基于内质网应激及GSK-3β/CREB信号通路探讨补肾化痰益智法对AD样学习记忆损伤的作用机制

Alzheimer’s Disease (AD) accounts for more than half of senile dementia which is aggravated by the growing population aging. Deficiency of kidney and obstruction of phlegm is one of the key pathogenesis of AD. The efficacy is precise but the exact mechanism is unclear by replenishing kidney-essence and removing phlegm with improving intelligence (improved prescription of wendan tang) to treat AD. Our preliminary study of AD like rats model injecting with tunicamycin induced the increasing of p-PERK, one of the marked protein of endoplasmic reticulum (ER) stress, as well as the lessening of dendritic spines in hippocampus with activating GSK-3β and transfer of CREB both inside and outside nucleus. These change could be reversed by treating with the prescription. These data highly supported the hypothesis that replenishing kidney-essence and removing phlegm with improving intelligence may play role through ER stress via GSK-3β/CREB signaling pathways to treat AD. Therefore, this programme will study the effection of the prescription on unfolded protein reaction of ER stress, phosphorylated Tau protein and beta amyloid protein (Aβ) in AD through western blot, immunohischemistry and real-time PCR. Simultaneously, co-immunoprecipitation, immunfluorescence are used to observe the interaction of GSK-3β/CREB and β2ARK1 pathway among primary neuron and cell passage culture to make clear the correlative mechanism mediated by ER stress. This study will provide the strong support to analyse molecular mechanism of replenishing kidney-essence and removing phlegm with improving intelligence, which can provide new ideas and methods of traditional Chinese medicine(TCM) to prevent and treat Alzheimer’s with developing the therapeutic principle and method of TCM.

阿尔茨海默病（AD)占老年痴呆的一半以上，人口老年化加剧了AD的发病，肾虚痰阻是AD关键病机之一，补肾化痰益智法（温胆汤改良方）治疗AD疗效确切但机制不明。前期发现TM诱导AD样模型大鼠内质网应激相关蛋白PERK等表达上升，海马树突棘减少并伴GSK-3β激活和CREB核内外转移，该方干预后内质网应激相关蛋白PERK水平下降、海马树突棘增加，提出补肾化痰益智法可能通过内质网应激及GSK-3β/CREB等信号通路对AD起治疗作用假说。运用WB、免疫组化和RT-PCR等法检测该方对AD内质网应激蛋白、磷酸化Tau和Aβ的影响。体外原代和传代细胞培养，运用IP、IF观察PERK、GSK-3β、CREB及β2ARK1等间的关系，以明确内质网应激介导的该方调节GSK-3β/CREB、β2ARK1通路的关联机制。该研究将为该法治疗AD的分子机制提供依据，同时为丰富和发展中医治则治法理论提供新思路和方法。

阿尔茨海默病（AD)占老年痴呆的一半以上，人口老年化加剧了AD的发病，肾虚痰阻是AD关键病机之一，补肾化痰益智法（温胆汤改良方）治疗AD疗效确切但机制不明。前期发现TM诱导AD样模型大鼠内质网应激相关蛋白PERK等表达上升，海马树突棘减少并伴GSK-3β激活和CREB核内外转移，该方干预后内质网应激相关蛋白PERK水平下降、海马树突棘增加，提出补肾化痰益智法可能通过内质网应激及GSK-3β/CREB等信号通路对AD起治疗作用假说。运用WB、免疫组化和RT-PCR等法检测该方对AD内质网应激蛋白、磷酸化Tau和Aβ的影响。体外原代和传代细胞培养，运用IP、IF观察PERK、GSK-3β、CREB等的关系，以明确内质网应激介导的该方调节PERK\GSK-3β\CREB通路的关联机制。研究结果提示依霉素（TM）能诱导出非GSK-3β依赖的内质网应激并介导AD样空间学习记忆障碍，补肾化痰益智方药能通过调控AD样模型大鼠海马树突棘的可塑性、降低磷酸化Tau蛋白水平及改善神经原纤维缠结等，继而发挥提高AD样模型大鼠空间学习记忆能力的作用，该作用与其抑制GSK-3β/CREB信号通路作用有关同时伴pSer133-CREB与pSer129-CREB核内外转移。此外，补肾化痰益智方药还能抑制pAPP668表达、增加α分泌酶活性、降低β和γ分泌酶活性、同时增加IDE的表达水平以减少Aβ生成并增加其降解，继而减少Aβ的聚集与沉积以缓解SP的形成，从而发挥其改善AD认知功能障碍的作用。该研究将为该法治疗AD的分子机制提供依据，为中医药防治老年痴呆提供新思路和方法。