经UPS蛋白降解途径探讨化痰开窍法抑制AD磷酸化tau蛋白聚集的分子机制

Neurofibrillary tangles formed by phosphorylated tau protein abnormal aggregation in nerve cell, is one of the typical pathological changes of Alzheimer's disease (AD.) The process are closely related with function impairment of the Ubiquitin proteasome system (UPS) and resulting in abnormal protein degradation. The elders’ weak zang fu-organs, fluid can not transportation and transformation then aggregated to phlegm. Phlegm obstructing orifices and mind disuse are the core link of AD attack. We found that Yuanzhi Powder within reducing phlegm and opening orifices function can effectively improve the elderly patients with memory impairment symptoms, also can improve memory and cognitive level in AD animal model, and reduced the expression of P-tau protein in hippocampes. Based on it, we put forward the hypothesis: "Yuanzhi powder in the treatment of AD playing a role in the regulation of multiple targetsa through UPS mediated by ubiquitin, may be related to the increasing of the 26S proteasome activity, and inhibition of phosphorylation of tau protein aggregation. This research discusses the mechanism of Yuanzhi Powder in treating AD under the guidance of the theory of traditional Chinese Medicine, around the core theory of "abnormal phosphorylation of tau aggregation" in the pathogenesis of AD, from the perspective of UPS dysfunction and excessive phosphorylation of tau degradation disorder. It takes 26S proteasome as the key point of entry, uses modern molecular biology techniques and methods, and provides objective scientific basis for reducing phlegm and open orifices treatment of AD.

磷酸化tau蛋白在神经元内异常聚集形成神经纤维缠结是阿尔茨海默病（AD）典型的病理改变之一，此过程与泛素-蛋白酶体系统（UPS）功能受损导致的异常蛋白质降解障碍密切相关。中医学认为，老年人脏腑功能虚弱，水湿津液不化，停聚为痰。痰浊蒙窍，神机失用是AD发病的核心环节。课题组前期研究发现，具有化痰开窍功效的远志散可以改善老年人记忆减退症状，提高认知水平，其益智作用在动物AD模型上也得到验证，能够降低海马中磷酸化tau蛋白的表达。基于此，我们提出假说：具有化痰开窍功效的远志散可能通过调控UPS蛋白降解途径，抑制磷酸化tau蛋白异常聚集，从而发挥治疗AD的作用”。本课题在中医理论指导下，围绕AD“磷酸化tau异常聚集”的发病机制，从UPS功能受损和磷酸化tau异常聚集的角度，以26S蛋白酶体为关键切入点，通过现代分子生物学技术和方法，探讨远志散治疗AD的作用机制，为化痰开窍法治疗AD提供科学依据。

泛素-蛋白酶体系统（Ubiquitin-proteasome system，UPS）是真核生物细胞中蛋白质降解的主要途径，参与细胞内 80％以上蛋白质的降解，其主要功能是选择性降解细胞内错误折叠、受损的蛋白，精确地调控细胞内蛋白降解，对维持细胞正常的生理功能有重要作用。作为阿尔茨海默病（Alzheimer’s Disease，AD）典型的病理改变之一，磷酸化tau蛋白在神经元内异常聚集形成神经纤维缠结与UPS功能受损导致异常蛋白质降解障碍密切相关。本项目基于AD“气虚生痰，蒙蔽清窍”的中医病机理论，结合现代医学的研究成果，以调控UPS功能和抑制磷酸化tau蛋白过度聚集为关键切入点，选用具有化痰开窍功效的远志散作为治疗药物，采用免疫荧光、免疫组化、RT-PCR、Western blot 等技术和方法，检测AD大鼠海马中磷酸化 tau 蛋白的聚集情况，以及泛素介导的UPS通路中关键酶（UbE1a/b、UbE2a、CHIP、UCH-L1、26s Proteasome）活性的变化，探讨远志散对 AD 的治疗作用和可能机制，为化痰开窍法治疗 AD提供科学依据。研究结果显示，远志散有较好的益智作用，能够提高AD大鼠的学习和记忆能力，改善脑组织形态和锥体神经元超微结构，增加海马CA1区树突棘密度，对PI3K/AKT/GSK-3β信号通路及tau蛋白磷酸化修饰关键蛋白酶有较好的调控作用，减轻海马CA1区tau蛋白与微管的解离，其治疗作用可能与调节UPS的功能，提高26S 蛋白酶体活性并抑制海马中磷酸化tau蛋白过度聚集有关。