Chinese formula Guben-Jiannao Ye alleviates the dysfunction of circadian and sleep rhythms in APP/PS1 mice implicated in activation of the PI3K/AKT/mTOR signaling pathway.

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. 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. 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 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. 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.

Mao JQ ，Cheng L ，Zhang YD ，Xie GJ ，Wang P ... -  《-》

Shenzhiling oral liquid protects the myelin sheath against Alzheimer's disease through the PI3K/Akt-mTOR pathway.

Shenzhiling oral liquid (SZL), a traditional Chinese medicine (TCM) compound, is firstly approved by the Chinese Food and Drug Administration (CFDA) for the treatment of mild to moderate Alzheimer's disease (AD). SZL is composed of ten Chinese herbs, and the precise therapy mechanism of its action to AD is far from fully understood. The purpose of this study was to observe whether SZL is an effective therapy for amyloid-beta (Aβ)-induced myelin sheath and oligodendrocytes impairments. Notably, the primary aim was to elucidate whether and through what underlying mechanism SZL protects the myelin sheath through the PI3K/Akt-mTOR signaling pathway in Aβ42-induced OLN-93 oligodendrocytes in vitro. APP/PS1 mice were treated with SZL or donepezil continuously for three months, and Aβ42-induced oligodendrocyte OLN-93 cells mimicking AD pathogenesis of myelin sheath impairments were incubated with SZL-containing serum or with donepezil. LC-MS/MS was used to analysis the active components of SZL and SZL-containing serum. The Y maze test was administered after 3 months of treatment, and the hippocampal tissues of the APP/PS1 mice were then harvested for observation of myelin sheath and oligodendrocyte morphology. Cell viability and toxicity were assessed using CCK-8 and lactate dehydrogenase (LDH) release assays, and flow cytometry was used to measure cell apoptosis. The expression of the myelin proteins MBP, PLP, and MAG and that of Aβ42 and Aβ40 in the hippocampi of APP/PS1 mice were examined after SZL treatment. Simultaneously, the expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were also examined. The expression of proteins, including CNPase, Olig2, NKX2.2, MBP, PLP, MAG, MOG, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR, was determined by immunofluorescence and Western blot, and the corresponding gene expression was evaluated by qPCR in Aβ42-induced OLN-93 oligodendrocytes. LC-MS/MS detected a total of 126 active compounds in SZL-containing serum, including terpenoids, flavones, phenols, phenylpropanoids and phenolic acids. SZL treatment significantly improved memory and cognition in APP/PS1 mice and decreased the G-ratio of myelin sheath, alleviated myelin sheath and oligodendrocyte impairments by decreasing Aβ42 and Aβ40 accumulation and increasing the expression of myelin proteins MBP, PLP, MAG, and PI3K/Akt-mTOR signaling pathway associated protein in the hippocampi of APP/PS1 mice. SZL-containing serum also significantly reversed the OLN-93 cell injury induced by Aβ42 by increasing cell viability and enhanced the expression of MBP, PLP, MAG, and MOG. Meanwhile, SZL-containing serum facilitated the maturation and differentiation of oligodendrocytes in Aβ42-induced OLN-93 cells by heightening the expression of CNPase, Olig2 and NKX2.2. SZL-containing serum treatment also fostered the expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR, indicating an activating PI3K/Akt-mTOR signaling pathway in OLN-93 cells. Furthermore, the effects of SZL on myelin proteins, p-Akt, and p-mTOR were clearly inhibited by LY294002 and/or rapamycin, antagonists of PI3K and m-TOR, respectively. Our findings indicate that SZL exhibits a neuroprotective effect on the myelin sheath by promoting the expression of myelin proteins during AD, and its mechanism of action is closely related to the activation of the PI3K/Akt-mTOR signaling pathway.

Zheng M ，Liu Z ，Mana L ，Qin G ，Huang S ，Gong Z ，Tian M ，He Y ，Wang P ... -  《-》

Qifu-yin activates the Keap1/Nrf2/ARE signaling and ameliorates synaptic injury and oxidative stress in APP/PS1 mice.

