从DJ-1介导JNK信号通路角度探讨知母宁对帕金森病线粒体功能的作用机制

Parkinson's disease (PD) is one of the most common neurodegenerative disease. DJ-1 is associated with PD closely. Moreover, mitochondrial dysfunction plays a key role in the pathogenesis of PD. Recent studies found that DJ-1-mediated JNK signaling pathway is related to PD. Our previous research demonstrated that Da-Bu-Yin Pill, a traditional Chinese formula, play a role in the protection of PD mitochondrial function. In additional, the herb Anemarrhena asphodeloides Bunge is one of the main ingredients of Da-Bu-Yin Pill, and mangiferin is the main polyphenolic compound extracted from the herb Anemarrhena asphodeloides Bunge. Based on our previous research, the present project intends to investigate that whether DJ-1-mediated JNK signaling pathway is a key mechanism for mangiferin to protect mitochondrial function in PD, with combined research methodology of neurobiology and pharmacology, using over expression and knock down of DJ-1 gene (both positive and negative aspects) in vitro and in vivo, respectively. DJ-1 is one of the current hot molecular targets, however, the study of DJ-1-mediated JNK signaling pathway to carry out anti-PD mechanism of mangiferin has not been reported. Therefore, it has important scientific value for this project to reveal whether the anti-PD signaling pathway of mangiferin, or to elucidate the mechanism of regulation of Chinese herbs on DJ-1-mediated JNK signaling pathway.

帕金森病（Parkinson's Disease, PD）是最常见的神经退行性疾病之一。DJ-1是较常见的与PD发病相关的基因，而线粒体功能障碍是PD发病中的关键环节。研究发现DJ-1介导JNK信号通路与PD密切相关。本课题组前期发现以中药知母为主的复方大补阴丸具有明确保护线粒体功能抗PD的作用，而知母宁是从知母中提取最主要的多酚类化合物。本项目拟在前期工作基础上，从DJ-1过表达和靶向敲减正反两方面，在离体细胞和整体动物两个水平，采用神经生物学和药理学相结合的研究手段，力争回答DJ-1介导JNK信号通路是否是知母宁保护线粒体功能抗PD作用的关键机制。DJ-1作为当前的热点分子靶标，围绕DJ-1介导JNK信号通路开展知母宁抗PD作用机制研究未见报道。因此，本项目的开展无论是对揭示知母宁抗PD作用的信号通路机制，还是对阐明中药调控DJ-1介导JNK信号通路的机理均具有重要的科学价值。

帕金森病（Parkinson's Disease, PD）是常见的神经退行性疾病之一。PD发病明确与线粒体功能失常有关系，DJ-1是较常见的与PD发病相关的基因，而研究发现DJ-1介导JNK信号通路与PD密切相关，而知母宁可以阻止PD模型小鼠DA神经元退行性变，改善运动障碍。本项目从DJ-1过表达和靶向敲减正反两方面，分别在离体细胞和整体动物两个水平，探讨知母宁影响DJ-1介导JNK信号通路在保护线粒体功能抗PD的作用机制。研究结果表明：1）离体细胞水平：A）对多巴胺能细胞SH-SY5Y和PC-12分别瞬时转染pDNA3-Flag-DJ-1质粒后，确定过表达DJ-1，没有影响Akt的表达，但至少通过Akt磷酸化的通路，可以提高线粒体活性及复合物I活性，对线粒体形态因子没有影响。B）在SH-SY5Y和PC-12细胞通过RNAi技术对DJ-1进行靶向敲减，确认DJ-1表达下降。DJ-1表达降低，也通过了Akt磷酸化、GSK3beta及JNK信号途径，导致线粒体数量减少、线粒体复合物I活性降低、ATP含量降低。2）整体动物水平：在PD模型动物靶向敲减DJ-1后，与DJ-1敲减后PD模型动物组比较，知母宁可以增加黑质线粒体产物ATP含量，而且知母宁可以改善行为学（阿扑吗啡诱导的旋转实验、转棒实验、旷场实验）的表现，增加黑质酪氨酸羟化酶表达，改善黑质、纹状体内线粒体超微结构、增强黑质、纹状体内线粒体膜电位，提高纹状体内ATP含量。知母宁治疗后显著上调DJ-1的表达，JNK磷酸化情况无显著改变，ERK的磷酸化显著增加，p38磷酸化显著降低，知母宁对ERK的磷酸化增加有显著调控作用，对JNK和p38磷酸化情况无显著调节。DJ-1作为当前的热点分子靶标，在调节和维持线粒体功能上发挥重要作用。因此，本项目的开展无论是对揭示知母宁抗PD作用的信号通路机制，还是对阐明中药调控DJ-1介导JNK信号通路的机理均不断深化和系统，为中药在临床上PD治疗的应用提供科学理论依据。