基于Pink1/Parkin介导线粒体自噬调控肠神经元变性探讨电针治疗帕金森病便秘的作用机制

Parkinson's disease (PD) is the second most common neurodegenerative disorder in the world, and constipation is one of PD’s main non-motor symptoms. A number of studies have shown that the culprit for PD-caused constipation is that while there are central nervous system degenerative changes there is also bowel nerve degeneration. ENS abnormalities may be related to the pathogenesis of PD, so constipation is not only an associated symptom of PD but also is closely related to the occurrence and progress of the disease.For PD constipation, there is always a lack of effective drug treatment. Acupuncture therapy is widely used to treat PD constipation and has reliable efficacy, but its mechanism of action is still unclear. The previous studies of our research group have found that electroacupuncture can improve the symptoms of constipation in clinical PD patients. By replicating the rat model of PD constipation, it has been verified that electroacupuncture can effectively improve the defecation function, and its mechanism may be related to ENS. . Therefore, this research project intends to confirm the effect of electroacupuncture on the treatment of PD constipation on animals. At the same time, a variety of molecular biological measures are applied, and in combination with the colonic electromyography monitoring and the gene knockout technique, to further explore the mechanism of electroacupuncture on ST25 in regulating mitochondrial autophagy and intestinal neuron protection from the Pink1/Parkin signaling pathway. The successful implementation of this research project will not just clarify part of the mechanism of electroacupuncture in the treatment of PD constipation, but also provide a new idea for the treatment of PD constipation.

便秘是帕金森病最主要的非运动症状之一，目前尚无有效的药物治疗手段。PD便秘可能与α -syn 聚集-线粒体自噬障碍导致的ENS变性有关。Pink1/Parkin是细胞内调控线粒体自噬的重要途径，能逆转pα-Syn高表达导致的线粒体功能障碍，可能是治疗PD便秘的潜在靶点。项目组前期通过动物实验发现其机制可能与促进ENS功能的恢复有关，既往研究显示电针对Pink1/Parkin信号及自噬相关通路具有一定调节作用，且能促进pα-Syn的清除。由此我们推断，电针天枢可通过上调Pink1/Parkin介导线粒体自噬途径清除pα-Syn，进而逆转肠神经元变性治疗PD便秘。为验证该假说，本项目拟在动物水平运用多种分子生物学手段，结合在体肌结肠肌电监测、基因敲除技术，从Pink1/Parkin信号通路深入探讨电针调控线粒体自噬和肠神经元保护的作用机制。本课题的顺利实施能够阐明电针治疗PD便秘的部分机制。

便秘是帕金森病最主要的非运动症状之一，本研究用电针干预野生型帕金森便秘大鼠和小鼠，从不同效应指标研究天枢穴对帕金森病便秘的治疗效应，结果表明电针天枢穴对帕金森病便秘疗效确切。在便秘症状方面，电针改善了粪便性状，增加了粪便排出量和含水量，并且加快了肠道转运。从结肠功能来看，电针有效改善了结肠蠕动节律的紊乱，加快的结肠蠕动并增加的结肠内压。为进一步探究其肠神经机制，本研究首先以结肠全层α-syn聚集、表达和磷酸化情况、ChAT和nNOS阳性神经元表达及肌间神经丛α-syn聚集、兴奋性和抑制性神经递质表达和无髓神经纤维密度，观察电针天枢对帕金森病便秘鼠肠神经系统的影响。结果表明电针天枢穴可降低帕金森便秘大鼠结肠α-syn 的聚集、表达和磷酸化水平，恢复结肠ChAT和nNOS阳性神经元表达。在自噬相关通路方面，以自噬必要蛋白ATG5、LC3II为指标，观察电针天枢穴对肠神经自噬功能的影响，结果表明电针天枢穴改善了结肠的自噬状态。为进一步验证电针疗效与线粒体自噬的关系，我们引进ATG5+/-小鼠以切断线粒体自噬通路，与野生型C57BL6/J小鼠进行对比。结果显示，ATG5基因敲除后，电针对于帕金森病运动和便秘症状的疗效失去显著性，α-syn聚集情况未改善，无法起到逆转肠神经元变性的作用，表明线粒体自噬是电针治疗帕金森运动和便秘症状必要途径。为进一步证明Pink1-Parkin通路是电针逆转肠神经变性的主要途径，采用腺相关病毒对Pink1和 Parkin进行基因敲低。运动和便秘症状评价显示，Pink1或Parkin基因敲低后，电针对于帕金森运动和便秘症状结无改善作用。Pink1基因敲低后，电针对运动症状的疗效丧失，对于便秘症状甚至产生恶化作用，提示Pink1蛋白对于研究电针对帕金森病运动症状的疗效更具价值；Parkin基因敲低后，电针对运动便秘的疗效丧失，对于运动症状产生恶化作用，提示Parkin蛋白与电针对帕金森病便秘症状的疗效更具相关性。肠神经元相关分子生物学结果显示，Pink1或 Parkin基因敲低后，电针均无法起到清除α-syn蛋白以逆转肠神经元变性的作用，验证了Pink1-Parkin是电针调控线粒体自噬逆转肠神经变性的主要途径之一。本课题阐明了电针治疗PD便秘的部分机制。