Application of mGluR5 antagonist MPEP in preparation of products for treating sepsis-related delirium

Abstract

The invention discloses an application of an mGluR5 antagonist MPEP in preparation of a sepsis-related delirium product, and relates to the technical field of biomedicine.According to the application, hippocampus mGluR5 signal transduction and cone neuron excitability are adjusted in an endotoxin-induced sepsis-related delirium mouse model to relieve delirium behaviors and recover inhibitory synaptic input, so that the sepsis-related delirium product can be prepared, and the delirium behavior can be relieved; neuron damage is reversed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an mGluR5 antagonist MPEP in the preparation of a product for treating sepsis-related delirium. Background Art

[0002] Sepsis-associated delirium (SAD) is an acute brain dysfunction caused by sepsis, with an incidence of up to 70% in sepsis patients and an increased mortality rate. It typically presents as a dramatic change in mental status, accompanied by a decline in cognitive function and attention. This neurological dysfunction can persist for months or even longer, impacting the long-term cognitive function of sepsis survivors.

[0003] In related technologies, treatments for sepsis-related delirium mainly focus on treating sepsis through anti-infection methods or controlling delirium-like behaviors through antipsychotic drugs, which can only relieve symptoms but cannot restore inhibitory synaptic input and reverse neuronal damage.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] The technical task of this application is to address the above shortcomings and provide an application of an mGluR5 antagonist MPEP in the preparation of a product for treating sepsis-related delirium. This application alleviates delirium-like behavior, restores inhibitory synaptic input, and reverses neuronal damage by regulating hippocampal mGluR5 signaling and pyramidal neuron excitability in an endotoxin-induced sepsis-related delirium mouse model.

[0006] To achieve the above objectives, this application provides the following technical solutions: According to one aspect of the present application, a use of an mGluR5 antagonist MPEP in preparing a product for treating sepsis-related delirium is provided.

[0007] In some embodiments, the MPEP alleviates delirium-like behaviors and reverses the suppression of sleep EEG activity in mice with sepsis-related delirium.

[0008] In some embodiments, the MPEP improves hippocampal glutamate metabolism imbalance and regional cerebral blood flow reduction in mice with sepsis-associated delirium.

[0009] In some embodiments, the MPEP reduces hippocampal mGluR5 expression and the number of neuronal apoptosis in the hippocampal CA1 region of mice with sepsis-related delirium.

[0010] In some embodiments, the MPEP reduces lipopolysaccharide-induced high expression of mGluR5 and the number of apoptotic cells in a mouse hippocampal neuronal cell line, wherein the mouse hippocampal neuronal cell line is an HT22 cell line.

[0011] In some embodiments, the MPEP improves neuronal damage and synaptic plasticity in the hippocampal CA1 region of mice with sepsis-associated delirium.

[0012] In some embodiments, the MPEP improves the abnormal Ca2+ activity of neurons in the hippocampal CA1 region of mice with sepsis-related delirium during free movement and Y-maze tests. 2+ Signal.

[0013] In some embodiments, the MPEP repairs the imbalance of excitatory and inhibitory neuronal activities in the CA1 region of the hippocampus of mice with sepsis-associated delirium.

[0014] In some embodiments, the MPEP reduces the excitability and synaptic transmission dysfunction of pyramidal neurons in the CA1 region of the hippocampus of mice with sepsis-related delirium.

[0015] In some embodiments, the product for treating sepsis-related delirium is a drug for treating sepsis-related delirium, the drug comprising an effective dose of the MPEP and pharmaceutically acceptable excipients, the drug being an injectable preparation, and the injectable preparation being administered by intraperitoneal infusion.

[0016] Compared with the existing technology, the advantages and positive effects of the present application are: the present application alleviates delirium-like behavior, restores inhibitory synaptic input, and reverses neuronal damage by regulating hippocampal mGluR5 signaling and pyramidal neuron excitability in an endotoxin-induced sepsis-related delirium mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The experimental results in the examples of the present application show that MPEP improves the EEG activity of LPS-induced SAD mice and alleviates delirium-like behavior.

[0019] Figure 2A schematic diagram showing that MPEP alleviates the delirium-like behavior of LPS-induced SAD mice in the examples of the present application.

[0020] Figure 3 It was shown that MPEP in the examples of the present application improved the LPS-induced Glu metabolic imbalance and blood hypoperfusion in the hippocampus of SAD mice.

[0021] Figure 4 The results show that MPEP reduces the expression of mGluR5 in the hippocampus and neuronal apoptosis in the hippocampal CA1 region of LPS-induced SAD mice in the examples of the present application.

[0022] Figure 5 The results show that MPEP reduces the expression of mGluR5 in hippocampal neuronal cells treated with LPS and inhibits cell apoptosis in the examples of the present application.

[0023] Figure 6 It was shown that MPEP in the examples of the present application alleviated LPS-induced neuronal damage in the hippocampal CA1 region of SAD mice and improved synaptic plasticity.

[0024] Figure 7 It is shown that MPEP reduces the spontaneous high Ca2+ activity of hippocampal CA1 neurons in free-moving SAD mice induced by LPS. 2+ Abnormal Ca during activity and Y-maze test 2+ Activity results.

[0025] Figure 8 The results in the examples of the present application show that MPEP reduces the spike discharges of excitatory pyramidal neurons and increases the spike discharges of inhibitory interneurons in the CA1 region of the hippocampus of SAD mice induced by LPS.

[0026] Figure 9 The results show that MPEP reduces the action potential (AP) frequency and excitatory synaptic transmission and improves inhibitory synaptic transmission in pyramidal neurons in the CA1 region of the hippocampus of LPS-induced SAD mice in the examples of the present application. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned objects, features and advantages of the present application, the present application is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other without conflict.

[0028] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0029] 1. Experimental Materials and Methods 1. Experimental Animals: 160 adult male SPFC57BL / 6J mice, aged 8 to 10 weeks and weighing 20 to 25 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in standard laboratory cages under a 12-h / 12-h light / dark cycle (lights on at 8:00 AM and off at 8:00 PM) with free access to food and water. This experiment was approved by the Committee on the Use of Laboratory Animals of Tianjin Medical University.

[0030] 2. Grouping and model building: Mice were randomly divided into four groups: control group, lipopolysaccharide (LPS, HY-D1056, MedChemExpress) group, vehicle (Vehicle) + LPS group, and MPEP (HY-14609, MedChemExpress) + LPS group, with 40 mice in each group. The treatment conditions of each group are as follows: Control group: SAD mice were intraperitoneally injected with normal saline, where the dose of normal saline was 10 ml / kg mouse body weight.

[0031] LPS group: SAD mice were intraperitoneally injected with 5 mg / kg LPS to establish an endotoxin-induced sepsis mouse model.

[0032] Vehicle+LPS group: SAD mice were treated with an intraperitoneal injection of 5 mg / kg LPS solution (endotoxin-induced sepsis mouse model), and a single intraperitoneal injection of normal saline was given 30 minutes before LPS injection, where the dose of normal saline was 10 ml / kg mouse body weight.

[0033] MPEP+LPS group: SAD mice were intraperitoneally injected with 5 mg / kg LPS (endotoxin-induced sepsis mouse model), and a single intraperitoneal injection of MPEP solution was given 30 minutes before LPS injection, where the MPEP concentration was 10 mg / kg.

[0034] It should be noted that the solvents for both the LPS solution and the MPEP solution were normal saline. The time at which the SAD mice in the control group were intraperitoneally injected with normal saline was the same as the time at which the other groups were administered LPS.

