Liraglutide alleviates sepsis-induced encephalopathy via attenuating neuronal damage, glial cell activation and mitochondrial dysfunction in a mouse model of sepsis

Liraglutide alleviates sepsis-induced encephalopathy via attenuating neuronal damage, glial cell activation and mitochondrial dysfunction in a mouse model of sepsis | 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 Liraglutide alleviates sepsis-induced encephalopathy via attenuating neuronal damage, glial cell activation and mitochondrial dysfunction in a mouse model of sepsis Hongyu Yi, Yan Jiao, Haoran He, Linjue Wang, Jie Hu, Yating Cui, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7340017/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 Sepsis-induced encephalopathy (SAE) affects about 70% of patients with sepsis and therefore leads to poor prognosis and long-term cognitive dysfunction. Unfortunately, there are still no effective pharmacological treatments to better manage this kind of encephalopathy. Recently, glucagon-like peptide 1 receptor (GLP-1R) agonists have gained much attention due to their neuroprotective effects in neurodegenerative diseases and brain injuries. In this study, we evaluated the potential effects of intracerebroventricular injection of Liraglutide, a kind of GLP-1R agonists, in SAE mice. We found that Liraglutide injected via intracerebroventricular improved neurological deficits and attenuated neuronal loss and degeneration, and glial cell activations in the hippocampus of septic mice. In vitro studies demonstrated that Liraglutide inhibited the interaction between microglia and neurons under LPS stimulation. Furthermore, Liraglutide restrained oxidative distress and mitochondria damage in hippocampal neurons. Mechanically, Liraglutide restored the downregulation of p-AKT but reversed the phosphorylation of STAT3 in hippocampal neurons. Collectively, these results indicated that the administration of GLP-1R agonist Liraglutide might exert neuroprotective effects on sepsis-induced brain impairments. Sepsis-induced encephalopathy Glucagon-like peptide-1 Liraglutide neuron damage mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Brain is one of the most vulnerable organs that is often affected in patients with sepsis. Up to 70% of sepsis patients underwent brain dysfunction ranging from confusion to delirium or even coma, which could be diagnosed as sepsis-induced encephalopathy (SAE)Czempik, Pluta (1). Although it was reported that SAE is closely associated with mortality risk and long-term cognitive impairment in septic patients[ 2 ], there are still no effective treatments to help reverse this sepsis-induced brain dysfunction. Systemic inflammation leads to blood brain barrier (BBB) disruption and therefore induces the activations of glial cells including microglia and astrocytes. During sepsis, activated microglia facilitate neuroinflammation and neuronal death, while astrocytes disrupt the neurotransmitter metabolism as well as the integrity of BBB[ 3 ]. Besides, oxidative stress and mitochondrial dysfunction also impair the homeostasis of the central nervous system (CNS) and thus exacerbate neural dysfunction in sepsis[ 4 ]. Glucagon-like peptide 1 (GLP-1) is a peptide hormone produced in enteroendocrine cells of the gastrointestinal tract, as well as other tissues such as the brain. GLP-1 exerts its biological action via binding to the GLP-1 receptor (GLP-1R) and it has broad pharmacological effects ranging from the glucose-dependent stimulation of insulin secretion, inhibition of food intake, and improvement of insulin resistance[ 5 ]. Besides, due to the widespread distribution of GLP-1R in the human and animal body, GLP-1 also contributes to the cardio- and neuroprotection, inhibition of inflammation and apoptosis, and amelioration of cognitive dysfunction especially in neurodegeneration diseases such as Alzheimer’s and Parkinson’s disease (AD and PD)[ 6 ]. It has been reported that the level of peripheral GLP-1 is increased in sepsis patients, which indicates a poor prognosis[ 7 ]. Additionally, GLP-1R agonists achieved promising therapeutic potential in pre-clinical and clinical studies of sepsis[ 5 , 7 ]. However, to our knowledge, the role of GLP-1 in SAE has not been reported to date. In the current study, we demonstrated that the central GLP-1R agonist administration (via Intracerebroventricular injection of Liraglutide) could alleviate the neural damage, glial activation, and mitochondrial dysfunction in the hippocampal of sepsis mice via mediating the AKT/STAT3 pathway. Materials and methods Animals Adult male C57BL/6J mice (8-12 weeks old, 20-25 g) were enrolled in this study. All mice were housed under specific pathogen-free conditions on a 12-h light/dark cycle with controlled temperature (22 2 ) and free access to food and water before the experiments. All experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital (Beijing, China). Experimental design of in vitro and in vivo studies The study design of in vivo experiments was presented in Fig. 1A. Briefly, C57BL/6J mice were randomly divided into three groups: the sham group, the CLP group, and the CLP+Lira (Liraglutide, Selleck) group. Mice in each group received PBS or Liraglutide (intracerebroventricular, i.c.v) 1 h before CLP. The mouse brain was obtained 1 d after CLP for western blotting, transmission electron microscopy, and immunohistochemistry analyses. A modified neurological severity score was performed at 0, 1, 3, and 5 d following the CLP procedure. Weight loss and food intake of mice in each group were monitored until the animals were sacrificed. In vitro experiments were conducted in BV2 and HT22 cells. For conditioned medium collection (Fig. 1B), BV2 cells were pre-treated with Liraglutide (500 nM) for 1 h, followed by exposure to LPS (1 μg/ml, sigma) for 24 h. Subsequently, the culture medium of BV2 cells in each group was collected as conditioned medium and then used to culture HT22 cells for another 24 h. For transwell assay (Fig. 1C), HT22 cells were seeded into the lower inserts (Corning) and pre-treated with Liraglutide (500 nM) for 1 h, followed by the exposure to LPS (1 μg/ml) for 48 h. BV2 cells were seeded in the upper chamber. After 48 h, the migration of BV2 cells was analyzed. Cecal ligation and puncture (CLP) procedure CLP was used to induce the mice model of sepsis as previously described[8]. Mice were anesthetized with 1% pentobarbital sodium (40 mg/kg) and then a midline incision (1 cm) was made in the abdomen. Murine cecum was ligated at half the distance between the distal pole of cecum and the ileocecal junction. Subsequently, the cecum was punctured with a 22-gauge needle and fecal contents were squeezed into the peritoneal cavity. Sham group mice underwent the same operation but without CLP. All animals received 40 ml/kg sterile saline via subcutaneous injection and returned to home cage with a warm cotton pad with free access to food and water. Intracerebroventricular injection The intracerebroventricular (i.c.v) injection was conducted using a stereotaxic apparatus as previously described[9]. The stereotaxic device was inserted into the lateral ventricle (the coordinates were 1.0 mm lateral and 0.5 mm posterior to the bregma over the left hemisphere, and 2.5 mm below the dural surface), then Liraglutide (2 μg/kg in 2 μl PBS) was injected slowly into the lateral ventricle. And mice in sham and CLP groups received the same volume of PBS via i.c.v administration. Modified neurological severity score test (mNSS) A modified neurological severity score (mNSS) was used to evaluate the neurological function of each group of mice at 0, 1, 3, and 5 d following sham or CLP operation as previously described[10]. Briefly, the severity score is composed of motor, sensory and reflex tests. The mice which cannot perform the tasks or lack a tested reflex get 1 point. A score of 10 to 14 represents severe injury; 5 to 9 represents moderate injury; 1 to 4 represents mild injury. TUNEL staining TUNEL assays were used to determine the apoptosis of the hippocampus in mice. Tissue slides were fixed with 4% paraformaldehyde and then the TUNEL staining was conducted according to the manufacturer's protocol (Beyotime). DAPI staining followed with TUNEL was used to assess the nuclear morphology. The slides were viewed using an OLYMPUS inverted microscope. Measurement of mitochondrial membrane potential (MMP) The mitochondrial membrane potential was determined using the JC-1 mitochondrial membrane potential assay kit (Beyotime) according to the manufacturer's instructions. Briefly, HT22 cells were incubated with JC-1 staining working solution for 20 min at 37 and washed with JC-1 staining wash buffer. The representative figures were obtained using an OLYMPUS fluorescence microscope, and fluorescence intensity was determined using ImageJ software (National Institutes of Health). Intracellular reactive oxygen species (ROS) detection The intracellular ROS of HT22 cells that underwent LPS stimulation with or without Liraglutide/Exendin-9 pre-treatment was measured using a ROS assay kit (Beyotime). Briefly, HT22 cells were incubated with 100 μM DCFH-DA for 20 min at room temperature to detect oxidation level of DCFH-DA into the fluorescent compound DCF. The cells were viewed using an OLYMPUS fluorescence microscope. Immunofluorescence staining All mice were anesthetized with 1% pentobarbital sodium and then intracardially perfused with saline solution and 4% paraformaldehyde. Afterward, the brain tissues were collected, post-fixed with 4% paraformaldehyde overnight, and sliced into 10-μm-thick sections using a cryostat. Being blocked with 3% BSA, the tissues were incubated with specific primary antibodies overnight at 4 , followed with the incubation of CoraLite488-conjugated or CoraLite594-conjugated secondary antibodies (proteintech) for 1 h at room temperature. The cell nuclei were stained with DAPI (Southern Biotech) and then view the sections using a confocal microscope (OLYMPUS). Immunohistochemistry Brain tissue sections were incubated for 1 h in 5% BSA with 0.3% Triton X-100 in double distilled water (ddH 2 O) and then incubated with specific primary antibodies overnight at 4 . Afterward, the slices were incubated with the given secondary antibodies for 1 h at room temperature. The positive cells were indicated by adding DAB to the tissues. Tissue images were captured using a light OLYMPUS microscope. Nissl staining Mouse brain tissues were fixed with 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, and paraffin-embedded and then brains were cut into 5 μm thick sections. Tissue sections were stained with 0.5% toluidine blue and observed using a light microscope (OLYMPUS). Fluoro-Jade B staining Brain sections underwent Fluoro-Jade B (FJB) staining to detect neuron degeneration following CLP. Briefly, slides were hydrated in graded ethanol and distilled water and incubated with 0.06% potassium permanganate