Morris Water Maze Experiment Research Articles

Chlorogenic Acid Mitigates Ferroptosis by Activating the Nrf2/GPX4 Pathway through Keap1 Blockade in Vascular Dementia Rats

Chlorogenic acid (CGA) is a dietary phenolic acid widely distributed in daily food and plants, but its role in vascular dementia (VaD) is still unclear. Hence, this study aimed to investigate whether CGA could rescue cognitive impairment in VaD, providing a new option for drug discovery. Novel object recognition and Morris water maze experiments revealed that CGA enhanced the learning and memory abilities in VaD rats. Nissl staining, Western blot, and transmission electron microscopy results demonstrated that CGA inhibited neuronal loss in the hippocampal CA1 region of VaD rats, increased the expression of synaptic‐related markers SYP and PSD95, thickened the postsynaptic density, and suppressed mitochondrial ridge rupture in neurons. GSH detection, MDA detection, and Western blot experiments indicated that CGA alleviated hippocampal GSH reduction and MDA elevation and increased the protein levels of GPX4 and SLC7A11. Cell activity, ROS detection, lipid peroxidation detection, intracellular Fe2+ level detection, GSH detection, and Western blot revealed that CGA inhibited the increase of intracellular Fe2+, ROS, and lipid peroxidation induced by OGD in PC12 cells, mitigated GSH reduction, and increased the protein levels of GPX4, SLC7A11, SYP, and PSD95. Western blot and immunofluorescence staining showed that CGA increased the expression of pSer40‐Nrf2 in the hippocampal tissue of VaD rats and PC12 cells, promoting Nrf2 nuclear translocation. Knockdown of Nrf2 prevented CGA from rescuing ferroptosis and synaptic damage induced by OGD in PC12 cells, as well as Nrf2 pathway activation. Molecular docking analysis suggested that CGA competitively bound to the Kelch domain of Keap1 with Nrf2 and then promoted Nrf2 release and activated downstream signaling pathways. In conclusion, our study suggests that CGA may activate the Nrf2 signaling pathway by disrupting the interaction between Nrf2 and Keap1 proteins, thereby inhibiting hippocampal neuronal ferroptosis and synaptic damage and ameliorating cognitive impairment in VaD. Practical Applications. Vascular dementia (VaD) stands as the second most prevalent type of dementia following Alzheimer’s disease. However, there is no effective clinical treatment drug available, and its pathogenic mechanisms remain elusive. This study proposed that CGA might activate the Nrf2‐GPX4 signaling pathway by competitively binding to Keap1, thereby attenuating hippocampal neuronal ferroptosis and synaptic damage and consequently ameliorating cognitive impairment in VaD. Our study may provide a novel option for drug research and development for VaD.

Keap1/Nrf2 signaling pathway participating in the progression of epilepsy via regulation of oxidative stress and ferroptosis in neurons

ObjectiveThis study aims to analyze the relationship between the Kelch-like ECH-associated protein 1 (Keap1)/Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Epilepsy (EP), as well as its mechanism of action. MethodsThirty Wistar rats were divided into a control group (without treatment), a model group (EP modeling), and an inhibition group (EP modeling + intervention by Keap1/Nrf2 signaling pathway inhibitor ATRA) and subject to Morris water maze experiment. Then, the expression of Oxidative Stress (OS) markers, ferroptosis-associated proteins and Keap1/Nrf2 pathway in rat hippocampus was measured. In addition, rat hippocampal neuronal cell HT22 was purchased and treated accordingly based on the results of grouping, and cell proliferation and apoptosis in the three groups were determined. ResultsCompared with rats in the model group, those in the inhibition group showed shorter escape latency and an increased number of platform crossings (p < 0.05). Significant OS and neuron ferroptosis, increased apoptosis rate, elevated Keap1 expression, and decreased Nrf2 expression were observed in the model group compared to the control group (p < 0.05). The inhibition group exhibited notably improved OS and ferroptosis, as well as enhanced neuronal viability (p < 0.05). ConclusionInhibition of the Keap1/Nrf2 pathway can reverse the OS and neuron viability in EP rats.

Systematic review and meta-analysis of the effects of exercise on cognitive impairment and neuroprotective mechanisms in diabetes mellitus animal models.

This study aims to assess the effects of exercise on cognitive impairment behavioral performance and neuroprotective mechanisms in diabetes mellitus (DM) animal models. PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang Database, VIP Database (VIP), and China Biomedical Literature Database (CBM) were systematically searched for studies investigating the impact of exercise on cognitive impairment in animal models of diabetes mellitus (DM) from the inception of these databases through July 2023. Rigorous quality assessments were conducted on the included literature. Primary outcome measures comprised fasting blood glucose (FBG) levels and performance in the Morris water maze test, while secondary outcomes focused on mechanisms related to neuroprotection. Statistical analysis of outcome data was conducted using RevMan 5.3 and R software. A total of 17 studies were included, encompassing 399 animals. The results of the meta-analysis of primary outcome measures revealed that, compared to the control group, exercise effectively reduced fasting blood glucose (FBG) levels in diabetic animal models. In the Morris water maze experiment, exercise also significantly decreased the escape latency of diabetic animal models, increased the number of platform crossings, improved the percentage of time spent in the target quadrant, extended the time spent in the target quadrant, and enhanced swimming speed. Meta-analysis of secondary outcome measures indicated that exercise effectively reduced Aβ deposition, attenuated oxidative stress, enhanced synaptic function, suppressed cellular apoptosis and neuroinflammation, and promoted neurogenesis. Exercise represents a promising non-pharmacological therapy with a positive impact on diabetes-related cognitive function and neuroprotection. Moreover, this study provides a theoretical foundation for further preclinical and clinical trials.