[0035] 3. Testing indicators: Sleep electroencephalogram (EEG) was recorded first, and behavioral tests were performed after sleep EEG recording. The recording time was from baseline (24 hours before LPS administration) to 24 hours after LPS administration. Magnetic resonance imaging (MRI), including proton magnetic resonance spectroscopy (1H-MRS) and arterial spin labeling (ASL), was used to evaluate changes in Glu metabolism and perfusion in the mouse hippocampus. Brain samples were collected for in vitro testing, including enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR (RT-qPCR), immunoblotting (WB), and immunofluorescence (IF). In addition, a cell counting kit (CCK-8) test of hippocampal neurons was also performed. Using fiber optic Ca 2+ Changes in neuronal activity and spike firing were assessed using imaging and a 16-channel microelectrode array (MEA) in the CA1 region of the hippocampus. Furthermore, action potentials (APs), excitability, and synaptic transmission in CA1 pyramidal cells were assessed using patch clamp techniques.

[0036] (1) Sleep electroencephalogram (EEG) recording EEG signals were recorded using a mouse electroencephalogram (EEG) recording system from 24 hours before to 24 hours after LPS administration. Specifically, mice were anesthetized with isoflurane, and their heads were fixed in a stereotaxic apparatus. After exposing the skull, holes were drilled 1.0 mm anterior / posterior and 1.5 mm lateral to the left / right sides of the bregma, and screws for connecting electrodes were fixed into the drilled holes in the skull. Furthermore, two collecting electromyogram (EMG) electrodes were embedded in the neck muscles. After 7 days of recovery, the mice underwent experimental procedures. Using Sirenia sleep software, EEG / EMG signals were analyzed according to a unified set of criteria, automatically identifying non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and wakefulness using a 10-second cycle. Furthermore, changes in the power of delta waves (1-4 Hz) during NREM sleep and theta waves (4-10 Hz) during REM sleep were analyzed.

[0037] (2) Behavioral testing The buried food test (BFT), open field test (OFT), and Y-maze test can assess SAD in the mouse model of sepsis. 24 hours before and 24 hours after LPS administration, these three tests are used to test the changes in autonomic state and cognitive behavior of mice. The BFT and OFT are used to assess changes in behavior under autonomic state, and the Y-maze test is used to assess changes in cognitive behavior. Behavioral tests can assess the core features of delirium, similar to the confusion assessment method. The specific methods are as follows: Two days before testing, each mouse was given two pieces of sweetened cereal. A test cage was set up with 3 cm of clean sawdust bedding, and one sweetened cereal piece was buried 0.5 cm below the bedding surface. The animal was then placed in the cage for 5 minutes, and the latency to detect the buried cereal piece was recorded using the SuperMaze animal behavior tracking system. The latency was defined as the time from the moment the mouse was placed in the cage to the moment it retrieved the cereal piece with its forepaws or teeth. After testing, the cage was cleaned with 75% ethanol and the bedding was replaced.

[0038] OFT: Animals were placed in an open field box and allowed to move freely for 5 minutes while their behavior was tracked. The box was divided into 25 squares, with the central 9 squares defining the center zone. The percentage of time spent in the center zone was recorded. After testing, the box was cleaned with 75% ethanol.

[0039] Y-maze: During training, the novel arm was blocked by a separator, and the animals were allowed to move freely in one of the other two arms for 10 minutes. Two hours later, during testing, the novel arms were opened, and the mice were allowed to freely explore all three arms. A behavioral tracking system recorded the time and number of entries into the novel arm within 5 minutes. Afterwards, the Y-maze was cleaned with 75% ethanol.

[0040] (3) Magnetic resonance imaging (MRI): Proton magnetic resonance spectroscopy (1H-MRS) and arterial spin labeling (ASL) data were acquired using a 9.4T small animal superconducting MRI system (BioSpec 94 / 30 USR, Bruker, Germany). Animals were anesthetized with 2% isoflurane mixed with oxygen and then secured to the MRI scanner bed. Respiratory rate was continuously monitored and maintained at 70–90 breaths / minute, and the temperature was maintained at 37°C with warm water circulation. T2-weighted imaging (T2WI) was performed using a rapid-acquisition relaxation-enhancement sequence to provide positioning of the hippocampus for 1H-MRS and ASL analysis.

[0041] 1H-MRS: 1H-MRS was performed using a point-resolved sequence with the following parameters: RT / TE = 1500 / 16.5 ms, average = 256. The target voxel of interest was located in the hippocampus with a thickness of 2 × 1 × 2 mm. 3 After extracting the magnetic resonance sequences, Mestrenova software was used to analyze and quantify the Glu, N-acetylaspartate (NAA), and creatine (Cr) peaks. In addition, the Glu / Cr and NAA / Cr ratios were determined.

[0042] ASL: ASL was performed using an echo planar imaging-fluid attenuated inversion recovery sequence with the following parameters: RT / TE = 4808 / 33 ms, image size = 128 × 128, slice = 1. The target voxel of interest was located in the hippocampus and had a size of 20 × 20 mm. 2 Regional cerebral blood flow (rCBF) in the hippocampus was analyzed and quantified using Bruker ParaVision 360 software.

[0043] (4) Biochemical assays: To collect samples, mice were deeply anesthetized with 5% isoflurane inhalation 24 hours after LPS administration. Hippocampal tissue was quickly collected on ice and stored at -80°C for further biochemical analysis. ELISA measured Glu levels in hippocampal tissue, RT-qPCR and WB measured mGluR5 expression in the CA1 region of the hippocampus, and IF (including TUNEL staining) measured GluR5 expression and the intensity of neuronal apoptosis in the CA1 region of the hippocampus. The specific methods are as follows: Enzyme-linked immunosorbent assay (ELISA): Hippocampal Glu levels were measured using a mouse Glu ELISA kit (MM-44113M1, Jiangsu Meimian Industrial Co., Ltd., China). Briefly, sample absorbance was measured at a wavelength of 450 nm using a microplate spectrometer according to the manufacturer's instructions. Sample concentrations were calculated based on a reference curve and the dilution ratio.

[0044] Real-time quantitative PCR (RT-qPCR): RNA was extracted from hippocampal tissue using a Trizol kit (#12183555, Thermo Fisher Scientific Inc.). The procedure was performed according to the instructions of the reverse transcription kit and the fluorescent dye kit. The upstream primer sequence for mGluR5 was 5'-CGAACGCTCATTGTCACACACTA-3', and the downstream primer sequence was 5'-TCAGCAGATCCAGGCAATATCC-3'. The upstream primer sequence for β-actin was 5'-GTACTCTGTGTGTGGATCGGTGG-3', and the downstream primer sequence was 5'-GCAGCTCAGTAACAGTCCG-3'. These primers were synthesized by Shanghai Bioengineering Co., Ltd. The relative cycle threshold (Ct) was normalized using β-actin, and the data were analyzed using the 2-ΔΔCt method.

[0045] Western blotting (WB): Total protein was extracted from the hippocampus using RIPA lysis buffer, and the protein concentration in the resulting lysate was measured using a BCA protein assay kit. After quantification, samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membranes were sealed with 5% skim milk for 2 hours at room temperature. The membranes were then incubated overnight at 4°C with a primary anti-mGluR5 rabbit polyclonal antibody (1:1000, #55920, Cell Signaling Technology). β-tubulin served as an internal reference. A secondary antibody conjugated to horseradish peroxidase was used to detect the bound primary antibody. mGluR5 and β-tubulin were visualized using enhanced chemiluminescence detection. mGluR5 protein expression was analyzed and quantified using ImageJ software.

[0046] Immunofluorescence staining (IF): Hippocampal slices were reheated at room temperature for 30 minutes, then 1% Triton X-100 solution was added at room temperature for 30 minutes, and 3% BSA solution was blocked at room temperature for 1 hour. Fluorescent primary anti-mGluR5 antibody (1:200, #55920, Cell Signaling Technology) was added to evenly cover the brain slices and incubated in a refrigerator at 4°C overnight. Fluorescent secondary antibody was added and protected from light at room temperature for 1 hour. DAPI was added to stain the cell nuclei for 5 minutes. Hippocampal slices were scanned under a microscope. ImageJ software was used to analyze and quantify the changes in the fluorescence intensity of mGluR5 in the CA1 region of the hippocampus.