for 10 min. Subsequently, 0.0025% FJB solution was added to the slides and incubated for 30 min in the darkness. Images were observed using a fluorescence microscope (Nikon ECLIPSE CI), and degenerated neurons in the DG region of the hippocampus (200 ) were counted in each slide. Transmission electron microscopy (TEM) The mouse hippocampus (1 mm 3 ) was fixed with 2.5% glutaraldehyde at 4 overnight, post-fixed in 1% osmium tetroxide for 2 h, and then dyed in 2% uranyl acetate and dehydrated in graded ethanol. The tissues were embedded in 100% acetone overnight, cut into 60-80 nm sections, and contrasted with 2% uranyl acetate for 15 min and lead citrate for 15 min. The images were captured using a transmission electron microscope (HT-7700, Hitachi Corporation). Cell culture BV2 cells (mouse microglia cell line) and HT22 cells (mouse hippocampus neuron cell line) were all purchased from American Type Culture Collection (ATCC) and cultured using DMEM (Gibco) supplemented with 100 U/ml penicillin/streptomycin and 10% fetal bovine serum (FBS, WISENT CORPORATION) in a humidified incubator at 37 with 5% CO 2 . The transwell assay HT22 (4 10 5 ) were seeded into the bottom chamber with an 8 μm pore transwell insert (Corning) in a 6-well plate. After 24 h, BV2 cells were resuspended in 1.5 ml FBS-free DMEM and seeded into the upper inserts. HT22 cells were divided into 3 groups: Control, LPS, and LPS+Lira groups. HT22 cells were pre-treated with Liraglutide for 1 h in the LPS+Lira group, then LPS was added into the lower chamber for an additional 48 h. HT22 cells in the control group were treated with PBS. After the co-culture, BV2 cells in the upper inserts were fixed with 4% paraformaldehyde (PFA, Servicebio) followed by the staining with crystal violet (Beyotime). Images were taken using an OLYMPUS microscope and the number of migrating cells was analyzed using ImageJ software (National Institutes of Health). Cell viability assay Cell viability was determined using a cell count kit-8 (CCK-8, Dojindo) according to the manufacturer’s protocol. Briefly, cells (4 10 3 ) were plated in a 96-well plate. After being treated with specific stimulations for 24 h, cells were washed twice with PBS. CCK-8 was added to the culture medium at a ratio of 1:10 and incubated at 37 for 1 h. The microplate reader (Tecan Spark) was utilized to measure the absorbance at 450 nm. Western blotting Whole mouse hippocampus was lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM NaN3, 5 mM EDTA, 0.1% SDS, and 2% Triton X-100) containing a mixture of protease inhibitors and PhosSTOP Phosphatase Inhibitor (Roche Molecular Biochemicals) for 30 min at 4 . The lysates were obtained after centrifugation at 12,000 × g to remove nuclei. Equal volumes and amounts of protein were electrophoresed and separated by an SDS-PAGE and transferred to a nitrocellulose membrane (Millipore). After the membrane was blocked using TBST containing 5% skim milk, the membrane was incubated with primary antibodies at 4 overnight followed by incubation with an HRP-conjugated secondary antibodies for 1 h at 37 . The membrane was visualized using ECL (Millipore) and analyzed by ImageJ software. Statistics Statistical analysis was performed using GraphPad Prism 8.0.0 (Graph Pad Software). All quantitative data in this study were presented as mean SEM. Data of Weight Loss, Food-intake and mNSS after CLP were analyzed using two-way ANOVA for repeated measures, followed by the Tukey post hoc test for multiple comparisons. All other variables were analyzed using the Student’s t test (for comparisons between two groups) or one-way ANOVA followed by Tukey’s multiple test (for comparisons among three or more groups). P < 0.05 was defined as statistically significant. Results GLP-1R agonist ameliorates neurological deficits in septic mice without affecting their weight and food-intake after CLP Studies found that GLP-1R expresses ubiquitously in normal human and mouse brains[11]. Here, we detected the level of GLP-1R in the septic animal model and found that CLP operation up-regulated the expression of GLP-1R in the mouse brain than that of in the sham group (Fig. 2A). GLP-1R agonists contribute to obesity control mainly via the regulation of food intake through the brain GLP-1 signaling[12]. However, we monitored the weight loss and food-intake of CLP mice received i.c.v administration of PBS or Liraglutide but did not find any difference between these two groups (Fig. 2B,C). We then evaluated the neurological deficits in each group of mice using mNSS. Results suggested that CLP mice exhibited more severe neurological damage compared to sham mice at 1, 3, and 5 d after CLP or sham procedure (Fig. 2D). Interestingly, Liraglutide administration via i.c.v successfully promoted neurological recovery at 1 and 3 d after CLP (Fig. 2D). Taken together, these findings reveal that sepsis up-regulated the GLP-1R expression in the mouse brain and the i.c.v administration of Liraglutide relieved the SAE-induced neurological deficits without influencing the food-intake of CLP mice. Liraglutide improves neuronal loss while reducing glial cell activation in the hippocampus of septic mice Hippocampus is known to express abundant pattern recognition receptors (PRRs) and therefore is vulnerable to systemic pro-inflammatory responses[13]. According to Nissl staining, we observed that the neuronal loss and degeneration within the ipsilateral CA1, CA3 and dentate gyrus (DG) regions of the hippocampus were more severe in CLP mice compared to sham group (Fig. 3A-E). Liraglutide administration partially reversed the neuronal damage in CLP+Lira group (Fig. 3A-E). Similarly, Fluoro-Jade B staining used to label the degenerated neurons reflected that Liraglutide partially diminished the number of degeneration neurons in the hippocampal DG section of SAE mice (Fig. 3F,G). We also assessed the glial cell activation in the hippocampus of each group. Results suggested that Liraglutide effectively reduced the number of Iba-1 positive microglia in the hippocampus of septic mice (Fig. 4A,B). Additionally, morphology analysis demonstrated that the hippocampal microglia in CLP mice showed an ameboid morphology with an enlarged cell body, thick and shrunk processes of activated microglia. In contrast, microglia in the hippocampus of mice in sham and CLP+Lira group exhibited more resting morphology with a thin cell body, fine and long processes (Fig. 4A). Similarly, Liraglutide significantly suppressed the activation of astrocytes as depicted by the diminished number of GFAP positive cells in the hippocampus of mice treated with Liraglutide, when compared to that of CLP group (Fig. 4C,D). Collectively, these results imply that Liraglutide exerted neuroprotective effects by mitigating the loss of neurons and glial cell activation in septic mice. Liraglutide treatment rescues neuronal death via mediating the interaction between microglia and neurons Next, we detected the apoptosis in the hippocampus of each group. Based on TUNEL staining, we found that DG, not CA1, CA2, or CA3 regions of the hippocampus in CLP mice, exhibited more significant neuronal death compared to sham group (Fig. 4A,B). Liraglutide administration reversed this effect, especially in the DG region of the hippocampus (Fig. 5A,B). The above results confirmed that GLP-1R agonist reduced microglial activation in septic mice, and we therefore investigated the possible effect of Liraglutide on the interplay between microglia and neuron via in vitro experiments. Firstly, we collected the conditioned medium (CM) of BV2 cells treated with LPS in the presence of Liraglutide or not (Fig. 5C). CCK-8 results showed that HT22 treated with LPS+Lira-CM showed improved cell viability compared to LPS-CM group (Fig. 5D). On the other hand, the migration of BV2 in the presence of HT22 was then evaluated using transwell assay. Results suggested that the BV2 migration was enhanced in LPS-treated HT22 group, which was hampered by the Liraglutide pre-treatment (Fig. 5E,F). Together, these results demonstrated that the activation of GLP-1R improved neuronal damage partially by intervening in the connection between microglia and neurons. Mitochondria damage and ROS production were relieved by GLP-1R agonist. One of the most representative characteristics of SAE is the morphological changes in mitochondria. According to the transmission electron microscope, we observed that in the hippocampus of mice that underwent CLP procedure, membrane rupture, cristae decreased, and membrane density compressed were more obvious. Whereas mitochondria damage was relatively slight in the hippocampus of CLP+Lira mice (Fig. 6A). Subsequently, we conducted in vitro studies and found that Liraglutide pre-treatment strongly reduced the LPS-induced ROS production in HT22 cells, which was abolished by GLP-1R antagonist Exendin-9 (Ex-9) (Fig. 6B). Furthermore, MMP ( m) level of mitochondria in each group was detected by JC-1 staining. As depicted in Fig. 6C-D, Liraglutide rescued the destruction of MMP ( m) level in LPS-stimulated HT22 cells, which was also reversed by Ex-9. GLP-1R agonist treatment mitigated the inflammatory responses in SAE mice and LPS-treated hippocampal cells via mediating the AKT/STAT3 activation. Excessive inflammation correlates with neuronal death, glial cell activation, and mitochondria destruction[14]. In the following studies, we assessed the possible mechanisms of Liraglutide in terms of its neuroprotective effects. Western blotting analysis showed that the phosphorylation of STAT3 was up-regulated in the hippocampus of CLP mice compared with that of sham and CLP+Lira mice (Fig. 7A,B), while the AKT phosphorylation was restored by Liraglutide in CLP mice (Fig. 7A,C). Consistently, in vitro experiments showed that Liraglutide pre-treatment up-regulated the phosphorylation of AKT, while inhibiting the level of p-STAT3 in LPS-stimulated HT22 cells (Fig. 7D,E). Discussion In the present study, we evaluated the therapeutic role of GLP-1R agonist Liraglutide in SAE mice and LPS-stimulated BV2 or HT22 cells. Firstly, we observed that GLP-1R is up-regulated in the brain of SAE mice and the i.c.v administration of Liraglutide alleviates neuronal damage and degeneration in the hippocampus, without affecting the weight and food-intake of septic mice. In addition, glial cell activation and neural apoptosis in the hippocampus are also partially inhibited in septic mice that received Liraglutide treatment. Then we performed in vitro experiments and found that Liraglutide exerts its neuroprotective effects via dampening the interplay between microglia and nerve cells. Furthermore, Liraglutide administration attenuates mitochondria damage and ROS production, while elevating the MMP level. Mechanically, Liraglutide restores the phosphorylation of AKT, whereas decreasing the STAT3 phosphorylation in the hippocampus of septic mice. Generally, brain is thought to be a relatively sterile