基于Akt/GSK3β/MEF2D/Beclin-1信号通路探讨定志小丸中人参-石菖蒲调节自噬体内Aβ转运的作用机制

Ginseng-Shichangpu in Dingzhi Xiaowan is a common drug pair for the treatment of dementia in traditional Chinese medicine. It is suitable for the type of pathogenesis of " Respiratory phlegm resuscitation, Yuan Qi hungry " in Alzheimer's disease (AD). It was found that this drug can improve the learning and memory ability and autophagy in mice with AD model; its active ingredient can reduce the expression of Aβ40 in AD transgenic mice by up-regulating the Akt/mTOR pathway. A significant correlation between MEF2D and Beclin-1 was also found in the 6-OHDA model. On the basis of the original project, the APP/PS1 transgenic mice were firstly used to investigate the effects of ginseng-Shichangpu active ingredients on learning and memory ability, Aβ metabolism and Aβ transport of AD mice. Then APP/PS1 transgenic cells were used to determine determine the targeting relationship between GSK3β and MEF2D. Akt inhibitors and autophagy inhibitors were used as controls, and Akt/GSK3β/MEF2D/Beclin-1 signaling pathway was used to explore the target of intervention of autophagy to prevent AD. This project cuts into the core problem of autophagy and AD. It systematically evaluates the efficacy of drugs in vivo and in vitro experiments, enriches the scientific connotation of TCM treatment of AD theory, and has broad clinical application prospects.

定志小丸中人参-石菖蒲是中医临床治疗痴呆类病症的常用药对，适用于阿尔茨海默病（AD）“痰阻清窍、元气虚馁”的病机类型。前期发现该药对可以提高拟AD模型小鼠学习记忆能力和调节自噬作用；其有效成分通过上调Akt/mTOR通路来减少AD转基因小鼠中Aβ40表达。前期还在6-OHDA模型中发现MEF2D与Beclin-1存在显著相关性。本项目在原基础上，先应用APP/PS1转基因小鼠，考察人参-石菖蒲有效成分对AD小鼠的学习记忆能力、Aβ代谢和Aβ转运的影响；再应用APP/PS1转基因细胞，确定GSK3β与MEF2D靶向关系，以Akt抑制剂和自噬抑制剂为对照，并通过Akt/GSK3β/MEF2D/Beclin-1信号通路，探讨其有效成分干预自噬防治AD的作用靶点。本项目切入自噬与AD的核心问题，在体内外实验中对药物的疗效进行系统性评价，丰富了中医药治疗AD理论的科学内涵，具有广阔的临床应用前景。

定志小丸中人参-石菖蒲是中医临床治疗痴呆类病症的常用药对，其有效成分（人参皂苷Rb3和β-细辛醚）的应用也适用于阿尔茨海默病（AD）“痰阻清窍、元气虚馁”的病机类型。本项目先应用APP/PS1转基因小鼠，考察人参-石菖蒲有效成分对AD小鼠的学习记忆能力、Aβ代谢和Aβ转运的影响；再应用APP/PS1转基因细胞，确定GSK3β与MEF2D靶向关系，探讨其有效成分干预自噬防治AD的作用机制。本研究结果发现人参皂苷Rb3和β-细辛醚联合对AD模型小鼠具有调节自噬保护神经元（减缓过度自噬和神经元损伤程度）、提高学习记忆能力（缩短潜伏期时间和减低Aβ42、p-tau、BACE1、AchE等表达）和增加Aβ外排（提高血脑屏障功能、增加LRP-1及减少RAGE表达）的药理作用。接着采用人参皂苷Rb3和β-细辛醚联合干预AD模型细胞，发现其调节自噬的作用效果与Akt抑制剂、MEF2D抑制剂趋势相似，通过现代分子生物学技术明确GSK3β与MEF2D在AD模型细胞中存在负向调控关系，在siGSK3β情况下MEF2D表达有所增加，但在siMEF2D情况下GSK3β表达无太大变化，提示GSK3β是MEF2D的上游效应因子。再应用MEF2D抑制剂和siMEF2D为对照，验证人参皂苷Rb3联合β-细辛醚后对AD模型细胞具有明显调节自噬的作用，进一步阐述人参皂苷Rb3和β-细辛醚联合对Akt/GSK3β/MEF2D/Beclin-1信号通路的调控而发挥减缓AD进程的作用。因此，本项目切入自噬与AD的核心问题，在体内外实验中对药物的疗效进行系统性评价，丰富了中医药治疗AD理论的科学内涵，为中医药相关新药研发提供了实验依据，具有广阔的临床应用前景。