Application of AZA1 in the preparation of drugs for treating sepsis and drugs

Abstract

The present invention discloses the use of AZA1 in the preparation of a drug for treating sepsis and the drug itself, belonging to the field of biomedicine. AZA1 of the present invention can mediate hypothalamic microglial homeostasis through the Arhgap24-Rac1 / Cdc42 signaling axis, regulating the septic immune response, thereby promoting sepsis recovery and improving prognosis, providing a potential new strategy for central nervous system immune regulation in the treatment of patients with systemic sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of AZA1 in preparing a medicament for treating sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS) triggered by infection, which can lead to multiple organ dysfunction and even death. It is a critical illness with extremely high morbidity and mortality worldwide, particularly common in intensive care units (ICUs). Sepsis is not only life-threatening but can also cause loss of productivity. According to statistics, approximately 49 million cases of sepsis occur worldwide each year, with 11 million deaths (accounting for 20% of all global deaths), making it a leading cause of death in ICU patients. Sepsis is a systemic inflammatory storm triggered by infection, a devastating condition that requires early identification and comprehensive treatment. Current treatments for sepsis include antimicrobial therapy, fluid resuscitation, vasopressors, mechanical ventilation (for patients with acute respiratory distress syndrome), renal replacement therapy (for patients with acute kidney injury), and corticosteroid shock. Despite continuous advances in treatment, sepsis' high mortality rate remains a major global health challenge. Some studies suggest that the immune response in the central nervous system plays a crucial role in the development of sepsis, but the specific mechanisms remain under investigation.

[0003] Therefore, in-depth exploration of the central nervous system immune regulation mechanism of systemic sepsis and the development of central nervous system immune regulation strategies targeting this mechanism have important clinical value and application prospects. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the use of AZA1 in the preparation of a drug for treating sepsis and the drug, aiming to provide a potential new strategy for central nervous system immune regulation for the treatment of patients with systemic sepsis.

[0005] The present invention found that AZA1 can mediate the homeostasis of hypothalamic microglia through the Arhgap24-Rac1 / Cdc42 signaling axis, regulate the immune response to sepsis, thereby promoting the recovery of sepsis and improving the prognosis, providing a potential new strategy for central nervous system immune regulation for the treatment of patients with systemic sepsis.

[0006] Among them, the CAS number of AZA1 is: 1071098-42-4, and the structural formula of AZA1 is as follows: .

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides use of AZA1 in the preparation of a medicament for treating sepsis.

[0009] Rac1 and Cdc42 belong to the Rho family of GTPases. These two proteins play a key role in cell migration, proliferation, cytoskeleton reorganization and other processes. Arhgap24 is a Rho family GAP enzyme that specifically acts on the GTPases Rac1 and Cdc42. It promotes the GTP hydrolysis of Rac1 / Cdc42 and the transition to the GDP-bound state, thereby negatively regulating cytoskeleton remodeling and downstream signaling pathways.

[0010] AZA1 is a dual inhibitor of Rac1 / Cdc42. AZA1 treatment can inhibit Rac1 / Cdc42, thereby significantly reducing sepsis-induced microglial migration and activation, alleviating neuroinflammation, and improving systemic inflammation and clinical prognosis.

[0011] AZA1 can mediate hypothalamic microglial homeostasis through the Arhgap24-Rac1 / Cdc42 signaling axis, regulate the immune response to sepsis, thereby promoting recovery from sepsis and improving prognosis, providing a potential new strategy for central nervous system immune regulation in the treatment of patients with systemic sepsis.

[0012] A second aspect of the present invention provides a drug for treating sepsis, wherein the drug comprises AZA1.

[0013] Optionally, the drug further comprises a pharmaceutically acceptable carrier.