organ being resistant to the circulating inflammatory toxins and infiltration of inflammatory cells due to the integrity of BBB. However, the dysregulation of systematic inflammation, elevation of permeability of BBB, microcirculation dysfunction, and activation of neuroendocrine system during sepsis conditions could ultimately induce brain dysfunction, that is so-called sepsis-induced encephalopathy (SAE)[15]. SAE is closely associated with higher mortality and long-term cognitive impairment. But unfortunately, no specific curative or preventive measure for SAE exists. GLP-1 is a kind of incretin and is mainly produced in enteroendocrine cells of the gastrointestinal tract, as well as in the medial nucleus of the solitary tract (NTS) of the medulla oblongata. Apart from the incretin effects, GLP-1 can also regulate inflammatory responses, improve cardiovascular function, and exert neuroprotection effects[16, 17]. It was reported that the peripheral gut-released GLP-1 or systemically administrated GLP-1R agonists can readily cross the BBB of the brain and abundant evidence have revealed the therapeutic potential of peripheral GLP-1R agonists administration for animal or patients with neurodegenerative diseases[18]. However, a clinical study in patients with Type 2 diabetes demonstrated that the cerebrospinal fluid (CSF) concentration of Liraglutide was lower than that of in plasma even following chronic peripheral administration of Liraglutide for 5 months[19]. Similarly, Williams et al. found that the effects of centrally administered GLP-1 can only be antagonized by central GLP-1 antagonist Ex-9, but not peripheral ones[20]. The above studies indicated that the access of peripheral administered GLP-1 into the CNS might be less than we anticipated[21]. Therefore, we treated septic mice with Liraglutide by intraventricular injection. Studies have found that GLP-1 is released in response to inflammatory stimuli, and many observational cohort studies demonstrated that GLP-1 levels are positively correlated with inflammatory markers and poor prognosis in patients with sepsis[22]. The activation of endogenous GLP-1 is thought to be a compensation in response to systemic inflammation in septic patients[7] and the therapeutic potential of GLP-1 agonists has now gained favorable results in a septic animal model[23, 24]. However, the role of GLP-1 in SAE has not been discovered. Studies have suggested that GLP-1R was up-regulated in several animal models of brain injury such as cerebral ischemia and mechanical lesion, especially in glial cells[24-26]. Consistently, we for the first time noticed that GLP-1R is also elevated in the brain of septic mice. It was reported that GLP-1 agonists could suppress the appetite and facilitate weight loss in obese people, without affecting healthy ones[27]. Similarly, our present data showed that Liraglutide administration did not alter the food-intake and weight loss of mice that underwent CLP, which confirmed the safety of GLP-1 agonists in a septic animal model. Neuron loss and damage are quite common in the brain of SAE patients. Brain, especially the hippocampus region has abundant pattern recognition receptors (PRRs) like Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which prompts the binding with inflammatory cytokines and other damage-associated molecular patterns (DAMPs). Sepsis-induced massive pro-inflammatory release into the brain and thus activates various intracellular pathways, leading to neuron dysfunction and death[28]. We found that GLP-1R agonist Liraglutide attenuated neuron loss and damage in the hippocampus of septic mice. Liraglutide administration also reduced the number of hippocampal degenerative neurons as revealed by FJB staining. Glial cells including microglia and astrocytes have vital roles in maintaining CNS homeostasis and normal function. Nevertheless, in patients with neurodegenerative diseases and brain injuries, microglia and astrocytes are overactivated and switch into neurotoxic phenotypes (namely M1 microglia and A1 astrocytes) facilitating neuronal damage[29]. Our present manuscript discovered that Liraglutide significantly impaired glial cell activation and therefore prevent neuronal death in both in vivo and in vitro models of sepsis. Besides, mitochondrial dysfunction has been reported in various brain regions of septic animals and patients, resulting in the outbreak of oxidative stress and DAMPs release[30]. Liraglutide has been reported to modulate mitochondria dynamics and therefore activate mitophagy to improve mitochondria quality in MPTP-induced PD mice[31]. Nevertheless, Liraglutide and exendin-4 may also alleviate the disruption of mitochondrial membrane potential and inhibit PINK1/Parkin-mediated mitophagy in diabetic retinopathy[32] and hypoxia-reoxygenation injury in cultured cardiomyocytes[33]. The above results suggested that GLP-1R agonists play an important role in maintaining mitochondrial function. Similarly, we discovered that Liraglutide significantly attenuated mitochondria disruption in the hippocampus of CLP mice. Furthermore, Liraglutide inhibited the production of ROS and alleviated mitochondria damage as depicted by the elevation of MMP in LPS-treated HT22 cells, which were reversed by Exendin-9, a GLP-1R antagonist. In terms of mechanism, we discovered that Liraglutide restored the phosphorylation of AKT, while down-regulating the level of p-STAT3 in both in vivo and in vitro septic models. It was reported that STAT3 activation correlates with sepsis pathophysiology and the suppression of STAT3 activity could ameliorate organ injuries in sepsis animal models [34]. In addition, STAT3 could induce the activation of the NF-κB pathway, triggering inflammatory responses in human monocytes[35]. Dipeptidyl peptidase-4 inhibitor linagliptin was reported to ease inflammation and improve survival in the CLP model of sepsis through the inhibition of IL-6/JAK/STAT3 signaling[36], while improving the cognitive dysfunction in neurodegeneration diseases via the activation of AKT/PI3K signaling[37, 38]. Conclusions Our present study suggested the possible role of GLP-1R agonists in attenuating SAE through the inhibition of neuronal damage, neuroinflammation, and mitochondrial dysfunction in the hippocampus. This study provides a potential therapeutic target for sepsis-induced encephalopathy. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Clinical trial number Not applicable. Competing Interests All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Funding This research was supported by the National Natural Science Foundation of China (No.81871581) and ‘Field Internal Science’ Army Key Discipline Construction. Authors’ contributions FZ and YW designed and directed the study. HY, YJ and HH performed experiments, wrote, and prepared the manuscript. LW, JH and YC provided materials and technical support and offered constructive proposals. All authors read and approved the final manuscript for publication. Acknowledgements We thank the General Hospital of Tibet Military Command and the First Medical Centre, Chinese PLA General Hospital for all the support. References Czempik PF, Pluta MP, Krzych Ł J. Sepsis-Associated Brain Dysfunction: A Review of Current Literature. International journal of environmental research and public health. 2020;17(16). Peng L, Peng C, Yang F, Wang J, Zuo W, Cheng C, et al. Machine learning approach for the prediction of 30-day mortality in patients with sepsis-associated encephalopathy. BMC medical research methodology. 2022;22(1):183. Denver P, Cunningham C. Microglial activation and neuroinflammation in acute and chronic cognitive deficits in sepsis. Neuropharmacology. 2025;267:110285. Clemente-Suárez VJ, Redondo-Flórez L, Beltrán-Velasco AI, Ramos-Campo DJ, Belinchón-deMiguel P, Martinez-Guardado I, et al. Mitochondria and Brain Disease: A Comprehensive Review of Pathological Mechanisms and Therapeutic Opportunities. Biomedicines. 2023;11(9). Fan W, Zhang Q, Wang C, Sun J, Zhang J, Yin Y. GLP-1 as a regulator of sepsis outcomes: Insights into cellular metabolism, inflammation, and therapeutic potential. International immunopharmacology. 2025;152:114390. Džidić-Krivić A, Fajkić A, Farhat EK, Lekić L, Ejubović A, Vukas SK, et al. Linking Metabolic Syndrome to Neurodegeneration Mechanisms and Potential Treatments. Molecular neurobiology. 2025. Shah FA, Mahmud H, Gallego-Martin T, Jurczak MJ, O'Donnell CP, McVerry BJ. Therapeutic Effects of Endogenous Incretin Hormones and Exogenous Incretin-Based Medications in Sepsis. The Journal of clinical endocrinology and metabolism. 2019;104(11):5274-84. Wang J, Zhu Q, Wang Y, Peng J, Shao L, Li X. Irisin protects against sepsis-associated encephalopathy by suppressing ferroptosis via activation of the Nrf2/GPX4 signal axis. Free Radic Biol Med. 2022;187:171-84. Yin J, Shen Y, Si Y, Zhang Y, Du J, Hu X, et al. Knockdown of long non-coding RNA SOX2OT downregulates SOX2 to improve hippocampal neurogenesis and cognitive function in a mouse model of sepsis-associated encephalopathy. J Neuroinflammation. 2020;17(1):320. Tang Y, Cai B, Yuan F, He X, Lin X, Wang J, et al. Melatonin Pretreatment Improves the Survival and Function of Transplanted Mesenchymal Stem Cells after Focal Cerebral Ischemia. Cell Transplant. 2014;23(10):1279-91. Rocha-Ferreira E, Poupon L, Zelco A, Leverin AL, Nair S, Jonsdotter A, et al. Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy. Brain. 2018;141(10):2925-42. Moiz A, Filion KB, Tsoukas MA, Yu OH, Peters TM, Eisenberg MJ. Mechanisms of GLP-1 Receptor Agonist-Induced Weight Loss: A Review of Central and Peripheral Pathways in Appetite and Energy Regulation. The American journal of medicine. 2025. Wu A, Zhang J. Neuroinflammation, memory, and depression: new approaches to hippocampal neurogenesis. Journal of neuroinflammation. 2023;20(1):283. Mazeraud A, Righy C, Bouchereau E, Benghanem S, Bozza FA, Sharshar T. Septic-Associated Encephalopathy: a Comprehensive Review. Neurotherapeutics. 2020;17(2):392-403. Jacob A, Brorson JR, Alexander JJ. Septic encephalopathy: inflammation in man and mouse. Neurochem Int. 2011;58(4):472-6. Insuela DBR, Carvalho VF. Glucagon and glucagon-like peptide-1 as novel anti-inflammatory and immunomodulatory compounds. European journal of pharmacology. 2017;812:64-72. Zafer M, Tavaglione F, Romero-Gómez M, Loomba R. Review Article: GLP-1 Receptor Agonists and Glucagon/GIP/GLP-1 Receptor Dual or Triple Agonists-Mechanism of Action and Emerging Therapeutic Landscape in MASLD. Alimentary pharmacology & therapeutics. 2025. Reich N, Holscher C. The neuroprotective effects of glucagon-like peptide 1 in Alzheimer's and Parkinson's disease: An in-depth review. Front Neurosci. 2022;16:970925. Christensen M, Sparre-Ulrich AH, Hartmann B, Grevstad U, Rosenkilde MM, Holst JJ, et al. Transfer of liraglutide from blood to cerebrospinal fluid is minimal in patients with type 2 diabetes. International journal of obesity (2005). 