Obesity exacerbates postoperative cognitive dysfunction by activating the PARP1/NAD+/SIRT1 axis through oxidative stress

The purposes of this study were to explore the impact of obesity on postoperative cognitive dysfunction (POCD) and to investigate the underlying mechanism by which obesity exacerbates POCD. In this study, fifteen-month-old male C57BL/6 J mice were fed a High-fat diet for three months to establish obesity models. Internal fixation of tibial fractures under isoflurane inhalation was performed to construct a POCD animal model. Three days after surgery, mice were subjected to the Morris water maze (MWM) experiment to evaluate their learning and memory abilities. The findings from the MWM experiment revealed that in comparison to the Ad Libitum Surgical group (ALS), mice in the High-fat Surgical group (HFS) exhibited prolonged escape latencies and reduced platform crossings. These outcomes suggest the potential exacerbating role of obesity in cognitive impairment within the POCD mouse models. Immunofluorescence (IF) findings demonstrate that obesity intensifies anesthesia and surgery-induced oxidative stress levels within the hippocampus. Compared to the Ad Libitum Control group (ALC), an elevation in PARP1 expression and a reduction in the NAD+/NADH ratio and SIRT1 expression were observed in the hippocampus of mice from the ALS. Moreover, when contrasting the HFS group with the ALS group, increased PARP1 expression along with decreased NAD+/NADH ratio and SIRT1 expression were evident. In vitro studies found that compared with the Control group (CON), oil red staining and BODIPY probe staining showed significant lipid droplet aggregation in the palmitic acid (PA) group. IF results demonstrated that HT22 cells in the PA group experienced oxidative stress and activation of the PARP1/NAD+/SIRT1 axis in contrast to the CON group. Moreover, manipulation of PARP1 expression in HT22 cells through PARP1 lentivirus-based silencing or overexpression revealed a converse relationship between PARP1 expression levels and the NAD+/NADH ratio as well as SIRT1 expression levels. This study concludes that obesity may exacerbate POCD by triggering activation of the oxidative stress-induced PARP1/NAD+/SIRT1 axis.

The study of therapeutic efficacy and mechanisms of Schisandra chinensis and Evodia rutaecarpa combined treatment in a rat model of Alzheimer's disease

Schisandra chinensis and Evodia rutaecarpa are traditional Chinese herbs used to treat neurodegenerative diseases. This study investigates the combined effects of SC and ER on learning and memory in an Alzheimer's disease rat model and their underlying mechanisms. MethodsHigh-performance liquid chromatography was employed to analyze the primary active constituents of Schisandra and Evodia. The effects of the combined treatment of Schisandra and Evodia on learning and memory in an Alzheimer's disease rat model were evaluated through Morris water maze and Hematoxylin-Eosin staining experiments. Immunohistochemical analysis was conducted to investigate the impact of S-E on Aβ1-42 and P-tau proteins. Western blotting and real-time quantitative polymerase chain reaction were utilized to quantify the expression of pivotal proteins and genes within the BDNF/TRKB/CREB and GSK-3β/Tau pathways. ResultsThe treatment group exhibited significant neuroprotective effects, ameliorating learning and memory impairments in the Alzheimer's disease rat model. The treatment regimen modulated the activity of the BDNF/TRKB/CREB and GSK-3β/Tau pathways by influencing the expression of relevant genes, thereby reducing the generation of Aβ1-42 and P-Tau proteins and inhibiting the deposition of senile plaques. Furthermore, among the three treatment groups, the combined treatment demonstrated notably superior therapeutic effects on Alzheimer's disease compared to the single-drug treatment groups.

Palmatine attenuates LPS-induced neuroinflammation through the PI3K/Akt/NF-κB pathway.

To investigate the key molecular mechanisms of palmatine for the treatment of neuroinflammation through modulation of a pathway using molecular docking, molecular dynamics (MD) simulation combined with network pharmacology, and animal experiments. Five alkaloid components were obtained from the traditional Chinese medicine Huangteng through literature mining. Molecular docking and MD simulation with acetylcholinesterase were used to screen palmatine. At the animal level, mice were injected with LPS intracerebrally to cause a neuroinflammatory model, and the Morris water maze experiment was performed to examine the learning memory of mice. Anxiety levels were tested using the autonomous activity behavior experiment with the open field and elevated behavior experiments. HE staining and Niss staining were performed on brain tissue sections to observe morphological lesions and apoptosis; serum was examined for inflammatory factors TNF-α, IL-6, and IL-1β; Western blot was performed to detect the protein expression. The expression of PI3K/AKT/NFkB signaling pathway-related proteins was examined by Western blot. The results of network pharmacology showed that the screening of palmatine activation containing the PI3K/Akt/NFkB signaling pathway exerts antineuroinflammatory effects. Results from behavioral experiments showed that Pal enhanced learning memory in model mice, improved anxiety behavior, and significantly improved brain damage caused by neuroinflammation. The results of HE staining and Niss staining of brain tissue sections showed that palmatine could alleviate morphological lesions and nucleus damage in brain tissue. Palmatine improved the levels of serum inflammatory factors TNF-α, IL-6, and IL-1β. SOD, MDA, CAT, ACH, and ACHE in the hippocampus were improved. Western blot results showed that palmatine administration ameliorated LPS-induced neuroinflammation through the PI3K/Akt/NFkB pathway.