[0014] Beneficial Effects: AZA1, a drug of the present invention, effectively inhibits excessive microglial activation and abnormal migration by targeting the Arhgap24-Rac1 / Cdc42 signaling axis in hypothalamic microglia, maintaining immune homeostasis. This, in turn, mitigates the inflammatory response in the central nervous system (particularly the median eminence-arcuate nucleus region) associated with sepsis, slowing the progression of systemic inflammatory storms and improving the body's tolerance to septic infection. Through this central regulatory mechanism, AZA1 significantly improved the survival rate, clinical scores, and systemic inflammation levels of septic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 2 shows the characteristic diagram of hypothalamic inflammatory factors and serum inflammatory factor storm caused by sepsis; A: protein electrophoresis results of hypothalamic inflammatory factors in the sham operation group and the cecal puncture and ligation (CLP) group at different time points; BD: comparison of the mRNA expression levels of hypothalamic pro-inflammatory factors in the sham operation group and the CLP group at different time points; EG: curve of changes in serum pro-inflammatory factor levels in the sham operation group and the CLP group over time; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0016] Figure 2 Figure 2 shows the characteristic diagram of hypothalamic microglial activation caused by sepsis; A: representative images of immunofluorescence staining of Iba1 (green), DAPI (blue) and merged images (Merge) in the hypothalamus of the sham group and the CLP group; B: statistical results of microglial cell density in the arcuate nucleus of the hypothalamus of the sham group and the CLP group; C: representative images of morphological analysis of single microglial cells in the arcuate nucleus of the hypothalamus of the sham group and the CLP group; DI: statistical results of morphological analysis of microglial cells in the hypothalamus of the sham group and the CLP group; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0017] Figure 3 The results show that the Rac1 / Cdc42 regulatory protein Arhgap24 is increased in hypothalamic microglia in sepsis; A: Representative images of immunofluorescence staining of Iba1 (green), Arhgap24 (red) and merged images (Merge) in the hypothalamus of the sham operation group and the CLP group; BC: Electrophoresis and statistical results of Arhgap24 protein in the hypothalamus of the sham operation group and the CLP group; **: P < 0.01.

[0018] Figure 4 The results show that lipopolysaccharide induces increased expression of Arhgap24 in BV2 cells; A: representative images of immunofluorescence staining of Iba1 (green), Arhgap24 (red) and merged images (Merge) in the control group BV2 cell line and after lipopolysaccharide treatment; BC: electrophoresis and statistical results of Arhgap24 protein in the control group and after lipopolysaccharide treatment in the BV2 cell line; **: P < 0.01.

[0019] Figure 5 This figure shows the dose-dependent inhibitory effect of AZA1 on BV2 cell viability.

[0020] Figure 6 Figure 2 shows the results of AZA1 blocking pseudopodia formation in BV2 cells; A: Representative images of F-actin (red) and DAPI (blue) immunofluorescence staining of BV2 cells in the control group, under AZA1 stimulation, LPS stimulation, and combined stimulation; BC: Statistical results of the length and number of pseudopodia of BV2 cells in each group; *: P < 0.05; **: P < 0.01.

[0021] Figure 7 Figure 2 shows the results of AZA1 inhibiting BV2 cell proliferation; A: representative images of BrdU (green) and DAPI (blue) immunofluorescence staining of BV2 cells in the control group, under AZA1 stimulation, LPS stimulation, and combined stimulation; B: statistical results of BrdU-positive cells in BV2 cells in each group; *: P < 0.05; **: P < 0.01.

[0022] Figure 8 Figure 1 shows that AZA1 improves the immune response of hypothalamic microglia in sepsis and improves prognosis; A: Representative images of immunofluorescence staining of Iba1 (green) and DAPI (blue) in the arcuate nucleus of mice treated with sham surgery + artificial cerebrospinal fluid (aCSF), sham surgery + AZA1 administration, CLP + artificial cerebrospinal fluid, and CLP + AZA1 administration; B: Statistical results of microglial cell density in the arcuate nucleus of the four groups of mice; CG: morphological analysis results of microglial cells in each group; H: Statistical results of survival of mice in each group; I: Hypothalamic inflammatory factors in each group Il1b mRNA quantitative results graph; J: Body weight change curve of each group of mice; K: Clinical score results of each group on day 1 of modeling; LN: Statistical results of serum pro-inflammatory factor levels of each group on day 1 of modeling; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001. DETAILED DESCRIPTION

[0023] The present invention provides the use of AZA1 in the preparation of a medicament for treating sepsis. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0024] The present invention is described in detail below through specific examples.