2015;39(11):1651-4. Williams DL, Baskin DG, Schwartz MW. Evidence that intestinal glucagon-like peptide-1 plays a physiological role in satiety. Endocrinology. 2009;150(4):1680-7. Trapp S, Brierley DI. Brain GLP-1 and the regulation of food intake: GLP-1 action in the brain and its implications for GLP-1 receptor agonists in obesity treatment. Br J Pharmacol. 2022;179(4):557-70. Lebherz C, Schlieper G, Möllmann J, Kahles F, Schwarz M, Brünsing J, et al. GLP-1 Levels Predict Mortality in Patients with Critical Illness as Well as End-Stage Renal Disease. The American journal of medicine. 2017;130(7):833-41.e3. Yang F, Zeng F, Luo X, Lei Y, Li J, Lu S, et al. GLP-1 Receptor: A New Target for Sepsis. Frontiers in pharmacology. 2021;12. Wong CK, McLean BA, Baggio LL, Koehler JA, Hammoud R, Rittig N, et al. Central glucagon-like peptide 1 receptor activation inhibits Toll-like receptor agonist-induced inflammation. Cell metabolism. 2024;36(1):130-43.e5. Lee CH, Yan B, Yoo KY, Choi JH, Kwon SH, Her S, et al. Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia. Journal of neuroscience research. 2011;89(7):1103-13. Chowen JA, de Fonseca FR, Alvarez E, Navarro M, García-Segura LM, Blázquez E. Increased glucagon-like peptide-1 receptor expression in glia after mechanical lesion of the rat brain. Neuropeptides. 1999;33(3):212-5. Pi-Sunyer X, Astrup A, Fujioka K, Greenway F, Halpern A, Krempf M, et al. A Randomized, Controlled Trial of 3.0 mg of Liraglutide in Weight Management. The New England journal of medicine. 2015;373(1):11-22. Gu M, Mei XL, Zhao YN. Sepsis and Cerebral Dysfunction: BBB Damage, Neuroinflammation, Oxidative Stress, Apoptosis and Autophagy as Key Mediators and the Potential Therapeutic Approaches. Neurotoxicity research. 2021;39(2):489-503. Xiao T, Ji H, Shangguan X, Qu S, Cui Y, Xu J. NLRP3 inflammasome of microglia promotes A1 astrocyte transformation, neo-neuron decline and cognition impairment in endotoxemia. Biochemical and biophysical research communications. 2022;602:1-7. Mantzarlis K, Tsolaki V, Zakynthinos E. Role of Oxidative Stress and Mitochondrial Dysfunction in Sepsis and Potential Therapies. Oxid Med Cell Longev. 2017;2017:5985209. Lin TK, Lin KJ, Lin HY, Lin KL, Lan MY, Wang PW, et al. Glucagon-Like Peptide-1 Receptor Agonist Ameliorates 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Neurotoxicity Through Enhancing Mitophagy Flux and Reducing α-Synuclein and Oxidative Stress. Frontiers in molecular neuroscience. 2021;14:697440. Zhou HR, Ma XF, Lin WJ, Hao M, Yu XY, Li HX, et al. Neuroprotective Role of GLP-1 Analog for Retinal Ganglion Cells via PINK1/Parkin-Mediated Mitophagy in Diabetic Retinopathy. Frontiers in pharmacology. 2020;11:589114. Kobara M, Toba H, Nakata T. A Glucagon-like Peptide 1 Analog Protects Mitochondria and Attenuates Hypoxia-Reoxygenation Injury in Cultured Cardiomyocytes. J Cardiovasc Pharmacol. 2022;79(4):568-76. Xu S, Pan X, Mao L, Pan H, Xu W, Hu Y, et al. Phospho-Tyr705 of STAT3 is a therapeutic target for sepsis through regulating inflammation and coagulation. Cell communication and signaling : CCS. 2020;18(1):104. Guzzo C, Che Mat NF, Gee K. Interleukin-27 induces a STAT1/3- and NF-kappaB-dependent proinflammatory cytokine profile in human monocytes. The Journal of biological chemistry. 2010;285(32):24404-11. Delic D, Klein T, Wohnhaas CT, Feng H, Lin X, Zhang JR, et al. Dipeptidyl peptidase-4 inhibitor linagliptin reduces inflammatory response, ameliorates tissue edema formation, and improves survival in severe sepsis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2025;182:117778. Saleh RA, Eissa TF, Abdallah DM, Saad MA, El-Abhar HS. Peganum harmala enhanced GLP-1 and restored insulin signaling to alleviate AlCl(3)-induced Alzheimer-like pathology model. Scientific reports. 2021;11(1):12040. Athauda D, Gulyani S, Karnati HK, Li Y, Tweedie D, Mustapic M, et al. Utility of Neuronal-Derived Exosomes to Examine Molecular Mechanisms That Affect Motor Function in Patients With Parkinson Disease: A Secondary Analysis of the Exenatide-PD Trial. JAMA neurology. 2019;76(4):420-9. 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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-7340017","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504922360,"identity":"779ce908-ce15-4c23-bd38-dc82fcea2b9a","order_by":0,"name":"Hongyu Yi","email":"","orcid":"","institution":"General Hospital of Tibet Military Command","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Yi","suffix":""},{"id":504922361,"identity":"75bd9f9a-2f34-4a9f-be75-a79023dc3fe2","order_by":1,"name":"Yan Jiao","email":"","orcid":"","institution":"Southwest Hospital, Army Medical University (Third Military Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Jiao","suffix":""},{"id":504922362,"identity":"c2aee413-fbad-434f-b458-a67b02e8241e","order_by":2,"name":"Haoran He","email":"","orcid":"","institution":"General Hospital of Tibet Military Command","correspondingAuthor":false,"prefix":"","firstName":"Haoran","middleName":"","lastName":"He","suffix":""},{"id":504922363,"identity":"8abee55f-b129-4020-aeb8-ea5689610ff7","order_by":3,"name":"Linjue Wang","email":"","orcid":"","institution":"General Hospital of Tibet Military Command","correspondingAuthor":false,"prefix":"","firstName":"Linjue","middleName":"","lastName":"Wang","suffix":""},{"id":504922364,"identity":"71289ee3-9bd7-4184-b462-89bdae3cb3c3","order_by":4,"name":"Jie Hu","email":"","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Hu","suffix":""},{"id":504922365,"identity":"c58183a3-df92-4277-a350-4248f0c0f997","order_by":5,"name":"Yating Cui","email":"","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Cui","suffix":""},{"id":504922366,"identity":"bf8d23df-13b7-4ca2-af3d-d53dbe691aeb","order_by":6,"name":"Yuliang Wang","email":"","orcid":"","institution":"General Hospital of Tibet Military Command","correspondingAuthor":false,"prefix":"","firstName":"Yuliang","middleName":"","lastName":"Wang","suffix":""},{"id":504922367,"identity":"5b32e571-3288-4593-8aa6-504ae4cec0c4","order_by":7,"name":"Feihu Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3PsYrCQBCA4REh2wym3SDEVxhYOK4Q7lWyCFsFEXyBiBAbH+CKewgfYcNwd41gm8JGBCuLlBYBTRArJdHOYv9mmvkYBsDlesNE0kmrYQH8WWILGoatBO2NSM6y74lRTxDwrgTMiLFg3U7EPN1juQ3JromH1I1A8O+qkWC2UIgHRbwkjskbAxqTN5EvqdM+Star+kpMOAWJH40EB7uKUE1i4k+SOmklslORqCYmYiB6gqBOgx/LKsjZZkuKlNf2C4r/gzyWHPY2s3lxKs+hL/ivkdznvbbucrlcrkddAEoqUQ56hng1AAAAAElFTkSuQmCC","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Feihu","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-08-10 16:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7340017/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7340017/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89975339,"identity":"f02db4da-3811-4419-8a22-064c5d387099","added_by":"auto","created_at":"2025-08-27 05:57:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":184888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy design. (A) \u003c/strong\u003eTimeline of mice that underwent sham or CLP surgery received the intraventricular injection of Liraglutide or PBS. \u003cstrong\u003e(B)\u003c/strong\u003e Timeline of the collection of conditioned medium from BV2 cells and cell viability assay of HT22 cell. \u003cstrong\u003e(C)\u003c/strong\u003e Timeline of the transwell assay between BV2 and HT22 cells.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/2c5de60b65ef3cfa18fd2473.png"},{"id":89975341,"identity":"a1564f70-d723-4e05-8206-93d41896bf25","added_by":"auto","created_at":"2025-08-27 05:57:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1R agonist ameliorates neurological deficits in septic mice without affecting the weight and food-intake after CLP.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eImmunofluorescence staining of GLP-1R (green) in the brain at 24 h after CLP (Scale bar=50 μm). \u003cstrong\u003e(B)\u003c/strong\u003e Weight loss of CLP or CLP+Lira mice was observed for 7 d after CLP (n=6 per group). \u003cstrong\u003e(C)\u003c/strong\u003eFood intake of CLP or CLP+Lira mice was observed for 7 d after CLP (n=16 per group). \u003cstrong\u003e(D)\u003c/strong\u003e Effects of Liraglutide on the neurological injury scores of each group were observed at 1, 3 and 5 d after CLP (n=6 for sham group, n=9 for CLP or CLP+Lira group). Results are shown as the mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/8ca4f29ff0cda2adc5e77ecb.png"},{"id":89976639,"identity":"66e3365a-e29c-4fa0-bbeb-c3600c2d3b8a","added_by":"auto","created_at":"2025-08-27 06:05:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":482547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLiraglutide reverses the neuronal loss and degeneration in hippocampus of septic mice. (A-E) \u003c/strong\u003eNissl staining of CA1, CA3 and DG regions (Scale bar=20 μm) in the hippocampus of mice in each group with the quantification representing average number of Nissl positive cells (B) and the number of dark staining neurons in CA1 (C), CA3 (D) and DG (E) regions. \u003cstrong\u003e(F-G) \u003c/strong\u003eRepresentative figures and quantifications of FJB staining in DG region of the hippocampus (Scale bar=100 μm, n=5 per group). Results are shown as the mean ±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/097a8e1aa92534583e8d6ee4.png"},{"id":89978011,"identity":"4b903f1c-515a-4049-8e87-5f9c4182ed1a","added_by":"auto","created_at":"2025-08-27 06:13:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLiraglutide alleviates the glial activation in hippocampus of septic mice. (A-B) \u003c/strong\u003eRepresentative figures and quantifications of immunohistochemistry of Iba-1 in the hippocampus (Scale bar=100 μm). Magnified Iba-1 positive cells were presented in the right side (Scale bar=20 μm). \u003cstrong\u003e(C-D) \u003c/strong\u003eRepresentative figures and quantifications of immunofluorescence staining of GFAP (red) in the hippocampus at 24 h after CLP (Scale bar=50 μm, n=5 per group). Results are shown as the mean \u0026nbsp;± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/81c0110e9c8bc0e471231143.png"},{"id":89975350,"identity":"b32ff95b-51f4-42dc-a8cd-04eafff4e712","added_by":"auto","created_at":"2025-08-27 05:57:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":474062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLiraglutide treatment rescues neuronal death via mediating interaction between microglia and neurons. (A-B) \u003c/strong\u003eRepresentative figures and quantifications of TUNEL staining in the hippocampus (Scale bar=100 μm). \u003cstrong\u003e(C-D)\u003c/strong\u003e HT22 cells were treated with conditioned medium (CM) derived from BV2 and then cell viability was determined using CCK-8 assay. \u003cstrong\u003e(E-F)\u003c/strong\u003eTranswell assay showing the migration of BV2 cells was visualized by crystal violet stain, using HT-22 cells pre-treated with Liraglutide followed with LPS or PBS stimulation for 48 h (Scale bar=50 μm, n=5 per group). Results are shown as the mean ±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***P \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/ca50503c1ebd512da6a86751.png"},{"id":89975347,"identity":"99993727-e665-4f90-9b4b-29b756c4488a","added_by":"auto","created_at":"2025-08-27 05:57:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":394501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1R agonist improves mitochondrial function. (A) \u003c/strong\u003eRepresentative transmission electron micrographs of mitochondria in hippocampus of septic mice (Scale bar=2000 nm). Black arrow: normal mitochondria; Red arrow: damaged mitochondria.