Abstract：
BACKGROUND: The traditional medicinal formulation, Qifu-yin (QFY), has been widely prescribed for Alzheimer\'s disease (AD) treatment in China, yet the comprehensive mechanisms through which QFY mitigates AD pathology remain to be fully delineated.
OBJECTIVE: This study aimed to explore the therapeutic implications of QFY on the synaptic injury and oxidative stress in the hippocampus of APPswe/PS1dE9 (APP/PS1) mice, with a concerted effort to elucidate the molecular mechanisms related to synaptic preservation and memory improvement.
METHODS: The components of QFY were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The neuroprotective effects of QFY was evaluated using six-month-old male APP/PS1 mice. Subsequent to a 15 days of QFY regimen, spatial memory was assessed utilizing the Morris water maze (MWM) test. Amyloid-beta (Aβ) aggregation was detected via immunostaining, while the quantification of Aβ1-40 and Aβ1-42 was achieved through enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to investigate the synaptic structure and mitochondrial morphology. Golgi staining was applied to examine dendritic spine density. Reactive oxygen species (ROS), 3-nitrotyrosine (3-NT) and 4-hydroxy-nonenal (4-HNE) assays were employed to assess oxidative stress. The expression profiles of Aβ metabolism-associated enzymes and the Keap1/Nrf2/ARE signaling pathway were determined by Western blot.
RESULTS: A total of 20 principal compounds in QFY were identified. QFY mitigated memory deficits of APP/PS1 mice, including reducing escape latency and search distance and increasing the time and distance spent in the target quadrant. In addition, QFY increased platform crossings of APP/PS1 mice in the probe trial of MWM tests. TEM analysis showed that QFY increased synapse number in the CA1 region of APP/PS1 mice. Further studies indicated that QFY elevated the expression levels of Post synaptic density protein 95 (PSD95) and synaptophysin, and mitigated the loss of dendritic spine density in the hippocampus of APP/PS1 mice. QFY has been shown to ameliorated the structural abnormalities of mitochondria, including mitochondrial dissolution and degradation, up-regulate ATP synthesis and membrane potential in the hippocampus of APP/PS1 mice. Moreover, QFY activated the Keap1/Nrf2/ARE signaling pathway in the hippocampus of APP/PS1 mice, which might contribute to the neuroprotective effects of QFY.
CONCLUSIONS: QFY activates the Keap1/Nrf2/ARE signaling, and protects against synaptic and mitochondrial dysfunction in APP/PS1 mice, proposing a potential alternative therapeutic strategy for AD management.
OBJECTIVE: This study aimed to explore the therapeutic implications of QFY on the synaptic injury and oxidative stress in the hippocampus of APPswe/PS1dE9 (APP/PS1) mice, with a concerted effort to elucidate the molecular mechanisms related to synaptic preservation and memory improvement.
METHODS: The components of QFY were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The neuroprotective effects of QFY was evaluated using six-month-old male APP/PS1 mice. Subsequent to a 15 days of QFY regimen, spatial memory was assessed utilizing the Morris water maze (MWM) test. Amyloid-beta (Aβ) aggregation was detected via immunostaining, while the quantification of Aβ1-40 and Aβ1-42 was achieved through enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to investigate the synaptic structure and mitochondrial morphology. Golgi staining was applied to examine dendritic spine density. Reactive oxygen species (ROS), 3-nitrotyrosine (3-NT) and 4-hydroxy-nonenal (4-HNE) assays were employed to assess oxidative stress. The expression profiles of Aβ metabolism-associated enzymes and the Keap1/Nrf2/ARE signaling pathway were determined by Western blot.
RESULTS: A total of 20 principal compounds in QFY were identified. QFY mitigated memory deficits of APP/PS1 mice, including reducing escape latency and search distance and increasing the time and distance spent in the target quadrant. In addition, QFY increased platform crossings of APP/PS1 mice in the probe trial of MWM tests. TEM analysis showed that QFY increased synapse number in the CA1 region of APP/PS1 mice. Further studies indicated that QFY elevated the expression levels of Post synaptic density protein 95 (PSD95) and synaptophysin, and mitigated the loss of dendritic spine density in the hippocampus of APP/PS1 mice. QFY has been shown to ameliorated the structural abnormalities of mitochondria, including mitochondrial dissolution and degradation, up-regulate ATP synthesis and membrane potential in the hippocampus of APP/PS1 mice. Moreover, QFY activated the Keap1/Nrf2/ARE signaling pathway in the hippocampus of APP/PS1 mice, which might contribute to the neuroprotective effects of QFY.