[0047] TUNEL staining of the hippocampus: Hippocampal sections were infiltrated with 0.1% Triton X-100 and incubated with TUNEL solution (ab66110, Abcam) for 60 minutes in dim light. Sections were then incubated with NeuN antibody (ab104224, Abcam) and mGluR5 antibody overnight at 4°C. The following day, sections were incubated with a fluorescent secondary antibody, goat anti-mouse IgG, for 60 minutes at room temperature. Finally, sections were sealed with DAPI-containing sealing solution. Apoptosis of pyramidal cells in the CA1 region of the hippocampus was observed using a fluorescence microscope.

[0048] (5) Hippocampal neuron culture and processing: Mouse HT22 hippocampal cell line cells (CL-0697, Wuhan Punosai Life Science Co., Ltd.) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in an incubator with 5% CO2. The cells were exposed to LPS (1 μg / ml) for 24 hours (LPS group). In the MPEP group, the mGluR5 antagonist MPEP (100 μM) was added to the cells 30 minutes before LPS administration. In the control group, saline at the same dose as LPS was added to the cells.

[0049] Glu levels in HT22 cells were measured by ELISA, mGluR5 expression in HT22 cells was measured by RT-qPCR and WB, and GluR5 expression and the intensity of neuronal apoptosis in HT22 cells were measured by IF (including TUNEL staining). The specific measurement methods are the same as those for hippocampal tissue in “(4) Biochemical Assays” and will not be repeated here.

[0050] HT22 cell viability was measured using the CCK-8 assay. HT22 cells were seeded into a 96-well cell culture plate. Following the kit instructions, CCK-8 solution (HY-K0301, MedChemExpress LLC) was added to each well, and the plate was incubated in an incubator for 2 hours. The optical density (OD) of each well was then measured at 450 nm using a microplate reader.

[0051] It should be noted that the Control group, LPS group and MPEP+LPS group measured in this part were treatments of the mouse HT22 hippocampal cell line.

[0052] (6) Fiber photometry Ca 2+ Imaging records: Fiber photometry Ca was performed according to the procedures of previous studies. 2+ Imaging recording. The mice were anesthetized with isoflurane, the skull was exposed, and 150nl of pAAV-Syn-GCaMP6f virus solution was injected into the CA1 region of the right hippocampus (AP: -1.8mm, ML: -1.50mm, DV: -1.25mm) using a brain stereotaxic instrument. A fiber-optic ceramic needle with a diameter of 200μm was inserted into the same position. The needle was fixed to the skull with dental cement and recovered for 3 weeks. Ca in hippocampal neurons was measured using a custom device 2+ Following previous research procedures, Ca 2+ signal and express the change as ΔF / F.

[0053] (7) Microelectrode array (MEA) recording: Mice were anesthetized and fixed on a stereotaxic apparatus with their skulls exposed. A 16-channel MEA was implanted into the CA1 region of the hippocampus using a micropositioner at a slow speed of 10 μm / s. Subsequently, the MEA was fixed to the mouse skull with dental cement. The mice were allowed to recover for 7 days before further experiments. When the mice were awake and moving freely, hippocampal neuronal signals were collected using a neurophysiological recording system (Jiangsu Yige Biotechnology Co., Ltd., China). Neuronal spike signals were analyzed and quantified using NeuroExplore software.

[0054] (8) Whole-cell patch clamp recording: Patch clamp electrophysiological recordings were performed on brain slices. The mouse brain tissue was transferred to a medium containing 120 NaCl, 2.5 KCl, and 2 CaCl2 . 2H2O, 1.25KH2PO4, 2MgSO4 . The brain sections were incubated in artificial cerebrospinal fluid (ACSF) containing a mixture of 7H2O, 26NaHCO3, and 10mM glucose in 95%O2+5%CO2 for 1 minute. Coronal sections containing the CA1 region of the hippocampus were then sliced ​​at 300 μm thickness using a vibrating microtome (CM1950, Leica, Wetzlar, Germany) in the same buffer. The brain sections were incubated in ACSF at 33°C for 30 minutes and then incubated with oxygenated ACSF at room temperature for at least 1 hour. Evoked action potentials (APs) were recorded from CA1 pyramidal cells in the whole-cell mode using a borosilicate glass pipette containing the following internal solution (mM): 140K gluconic acid, 10KCl, 10HEPES, 0.25EGTA, 5Mg-ATP, pH 7.3, with KOH. Additionally, spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from CA1 pyramidal cells in the voltage-clamp mode. For sIPSC recordings, borosilicate glass pipettes (3-6 MΩ resistance) were filled with an internal solution consisting of (mM): 140 CsCl, 0.5 EGTA, 10 HEPES, 2 MgCl2, 2 Mg-ATP, 12 creatinine phosphate, 0.5 Na3GTP, and 30 N-methyl-D-glucamine (NMG), adjusted to pH 7.2 with CsOH, with an osmotic coefficient of 280-290 mOsm. Whole-cell voltage-clamp recordings were performed on CA1 pyramidal neurons in the hippocampus at a holding potential of -60 mV. To isolate inhibitory postsynaptic currents, perfused ACSF was supplemented with 1 mM kynurenic acid to block ionotropic Glu receptors. Data were analyzed using pCLAMP10 software (Molecular Devices, Sunnyvale, CA, USA).

[0055] 4. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.0 Software (GraphPad Software, Inc.). All data are presented as mean ± standard error (SME). After verifying the normal distribution of the data, one-way or two-way analysis of variance (ANOVA) was used, followed by Tukey post hoc test or Bonferroni correction for multiple comparisons as appropriate. Paired t-test was used to compare Ca within the group. 2+ Signal and MEA data. P values ​​< 0.05 were considered statistically significant.

[0056] 5. Result analysis: (1) MPEP improved the EEG activity suppression, increased NREM sleep, and delirium-like behavior in LPS-induced SAD mice.

[0057] To investigate the role of MPEP in LPS-induced SAD, we performed a series of behavioral tests combined with EEG monitoring. Figure 1 The results of the experiment in the embodiment of the present application show that MPEP improves the EEG activity of LPS-induced SAD mice and alleviates delirium-like behavior, wherein, Figure 1 A is a schematic timeline of four sets of EEG / EMG recordings and representative signals of delta and theta waves extracted from EEG. Figure 1 B Representative spectra of EEG activities of mice in four groups, Figure 1 C is the average power percentage of delta waves in EEG analysis of the four groups of mice, Figure 1 D is the average power percentage of theta waves in the EEG analysis of the four groups of mice, Figure 1 E is the non-rapid eye movement (NREM) sleep time in the sleep stages of the four groups of mice, Figure 1 F is the rapid eye movement (REM) sleep time in the sleep stages of the four groups of mice. Figure 1 G is the wakefulness (WAKE) time in the sleep stages of the four groups of mice. Figure 1 A- Figure 1 The sample size of each group in G n =6; Figure 1 H is a schematic timeline of the four behavioral tests and a legend of the buried test (BFT), open field test (OFT) and Y maze test. Figure 1 I is a representative trajectory diagram of the BFT, OFT and Y maze tests of four groups of mice, Figure 1 J is the latency to eat in the BFT of the four groups of mice, Figure 1 K is the percentage of time spent in the central area in the OFT of the four groups of mice, Figure 1 L is the time it takes for the four groups of mice to enter the new arm in the Y maze test, Figure 1 M is the duration of time that the four groups of mice stayed in the new arm, Figure 1 H- Figure 1 The sample size of each group in M n =10, data are expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey's post hoc test. ns: no difference, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. Figure 2 A schematic diagram showing that MPEP alleviates the delirium-like behavior of LPS-induced SAD mice in the examples of the present application.

