Semaglutide Ameliorates Neuroinflammation Caused by Enterogenous Pyrogen in APP/PS1 Mice

Semaglutide Ameliorates Neuroinflammation Caused by Enterogenous Pyrogen in APP/PS1 Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Semaglutide Ameliorates Neuroinflammation Caused by Enterogenous Pyrogen in APP/PS1 Mice Yuan Yuan, Jiawei Zhang, Ziyao Zhang, Yanyu Zhai, Xiaojuan Cheng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6098406/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and purpose: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction, which is common in the elderly. In recent years, it has been reported that glucagon-like peptide 1 (GLP-1) analogues have neuroprotective function. However, the mechanism of GLP-1 analogues improving neurological function has not been fully clarified. This study attempts to clarify the mechanism of GLP-1 alleviating AD phenotype. Methods: In this study, a modified once-weekly GLP-1 analogue, Semaglutide, was used to treat 8-month-old amyloid precursor protein / presenilin 1 (APP/PS1) transgenic mice. By means of ethology, molecular biology and 16s rRNA amplicon sequencing, it was confirmed that Semaglutide alleviated the disease phenotype of APP/PS1 mice. Results: GLP-1 improved the behavioral performance of APP/PS1 mice, reduced neuronal damage and aggregation of amyloid-β (Aβ) plaques, and enhanced synaptic plasticity. GLP-1 also attenuated pyroptosis mediated by NOD-like receptor thermal protein domain associated protein 3 (NLRP3), inflammatory reaction mediated by toll-like receptor 4 (TLR4) and mitochondrial damage of microglia as well as improved the structure and function of blood-brain barrier (BBB) in AD mice. Conclusion: GLP-1 may repair the blood-brain barrier to alleviate the central nervous system injury caused by the displacement of pyrogen in gut of AD mice. Alzheimer's disease Semaglutide APP/PS1 inflammation gut microbiota blood brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Alzheimer's disease (AD), progressing from mild memory impairment in the early stages to advanced dementia, is a highly inherited neurodegenerative disease, which is affecting tens of millions of people worldwide 1 . Nevertheless, many studies so far have found that exogenous regulation can delay the development of AD 2 – 5 . Our AD research team turned to the latest star drug, the glucagon-like peptide 1 (GLP-1) analogue, Semaglutide. GLP-1 is an incretin hormone that controls insulin and glucose homeostasis, which is successfully employed for type 2 diabetes mellitus (T2DM) treatment 6 . As a gastrointestinal hormone, GLP-1 has been widely discussed in the field of brain-gut axis. Researchers generally believe that GLP-1 can be transported from the peripheral to the brain through blood and cross the blood-brain barrier, thereby exerting its effects in the central nervous system (CNS) 7 , 8 . A recent study demonstrated the anti-inflammatory effects of GLP-1 in animal model systematically, which coincides with the current research 8 . In this study, we conducted long-term intervention with GLP-1 on an amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mouse model, mainly observing changes in AD pathology, inflammation related signaling pathways and blood brain barrier (BBB) in the central nervous system (CNS). In addition, we also investigated the composition of intestinal microbiota in APP/PS1 mice via 16S rRNA. Methods Animals and drug treatment Male specific-pathogen-free (SPF) APP/PS1 mice were purchased from the Shanghai Model Organisms Center (Shanghai, China), and housed in SPF-barrier environment under standard temperature (20–25 ℃) and light (12 h light/12 h dark) conditions, with water and food available ad libitum. The genotype was confirmed by PCR using tail tissue DNA. Age-matched wild-type (WT) littermates were used as controls. All procedures were approved by the ethical committee on animal welfare of Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in accordance with the principles outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The mice were randomly divided into 3 groups (n = 15 in each group): (1) wild-type (WT), (2) APP/PS1 (AD), (3) APP/PS1 + Semaglutide (AD + GLP-1). Mice in the AD + GLP-1 group were subcutaneously administered with Semaglutide (6.7 µg/kg body weight) 2 times each week for 8 weeks, and monitor the weight of mice every week. A workflow of the experiment procedure was shown in Fig. 1 A. Enzyme-linked immunosorbent assay (ELISA) Prior to the collection of blood, mice were given a 4-h fast and had free access to water. The serum was extracted by centrifuging the blood mouse, and kept at – 80 ℃. The levels of serum insulin were determined through enzyme linked immunosorbent assay (ELISA) kits (Shanghai Lizhi Biotechnology Co., LTD). The sensitivity of the ELISA for insulin is 0.1 mU/L. Morris water maze (MWM) test MWM test was carried to determine the spatial learning and memory as described in previous research 9 with minor modifications. Shortly, a circular pool with a height of 50 cm and a diameter of 120 cm was employed in current study, and it also had an escape platform with a radius of 5 cm, which was placed 1 cm underwater. The temperature of water in the pool was kept at room temperature (25 ± 2 ℃) during the experiment, and the pool was surrounded by four blue curtains, each with a striking and completely different visual logo. Mice were placed in one of the four quadrants to find for the hidden platform for 60 s during the orientation navigation test phase lasted for 5 days. For each day of the acquisition test, the maximum swimming time of the mice was 60 s, and remained on the platform for at least 10 seconds after climbing it was considered to have found the platform. If mice did not find the platform within 60 successfully, they will be guided to the platform using a pole and kept there for 10 s. Escape latency was defined as the latency required to reach the platform in each trial. There would be a probe trail on the 6th day. Mice were allowed to swim freely for 60 s without the platform during the probe trail. The entire experimental processes were monitored by a video tracking system EthoVision® XT, which is able to record and measure the number of platform crossings, the percentage of time spent in the target quadrant, and the average swimming speed automatically. Nissl Staining Mice were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde after the MWM test. The brains were then encased in paraffin wax and sliced through a microtome into 8 mm thick slices for further staining. Briefly, the slices were deparaffinized, followed by rehydrated in graded concentrations of ethanol step by step, and then treated with Nissl staining solution. The images of the brain sections were finally observed using an optical microscope (× 10 and × 100). Immunofluorescence Staining For fluorescence staining of brain tissue, after deparaffinization and antigen retrieval (0.05% citraconic acid), the paraffin-embedded sections were treated with endogenous peroxidase (3% H 2 O 2 in PBS) for 10 min, and then incubated with 5% donkey serum containing 0.1% Triton X-100 in PBS for 1 h. Next, the sections were incubated at 4°C for at least 12 h with the following primary antibodies: 6E10 (1:10000, No. 803014, SanDiego, CA, USA), and NeuN (1:800, GB11138-100, Servicebio, Wuhan, China), followed by incubation with the corresponding fluorescent secondary antibody at room temperature for 2 h. Photographs were taken with a fluorescence microscope (IX53, Olympus, Tokyo, Japan). Transmission electron microscopy (TEM) According to our previously published method 9 , the CA1 subregion of the hippocampus was dissected and cut into 1mm sections in electron microscope fixative as soon as possible after transcardial perfusion with paraformaldehyde. Then, after fixation in electron microscopy fixative (Servicebio, China) made of 2.5% glutaraldehyde overnight at 4°C, the sections were washed three times using 0.1 M PBS and postfixed the brain tissues using OsO 4 for 2 h at 25 ℃. Then, the tissues were dehydrated by graded ethanol (30, 50, 70, 80, 95, and 100%), embedded in resin, and polymerized in a 60 ℃ oven for 48 h. Finally, the tissues were cut into ultrathin (60–80 nm) slices, which were stained with uranyl acetate and lead citrate. The sections were then taken images using a transmission electron microscope (H-7800, Hitachi, Japan). Western blotting analysis Western blotting analysis of protein extract from the brain tissue was performed as we described previously 9 with minor modifications. In short, unilateral brain tissues were lysed and homogenized with 1 ml RIPA buffer contained with protease and phosphatase inhibitors through sonication, followed by centrifugation at 12,000 rpm for 30 min at 4°C, and then collect supernatant. The concentration of protein was determined using the BCA kit (Epizyme, Shanghai, China). Adjusting the protein samples with different concentrations to the same concentration according to the detected concentration. Each sample with an equal volume of protein was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to polyvinylidene fluoride (PVDF) membranes. Membranes containing protein were blocked with 5% non-fat milk (Epizyme, Shanghai, China) or protein free rapid blocking buffer (Epizyme, Shanghai, China) and then incubated with the following primary antibodies overnight at 4 ℃, including nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) (P60622R3, 1:1000, Abmart, Shanghai, China); apoptosis-associated speck-like protein containing a CARD (ASC) (10500-1-AP, 1:5000, Proteintech, Wuhan, China); gasdermin-D (GSDMD) (GB114198-100, 1:500, Servicebio, Wuhan, China); caspase1 (22915-1-AP, 1:2000, Proteintech, Wuhan, China); caspase11 (647201, 1:500, BioLegend, San Diego, USA); interleukin-1β (IL-1β) (16806-1-AP, 1:2000, Proteintech, Wuhan, China); interleukin-18 (IL-18) (60070-1-Ig, 1:2000, Proteintech, Wuhan, China); tumor necrosis factor-α (TNF-α) (PY19810S, 1:1000, Abmart, Shanghai, China); toll-like receptor 4 (TLR4) (A5258, 1:1000, Abclonal, Wuhan, China); nuclear factor kappa B (NF-κb) p65 (MA9199, 1:500, Abmart, Shanghai, China); p-NF-κB p65 (ab76302, 1:20000; Abcam, Cambrige, UK); claudin-5 (29767-1-AP, 1:5000, Proteintech, Wuhan, China); zona occludens 1 (ZO-1) (21773-1-AP, 1:5000, Proteintech, Wuhan, China); albumin (66051-1-Ig, 1:5000, Proteintech, Wuhan, China); low density lipoprotein-related protein 1 (LRP-1) (26106-1-AP, 1:500, Proteintech, Wuhan, China); p-glycoprotein (P-gp) (22336-1-AP, 1:500, Proteintech, Wuhan, China). Washing with TBS-T for 10 min 3 times, followed by incubating membranes with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) secondary anti-rabbit antibody (GB23303, 1:1000, Servicebio, Wuhan, China), anti-mouse antibody (A0216, 1:1000, Beyotime, Nantong, China), and anti-rat antibody (SA00001-15, 1:1000, Proteintech, Wuhan, China). Immunoblots were observed on the gel imaging system using the enhanced chemiluminescence (ECL) kit (Yeasen, Shanghai, China) and the blots were quantified using ImageJ software to calculate the grayscale value of signals. 