\u003cstrong\u003e (B)\u003c/strong\u003e Representative figures of ROS production in LPS-stimulated HT22 cells, with or without the pre-treatment of Liraglutide and Ex-9 (Scale bar=100 μm). \u003cstrong\u003e(C-D)\u003c/strong\u003e Representative figures of JC-1 and quantifications of MMP level in LPS-stimulated HT22 cells, with or without the pre-treatment of Liraglutide and Ex-9 (Scale bar=20 μm, n=5 per group). Results are shown as the mean \u0026nbsp;±SEM. ***P \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/9f8616619632de4cd567e678.png"},{"id":89975349,"identity":"6f607fba-ce18-452d-934d-53583c450ef3","added_by":"auto","created_at":"2025-08-27 05:57:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":279525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1R agonist treatment mitigated the inflammatory responses in SAE mice and LPS-treated HT22 cells via inhibiting the NF-κB/STAT3 pathway. (A-C)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative figures and quantifications of hippocampal p-STAT3, STAT3, p-NF-κB, and NF-κB (n=5 per group). \u003cstrong\u003e(D)\u003c/strong\u003e Representative figures of immunofluorescence staining of p-AKT (red) in LPS-stimulated HT22 cells, with or without the pre-treatment of Liraglutide (Scale bar=10 μm).\u003cstrong\u003e(E)\u003c/strong\u003e Representative figures of immunofluorescence staining of p-STAT3 (green) in LPS-stimulated HT22 cells, with or without the pre-treatment of Liraglutide (Scale bar=10 μm). Results are shown as the mean ±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/f2bcd0ad525ed4b2e7c98656.png"},{"id":91004103,"identity":"fdb68ea5-c7b0-43a6-ab32-142ea3a39ca6","added_by":"auto","created_at":"2025-09-10 14:17:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3690276,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7340017/v1/0a67b637-fd4f-411d-af52-c1f5b3434b9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Liraglutide alleviates sepsis-induced encephalopathy via attenuating neuronal damage, glial cell activation and mitochondrial dysfunction in a mouse model of sepsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBrain is one of the most vulnerable organs that is often affected in patients with sepsis. Up to 70% of sepsis patients underwent brain dysfunction ranging from confusion to delirium or even coma, which could be diagnosed as sepsis-induced encephalopathy (SAE)Czempik, Pluta (1). Although it was reported that SAE is closely associated with mortality risk and long-term cognitive impairment in septic patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], there are still no effective treatments to help reverse this sepsis-induced brain dysfunction.\u003c/p\u003e\u003cp\u003eSystemic inflammation leads to blood brain barrier (BBB) disruption and therefore induces the activations of glial cells including microglia and astrocytes. During sepsis, activated microglia facilitate neuroinflammation and neuronal death, while astrocytes disrupt the neurotransmitter metabolism as well as the integrity of BBB[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Besides, oxidative stress and mitochondrial dysfunction also impair the homeostasis of the central nervous system (CNS) and thus exacerbate neural dysfunction in sepsis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGlucagon-like peptide 1 (GLP-1) is a peptide hormone produced in enteroendocrine cells of the gastrointestinal tract, as well as other tissues such as the brain. GLP-1 exerts its biological action via binding to the GLP-1 receptor (GLP-1R) and it has broad pharmacological effects ranging from the glucose-dependent stimulation of insulin secretion, inhibition of food intake, and improvement of insulin resistance[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Besides, due to the widespread distribution of GLP-1R in the human and animal body, GLP-1 also contributes to the cardio- and neuroprotection, inhibition of inflammation and apoptosis, and amelioration of cognitive dysfunction especially in neurodegeneration diseases such as Alzheimer\u0026rsquo;s and Parkinson\u0026rsquo;s disease (AD and PD)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt has been reported that the level of peripheral GLP-1 is increased in sepsis patients, which indicates a poor prognosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, GLP-1R agonists achieved promising therapeutic potential in pre-clinical and clinical studies of sepsis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, to our knowledge, the role of GLP-1 in SAE has not been reported to date. In the current study, we demonstrated that the central GLP-1R agonist administration (via Intracerebroventricular injection of Liraglutide) could alleviate the neural damage, glial activation, and mitochondrial dysfunction in the hippocampal of sepsis mice via mediating the AKT/STAT3 pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male C57BL/6J mice (8-12 weeks old, 20-25 g) were enrolled in this study. All mice were housed under specific pathogen-free conditions on a 12-h light/dark cycle with controlled temperature (22 2 ) and free access to food and water before the experiments. All experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital (Beijing, China). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eExperimental design of in vitro and in vivo studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study design of in vivo experiments was presented in Fig. 1A. Briefly, C57BL/6J mice were randomly divided into three groups: the sham group, the CLP group, and the CLP+Lira (Liraglutide, Selleck) group. Mice in each group received PBS or Liraglutide (intracerebroventricular, i.c.v) 1 h before CLP. The mouse brain was obtained 1 d after CLP for western blotting, transmission electron microscopy, and immunohistochemistry analyses. A modified neurological severity score was performed at 0, 1, 3, and 5 d following the CLP procedure. Weight loss and food intake of mice in each group were monitored until the animals were sacrificed. \u003c/p\u003e\n\u003cp\u003eIn vitro experiments were conducted in BV2 and HT22 cells. For conditioned medium collection (Fig. 1B), BV2 cells were pre-treated with Liraglutide (500 nM) for 1 h, followed by exposure to LPS (1 \u0026mu;g/ml, sigma) for 24 h. Subsequently, the culture medium of BV2 cells in each group was collected as conditioned medium and then used to culture HT22 cells for another 24 h. For transwell assay (Fig. 1C), HT22 cells were seeded into the lower inserts (Corning) and pre-treated with Liraglutide (500 nM) for 1 h, followed by the exposure to LPS (1 \u0026mu;g/ml) for 48 h. BV2 cells were seeded in the upper chamber. After 48 h, the migration of BV2 cells was analyzed.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCecal ligation and puncture (CLP) procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCLP was used to induce the mice model of sepsis as previously described[8]. Mice were anesthetized with 1% pentobarbital sodium (40 mg/kg) and then a midline incision (1 cm) was made in the abdomen. Murine cecum was ligated at half the distance between the distal pole of cecum and the ileocecal junction. Subsequently, the cecum was punctured with a 22-gauge needle and fecal contents were squeezed into the peritoneal cavity. Sham group mice underwent the same operation but without CLP. All animals received 40 ml/kg sterile saline via subcutaneous injection and returned to home cage with a warm cotton pad with free access to food and water. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIntracerebroventricular injection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intracerebroventricular (i.c.v) injection was conducted using a stereotaxic apparatus as previously described[9]. The stereotaxic device was inserted into the lateral ventricle (the coordinates were 1.0 mm lateral and 0.5 mm posterior to the bregma over the left hemisphere, and 2.5 mm below the dural surface), then Liraglutide (2 \u0026mu;g/kg in 2 \u0026mu;l PBS) was injected slowly into the lateral ventricle. And mice in sham and CLP groups received the same volume of PBS via i.c.v administration. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eModified neurological severity score test (mNSS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA modified neurological severity score (mNSS) was used to evaluate the neurological function of each group of mice at 0, 1, 3, and 5 d following sham or CLP operation as previously described[10]. Briefly, the severity score is composed of motor, sensory and reflex tests. The mice which cannot perform the tasks or lack a tested reflex get 1 point. A score of 10 to 14 represents severe injury; 5 to 9 represents moderate injury; 1 to 4 represents mild injury.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTUNEL staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTUNEL assays were used to determine the apoptosis of the hippocampus in mice. Tissue slides were fixed with 4% paraformaldehyde and then the TUNEL staining was conducted according to the manufacturer\u0026apos;s protocol (Beyotime). DAPI staining followed with TUNEL was used to assess the nuclear morphology. The slides were viewed using an OLYMPUS inverted microscope. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMeasurement of mitochondrial membrane potential (MMP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitochondrial membrane potential was determined using the JC-1 mitochondrial membrane potential assay kit (Beyotime) according to the manufacturer\u0026apos;s instructions. Briefly, HT22 cells were incubated with JC-1 staining working solution for 20 min at 37 and washed with JC-1 staining wash buffer. The representative figures were obtained using an OLYMPUS fluorescence microscope, and fluorescence intensity was determined using ImageJ software (National Institutes of Health). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIntracellular reactive oxygen species (ROS) detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intracellular ROS of HT22 cells that underwent LPS stimulation with or without Liraglutide/Exendin-9 pre-treatment was measured using a ROS assay kit (Beyotime). Briefly, HT22 cells were incubated with 100 \u0026mu;M DCFH-DA for 20 min at room temperature to detect oxidation level of DCFH-DA into the fluorescent compound DCF. The cells were viewed using an OLYMPUS fluorescence microscope.