The effects of protein-rich extract from Rhizoma Gastrodiae against cerebral ischemia/reperfusion injury via regulating MAPK and PI3K/AKT signaling pathway

BackgroundRhizoma Gastrodiae is a highly valuable traditional Chinese medicine and functional health food that has been used in China to treat neurological disorders for thousands of years. Rhizoma Gastrodiae contains various of biological activities, such as antioxidative, neuroprotective, learning improvement, anxiolytic, and antidepressant effects. However, no studies have been conducted to explore the effects of the protein components in Rhizoma Gastrodiae (GEPS) and its potential protective effects against ischemic stroke.Our main goal was to investigate the effects of GEPS on ischemia/reperfusion (I/R) injury and its possible mechanisms. MethodsA middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia mouse model and an oxygen-glucose deprivation (OGD/R) injury model in HT22 cells were established. A neurobehavioral test was performed 24 h after MCAO, and brain infarction was measured. A Morris water maze experiment was conducted on Day 14 after reperfusion in mice. Hematoxylin and eosin (HE) and TUNEL staining were performed to assess apoptotic neuronal death. Immunohistochemical analysis was used to detect BDNF and GAP43 expression. The content of SOD, MDA, GSH-PX and ROS were detected. The protein expression was analyzed using Western blotting. Cell viability was determined by MTT assay. Cell apoptosis was examined by flow cytometry. ResultsGEPS reduced apoptosis, decreased cerebral infarction, improved neurological defects, and ameliorated oxidative stress in the ischemic penumbra. In addition, GEPS increased the expression of BDNF and GA43 in the penumbra. Mechanistically, GEPS counteracted MCAO-induced PI3K/AKT inhibition and activation of MAPK signaling pathways. ConclusionGEPS has a clear neuroprotective effect on I/R injury, and its mechanism may be linked to the PI3K/AKT and MAPK signaling pathways.

Iron-overload-induced ferroptosis in mouse cerebral toxoplasmosis promotes brain injury and could be inhibited by Deferiprone.

Iron is a trace metal element that is essential for the survival of cells and parasites. The role of iron in cerebral toxoplasmosis (CT) is still unclear. Deferiprone (DFP) is the orally active iron chelator that binds iron in a molar ratio of 3:1 (ligand:iron) and promotes urinary iron excretion to remove excess iron from the body. The aims of this experiment were to observe the alterations in iron in brains with Toxoplasma gondii (T. gondii) acute infections and to investigate the mechanism of ferroptosis in CT using DFP. We established a cerebral toxoplasmosis model in vivo using TgCtwh3, the dominant strains of which are prevalent in China, and treated the mice with DFP at a dose of 75 mg/kg/d. Meanwhile, we treated the HT-22 cells with 100 μM DFP for half an hour and then infected cells with TgCtwh3 in vitro. A qRT-PCR assay of TgSAG1 levels showed a response to the T. gondii burden. We used inductively coupled plasma mass spectrometry, an iron ion assay kit, Western blot analysis, glutathione and glutathione disulfide assay kits, a malonaldehyde assay kit, and immunofluorescence to detect the ferroptosis-related indexes in the mouse hippocampus and HT-22 cells. The inflammatory factors interferon-γ, tumor necrosis factor-α, transforming growth factor-β, and arginase 1 in the hippocampus and cells were detected using the Western blot assay. Hematoxylin and eosin staining, electron microscopy, and the Morris water maze experiment were used to evaluate the brain injuries of the mice. The results showed that TgCtwh3 infection is followed by the activation of ferroptosis-related signaling pathways and hippocampal pathological damage in mice. The use of DFP led to ferroptosis resistance and attenuated pathological changes, inflammatory reactions and T. gondii burden of the mice, prolonging their survival time. The HT-22 cells with TgCtwh3 activated the ferroptosis pathway and was inhibit by DFP in vitro. In TgCtwh3-infected cells, inflammatory response and mitochondrial damage were severe, but these effects could be reduced by DFP. Our study elucidates the mechanism by which T. gondii interferes with the host's iron metabolism and activates ferroptosis, complementing the pathogenic mechanism of CT and further demonstrating the potential value of DFP for the treatment of CT.

A novel Vestitain A from the ripe fruits of Embelia vestita Roxb.

A new compound, vestitain A (1), together with 11 known compounds were isolated from the ripe fruits of Embelia vestita Roxb., among them compounds 5,10-12 were isolated from this plant for the first time. Their structures were elucidated and characterized by detailed spectroscopic analysis. Further, the isolated new compound 1 was evaluated for its hypoglycemic effects in vivo. Our research showed that compound 1 could decrease the fasting blood glucose (FBG) by approximately 36.31% in diabetic rats at the high dose (800 mg/kg). By the Morris Water Maze experiments, we found that compound 1 had the effect of intervention on social behavior in diabetic rats, which might provide a reference basis for its development and utilization as a potential hypoglycemic drug.

