栀子苷干预ROS介导mTORC1-Nrf2信号通路防治帕金森病发生分子机理研究

Gardenia jasminoides J. Ellis, with a kind of bitter and cool character, could suppress the evil fire, relieve internal heat. Geniposide is one of the main bioactive constituents of Gardenia fruit, which play a role in prevention and treatment of Parkinson’s disease (PD). Accumulated data show that mTORC1 pathway and Nrf2 signaling may have a crosstalk. Recently, the applicant discovered that ROS overproduction mediated the inhibition of mTORC1 pathway leading to neuronal apoptosis in PD, geniposide prevented from genesis of PD in vivo and in vitro via intervening the inhibition of mTORC1 pathway and involving the Nrf2 activity to fight back neuronal apoptosis. Our study will deeply explore how geniposide influence ROS and the relationship between mTORC1 pathway and Nrf2 signaling, analyze the change of ROS level, the expression of Nrf2 and neuronal viabilities after up- or down- regulation of mTORC1 pathway including mTORC1, S6K1 and 4E-BP1, and define the functional status and the role of Nrf2 signaling in geniposide intervention of ROS-mediated mTORC1 pathway in preventing from PD. The present study will help to reveal the molecular mechanisms, theoretical and practical value in geniposide preventing and treating PD, and to propose new thoughts and theoretical basis for traditional medicine in prevention and treatment PD.

栀子味苦性清降，主治火毒热炽，栀子有效活性成分栀子苷在防治帕金森病（PD）中有作用。最近有研究指出，mTORC1通路与Nrf2信号之间存在网络联系。申请人前期工作发现，PD发生中ROS水平高涨介导mTORC1通路抑制导致神经细胞凋亡，栀子苷干预在体/离体PD模型时削弱神经细胞mTORC1通路抑制并涉及Nrf2活性抗细胞凋亡。本项目拟深入研究栀子苷对ROS影响及其与mTORC1通路和Nrf2信号的相互关系，分析ROS改变、mTORC1通路中mTORC1及其下游分子S6K1和4E-BP1在上调或下调后，栀子苷干预PD模型中神经细胞Nrf2表达和细胞凋亡表现，明确Nrf2功能活性状态在栀子苷影响ROS介导的mTORC1通路干预PD模型中的重要性及意义，深入揭示栀子苷干预ROS介导mTORC1-Nrf2信号通路防治帕金森病发生的分子机理及其理论意义和应用价值，为中医药防治PD提供新思路和理论依据。

积累的研究资料显示栀子苷可能参与了帕金森病（PD）的预防和治疗，然而目前，栀子苷预防和治疗PD涉及的具体的分子机制尚不清楚。本研究通过细胞培养、荧光染色、Western Blot、免疫组化、流式细胞仪检测、双荧光素酶报告基因系统、高效液相检测法等分子生物学技术，以栀子苷为工具药，首先使用鱼藤酮持续灌胃制备小鼠PD模型，检测实验动物血浆多巴胺（DA）及代谢产物3，4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）的含量、观察PD相关运动行为、检测小鼠纹状体区酪氨酸羟化酶（TH+）的表达和生化指标，评价栀子苷对PD小鼠的保护效应；检测分析纹状体区ROS水平及mTORC1和Nrf2信号网络中关键蛋白mTORC1，S6K1，4E-BP1，Nrf2，HO-1和NQO-1的表达，提出栀子苷干预PD的分子靶点；然后，以SH-SY5Y人源神经细胞为研究对象，栀子苷单独或联合鱼藤酮共处理，检测栀子苷诱导ARE-Luc荧光素酶表达情况，检测神经细胞活力、神经细胞凋亡、ROS强度、mTORC1和Nrf2信号通路中关键分子表达，论证栀子苷对离体PD模型的保护作用和靶点。本项目揭示了栀子苷通过抑制ROS调控mTORC1和Nrf2信号抗PD的分子机理，为栀子苷防治鱼藤酮诱导的PD等神经变性疾病提供理论依据和科学指导。