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mice were anesthetized with 1% pentobarbital sodium and then intracardially perfused with saline solution and 4% paraformaldehyde. Afterward, the brain tissues were collected, post-fixed with 4% paraformaldehyde overnight, and sliced into 10-\u0026mu;m-thick sections using a cryostat. Being blocked with 3% BSA, the tissues were incubated with specific primary antibodies overnight at 4 , followed with the incubation of CoraLite488-conjugated or CoraLite594-conjugated secondary antibodies (proteintech) for 1 h at room temperature. The cell nuclei were stained with DAPI (Southern Biotech) and then view the sections using a confocal microscope (OLYMPUS). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissue sections were incubated for 1 h in 5% BSA with 0.3% Triton X-100 in double distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO) and then incubated with specific primary antibodies overnight at 4 . Afterward, the slices were incubated with the given secondary antibodies for 1 h at room temperature. The positive cells were indicated by adding DAB to the tissues. Tissue images were captured using a light OLYMPUS microscope. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNissl staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse brain tissues were fixed with 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, and paraffin-embedded and then brains were cut into 5 \u0026mu;m thick sections. Tissue sections were stained with 0.5% toluidine blue and observed using a light microscope (OLYMPUS). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFluoro-Jade B staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain sections underwent Fluoro-Jade B (FJB) staining to detect neuron degeneration following CLP. Briefly, slides were hydrated in graded ethanol and distilled water and incubated with 0.06% potassium permanganate for 10 min. Subsequently, 0.0025% FJB solution was added to the slides and incubated for 30 min in the darkness. Images were observed using a fluorescence microscope (Nikon ECLIPSE CI), and degenerated neurons in the DG region of the hippocampus (200 ) were counted in each slide. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse hippocampus (1 mm\u003csup\u003e3\u003c/sup\u003e) was fixed with 2.5% glutaraldehyde at 4 overnight, post-fixed in 1% osmium tetroxide for 2 h, and then dyed in 2% uranyl acetate and dehydrated in graded ethanol. The tissues were embedded in 100% acetone overnight, cut into 60-80 nm sections, and contrasted with 2% uranyl acetate for 15 min and lead citrate for 15 min. The images were captured using a transmission electron microscope (HT-7700, Hitachi Corporation). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBV2 cells (mouse microglia cell line) and HT22 cells (mouse hippocampus neuron cell line) were all purchased from American Type Culture Collection (ATCC) and cultured using DMEM (Gibco) supplemented with 100 U/ml penicillin/streptomycin and 10% fetal bovine serum (FBS, WISENT CORPORATION) in a humidified incubator at 37 with 5% CO\u003csub\u003e2\u003c/sub\u003e. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eThe transwell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHT22 (4 10\u003csup\u003e5\u003c/sup\u003e) were seeded into the bottom chamber with an 8 \u0026mu;m pore transwell insert (Corning) in a 6-well plate. After 24 h, BV2 cells were resuspended in 1.5 ml FBS-free DMEM and seeded into the upper inserts. HT22 cells were divided into 3 groups: Control, LPS, and LPS+Lira groups. HT22 cells were pre-treated with Liraglutide for 1 h in the LPS+Lira group, then LPS was added into the lower chamber for an additional 48 h. HT22 cells in the control group were treated with PBS. After the co-culture, BV2 cells in the upper inserts were fixed with 4% paraformaldehyde (PFA, Servicebio) followed by the staining with crystal violet (Beyotime). Images were taken using an OLYMPUS microscope and the number of migrating cells was analyzed using ImageJ software (National Institutes of Health). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was determined using a cell count kit-8 (CCK-8, Dojindo) according to the manufacturer\u0026rsquo;s protocol. Briefly, cells (4 10\u003csup\u003e3\u003c/sup\u003e) were plated in a 96-well plate. After being treated with specific stimulations for 24 h, cells were washed twice with PBS. CCK-8 was added to the culture medium at a ratio of 1:10 and incubated at 37 for 1 h. The microplate reader (Tecan Spark) was utilized to measure the absorbance at 450 nm. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole mouse hippocampus was lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM NaN3, 5 mM EDTA, 0.1% SDS, and 2% Triton X-100) containing a mixture of protease inhibitors and PhosSTOP Phosphatase Inhibitor (Roche Molecular Biochemicals) for 30 min at 4 . The lysates were obtained after centrifugation at 12,000 \u0026times; g to remove nuclei. Equal volumes and amounts of protein were electrophoresed and separated by an SDS-PAGE and transferred to a nitrocellulose membrane (Millipore). After the membrane was blocked using TBST containing 5% skim milk, the membrane was incubated with primary antibodies at 4 overnight followed by incubation with an HRP-conjugated secondary antibodies for 1 h at 37 . The membrane was visualized using ECL (Millipore) and analyzed by ImageJ software. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.0.0 (Graph Pad Software). All quantitative data in this study were presented as mean SEM. Data of Weight Loss, Food-intake and mNSS after CLP were analyzed using two-way ANOVA for repeated measures, followed by the Tukey post hoc test for multiple comparisons. All other variables were analyzed using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test (for comparisons between two groups) or one-way ANOVA followed by Tukey\u0026rsquo;s multiple test (for comparisons among three or more groups). \u003cem\u003eP\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05 was defined as statistically significant. \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGLP-1R agonist ameliorates neurological deficits in septic mice without affecting their weight and food-intake after CLP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies found that GLP-1R expresses ubiquitously in normal human and mouse brains[11]. Here, we detected the level of GLP-1R in the septic animal model and found that CLP operation up-regulated the expression of GLP-1R in the mouse brain than that of in the sham group (Fig. 2A). GLP-1R agonists contribute to obesity control mainly via the regulation of food intake through the brain GLP-1 signaling[12]. However, we monitored the weight loss and food-intake of CLP mice received i.c.v administration of PBS or Liraglutide but did not find any difference between these two groups (Fig. 2B,C). We then evaluated the neurological deficits in each group of mice using mNSS. Results suggested that CLP mice exhibited more severe neurological damage compared to sham mice at 1, 3, and 5 d after CLP or sham procedure (Fig. 2D). Interestingly, Liraglutide administration via i.c.v successfully promoted neurological recovery at 1 and 3 d after CLP (Fig. 2D). Taken together, these findings reveal that sepsis up-regulated the GLP-1R expression in the mouse brain and the i.c.v administration of Liraglutide relieved the SAE-induced neurological deficits without influencing the food-intake of CLP mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiraglutide improves neuronal loss\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;while reducing glial cell activation in the hippocampus of septic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHippocampus is known to express abundant pattern recognition receptors (PRRs) and therefore is vulnerable to systemic pro-inflammatory responses[13]. According to Nissl staining, we observed that the neuronal loss and degeneration within the ipsilateral CA1, CA3 and dentate gyrus (DG) regions of the hippocampus were more severe in CLP mice compared to sham group (Fig. 3A-E). Liraglutide administration partially reversed the neuronal damage in CLP+Lira group (Fig. 3A-E). Similarly, Fluoro-Jade B staining used to label the degenerated neurons reflected that Liraglutide partially diminished the number of degeneration neurons in the hippocampal DG section of SAE mice (Fig. 3F,G).\u003c/p\u003e\n\u003cp\u003eWe also assessed the glial cell activation in the hippocampus of each group. Results suggested that Liraglutide effectively reduced the number of Iba-1 positive microglia in the hippocampus of septic mice (Fig. 4A,B). Additionally, morphology analysis demonstrated that the hippocampal microglia in CLP mice showed an ameboid morphology with an enlarged cell body, thick and shrunk processes of activated microglia. In contrast, microglia in the hippocampus of mice in sham and CLP+Lira group exhibited more resting morphology with a thin cell body, fine and long processes (Fig. 4A). Similarly, Liraglutide significantly suppressed the activation of astrocytes as depicted by the diminished number of GFAP positive cells in the hippocampus of mice treated with Liraglutide, when compared to that of CLP group (Fig. 4C,D). Collectively, these results imply that Liraglutide exerted neuroprotective effects by mitigating the loss of neurons and glial cell activation in septic mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiraglutide treatment rescues neuronal death via mediating the interaction between microglia and neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we detected the apoptosis in the hippocampus of each group. Based on TUNEL staining, we found that DG, not CA1, CA2, or CA3 regions of the hippocampus in CLP mice, exhibited more significant neuronal death compared to sham group (Fig. 4A,B). Liraglutide administration reversed this effect, especially in the DG region of the hippocampus (Fig. 5A,B). The above results confirmed that GLP-1R agonist reduced microglial activation in septic mice, and we therefore investigated the possible effect of Liraglutide on the interplay between microglia and neuron via in vitro experiments. Firstly, we collected the conditioned medium (CM) of BV2 cells treated with LPS in the presence of Liraglutide or not (Fig. 5C). CCK-8 results showed that HT22 treated with LPS+Lira-CM showed improved cell viability compared to LPS-CM group (Fig. 5D). On the other hand, the migration of BV2 in the presence of HT22 was then evaluated using transwell assay. Results suggested that the BV2 migration was enhanced in LPS-treated HT22 group, which was hampered by the Liraglutide pre-treatment (Fig. 5E,F). Together, these results demonstrated that the activation of GLP-1R improved neuronal damage partially by intervening in the connection between microglia and neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondria damage and ROS production were relieved by GLP-1R agonist.