基于LKB1-AMPK自噬调控通路的通腑养髓方对高铜诱导的多巴胺能神经元损伤的保护机制研究

Wilson disease (WD) is an autosomal recessively inherited disorder of copper metabolism, and dopaminergic neurons damage induced by high copper in basal ganglia is main pathogenesis for WD patients with brain injury. For this reason, it’s an important scientific question for treatment of WD patient with cerebral type to study the mechanism of dopaminergic neurons damage induced by high copper and possible intervention approaches. In this project, the applicant and his team members assume that Tongfu-Yangsui recipe had the function of neuron protection which could block dopaminergic neurons damage induced by high copper through LKB1-AMPK autophagy regulation pathways and carry out the following works: study the protection mechanism of Tongfu-Yangsui recipe on dopaminergic neurons damage induced by high copper through LKB1-AMPK autophagy regulation pathways, ascertain the specific protection aspects and targets of Tongfu-Yangsui recipe on dopaminergic neurons, based on the foundation of clinical and research on treatment of patients with WD and the research progress of neurons autophagy related pathways in recently years. Therefore, this project will provide theoretical basis of Tongfu-Yangsui Recipe for treatment of WD patient with cerebral type and establish the foundation of formulating effective and less side effects integrated treatment solutions combined by Western and Chinese medicine. Furthermore, the results of therapeutic mechanism research of Tongfu-Yangsui Recipe based on the mechanism of autophagy in this project will provide new therapeutic targets and the research ideas for the prevention and treatment of nerve genetic degeneration diseases with traditional Chinese medicine.

Wilson病(WD)是一种常染色体隐性遗传铜代谢障碍疾病，高铜诱导的基底神经节多巴胺能神经元损伤是WD患者脑损伤的重要机制，研究高铜诱导的多巴胺能神经元损伤机制及可能的干预途径，是脑型WD患者治疗学研究的一个重要科学问题。本项目基于申请人及其团队多年来应用通腑养髓方治疗WD所积累的临床和研究基础及神经元自噬相关通路的研究进展，假设通腑养髓方能通过LKB1-AMPK自噬调控通路阻断高铜诱导的多巴胺能神经元的损伤而具有神经元保护作用，开展基于LKB1-AMPK自噬调控通路的通腑养髓方对高铜诱导的多巴胺能神经元损伤的保护机制研究，明确通腑养髓方对多巴胺能神经元保护的具体环节和作用靶点，为通腑养髓方治疗脑型WD患者提供理论依据，并为制定疗效好、副作用小的WD中西医结合治疗方案奠定基础。本项目基于自噬机制研究通腑养髓方疗效机制的研究结果，将为中医药防治神经遗传变性疾病提供新的治疗靶点和研究思路。

Wilson病(WD)是一种常染色体隐性遗传铜代谢障碍疾病，高铜诱导的基底神经节多巴胺能神经元损伤是WD患者脑损伤的重要机制，研究高铜诱导的多巴胺能神经元损伤机制及可能的干预途径，是脑型WD患者治疗学研究的一个重要科学问题。.本项目基于申请人及其团队多年来应用通腑养髓方治疗WD所积累的临床和研究基础及神经元自噬相关通路的研究进展，假设通腑养髓方能通过LKB1-AMPK自噬调控通路阻断高铜诱导的多巴胺能神经元的损伤而具有神经元保护作用，开展基于LKB1-AMPK自噬调控通路的通腑养髓方对高铜诱导的多巴胺能神经元损伤的保护机制研究，明确通腑养髓方对多巴胺能神经元保护的具体环节和作用靶点，为通腑养髓方治疗脑型WD患者提供理论依据，并为制定疗效好、副作用小的WD中西医结合治疗方案奠定基础。. 实验表明：高铜可通过诱导SH-SY5Y细胞内线粒体氧化应激，上调自噬相关蛋白及基因LKB1、AMPK、p-AMPK、TSC2、LC3A/B、P27及ULK1的表达，下调自噬相关蛋白及基因mTOR、p-ULK的表达，导致SH-SY5Y细胞发生自噬性死亡，而通腑养髓方可通过调控LKB1-AMPK信号通路，下调自噬相关蛋白及基因LKB1、AMPK、p-AMPK、TSC2、LC3A/B、P27、ULK1的表达，上调自噬相关蛋白及基因mTOR、p-ULK的表达，抑制自噬的发生，阻断高铜诱导的神经元损伤，从而发挥神经保护作用。.研究结果显示：通腑养髓方可显著降低AMPK基因过表达稳转细胞内LDH漏出率，增加SOD表达量，上调LKB1-AMPK自噬信号通路相关蛋白及基因AMPK、p-AMPK、LC3Ⅱ的表达，下调自噬相关蛋白及基因mTOR的表达，表明高铜诱导多巴胺能神经元损伤的氧化应激及分子信号机制与诱导自噬死亡的LKB1-AMPK信号通路有密切关联；同时表明通腑养髓方具有一定的神经保护作用，为临床上WD特别是脑型的治疗提供重要依据。.本研究从分子水平初步阐明了通腑养髓方调控LKB1-AMPK自噬通路阻断铜诱导多巴胺能神经元损伤的机制，为从现代医学角度对中医通腑利湿、活血养髓法防治WD的治法提供理论依据，并为中医药防治神经遗传变性疾病提供新的治疗靶点和研究思路。