Learning-memory Ability Research Articles

Effect of Huayu Tongluo moxibustion on learning-memory ability in rats with vascular dementia based on hippocampal Mst1/NF-κB p65 pathway

To observe the effects of Huayu Tongluo (transforming stasis and unblocking collaterals) moxibustion on learning-memory ability and hippocampal mammalian sterile 20-like kinase 1 (Mst1)/nuclear factor κB (NF-κB) p65 pathway related to inflammatory response in rats with vascular dementia (VD). A total of 60 male Wistar rats of SPF grade were randomly divided into a sham operation group (12 rats) and a modeling group (48 rats). VD model was established by the method of modified bilateral common carotid artery permanent ligation in the modeling group. Thirty-six rats with successful modeling were randomly divided into a model group, a moxibustion group and a western medication group, with 12 rats in each group. Huayu Tongluo moxibustion was applied at "Dazhui" (GV14), "Baihui" (GV20) and "Shenting" (GV24) in the moxibustion group, 20 min each time, once a day, 7 day-intervention was as one course, and 1 day-interval was taken between two courses, for a total of 3 courses. In the western medication group, piracetam was given 0.72 mg/kg by intragastric administration, twice a day, the course of intervention was same as that of the moxibustion group. The learning-memory ability was detected by Morris water maze test; the morphology of hippocampal CA1 region was observed by HE staining; the mRNA expression of Mst1, M1 microglia markers CD86, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected by real-time PCR; the levels of IL-6 and TNF-α in hippocampus were detected by ELISA; and the protein expression of Mst1 and NF-κB p65 in hippocampus was detected by Western blot in rats of each group. Compared with the sham operation group, the escape latency was prolonged in the model group (P<0.05); compared with the model group, the escape latency was shortened in the moxibustion group and the western medication group (P<0.05). The cells in the CA1 region of hippocampus were disordered, cell collapse and irregular nuclei could be observed in the model group; compared with the model group, the cell arrangement in the CA1 region of hippocampus was more regular, and the damage was improved in the moxibustion group and the western medication group. Compared with the sham operation group, the mRNA expression of Mst1, CD86, IL-6 and TNF-α, as well as the protein expression of Mst1, NF-κB p65 in hippocampus were increased in the model group (P<0.05). Compared with the model group, the mRNA expression of Mst1, CD86, IL-6 and TNF-α, as well as the protein expression of Mst1, NF-κB p65 in hippocampus were decreased in the moxibustion group and the western medication group (P<0.05). Compared with the sham operation group, the levels of IL-6 and TNF-α in hippocampus were increased in the model group (P<0.05). Compared with the model group, the levels of IL-6 and TNF-α in hippocampus were decreased in the moxibustion group and the western medication group (P<0.05). Huayu Tongluo moxibustion can improve the learning-memory ability of VD rats, the mechanism may be related to regulating the activation of microglia through Mst1/NF-κB p65 pathway, reducing the release of pro-inflammatory factors i.e. IL-6 and TNF-α, so as to alleviating the damage of inflammatory factors in the hippocampus of VD rats.

Downregulation of circTLK1 improves the impairments in learning and memory induced by anesthetics via regulating miR-374b-5p expression and reducing neuroinflammation.

Sevoflurane (Sev) is a common anesthetic used during surgery, but research on its induction of neurotoxicity and learning memory impairment is insufficient. This study aimed to explore the role of Circular RNA tousled like kinase 1 (circTLK1) and its target microRNA (miR)-374b-5p in Sev-induced neurotoxicity and learning memory impairment. Mouse hippocampal neuronal HT22 cells and SD rats were treated with Sev. Levels of circTLK1 and miR-374b-5p were detected using RT-qPCR. The concentration of inflammatory factors was determined using ELISA. Cell viability and apoptosis were analyzed using CCK-8 and flow cytometry. Targeting relationship between circTLK1 and miR-374b-5p was validated using dual-luciferase reporter assays and RIP experiments. The Morris water maze test was used to assess the learning and spatial memory abilities of rats. The results indicated that Sev treatment stimulated neuroinflammation and oxidative stress while increasing circTLK1 levels and decreasing miR-374b-5p levels in both rats and HT22 cells. Silencing circTLK1 alleviated the decrease in cell viability, increased apoptosis rates, and raised concentrations of inflammatory factors caused by Sev treatment. In in vivo experiments, silencing circTLK1 was also found to counteract the oxidative stress, neuroinflammation, and learning and memory impairment induced by Sev treatment in rats. Additionally, circTLK1 was shown to interact with miR-374b-5p, and inhibiting miR-374b-5p could counteract the neuroprotective effects of si-circTLK1. This research suggested that silencing circTLK1 can mitigate Sev-induced neurotoxicity and learning memory impairment by modulating miR-374b-5p.

Long-term aerobic exercise improves learning memory capacity and effects on oxidative stress levels and Keap1/Nrf2/GPX4 pathway in the hippocampus of APP/PS1 mice.

