徐燕,孟雪,赵文学,刘冬光,朱建国,姚茹,姚景春,张贵民
XU Yan,MENG Xue,ZHAO Wen-xue,LIU Dong-guang,ZHU Jian-guo,YAO Ru,YAO Jing-chun,ZHANG Gui-min

• 1:鲁南制药集团股份有限公司新药药理中心
• 2:中药制药共性技术国家重点实验室

摘要(Abstract)：

探究人参皂苷Re(ginsenoside Re, GS-Re)对鱼藤酮(rotenone, Rot)诱导果蝇帕金森病(Parkinson′s disease, PD)模型脑神经的保护作用机制。利用Rot建立果蝇PD模型，对其进行分组给药(0.1、0.4、1.6 mmol·L~(-1 )GS-Re; 80μmol·L~(-1 )L-dopa),通过功能表型实验检测果蝇寿命及爬行能力，ELISA检测果蝇大脑抗氧化活力(CAT、MDA、ROS、SOD含量)、多巴胺(dopamine, DA)含量及线粒体功能(ATP含量、NDUFB8Ⅰ活性、SDHBⅡ活性),免疫荧光检测果蝇大脑DA神经元数量；Western blot检测果蝇大脑NDUFB8Ⅰ、SDHBⅡ、Cyt C、Nrf2、HO-1、Bcl-2/Bax、cleaved caspase-3/caspase-3表达水平的变化。结果显示，与正常组相比，以475μmol·L~(-1) Rot(IC_(50))建立果蝇PD模型，果蝇存活率显著下降，并表现出明显的运动功能障碍，果蝇脑内DA含量及神经元数量均显著降低，脑内ROS生成量、MDA含量升高，SOD含量、CAT含量下降，ATP含量、NDUFB8Ⅰ活性、SDHBⅡ活性显著下降，NDUFB8Ⅰ、SDHBⅡ、Bcl-2/Bax表达显著下调，Cyt C从线粒体向胞浆大量释放，Nrf2核转移降低，cleaved caspase-3/caspase-3表达显著上调，给予GS-Re(0.1、0.4、1.6 mmol·L~(-1))干预后，显著提高了PD果蝇的存活率，改善了果蝇的运动功能障碍，增加了脑内DA含量，减轻了DA神经元的丢失，降低脑内ROS生成量及MDA含量，增加SOD含量及CAT含量，提高脑内抗氧化活力，维护了线粒体功能稳态，表现为ATP含量、NDUFB8Ⅰ活性、SDHBⅡ活性显著增加，NDUFB8Ⅰ、SDHBⅡ、Bcl-2/Bax表达显著上调，显著降低胞浆中Cyt C的表达水平，Nrf2核转移增加，cleaved caspase-3/caspase-3表达显著下调。综上表明，GS-Re可明显改善Rot诱导的果蝇脑神经毒性作用，其机制可能是GS-Re通过维护线粒体功能稳态，激活Keap1-Nrf2-ARE信号通路，提高脑神经元抗氧化能力，进而抑制线粒体介导的caspase-3信号通路，抑制神经元细胞的凋亡，从而发挥神经保护作用。
This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson′s disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.

关键词(KeyWords)： 人参皂苷Re;帕金森病;果蝇;脑神经;氧化应激;线粒体稳态
ginsenoside Re;Parkinson′s disease;drosophila;cerebral neuron;oxidative stress;mitochondrial homeostasis

Abstract:

Keywords:

基金项目(Foundation): 山东省重大科技创新工程项目(2021CXGC010508);; 山东省自然科学基金项目(ZR2021QH289);; 国家重点研发计划项目(2019YFC1711205,2019YFC1711200)

作者(Author): 徐燕,孟雪,赵文学,刘冬光,朱建国,姚茹,姚景春,张贵民
XU Yan,MENG Xue,ZHAO Wen-xue,LIU Dong-guang,ZHU Jian-guo,YAO Ru,YAO Jing-chun,ZHANG Gui-min

DOI: 10.19540/j.cnki.cjcmm.20230103.705

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