[0058] like Figure 1, EEG spectrograms showed that LPS reduced cortical EEG activity, while MPEP reversed the inhibition of EEG activity in septic mice ( Figure 1 B). Power spectrum analysis showed that the delta power ratio of septic mice was higher than that of normal mice (LPS: 46.02±2.135 vs Control: 27.91±2.330, P =0.0003; Figure 1 C), while MPEP-treated septic mice showed a decrease in delta power ratio (MPEP+LPS: 30.13±2.795 vs Vehicle+LPS: 46.45±2.654, P=0.0009; Figure 1 C), compared with the mice treated with vehicle for sepsis. There was no significant difference in EEG theta power among the four groups (F(3,20)=1.555, P =0.2314, Figure 1 D). In addition, LPS caused an increase of approximately 52% in NREM sleep compared with normal mice (LPS: 66.42±1.017 vs Control: 43.81±1.652, P <0.0001; Figure 1 E), REM sleep decreased by about 44% (LPS: 5.673±0.6414 vs Control: 10.05±0.7797, P=0.0009; Figure 1 F), the awake time ratio decreased by about 40% (LPS: 27.91 ± 1.312 vs Control: 46.15 ± 1.987, P <0.0001; Figure 1 G). In septic mice treated with Vehicle, sleep stage changes similar to those in sepsis were found (NREM sleep: P =0.9368; REM sleep: P =0.9998; awake state: P =0.9603, but MPEP treatment reversed the NREM sleep induced by sepsis (MPEP+LPS: 47.87±1.683 vs Vehicle+LPS: 65.25±1.266, P <0.0001; Figure 1 E), REM sleep (MPEP+LPS: 10.66±0.4925 vs Vehicle+LPS: 5.752±0.7295, P =0.0002; Figure 1 F) and awake state (MPEP+LPS: 41.48±1.852 vs Vehicle+LPS: 29.00±0.9481,P =0.0001; Figure 1 G) changes.

[0059] The main features of SAD in animal models are olfactory impairment, anxiety, and cognitive deficits. Figure 1 H shows the procedure for evaluating SAD, Figure 1 I shows representative behavioral traces of the four groups: BFT (assessing olfactory impairment), OFT (assessing anxiety), and Y-maze test (assessing cognitive deficits). Compared with normal mice, LPS mice took longer to find the buried food in the BFT test (LPS: 1.373±0.1323 vs Control: 0.5494±0.05995, P <0.0001; Figure 1 J), the time spent in the central area of ​​OFT test was shorter (LPS: 0.3687±0.05030 vs Control: 0.7394±0.05814, P <0.0001, Figure 1 K), and the time to enter the novel arm in the Y-maze test was significantly reduced (LPS: 0.3137±0.03913 vs Control: 0.9563±0.07656, P <0.0001; Figure 1 L) and the duration in the novel arm was also reduced (LPS: 0.2634±0.03034 vs Control: 1.159±0.09683, P<0.0001, Figure 1 M), indicating that LPS successfully induced the SAD model. Compared with LPS mice treated with Vehicle, LPS mice treated with MPEP significantly improved the olfactory damage caused by sepsis (MPEP+LPS: 0.8292±0.08921 vs Vehicle+LPS: 1.315±0.1029, P=0.0076; Figure 1 J), anxiety (MPEP+LPS: 0.6820±0.05289 vs Vehicle+LPS: 0.3456±0.04552, P=0.0003; Figure 1 K) and cognitive deficits (time to enter the novel arm: MPEP+LPS: 0.8619±0.0777 vs Vehicle+LPS: 0.2917±0.03813, P <0.0001, Figure 1 L; duration of new arm: MPEP+LPS: 0.9213±0.06993 vs Vehicle+LPS: 0.2509±0.03426, P <0.0001, Figure 1These results indicate that MPEP significantly alleviated LPS-induced SAD in a mouse sepsis model.

[0060] (2) MPEP restored the LPS-induced glutamate metabolism disorder and regional cerebral blood flow (rCBF) reduction in the hippocampus of SAD mice.

[0061] Figure 3 It is shown that MPEP improves LPS-induced hippocampal Glu metabolism imbalance and blood hypoperfusion in SAD mice in the examples of this application, wherein, Figure 3 A is a schematic timeline of four sets of 1H-MRS and ASL, as well as the analysis process of hippocampal 1H-MRS and ASL. Figure 3 B is the representative spectra of glutamate (Glu), creatine (Cr), and N-acetylaspartate (NAA) products in the hippocampus of the four groups of mice. Figure 3 C is the quantitative results of 1H-MRS analysis of hippocampal Glu / Cr in four groups of mice. Figure 3 D is the quantitative results of 1H-MRS analysis of hippocampal NAA / Cr in four groups of mice. Figure 3 E is a representative heat map of regional cerebral blood flow (rCBF) in the hippocampus of four groups of mice. Figure 3 F is the quantitative results of hippocampal rCBF analysis by ASL in four groups of mice. Data are presented as mean ± SEM ( n =6) and analyzed by one-way ANOVA with Tukey's post hoc test. ns: no difference, *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001.

[0062] like Figure 3 To observe the changes in hippocampal excitatory metabolite levels and corresponding rCBF, we used in vivo 1H-MRS to detect Glu and NAA levels in hippocampal neurons and ASL to detect hippocampal perfusion rCBF ( Figure 3 A). Figure 3 B shows representative 1H-MRS spectra of Glu and NAA in the four groups. Compared with the control group, the Glu / Cr ratio in the hippocampus of the LPS group increased (LPS: 0.5550±0.02975 vs Control: 0.4033±0.02824, P =0.0080; Figure 3 C), while NAA / Cr was reduced (LPS: 1.772±0.1154 vs Control: 2.632±0.1592, P =0.0015; Figure 3D). MPEP treatment significantly reversed the Glu / Cr ratio in the hippocampus of LPS-induced SAD mice (MPEP+LPS: 0.4333±0.02333 vs Vehicle+LPS: 0.5583±0.03506, P =0.0325; Figure 3 C) and NAA / Cr changes (MPEP+LPS: 2.500±0.1679 vs Vehicle+LPS: 1.887±0.1027, P =0.0256; Figure 3 D). In the hippocampal ASL test ( Figure 3 E), hippocampal rCBF in the LPS group was reduced compared with that in the control group (LPS: 74.85±4.423 vs Control: 115.2±5.620, P=0.0001; Figure 3 F). However, after MPEP treatment, hippocampal rCBF in SAD mice was restored (MPEP+LPS: 102.4±5.520 vs Vehicle+LPS: 73.47±4.876, P =0.0037; Figure 3 F). These findings indicate that MPEP treatment rescues Glu-induced neuronal damage and hypoperfusion in the hippocampus of SAD mice.

[0063] (3) MPEP alleviated SAD-induced hippocampal mGluR5 expression and hippocampal CA1 neuronal apoptosis.

[0064] Figure 4 The results of the present application show that MPEP reduces the expression of mGluR5 in the hippocampus and the apoptosis of neurons in the hippocampal CA1 region of LPS-induced SAD mice, wherein: Figure 4 A is a schematic timeline of the four in vitro tests and the analysis process of hippocampal ELISA, RT-qPCR, WB and IF staining. Figure 4 B is the quantitative results of hippocampal Glu levels in the four groups. Figure 4 C is the quantitative results of the relative mRNA expression levels of hippocampal mGluR5 in the four groups. Figure 4 D is a representative WB bar of the hippocampal mGluR5 protein expression levels in the four groups ( Figure 4 D1) and quantitative results of hippocampal mGluR5 / β-tubulin in four groups ( Figure 4 D2), Figure 4 E is the IF staining image of mGluR5 (green) in the hippocampal CA1 region of the four groups of mouse brain slices (40 times microscope, scale bar: 20 μm, Figure 4 E1) and quantitative results of mGluR5 fluorescence intensity in the hippocampal CA1 region of the four groups ( Figure 4 E2), Figure 4F is the IF staining image of NeuN (red) and TUNEL (green) in the hippocampal CA1 region of the four groups of mouse brain sections (40 times microscope, scale bar: 20 μm, Figure 4 F1) and the quantitative results of the percentage of TUNEL+ / NeuN+ in the hippocampal CA1 region of the four groups of mice ( Figure 4 F2). Data are presented as mean ± SEM ( n =6) and one-way ANOVA with Tukey's post hoc test. ns: no difference, *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001.