Exploration of the mechanisms of improving learning and memory in the offspring of aging pregnant mice by supplementation with Paris polyphylla polysaccharide based on the P19-P53-P21 and Wnt/β-catenin signaling pathways

Ethnopharmacological relevanceFirst recorded in “Sheng Nong's herbal classic”, Paris polyphylla is used to treat diseases, such as convulsions, head shaking and tongue fiddling, and epilepsy. Studies have shown that the ability of three Liliaceae polysaccharides in improving learning and memory may be related to the P19-P53-P21 and Wnt/β-catenin signaling pathways. Moreover, a link between these two signaling pathways and the possible neuroprotective impact of Paris polyphylla polysaccharide has been proposed. Aim of the studyWe explored the mechanisms of improving learning and memory in the offspring of pre-pregnant parental mice and D-galactose–induced aging pregnant mice by supplementation with P. polyphylla polysaccharide based on the P19-P53-P21 and Wnt/β-catenin signaling pathways. Study design and methodsAfter 3 weeks of supplementation of D-galactose–induced pre-pregnant parental mice with P. polyphylla polysaccharide component 1 (PPPm-1), the male and female parental mice mated in cages. The D-galactose–induced pregnant mice were continued to be supplemented with PPPm-1 for 18 days before delivery of the offspring. Behavioral experiments (Morris water maze and dark avoidance experiments) were conducted on the offspring mice born 48 days later to determine whether PPPm-1 had the effect of improving their learning and memory. Based on the P19/P53/P21 and Wnt/β-catenin signaling pathways, the mechanisms of PPPm-1 in improving learning and memory in offspring mice were further investigated. ResultsOffspring mice administered low- or high-dose PPPm-1 exhibited stronger motor and memory abilities in behavioral experiments than the aging model of offspring mice. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction revealed that the expressions of P19 and P21 mRNA and protein were inhibited in offspring mice administered low- and high-dose PPPm-1. However, P53 expression was inhibited in the low-dose PPPm-1 offspring group but promoted in the high-dose PPPm-1 offspring group. Additionally, PPPm-1 could effectively activate the Wnt/β-catenin signaling pathway, promote the expressions of Wnt/1, β-catenin, CyclinD1, and TCF-4 mRNA and protein, and inhibit GSK-3β mRNA and protein expression to improve the learning and memory abilities of offspring mice. ConclusionThus, PPPm-1 improved the learning and memory abilities in the offspring of aging pregnant mice by acting on the P19-P53-P21 and Wnt/β-catenin signaling pathways.

Ameliorative effect of scopolamine-induced cognitive dysfunction by Fufangmuniziqi formula: The roles of alkaloids, saponins, and flavonoids

Ethnopharmacological relevanceFufangmuniziqi formula (FFMN), a traditional Uyghur medicine used in China, is derived from an ancient Uyghur medical book and consists of 13 herbs. The herbs of FFMN, such as Peganum harmala L., Glycyrrhiza uralensis Fisch., and Nigella glandulifera, have been demonstrated to have acetylcholinesterase (AChE) inhibitory, anti-neuroinflammatory, or antioxidant effects. Therefore, FFMN may have a good anti-Alzheimer's disease (AD) effect, but its specific action and mechanism need to be further proven. Aim of the studyThis study aims to investigate the anti-AD effects of FFMN and the role played by alkaloids, flavonoids, and saponins in anti-AD. Materials and methodsThe alkaloids, flavonoids, and saponins fractions of FFMN were prepared by macroporous resin chromatography. The absorbed ingredients in the drug-containing serum were identified by UPLC⁃Q⁃TOF⁃MS. An AD mouse model was established by intraperitoneal injection of scopolamine (SCO). The role of different fractions of FFMN in the anti-AD process was examined by Morris water maze (MWM), in-vitro cell, and AChE inhibition assay. ResultsA total of 20 ingredients were identified in the serum samples collected after oral administration of FFMN, and seven compounds were selected as candidate active compounds. MWM experiments showed that different fractions of FFMN could significantly improve SCO-induced learning memory impairment in mice. The alkaloids fraction (ALK) regulated cholinergic function by inhibiting AChE activity, activating choline acetyltransferase activity, and protein expression. Flavonoids and saponins were more potent than the ALK in downregulating pro-inflammatory factors or inflammatory mediators, such as TNF-α, MPO, and nitric oxide. Western blot results further confirmed that flavonoids and saponins attenuated neuroinflammation by inhibiting the phosphorylation of IκB and NF-κB p65. This result was also verified by in-vitro cellular assays. FFMN enhanced antioxidant defense by increasing the activity of superoxide dismutase and reducing the production of MDA. Combined with cellular experiments, flavonoids and saponins were proven more protective against oxidative damage. ConclusionFFMN improved cognitive and memory impairment in the SCO-induced AD mouse model. ALK mainly enhanced the function of the cholinergic system. Flavonoid and saponin fractions mainly attenuated neuroinflammation and oxidative stress by modulating the NF-κB pathway. All these findings strongly suggested that the combination of alkaloid, flavonoid, and saponin fractions derived from FFMN is a promising anti-AD agent that deserves further development.

