基于AMPK-PINK1介导的线粒体自噬通路探讨海昆肾喜胶囊改善AD认知障碍的作用机制

Alzheimer's disease (AD) is one of the most prevalent age-dependent neurodegenerative disorders. The pathogenesis hypothesis of “toxin damaging brain collaterals”was put forward by Academician WANG Yong-yan and plays an important role in the pathology of AD, which is also closely related with autophagy. PTEN-induced putative kinase 1 (PINK1) is critical to the maintenance of mitochondrial function and quality control by promoting the removal of damaged mitochondria via mitophagy, and activation of PINK1 represents a new therapeutic avenue for combating AD.. HKSX capsule can remove turbidites and detoxication, which is associated with a reduced risk of chronic renal failure (CRF) in clinical application. Besides, "turbidites and detoxication" is a common pathogenesis of CRF and AD. Based on "the same treatment for different diseases", the neuroprotective effect of HKSX was studied. Our research showed that HKSX could improve the learning and memory behavior of zebrafish, with an increase of expression of PINK1 and phosphorylaton of AMPK in Thr172. However, the mechanism remains unclear.. Therefore, in this study, we take the AMPK-PINK1 signal pathway as breakthrough point to illustrate the neuroprotective effect and mechanism in SAMP8 mice and Neuro2a cells stably overexpressed APPsw (N2a-APPsw), which are excellent models for studying the neurodegenerative changes associated with AD. ① we investigate the effect and mechanism of HKSX protecting against cognitive dysfunction in SAMP8 mice, with such method as Morris water maze、LTP、ELISA、Tunnel、transmission electron microscopy、immunofluorescence、confocal、QPCR and Western blot.②To determine whether HKSX ecerts neuroprotective effect through AMPK-PINK1 mediated mitophagy, N2a-APPsw cells were pretreated with CsA、siRNA-AMPK、or siRNA-PINK1, respectively, and then the effect of HKSX was investigated using flow cytometry、ELISA、transmission electron microscopy、immunofluorescence、QPCR and Western blot. . The experimental results of present study support the neuroprotection of HKSX and provide a new strategy of preventing and curing AD. Moreover, it could provide ideas for the mechanism study of "removing turbidites and detoxication" therapy in AD.

“浊毒损脑”是AD的重要病机，且与自噬障碍引起的Aβ沉积密切相关。PINK1介导的线粒体自噬是AD防治的研究热点。海昆肾喜胶囊（HKSX）化浊排毒，改善肾衰竭。而浊毒为肾衰和AD的共同病机，遵异病同治之理，申请人对HKSX的抗AD作用进行研究，发现HKSX可以显著改善AD斑马鱼的学习记忆能力，提高AMPK的磷酸化和PINK1的表达，但作用机制尚不清楚。因此，本项目拟开展以下研究：①以SAMP8小鼠为AD模型，采用Morris水迷宫、LTP、线粒体功能等检测，探讨HKSX的神经保护作用，应用透射电镜、激光共聚焦、WB和qPCR探讨其对线粒体自噬、神经元凋亡和突触可塑性的调节机制；②以N2a-APPsw细胞为模型，利用线粒体自噬抑制剂和siRNA抑制AMPK-PINK1通路，明确此通路在HKSX干预AD中的作用。以阐明HKSX化浊排毒，改善AD认知障碍的作用机制，为其临床应用提供科学依据。

“浊毒损脑”是AD的重要病机，且与自噬障碍密切相关。PINK1介导的线粒体自噬是AD防治的研究热点。本项目基于AD、浊毒损脑及线粒体自噬三者之间的关系，以化浊排毒药物海昆肾喜胶囊（HKSX）为研究对象，以SAMP8小鼠和Aβ诱导的SH-SY5Y细胞为AD模型，探索HKSX对AD的干预作用，并以线粒体自噬为切入点，对其作用机制进行探究。.实验结果表明，HKSX可以改善SAMP8小鼠的学习记忆障碍，包括提高对新物体的识别指数，降低水迷宫中寻找平台所需的逃避潜伏期，提高在目标象限的停留时间和穿越平台的次数；降低小鼠海马内Aβ和磷酸化Tau蛋白的异常沉积及凋亡基因的表达,并上调Syn、PSD95的表达。线粒体方面，HKSX改善SAMP8小鼠中出现的线粒体重度肿胀，嵴破裂、消失等现象，明显提升ATP和MMP的水平及Complex Ⅳ的活性，显著降低ROS水平，并可以观察到明显的自噬溶酶体，说明可以显著改善线粒体结构、功能和自噬状态；另一方面，HKSX明显提高线粒体自噬关键蛋白PINK1、Parkin、OPTN、TOM20、LC3-Ⅱ的表达，降低p62的表达，说明可以激活线粒体自噬。免疫荧光双标结果表明，HKSX能促进Parkin/Tom20及LAMP/LC-3的共定位，说明其可以促进Parkin到线粒体的转位，刺激线粒体自噬，并激活自噬小体及与溶酶体的融合，对其进行降解。此外，AMPKα在Thr172位点的磷酸化程度明显增加，提示，HKSX有可能是通过调控AMPK-PINK1介导的线粒体自噬改善AD认知障碍。对于Aβ诱导的SH-SY5Y细胞，HKSX可以提高细胞活力，降低细胞凋亡率，并降低Aβ的含量，而siRNA-PINK1或AMPK抑制剂CC的干预，在很大程度上抑制了HKSX对AMPK-PINK1信号通路相关蛋白的回调作用，并降低HKSX对细胞活力的提升，说明HKSX对SH-SY5Y细胞的神经保护作用依赖于AMPK-PINK1信号通路。.综上，本研究结果表明，HKSX可激活AMPK-PINK1介导的线粒体自噬，并通过自噬溶酶体将受损线粒体及Aβ、Tau等异常蛋白清除，改善线粒体结构与功能障碍，抑制神经毒性损伤，缓解神经凋亡现象，重塑神经突触功能，最终改善AD认知障碍。这些结果为HKSX在防治AD的临床应用提供科学依据，也为其他化浊排毒中药防治AD的机制研究提供思路。