基于LncBACE1-AS介导的线粒体自噬探讨β-细辛醚对AD的作用机制

LncBACE1 and mitophagy are closely related to the metabolism of APP, and play an important role in the pathogenesis of AD. BACE1 can promote the degradation of APP to Aβ, leading to protein precipitation and impaired mitochondrial function. Autophagy can remove deposited proteins and damaged mitochondria, but the effect of the two and their effects on AD are still unclear. The bottleneck of AD research is that the drug is difficult to pass the blood-brain barrier (BBB) and the mechanism is not clear. The main component of Acorus tatarinowii Schott volatile oil is β-asarone. It can regulate the autophagy of neurons through the BBB, inhibit the production and promote the deposition of Aβ, thus treating the AD model cells and animals. This project based on the previous work,1) in APP/PS1 transgenic mouse model, we determine the effects of β-asarone on learning and memory, brain patholigical features and neuronal structure; 2) in mice and Aβ1-42 induced PC12 cell model, we detect the effects on mitochondrial fuction, autophagosomes, autophagy protein/mRNA and MMP; 3) then further examine the relationship between BACE1-AS and mitophagy. The point of penetration is mitophagy and AD-APP metabolism, and LncRNA BACE1-AS. Thus to elucidate the mechanism of β-asarone, which is important to theoretical significance and broad application prospets, for enriching and developing the scientific connotation of TCM prevention and treatment of AD.

LncBACE1和线粒体自噬与APP 的代谢密切相关，在AD 发病过程中起重要作用，是当前的研究热点。BACE1可促进APP降解为Aβ，导致蛋白沉淀和线粒体功能受损；而自噬可清除沉积的蛋白和受损的线粒体，但两者之间的作用及对AD的影响尚不明确。发病机制复杂及药物难以通过BBB是AD研究的瓶颈，开窍药石菖蒲的主要成分β-细辛醚分子量小、脂溶性高，能透过BBB，调节自噬，促进沉积的Aβ清除，对AD模型细胞和动物有治疗作用。本项目以前期工作为基础，1)在APP/PS1小鼠上，明确β-细辛醚对学习记忆能力、脑内病理征和AchE的影响；2)在小鼠和细胞模型上，考察其对线粒体、自噬体、自噬蛋白/mRNA、MMP的影响；3)进一步在细胞模型上，考察此作用与BACE1-AS和线粒体自噬的关系。旨在切入线粒体自噬与AD 的核心问题—APP 的代谢与LncBACE1，阐明β细辛醚治疗AD 的作用机理及靶点。

阿尔茨海默病（Alzheimer disease，AD）是最常见的神经系统退行性疾病，对该疾病的防治研究仍面临巨大挑战。在明确AD动物和细胞模型中线粒体功能和自噬的变化的基础上，我们以补肾开窍法对AD的作用机理出发，丰富和发展中医药防治AD 的科学内涵。.结果：1.AD模型小鼠、细胞模型中海马内线粒体肿胀、形态受损，PINK1、LC3 Ⅰ/Ⅱ、Beclin-1表达降低，细胞内Ca2+流动增加、MMP较正常细胞降低。2.淫羊藿苷联合β-细辛醚能降低APP/PS1小鼠的水迷宫潜伏时间、减低跳台和避暗实验错误次数，降低海马和PFC中Aβ42的浓度，抑制海马/PFC中Aβ、APP、PS1和BACE1的表达、促进Syn的表达。3.淫羊藿苷联合β-细辛醚能降低Aβ42引起的细胞毒性、提高细胞活力，抑制Aβ、APP、PS1和BACE1的表达、促进Syn的表达。4.淫羊藿苷联合β-细辛醚可促进APP/PS1小鼠和PC12细胞模型Beclin-1、LC3 Ⅰ/Ⅱ、PINK1、Parkin、p-Parkin的表达、抑制p62的表达，促进DAL-Autophagy/Mitophagy的荧光表达。5.淫羊藿苷联合β-细辛醚可促进APP/PS1小鼠和PC12细胞模型的自噬功能，这种促进结果可以抑制海马/PFC和PC12中Aβ和BACE1的表达。6.淫羊藿苷联合β-细辛醚可抑制PC12细胞lncRNA BACE1-AS对BACE1的作用、降低Aβ、APP和PS1并提高syn水平，促进Beclin-1、LC3 Ⅰ/Ⅱ并抑制p62的表达。.结论：1.AD模型小鼠和细胞线粒体功能和自噬功能降低。2.淫羊藿苷联合β-细辛醚可提高双转基因AD模型小鼠的学习记忆能力，保护神经元和突触；改善Aβ42引起的细胞毒性，补肾开窍对其改善作用优于单独用药。3.淫羊藿苷联合β-细辛醚可改善AD小鼠和PC12细胞模型的损伤的自噬和线粒体自噬功能。4.淫羊藿苷联合β-细辛醚对AD小鼠和PC12细胞模型的损伤有修复功能，且通过激活自噬抑制Aβ的产生、促进其降解。5.shRNA BACE1-AS可抑制BACE1的表达，淫羊藿苷联合β-细辛醚可抑制BACE1并促进自噬的发生。6.补肾开窍中药单体联合对AD有良好的治疗作用，优于单独使用淫羊藿苷或β-细辛醚，其机制可能是通过激活自噬并抑制BACE1发挥作用的。