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the most representative characteristics of SAE is the morphological changes in mitochondria. According to the transmission electron microscope, we observed that in the hippocampus of mice that underwent CLP procedure, membrane rupture, cristae decreased, and membrane density compressed were more obvious. Whereas mitochondria damage was relatively slight in the hippocampus of CLP+Lira mice (Fig. 6A). Subsequently, we conducted in vitro studies and found that Liraglutide pre-treatment strongly reduced the LPS-induced ROS production in HT22 cells, which was abolished by GLP-1R antagonist Exendin-9 (Ex-9) (Fig. 6B). Furthermore, MMP ( m) level of mitochondria in each group was detected by JC-1 staining. As depicted in Fig. 6C-D, Liraglutide rescued the destruction of MMP ( m) level in LPS-stimulated HT22 cells, which was also reversed by Ex-9.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP-1R agonist treatment mitigated the inflammatory responses in SAE mice and LPS-treated hippocampal cells via mediating the AKT/STAT3 activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcessive inflammation correlates with neuronal death, glial cell activation, and mitochondria destruction[14]. In the following studies, we assessed the possible mechanisms of Liraglutide in terms of its neuroprotective effects. Western blotting analysis showed that the phosphorylation of STAT3 was up-regulated in the hippocampus of CLP mice compared with that of sham and CLP+Lira mice (Fig. 7A,B), while the AKT phosphorylation was restored by Liraglutide in CLP mice (Fig. 7A,C). Consistently, in vitro experiments showed that Liraglutide pre-treatment up-regulated the phosphorylation of AKT, while inhibiting the level of p-STAT3 in LPS-stimulated HT22 cells (Fig. 7D,E).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we evaluated the therapeutic role of GLP-1R agonist Liraglutide in SAE mice and LPS-stimulated BV2 or HT22 cells. Firstly, we observed that GLP-1R is up-regulated in the brain of SAE mice and the i.c.v administration of Liraglutide alleviates neuronal damage and degeneration in the hippocampus, without affecting the weight and food-intake of septic mice. In addition, glial cell activation and neural apoptosis in the hippocampus are also partially inhibited in septic mice that received Liraglutide treatment. Then we performed in vitro experiments and found that Liraglutide exerts its neuroprotective effects via dampening the interplay between microglia and nerve cells. Furthermore, Liraglutide administration attenuates mitochondria damage and ROS production, while elevating the MMP level. Mechanically, Liraglutide restores the phosphorylation of AKT, whereas decreasing the STAT3 phosphorylation in the hippocampus of septic mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenerally, brain is thought to be a relatively sterile organ being resistant to the circulating inflammatory toxins and infiltration of inflammatory cells due to the integrity of BBB. However, the dysregulation of systematic inflammation, elevation of permeability of BBB, microcirculation dysfunction, and activation of neuroendocrine system during sepsis conditions could ultimately induce brain dysfunction, that is so-called sepsis-induced encephalopathy (SAE)[15]. SAE is closely associated with higher mortality and long-term cognitive impairment. But unfortunately, no specific curative or preventive measure for SAE exists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGLP-1 is a kind of incretin and is mainly produced in enteroendocrine cells of the gastrointestinal tract, as well as in the medial nucleus of the solitary tract (NTS) of the medulla oblongata. Apart from the incretin effects, GLP-1 can also regulate inflammatory responses, improve cardiovascular function, and exert neuroprotection effects[16, 17]. It was reported that the peripheral gut-released GLP-1 or systemically administrated GLP-1R agonists can readily cross the BBB of the brain and abundant evidence have revealed the therapeutic potential of peripheral GLP-1R agonists administration for animal or patients with neurodegenerative diseases[18]. However, a clinical study in patients with Type 2 diabetes demonstrated that the cerebrospinal fluid (CSF) concentration of Liraglutide was lower than that of in plasma even following chronic peripheral administration of Liraglutide for 5 months[19]. Similarly, Williams et al. found that the effects of centrally administered GLP-1 can only be antagonized by central GLP-1 antagonist Ex-9, but not peripheral ones[20]. The above studies indicated that the access of peripheral administered GLP-1 into the CNS might be less than we anticipated[21]. Therefore, we treated septic mice with Liraglutide by intraventricular injection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies have found that GLP-1 is released in response to inflammatory stimuli, and many observational cohort studies demonstrated that GLP-1 levels are positively correlated with inflammatory markers and poor prognosis in patients with sepsis[22]. The activation of endogenous GLP-1 is thought to be a compensation in response to systemic inflammation in septic patients[7] and the therapeutic potential of GLP-1 agonists has now gained favorable results in a septic animal model[23, 24]. However, the role of GLP-1 in SAE has not been discovered. Studies have suggested that GLP-1R was up-regulated in several animal models of brain injury such as cerebral ischemia and mechanical lesion, especially in glial cells[24-26]. Consistently, we for the first time noticed that GLP-1R is also elevated in the brain of septic mice. It was reported that GLP-1 agonists could suppress the appetite and facilitate weight loss in obese people, without affecting healthy ones[27]. Similarly, our present data showed that Liraglutide administration did not alter the food-intake and weight loss of mice that underwent CLP, which confirmed the safety of GLP-1 agonists in a septic animal model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeuron loss and damage are quite common in the brain of SAE patients. Brain, especially the hippocampus region has abundant pattern recognition receptors (PRRs) like Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which prompts the binding with inflammatory cytokines and other damage-associated molecular patterns (DAMPs). Sepsis-induced massive pro-inflammatory release into the brain and thus activates various intracellular pathways, leading to neuron dysfunction and death[28]. We found that GLP-1R agonist Liraglutide attenuated neuron loss and damage in the hippocampus of septic mice. Liraglutide administration also reduced the number of hippocampal degenerative neurons as revealed by FJB staining. Glial cells including microglia and astrocytes have vital roles in maintaining CNS homeostasis and normal function. Nevertheless, in patients with neurodegenerative diseases and brain injuries, microglia and astrocytes are overactivated and switch into neurotoxic phenotypes (namely M1 microglia and A1 astrocytes) facilitating neuronal damage[29]. Our present manuscript discovered that Liraglutide significantly impaired glial cell activation and therefore prevent neuronal death in both in vivo and in vitro models of sepsis. Besides, mitochondrial dysfunction has been reported in various brain regions of septic animals and patients, resulting in the outbreak of oxidative stress and DAMPs release[30]. Liraglutide has been reported to modulate mitochondria dynamics and therefore activate mitophagy to improve mitochondria quality in MPTP-induced PD mice[31]. Nevertheless, Liraglutide and exendin-4 may also alleviate the disruption of mitochondrial membrane potential and inhibit PINK1/Parkin-mediated mitophagy in diabetic retinopathy[32] and hypoxia-reoxygenation injury in cultured cardiomyocytes[33]. The above results suggested that GLP-1R agonists play an important role in maintaining mitochondrial function. Similarly, we discovered that Liraglutide significantly attenuated mitochondria disruption in the hippocampus of CLP mice. Furthermore, Liraglutide inhibited the production of ROS and alleviated mitochondria damage as depicted by the elevation of MMP in LPS-treated HT22 cells, which were reversed by Exendin-9, a GLP-1R antagonist.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of mechanism, we discovered that Liraglutide restored the phosphorylation of AKT, while down-regulating the level of p-STAT3 in both in vivo and in vitro septic models. It was reported that STAT3 activation correlates with sepsis pathophysiology and the suppression of STAT3 activity could ameliorate organ injuries in sepsis animal models [34]. In addition, STAT3 could induce the activation of the NF-\u0026kappa;B pathway, triggering inflammatory responses in human monocytes[35]. Dipeptidyl peptidase-4 inhibitor linagliptin was reported to ease inflammation and improve survival in the CLP model of sepsis through the inhibition of IL-6/JAK/STAT3 signaling[36], while improving the cognitive dysfunction in neurodegeneration diseases via the activation of AKT/PI3K signaling[37, 38].\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur present study suggested the possible role of GLP-1R agonists in attenuating SAE through the inhibition of neuronal damage, neuroinflammation, and mitochondrial dysfunction in the hippocampus. This study provides a potential therapeutic target for sepsis-induced encephalopathy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No.81871581) and \u0026lsquo;Field Internal Science\u0026rsquo; Army Key Discipline Construction. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFZ and YW designed and directed the study. HY, YJ and HH performed experiments, wrote, and prepared the manuscript. LW, JH and YC provided materials and technical support and offered constructive proposals. All authors read and approved the final manuscript for publication. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the General Hospital of Tibet Military Command and the First Medical Centre, Chinese PLA General Hospital for all the support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCzempik PF, Pluta MP, Krzych Ł J. Sepsis-Associated Brain Dysfunction: A Review of Current Literature. International journal of environmental research and public health. 2020;17(16).\u003c/li\u003e\n\u003cli\u003ePeng L, Peng C, Yang F, Wang J, Zuo W, Cheng C, et al. Machine learning approach for the prediction of 30-day mortality in patients with sepsis-associated encephalopathy. BMC medical research methodology. 2022;22(1):183.\u003c/li\u003e\n\u003cli\u003eDenver P, Cunningham C. Microglial activation and neuroinflammation in acute and chronic cognitive deficits in sepsis. Neuropharmacology. 