Abstract：
BACKGROUND: The Chinese formula Guben-Jiannao Ye (GBJNY) formula has a long history of usage in traditional Chinese medicine (TCM) for the treatment of learning and memory disorders as well as senile insomnia. This formulation is derived from Sun Simiao\'s five tonic pills. Furthermore, modern pharmacological investigations have revealed its ability to improve cognitive impairment and ameliorate sleep-wake circadian rhythm disorders. However, the precise mechanism underlying its efficacy remains elusive.
OBJECTIVE: The current research explored the modulatory effects and possible mechanisms of GBJNY in circadian rhythm sleep-wake disorders and cognitive dysfunction in Alzheimer\'s disease using transcriptome sequencing and experimental validation.
METHODS: The LC-MS/MS tandem technology was utilized to qualitatively discern the active components present in GBJNY. The APP/PS1 mice received continuous treatment with GBJNY or Melatonin for 3 months. The learning and memory abilities of mice were assessed utilizing the Morris water maze (MWM) test, while sleep changes were studied utilizing the electroencephalogram (EEG) and electromyogram (EMG). Concurrently, mice\'s hippocampus clock gene rhythmicity was investigated. Subsequently, we employed HE staining, Golgi staining, and immunofluorescence to observe GBJNY\'s impact on synaptic damage and neuronal loss. We performed high-throughput sequencing to analyze the mRNA expression profiles of mice, aiming to identify differentially expressed genes (DEGs). Subsequently, we conducted GO and KEGG enrichment analyses to explore associated signaling pathways. Furthermore, we evaluated the expression levels of proteins involved in the PI3K/AKT/mTOR pathway and Aβ deposition in the hippocampus of mice. Through this comprehensive approach, we sought to elucidate and validate the potential mechanisms of action of GBJNY in APP/PS1 mice.
RESULTS: Results showed 216 DEGs. Following this, we conducted GO enrichment and KEGG pathway analyses to delve deeper into the distinctions and fundamental functions of the mRNA target genes. The enrichment analysis underscored the prominence of the PI3K/Akt/mTOR signaling pathway as the most pivotal among them. Through in vivo experiments, it was further demonstrated that the administration of GBJNY enhanced memory and learning capacities in APP/PS1 mice. Additionally, GBJNY treatment resulted in alterations in the sleep-wake circadian rhythm, characterized by reduced wakefulness and an increase in non-rapid eye movement (NREM) sleep. Moreover, alterations in the peak expression of Per1, Per2, Clock, Cry1, Cry2, and Bmal1 mRNA were noted in the hippocampus of treated mice. Particularly noteworthy were the observed reductions in amyloid-beta (Aβ) deposition within the hippocampus, improvements in neuronal synaptic integrity, and upregulation of mTOR, Akt, and PI3K protein expression in the hippocampal region. These findings underscore the critical involvement of the PI3K/Akt/mTOR signaling pathway in mitigating disturbances in sleep-wake circadian rhythms.
CONCLUSIONS: GBJNY enhanced the cognitive performance of APP/PS1 mice and altered clock gene expression patterns, alleviating sleep-wake circadian rhythm disruptions. The fundamental mechanism appears to be linked to the PI3K/Akt/mTOR pathway regulation, offering a foundation for potential clinical applications.
OBJECTIVE: The current research explored the modulatory effects and possible mechanisms of GBJNY in circadian rhythm sleep-wake disorders and cognitive dysfunction in Alzheimer\'s disease using transcriptome sequencing and experimental validation.
METHODS: The LC-MS/MS tandem technology was utilized to qualitatively discern the active components present in GBJNY. The APP/PS1 mice received continuous treatment with GBJNY or Melatonin for 3 months. The learning and memory abilities of mice were assessed utilizing the Morris water maze (MWM) test, while sleep changes were studied utilizing the electroencephalogram (EEG) and electromyogram (EMG). Concurrently, mice\'s hippocampus clock gene rhythmicity was investigated. Subsequently, we employed HE staining, Golgi staining, and immunofluorescence to observe GBJNY\'s impact on synaptic damage and neuronal loss. We performed high-throughput sequencing to analyze the mRNA expression profiles of mice, aiming to identify differentially expressed genes (DEGs). Subsequently, we conducted GO and KEGG enrichment analyses to explore associated signaling pathways. Furthermore, we evaluated the expression levels of proteins involved in the PI3K/AKT/mTOR pathway and Aβ deposition in the hippocampus of mice. Through this comprehensive approach, we sought to elucidate and validate the potential mechanisms of action of GBJNY in APP/PS1 mice.
RESULTS: Results showed 216 DEGs. Following this, we conducted GO enrichment and KEGG pathway analyses to delve deeper into the distinctions and fundamental functions of the mRNA target genes. The enrichment analysis underscored the prominence of the PI3K/Akt/mTOR signaling pathway as the most pivotal among them. Through in vivo experiments, it was further demonstrated that the administration of GBJNY enhanced memory and learning capacities in APP/PS1 mice. Additionally, GBJNY treatment resulted in alterations in the sleep-wake circadian rhythm, characterized by reduced wakefulness and an increase in non-rapid eye movement (NREM) sleep. Moreover, alterations in the peak expression of Per1, Per2, Clock, Cry1, Cry2, and Bmal1 mRNA were noted in the hippocampus of treated mice. Particularly noteworthy were the observed reductions in amyloid-beta (Aβ) deposition within the hippocampus, improvements in neuronal synaptic integrity, and upregulation of mTOR, Akt, and PI3K protein expression in the hippocampal region. These findings underscore the critical involvement of the PI3K/Akt/mTOR signaling pathway in mitigating disturbances in sleep-wake circadian rhythms.