To examine the effects of long-term aerobic exercise on oxidative stress and learning memory ability of APP/PS1 mice, focusing on the hippocampal Keap1, Nrf2, HO-1, and GPX4 proteins. Thirty APP/PS1 double transgenic AD mice were randomly divided into three groups: model group, short-term exercise group, and long-term exercise group, with 10 mice in each group. Male non-transgenic mice of the same age served as the control group. The groups underwent swimming training for 6 weeks and 12 weeks, respectively. After the intervention, cognitive abilities were assessed using the Morris water maze test. Hippocampal tissue samples were analyzed for changes in superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. ROS expression was observed using dihydroethidium probe, and Keap1, Nrf2, HO-1, and GPX4 protein levels were detected by Western blot analysis. Aerobic exercise significantly reduced the escape latency and increased both the time spent in the target quadrant and the number crossing the platform compared to the model group (p < 0.05). In the hippocampus, aerobic exercise significantly reduced the MDA content, while significantly increased SOD activity (p < 0.05). The level of ROS in the hippocampal region was significantly reduced by aerobic exercise (p < 0.05), with decreased Keap1 protein expression of and increased Nrf2, HO-1, GPX4 protein expression (p < 0.05). Aerobic exercise enhances memory and learning abilities, improves cognitive function, and reduces the oxidative stress levels in the hippocampus of AD mice, which involves in the activation of Keap1/Nrf2/GPX4 pathway.

Electroacupuncture improves cognitive function by modulating hippocampal lactate-mediated HIF-1α signaling pathway and inhibiting inflammation response in vascular dementia rats

To observe the effects of electroacupuncture (EA) stimulation of "Sishencong"(EX-HN1) and "Fengchi"(GB20) on lactate (Lac) content, expression of proline hydroxylase 2 (PHD2), hypoxia-inducible factor-1α (HIF-1α)/nuclear transcription factor- κB (NF- κB)/NOD-like receptor thermoprotein structural domain-associated protein 3 (NLRP3) signaling pathway, and inflammatory factors in hippocampal tissue of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. Male SD rats screened by Morris water maze tests were randomly divided into blank control, sham-operation, VD model and EA groups (12 rats in each group). The VD model was replicated using the 4-vessel occlusion (VO) method. EA (2 Hz, 1 mA) was applied to EX-HN1 and bilateral GB20 for 30 min, once daily for consecutive 21 days. Morris water maze test was employed to test the rat's memory learning ability before and after modeling and after the intervention. The hippocampal tissue was sampled for observing histopathologic changes with H.E. staining；and detecting Lac content with colorimetric method, and the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-18 (also in serum) by using ELISA, respectively. The immunoactivity levels of PHD2, HIF-1α, and p-NF-κB p65 in hippocampal tissue were detected by immunohistochemistry, and the expression levels of PHD2, HIF-1α, NF-κB p65, p-NF-κB p65 and NLRP3 proteins in hippocampal tissue were detected by Western blot. Compared with the blank control and sham-operation groups, the escaping latency, Lac content in hippocampus, the TNF-α, IL-1β, and IL-18 contents in both hippocampus and serum, the immunoactivity of HIF-1α and p-NF-κB p65 and expression levels of HIF-1α, NF-κB p65, p-NF-κB p65, and NLRP3 proteins were significantly increased (P<0.01), while the number of original platform crossing, and PHD2 immunoactivity and protein expression level were significantly decreased (P<0.05, P<0.01) in the model group. Following EA intervention, modeling induced increase and decrease of the indexes mentioned above were all reversed in the EA group (P<0.05, P<0.01). H.E. staining showed disordered arrangement of neurons, uneven cytoplasm stain, blurred nucleolus or disappearance of nucleolus, dilated capillaries, many apoptotic bodies and increased inflammatory cells in the hippocampus tissue of the model group, which was improved to a certain extent in the EA group, including relatively regular arrangement of neurons, reduced apoptotic bodies and inflammatory cells, etc. in the hippocampus. EA stimulation of EX-HN1 and GB20 can improve the cognitive function in VD rats, which may be related to its functions in reducing Lac content, regulating the expression of HIF-1α pathway related proteins, and inhibiting inflammatory responses in the hippocampus tissue.

Regulation of the NF-κB/NLRP3 signalling pathway by Shenghui Yizhi decoction reduces neuroinflammation in mice with Alzheimer’s disease

Background Shenghui Yizhi Decoction (SHYZD) has exhibited the capacity to enhance cognitive function and learning abilities in individuals diagnosed with Alzheimer’s disease (AD) while ameliorating pre-existing neuroinflammation. Nevertheless, the precise mechanism underlying its therapeutic effects on AD remains to be elucidated. Methods Twenty-four male SAMP8 mice were randomly divided into three groups, and eight male SAMR1 mice were used as a blank control, to examine their learning and spatial memory abilities. The expression of amyloid β1-42 (Aβ1-42) was detected by immunohistochemical staining of hippocampal tissue. ELISA was used to detect the interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) expressions. Real time PCR was used to detect NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteine protease-1 (Caspase-1), and IL-1β mRNA expression. Western blot was used to detect nuclear factor kappa-B (NF-κB), inhibitor of NF-κB α (IκBα), IκB kinase α (IKKα), NLRP3, Caspase-1, and IL-1β protein expression. Results In this study, SAMP8 mice, employed as an AD model, displayed markedly diminished abilities in terms of spatial localization, navigation, and spatial exploration when compared to the blank control group. Additionally, there was a substantial upregulation of Aβ1-42 expression in the hippocampus of these mice, along with a significant increase in the levels of inflammation-associated factors, including IL-1β, IL-6, TNF-α, NLRP3, Caspase-1, as well as the NF-κB pathway-related proteins, namely, NF-κB, IκBα, and IKKα. Moreover, after treatment with positive drugs (donepezil hydrochloride) and SHYZD, the learning abilities of the mice exhibited significant improvements. Furthermore, the hallmark AD protein Aβ1-42, inflammatory factors, and NF-κB/NLRP3 signalling pathway proteins were significantly reduced. These findings collectively suggest that SHYZD exerts a therapeutic effect on AD. Conclusion In summary, the specific molecular mechanisms through which SHYZD alleviates AD and the potential role for SHYZD in the NF-κB/NLRP3 signalling pathway are identified in this study.