16S rRNA Gene Sequencing of Fecal Samples 16s rRNA amplicon sequencing was performed as we described previously with minor modifications 10 . Total microbiome DNA was extracted from microbiome samples from different sources by CTAB method. The quality of DNA extraction was detected by agarose gel electrophoresis, and the DNA was quantified by ultraviolet spectrophotometer. The V3-V4 region of the small subunit 16S rDNA gene of prokaryotes (bacteria and archaea) was amplified with universal primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-CCTACGGGNGGCWGCAG-3'). PCR products were purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). PCR amplification products were detected by 2% agarose gel electrophoresis, and AMPureXTbeads recovery kit was used for recovery. Purified PCR products were evaluated using the Agilent 2100 Bioanalyzer (Agilent, USA) and Illumina's library quantification kit (Kapa Biosciences, Woburn, MA, USA). The qualified library concentration should be above 2 nM. The qualified online sequencing libraries (Index sequence is not repeatable) were diluted in gradient, mixed in proportion according to the required sequencing amount, and denatured into single strand by NaOH for on-machine sequencing. The NovaSeq 6000 sequencer was used for 2 × 250 bp double-ended sequencing with the NovaSeq 6000 SP Reagent Kit (500 cycles). For the double-ended data obtained by sequencing, the sample should first be separated according to barcode information, and the joint and barcode sequence should be removed. Then data splicing and filtering are carried out. The filtered data were denoised by DADA2 to generate an amplicon sequence variation (ASV) feature table 11 , 12 . Alpha diversity (α-diversity) analysis mainly evaluates the diversity in the living area through six indexes: observed otus, chao1, simpson, shannon 10 . Beta diversity (β-diversity) mainly evaluates and analyzes the diversity among habitats (between samples/groups) by calculating distances of jaccard 10 . According to the ASV feature table sequence file, we annotated the taxonomy with the database of SILVA v138 11,13 , and made statistics on the abundance of each taxonomy in each sample according to the ASV (feature) abundance table. Mann-Whitney U test was used to compare the differences between two groups of samples with biological duplication. Statistical Analysis The statistical analyses were performed with GraphPad Prism 9 software. The differences of data among groups were analysed through one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test or Mann-Whitney U test. The data are shown as the mean ± standard error of the mean (SEM) or median (interquartile range). P < 0.05 was regarded to be statistically significant. Results GLP-1 treatment improved cognitive deficits in APP/PS1 mice In this study, the effects of GLP-1 on the cognition of APP/PS1 mice at 10 months of age were tested by MWM experiment, which is a very popular method for evaluating spatial learning and memory 14 . We observed that on the 4th and 5th day of the orientation navigation test phase, the escape latency of AD group mice was significantly longer than that of WT group and GLP-1 treatment group (Fig. 1 B and G). We then performed a probe trial on 6th day of MWM test where the number of times across the withdrawn platform, the percentage of time spent in the target quadrant and the mean distance to withdrawn platform were logged to estimate spatial memory. The times crossing the platform (Fig. 1 C and H) and the time spent in the target quadrant (Fig. 1 D and H) of mice in AD group were significantly reduced while the mean distance to platform (Fig. 1 E and H) were increased compared with WT group and AD + GLP-1 group. Besides, there was no significant difference in average swimming speed among the three groups (Fig. 1 F). In other words, there was no severe visual or movement aberration among the mice. Furthermore, we also recorded the body weight (BW) of mice every week and determined the serum insulin level after 4-h fast given that GLP-1 has the effect of improving metabolism and reducing BW 15 . Interestingly, the average BW of mice in the AD group was lower than the WT group, while the BW was further reduced after GLP-1 treatment (Fig. 1 I). We found that the insulin level of mice in AD group was higher than WT, while the insulin level was reduced after GLP-1 treatment, although there were no significant differences between all groups (Fig. 1 J). Taken together, the results of MWM test uncovered a beneficial effect of GLP-1 on cognitive performance of APP/PS1 mice. The data of metabolism in this study are basically consistent with previous studies on GLP-1, that is GLP-1 reduces body weight and insulin resistance. Although the difference is not significant, it is confirmed that GLP-1 does play a role in vivo. GLP-1 treatment saved dying neurons in hippocampus of APP/PS1 mice Given the results of the above behavioral experiments, we speculated that GLP-1 protected hippocampal neurons in AD group. In order to verify our conjecture, the Nissl staining and immunofluorescence were employed to observe the morphology and quantity of hippocampal neurons. As we predicted, Nissl staining showed that the hippocampal neurons in CA1 region of mice in AD group were stained deeper, shrunk and showed necrotic pathological morphology compared to the WT group, while the morphology of neurons were recovered to normal after treating with GLP-1(Fig. 2 A, B and C). Consistent with the results of Nissl staining, immunofluorescence displayed that GLP-1 completely reversed the loss of NeuN positive neurons (Fig. 2 D) in the hippocampal CA1 (Fig. 2 E), CA2 (Fig. 2 F), CA3 (Fig. 2 G) and DG (Fig. 2 H) regions of AD group mice. In short, the morphological results revealed that GLP-1 saved the dying neurons in the hippocampus of mice in AD group. GLP-1 treatment reversed AD pathological phenotypes and improved the synaptic plasticity According to previous reports, the deposition of β-amyloid (Aβ) peptide is a pathological hallmark of AD 16 . In addition, abnormal morphology and reduced number of synaptic in AD have also been widely reported 17 , 18 . To further verify the protective effect of GLP-1 on the central nervous system, we observed Aβ plaques and synapses in the mice brain by immunofluorescence and TEM, respectively. Interestingly, the present study found that GLP-1 not only reduced the number of Aβ plaques in the cortex of APP/PS1 mice, but also inhibited the deposition of Aβ in the hippocampus (Fig. 3 A and B). In addition, we observed that the number of synapses in hippocampus of mice in AD group significantly decreased when compared with WT group via TEM, while the number of synapses in hippocampus of APP/PS1 mice significantly increased after GLP-1 treatment (Fig. 3 C and D). We further analyzed the synaptic morphology, calculated the length of active zone, thickness of post synaptic density (PSD), width of synaptic cleft, and synaptic curvature according to the previous research 19 . The present study demonstrated that the treatment of GLP-1 significantly increased the length of active zone (Fig. 3 E, and F), PSD thickness (Fig. 3 E, and G) and synaptic curvature (Fig. 3 E, and I) of hippocampal synapses in AD mice, while reduced the width of synaptic cleft (Fig. 3 E, and H). In a word, our research clarified that GLP-1 treatment increased synaptic plasticity and reversed the pathological phenotype of AD in APP/PS1 mice. GLP-1 treatment attenuated neuroinflammation in APP/PS1 mice The current study further explored the upstream mechanism of GLP-1 reversing the AD phenotype of APP/PS1 mice by western blotting. Given that neuroinflammation has been widely reported in AD 20 , 21 , we examined the expression levels of several inflammation-associated proteins. Consistent with previous studies 22 , 23 , we found that canonical pyroptosis pathway signaling, such as NLRP3 (Fig. 4 A and B), ASC (Fig. 4 A and C), GSDMD (Fig. 4 A and D) and caspase-1 (Fig. 4 E-H), were all significantly upregulated in AD mice brain tissue compared to WT. Interestingly, expression levels of these proteins were significantly down-regulated after GLP-1 treatment. In addition, this research also detected caspase11, a caspase-1-independent pyroptosis pathway in mice, which recognizes lipopolysaccharide (LPS) in cytoplasm 24 , 25 . What fascinating is that compared with WT group, the protein was significantly up-regulated in the brain tissue of AD group mice, and after GLP-1 treatment, the protein expression level returned to WT level (Fig. 4 I-L). It may be due to the existence of LPS in the cytoplasm of brain tissue that can be recognized by caspase11. Next, we explored downstream signals of the pyroptosis pathway, interleukin-1β (IL-1β) and interleukin-18 (IL-18). The two proinflammatory factors were significantly increased in the brain tissue of mice in AD group, and the levels of IL-1β and IL-18 factors were obviously decreased after GLP-1 treatment (Fig. 4 M-O). Since the elevation of caspase11 in AD mice suggests that exogenous LPS may enter brain tissue, we further examined another inflammatory signaling pathway in vivo that recognizes LPS, which is TLR4 and downstream tumor necrosis factor-α (TNF-α), NF-κb p65 and p-NF-κb p65. Consistent with above results, these proinflammatory signaling proteins were significantly upregulated in the brain tissue of AD group mice compared to WT, and treatment with GLP-1 reversed the upregulation of this pro-inflammatory mediator (Fig. 5 A-E). GLP-1 treatment improved the blood-brain barrier function of APP/PS1 mice Given the above results and previous reports on the gut-brain axis, we speculate that LPS in the gut may be ectopic in the brain tissue of AD mice through damaged blood-brain barrier and intestinal barrier 26 . So, we determined the integrity of the blood-brain barrier in mice. We detected the tight junction proteins Claudin-5 and ZO-1 of BBB. As expected, the expressions of Claudin-5 (Fig. 6 A and B) and ZO-1 (Fig. 6 A and C) in AD group were significantly decreased compared with WT, but they were increased after GLP-1 treatment. Next, we determined whether there is blood-brain barrier leakage by detecting albumin level in brain tissue. We found that the albumin in brain tissue of mice in AD group increased, which proved that the blood-brain barrier was damaged and protein leakage occurred (Fig. 6 A and D). After GLP-1 treatment, the blood-brain barrier was repaired and the blood-brain barrier leakage was reduced. In view of the fact that the damage of blood-brain barrier has occurred in AD mice, we detected the expression level of low-density lipoprotein receptor-related protein 1 (LRP1) and P-glycoprotein (P-gp). It is reported that this protein located in endothelial cells mediates the clearance of Aβ protein in brain tissue 27 , 28 , 29 . We found that the expression of these two proteins was down-regulated in the brain tissue of AD mice compared with WT, and GLP-1 treatment saved their loss (Fig. 6 A, E and F). It can be seen that the mice in AD group not only face the invasion of enterogenous LPS, but also suffer the damage caused by the accumulation of Aβ in the brain. GLP-1 treatment alleviated the mitochondrial damage of microglia In view of the severe inflammatory attack in the brain tissue of AD mice, we observed the morphology of microglial cells in the hippocampus of mice by TEM. Of course, we do not rule out the