[0025] 1. Materials and Methods

[0026] 1. Experimental Animals

[0027] Male SPF-grade C57BL / 6 mice, 6-8 weeks old, were purchased from the Animal Experimental Center of Southern Medical University. They were housed in a standard SPF-grade laboratory animal room at the Nanfang Hospital of Southern Medical University (humidity maintained at approximately 45-60%, ambient temperature maintained at approximately 22-24°C, daily light and dark cycles of 12 h each, background noise at 30-45 decibels, and purified air). Mice were acclimated to the experimental environment 3 days before the experiment. The use and maintenance conditions of experimental animals complied with the regulations and guidelines of the Animal Experimental Center of Southern Medical University. All experimental procedures and experimental procedures were reviewed and approved by the Ethics Committee of Southern Medical University.

[0028] 2. Survival rate, body weight and clinical scores

[0029] Three days before surgery and one week after surgery, mouse survival rate, body weight, and clinical scores were recorded daily at the same time (9:00 AM ± 1 hour). Clinical scores were based on a modified SHIRPA (SmithKline, Harwell, Imperial College, Royal Hospital, Phenotype Assessment) protocol. Specific components include piloerection, abdominal distortion, altered muscle tone, stool consistency, lacrimation, palpebral closure, motor activity, tactile avoidance reflex, spontaneous behavior, grip strength, kyphosis, and respiratory rate. Each abnormality was scored 1 point, with a maximum score of 12. Death on the same day was scored 12 points. These data were used to assess disease progression and severity.

[0030] 3. Mouse Perfusion Fixation and Brain Removal

[0031] Mice were perfused, fixed, and brain tissue extracted. The specific steps were as follows: Mice were deeply anesthetized by intraperitoneal injection of a 1% sodium pentobarbital solution (80 mg / kg, representing 80 mg of sodium pentobarbital per kg of animal body weight) at a mass / volume (% w / v) concentration. Mice were placed in a supine position with their limbs secured and moderate tension maintained on the abdominal skin. Heart exposure: The skin and peritoneum were incised along the midline of the abdomen, and the ribs were carefully dissected to fully expose the thoracic cavity and heart, avoiding damage to the lungs. Blood collection: Blood samples were drawn from the left ventricle using a 1 mL syringe. After centrifugation, serum was collected for subsequent analysis of inflammatory factors. Perfusion preparation: A purple scalp needle was inserted into the left ventricle and secured. A small amount of 4°C pre-cooled saline was injected to inflate the left ventricle. The right atrial appendage was opened, and 4°C pre-cooled saline was perfused through the left ventricle using an automated micropump until the liver turned pale, indicating complete depletion of peripheral blood. Replace the perfusion solution with 4% paraformaldehyde solution (prepared by dissolving 4 g of paraformaldehyde in 100 mL of phosphate buffered saline) pre-cooled at 4°C for fixation. Brain tissue extraction: After perfusion is complete, remove the brain tissue as per conventional methods for subsequent sectioning.

[0032] 4. Mouse Serum Enzyme-Linked Immunosorbent Assay (ELISA)

[0033] Inflammatory cytokine levels were measured using ELISA kits according to the kit instructions, and the results were read using a multi-functional microplate reader. The specific steps were as follows: All experimental groups were assigned and equilibrated at room temperature for 2 h. Calibrator wells, sample diluent wells, blank wells, and sample wells were set up. 50 μL of calibrator of varying concentrations was added to each calibrator well, 50 μL of sample diluent was added to the sample diluent wells, and nothing was added to the blank wells. 50 μL of the sample to be tested was added to the sample wells. 100 μL of biotin antibody was added to each well except the blank wells. The reaction plate was covered with a sealing film and incubated in a 37°C incubator in the dark for 60 min. The plate was washed five times. 100 μL of horseradish peroxidase (HRP)-conjugated avidin was added to each well except the blank wells. The plate was covered with a sealing film and incubated in a 37°C incubator in the dark for 20 min. The plate was washed five times. 100 μL of 3,3',5,5'-tetramethylbenzidine colorimetric solution (TMB colorimetric solution) was added to all wells. Cover the reaction plate with sealing film and incubate in a 37°C incubator in the dark for 15 minutes. Add 50 μL of stop solution to each well and read the absorbance (OD) of each well on a multi-function microplate reader at a wavelength of 450 nm.