Untargeted metabolomics and mendelian randomization analysis identify alpha-linolenic acid and linoleic acid as novel biomarkers of perioperative neurocognitive dysfunction

Perioperative neurocognitive dysfunction (PND) occurs in elderly individuals undergoing anesthesia and surgery. To explore the potential molecular mechanisms, we performed right-sided cervical exploratory surgery under sevoflurane anesthesia in 18-month-old male Sprague-Dawley rats. Anxiety-depression-like behaviors and learning memory abilities were assessed using the Open Field Test (OFT) and Novel Object Recognition (NOR). Additionally, the hippocampus was collected one day after surgery for inflammatory factor detection, TUNEL staining, and metabolomics analysis. Mendelian randomization (MR) analyses were subsequently conducted to validate the causal relationships by using a series of GWAS datasets related to representative differential metabolites as exposures and cognitive impairment as endpoints. The results indicated that rats exposed to anesthesia and surgery exhibited poorer cognitive performance, significant elevations in hippocampal inflammatory factors such as IL-1β and TNF-α, and extensive neuronal apoptosis. LC-MS/MS-based untargeted metabolomics identified 19 up-regulated and 32 down-regulated metabolites in the test group, with 6 differential metabolites involved in metabolic pathways enriched according to the KEGG database. ROC analysis revealed a correlation between α-linolenic acid (ALA) and linoleic acid (LA) and the development of PND. Further MR analysis confirmed that ALA was significantly associated with cognitive performance and the risk of depression, while LA was significantly associated with the risk of memory loss. Taken together, our results identified ALA and LA as potentially powerful biomarkers for PND.

Effect of artificial sugar supplement on the lifespan and learning memory ability of honey bee (Apis cerana) worker bee offspring.

Honeybees maintain their growth and reproduction mainly by collecting nutrients from nectar-source plants. Apis cerana, a unique species of honeybee in China, is capable of sporadically collecting nectar. In traditional beekeeping, sugar syrup or a honey-water solution must be artificially fed to bees to supplement their diet during rainy weather or nectar-deficient periods. In this study, 2 groups of honeybee colonies were each fed sugar syrup or a honey-water solution, and a third group consisting of colonies that were allowed to naturally forage without any dietary supplement was used as the control. The effects of the 2 sugar sources on A. cerana worker bee offspring were compared. The results showed that the sugar source affected the lifespan and learning memory of the worker bee offspring. The lifespan, learning memory ability, and expression of related genes in the sugar syrup group were significantly lower than those in the honey-water solution and natural nectar foraging groups (P < 0.05). A honey-water solution supplement was more beneficial to the healthy development of worker bee offspring than a sugar syrup supplement when the colonies lacked dietary resources. These findings provide a theoretical basis that can guide beekeepers in choosing the appropriate dietary supplements for honeybees.

Topical application of daphnetin hydrogel for traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal cell damage and dysfunction. According to previous studies, daphnetin (Dap) has a protective effect in neurological injury. However, the in vivo bioavailability of daphnetin is not high. The purpose of this study was to determine whether administering daphnetin directly into the site of injury via a hydrogel drug carrier could improve its therapeutic impact. Tripolycerol monostearates / daphnetin (TM/Dap) hydrogels were prepared and characterised using water bath heating, scanning electron microscopy (SEM) and small animal in vivo imaging techniques. The TBI model was established using the Feeney free fall impact method. Using the Morris water maze test, the mNSS neurological deficit rating scale, haematoxylin-eosin staining, and liver and kidney function tests, the therapeutic benefit of TM/Dap and its toxic side effects were assessed. The therapeutic effects of TM/Dap were further investigated using wet and dry gravimetric methods, Evans blue staining, protein immunoblotting, immunofluorescence staining techniques and ELISA. The efficacy of the TM/Dap hydrogel in gradually releasing daphnetin in the context of traumatic brain damage was shown by both in vitro and in vivo tests. Behavioral experiments showed that the learning and spatial memory abilities of TM/Dap hydrogel treated mice were significantly improved in the water maze experiment. And TM/Dap hydrogel has high biosafety for organisms. The results of the therapeutic mechanism of action showed that TM/Dap hydrogel showed more significant efficacy in reducing the neuroinflammatory response caused by TNF-α, IL-6 and other factors, as well as promoting the recovery of post-traumatic neurological function. The use of hydrogel as a drug carrier for daphnetin showed more significant efficacy in reducing neuroinflammatory response, protecting nerve tissue and promoting post-traumatic neurological recovery compared with traditional drug delivery methods.