possibility that other cells, such as astrocytes and even neurons themselves, are involved in the inflammatory response. However, as resident macrophages in brain tissue, microglia respond most sensitively and strongly to injury factors. As expected, compared with the WT group (Fig. 7 A), the mitochondria in the microglia of the mice in AD group were severely damaged, characterized by mitochondrial fragmentation, vacuolation and a decrease in number (Fig. 7 B). Interestingly, the mitochondrial morphology and number of hippocampal microglia returned to normal after GLP-1 treatment (Fig. 7 C). Neuroinflammation in APP/PS1 mice may be due to their disturbed gut flora In view of the upregulation of LPS-recognizing proinflammatory mediators in the brain tissue of AD mice, we examined the composition of the intestinal microbiota of mice by 16srRNA amplicon sequencing. Firstly, we observed differences in the composition of gut microbiota between AD and WT mice by determining α and β diversity. α diversity is used to assess the diversity of community composition within a sample 10 . The α diversity analysis did not show a significant difference in gut microbial community evenness and richness between the two groups of mice based on Chao 1 (Fig. 8 A), Observed-otus (Fig. 8 B), Shannon (Fig. 8 C) and Simpson indices (Fig. 8 D). Beta diversity was used to assess differences in the overall composition of microbiota between groups 10 . In this study, principal coordinate analysis (PCoA) based jaccard was used to measure β diversity, but no significant differences were found (Fig. 8 E). Next, we analyzed the differential abundance microbiota between the WT and AD group. First, we analyzed the differential abundance of gut microbes between the two groups at the phylum level (Fig. 9 A). We focused on the Firmicutes and Bacteroides ratio (F/B), which is reported to reflect different disease states 30 , 31 . Compared with WT group, F/B value in AD group was lower but not significant (Fig. 9 B). Subsequently, we analyzed the differential abundance taxa by method of Linear discriminant analysis Effect Size (LEfSe) and heatmap. At genus level, Akkermansia and Herminiimonas was abundant in WT group (Fig. 9 C). While Erysipelotrichaceae_unclassified, Alistipes, Desulfovibrionaceae_unclassified, Candidatus_Saccharimonas , etc., were enriched in AD group (Fig. 9 D). Discussion In this study, APP/PS1 mice were treated with GLP-1 analog Semaglutide. Through behavioral experiments, we found that GLP-1 improved the cognitive function of AD mice. Furthermore, we observed the morphology of brain tissue of AD mice, and found that GLP-1 treatment reduced damage of neuron, aggregation of Aβ plaques in hippocampus, and increased synaptic plasticity of hippocampal neurons. We further detected the expression of inflammatory factors in the brain tissue of mice, and found that GLP-1 reversed the canonical caspase-1-dependent pyroptosis pathway, the caspase-11-dependent noncanonical pyroptosis pathway and the TLR4/NF-κb pathway activated in the brain tissue of AD mice. In addition, this study also indicated that GLP-1 treatment reduced the damage of blood-brain barrier and microglia mitochondria in AD mice. Finally, we found by amplicon sequencing that compared with WT mice, the abundance of pro-inflammatory bacteria such as Erysipelotrichaceae_unclassified, Alistipes, Desulfovibrionaceae_unclassified, Candidatus_Saccharimonas , etc. increased, while the abundance of anti-inflammatory bacteria such as Akkermansia and Herminiimonas , etc. decreased. We speculated that the increase of pro-inflammatory bacteria in AD mice may damage the intestinal barrier and cause intestinal leakage, and the pyrogen in the intestine enters the circulation and leads to low-grade inflammatory reaction in the whole body, and at the same time enters the center never system through the damaged blood-brain barrier of AD mice, causing neuroinflammation. GLP-1 may reduce the systemic inflammatory response of AD mice by direct inhibition or by repairing the damaged intestinal barrier and blood-brain barrier. Researchers have found that the brain of AD showed increased levels of lipid peroxidation, microglia/astrocyte activation, and overexpression of pro-inflammatory cytokines (TNF-α, IL- 6) through autopsies of AD patients 24 . Studies have shown that the pyroptosis pathway is activated in AD mice 25 . The present research found that not only the canonical pyroptosis pathway dependent on caspase1 was activated, but also the noncanonical pyroptosis pathway dependent on caspase11 was activated. Caspase-11 in mice was reported to respond to various bacterial infections, and could respond to the stress of cytoplasmic LPS, and then activating pyroptosis 32 , 33 . Although previous studies have found that the expression of caspase11 is increased in aging mice 34 , the up-regulation of caspase11 in AD in vivo model has not been reported before. Therefore, we speculated that there is a low-grade inflammatory reaction induced by LPS in the brain tissue of AD mice. In order to verify our conjecture further, the current study detected the level of toll-like receptor 4 and its downstream inflammatory signal. In fact, the up-regulation of TLR4 and its downstream signals NF-κb and TNF-α in AD mice has been widely reported 20 , 35 , 36 . Previous reports showed that intestinal dysfunction and pyrogen displacement in AD mice led to the increase of LPS in circulation and activated inflammatory signaling pathway in vivo 26 , 35 . Therefore, we sequenced 16srRNA amplicon in mouse feces and found that Erysipelotrichaceae_unclassified , Alistipes, Desulfovibrionaceae_unclassified , and Candidatus_Saccharimonas , etc. increased, while the abundance of Akkermansia and Herminiimonas , etc. decreased. Previous reports generally believed that Alistipes is associated with anxiety and depression 37 , and is enriched in AD mice 38 , 39 . Interestingly, Zihao Ou et al. found that APP/PS1 mice supplemented with AKK significantly improved the disease phenotype 40 . In view of the fact that enterogenous pyrogens in blood still need to permeate the blood-brain barrier (BBB) to enter the central nervous system, we also detected the expression of BBB-related proteins. It was found that the blood-brain barrier of AD mice was damaged. In addition, we also found that the expression of LPR1 27 and P-gp 28 , 29 related to Aβ clearance also decreased. Glucagon like peptide-1 (GLP-1) is a kind of peptide gastrointestinal hormone secreted by the intestinal tract, which plays an important role in the regulating of postprandial glucose homeostasis via acting upon insulin secretion, food intake and gastrointestinal motility 41 . Inadequate secretion of GLP-1 increases the risk of metabolic diseases, such as type 2 diabetes and obesity 42 . Studies have shown that GLP-1 is able to cross the blood-brain barrier and act on GLP-1 receptors in the central nervous system (CNS) 43 . Semaglutide, a GLP-1 analogue of a modified once-weekly agent based on liraglutide, has become a first-line drug in the clinical treatment of obesity and type 2 diabetes in the past few years 15 , 44 , in addition to its excellent performance in improving cardiovascular and cerebrovascular outcomes in metabolic diseases 45 – 47 . Interestingly, more and more researchers have shown great interest in the role of GLP-1 in non-metabolic diseases such as nervous system diseases in recent years 48 , 49 . The clinical research published in 2017 suggested that GLP-1 analogue exenatide may benefits motor function in patients with Parkinson's disease (PD) 50 . However, two recent studies have come to conflicting conclusions. Studies by Wassilios G Meissner et al. suggested that Lixisenatide group had a lower progression of dyskinesia than placebo group in patients with early PD 12 months after treatment 51 , while Andrew Mc Garry et al. demonstrated that another GLP-1 analogue, NLY01-a, did not improve the dyskinesia symptoms of PD patients 52 . This indicates that not all GLP-1 analogues can play a neuroprotective role, and of course, it does not rule out the individual differences of patients in different cohorts. A randomized controlled trial (RCT) published in 2023 showed that liraglutide could restore the impaired associative learning of obesity through mesoaccumbens pathway 53 . In fact, it has been widely reported that GLP-1 can alleviate neuroinflammation and pathological phenotype; improve neurological function as well as delay the progress of neurodegenerative diseases through anti-inflammation and antioxidation 48 , 49 , 54 – 56 . However, the researches on the role of GLP-1, especially Semaglutide, in the treatment of AD is limited so far. A study in vitro showed that Semaglutide treatment reduced the damage of Aβ 25−35 to SH-SY5Y cells by promoting autophagy suppressing apoptosis 57 . A recent study confirmed in vivo and in vitro that Semaglutide treatment can improve glucose metabolism in brain tissue and cells through GLP-1R/SIRT1/GLUT4 pathway to alleviate the disease phenotype of AD mice and the damage of Aβ 1−42 to hippocampal neurons HT22 cells 58 . Interestingly, Joseph Bailey et al recently published a study showing that GLP-1 receptor activation can improve brain pericyte function, thus restore vascular integrity and blood-brain barrier function in diabetic patients, and reduce diabetes-induced cognitive impairment in mice 59 . On the basis of previous researches, the current study confirmed for the first time that GLP-1 analogue Semaglutide restored the blood-brain barrier of APP/PS1 mice, and alleviated the neuroinflammation in the brain tissue of APP/PS1 mice, which may be caused by enterogenous pyrogen. In addition, GLP-1 also upregulated LRP1 and P-gp regarded as Aβ clearance-associating protein in the blood-brain barrier of AD mice. To sum up, this study found that the pyrogen in the intestine of APP/PS1 mice shifted to the circulation and entered the CNS through the damaged BBB, which led to severe neuroinflammation and aggravated the phenotype of AD. However, the treatment of GLP-1 analogue Semaglutide improved the structure and function of BBB in APP/PS1 mice, reduced the damage of microglia mitochondria, neuroinflammation and aggregation of Aβ plaques, increased the number of normal neurons and synaptic plasticity, and further improved the cognitive function of APP/PS1 mice. There are some limitations in the present study. First, we did not observe whether the intestinal barrier of APP/PS1 mice was damaged. Secondly, we did not investigate the effect of GLP-1 on gut microbiota of AD mice. In addition, we did not detect the inflammatory state in peripheral circulation. Finally, this study is only performed in animal models, and has not been extended to clinical research, nor has it been explored in depth in vitro. Conclusion GLP-1 Semaglutide repaired the structure and function of BBB in APP/PS1 mice, reduced neuroinflammation and AD pathological phenotype of APP/PS1 mice, and improved the cognitive function of APP/PS1 mice. Abbreviations AD, Alzheimer's disease; GLP-1, glucagon-like peptide 1; APP/PS1, amyloid precursor protein / presenilin 1; Aβ, amyloid-β; NLRP3, NOD-like receptor thermal protein domain associated protein 3; TLR4, toll-like receptor 4; BBB, blood-brain