基于Akt/mTOR/70S6K通路雷公藤甲素调控自噬防治Alzheimer病机制的研究

Current evidence has shown that autophagy is critical for the pathogenesis of Alzheimer’s disease (AD) and the deposition of β-amyloid protein (Aβ). Therefore, autophagy has been the direction of research in AD in recent years. And accumulating evidence has shown that protein kinase B/ mammalian target of rapamycin/ ribosomal protein S6 kinase (Akt/mTOR/70S6K) signal transduction pathway is a classic pathway related to the regulation of autophagy. Triptolide (TP) has been shown to possess a broad biological profile including neurotrophic and neuroprotective effects. However, little is known about the mechanism of TP on Aβ metabolism and cytotoxicity of AD. Our preliminary experiments showed that TP could protect PC12 against Aβ-induced cytotoxicity and attenuate Aβ-induced PC12 apoptosis. In addition, TP could also suppress the level of autophagy. Therefore, in this study, we speculate that “TP will repair the neurological damage and cognitive impairment of AD through activating the Akt/mTOR/70S6K signaling pathway”. We plan to prove our hypothesis in animal and cell levels. Then we detect the effect and mechanism of TP in process of amyloid-peptide metabolism, also the mechanism on learning and memory of Alzheimer’s disease mice based on the autophagy regulated by the Akt/mTOR/70S6K signaling pathway. The results may provide an interesting view of the potential application of TP or TWHF in future. More importantly, it is hoped that this research can enlighten a new possible drug target of AD.

近期研究指出自噬参与了Aβ沉积及阿尔兹海默病(AD)的发病，因此调控自噬逐渐成为研究AD的重要方向，而Akt/mTOR/70S6K作为抑制自噬的经典途径被广泛关注。尽管众多实验都证实雷公藤甲素(TP）具有神经保护作用，但其对AD病程中Aβ代谢及神经损伤的修复机制尚未阐明。课题组前期发现TP可以改善Aβ造成的神经损伤、减少自噬，且此作用可被Akt/mTOR/70S6K途径抑制剂雷帕霉素阻断，据此提出 “TP通过激活Akt/mTOR/70S6K途径抑制自噬调节Aβ代谢并进而修复神经损伤，改善AD的认知障碍” 的假说。拟以APP/PS1痴呆小鼠及Aβ处理的海马神经元为对象，基于Akt/mTOR/70S6K途径对自噬的调节，从在体和离体层次阐明TP防治AD的机制，明确TP修复AD神经损伤的具体靶点，充实传统医药理论，并为临床开发TP治疗AD提供科学依据，对缓解AD患者造成的社会压力起到积极作用。

雷公藤甲素是从雷公藤中提取的主要天然活性成分，具有良好的神经保护作用。目前发现其作为一种天然药物成分具有很好的治疗阿尔茨海默病（AD）的前景。最近研究表明，自噬在AD中起主要作用，而Akt/mTOR/p70S6K途径是调控自噬的经典途径。本研究的目的就是为了评价雷公藤甲素对AD的防治作用以及Akt/mTOR/p70S6K通路调节的自噬是否参与雷公藤甲素抗AD的神经保护作用。本研究从动物和细胞水平以行为学、电生理、Elisa试剂盒、细胞存活率、细胞凋亡以及Western blot等技术评价雷公藤甲素对AD的治疗作用及其机制。Morris水迷宫实验的结果表明，与AD小鼠相比，雷公藤甲素干预组小鼠表现出更好的空间学习和记忆能力，尤其以低浓度组更为显著。神经电生理实验结果也显示雷公藤甲素提高了AD小鼠海马脑区PP-DG通路的突触可塑性。雷公藤甲素对突触可塑性和学习记忆能力的改善主要是由于小鼠海马组织中SYP和PSD 95蛋白表达的明显升高及Aβ1-40、Aβ1-42生成的量显著降低。同时，伴随着雷公藤甲素的神经保护作用我们还发现雷公藤甲素可以降低AD小鼠海马及Aβ1-42孵育的PC12细胞内过度自噬，提高Akt/mTOR/p70S6K通路的表达。说明雷公藤甲素的自噬降低可能与Akt/mTOR/p70S6K通路的增加有关。为了进一步验证其相关性，我们选择性抑制Akt/mTOR/p70S6K通路的各个环节发现雷公藤甲素的神经保护作用明显降低，证实结果雷公藤甲素通过激活Akt/mTOR/p70S6K途径减少自噬，从而发挥对AD小鼠的神经保护作用，同时缓解Aβ1-42-诱导的细胞毒性。本课题结果为开发雷公藤甲素防治老年性痴呆提供更多的科学依据，更为预防和治疗AD提供了新的思路。