[0065] like Figure 4 As shown in Figure 2, we investigated the effects of MPEP on SAD-induced excitotoxicity in hippocampal neurons, particularly its associated changes in mGluR5 signaling. We performed RT-qPCR, WB and IF staining, and ELISA to assess Glu levels ( Figure 4 A). ELISA showed that the Glu level in the hippocampus of LPS mice was significantly increased compared with that of control mice (LPS: 14.31±0.7773 vs Control: 7.351±0.5766, P <0.0001; Figure 4 B). In LPS mice treated with MPEP, Glu levels in the hippocampus were significantly decreased (MPEP+LPS: 15.28±0.6095 vs Vehicle+LPS: 15.04±0.9015, P =0.0009; Figure 4 B). RT-qPCR showed that the relative mRNA level of mGluR5 in the hippocampus of LPS mice was significantly higher than that in control mice (LPS: 1.700±0.1295 vs Control: 1.003±0.0099, P =0.0007; Figure 4 C). In contrast, the relative mRNA expression of mGluR5 was decreased in LPS mice treated with MPEP (MPEP+LPS: 1.242±0.1029 vs Vehicle+LPS: 1.767±0.1286, P =0.0102; Figure 4 C). Compared with control mice, the expression of mGluR5 protein in the hippocampus of LPS mice was increased (LPS: 0.9716±0.0562 vs Control: 0.4555±0.0324, P <0.0001; Figure 4 D), while MPEP-treated LPS mice had decreased hippocampal mGluR5 protein expression (MPEP+LPS: 0.6392±0.0453 vs Vehicle+LPS: 1.083±0.0419, P <0.0001; Figure 4 D). IF staining showed that the fluorescence intensity of mGluR5 in the hippocampal CA1 region of LPS mice was significantly increased compared with that of control mice (LPS: 51.57±2.030 vs Control: 37.56±1.691, P <0.0001, Figure 4 E). The fluorescence intensity of hippocampal mGluR5 in MPEP-treated LPS mice was significantly decreased (MPEP+LPS: 43.64±1.465 vs Vehicle+LPS: 50.61±1.527, P =0.0396; Figure 4 E). In addition, TUNEL staining showed that the number of apoptotic neurons in the CA1 region of the hippocampus of LPS mice increased compared with that of control mice (LPS: 10.10±0.6001 vs Control: 3.537±0.5455, P <0.0001; Figure 4 F). Neuronal apoptosis in the hippocampal CA1 region of LPS mice treated with MPEP was significantly reduced (MPEP+LPS: 6.420±0.5060 vs Vehicle+LPS: 11.09±1.121, P =0.0012; Figure 4 F).

[0066] (4) MPEP reduced LPS-induced mGluR5 overexpression and apoptosis in hippocampal neurons.

[0067] Figure 5 The results show that MPEP reduces the expression of mGluR5 in hippocampal neurons treated with LPS and inhibits cell apoptosis. Figure 5 A shows the preparation scheme of the hippocampal SAD model and the MPEP treatment method, as well as the analysis process of biochemical tests including ELISA, RT-qPCR, WB, IF staining, and CCK-8 assay. Figure 5 B is a representative WB band of mGluR5 protein expression level in HT22 cells ( Figure 5 B1) and quantitative results of mGluR5 / β-tubulin in HT22 cells between different groups ( Figure 5 B2), Figure 5 C is the quantitative results of relative mRNA expression levels of mGluR5 in HT22 cells among different groups. Figure 5 D is the quantitative results of Glu levels in HT22 cells among different groups. Figure 5 E is the OD value of HT22 cells in CCK-8 assay, Figure 5 F is the quantitative analysis of HT22 cell viability in CCK-8 assay, Figure 5 G is the IF staining image of mGluR5 (green) in HT22 cells ( Figure 5 G1, scale bar: 50 μm) and quantitative results of mGluR5 intensity in HT22 cells between different groups ( Figure 5 G2), Figure 5 H is the IF staining image of mGluR5 (green) and TUNEL (red) in HT22 cells ( Figure 5 H1, scale bar: 50 μm) and quantitative results of the percentage of TTUNEL+ / mGluR5+ in HT22 cells among different groups ( Figure 5 H2). Data are presented as mean ± SEM ( n =3) and analyzed by one-way ANOVA with Tukey's post hoc test. ns: no difference, *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001.

[0068] like Figure 5 To further evaluate the expression of mGluR5 in hippocampal neurons, RT-qPCR, WB and IF staining, as well as CCK-8 assay reflecting cell viability were performed in cultured hippocampal neurons ( Figure 5 A). Compared with the control group, the mGluR5 protein in hippocampal neurons treated with LPS (LPS: 0.9749±0.0121 vs Control: 0.6700±0.0571, P =0.0054; Figure 5 B) and relative mRNA levels (LPS: 2.166 ± 0.0425 vs Control: 1.000 ± 0.0243, P <0.0001; Figure 5 C) significantly increased, and the level of Glu in hippocampal neurons also increased significantly (LPS: 23.74±1.346 vs Control: 15.17±0.4772, P =0.0015; Figure 5 D). The MPEP treatment group ( Figure 5 BD) inhibited the effect of LPS on mGluR5 protein in hippocampal neurons ( P =0.0443), relative mRNA (P <0.0001) and Glu ( P =0.0291). The results of CCK-8 assay showed that LPS reduced the proliferation of hippocampal neurons (LPS: 0.9350±0.01510 vs Control: 1.365±0.0147, P <0.0001; Figure 5 E) and vitality (LPS: 63.80±1.271 vs Control: 99.97±1.234, P <0.0001; Figure 5 F). MPEP treatment significantly improved LPS-induced decrease in hippocampal neuronal cell viability ( P <0.0001) ( Figure 5 EF). IF staining results ( Figure 5 G) showed that the expression intensity of mGluR5 in hippocampal neurons in the LPS group was increased compared with that in the control group (LPS: 14.27±1.116 vs Control: 7.792±0.5569, P =0.0032), but MPEP treatment reduced the effect of LPS on mGluR5 expression in hippocampal neurons ( P =0.0438). In addition, TUNEL staining ( Figure 5 H) showed that LPS increased the apoptosis of hippocampal neurons (LPS: 15.82±1.455 vs Control: 4.920±0.9212, P =0.0017), while MPEP treatment alleviated the effect of LPS on apoptosis ( P =0.0244).

[0069] (5) MPEP alleviated LPS-induced neuronal and synaptic plasticity damage in the CA1 region of the hippocampus of SAD mice.

[0070] Figure 6 It was shown that MPEP in the examples of the present application alleviated LPS-induced neuronal damage in the hippocampal CA1 region of SAD mice and improved synaptic plasticity. Figure 6 A is a schematic timeline of Nissl staining and Golgi staining in the four groups and the analysis process of Nissl staining and Golgi staining in the hippocampal CA1 region. Figure 6 B is a representative Nissl staining of the hippocampal CA1 region ( Figure 6 B1, scale bar: 20 μm) and Nissl in the hippocampal CA1 region between different groups + Quantitative results of cell number ( Figure 6 B2), Figure 6 C is a representative dendritic spine in the CA1 region of the hippocampus ( Figure 6 C1, scale bar: 5 μm) and quantitative results of the number of dendritic spines / 10 μm in the hippocampal CA1 region between different groups ( Figure 6 C2), Figure 6 D is the sholl diagram of dendritic complexity of neurons in the hippocampal CA1 region ( Figure 6 D1) and statistical analysis of dendritic complexity in the hippocampal CA1 region between different groups ( Figure 6 D2). # indicates significant differences among the four groups. Data are expressed as mean ± SEM ( n =6) and were analyzed by one-way ANOVA with Tukey's post hoc test (BC) or two-way ANOVA with Bonferroni correction (D). ns: no difference, *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001.