Sanwei DouKou Decoction ameliorate Alzheimer disease by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway.

Sanwei DouKou decoction (SDKD) is a traditional Chinese medicine (TCM) prescription derived from the Tibetan medical book "Si Bu Yi Dian" and is clinically used for the treatment of Alzheimer's disease (AD). However, the potential mechanism of SDKD treatment for AD remains elusive. This study aims to explore the potential mechanism by which SDKD alleviates AD. Extracts of SDKD were identified with Gas chromatograph-mass spectrometer (GC-MS). 5×FAD mice were treated with SDKD for 8 weeks. The efficacy of SDKD against AD was evaluated by in-vivo experiments. Morris water maze and contextual fear conditioning tests were used to detect the learning and memory ability of mice. Hematoxylin-eosin staining (H&E) staining was used to observe the pathological changes of brain tissue. Immunohistochemistry was used to detect the positive expression of Nestin in hippocampus. In in-vitro experiments, the Cell Counting Kit 8 (CCK-8) technique was used to detect cell viability, the proliferation of neural stem cells was detected by immunofluorescence staining, the intracellular protein expression was detected by Western Blot. The results of this study suggested that SDKD may ameliorate AD. SDKD significantly shortened the escape latency of mice in the Morris water maze experiment, increased the number of times the mice crossed the target quadrant, and prolonged freezing time in the contextual fear memory experiment. SDKD also improved neuronal pathology in the hippocampus, decreased neuronal loss, and increased Nestin protein levels. Furthermore, in in-vitro experiments, SDKD could significantly increase Neural stem cells (NSCs) viability, promoted NSCs proliferation, and also effectively activated the Wnt/β-catenin signalling pathway, increased Wnt family member 3A (Wnt3a), β-catenin and CyclinD1 protein levels, activated the NSCs proliferation pathways in AD model mouse brain tissue. The present study demonstrated that sanwei doukou decoction can ameliorate AD by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway. Our observations justify the traditional use of SDKD for a treatment of AD in nervous system.

Effects of Dl-3-n-butylphthalide on cognitive functions and blood–brain barrier in chronic cerebral hypoperfusion rats

Vascular cognitive impairment (VCI) has been one of the major types of cognitive impairment. Blood–brain barrier damage plays an essential part in the pathogenesis of VCI. At present, the treatment of VCI is mainly focused on prevention, with no drug clinically approved for the treatment of VCI. This study aimed to investigate the effects of DL-3-n-butylphthalide (NBP) on VCI rats. A modified bilateral common carotid artery occlusion (mBCCAO) model was applied to mimic VCI. The feasibility of the mBCCAO model was verified by laser Doppler, 13N-Ammonia-Positron Emission Computed Tomography (PET), and Morris Water Maze. Subsequently, the Morris water maze experiment, Evans blue staining, and western blot of tight junction protein were performed to evaluate the effect of different doses of NBP (40 mg/kg, 80 mg/kg) on the improvement of cognitive impairment and BBB disruption induced by mBCCAO. Immunofluorescence was employed to examine the changes in pericyte coverage in the mBCCAO model and the effect of NBP on pericyte coverage was preliminarily explored. mBCCAO surgery led to obvious cognitive impairment and the decrease of whole cerebral blood flow, among which the blood flow in the cortex, hippocampus and thalamus brain regions decreased more significantly. High-dose NBP (80 mg/kg) improved long-term cognitive function in mBCCAO rats, alleviated Evans blue leakage and reduced the loss of tight junction proteins (ZO-1, Claudin-5) in the early course of the disease, thereby exerting a protective effect on the blood–brain barrier. No significant changes in pericyte coverage were observed after mBCCAO. High-dose NBP improved cognitive function in mBCCAO rats. High-dose NBP protected the integrity of BBB by upregulating TJ protein expression, rather than regulating pericyte coverage ratio. NBP could be a potential drug for the treatment of VCI.

Wuzang Wenyang Huayu Tang promoting learning-memory ability in vascular dementia rats via brain-gut-microbiome axis