Huanglian Wendan Decoction Improves Insomnia in Rats by Regulating BDNF/TrkB Signaling Pathway Through Gut Microbiota-Mediated SCFAs and Affecting Microglia Polarization.

Insomnia is a typical type of sleep disorder. Huanglian Wendan Decoction (HWD) is a traditional Chinese medicine (TCM) with the effects of regulating Qi, drying dampness and resolving phlegm, calming the mind, and relieving irritation. This study aims to investigate the effect of HWD on insomnia in rats and its mechanism. Para-chlorophenylalanine (PCPA)-induced insomnia in rats was used for in vivo experiments and then treated with HWD. Behavioral tests, Western blot, real-time PCR, immunofluorescent staining, 16S rRNA sequencing were conducted. The content of SCFAs was determined by GC-MS. Acetic acid-pretreated rat hippocampal nerve cells were used for in vitro experiments. The results showed that HWD significantly improved the learning memory ability, decreased sleep latency, and prolonged sleep duration in insomniac rats. HWD reduced TNF-α and IL-6 levels and increased IL-10 and Foxp3 levels. HWD also promoted the polarization of macrophages from M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype. In addition, HWD increased the expression levels of BDNF and TrkB in the hippocampus. Administration of the TrkB receptor agonist 7,8-dihydroxyflavone (7,8-DHF) confirmed the mechanism by which HWD activates BDNF/TrkB signaling to ameliorate insomnia. Furthermore, HWD restored gut microbiota richness and diversity and promoted short-chain fatty acid (SCFA) production in insomniac rats. In vitro experiments confirmed that the acetic acid-treated SCFA group could activate the BDNF/TrkB signaling pathway in neuronal cells, further promoting neuronal cell growth. In conclusion, HWD alleviated insomnia by maintaining gut microbiota homeostasis, promoting SCFA production, reducing neuroinflammatory response and microglia activation, and activating BDNF/TrkB signaling pathway.

Caloric restriction leading to attenuation of experimental Alzheimer's disease results from alterations in gut microbiome.

Caloric restriction (CR) might be effective for alleviating/preventing Alzheimer's disease (AD), but the biological mechanisms remain unclear. In the current study, we explored whether CR caused an alteration of gut microbiome and resulted in the attenuation of cognitive impairment of AD animal model. Thirty-week-old male APP/PS1 transgenic mice were used as AD models (AD mouse). CR was achieved by 30% reduction of daily free feeding (ad libitum, AL) amount. The mice were fed with CR protocol or AL protocol for six consecutive weeks. We found that with CR treatment, AD mice showed improved ability of learning and spatial memory, and lower levels of Aβ40, Aβ42, IL-1β, TNF-α, and ROS in the brain. By sequencing 16S rDNA, we found that CR treatment resulted in significant diversity in composition and abundance of gut flora. At the phylum level, Deferribacteres (0.04%), Patescibacteria (0.14%), Tenericutes (0.03%), and Verrucomicrobia (0.5%) were significantly decreased in CR-treated AD mice; at the genus level, Dubosiella (10.04%), Faecalibaculum (0.04%), and Coriobacteriaceae UCG-002 (0.01%) were significantly increased in CR-treated AD mice by comparing with AL diet. Our results demonstrate that the attenuation of AD following CR treatment in APP/PS1 mice may result from alterations in the gut microbiome. Thus, gut flora could be a new target for AD prevention and therapy.

NEAT1 Promotes Valproic Acid-Induced Autism Spectrum Disorder by Recruiting YY1 to Regulate UBE3A Transcription.

Evidence suggests that long non-coding RNAs (lncRNAs) play a significant role in autism. Herein, we explored the functional role and possible molecular mechanisms of NEAT1 in valproic acid (VPA)-induced autism spectrum disorder (ASD). A VPA-induced ASD rat model was constructed, and a series of behavioral tests were performed to examine motor coordination and learning-memory abilities. qRT-PCR and western blot assays were used to evaluate target gene expression levels. Loss-and-gain-of-function assays were conducted to explore the functional role of NEAT1 in ASD development. Furthermore, a combination of mechanistic experiments and bioinformatic tools was used to assess the relationship and regulatory role of the NEAT1-YY1-UBE3A axis in ASD cellular processes. Results showed that VPA exposure induced autism-like developmental delays and behavioral abnormalities in the VPA-induced ASD rat model. We found that NEAT1 was elevated in rat hippocampal tissues after VPA exposure. NEAT1 promoted VPA-induced autism-like behaviors and mitigated apoptosis, oxidative stress, and inflammation in VPA-induced ASD rats. Notably, NEAT1 knockdown improved autism-related behaviors and ameliorated hippocampal neuronal damage. Mechanistically, it was observed that NEAT1 recruited the transcription factor YY1 to regulate UBE3A expression. Additionally, in vitro experiments further confirmed that NEAT1 knockdown mitigated hippocampal neuronal damage, oxidative stress, and inflammation through the YY1/UBE3A axis. In conclusion, our study demonstrates that NEAT1 is highly expressed in ASD, and its inhibition prominently suppresses hippocampal neuronal injury and oxidative stress through the YY1/UBE3A axis, thereby alleviating ASD development. This provides a new direction for ASD-targeted therapy.

