地黄饮子及其有效成分五味子醇甲通过PI3K/Akt/mTOR通路诱导自噬修复神经细胞氧化损伤机制研究

Autophagy plays an important role in the pathogenesis of AD. Oxidative stress, mitochondrial dysfunction and autophagy have complex relationship. We include an antioxidant action of Dihuangyinzi, concerned with inhibition of RAGE, oxidative stress and regulation of p38 signaling pathway. Dihuangyinzi improve the learning and memory of tau gene transfer in Drosophila by PI3K/Akt /mTOR Signal Pathway. We put forward the hypothesis that Dihuangyinzi-induced autophagy attenuates oxidative damage by PI3K/Akt /mTOR signal pathway in nerve cells. For the first time we investigate Dihuangyinzi and its active ingredients of schisandrin will induce autophagy by PI3K/Akt /mTOR signal pathway in SH-SY5Y damaged by Aβ and APP/PS1 double transgenic mice. Dihuangyinzi and schisandrin improve mitochondrial dysfunction and apoptosis. Dihuangyinzi and schisandrin remove of abnormal protein aggregation and repair oxidative damage of nerve cells. We use electron microscope and techniques of single fluorescent GFP-LC3 system stable transfection combined with fluorescence microscopy. We discuss that Dihuangyinzi have the molecular mechanism of regulate autophagy and antioxidant. We confirmed that schisandrin is harmacodynamic material basis of anti oxidation. We further reveal the biological basis for the prevention and treatment of kidney essence. We provide the basis for the study of drug development of Dihuangyinzi.

自噬在AD发病中起着重要作用，氧化应激、线粒体功能障碍、自噬等存在复杂交互关系。前期工作证实地黄饮子防治AD抗氧化作用与抑制RAGE、氧化应激反应及调节p38信号通路有关；调控PI3K/AKT/mTOR信号通路提高转tau基因果蝇学习记忆。因此提出地黄饮子及有效成分五味子醇甲是否通过PI3K/Akt/mTOR信号通路调节自噬以缓解氧化应激导致的神经细胞损伤？课题采用Aβ诱导SH-SY5Y和APP/PS1双转基因鼠，运用自噬抑制剂和诱导剂,通过电镜、Westernblot、GFP-LC3体系稳定转染结合荧光显微镜等技术，首次探讨地黄饮子及其有效成分五味子醇甲调控PI3K/Akt/mTOR信号通路诱导自噬，改善线粒体功能障碍和凋亡，清除异常蛋白聚集，修复神经细胞氧化损伤机制。深入阐述地黄饮子调节自噬和抗氧化作用分子机制，证实五味子醇甲是防治AD药效物质基础，揭示补肾填精法防治AD生物学基础。

自噬在AD发病中起着重要作用，氧化应激、线粒体功能障碍、自噬等存在复杂交互关系。基于前期工作，我们提出地黄饮子及有效成分五味子醇甲是否通过PI3K/Akt/mTOR信号通路调节自噬以缓解氧化应激导致的神经细胞损伤？课题动态研究地黄饮子及五味子醇甲Aβ诱导的SH-SY5Y的细胞活力、ROS以及自噬的影响，结果证实地黄饮子可以通过激活自噬，下调ROS发挥抗氧化作用，增强细胞活力。在电镜下地黄饮子组的自噬现象增加，细胞状态较好，线粒体变性少。地黄饮子组及五味子醇甲处理Aβ1-42诱导SH-SY5Y细胞后，LC3II和Beclin-1均增加。地黄饮子组的mRFP-GFP-LC3斑点增加，自噬流较增强,表示从自噬体到自噬溶酶体过程较为顺畅。地黄饮子及五味子醇甲可抑制PI3K/Akt/mTOR信号通路的异常激活，发挥增强或恢复细胞自噬水平，促进对机体异常Aβ沉积清除的作用。地黄饮子及五味子醇甲可以改善APP/PS1小鼠空间探索记忆能力，地黄饮子可能是通过抑制APP/PS1小鼠脑组织ROS产生和Aβ1-42蛋白，下调海马自噬相关蛋白beclin1和LC3Ⅰ/Ⅱ及激活小鼠脑组织mTOR蛋白，抑制细胞凋亡。综上，本课题采用Aβ诱导SH-SY5Y和APP/PS1双转基因鼠，运用自噬抑制剂和诱导剂,通过电镜、Westernblot、GFP-LC3体系稳定转染结合荧光显微镜等技术，探讨地黄饮子及其有效成分五味子醇甲调控PI3K/Akt/mTOR信号通路诱导自噬，改善线粒体功能障碍和凋亡，清除异常蛋白聚集，修复神经细胞氧化损伤机制。深入阐述地黄饮子调节自噬和抗氧化作用分子机制，证实五味子醇甲是防治AD药效物质基础，揭示补肾填精法防治AD生物学基础。