IntroductionLots of research has revealed that vascular dementia (VD), which is closely related to the metabolic disorders of intestinal microbiota, can be treated through traditional Chinese drug by regulating the brain-gut-microbiome axis. However, the specific interaction between VD and intestinal microflora, as well as the mechanism of Wuzang Wenyang Huayu Tang (WWHT) in treating VD by regulating the brain-gut-microbiome axi remains unknown on the whole and needs to be further studied. ObjectivesTo explore the effect of WWHT on the learning and memory ability of rats with VD based on brain-gut-microbiome axis. MethodsIn this study, taking the specific pathogen-free (SPF) SD rats as the subjects, a vascular dementia rat model was created by using the two-vessel occlusion model (2-VO). The subjects were divided into six groups, namely control group, sham group, VD model group, the piracetam group, the WWHT group, and the WWHT combined with piracetam (WCP) group. After 22 days of the gavage administration method, the Morris water maze experiment was adopted to determine the learning and memory abilities of each group through the water maze, observe the effects of hematoxylin and eosin (HE) staining and transmission electron microscopy in the hippocampal area of each group, and analyze the relevant proteins in the whole brain by Western Blot (WB). In addition, the feces of each group were collected and analyzed by fecal metabolomics combined with 16S rRNA gene sequencing to explore the interaction between VD and intestinal microbiota. ResultsThrough Morris water maze experiment, it was found that the learning and memory ability of VD rats in WWHT, piracetam and WCP groups was improved. The morphology of hippocampal neuron cells were improved in the WWHT group and the number of hippocampal neuron cells were increased in the VD rats by HE. By observing the ultrastructure of hippocampal cells, it can be found that the VD model group showed cell membrane edema and large organelle lysis. The ultrastructure of the hippocampus of all the administered groups was improved. In terms of the effect on the expression levels of related proteins in the whole brain of rats, compared with the VD model group, the expression levels of Caspase-3 protein decreased in all groups after drug administration. The protein expression levels of APG5L/ATG5, Beclin-1 and LC3A/B were upregulated, indicating that the drug administration group could regulate cellular autophagy in the whole brain to further protect brain tissue. Fecal metabolite profiles revealed 27 different metabolites related to VD. These metabolites are mainly involved in related metabolic pathways, such as vitamin B metabolism, purine metabolism, pyrimidine metabolism, and histidine metabolism. Further analysis of the gut microbiota by 16S rRNA gene sequencing data showed that WWHT-treated VD rats were different in gut microbiota composition. DiscussionThrough the effect of brain-gut-microbiome axis, WWHT regulates the metabolism of vitamin B6 and tryptophan in VD rats, which can improve the intestinal microbiota of VD rats and regulate autophagy in the brain to improve cell morphology in the hippocampus, thus increasing the number of hippocampal cells to protect hippocampal neuron cells2510100, and finally improving the learning and memory ability of VD rats.

Repeated neonatal Needle-pricking stimulation alter neurodevelopment in adolescent rats

ObjectiveTo explore the repeated pain stimulation in neonatal rats affects their cognitive and memory abilities during puberty, and the proliferation expression of hippocampal neurons. MethodsPostnatal 1 day (P1) SD rats were randomly divided into two groups, and the skin of the needle group was pricked for seven days consecutively while the skin of the control group was stroked for the same period of time. The rats in both groups were weighed every week, and the Morris water maze experiment was performed from P44 to P49 to test the cognitive and memory abilities of the rats. On P50, the hippocampal tissue was extracted for observation of pathological features and the expressions of Ki-67 and caspase 3 were determined. ResultsWith the increase of the days, the body weight of the rats in the needle group increased slightly slower than that of the control group. The escape latency of the needle group was significantly higher than that of the control group in the water maze test at P45 and P48, and the number of times the rats crossing the platform in the needle group was lower than that of the control group. The HE staining of the hippocampal tissue showed that the cells in the needle group were disorganized, with irregular morphology. Under the electron microscope, the structure of neuron cells and organelles is changed in the hippocampal CA1 region of rats. It showed a decrease in the Ki-67 expression and an increase in caspase 3 in the needle group. ConclusionRepeated experience of needle-pricking stimulation in neonatal rats can cause cognitive impairment and memory loss in puberty, disrupt hippocampal organization, and diminish neuronal proliferation.

Multi-pathway neuroprotective effects of a novel salidroside derivative SHPL-49 against acute cerebral ischemic injury

SHPL-49 ((2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-(4-(4-methoxyphenyl) butoxy) tetrahydro-2H-pyran-3,4,5-triol) is a novel glycoside derivative obtained from structural modification of salidroside, which is isolated from the medicinal plant Rhodiola rosea L. SHPL-49 was administered to rats with permanent middle cerebral artery occlusion (pMCAO) for 5 days, and it was found that SHPL-49 could alleviate the cerebral infarct volume and reduce the neurological deficit score. Moreover, the effective time window of SHPL-49 in the pMCAO model was from 0.5 to 8 h after embolization. In addition, the result of immunohistochemistry showed that SHPL-49 could increase the number of neurons in the brain tissue and reduce the occurrence of apoptosis. Morris water maze and Rota-rod experiments showed that SHPL-49 could improve neurological deficits, repair neurocognitive and motor dysfunction, and enhance learning and memory ability in the pMCAO model after 14 days of SHPL-49 treatment. Further in vitro experiments showed that SHPL-49 significantly reduced the calcium overload of PC-12 cells and the production of reactive oxygen species (ROS) induced by oxygen and glucose deprivation (OGD), and increased the levels of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), decreased the production of malondialdehyde (MDA). Furthermore, SHPL-49 could reduce cell apoptosis by increasing protein expression ratio of anti-apoptotic factor Bcl-2 to pro-apoptotic factor Bax in vitro. SHPL-49 also regulated the expression of Bcl-2 and Bax in ischemic brain tissue, and even inhibited the caspase cascade of pro-apoptotic proteins Cleaved-caspase 9 and Cleaved-caspase 3. Taken together, SHPL-49 exhibited neuroprotective effects against cerebral ischemic injury through multiple pathways, such as alleviating calcium overload, reducing oxidative stress damage, and inhibiting apoptosis.