Effect of electroacupuncture on expression of protein phosphorylation in hippocampus tissues of rats with chronic fatigue syndrome.

To observe the effect of electroacupuncture (EA) on behavior and hippocampal protein phosphorylation in rats with chronic fatigue syndrome (CFS), so as to explore its mechanisms underlying improvement of CFS. Male SD rats were randomly divided into control, model and EA groups (n=12 rats in each group). The CFS model was established by chronic multifactor combined with stress stimulation (treadmill training + restraint stress + sleep disturbance + crowded environment). For rats of the EA group, EA (1 mA, frequency of 10 Hz) was applied to "Shenting" (GV24) (with an acupuncture needle penetrated from GV24 to "Baihui" ［GV20］) and "Dazhui" (GV14) for 15 min, once daily for 28 days. After treatment, the body weight, food intake and water intake of rats in each group were observed. The fatigue degree of rats was evaluated by Semi-quantitative score observation table of the general condition of experimental rats.The open field test (OFT) was used to assess the rats'anxiety severity by detecting the total number of grid-crossing and the times of the central area entered in 5 min, and Morris water maze test was employed to assess the rats' learning-memory ability by detecting the escape latency in 1 min, and the times of the original platform quadrant crossing in 1 min. The hippocampaus was taken for phosphorylated Label-free quantitative proteomics analysis by using Maxquant technology based on full scan mode to calculate the integral of each peptide signal of liquid chromatography-mass spectrometry(LC-MS). The differentially-expressed proteins (>1.5 folds for up-regulation or <0.67 folds for down-regulation) were evaluated by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Compared with the control group, the body weight, food intake, and the times of original-platform quadrant crossing of spatial exploring of Morris water maze test were significantly decreased (P<0.01, P<0.05) , and the score of general conditions, times of grid-crossing and center area-entering of OFT, and the escape latency of navigation task were apparently increased (P<0.01) in rats of the model group. After EA intervention, the decreased original-platform quadrant crossing, and the increased score of general conditions, times of grid-crossing and the escape latency of navigation task were all reversed (P<0.01, P<0.05). Outcomes of proteomics analysis indicated that compared with the model group, there were 297 differentially expressed peptide (48 up-regulated and 249 down-regulated) segments in the control group, and there were 245 differentially expressed peptide (185 up-regulated and 60 down-regulated) segments in the EA group, in which, 25 overlapping peptide segments were reversed after EA treatment, corresponding to 24 proteins, mainly involving cytoskeletal structure. GO function annotation analysis showed that the top three differentially expressed phosphorylated proteins involved in the effect of EA intervention were the actin filament polymerization, protein depolymerization and cytoskeletal tissue in the biological process, the actin binding, structural molecular activity and cytoskeletal protein binding in the molecular function, and the cytoskeleton, dendrites and dendritic trees in the cellular component, respectively. The KEGG pathway annotation analysis for differentially expressed phosphorylated proteins showed that theinsulin secretion, axon guidance, phosphatidylinositol signaling system and lysine biosynthesis, etc. were involved in the effect of EA intervention. EA of GV24-GV20 and GV14 can improve the general state, anxiety and learning-memory ability of CFS model rats, which may be related to its functions in regulating the hippocampal protein phosphorylation level, and repairing the structure and function of synapses in hippocampus.

Dexmedetomidine improves the circulatory dysfunction of the glymphatic system induced by sevoflurane through the PI3K/AKT/ΔFosB/AQP4 pathway in young mice

Multiple sevoflurane exposures may damage the developing brain. The neuroprotective function of dexmedetomidine has been widely confirmed in animal experiments and human studies. However, the effect of dexmedetomidine on the glymphatic system has not been clearly studied. We hypothesized that dexmedetomidine could alleviate sevoflurane-induced circulatory dysfunction of the glymphatic system in young mice. Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 2 h daily, continuously for 3 days. Intraperitoneal injection of either normal saline or dexmedetomidine was administered before every anaesthesia. Meanwhile the circulatory function of glymphatic system was detected by tracer injection at P8 and P32. On P30-P32, behavior tests including open field test, novel object recognition test, and Y-maze test were conducted. Primary astrocyte cultures were established and treated with the PI3K activator 740Y-P, dexmedetomidine, and small interfering RNA (siRNA) to silence ΔFosB. We propose for the first time that multiple exposure to sevoflurane induces circulatory dysfunction of the glymphatic system in young mice. Dexmedetomidine improves the circulatory capacity of the glymphatic system in young mice following repeated exposure to sevoflurane through the PI3K/AKT/ΔFosB/AQP4 signaling pathway, and enhances their long-term learning and working memory abilities.