[0071] like Figure 6 As shown, Nissl staining was used to determine the number of neurons in the hippocampal CA1 region, and Golgi staining was used to observe changes in neuronal dendritic spine density and dendritic complexity ( Figure 6 A). Nissl staining showed neuronal damage in the CA1 region of the hippocampus of LPS mice, which is characterized by Nissl staining. + The number of cells was significantly reduced (LPS: 107.0±7.132 vs Control: 143.8±4.408, P =0.0002; Figure 6 B). Similarly, Golgi staining showed that the density of dendritic spines in the CA1 region of the hippocampus of LPS mice was significantly reduced (LPS: 7.623±0.5638 vs Control: 11.40±0.4624, P =0.0002; Figure 6 C). Sholl's analysis showed that the dendrite formation, spine formation and maturation of neurons in the CA1 region of the hippocampus of LPS mice were significantly reduced compared with those of control mice, indicating that postsynaptic plasticity was impaired in LPS mice. In LPS mice treated with MPEP, the number of neurons (MPEP+LPS: 129.8±3.754 vs Vehicle+LPS: 109.3±4.047, P =0.0416; Figure 6 B) and dendritic spine density (MPEP+LPS: 10.26±0.3849 vs Vehicle+LPS: 7.535±0.6212, P =0.0066; Figure 6 C) Increased dendritic complexity Sholl analysis ( Figure 6 D) showed that LPS significantly reduced the dendritic complexity at different distances, such as 80 μm (LPS: 9.500±1.607 vs Control: 16.333±1.333, P =0.0009), 100μm (LPS: 12.333±1.116 vs Control: 18.167±1.327, P =0.0070), 120μm (LPS: 13.167±1.470 vs Control: 21.500±1.258, P <0.0001), 140μm (LPS: 15.333±1.333 vs Control: 23.000±1.065, P =0.0001), 160μm (LPS: 15.500±1.607 vsControl: 25.000±1.000, P <0.0001), 180μm (LPS: 14.500±1.727 vs Control: 25.167±1.167, P <0.0001), 200μm (LPS: 14.000±1.932 vs Control: 23.167±0.654, P <0.0001) and 220 μm (LPS: 11.000 ± 2.082 vs Control: 20.667 ± 1.542, P <0.0001). However, sholl analysis of MPEP-treated LPS mice showed that the complexity of damaged dendritic spines was 100 μm (MPEP+LPS: 17.333±1.333 vs Vehicle+LPS: 11.500±1.607, P =0.0070), 120μm (MPEP+LPS: 21.833±1.138 vs Vehicle+LPS: 14.333±1.430, P =0.0002), 140μm (MPEP+LPS: 20.500±1.765 vs Vehicle+LPS: 13.833±1.138, P =0.0013), 160μm (MPEP+LPS: 23.000±1.211 vs Vehicle+LPS: 16.000±1.770, P =0.0006), 180μm (MPEP+LPS: 22.333±0.919 vs Vehicle+LPS: 14.667±1.994, P=0.0001), 200μm (MPEP+LPS: 2O.333±0.882 vs Vehicle+LPS: 14.000±2.280, P =0.0026) and 220μm (MPEP+LPS: 18.000±1.265 vs Vehicle+LPS: 11.167±1.195, P =0.0009). These results indicate that MPEP treatment improves neuronal damage and synaptic plasticity impairment in the CA1 region of the hippocampus in SAD mice.

[0072] (6) MPEP improved the abnormal Ca2+ levels in hippocampal CA1 neurons in LPS-induced SAD mice during free movement and Y-maze tests. 2+ Signal.

[0073] Figure 7 It is shown that MPEP reduces the spontaneous high Ca2+ activity of hippocampal CA1 neurons in free-moving SAD mice induced by LPS. 2+ Abnormal Ca during activity and Y-maze test 2+ Activity results, including Figure 7 A is the recording of Ca in hippocampal CA1 neurons by fiber photometry 2+ Schematic diagram of the activity program. Figure 7 B is the representative Ca of neurons in the hippocampal CA1 region of the Control group at baseline and 24 hours of free movement. 2+ signal wave ( Figure 7 B1) and its corresponding statistical data ( Figure 7 B2), Figure 7 C is the representative Ca level of hippocampal CA1 neurons in the LPS group at baseline and 24 hours of free movement. 2+ signal wave ( Figure 7 C1) and its corresponding statistics ( Figure 7 C2), Figure 7 D is the representative Ca2+ levels of neurons in the hippocampal CA1 region in the Vehicle+LPS group at baseline and 24 hours of free movement. 2+ signal wave ( Figure 7 D1) and its corresponding statistics ( Figure 7 D2), Figure 7 E is the representative Ca2+ / -values ​​of hippocampal CA1 neurons in the MPEP+LPS group at baseline and 24 h of free movement. 2+ signal wave ( Figure 7 E1) and its corresponding statistics ( Figure 7 E2), Figure 7 F is the Ca distribution of the control group during the Y-maze test when exploring the new arm and the other arms. 2+Representative examples of signals ( Figure 7 F1) and representative examples of heat maps ( Figure 7 F2) and its corresponding statistics ( Figure 7 F3), Figure 7 G is the Ca distribution when exploring the new arm and the other arms in the LPS group during the Y-maze test. 2+ Representative examples of signals ( Figure 7 G1) and representative examples of heat maps ( Figure 7 G2) and its corresponding statistics ( Figure 7 G3), Figure 7 H is the Ca distribution when exploring the new arm and the other arms in the Vehicle+LPS group during the Y-maze test. 2+ Representative example diagram of the signal ( Figure 7 H1) and representative examples of heat maps ( Figure 7 H2) and its corresponding statistics ( Figure 7 H3), Figure 7 I is the Ca distribution when exploring the new arm and the other arms in the MPEP+LPS group during the Y-maze test. 2+ Representative examples of signals ( Figure 7 I1) and representative examples of heat maps ( Figure 7 I2) and its corresponding statistics ( Figure 7 I3). Data are presented as mean ± SEM ( n =6) and by two-sided pairing t ns: no difference, **: P <0.01,***: P <0.001.