barrier; CNS, central nervous system; T2DM, type 2 diabetes mellitus; SPF, specific-pathogen-free; WT, wild-type; NIH, National Institutes of Health; ELISA, Enzyme-linked immunosorbent assay; MWM, Morris water maze; TEM, Transmission electron microscopy; SDS-PAGE, sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; ASC, apoptosis-associated speck-like protein containing a CARD; GSDMD, gasdermin-D; IL-1β, interleukin-1β; IL-18, interleukin-18; TNF-α, tumor necrosis factor-α; NF-κb, nuclear factor kappa B; ZO-1, zona occludens 1; LRP-1, low density lipoprotein-related protein 1; P-gp, p-glycoprotein; HRP, horseradish peroxidase; IgG, immunoglobulin G; ECL, enhanced chemiluminescence; ASV, amplicon sequence variation; α-diversity, Alpha diversity; β-diversity, Beta diversity; ANOVA, analysis of variance; SEM, standard error of the mean; ASV, amplicon sequence variation; α-diversity, Alpha diversity; β-diversity, Beta diversity; SEM, standard error of the mean; BW, body weight; PSD, post synaptic density; LPS, lipopolysaccharide; principal coordinate analysis (PCoA); Linear discriminant analysis Effect Size (LEfSe); Parkinson's disease (PD); randomized controlled trial (RCT). Declarations Funding This work was supported by grants from the National Natural Science Foundation of China (81801169, 81870952, 82001303, 82371255, 82071258), Shanghai Science and Technology Innovation Action Plan (23DZ2291500), Natural Science Foundation of Shanghai (24ZR1457100), Project of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (ynnkxyb202412), Program for Shanghai Outstanding Academic Leaders(23XD1402500), Program for Outstanding Medical Academic Leader of Shanghai (2022LJ011), and Training program for research physicians of innovative translational ability (SHDC2022CRD037). Conflicts of Interest: The authors declare no conflict of interest. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. 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GLP-1 receptor nitration contributes to loss of brain pericyte function in a mouse model of diabetes. Diabetologia. 2022;65:1541–54. 10.1007/s00125-022-05730-5 . Supplementary Files supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6098406","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422183105,"identity":"a37e8a62-7d92-4a02-8f3d-ec5529387b4b","order_by":0,"name":"Yuan Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIie3Rv0rEMBzA8V8I5BzCzSnRim/wk6wH56OkFG66oSCIm5FCpwPX3Fv4CC03ZCnOHeuia+UGHQTNHQ5OaUfBfJeEkA/kD0As9gdbuof3jwEX6dIZwONSPWbaGhNbrJSfANaTSHeF8mTYZabTP7vHCLGgFSDN7rev/fVbBem802RfBAiVps4LZKqUa8SmApV0mkobIOy0MTuL/KySaziQ7LHTjPIA4SInJUdBNonrD+RulAixopQjXlgBx4NpHCPIW0YsaoXc36V9Epfb9rmUQTLbMBg+v1KcuRe8vVmcz13e7EPkd8z/vvAjMROBf/B+8tZYLBb7V30Dvo5OyY3MCUAAAAAASUVORK5CYII=","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yuan","suffix":""},{"id":422183106,"identity":"744e073e-fbd9-402d-bc11-7a5d2bab475b","order_by":1,"name":"Jiawei Zhang","email":"","orcid":"","institution":"Department of Neurology and Department of Neuroscience, the First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Zhang","suffix":""},{"id":422183107,"identity":"595aaf42-a5f3-4d01-957e-0433dece110b","order_by":2,"name":"Ziyao Zhang","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ziyao","middleName":"","lastName":"Zhang","suffix":""},{"id":422183108,"identity":"0b9924d5-f507-4ed1-93bd-2960f8858221","order_by":3,"name":"Yanyu Zhai","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanyu","middleName":"","lastName":"Zhai","suffix":""},{"id":422183109,"identity":"ab8fe8a0-ef99-48f2-9f62-373b159d296e","order_by":4,"name":"Xiaojuan Cheng","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Cheng","suffix":""},{"id":422183110,"identity":"b6a854f7-c7a8-4176-9a1b-369ca8346751","order_by":5,"name":"Lixia Xue","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lixia","middleName":"","lastName":"Xue","suffix":""},{"id":422183111,"identity":"f9cb2776-66da-482c-922a-b9e68f5d8f72","order_by":6,"name":"Fei Zhao","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Zhao","suffix":""},{"id":422183112,"identity":"5068218f-aa07-4d5d-80df-bbd5c1439189","order_by":7,"name":"Li Cao","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Cao","suffix":""},{"id":422183113,"identity":"a2536c69-182b-477d-b800-9cc1d1127c2d","order_by":8,"name":"Hongmei Wang","email":"","orcid":"","institution":"Department of Neurology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongmei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-02-24 15:51:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6098406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6098406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77628754,"identity":"8f16fcd2-1126-4c63-b6ec-f8ea64b92824","added_by":"auto","created_at":"2025-03-03 17:07:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment improved cognitive deficits in APP/PS1 mice.\u003c/strong\u003e A. Schematic diagram of the experimental procedure. B. The escape latency of mice to reach the platform during the acquisition test. n ≥ 5 per group. C. The numbers of platform crossings in the probe trial, n ≥ 5 per group. D. Percentage of time spent in the target quadrant of mice in the probe trial, n ≥ 5 per group. E. The mean distance to platform of mice in probe trial, n ≥ 5 per group. F. Average swimming speeds of mice in different groups during the probe trial, n ≥ 5 per group. G. Representative exploring traces of mice in different groups during the orientation navigation test phase, n ≥ 5 per group. H. Representative exploring traces of mice in different groups during the probe trial, n ≥ 5 per group. I. Trend of body weight change of mice in each group during GLP-1 treatment, n = 8 per group. J. Fasting insulin level of mice in each group, n = 8 per group. Data are presented as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns means not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/27fc082fbdb7346bfc72b935.png"},{"id":77628289,"identity":"2b012bb9-4aaa-412f-b939-76af96c9f410","added_by":"auto","created_at":"2025-03-03 16:59:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":285147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment saved dying neurons in hippocampus of APP/PS1 mice.\u003c/strong\u003e A. Representative Nissl staining of the mice hippocampus. Scale bar: 500 μm. n = 3 per group. B. Representative Nissl staining in the mice CA1 region of hippocampus. Scale bar: 50 μm. n = 3 per group. C. Quantification of Nissl positive cells in the mice CA1 region of hippocampus. n = 3 per group. D. Representative immunofluorescence images of Neun (left penal), DAPI (middle penal) and overlay (right penal) in the hippocampal. Scale bar: 500 μm. n = 3 per group. E-H. Quantification of fluorescence intensity of NeuN in the mice CA1 (E), CA2 (F), CA3(G), and DG (H) region of hippocampus, n = 3 per group. Data are presented as the mean ± SEM. n = 3 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/d75f86dee0a65bfd0287aa2d.png"},{"id":77628296,"identity":"dc1e718c-9442-4531-aa12-590e38fff2f1","added_by":"auto","created_at":"2025-03-03 16:59:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment reversed AD pathological phenotypes and improved the synaptic plasticity.\u003c/strong\u003e A. Representative immunofluorescence images of overlayed Aβ and DAPI in the hippocampal (right penal) and cortex (left penal). Scale bar: 200 μm (middle penal) and 100 μm (right and left penal). n = 3 per group. B. Quantification of fluorescence intensity of NeuN in the mice hippocampus and cortex. n = 3 per group. C. Representative transmission electron micrographs of hippocampal synapse numbers in different groups. Red arrowhead: synaptic junction. Scale bar: 2 μm. n = 3 per group. D. Quantitative analysis of synapse number in hippocampus. n = 3 per group. E. Representative transmission electron micrographs of hippocampal synapse structure in different groups, scale bar: 1 μm. Red circle: synaptic structure, scale bar: 0.1 μm. n = 3 per group. F. Quantitative analysis of length of active zone in hippocampus. n = 3 per group. G. Quantitative analysis of thickness of PSD in hippocampus. n = 3 per group. H. Quantitative analysis of width of synaptic cleft in hippocampus. n = 3 per group. I. Quantitative analysis of synaptic curvature in hippocampus. n = 3 per group. Data are presented as the mean ± SEM. n = 3 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/2b151a1e52ec878863a00984.png"},{"id":77628752,"identity":"a5004054-0dc5-4a51-8c3f-0fd1927fb1f1","added_by":"auto","created_at":"2025-03-03 17:07:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment reduced pyroptosis in APP/PS1 mice. \u003c/strong\u003eA. Representative immunoblotting bands of NLRP3, ASC and GSDMD in the brain tissue of mice in different group. n=3 per group. B-D. Quantitative analysis of NLRP3/actin, ASC/actin and GSDMD/actin. n=3 per group. E. Representative immunoblotting bands of caspase1 in the brain tissue of mice in different group. n=3 per group. F-H. Quantitative analysis of pro-caspase1 (45KDa) /actin, cl-caspase1 p35 (35KDa) / actin and cl-caspase1 p20 (20KDa) /actin. n=3 per group. I. Representative immunoblotting bands of caspase11 in the brain tissue of mice in different group. n=3 per group. J-L. Quantitative analysis of pro-caspase11 (45KDa) /actin, cl-caspase11 p30 (30KDa) / actin and cl-caspase11 p15 (15KDa) /actin. n=3 per group. M. Representative immunoblotting bands of IL-1β and IL-18 in the brain tissue of mice in different group. n=3 per group. N-O. Quantitative analysis of IL-1β/actin and IL-18/actin. n=3 per group. Data are presented as the mean ± SEM. n = 3 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/fd41a004884e137e8510e297.png"},{"id":77628293,"identity":"ed657b2b-b5ba-4069-87f2-cf2c20023ee0","added_by":"auto","created_at":"2025-03-03 16:59:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment reduced inflammation mediated by TLR4 in APP/PS1 mice. \u003c/strong\u003eA. Representative immunoblotting bands of TNF-α, TLR4, NF-κB p65 and p-NF-κB p65 in the brain tissue of mice in different group. n=3 per group. B-E. Quantitative analysis of membrane-TNF-α/actin, soluble-TNF-α/ actin, TLR4/actin and p-NF-κB p65/ NF-κB p65. n=3 per group. Data are presented as the mean ± SEM. n = 3 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/fad75c52dfe6aabe82d42f89.png"},{"id":77628290,"identity":"21165de6-0eb6-43f5-80ce-cb565c4c2278","added_by":"auto","created_at":"2025-03-03 16:59:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":129211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment improved the blood-brain barrier function of APP/PS1 mice.