KeYu ShuShen Granule alleviates symptoms of depression by targeting the mTOR pathway in a rat model of depression induced by chronic unpredictable mild stress

BackgroundKeYu ShuoShen Granules (KSG) is a traditional Chinese medicine formula with radix bupleuri as the kingpin, which is widely used in the treatment of depression, but its exact mechanism of action is not yet fully understood. The aim of this study is to investigate the therapeutic mechanisms of KSG in a model of depression in rats. MethodsRats were equally divided into 5 groups including control, model, fluoxetine, KSG at high and low doses, chronic unpredictable mild stress combined with solitary rearing for 28 days was used to prepare a rat model of depression followed by 14 days of continuous gavage and modelling, and the antidepressant effects of KSG were analyzed using behavioral tests, proteomic techniques and pharmacological methods. ResultsAfter KGS treatment, the body weight, Sucrose Preference of the model rats increased significantly, and their motor and learning memory abilities were significantly improved in the open field test and Morris water maze (P<0.01 or P<0.05); At the same time, proteomics revealed a total of 7 differential proteins, 3 differential pathways, including Agps, Eif4e2, Pter, Sys1, Rgcc, Polr1d and Npy1r, mTOR signalling pathway, insulin signalling pathway, and human papillomavirus infection; the expression of p-mTOR, mTOR, and Eif4e2 was significantly up-regulated after drug administration (P<0.01 or P<0.05), and neuronal damage in the cerebral cortex was significantly restored. ConclusionKSG improved depression symptoms in model rats by modulating protein synthesis processes involved in the mTOR pathway and Eif4e2, producing a combined effect on the nervous system. This finding provides new theoretical support and a mechanistic explanation for the use of KSG in the treatment of depression.

Bu zhong Yiqi Decoction ameliorates mild cognitive impairment by improving mitochondrial oxidative stress damage via the SIRT3/MnSOD/OGG1 pathway

Ethnopharmacological relevanceBu-Zhong-Yi-Qi Decoction(BZYQD) is a traditional formula commonly used in China, known for its effects in tonifying Qi and raising Yang. It can relieve symptoms of cognitive impairment such as forgetfulness and lack of concentration caused by qi deficiency, which is common in aging and debilitating. However, much of the current research on BZYQD has been focused on its impact on the digestive system, leaving its molecular mechanisms in improving cognitive function largely unexplored. Aim of the studyCognitive decline in the aging central nervous system is intrinsically linked to oxidative damage. This study aims to investigate the therapeutic mechanism of BZYQD in treating mild cognitive impairment caused by qi deficiency, particularly through repair of mitochondrial oxidative damage. Materials and methodsA rat model of mild cognitive impairment (MCI) was established by administering reserpine subcutaneously for two weeks, followed by a two-week treatment with BZYQD/GBE. In vitro experiments were conducted to assess the effects of BZYQD on neuronal cells using a H2O2-induced oxidative damage model in PC12 cells. The open field test and the Morris water maze test evaluated the cognitive and learning memory abilities of the rats. HE staining and TEM were employed to observe morphological changes in the hippocampus and its mitochondria. Mitochondrial activity, ATP levels, and cellular viability were measured using assay kits. Protein expression in the SIRT3/MnSOD/OGG1 pathway was analyzed in tissues and cells through western blotting. Levels of 8-OH-dG in mitochondria extracted from tissues and cells were quantified using ELISA. Mitochondrial morphology in PC12 cells was visualized using Mito Red, and mitochondrial membrane potential was assessed using the JC-1 kit. ResultsBZYQD treatment significantly improved cognitive decline caused by reserpine in rats, as well as enhanced mitochondrial morphology and function in the hippocampus. Our findings indicate that BZYQD mitigates mtDNA oxidative damage in rats by modulating the SIRT3/MnSOD/OGG1 pathway. In PC12 cells, BZYQD reduced oxidative damage to mitochondria and mtDNA in H2O2-induced conditions and was associated with changes in the SIRT3/MnSOD/OGG1 pathway. ConclusionBZYQD effectively counteracts reserpine-induced mild cognitive impairment and ameliorates mitochondrial oxidative stress damage through the SIRT3/MnSOD/OGG1 pathway.

The phenylpropionamide extract of hemp (Cannabis sativa L.) seed improves learning memory ability and survival rate by attenuating microglia activation and reducing amyloid deposition via the SIRT1-AMAD10 pathway in APP/PS1 mice

BackgroundFructus cannabis (Hemp seed), the dried and mature fruit of Cannabis sativa L., was reported to have effects against Alzheimer's disease (AD) in different experimental models. However, either chemical composition or mechanisms of action was not clear. Our previous work isolated and identified diverse phenylpropionamides such as cannabisin A-F, caffeoyltyramine and feryroyltyramine from hemp seed, which showed antioxidant and anti-neuroinflammatory activity in cell models. PurposeTo study the anti-AD effect and mechanism of the total phenylpropionamides (TPA) extract from hemp seed using APPswe/PS1dE9 mice model. MethodsTPA extract was prepared via extraction and column chromatography techniques, then analyzed and measured using HPLC, HPLC-MS and Folin-Ciocalteu method. The extract was administered by gavage to APPswe/PS1dE9 mice. The learning memory ability of mice were examined using new object recognition test and Morris water maze. Hematoxylin and Eosin staining was used to observe the morphology of neuronal cells in the hippocampal region of brain tissue; ELISA was used to measure the content of IL-1β, TNF-α, IL-6, superoxide dismutase (SOD), malondialdehyde (MDA) in the brain tissue; the deposition of Aβ protein was measured by immunohistochemistry and Western blotting; the activation of microglia was measured by immunofluorescence. The expression of APP, PS1, SIRT1, and ADAM10 proteins was measured by Western blotting. ResultsFolin-Ciocalteu method showed the total phenylpropionamides content was 238.03 μg caffeic acid equivalent /mg TPA. HPLC and HPLC-MS analysis showed the contents of N-trans-caffeoyltyramine, cannabisin A and B were 20.16 μg/mg, 32.16 μg/mg and 214.35 μg/mg, respectively, in TPA extract. TPA treatment significantly increased the survival rate of APP/PS1 mice, improved the learning and memory ability, reversed the neural cell damage in the hippocampal region, and reduced Aβ deposition and activated microglia in brain tissue. Meanwhile, the administration decreased the neuroinflammatory factors TNF-α, IL-6, IL-1β expression, increased antioxidant enzyme SOD activity, and attenuated MDA expression. Western blotting showed that TPA administration increased SIRT1 and ADAM10 protein expression and decreased APP and PS1 protein expression. ConclusionTPA improved learning memory capacity, increased survival rate, attenuated neuroinflammation, oxidative stress, microglia activation and hippocampal apoptosis, and reduced amyloid deposition via the SIRT1-ADAM10 pathway in APP/PS1 mice.