[0074] like Figure 7 As shown in the results, a fiber optic photometry recording device was used to observe the effects of MPEP on the spontaneous activity of neurons in the CA1 region of the hippocampus of SAD mice, as well as the neuronal activity in the Y-maze test. This required unilateral injection of pAAV-Syn-GCaMP6f into the CA1 region of the hippocampus and the expression of GCaMP6f ( Figure 7 A) Ca levels in hippocampal CA1 neurons in control mice at baseline and 24 hours after exercise. 2+ There was no significant change in signal amplitude (24Hr: 0.3525±0.0154 vs baseline: 0.3753±0.0156, t=0.7926, P =0.4639; Figure 7 B) The Ca of neurons in the CA1 region of the hippocampus of LPS mice 2+The signal showed a significant increasing trend at 24 hours (24Hr: 0.4411±0.0203 vs baseline: 0.7597±0.0141, t=4.685, P =0.0054; Figure 7 C) Ca2+ expression in neurons of the hippocampal CA1 region of LPS-treated mice 2+ The signal showed the same trend as that of LPS mice (24Hr: 0.4603±0.0186 vs baseline: 0.3532±0.0158, t=4.564, P =0.0060; Figure 7 D) However, in LPS mice treated with MPEP, Ca 2+ The signal increased at 24 hours and did not return to the baseline level, but there was no statistical difference compared with the baseline (24Hr: 0.3797±0.0182 vs baseline: 0.3389±0.0179, t=1.784, P =0.1345; Figure 7 E). During the Y-maze test, we measured changes in hippocampal CA1 neuronal activity in four groups. In the control group, when mice explored the new arm, the Ca activity of hippocampal CA1 neurons increased. 2+ The amplitude of the signal increased significantly (Novelarm: 1.112 ± 0.0941 vs oherarm: 0.4060 ± 0.0305, t = 10.16, P =0.0002; Figure 7 F). It is worth noting that the LPS group (Novelarm: 0.5894±0.0341 vs oherarm: 0.5319±0.0308, t=2.488, P =0.0553; Figure 7 G) and Vehicle+LPS group (Novelarm: 0.6340±0.0518 vs oherarm: 0.5171±0.0311, t=1.623, P =0.1656; Figure 7 H) No Ca2+ expression was observed in hippocampal CA1 neurons during exploration of the novel arm. 2+ However, in LPS mice treated with MPEP, Ca signals in the CA1 region of the hippocampus were significantly altered. 2+ The amplitude of the signal was enhanced (Novelarm: 1.009±0.0867 vs oherarm: 0.4721±0.0271, t=7.189, P =0.0008; Figure 7These results indicate that MPEP treatment reduces the Ca2+ level in hippocampal CA1 neurons in SAD mice during free movement. 2+ Abnormal activation of Ca signals and restoration of disturbed Ca signaling during the Y-maze spatial memory test 2+ Signal.

[0075] (7) MPEP restored the imbalance of excitatory and inhibitory neuronal activity in the CA1 region of the hippocampus of SAD mice induced by LPS.

[0076] Figure 8 The results show that MPEP reduced the spike discharge of excitatory pyramidal neurons in the hippocampal CA1 region of SAD mice induced by LPS and increased the spike discharge of inhibitory interneurons in the examples of the present application, wherein, Figure 8 A is the schematic schedule of EMA implantation in the CA1 region of the hippocampus for the four groups and the analysis process of MEA in the CA1 region of the hippocampus; Figure 8 B is the ISI histogram (ISI, Figure 8 B1) and auto-correlograms ( Figure 8 B2) Putative pyramidal neurons and their interactions with interneurons via cross-correlograms (Cross-correlograms, Figure 8 B3); Figure 8 C is n = 18 cells in the control group, representative spike discharge signals of pyramidal cells in the CA1 region of the mouse hippocampus at baseline and 24 hours (24Hr) of free movement ( Figure 8 C1) and its corresponding statistics ( Figure 8 C2); Figure 8 D is n =24 cells in the LPS group, representative spike discharge signals of pyramidal cells in the CA1 region of the mouse hippocampus at baseline and 24 hours (24 hr) of free movement ( Figure 8 D1) and its corresponding statistics ( Figure 8 D2); Figure 8 E is n = 22 cells in the Vehicle+LPS group, representative spike discharge signals of pyramidal cells in the CA1 region of the mouse hippocampus at baseline and 24 hours (24 hr) of free movement ( Figure 8 E1) and its corresponding statistics ( Figure 8 E2); Figure 8 F is n=21 cells in the MPEP+LPS group, representative spike discharge signals of pyramidal cells in the CA1 region of the mouse hippocampus at baseline and 24 hours (24 hr) of free movement ( Figure 8 F1) and its corresponding statistics ( Figure 8 F2); Figure 8 G is the ISI histogram (ISI, Figure 8 G1) and auto-correlograms ( Figure 8 G2) putative interneurons and their interactions with pyramidal cells through cross-correlograms (Cross-correlograms, Figure 8 G3); Figure 8 H is n = 6 cells in the Control group, representative spike discharge signals of hippocampal CA1 interneurons at baseline and 24 hours (24Hr) of free movement ( Figure 8 H1) and its corresponding statistics ( Figure 8 H2); Figure 8 I is n =7 cells in the LPS group, representative spike discharge signals of hippocampal CA1 interneurons at baseline and 24 hours (24 hr) of free movement ( Figure 8 I1) and its corresponding statistics ( Figure 8 I2); Figure 8 J is n =8 cells in the Vehicle+LPS group, representative spike discharge signals of hippocampal CA1 interneurons at baseline and 24 hours (24 hr) of free movement ( Figure 8 J1) and its corresponding statistics ( Figure 8 J2); Figure 8 K is n = 8 cells in the MPEP+LPS group, representative spike discharge signals of hippocampal CA1 interneurons at baseline and 24 hours (24 hr) of free movement ( Figure 8 K1) and its corresponding statistics ( Figure 8 K2). Data are presented as mean ± SEM ( n =4) and by two-sided pairing t ns: no difference, ***: P <0.001, ****: P <0.0001.

[0077] like Figure 8As shown, MEAs were implanted into the CA1 region of the mouse hippocampus, and spike discharge signals of excitatory and inhibitory neurons were recorded before and 24 hours after group treatment ( Figure 8 A). Excitatory and inhibitory neurons in the CA1 region of the hippocampus are interconnected and work together to play a key role in learning and memory. Spikes, often considered APs, are crucial for regulating cognitive function within the hippocampal neuronal system. Therefore, analyzing the spike discharges of excitatory and inhibitory neurons provides insight into the neural activity characteristics of MPEP-treated SAD mice. By analyzing inter-spike interval (ISI) histograms and autocorrelograms, we were able to roughly distinguish between pyramidal neurons in the CA1 region of the hippocampus ( Figure 8 B) and interneurons ( Figure 8 G) discharge activity. In addition, the cross-correlograms of different neurons ( Figure 8 B and Figure 8 G) Demonstrates excitatory monosynaptic connections between pyramidal neurons and interneurons. Figure 8 CF shows the spike firing rate of pyramidal neurons in the hippocampal CA1 region of the four groups at baseline and 24 hours. Specifically, the spike firing rate of pyramidal neurons in the control group did not change significantly between baseline and 24 hours (24 Hr: 102.8 ± 5.819 vs baseline: 99.99 ± 4.812, t = 0.8312, P =0.4174; Figure 8 C). The spike discharge of pyramidal neurons in the LPS group increased significantly at 24 hours (24Hr: 118.8 ± 4.705 vs baseline: 100.0 ± 4.298, t = 4.957, P <0.0001; Figure 8 D), similar to the changes in the Vehicle+LPS group (24Hr: 115.2±3.539 vs baseline: 100.0±4.145, t=7.058, P <0.0005; Figure 8 E). There was no statistical difference in the spike discharge level between the LPS mice treated with MPEP at 24 hours and the baseline level (24Hr: 107.1 ± 3.303 vs baseline: 100.0 ± 3.561, t = 1.841, P =0.0805; Figure 8 F). Figure 8 HK shows the spike discharge rate of hippocampal CA1 interneurons in the four groups at baseline and 24 hours. In the control group, there was no significant difference in the peak discharge of interneurons between baseline and 24 hours (24Hr: 103.0±9.774 vs baseline: 99.91±12.72, t=0.8484, P=0.4349; Figure 8 H). LPS group (24Hr: 73.04±5.598 vs baseline: 99.98±6.599, t=6.702, P =0.0005; Figure 8 I) and Vehicle+LPS group (24Hr: 67.16±7.022 vs baseline: 100.1±8.793, t=6.277, P =0.0004; Figure 8 J) showed that the spike discharge of interneurons at 24 hours was significantly lower than the baseline level. Compared with the baseline, the spike discharge of interneurons in the MPEP+LPS group at 24 hours decreased slightly, and the difference was not significant (24Hr: 93.28±4.638 vs baseline: 100.1±4.994, t=1.509, P =0.1751; Figure 8 K). These results indicate that LPS activates excitatory neurons and inhibits inhibitory neurons in the CA1 region of the hippocampus, and MPEP treatment repairs the LPS-induced imbalance in excitatory and inhibitory neuronal activity in the CA1 region of the hippocampus of SAD mice.