\u003c/strong\u003e A. Representative immunoblotting bands of Claudin-5, ZO-1, Albumin, LRP-1, and P-gp in the brain tissue of mice in different group. n=3 per group. B. Quantitative analysis of Claudin-5/actin. n=3 per group. C. Quantitative analysis of ZO-1/actin. n=3 per group. D. Quantitative analysis of albumin/actin. n=3 per group. E. Quantitative analysis of LRP-1/actin. n=3 per group. F. Quantitative analysis of P-gp/actin. n=3 per group. Data are presented as the mean ± SEM. n = 3 per group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/12377dbd02d774e29e69acce.png"},{"id":77629622,"identity":"594b72b3-6eac-499e-b38f-58d17842375a","added_by":"auto","created_at":"2025-03-03 17:15:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":261192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 treatment alleviated the mitochondrial damage of microglia. \u003c/strong\u003eA. Representative transmission electron micrographs of hippocampal microglia in WT group. B. Representative transmission electron micrographs of hippocampal microglia in AD group. C. Representative transmission electron micrographs of hippocampal microglia in AD+GLP-1 group. Blue shadow: microglia, red arrowhead: mitochondria. Scale bar: 0.1 μm (upper panel) and 0.5 μm (lower panel). N = 3 per group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/739ab36c2fbeea6134ad2b8d.png"},{"id":77628756,"identity":"e7216dbf-52a0-4a96-a516-6e07c195b1e6","added_by":"auto","created_at":"2025-03-03 17:07:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":77613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiversity analysis of microbiota in fecal samples.\u003c/strong\u003e A-D Variation in alpha diversity (Observed otus (A), Chao1 (B), Simpson (C) and Shannon (D)) in mice of different group n = 8 per group. E. PCoA analysis of gut microbiota. n = 8 per group. Data are presented as the median (interquartile range). ns means not significant.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/13730ba3e4bcf6f20543ce9f.png"},{"id":77628757,"identity":"b7acd70d-ee33-4ec7-b771-482b649e0713","added_by":"auto","created_at":"2025-03-03 17:07:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":223152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of differential taxon of microbiota in fecal samples of mice.\u003c/strong\u003e A. Cluster diagram of microbiota abundance at phylum level. n=8 per group. B. Ratio of Firmicutes to Bacteroides (F/B). n=8 per group. C. Differential taxa in mice of different groups. n=8 per group. D. Community heatmap at the genus level. n=8 per group. Data are presented as the median (interquartile range). n = 8 per group. ns means not significant.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/e4dc9187f8a484ea20667537.png"},{"id":78784748,"identity":"2972bdc4-045a-4ab1-8264-e85cc5f6502d","added_by":"auto","created_at":"2025-03-19 00:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2492867,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/925868da-f55b-4112-b8aa-28214461bf93.pdf"},{"id":77628768,"identity":"5ce1330b-94b2-462d-bce8-b52ec836c524","added_by":"auto","created_at":"2025-03-03 17:07:17","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":2064391,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6098406/v1/e1b65ca59fefd448f5942624.docx"}],"financialInterests":"","formattedTitle":"Semaglutide Ameliorates Neuroinflammation Caused by Enterogenous Pyrogen in APP/PS1 Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD), progressing from mild memory impairment in the early stages to advanced dementia, is a highly inherited neurodegenerative disease, which is affecting tens of millions of people worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Nevertheless, many studies so far have found that exogenous regulation can delay the development of AD\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur AD research team turned to the latest star drug, the glucagon-like peptide 1 (GLP-1) analogue, Semaglutide. GLP-1 is an incretin hormone that controls insulin and glucose homeostasis, which is successfully employed for type 2 diabetes mellitus (T2DM) treatment\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As a gastrointestinal hormone, GLP-1 has been widely discussed in the field of brain-gut axis. Researchers generally believe that GLP-1 can be transported from the peripheral to the brain through blood and cross the blood-brain barrier, thereby exerting its effects in the central nervous system (CNS)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A recent study demonstrated the anti-inflammatory effects of GLP-1 in animal model systematically, which coincides with the current research\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we conducted long-term intervention with GLP-1 on an amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mouse model, mainly observing changes in AD pathology, inflammation related signaling pathways and blood brain barrier (BBB) in the central nervous system (CNS). In addition, we also investigated the composition of intestinal microbiota in APP/PS1 mice via 16S rRNA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAnimals and drug treatment\u003c/p\u003e \u003cp\u003eMale specific-pathogen-free (SPF) APP/PS1 mice were purchased from the Shanghai Model Organisms Center (Shanghai, China), and housed in SPF-barrier environment under standard temperature (20\u0026ndash;25 ℃) and light (12 h light/12 h dark) conditions, with water and food available ad libitum. The genotype was confirmed by PCR using tail tissue DNA. Age-matched wild-type (WT) littermates were used as controls. All procedures were approved by the ethical committee on animal welfare of Shanghai Sixth People\u0026rsquo;s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in accordance with the principles outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003cp\u003eThe mice were randomly divided into 3 groups (n\u0026thinsp;=\u0026thinsp;15 in each group): (1) wild-type (WT), (2) APP/PS1 (AD), (3) APP/PS1\u0026thinsp;+\u0026thinsp;Semaglutide (AD\u0026thinsp;+\u0026thinsp;GLP-1). Mice in the AD\u0026thinsp;+\u0026thinsp;GLP-1 group were subcutaneously administered with Semaglutide (6.7 \u0026micro;g/kg body weight) 2 times each week for 8 weeks, and monitor the weight of mice every week. A workflow of the experiment procedure was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e \u003cp\u003ePrior to the collection of blood, mice were given a 4-h fast and had free access to water. The serum was extracted by centrifuging the blood mouse, and kept at \u0026ndash; 80 ℃. The levels of serum insulin were determined through enzyme linked immunosorbent assay (ELISA) kits (Shanghai Lizhi Biotechnology Co., LTD). The sensitivity of the ELISA for insulin is 0.1 mU/L.\u003c/p\u003e \u003cp\u003eMorris water maze (MWM) test\u003c/p\u003e \u003cp\u003eMWM test was carried to determine the spatial learning and memory as described in previous research\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e with minor modifications. Shortly, a circular pool with a height of 50 cm and a diameter of 120 cm was employed in current study, and it also had an escape platform with a radius of 5 cm, which was placed 1 cm underwater. The temperature of water in the pool was kept at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃) during the experiment, and the pool was surrounded by four blue curtains, each with a striking and completely different visual logo. Mice were placed in one of the four quadrants to find for the hidden platform for 60 s during the orientation navigation test phase lasted for 5 days. For each day of the acquisition test, the maximum swimming time of the mice was 60 s, and remained on the platform for at least 10 seconds after climbing it was considered to have found the platform. If mice did not find the platform within 60 successfully, they will be guided to the platform using a pole and kept there for 10 s. Escape latency was defined as the latency required to reach the platform in each trial. There would be a probe trail on the 6th day. Mice were allowed to swim freely for 60 s without the platform during the probe trail. The entire experimental processes were monitored by a video tracking system EthoVision\u0026reg; XT, which is able to record and measure the number of platform crossings, the percentage of time spent in the target quadrant, and the average swimming speed automatically.\u003c/p\u003e \u003cp\u003eNissl Staining\u003c/p\u003e \u003cp\u003eMice were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde after the MWM test. The brains were then encased in paraffin wax and sliced through a microtome into 8 mm thick slices for further staining. Briefly, the slices were deparaffinized, followed by rehydrated in graded concentrations of ethanol step by step, and then treated with Nissl staining solution. The images of the brain sections were finally observed using an optical microscope (\u0026times; 10 and \u0026times; 100).\u003c/p\u003e \u003cp\u003eImmunofluorescence Staining\u003c/p\u003e \u003cp\u003eFor fluorescence staining of brain tissue, after deparaffinization and antigen retrieval (0.05% citraconic acid), the paraffin-embedded sections were treated with endogenous peroxidase (3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS) for 10 min, and then incubated with 5% donkey serum containing 0.1% Triton X-100 in PBS for 1 h. Next, the sections were incubated at 4\u0026deg;C for at least 12 h with the following primary antibodies: 6E10 (1:10000, No. 803014, SanDiego, CA, USA), and NeuN (1:800, GB11138-100, Servicebio, Wuhan, China), followed by incubation with the corresponding fluorescent secondary antibody at room temperature for 2 h. Photographs were taken with a fluorescence microscope (IX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM)\u003c/p\u003e \u003cp\u003eAccording to our previously published method\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, the CA1 subregion of the hippocampus was dissected and cut into 1mm sections in electron microscope fixative as soon as possible after transcardial perfusion with paraformaldehyde. Then, after fixation in electron microscopy fixative (Servicebio, China) made of 2.5% glutaraldehyde overnight at 4\u0026deg;C, the sections were washed three times using 0.1 M PBS and postfixed the brain tissues using OsO\u003csub\u003e4\u003c/sub\u003e for 2 h at 25 ℃. Then, the tissues were dehydrated by graded ethanol (30, 50, 70, 80, 95, and 100%), embedded in resin, and polymerized in a 60 ℃ oven for 48 h. Finally, the tissues were cut into ultrathin (60\u0026ndash;80 nm) slices, which were stained with uranyl acetate and lead citrate. The sections were then taken images using a transmission electron microscope (H-7800, Hitachi, Japan).