Melatonin is a Neuroprotective and Antioxidant Agent against Neurotoxicity Induced by an Intrahippocampal Injection of Nickel in Rats.

The investigation into the hippocampal function and its response to heavy metal exposure is crucial for understanding the mechanisms underlying neurotoxicity, this can potentially inform strategies for mitigating the adverse effects associated with heavy metal exposure. Melatonin is an essential neuromodulator known for its efficacy as an antioxidant. In this study, we aimed to determine whether melatonin could protect against Nickel (Ni) neurotoxicity. To achieve this, we performed an intracerebral injection of Ni (300 µM NiCl2) into the right hippocampus of male Wistar rats, followed by melatonin treatment. Based on neurobehavioral and neurobiochemical assessments, our results demonstrate that melatonin efficiently enhances Ni-induced behavioral dysfunction and cognitive impairment. Specifically, melatonin treatment positively influences anxious behavior, significantly reduces immobility time in the forced swim test (FST), and improves learning and spatial memory abilities. Moreover, neurobiochemical assays revealed that melatonin treatment modulates the Ni-induced alterations in oxidative stress balance by increasing antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT). Additionally, we observed that melatonin significantly attenuated the increased levels of lipid peroxidation (LPO) and nitric oxide (NO). In conclusion, the data from this study suggests that melatonin attenuates oxidative stress, which is the primary mechanism responsible for Ni-induced neurotoxicity. Considering that the hippocampus is the main structure involved in the pathology associated with heavy metal intoxication, such as Ni, these findings underscore the potential therapeutic efficacy of melatonin in mitigating heavy metal-induced brain damage.

Integrated serum, urine metabolomics and 16S rRNA sequencing to investigate the modulatory effect of Ziziphi Spinosae Folium flavonoid on D-galactose-induced aging of rats

Ziziphi Spinosae Folium, the leaf of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou (LZJS), was widely distributed in northern China. Our previous study showed that LZJS flavonoid (LZJSF) exerted antioxidant activity at chemical and PC12 cell levels, respectively. Here, we further studied the function of LZJSF on aging-related oxidative stress in D-galactose (D-gal)-induced rats and the potential mechanisms were explored. The aging model was established by intraperitoneal injection of D-gal (300 mg·kg−1·day−1) for 36 days, the low-dose of LZJSF (LLZJSF 100 mg·kg−1·day−1) and the high-dose of LZJSF (HLZJSF, 200 mg·kg−1·day−1) were administered daily by oral gavage at the same time, respectively. Their spatial and learning memory abilities were evaluated by the Morris water maze test. HLZJSF was observed to significantly ameliorate cognitive ability and oxidative indexes, such as reduction of MDA and augmentation of SOD, GSH-Px, T-AOC and CAT, and the effect of HLZJSF on these indexes was stronger than that of LLZJSF. A precise and well-defined research strategy was successfully applied to screen serum differential metabolites. Furthermore, integrated serum and urine metabolomics showed that the derivations of nine significant differential metabolites could be regulated by administration of HLZJSF, which suggested that the intervention effect of HLZJSF against aging may involve in the amino acid metabolism including D-Glutamine and D-glutamate, alanine, aspartate and glutamate, arginine and proline metabolism and arginine biosynthesis. 16S rRNA sequencing analysis showed that HLZJSF could descend the relative abundance of Firmicutes and Actinobacteria, as well as the ratio of Firmicutes/Bacteroidetes, and increase the relative abundance of Bacteroidetes and Proteobacteria. Finally, correlation analysis further showed that the beneficial effects of HLZJSF resulted from the regulation of the disturbance of gut microbiota through the “microbiota-gut-brain” axis to affect amino acid metabolism and regulate oxidative stress. This study will provide a foundation for the development and utilization of LZJSF as functional foods and healthy beneficial food products.

Effect of "Tongdu Qishen" acupuncture on expressions of ferroptosis-related factors in the hippocampus of APPswe/PS1dE9 Mice.