CONCLUSIONS: GBJNY enhanced the cognitive performance of APP/PS1 mice and altered clock gene expression patterns, alleviating sleep-wake circadian rhythm disruptions. The fundamental mechanism appears to be linked to the PI3K/Akt/mTOR pathway regulation, offering a foundation for potential clinical applications.
摘要：
背景：中药固本健脑叶方（GBJNY）在中医治疗学习记忆障碍和老年失眠方面有着悠久的历史。该配方来源于孙思淼的五剂补药。此外，现代药理学研究揭示了其改善认知障碍和改善睡眠-觉醒昼夜节律紊乱的能力。然而,其疗效的确切机制仍然难以捉摸。
目的：目前的研究通过转录组测序和实验验证，探讨了GBJNY在阿尔茨海默病昼夜节律睡眠-觉醒障碍和认知功能障碍中的调节作用和可能的机制。
方法:利用LC-MS/MS串联技术定性地辨别存在于GBJNY中的活性组分。APP/PS1小鼠接受GBJNY或褪黑素连续治疗3个月。利用Morris水迷宫（MWM）测试对小鼠的学习和记忆能力进行评估，同时利用脑电图（EEG）和肌电图（EMG）研究睡眠变化。同时,研究了小鼠海马时钟基因的节律性。随后，我们用HE染色,高尔基染色,和免疫荧光观察GBJNY对突触损伤和神经元丢失的影响。我们进行了高通量测序以分析小鼠的mRNA表达谱，旨在鉴定差异表达基因(DEGs)。随后，我们进行了GO和KEGG富集分析，以探索相关的信号通路。此外，我们评估了小鼠海马中PI3K/AKT/mTOR通路和Aβ沉积相关蛋白的表达水平。通过这种全面的方法，我们试图阐明和验证GBJNY在APP/PS1小鼠中的潜在作用机制.
结果：结果显示216个DEG。在此之后,我们进行了GO富集和KEGG通路分析，以更深入地研究mRNA靶基因的区别和基本功能。富集分析强调了PI3K/Akt/mTOR信号通路作为其中最关键的信号通路的重要性。通过体内实验,进一步证明,给予GBJNY可增强APP/PS1小鼠的记忆和学习能力.此外,GBJNY治疗导致睡眠-觉醒昼夜节律的改变，其特征在于觉醒减少和非快速眼动(NREM)睡眠增加。此外，Per1、Per2、Clock、在处理的小鼠的海马中注意到Cry1、Cry2和Bmal1mRNA。特别值得注意的是观察到海马内淀粉样β（Aβ）沉积的减少，神经元突触完整性的改善，和mTOR的上调，Akt,海马区PI3K蛋白表达。这些发现强调了PI3K/Akt/mTOR信号通路在减轻睡眠-觉醒昼夜节律紊乱中的关键参与。
结论：GBJNY增强了APP/PS1小鼠的认知能力，改变了时钟基因表达模式，减轻睡眠-觉醒昼夜节律中断。基本机制似乎与PI3K/Akt/mTOR通路调节有关，为潜在的临床应用奠定了基础。
目的：目前的研究通过转录组测序和实验验证，探讨了GBJNY在阿尔茨海默病昼夜节律睡眠-觉醒障碍和认知功能障碍中的调节作用和可能的机制。
方法:利用LC-MS/MS串联技术定性地辨别存在于GBJNY中的活性组分。APP/PS1小鼠接受GBJNY或褪黑素连续治疗3个月。利用Morris水迷宫（MWM）测试对小鼠的学习和记忆能力进行评估，同时利用脑电图（EEG）和肌电图（EMG）研究睡眠变化。同时,研究了小鼠海马时钟基因的节律性。随后，我们用HE染色,高尔基染色,和免疫荧光观察GBJNY对突触损伤和神经元丢失的影响。我们进行了高通量测序以分析小鼠的mRNA表达谱，旨在鉴定差异表达基因(DEGs)。随后，我们进行了GO和KEGG富集分析，以探索相关的信号通路。此外，我们评估了小鼠海马中PI3K/AKT/mTOR通路和Aβ沉积相关蛋白的表达水平。通过这种全面的方法，我们试图阐明和验证GBJNY在APP/PS1小鼠中的潜在作用机制.
结果：结果显示216个DEG。在此之后,我们进行了GO富集和KEGG通路分析，以更深入地研究mRNA靶基因的区别和基本功能。富集分析强调了PI3K/Akt/mTOR信号通路作为其中最关键的信号通路的重要性。通过体内实验,进一步证明,给予GBJNY可增强APP/PS1小鼠的记忆和学习能力.此外,GBJNY治疗导致睡眠-觉醒昼夜节律的改变，其特征在于觉醒减少和非快速眼动(NREM)睡眠增加。此外，Per1、Per2、Clock、在处理的小鼠的海马中注意到Cry1、Cry2和Bmal1mRNA。特别值得注意的是观察到海马内淀粉样β（Aβ）沉积的减少，神经元突触完整性的改善，和mTOR的上调，Akt,海马区PI3K蛋白表达。这些发现强调了PI3K/Akt/mTOR信号通路在减轻睡眠-觉醒昼夜节律紊乱中的关键参与。
结论：GBJNY增强了APP/PS1小鼠的认知能力，改变了时钟基因表达模式，减轻睡眠-觉醒昼夜节律中断。基本机制似乎与PI3K/Akt/mTOR通路调节有关，为潜在的临床应用奠定了基础。