\u003c/p\u003e \u003cp\u003eWestern blotting analysis\u003c/p\u003e \u003cp\u003eWestern blotting analysis of protein extract from the brain tissue was performed as we described previously\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e with minor modifications. In short, unilateral brain tissues were lysed and homogenized with 1 ml RIPA buffer contained with protease and phosphatase inhibitors through sonication, followed by centrifugation at 12,000 rpm for 30 min at 4\u0026deg;C, and then collect supernatant. The concentration of protein was determined using the BCA kit (Epizyme, Shanghai, China). Adjusting the protein samples with different concentrations to the same concentration according to the detected concentration. Each sample with an equal volume of protein was loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to polyvinylidene fluoride (PVDF) membranes. Membranes containing protein were blocked with 5% non-fat milk (Epizyme, Shanghai, China) or protein free rapid blocking buffer (Epizyme, Shanghai, China) and then incubated with the following primary antibodies overnight at 4 ℃, including nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) (P60622R3, 1:1000, Abmart, Shanghai, China); apoptosis-associated speck-like protein containing a CARD (ASC) (10500-1-AP, 1:5000, Proteintech, Wuhan, China); gasdermin-D (GSDMD) (GB114198-100, 1:500, Servicebio, Wuhan, China); caspase1 (22915-1-AP, 1:2000, Proteintech, Wuhan, China); caspase11 (647201, 1:500, BioLegend, San Diego, USA); interleukin-1β (IL-1β) (16806-1-AP, 1:2000, Proteintech, Wuhan, China); interleukin-18 (IL-18) (60070-1-Ig, 1:2000, Proteintech, Wuhan, China); tumor necrosis factor-α (TNF-α) (PY19810S, 1:1000, Abmart, Shanghai, China); toll-like receptor 4 (TLR4) (A5258, 1:1000, Abclonal, Wuhan, China); nuclear factor kappa B (NF-κb) p65 (MA9199, 1:500, Abmart, Shanghai, China); p-NF-κB p65 (ab76302, 1:20000; Abcam, Cambrige, UK); claudin-5 (29767-1-AP, 1:5000, Proteintech, Wuhan, China); zona occludens 1 (ZO-1) (21773-1-AP, 1:5000, Proteintech, Wuhan, China); albumin (66051-1-Ig, 1:5000, Proteintech, Wuhan, China); low density lipoprotein-related protein 1 (LRP-1) (26106-1-AP, 1:500, Proteintech, Wuhan, China); p-glycoprotein (P-gp) (22336-1-AP, 1:500, Proteintech, Wuhan, China). Washing with TBS-T for 10 min 3 times, followed by incubating membranes with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) secondary anti-rabbit antibody (GB23303, 1:1000, Servicebio, Wuhan, China), anti-mouse antibody (A0216, 1:1000, Beyotime, Nantong, China), and anti-rat antibody (SA00001-15, 1:1000, Proteintech, Wuhan, China). Immunoblots were observed on the gel imaging system using the enhanced chemiluminescence (ECL) kit (Yeasen, Shanghai, China) and the blots were quantified using ImageJ software to calculate the grayscale value of signals.\u003c/p\u003e \u003cp\u003e16S rRNA Gene Sequencing of Fecal Samples\u003c/p\u003e \u003cp\u003e16s rRNA amplicon sequencing was performed as we described previously with minor modifications\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Total microbiome DNA was extracted from microbiome samples from different sources by CTAB method. The quality of DNA extraction was detected by agarose gel electrophoresis, and the DNA was quantified by ultraviolet spectrophotometer. The V3-V4 region of the small subunit 16S rDNA gene of prokaryotes (bacteria and archaea) was amplified with universal primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-CCTACGGGNGGCWGCAG-3'). PCR products were purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). PCR amplification products were detected by 2% agarose gel electrophoresis, and AMPureXTbeads recovery kit was used for recovery. Purified PCR products were evaluated using the Agilent 2100 Bioanalyzer (Agilent, USA) and Illumina's library quantification kit (Kapa Biosciences, Woburn, MA, USA). The qualified library concentration should be above 2 nM. The qualified online sequencing libraries (Index sequence is not repeatable) were diluted in gradient, mixed in proportion according to the required sequencing amount, and denatured into single strand by NaOH for on-machine sequencing. The NovaSeq 6000 sequencer was used for 2 \u0026times; 250 bp double-ended sequencing with the NovaSeq 6000 SP Reagent Kit (500 cycles).\u003c/p\u003e \u003cp\u003eFor the double-ended data obtained by sequencing, the sample should first be separated according to barcode information, and the joint and barcode sequence should be removed. Then data splicing and filtering are carried out. The filtered data were denoised by DADA2 to generate an amplicon sequence variation (ASV) feature table\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Alpha diversity (α-diversity) analysis mainly evaluates the diversity in the living area through six indexes: observed otus, chao1, simpson, shannon\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Beta diversity (β-diversity) mainly evaluates and analyzes the diversity among habitats (between samples/groups) by calculating distances of jaccard\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. According to the ASV feature table sequence file, we annotated the taxonomy with the database of SILVA v138\u003csup\u003e11,13\u003c/sup\u003e, and made statistics on the abundance of each taxonomy in each sample according to the ASV (feature) abundance table. Mann-Whitney U test was used to compare the differences between two groups of samples with biological duplication.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analyses were performed with GraphPad Prism 9 software. The differences of data among groups were analysed through one-way or two-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test or Mann-Whitney U test. The data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) or median (interquartile range). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGLP-1 treatment improved cognitive deficits in APP/PS1 mice\u003c/h2\u003e \u003cp\u003eIn this study, the effects of GLP-1 on the cognition of APP/PS1 mice at 10 months of age were tested by MWM experiment, which is a very popular method for evaluating spatial learning and memory\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We observed that on the 4th and 5th day of the orientation navigation test phase, the escape latency of AD group mice was significantly longer than that of WT group and GLP-1 treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and G). We then performed a probe trial on 6th day of MWM test where the number of times across the withdrawn platform, the percentage of time spent in the target quadrant and the mean distance to withdrawn platform were logged to estimate spatial memory. The times crossing the platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and H) and the time spent in the target quadrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and H) of mice in AD group were significantly reduced while the mean distance to platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and H) were increased compared with WT group and AD\u0026thinsp;+\u0026thinsp;GLP-1 group. Besides, there was no significant difference in average swimming speed among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In other words, there was no severe visual or movement aberration among the mice.\u003c/p\u003e \u003cp\u003eFurthermore, we also recorded the body weight (BW) of mice every week and determined the serum insulin level after 4-h fast given that GLP-1 has the effect of improving metabolism and reducing BW\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Interestingly, the average BW of mice in the AD group was lower than the WT group, while the BW was further reduced after GLP-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). We found that the insulin level of mice in AD group was higher than WT, while the insulin level was reduced after GLP-1 treatment, although there were no significant differences between all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eTaken together, the results of MWM test uncovered a beneficial effect of GLP-1 on cognitive performance of APP/PS1 mice. The data of metabolism in this study are basically consistent with previous studies on GLP-1, that is GLP-1 reduces body weight and insulin resistance. Although the difference is not significant, it is confirmed that GLP-1 does play a role in vivo.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGLP-1 treatment saved dying neurons in hippocampus of APP/PS1 mice\u003c/h3\u003e\n\u003cp\u003eGiven the results of the above behavioral experiments, we speculated that GLP-1 protected hippocampal neurons in AD group. In order to verify our conjecture, the Nissl staining and immunofluorescence were employed to observe the morphology and quantity of hippocampal neurons. As we predicted, Nissl staining showed that the hippocampal neurons in CA1 region of mice in AD group were stained deeper, shrunk and showed necrotic pathological morphology compared to the WT group, while the morphology of neurons were recovered to normal after treating with GLP-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B and C). Consistent with the results of Nissl staining, immunofluorescence displayed that GLP-1 completely reversed the loss of NeuN positive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) in the hippocampal CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), CA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), CA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) regions of AD group mice. In short, the morphological results revealed that GLP-1 saved the dying neurons in the hippocampus of mice in AD group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGLP-1 treatment reversed AD pathological phenotypes and improved the synaptic plasticity\u003c/h3\u003e\n\u003cp\u003eAccording to previous reports, the deposition of β-amyloid (Aβ) peptide is a pathological hallmark of AD\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, abnormal morphology and reduced number of synaptic in AD have also been widely reported\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To further verify the protective effect of GLP-1 on the central nervous system, we observed Aβ plaques and synapses in the mice brain by immunofluorescence and TEM, respectively. Interestingly, the present study found that GLP-1 not only reduced the number of Aβ plaques in the cortex of APP/PS1 mice, but also inhibited the deposition of Aβ in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). In addition, we observed that the number of synapses in hippocampus of mice in AD group significantly decreased when compared with WT group via TEM, while the number of synapses in hippocampus of APP/PS1 mice significantly increased after GLP-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). We further analyzed the synaptic morphology, calculated the length of active zone, thickness of post synaptic density (PSD), width of synaptic cleft, and synaptic curvature according to the previous research\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The present study demonstrated that the treatment of GLP-1 significantly increased the length of active zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and F), PSD thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and G) and synaptic curvature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and I) of hippocampal synapses in AD mice, while reduced the width of synaptic cleft (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, and H). In a word, our research clarified that GLP-1 treatment increased synaptic plasticity and reversed the pathological phenotype of AD in APP/PS1 mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGLP-1 treatment attenuated neuroinflammation in APP/PS1 mice\u003c/h2\u003e \u003cp\u003eThe current study further explored the upstream mechanism of GLP-1 reversing the AD phenotype of APP/PS1 mice by western blotting. Given that neuroinflammation has been widely reported in AD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we examined the expression levels of several inflammation-associated proteins. Consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we found that canonical pyroptosis pathway signaling, such as NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B), ASC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and C), GSDMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and D) and caspase-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H), were all significantly upregulated in AD mice brain tissue compared to WT. Interestingly, expression levels of these proteins were significantly down-regulated after GLP-1 treatment. In addition, this research also detected caspase11, a caspase-1-independent pyroptosis pathway in mice, which recognizes lipopolysaccharide (LPS) in cytoplasm\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. What fascinating is that compared with WT group, the protein was significantly up-regulated in the brain tissue of AD group mice, and after GLP-1 treatment, the protein expression level returned to WT level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). It may be due to the existence of LPS in the cytoplasm of brain tissue that can be recognized by caspase11. Next, we explored downstream signals of the pyroptosis pathway, interleukin-1β (IL-1β) and interleukin-18 (IL-18). The two proinflammatory factors were significantly increased in the brain tissue of mice in AD group, and the levels of IL-1β and IL-18 factors were obviously decreased after GLP-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM-O). Since the elevation of caspase11 in AD mice suggests that exogenous LPS may enter brain tissue, we further examined another inflammatory signaling pathway in vivo that recognizes LPS, which is TLR4 and downstream tumor necrosis factor-α (TNF-α), NF-κb p65 and p-NF-κb p65. Consistent with above results, these proinflammatory signaling proteins were significantly upregulated in the brain tissue of AD group mice compared to WT, and treatment with GLP-1 reversed the upregulation of this pro-inflammatory mediator (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGLP-1 treatment improved the blood-brain barrier function of APP/PS1 mice\u003c/h3\u003e\n\u003cp\u003eGiven the above results and previous reports on the gut-brain axis, we speculate that LPS in the gut may be ectopic in the brain tissue of AD mice through damaged blood-brain barrier and intestinal barrier\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. So, we determined the integrity of the blood-brain barrier in mice. We detected the tight junction proteins Claudin-5 and ZO-1 of BBB. As expected, the expressions of Claudin-5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B) and ZO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and C) in AD group were significantly decreased compared with WT, but they were increased after GLP-1 treatment. Next, we determined whether there is blood-brain barrier leakage by detecting albumin level in brain tissue. We found that the albumin in brain tissue of mice in AD group increased, which proved that the blood-brain barrier was damaged and protein leakage occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and D). After GLP-1 treatment, the blood-brain barrier was repaired and the blood-brain barrier leakage was reduced. In view of the fact that the damage of blood-brain barrier has occurred in AD mice, we detected the expression level of low-density lipoprotein receptor-related protein 1 (LRP1) and P-glycoprotein (P-gp). It is reported that this protein located in endothelial cells mediates the clearance of Aβ protein in brain tissue\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We found that the expression of these two proteins was down-regulated in the brain tissue of AD mice compared with WT, and GLP-1 treatment saved their loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, E and F). It can be seen that the mice in AD group not only face the invasion of enterogenous LPS, but also suffer the damage caused by the accumulation of Aβ in the brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eGLP-1 treatment alleviated the mitochondrial damage of microglia\u003c/b\u003e\u003c/div\u003e \u003cp\u003eIn view of the severe inflammatory attack in the brain tissue of AD mice, we observed the morphology of microglial cells in the hippocampus of mice by TEM. Of course, we do not rule out the possibility that other cells, such as astrocytes and even neurons themselves, are involved in the inflammatory response. However, as resident macrophages in brain tissue, microglia respond most sensitively and strongly to injury factors. As expected, compared with the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), the mitochondria in the microglia of the mice in AD group were severely damaged, characterized by mitochondrial fragmentation, vacuolation and a decrease in number (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Interestingly, the mitochondrial morphology and number of hippocampal microglia returned to normal after GLP-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNeuroinflammation in APP/PS1 mice may be due to their disturbed gut flora\u003c/h2\u003e \u003cp\u003eIn view of the upregulation of LPS-recognizing proinflammatory mediators in the brain tissue of AD mice, we examined the composition of the intestinal microbiota of mice by 16srRNA amplicon sequencing. Firstly, we observed differences in the composition of gut microbiota between AD and WT mice by determining α and β diversity. α diversity is used to assess the diversity of community composition within a sample\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The α diversity analysis did not show a significant difference in gut microbial community evenness and richness between the two groups of mice based on Chao 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), Observed-otus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), Shannon (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) and Simpson indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Beta diversity was used to assess differences in the overall composition of microbiota between groups\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In this study, principal coordinate analysis (PCoA) based jaccard was used to measure β diversity, but no significant differences were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we analyzed the differential abundance microbiota between the WT and AD group. First, we analyzed the differential abundance of gut microbes between the two groups at the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). We focused on the Firmicutes and Bacteroides ratio (F/B), which is reported to reflect different disease states\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Compared with WT group, F/B value in AD group was lower but not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Subsequently, we analyzed the differential abundance taxa by method of Linear discriminant analysis Effect Size (LEfSe) and heatmap. At genus level, \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eHerminiimonas\u003c/em\u003e was abundant in WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). While \u003cem\u003eErysipelotrichaceae_unclassified, Alistipes, Desulfovibrionaceae_unclassified, Candidatus_Saccharimonas\u003c/em\u003e, etc., were enriched in AD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, APP/PS1 mice were treated with GLP-1 analog Semaglutide. Through behavioral experiments, we found that GLP-1 improved the cognitive function of AD mice. Furthermore, we observed the morphology of brain tissue of AD mice, and found that GLP-1 treatment reduced damage of neuron, aggregation of Aβ plaques in hippocampus, and increased synaptic plasticity of hippocampal neurons. We further detected the expression of inflammatory factors in the brain tissue of mice, and found that GLP-1 reversed the canonical caspase-1-dependent pyroptosis pathway, the caspase-11-dependent noncanonical pyroptosis pathway and the TLR4/NF-κb pathway activated in the brain tissue of AD mice. In addition, this study also indicated that GLP-1 treatment reduced the damage of blood-brain barrier and microglia mitochondria in AD mice. Finally, we found by amplicon sequencing that compared with WT mice, the abundance of pro-inflammatory bacteria such as \u003cem\u003eErysipelotrichaceae_unclassified, Alistipes, Desulfovibrionaceae_unclassified, Candidatus_Saccharimonas\u003c/em\u003e, etc. increased, while the abundance of anti-inflammatory bacteria such as \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eHerminiimonas\u003c/em\u003e, etc. decreased. We speculated that the increase of pro-inflammatory bacteria in AD mice may damage the intestinal barrier and cause intestinal leakage, and the pyrogen in the intestine enters the circulation and leads to low-grade inflammatory reaction in the whole body, and at the same time enters the center never system through the damaged blood-brain barrier of AD mice, causing neuroinflammation. GLP-1 may reduce the systemic inflammatory response of AD mice by direct inhibition or by repairing the damaged intestinal barrier and blood-brain barrier.\u003c/p\u003e \u003cp\u003eResearchers have found that the brain of AD showed increased levels of lipid peroxidation, microglia/astrocyte activation, and overexpression of pro-inflammatory cytokines (TNF-α, IL- 6) through autopsies of AD patients\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the pyroptosis pathway is activated in AD mice\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The present research found that not only the canonical pyroptosis pathway dependent on caspase1 was activated, but also the noncanonical pyroptosis pathway dependent on caspase11 was activated. Caspase-11 in mice was reported to respond to various bacterial infections, and could respond to the stress of cytoplasmic LPS, and then activating pyroptosis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Although previous studies have found that the expression of caspase11 is increased in aging mice\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, the up-regulation of caspase11 in AD in vivo model has not been reported before. Therefore, we speculated that there is a low-grade inflammatory reaction induced by LPS in the brain tissue of AD mice. In order to verify our conjecture further, the current study detected the level of toll-like receptor 4 and its downstream inflammatory signal. In fact, the up-regulation of TLR4 and its downstream signals NF-κb and TNF-α in AD mice has been widely reported\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Previous reports showed that intestinal dysfunction and pyrogen displacement in AD mice led to the increase of LPS in circulation and activated inflammatory signaling pathway in vivo\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, we sequenced 16srRNA amplicon in mouse feces and found that \u003cem\u003eErysipelotrichaceae_unclassified\u003c/em\u003e, \u003cem\u003eAlistipes, Desulfovibrionaceae_unclassified\u003c/em\u003e, and \u003cem\u003eCandidatus_Saccharimonas\u003c/em\u003e, etc. increased, while the abundance of \u003cem\u003eAkkermansia\u003c/em\u003e and \u003cem\u003eHerminiimonas\u003c/em\u003e, etc. decreased. Previous reports generally believed that \u003cem\u003eAlistipes\u003c/em\u003e is associated with anxiety and depression\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and is enriched in AD mice\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Interestingly, Zihao Ou et al. found that APP/PS1 mice supplemented with AKK significantly improved the disease phenotype\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In view of the fact that enterogenous pyrogens in blood still need to permeate the blood-brain barrier (BBB) to enter the central nervous system, we also detected the expression of BBB-related proteins. It was found that the blood-brain barrier of AD mice was damaged. In addition, we also found that the expression of LPR1\u003csup\u003e27\u003c/sup\u003e and P-gp\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e related to Aβ clearance also decreased.