[0078] (8) MPEP alleviated LPS-induced dysfunction of excitability and synaptic transmission in pyramidal neurons in the CA1 region of the hippocampus of SAD mice.

[0079] Figure 9 The results show that MPEP in the present application reduces the action potential (AP) frequency and excitatory synaptic transmission of pyramidal neurons in the hippocampal CA1 region of LPS-induced SAD mice and improves inhibitory synaptic transmission. Figure 9 A is a representative illustration of APs evoked by pyramidal neurons in the hippocampal CA1 region in four groups; Figure 9 B is the quantitative results of AP induced by depolarizing current in hippocampal CA1 pyramidal neurons in different groups. Figure 9 A and Figure 9 The data in B are presented as mean ± SEM ( n =6) and analyzed using two-way ANOVA with Bonferroni correction. # indicates significant differences among the four groups. Figure 9 C is the representative signal of sEPSC amplitude and frequency of pyramidal neurons in the hippocampal CA1 region of four groups; Figure 9 D is the quantitative results of sEPSC amplitude in pyramidal neurons in the hippocampal CA1 region of the four groups; Figure 9 E is the quantitative results of sEPSC frequency in pyramidal neurons in the hippocampal CA1 region of the four groups; Figure 9 F is the representative signal of sIPSC amplitude and frequency of pyramidal neurons in the hippocampal CA1 region of the four groups; Figure 9H is the quantitative results of sIPSC amplitude and frequency in hippocampal CA1 pyramidal neurons of the four groups. Figure 9 C- Figure 9 H data are presented as mean ± SEM ( n =6) and analyzed using one-way ANOVA with Tukey's post hoc test. ns: no difference, *: P <0.05,**: P <0.01,***: P <0.001, ****: P <0.0001.

[0080] like Figure 9 To further investigate the cellular mechanism by which MPEP improves sepsis-induced delirium-like behavior, electrophysiological recordings were performed on pyramidal neurons in the CA1 region of the hippocampus. The changes in intrinsic excitability APs of pyramidal neurons in the CA1 region of the hippocampus in four groups were detected ( Figure 9 A). The results showed that the pyramidal neurons in the CA1 region of the hippocampus in the LPS group were at 150pA (LPS: 9.167±0.833 vs. Control: 5.000±0.577, P =0.0002), 200 (LPS: 12.500±0.764 vs control: 8.500±0.764, P =0.0003), 250pA (LPS: 14.677±0.715 vs Control: 9.500±0.764, P <0.0001) and 300pA (LPS: 15.167±1.138 vs control: 9.833±0.946, P <0.0002) at 24 h, the AP induced by the sham group was significantly higher than that of the control group ( Figure 9 B). MPEP treatment reversed LPS-induced AP changes in hippocampal CA1 pyramidal neurons (150pA: MPEP+LPS: 6.167±0.946 vs Vehicle+LPS: 9.833±1.108, P =0.0013; 200pA: MPEP+LPS: 9.333±0.715vs Vehicle+LPS: 12.000±0.730, P =0.0388; 250pA: MPEP+LPS: 10.500±0.847 vs Vehicle+LPS: 14.000±0.730, P =0.0024; 300pA: MPEP+LPS: 11.000±0.894 vs Vehicle+LPS: 15.000±0.966, P=0.0003)( Figure 9 B). In addition, to determine whether LPS could alter excitatory and inhibitory synaptic transmission and the restorative effects of MPEP treatment, sEPSCs were recorded from four groups of hippocampal CA1 pyramidal neurons ( Figure 9 C) and sEPSC ( Figure 9 F). Compared with the control group, the amplitude of sEPSC in the CA1 pyramidal neurons of the hippocampus in the LPS group at 24 hours (LPS: 22.64±1.291 vs Control: 14.98±0.8047, P =0.0003; Figure 9 D) and frequency (LPS: 5.400 ± 0.4091 vs Control: 2.667 ± 0.2728, P <0.0001; Figure 9 E) significantly increased, indicating that LPS enhanced excitatory synaptic input and output. Compared with the Vehicle+LPS group, MPEP treatment significantly reduced the amplitude of sIPSCs in hippocampal CA1 pyramidal neurons (MPEP+LPS: 18.53±0.8625 vs Vehicle+LPS: 23.09±1.231, P =0.0318; Figure 9 D) and frequency (MPEP+LPS: 3.667±0.2472 vs Vehicle+LPS: 5.500±0.3812, P =0.0048; Figure 9 E). In terms of inhibitory synaptic transmission, the LPS group showed an increase in the amplitude of sIPSC in hippocampal CA1 pyramidal neurons compared with the control group (LPS: 7.690±0.9583 vs Control: 13.990±1.1580, P =0.0004; Figure 9 G) and frequency (LPS: 1.800 ± 0.2394 vs Control: 3.333 ± 0.2459, P =0.0001; Figure 9 H) were significantly decreased, while MPEP treatment reversed the LPS-induced sIPSC amplitude of pyramidal neurons in the hippocampal CA1 region of SAD mice (MPEP+LPS: 11.29±0.7328 vs Vehicle+LPS: 7.453±0.6061, P =0.0296; Figure 9 G) and frequency (MPEP+LPS: 2.600±0.1155 vs Vehicle+LPS: 1.517±0.1641, P =0.0050; Figure 9H) decreased, indicating that MPEP treatment restored the inhibitory synaptic input and output damaged by LPS. These results indicate that LPS causes abnormal excitation and impaired excitatory and inhibitory synaptic transmission in pyramidal neurons in the CA1 region of the hippocampus of SAD mice, while MPEP treatment significantly reversed the functional impairment and synaptic transmission impairment of pyramidal neurons in the CA1 region of the hippocampus of SAD mice.

[0081] Through the above specific embodiments, those skilled in the art can easily implement the present application. However, it should be understood that the present application is not limited to the above specific embodiments. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to implement different technical solutions.

Claims

1. Application of the mGluR5 antagonist MPEP in the preparation of products for the treatment of sepsis-related delirium.

2. The use according to claim 1, characterized in that The MPEP alleviated the delirium-like behavior of mice with sepsis-related delirium and reversed the suppression of sleep EEG activity in mice with sepsis-related delirium.

3. The use according to claim 1, characterized in that The MPEP improved the imbalance of hippocampal glutamate metabolism and the reduction of regional cerebral blood flow in mice with sepsis-related delirium.

4. The use according to claim 1, characterized in that The MPEP reduces the expression of mGluR5 in the hippocampus and the number of neuronal apoptosis in the hippocampal CA1 region of mice with sepsis-related delirium.

5. The use according to claim 1, characterized in that The MPEP reduces the high expression of mGluR5 and the number of cell apoptosis in a mouse hippocampal neuron cell line induced by lipopolysaccharide, wherein the mouse hippocampal neuron cell line is an HT22 cell line.

6. The use according to claim 1, characterized in that The MPEP improved neuronal damage and synaptic plasticity in the hippocampal CA1 region of mice with sepsis-related delirium.

7. The use according to claim 1, characterized in that MPEP improved the abnormal Ca2+ activity in hippocampal CA1 neurons of sepsis-related delirium mice during free movement and Y-maze tests. 2+ Signal.

8. The use according to claim 1, characterized in that The MPEP repaired the imbalance of excitatory and inhibitory neuronal activities in the CA1 region of the hippocampus of mice with sepsis-related delirium.

9. The use according to claim 1, characterized in that The MPEP alleviated the excitability and synaptic transmission dysfunction of pyramidal neurons in the hippocampal CA1 region of mice with sepsis-related delirium.

10. The use according to any one of claims 1 to 9, characterized in that The product for treating sepsis-related delirium is a drug for treating sepsis-related delirium. The drug comprises an effective dose of the MPEP and pharmaceutically acceptable excipients. The drug is an injection preparation, and the injection preparation is administered by intraperitoneal infusion.

Images