Phosphate and tension homology-induced kinase 1/Parkin signaling mediates cognitive dysfunction in sepsis-associated encephalopathy through activation of hippocampal mitochondrial autophagy

To investigate the effects of gene of phosphate and tension homology (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway on hippocampal mitophagy and cognitive function in mice with sepsis-associated encephalopathy (SAE) and its possible mechanism. A total of 80 male C57BL/6J mice were randomly divided into Sham group, cecal ligation puncture (CLP) group, PINK1 plasmid transfection pretreatment groups (p-PINK1+Sham group, p-PINK1+CLP group), empty vector plasmid transfection control group (p-vector+CLP group), with 16 mice in each group. The mice in CLP groups were treated with CLP to reproduce SAE models. The mice in the Sham groups were performed laparotomy only. Animals in the p-PINK1+Sham and p-PINK1+CLP groups were transfected with PINK1 plasmid through the lateral ventricle at 24 hours before surgery, while mice in the p-vector+CLP group were transfected with the empty plasmid. Morris water maze experiment was performed 7 days after CLP. The hippocampal tissues were collected, the pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and the mitochondrial autophagy was observed under a transmission electron microscopy after uranyl acetate and lead citrate staining. The expressions of PINK1, Parkin, Beclin1, interleukins (IL-6, IL-1β) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. Compared with the Sham group, CLP group mice in Morris water maze experiment had longer escape latency, shorter target quadrant residence time, and fewer times of crossing the platform at 1-4 days. Under the light microscope, the hippocampal structure of the mouse was injured, the neuronal cells were arranged in disorder, and the nuclei were pyknotic. Under the electron microscope, the mitochondria appeared swollen, round, and wrapped by bilayer or multilayer membrane structures. Compared with the Sham group, CLP group had higher expressions of PINK1, Parkin, Beclin1, LC3II/LC3I ratio, IL-6 and IL-1β in hippocampus, indicating that sepsis induced by CLP could activated inflammatory response and caused PINK1/Parkin-mediated mitophagy. Compared with the CLP group, p-PINK1+CLP group had shorter escape latencies, spent more time in the target quadrant and had more number of crossings in the target quadrant at 1-4 days. Under the light microscope, the hippocampal structures of mice was destroyed, the neurons were arranged disorderly, and the nuclei were pyknotic. Under transmission electron microscope, swollen and rounded mitochondria and mitochondrial structure wrapped by double membrane or multilayer membrane structure were observed. Compared with the CLP group, the levels of PINK1, Parkin, Beclin1 and LC3II/LC3 ratio in the p-PINK1+CLP group were significantly increased [PINK1 protein (PINK1/β-actin): 1.95±0.17 vs. 1.74±0.15, Parkin protein (Parkin/β-actin): 2.06±0.11 vs. 1.78±0.12, Beclin1 protein (Beclin1/β-actin): 2.11±0.12 vs. 1.67±0.10, LC3II/LC3I ratio: 3.63±0.12 vs. 2.27±0.10, all P < 0.05], while the levels of IL-6 and IL-1β were significantly decreased [IL-6 protein (IL-6/β-actin): 1.69±0.09 vs. 2.00±0.11, IL-1β protein (IL-1β/β-actin): 1.11±0.12 vs. 1.65±0.12, both P < 0.05], suggesting that overexpression of PINK1 protein could further activate mitophagy and reduce the inflammatory response caused by sepsis. There was no statistically significant difference in the above pathological changes and related indicators between Sham group and p-PINK1+Sham group, CLP group and p-vector+CLP group. PINK1 overexpression can further activate CLP-induced mitophagy by upregulating Parkin, thereby inhibiting inflammation response and alleviate cognitive function impairment in SAE mice.

Scalp acupuncture Yikang therapy on Baihui (GV20), Sishencong (EX-HN1), Zhisanzhen, Niesanzhen improves neurobehavior in young rats with cerebral palsy through Notch signaling pathway.

To investigate the efficacy of scalp acupuncture Yikang therapy on Baihui (GV20), Sishencong (EX-HN1), Zhisanzhen, Niesanzhen, on neurobehavior in young rats with cerebral palsy based on Notch signaling pathway. Thirty 7-day-old rats were randomly divided into sham, model and acupuncture, 10 rats in each group. The cerebral palsy model was established by the accepted modeling method, the acupuncture group selected "Baihui (GV20)", "Sishencong (EX-HN1)", "Zhisanzhen" and "Niesanzhen" for intervention 24 h after the model was made. The body masses were recorded before and after the treatment, respectively. After the intervention, the rats were subjected to suspension experiment, slope experiment, tactile stimulation experiment and Morris water maze experiment. After the end of the experiment, the morphological changes of hippocampal histology were observed by hematoxylin-eosin (HE) staining under light microscope, and the expression of Notch1, Notch3 and Hes5 were detected by Western blot and quantitative real-time polymerase chain reaction (PCR). The changes in body mass of the rats in each group were different; in behavioral experiments, compared with the sham, the suspension time of the model was shortened, the slope experiment, tactile stimulation experiment, and escape latency time were prolonged, and the number of platform crossing was reduced in the model, compared with the model, the suspension time of the acupuncture was prolonged, the slope experiment, tactile stimulation experiment, and escape latency time were shortened, and the number of platform crossing times was increased; HE staining showed severe hippocampal damage in the model and reduced hippocampal damage in the acupuncture. Western Blot and real-time fluorescence quantitative PCR showed that the expression of Notch1, Notch3 and Hes5 were increased in the model and the expression of Notch1, Notch3, Hes5 in acupuncture were decreased. Scalp acupuncture Yikang therapy may improve neurobehavior and reduce brain injury in rats with cerebral palsy by downregulating the expression of Notch1, Notch3, and Hes5.