2025;267:110285.\u003c/li\u003e\n\u003cli\u003eClemente-Su\u0026aacute;rez VJ, Redondo-Fl\u0026oacute;rez L, Beltr\u0026aacute;n-Velasco AI, Ramos-Campo DJ, Belinch\u0026oacute;n-deMiguel P, Martinez-Guardado I, et al. Mitochondria and Brain Disease: A Comprehensive Review of Pathological Mechanisms and Therapeutic Opportunities. Biomedicines. 2023;11(9).\u003c/li\u003e\n\u003cli\u003eFan W, Zhang Q, Wang C, Sun J, Zhang J, Yin Y. GLP-1 as a regulator of sepsis outcomes: Insights into cellular metabolism, inflammation, and therapeutic potential. International immunopharmacology. 2025;152:114390.\u003c/li\u003e\n\u003cli\u003eDžidić-Krivić A, Fajkić A, Farhat EK, Lekić L, Ejubović A, Vukas SK, et al. Linking Metabolic Syndrome to Neurodegeneration Mechanisms and Potential Treatments. Molecular neurobiology. 2025.\u003c/li\u003e\n\u003cli\u003eShah FA, Mahmud H, Gallego-Martin T, Jurczak MJ, O\u0026apos;Donnell CP, McVerry BJ. Therapeutic Effects of Endogenous Incretin Hormones and Exogenous Incretin-Based Medications in Sepsis. The Journal of clinical endocrinology and metabolism. 2019;104(11):5274-84.\u003c/li\u003e\n\u003cli\u003eWang J, Zhu Q, Wang Y, Peng J, Shao L, Li X. Irisin protects against sepsis-associated encephalopathy by suppressing ferroptosis via activation of the Nrf2/GPX4 signal axis. Free Radic Biol Med. 2022;187:171-84.\u003c/li\u003e\n\u003cli\u003eYin J, Shen Y, Si Y, Zhang Y, Du J, Hu X, et al. Knockdown of long non-coding RNA SOX2OT downregulates SOX2 to improve hippocampal neurogenesis and cognitive function in a mouse model of sepsis-associated encephalopathy. J Neuroinflammation. 2020;17(1):320.\u003c/li\u003e\n\u003cli\u003eTang Y, Cai B, Yuan F, He X, Lin X, Wang J, et al. Melatonin Pretreatment Improves the Survival and Function of Transplanted Mesenchymal Stem Cells after Focal Cerebral Ischemia. Cell Transplant. 2014;23(10):1279-91.\u003c/li\u003e\n\u003cli\u003eRocha-Ferreira E, Poupon L, Zelco A, Leverin AL, Nair S, Jonsdotter A, et al. Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy. Brain. 2018;141(10):2925-42.\u003c/li\u003e\n\u003cli\u003eMoiz A, Filion KB, Tsoukas MA, Yu OH, Peters TM, Eisenberg MJ. Mechanisms of GLP-1 Receptor Agonist-Induced Weight Loss: A Review of Central and Peripheral Pathways in Appetite and Energy Regulation. The American journal of medicine. 2025.\u003c/li\u003e\n\u003cli\u003eWu A, Zhang J. Neuroinflammation, memory, and depression: new approaches to hippocampal neurogenesis. Journal of neuroinflammation. 2023;20(1):283.\u003c/li\u003e\n\u003cli\u003eMazeraud A, Righy C, Bouchereau E, Benghanem S, Bozza FA, Sharshar T. Septic-Associated Encephalopathy: a Comprehensive Review. Neurotherapeutics. 2020;17(2):392-403.\u003c/li\u003e\n\u003cli\u003eJacob A, Brorson JR, Alexander JJ. Septic encephalopathy: inflammation in man and mouse. Neurochem Int. 2011;58(4):472-6.\u003c/li\u003e\n\u003cli\u003eInsuela DBR, Carvalho VF. Glucagon and glucagon-like peptide-1 as novel anti-inflammatory and immunomodulatory compounds. European journal of pharmacology. 2017;812:64-72.\u003c/li\u003e\n\u003cli\u003eZafer M, Tavaglione F, Romero-G\u0026oacute;mez M, Loomba R. Review Article: GLP-1 Receptor Agonists and Glucagon/GIP/GLP-1 Receptor Dual or Triple Agonists-Mechanism of Action and Emerging Therapeutic Landscape in MASLD. Alimentary pharmacology \u0026amp; therapeutics. 2025.\u003c/li\u003e\n\u003cli\u003eReich N, Holscher C. The neuroprotective effects of glucagon-like peptide 1 in Alzheimer\u0026apos;s and Parkinson\u0026apos;s disease: An in-depth review. Front Neurosci. 2022;16:970925.\u003c/li\u003e\n\u003cli\u003eChristensen M, Sparre-Ulrich AH, Hartmann B, Grevstad U, Rosenkilde MM, Holst JJ, et al. Transfer of liraglutide from blood to cerebrospinal fluid is minimal in patients with type 2 diabetes. International journal of obesity (2005). 2015;39(11):1651-4.\u003c/li\u003e\n\u003cli\u003eWilliams DL, Baskin DG, Schwartz MW. Evidence that intestinal glucagon-like peptide-1 plays a physiological role in satiety. Endocrinology. 2009;150(4):1680-7.\u003c/li\u003e\n\u003cli\u003eTrapp S, Brierley DI. Brain GLP-1 and the regulation of food intake: GLP-1 action in the brain and its implications for GLP-1 receptor agonists in obesity treatment. Br J Pharmacol. 2022;179(4):557-70.\u003c/li\u003e\n\u003cli\u003eLebherz C, Schlieper G, M\u0026ouml;llmann J, Kahles F, Schwarz M, Br\u0026uuml;nsing J, et al. GLP-1 Levels Predict Mortality in Patients with Critical Illness as Well as End-Stage Renal Disease. The American journal of medicine. 2017;130(7):833-41.e3.\u003c/li\u003e\n\u003cli\u003eYang F, Zeng F, Luo X, Lei Y, Li J, Lu S, et al. GLP-1 Receptor: A New Target for Sepsis. Frontiers in pharmacology. 2021;12.\u003c/li\u003e\n\u003cli\u003eWong CK, McLean BA, Baggio LL, Koehler JA, Hammoud R, Rittig N, et al. Central glucagon-like peptide 1 receptor activation inhibits Toll-like receptor agonist-induced inflammation. Cell metabolism. 2024;36(1):130-43.e5.\u003c/li\u003e\n\u003cli\u003eLee CH, Yan B, Yoo KY, Choi JH, Kwon SH, Her S, et al. Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia. Journal of neuroscience research. 2011;89(7):1103-13.\u003c/li\u003e\n\u003cli\u003eChowen JA, de Fonseca FR, Alvarez E, Navarro M, Garc\u0026iacute;a-Segura LM, Bl\u0026aacute;zquez E. Increased glucagon-like peptide-1 receptor expression in glia after mechanical lesion of the rat brain. Neuropeptides. 1999;33(3):212-5.\u003c/li\u003e\n\u003cli\u003ePi-Sunyer X, Astrup A, Fujioka K, Greenway F, Halpern A, Krempf M, et al. A Randomized, Controlled Trial of 3.0 mg of Liraglutide in Weight Management. The New England journal of medicine. 2015;373(1):11-22.\u003c/li\u003e\n\u003cli\u003eGu M, Mei XL, Zhao YN. Sepsis and Cerebral Dysfunction: BBB Damage, Neuroinflammation, Oxidative Stress, Apoptosis and Autophagy as Key Mediators and the Potential Therapeutic Approaches. Neurotoxicity research. 2021;39(2):489-503.\u003c/li\u003e\n\u003cli\u003eXiao T, Ji H, Shangguan X, Qu S, Cui Y, Xu J. NLRP3 inflammasome of microglia promotes A1 astrocyte transformation, neo-neuron decline and cognition impairment in endotoxemia. Biochemical and biophysical research communications. 2022;602:1-7.\u003c/li\u003e\n\u003cli\u003eMantzarlis K, Tsolaki V, Zakynthinos E. Role of Oxidative Stress and Mitochondrial Dysfunction in Sepsis and Potential Therapies. Oxid Med Cell Longev. 2017;2017:5985209.\u003c/li\u003e\n\u003cli\u003eLin TK, Lin KJ, Lin HY, Lin KL, Lan MY, Wang PW, et al. Glucagon-Like Peptide-1 Receptor Agonist Ameliorates 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Neurotoxicity Through Enhancing Mitophagy Flux and Reducing \u0026alpha;-Synuclein and Oxidative Stress. Frontiers in molecular neuroscience. 2021;14:697440.\u003c/li\u003e\n\u003cli\u003eZhou HR, Ma XF, Lin WJ, Hao M, Yu XY, Li HX, et al. Neuroprotective Role of GLP-1 Analog for Retinal Ganglion Cells via PINK1/Parkin-Mediated Mitophagy in Diabetic Retinopathy. Frontiers in pharmacology. 2020;11:589114.\u003c/li\u003e\n\u003cli\u003eKobara M, Toba H, Nakata T. A Glucagon-like Peptide 1 Analog Protects Mitochondria and Attenuates Hypoxia-Reoxygenation Injury in Cultured Cardiomyocytes. J Cardiovasc Pharmacol. 2022;79(4):568-76.\u003c/li\u003e\n\u003cli\u003eXu S, Pan X, Mao L, Pan H, Xu W, Hu Y, et al. Phospho-Tyr705 of STAT3 is a therapeutic target for sepsis through regulating inflammation and coagulation. Cell communication and signaling : CCS. 2020;18(1):104.\u003c/li\u003e\n\u003cli\u003eGuzzo C, Che Mat NF, Gee K. Interleukin-27 induces a STAT1/3- and NF-kappaB-dependent proinflammatory cytokine profile in human monocytes. The Journal of biological chemistry. 2010;285(32):24404-11.\u003c/li\u003e\n\u003cli\u003eDelic D, Klein T, Wohnhaas CT, Feng H, Lin X, Zhang JR, et al. Dipeptidyl peptidase-4 inhibitor linagliptin reduces inflammatory response, ameliorates tissue edema formation, and improves survival in severe sepsis. Biomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie. 2025;182:117778.\u003c/li\u003e\n\u003cli\u003eSaleh RA, Eissa TF, Abdallah DM, Saad MA, El-Abhar HS. Peganum harmala enhanced GLP-1 and restored insulin signaling to alleviate AlCl(3)-induced Alzheimer-like pathology model. Scientific reports. 2021;11(1):12040.\u003c/li\u003e\n\u003cli\u003eAthauda D, Gulyani S, Karnati HK, Li Y, Tweedie D, Mustapic M, et al. Utility of Neuronal-Derived Exosomes to Examine Molecular Mechanisms That Affect Motor Function in Patients With Parkinson Disease: A Secondary Analysis of the Exenatide-PD Trial. JAMA neurology. 2019;76(4):420-9.\u003c/li\u003e\n\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":"Sepsis-induced encephalopathy, Glucagon-like peptide-1, Liraglutide, neuron damage, mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-7340017/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7340017/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis-induced encephalopathy (SAE) affects about 70% of patients with sepsis and therefore leads to poor prognosis and long-term cognitive dysfunction. Unfortunately, there are still no effective pharmacological treatments to better manage this kind of encephalopathy. Recently, glucagon-like peptide 1 receptor (GLP-1R) agonists have gained much attention due to their neuroprotective effects in neurodegenerative diseases and brain injuries. In this study, we evaluated the potential effects of intracerebroventricular injection of Liraglutide, a kind of GLP-1R agonists, in SAE mice. We found that Liraglutide injected via intracerebroventricular improved neurological deficits and attenuated neuronal loss and degeneration, and glial cell activations in the hippocampus of septic mice. In vitro studies demonstrated that Liraglutide inhibited the interaction between microglia and neurons under LPS stimulation. Furthermore, Liraglutide restrained oxidative distress and mitochondria damage in hippocampal neurons. Mechanically, Liraglutide restored the downregulation of p-AKT but reversed the phosphorylation of STAT3 in hippocampal neurons. Collectively, these results indicated that the administration of GLP-1R agonist Liraglutide might exert neuroprotective effects on sepsis-induced brain impairments.\u003c/p\u003e","manuscriptTitle":"Liraglutide alleviates sepsis-induced encephalopathy via attenuating neuronal damage, glial cell activation and mitochondrial dysfunction in a mouse model of sepsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:57:08","doi":"10.21203/rs.3.rs-7340017/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":"b3a632fb-e48b-4194-853a-1fec7ddad7ef","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T14:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 05:57:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7340017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7340017","identity":"rs-7340017","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}