA均可通过自噬调控机制、不同程度地改善AD认知障碍或发挥神经保护作用；②Cy3G是蓝莓提取物发挥自噬调控机制的主要活性成分；③蓝莓提取物的有效干预剂量为150mg/kg•bw，而Cy3G为50mg/kg·bw。研究结果对于从自噬调控角度认识花色苷改善AD认知功能障碍的新机制，制定改善老年人脑功能、防治AD的营养防治措施具有重要科学意义和广泛实用价值。