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. 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. 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. 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. 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.

Wang S ，Huang J ，Chen Y ，Liang Y ，Chen L ，Ye D ，Yang H ，Hui Z ，Wang X ，Zhang Z ，Zhu X ... -  《-》

Downregulation of PI3K/Akt/mTOR signaling pathway in curcumin-induced autophagy in APP/PS1 double transgenic mice.

Autophagy is a lysosomal degradation pathway, which is essential for cell survival, proliferation, differentiation and homeostasis. It is well known that beta-amyloid (Aβ) aggregation is one of key characteristics for Alzheimer's disease (AD), which triggers a complex pathological cascade, leading to neurodegeneration. Recent studies have shown that Aβ peptide is generated from amyloid β precursor protein (APP) during autophagic turnover of APP-rich organelles by autophagy. Aβ generation during normal autophagy is subsequently degraded by lysosomes. Curcumin, a nature plant extraction, has been reported to inhibit the generation and deposition of Aβ; however, the underlying mechanisms are not fully understood yet. In the present study, we reported that curcumin treatment not only attenuated cognitive impairment detected by Morris water maze test, but also inhibited the generation of Aβ investigated by immunohistochemistry in APP/PS1 double transgenic AD mice. Moreover, curcumin induced autophagy in the mice, evidenced by LC3 immunofluorescence analysis and western blot assays on LC3. Furthermore, we found that curcumin significantly decreased the expression of Phosphatidylinositol 3-Kinase (PI3K), phosphorylated Akt and rapamycin (mTOR) at protein levels, respectively. Taken together, our data suggests that curcumin inhibits Aβ generation and induces of autophagy by downregulating PI3K/Akt/mTOR signaling pathway, and further shows a neuroprotective effect. Meanwhile curcumin might be a candidate neuroprotective agent for AD patients treatment by inducing autophagy.

Wang C ，Zhang X ，Teng Z ，Zhang T ，Li Y ... -  《-》

β-asarone improves learning and memory and reduces Acetyl Cholinesterase and Beta-amyloid 42 levels in APP/PS1 transgenic mice by regulating Beclin-1-dependent autophagy.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, and studies have suggested that β-asarone has pharmacological effects on beta-amyloid (Aβ) injected in the rat hippocampus. However, the effect of β-asarone on autophagy in the APP/PS1 transgenic mouse is unreported. APP/PS1 transgenic mice were randomly divided into six groups (n=10/group): an untreated group, an Aricept-treated group, a 3-MA-treated group, a rapamycin-treated group, an LY294002-treated group, a β-asarone-treated group. The control group consisted of wild-type C57BL/6 mice. All treatments were administered to the mice for 30 days. Spatial learning and memory were assessed by water maze, passive avoidance, and step-down tests. AChE and Aβ42 levels in the hippocampus were determined by ELISA. p-Akt, p-mTOR, and LC3B expression were detected by flow cytometry. The expression of p-Akt, p-mTOR, Beclin-1, and p62 proteins was assessed by western blot. Changes in autophagy were viewed using a transmission electron microscope. APP and Beclin-1 mRNA levels were measured by Real-Time PCR. The learning and memory of APP/PS1 transgenic mice were improved significantly after β-asarone treatment compared with the untreated group. In addition, β-asarone treatment reduced AChE and Aβ42 levels, increased p-mTOR and p62 expression, decreased p-Akt, Beclin-1, and LC3B expression, decreased the number of autophagosomes and reduced APP mRNA and Beclin-1 mRNA levels compared with the untreated group. That is, β-asarone treatment can improve the learning and memory abilities of APP/PS1 transgenic mouse by inhibiting Beclin-1-dependent autophagy via the PI3K/Akt/mTOR pathway.

Deng M ，Huang L ，Ning B ，Wang N ，Zhang Q ，Zhu C ，Fang Y ... -  《-》