Maresin1 Ameliorates Sepsis-Induced Microglial Neuritis Induced through Blocking TLR4-NF-κ B-NLRP3 Signaling Pathway.

Objective: Neuroinflammation is a major etiology of cognitive dysfunction due to sepsis. Maresin1 (MaR1), identified as a docosahexaenoic acid (DHA)-derived metabolite from macrophages, has been demonstrated to exhibit potent neuroprotective and anti-inflammatory effects. Nevertheless, detailed functions and molecular mechanism of MaR1 in sepsis-induced cognitive dysfunction has not been fully elucidated. Here, we aimed to investigate potential neuroprotective effects of MaR1 on microglia-induced neuroinflammation in sepsis-induced cognitive impairment and to explore its anti-inflammatory mechanism. Methods: Different doses of MaR1 were administered to septic rats by via tail vein injection. The optimal dose was determined based on the 7-day survival rate of rats from each group. derived from macrophages with both anti-inflammatory to observe the ameliorative effects of MaR1 at optimal doses on cognitive dysfunction in septic rats. The effects of MaR1 on neuroinflammation-mediated microglial activation, neuronal apoptosis, and pro-inflammatory cytokine productions were in vivo and in vitro assayed, using Western blot, ELISA, TUNEL staining, Nissl staining, and the immunofluorescence method. To further elucidate anti-inflammatory machinery of MaR1, protein expressions of NLRP3 inflammatory vesicles and TLR4-NF-κB pathway-related proteins were subjected to Western blot assay. Results: After tail vein injection of MaR1 with different doses (2 ng/g, 4 ng/g, 8 ng/g), the results showed that 4 ng/g MaR1 treatment significantly increased the rats' 7-day survival rate compared to the CLP controls. Therefore, subsequent experiments set 4 ng/g MaR1 as the optimal dose. Morris water maze experiments confirmed that MaR1 significantly reduced space memory dysfunction in rats. In addition, in CLP rats and LPS-stimulated BV2 microglia, MaR1 significantly reduced activated microglia and pro-inflammatory cytokines levels and neuronal apoptosis. Mechanically, MaR1 inhibits microglia-induced neuroinflammation through suppressing activations of NLRP3 inflammatory vesicles and TLR4-NF-κB signal pathway. Conclusion: Collectively, our findings suggested that MaR1 might be a prospective neuroprotective compound for prevention and treatment in the sepsis process.

Effect of Sinapic Acid on Scopolamine-Induced Learning and Memory Impairment in SD Rats

The seriousness of the diseases caused by aging have recently gained attention. Alzheimer's disease (AD), a chronic neurodegenerative disease, accounts for 60-80% of senile dementia cases. Continuous research is being conducted on the cause of Alzheimer's disease, and it is believed to include complex factors, such as genetic factors, the accumulation of amyloid beta plaques, a tangle of tau protein, oxidative stress, cholinergic dysfunction, neuroinflammation, and cell death. Sinapic acid is a hydroxycinnamic acid found in plant families, such as oranges, grapefruit, cranberry, mustard seeds, and rapeseeds. It exhibits various biological activities, including anti-inflammatory, anti-oxidant, anti-cancer, and anti-depressant effects. Sinapic acid is an acetylcholine esterase inhibitor that can be applied to the treatment of dementia caused by Alzheimer's disease and Parkinson's disease. However, electrophysiological studies on the effects of sinapic acid on memory and learning must still be conducted. Therefore, it was confirmed that sinapic acid was effective in long-term potentiation (LTP) using organotypic hippocampal segment tissue. In addition, the effect on scopolamine-induced learning and memory impairment was measured by oral administration of sinapic acid 10 mg/kg/day for 14 days, and behavioral experiments related to short-term and long-term spatial memory and avoidance memory were conducted. Sinapic acid increased the activity of the field excitatory postsynaptic potential (fEPSP) in a dose-dependent manner after TBS, and restored fEPSP activity in the CA1 region suppressed by scopolamine. The scopolamine-induced learning and memory impairment group showed lower results than the control group in the Y-maze, Passive avoidance (PA), and Morris water maze (MWM) experiments. Sinapic acid improved avoidance memory, short and long-term spatial recognition learning, and memory. In addition, sinapic acid weakened the inhibition of the brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and the activation of prostaglandin-endoperoxide synthase 2 (COX-2) and interleukin 1 beta (IL-1β) induced by scopolamine in the hippocampus. These results show that sinapic acid is effective in restoring LTP and cognitive impairment induced by the cholinergic receptor blockade. Moreover, it showed the effect of alleviating the reduction in scopolamine-induced BDNF and TrkB, and alleviated neuroinflammatory effects by inhibiting the increase in COX-2 and IL-1β. Therefore, we showed that sinapic acid has potential as a treatment for neurodegenerative cognitive impairment.