To investigate the effect of "Tongdu Qishen" acupuncture［dredging the Govern Vessel and normalizing mental activities, electroacupuncture (EA) of "Baihui" (GV20), "Yintang" (EX-HN3) and "Shuigou" (GV26) needling］on the learning-memory ability and the mechanism of ferroptosis in Alzheimer's disease (AD) mice. Twenty-four male APPswe/PS1dE9 mice were randomly and equally divided into model group and EA group, and 12 normal C57BL/6 mice were used as the control group. In the EA group, EA (2 Hz/100 Hz, 20 min) was applied to GV20 and EX-HN3 in combination with manual acupuncture stimulation of GV26. The treatment was performed once a day, for a total of 28 days. The mice in the three groups were given the same fixation and grasping operation. Morris water maze swimming tests were used to assess the mice's learning-memory ability. Nissl staining and transmission electron microscopy were used to observe the morphological changes of neurons in the hippocampus. The activity of superoxide dismutase (SOD) in the hippocampus tissue was detected by superoxide anionic colorimetric assay kit (WST-1), and malondialdehyde (MDA) contents were detected by thiobarbituric acid (TBA) method. The expression levels of prostaglandin endoperoxide synthase 2 (ptgs2) and glutathione peroxidase 4 (GPX4) mRNA in the hippocampus were detected by fluorescent quantitative real-time PCR. Behavioral results showed that compared with the control group, the escape latencies at the 2nd, 3rd, 4th and 5th day of Morris water maze swimming test were significantly increased (P<0.05), and the swimming time in the original platform quadrant and the times of cros-sing the original platform were considerably decreased (P<0.05) in the model group. In comparison with the model group, the escape latencies at the 4th and 5th day were strikingly decreased (P<0.05), and the swimming time in the original platform quadrant and times of crossing the original platform significantly increased (P<0.05) in the EA group. Following modeling, the SOD activity and the expression of GPX4 mRNA were obviously down-regulated (P<0.05), and the content of MDA and the expression of ptgs2 mRNA significantly up-regulated (P<0.05) in the model group rele-vant to the control group. The SOD activity and the expression of GPX4 mRNA were apparently increased (P<0.05), and the content of MDA and the expression of ptgs2 mRNA remarkably down-regulated (P<0.05) in the EA group rele-vant to the model group. Histopathological and ultrastructural results showed scattered arrangement of cells, widened space among cells, reduction in the number of cells, and many shrunk of dissolved nucleoli, shrunking and incomplete mitochondria, and high membrane electron density in the hippocampus of the model group, which was relatively milder in the EA group. "Tongdu Qishen" acupuncture can improve the learning-memory ability of AD mice, which may be related to its functions in up-regulating SOD activity and GPX4 mRNA expression, and down-regulating MDA content and ptgs2 mRNA expression to reduce the lipid peroxidation in the process of ferroptosis.

Moxibustion influences hippocampal microglia polarization via IL-33/ST2 pathway in Alzheimer's disease mice.

To observe the effect of moxibustion on the polarization of microglia towards M2 direction in Alzheimer's disease (AD) mice through the interleukin-33 (IL-33)/growth stimulating gene 2 protein (ST2) signaling pathway. Five-month-old APP/PS1 male mice were randomly divided into model and moxibustion (Moxi) groups, and C57BL/6J mice of the same age were as the control group, with 9 mice in each group. In the Moxi group, moxibustion was applied at "Baihui" (GV20) and "Yongquan" (KI1) for 30 min, once a day, 5 days a week for 4 weeks. The spatial learning memory ability was observed by the Morris water maze test. The relative expressions of IL-33 and ST2 in hippocampus were detected by Western blot. The positive expression of amyloid-β (Aβ), phosphorylated Tau (p-Tau), IL-33/ionized calcium binding adapter molecule 1(Iba-1), ST2/Iba-1, arginase 1 (Arg1)/Iba-1 and indu-cible nitric oxide synthase (iNOS)/Iba-1 in hippocampal CA1 region were detected by immunofluorescence. Compared with the control group, the escape latency of the mice in the model group was prolonged (P<0.001, P<0.01), the number of times to enter the effective area and the percentage of target quadrant swimming time were reduced (P<0.001), the positive expression of both Aβ and p-Tau, the positive expression of iNOS/Iba-1 in the hippocampal CA1 region was increased (P<0.001), while the expression of IL-33 and ST2 protein in hippocampal tissue, the positive expression levels of IL-33/Iba-1, ST2/Iba-1 and Arg1/Iba-1 in hippocampal CA1 region were all decreased (P<0.05, P<0.001). After treatment, compared with the model group, the escape latency of the mice in the moxibustion group was shortened (P<0.001, P<0.01), the number of entries into the effective area and the percentage of target quadrant swimming time were increased (P<0.001), the positive expression of Aβ and p-Tau in the hippocampal CA1 region, and the positive expression of iNOS/Iba-1 were decreased (P<0.001), while the expression of IL-33 and ST2 protein in the hippocampal tissue, the positive expression of IL-33/Iba-1, ST2/Iba-1 and Arg1/Iba-1 in hippocampal CA1 region were all increased (P<0.05, P<0.01, P<0.001). Moxibustion can improve the spatial learning and memory abilities, reduce the pathological deposition of Aβ and p-Tau in APP/PS1 mice, which may be related to its function in up-regulating the IL-33/ST2 signaling pathway to regulate the polarization of microglia towards M2 direction.