\u003c/p\u003e \u003cp\u003eGlucagon like peptide-1 (GLP-1) is a kind of peptide gastrointestinal hormone secreted by the intestinal tract, which plays an important role in the regulating of postprandial glucose homeostasis via acting upon insulin secretion, food intake and gastrointestinal motility\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Inadequate secretion of GLP-1 increases the risk of metabolic diseases, such as type 2 diabetes and obesity\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Studies have shown that GLP-1 is able to cross the blood-brain barrier and act on GLP-1 receptors in the central nervous system (CNS)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Semaglutide, a GLP-1 analogue of a modified once-weekly agent based on liraglutide, has become a first-line drug in the clinical treatment of obesity and type 2 diabetes in the past few years\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, in addition to its excellent performance in improving cardiovascular and cerebrovascular outcomes in metabolic diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Interestingly, more and more researchers have shown great interest in the role of GLP-1 in non-metabolic diseases such as nervous system diseases in recent years\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The clinical research published in 2017 suggested that GLP-1 analogue exenatide may benefits motor function in patients with Parkinson's disease (PD)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, two recent studies have come to conflicting conclusions. Studies by Wassilios G Meissner et al. suggested that Lixisenatide group had a lower progression of dyskinesia than placebo group in patients with early PD 12 months after treatment\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, while Andrew Mc Garry et al. demonstrated that another GLP-1 analogue, NLY01-a, did not improve the dyskinesia symptoms of PD patients\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. This indicates that not all GLP-1 analogues can play a neuroprotective role, and of course, it does not rule out the individual differences of patients in different cohorts. A randomized controlled trial (RCT) published in 2023 showed that liraglutide could restore the impaired associative learning of obesity through mesoaccumbens pathway\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In fact, it has been widely reported that GLP-1 can alleviate neuroinflammation and pathological phenotype; improve neurological function as well as delay the progress of neurodegenerative diseases through anti-inflammation and antioxidation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. However, the researches on the role of GLP-1, especially Semaglutide, in the treatment of AD is limited so far. A study in vitro showed that Semaglutide treatment reduced the damage of Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e to SH-SY5Y cells by promoting autophagy suppressing apoptosis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. A recent study confirmed in vivo and in vitro that Semaglutide treatment can improve glucose metabolism in brain tissue and cells through GLP-1R/SIRT1/GLUT4 pathway to alleviate the disease phenotype of AD mice and the damage of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e to hippocampal neurons HT22 cells\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Interestingly, Joseph Bailey et al recently published a study showing that GLP-1 receptor activation can improve brain pericyte function, thus restore vascular integrity and blood-brain barrier function in diabetic patients, and reduce diabetes-induced cognitive impairment in mice\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. On the basis of previous researches, the current study confirmed for the first time that GLP-1 analogue Semaglutide restored the blood-brain barrier of APP/PS1 mice, and alleviated the neuroinflammation in the brain tissue of APP/PS1 mice, which may be caused by enterogenous pyrogen. In addition, GLP-1 also upregulated LRP1 and P-gp regarded as Aβ clearance-associating protein in the blood-brain barrier of AD mice.\u003c/p\u003e \u003cp\u003eTo sum up, this study found that the pyrogen in the intestine of APP/PS1 mice shifted to the circulation and entered the CNS through the damaged BBB, which led to severe neuroinflammation and aggravated the phenotype of AD. However, the treatment of GLP-1 analogue Semaglutide improved the structure and function of BBB in APP/PS1 mice, reduced the damage of microglia mitochondria, neuroinflammation and aggregation of Aβ plaques, increased the number of normal neurons and synaptic plasticity, and further improved the cognitive function of APP/PS1 mice.\u003c/p\u003e \u003cp\u003eThere are some limitations in the present study. First, we did not observe whether the intestinal barrier of APP/PS1 mice was damaged. Secondly, we did not investigate the effect of GLP-1 on gut microbiota of AD mice. In addition, we did not detect the inflammatory state in peripheral circulation. Finally, this study is only performed in animal models, and has not been extended to clinical research, nor has it been explored in depth in vitro.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGLP-1 Semaglutide repaired the structure and function of BBB in APP/PS1 mice, reduced neuroinflammation and AD pathological phenotype of APP/PS1 mice, and improved the cognitive function of APP/PS1 mice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, Alzheimer\u0026apos;s disease; GLP-1, glucagon-like peptide 1; APP/PS1, amyloid precursor protein / presenilin 1; A\u0026beta;, amyloid-\u0026beta;; NLRP3, NOD-like receptor thermal protein domain associated protein 3; TLR4, toll-like receptor 4; BBB, blood-brain barrier; CNS, central nervous system; T2DM, type 2 diabetes mellitus; SPF, specific-pathogen-free; WT, wild-type; NIH, National Institutes of Health; ELISA, Enzyme-linked immunosorbent assay; MWM, Morris water maze; TEM, Transmission electron microscopy; SDS-PAGE, sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; ASC, apoptosis-associated speck-like protein containing a CARD; GSDMD, gasdermin-D; IL-1\u0026beta;, interleukin-1\u0026beta;; IL-18, interleukin-18; TNF-\u0026alpha;, tumor necrosis factor-\u0026alpha;; NF-\u0026kappa;b, nuclear factor kappa B; ZO-1, zona occludens 1; LRP-1, low density lipoprotein-related protein 1; P-gp, p-glycoprotein; HRP, horseradish peroxidase; IgG, immunoglobulin G; ECL, enhanced chemiluminescence; ASV, amplicon sequence variation; \u0026alpha;-diversity, Alpha diversity; \u0026beta;-diversity, Beta diversity; ANOVA, analysis of variance; SEM, standard error of the mean; ASV, amplicon sequence variation; \u0026alpha;-diversity, Alpha diversity; \u0026beta;-diversity, Beta diversity; SEM, standard error of the mean; BW, body weight; PSD, post synaptic density; LPS, lipopolysaccharide; principal coordinate analysis (PCoA); Linear discriminant analysis Effect Size (LEfSe); Parkinson\u0026apos;s disease (PD); randomized controlled trial (RCT).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81801169, 81870952, 82001303, 82371255, 82071258), Shanghai Science and Technology Innovation Action Plan (23DZ2291500), Natural Science Foundation of Shanghai (24ZR1457100), Project of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (ynnkxyb202412), Program for Shanghai Outstanding Academic Leaders(23XD1402500), Program for Outstanding Medical Academic Leader of Shanghai (2022LJ011), and Training program for research physicians of innovative translational ability (SHDC2022CRD037).\u003c/p\u003e \u003cp\u003eConflicts of Interest: The authors declare no conflict of interest.\u003c/p\u003e \u003cp\u003eData Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003cp\u003e Ethics declaration: All procedures were approved by the ethical committee on animal welfare of Shanghai Sixth People\u0026rsquo;s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in accordance with the principles outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXiong X, et al. 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Diabetologia. 2022;65:1541\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00125-022-05730-5\u003c/span\u003e\u003cspan address=\"10.1007/s00125-022-05730-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, Semaglutide, APP/PS1, inflammation, gut microbiota, blood brain barrier","lastPublishedDoi":"10.21203/rs.3.rs-6098406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6098406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and purpose:\u003c/h2\u003e \u003cp\u003eAlzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction, which is common in the elderly. In recent years, it has been reported that glucagon-like peptide 1 (GLP-1) analogues have neuroprotective function. However, the mechanism of GLP-1 analogues improving neurological function has not been fully clarified. This study attempts to clarify the mechanism of GLP-1 alleviating AD phenotype.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eIn this study, a modified once-weekly GLP-1 analogue, Semaglutide, was used to treat 8-month-old amyloid precursor protein / presenilin 1 (APP/PS1) transgenic mice. By means of ethology, molecular biology and 16s rRNA amplicon sequencing, it was confirmed that Semaglutide alleviated the disease phenotype of APP/PS1 mice.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eGLP-1 improved the behavioral performance of APP/PS1 mice, reduced neuronal damage and aggregation of amyloid-β (Aβ) plaques, and enhanced synaptic plasticity. GLP-1 also attenuated pyroptosis mediated by NOD-like receptor thermal protein domain associated protein 3 (NLRP3), inflammatory reaction mediated by toll-like receptor 4 (TLR4) and mitochondrial damage of microglia as well as improved the structure and function of blood-brain barrier (BBB) in AD mice.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eGLP-1 may repair the blood-brain barrier to alleviate the central nervous system injury caused by the displacement of pyrogen in gut of AD mice.\u003c/p\u003e","manuscriptTitle":"Semaglutide Ameliorates Neuroinflammation Caused by Enterogenous Pyrogen in APP/PS1 Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 16:59:11","doi":"10.21203/rs.3.rs-6098406/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"287c7436-6a75-41f9-b32a-4a44b65751c2","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-19T00:00:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 16:59:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6098406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6098406","identity":"rs-6098406","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}