CONCLUSIONS: QFY activates the Keap1/Nrf2/ARE signaling, and protects against synaptic and mitochondrial dysfunction in APP/PS1 mice, proposing a potential alternative therapeutic strategy for AD management.
摘要：
背景：传统的药物配方，启复(QFY)，在中国已被广泛用于阿尔茨海默病（AD）的治疗，然而，QFY缓解AD病理的综合机制仍有待完全描述。
目的：本研究旨在探讨QFY对APPswe/PS1dE9（APP/PS1）小鼠海马突触损伤和氧化应激的治疗意义，共同努力阐明与突触保存和记忆改善有关的分子机制。
方法：采用超高效液相色谱-串联质谱(UHPLC-MS/MS)对QFY的成分进行鉴定。使用6个月大的雄性APP/PS1小鼠评价QFY的神经保护作用。在15天的QFY方案之后，利用莫里斯水迷宫（MWM）测试评估空间记忆。通过免疫染色检测到淀粉样β（Aβ）聚集，而Aβ1-40和Aβ1-42的定量是通过酶联免疫吸附测定（ELISA）实现的。透射电子显微镜（TEM）用于研究突触结构和线粒体形态。应用高尔基染色检查树突棘密度。活性氧（ROS），采用3-硝基酪氨酸（3-NT）和4-羟基-壬烯醛（4-HNE）测定来评估氧化应激。Westernblot检测Aβ代谢相关酶的表达谱和Keap1/Nrf2/ARE信号通路。
结果:在QFY中鉴定出总共20个主要化合物。QFY减轻APP/PS1小鼠的记忆缺陷，包括减少逃避延迟和搜索距离，并增加在目标象限中花费的时间和距离。此外,在MWM测试的探针试验中,QFY增加APP/PS1小鼠的平台交叉。TEM分析显示QFY增加APP/PS1小鼠CA1区的突触数量。进一步的研究表明,QFY能提高突触后密度蛋白95(PSD95)和突触素的表达水平,并减轻APP/PS1小鼠海马树突棘密度的损失。QFY已被证明可以改善线粒体的结构异常，包括线粒体溶解和降解，上调APP/PS1小鼠海马ATP合成和膜电位。此外，QFY激活APP/PS1小鼠海马Keap1/Nrf2/ARE信号通路,这可能有助于QFY的神经保护作用。
结论：QFY激活Keap1/Nrf2/ARE信号，并防止APP/PS1小鼠的突触和线粒体功能障碍，提出了AD管理的潜在替代治疗策略。
目的：本研究旨在探讨QFY对APPswe/PS1dE9（APP/PS1）小鼠海马突触损伤和氧化应激的治疗意义，共同努力阐明与突触保存和记忆改善有关的分子机制。
方法：采用超高效液相色谱-串联质谱(UHPLC-MS/MS)对QFY的成分进行鉴定。使用6个月大的雄性APP/PS1小鼠评价QFY的神经保护作用。在15天的QFY方案之后，利用莫里斯水迷宫（MWM）测试评估空间记忆。通过免疫染色检测到淀粉样β（Aβ）聚集，而Aβ1-40和Aβ1-42的定量是通过酶联免疫吸附测定（ELISA）实现的。透射电子显微镜（TEM）用于研究突触结构和线粒体形态。应用高尔基染色检查树突棘密度。活性氧（ROS），采用3-硝基酪氨酸（3-NT）和4-羟基-壬烯醛（4-HNE）测定来评估氧化应激。Westernblot检测Aβ代谢相关酶的表达谱和Keap1/Nrf2/ARE信号通路。
结果:在QFY中鉴定出总共20个主要化合物。QFY减轻APP/PS1小鼠的记忆缺陷，包括减少逃避延迟和搜索距离，并增加在目标象限中花费的时间和距离。此外,在MWM测试的探针试验中,QFY增加APP/PS1小鼠的平台交叉。TEM分析显示QFY增加APP/PS1小鼠CA1区的突触数量。进一步的研究表明,QFY能提高突触后密度蛋白95(PSD95)和突触素的表达水平,并减轻APP/PS1小鼠海马树突棘密度的损失。QFY已被证明可以改善线粒体的结构异常，包括线粒体溶解和降解，上调APP/PS1小鼠海马ATP合成和膜电位。此外，QFY激活APP/PS1小鼠海马Keap1/Nrf2/ARE信号通路,这可能有助于QFY的神经保护作用。
结论：QFY激活Keap1/Nrf2/ARE信号，并防止APP/PS1小鼠的突触和线粒体功能障碍，提出了AD管理的潜在替代治疗策略。