[0034] 5. Immunofluorescence Staining

[0035] Washing: Select frozen sections and cell slides of the corresponding layer, place them in a 6-well plate, and wash three times with phosphate-buffered saline (PBS), each time for 5-10 minutes. Non-specific antigen blocking: Place the sections in a humidified chamber containing 5wt% donkey serum and 0.2wt% Triton 100 in phosphate buffered saline, and block in a 37°C oven for 1 hour. Primary antibody incubation: Prepare the primary antibody solution according to the concentration recommended in the instructions, add it to the antibody incubation plate, transfer the sections and cell slides to the primary antibody, and incubate overnight at 4°C. Secondary antibody incubation: The next day, transfer the sections and cell slides to phosphate-buffered saline containing 0.2wt% Triton X-100 (abbreviated as 0.2wt% PBS-T buffer), wash three times, add the corresponding species of fluorescent-labeled secondary antibody, and incubate at 37°C in the dark for 1 hour. 4',6-diamidino-2-phenylindole (DAPI) staining and mounting: After incubation, wash three times with 0.2wt% PBS-T buffer for 5-10 minutes each. Sections and cell slides were incubated with DAPI for 5 minutes at room temperature in the dark. After washing, they were transferred to glass slides, and excess liquid was removed. The sections were mounted with antifade reagent and covered with a coverslip. Images were captured using a laser confocal microscope (Carl Zeiss).

[0036] 6. Microglial Morphological Analysis and 3D Reconstruction

[0037] Imaris 9.9 software (Bitplane) was used to perform morphological three-dimensional reconstruction and quantitative analysis of Iba1-positive microglia. First, the Z-stack images of the Iba1 channel acquired by laser confocal microscopy were imported into Imaris, and the background correction module was used to remove non-specific signals and uneven illumination. Subsequently, an automatic threshold algorithm was applied in the "Surface" tool to identify the outline of the microglial cell body, and dynamic calculations were performed based on the k-means statistical clustering method to take into account the integrity of the cell body and slender protrusions; manual fine-tuning could be performed if necessary to ensure the complete capture of pseudopodia. After the cell body segmentation was completed, the surface was used as a mask to further extract the Iba1 signal channel outside the cell body and generate an independent protrusion network model; in the "FilamentTracer" module, parameters such as the minimum diameter and branch length were set to automatically track and reconstruct all pseudopodia and branches. Finally, the three-dimensional volume (µm) of each cell was obtained through the "Statistics" output module of Imaris. 3 ), total length of protrusions, number of branches and other morphological indicators.

[0038] 7. BV2 Cell Culture and Drug Stimulation

[0039] BV2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. When cells reached approximately 80% confluence, they were gently washed three times with PBS, harvested by pipetting, centrifuged at 300×g for 5 minutes, resuspended in fresh culture medium, and expanded at a 1:3 ratio. Medium was changed every 2-3 days. Experiments were performed on healthy cells at passages 5-15. For stimulation experiments, cells were starved with serum-free DMEM for 1 hour and then stimulated with 1µg / mL lipopolysaccharide or 3µM / mL AZA1 for 24 hours. After stimulation, cells were harvested for immunofluorescence and protein electrophoresis.

[0040] 8.BrdU proliferation assay

[0041] Cells were seeded on slides, 10 μM 5-bromodeoxyuridine (BrdU) was added to the culture medium and the corresponding drugs were treated at the same time. After incubation at 37 °C, 5% CO2 for 6 h, the cells were fixed with paraformaldehyde and treated with 2 M hydrochloric acid for 10 min for antigen retrieval. They were then neutralized with 0.1 M boric acid buffer and finally subjected to BrdU immunofluorescence staining.

[0042] 9.CCK-8 cell viability assay

[0043] BV2 cells were collected at 1×10 4Cells were seeded / well in a 96-well plate and incubated at 37°C, 5% CO2 for 24 h. The old solution was discarded and treated with 0.1-10 μM gradient AZA1 or an equal volume of DMSO (dimethyl sulfoxide) as a control for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 1 h. The absorbance (OD) was measured at 450 nm on a microplate reader. The cell viability (%) was calculated as [(OD treatment group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0044] 10. Tissue Protein Extraction

[0045] After the postoperative observation period, the animals were deeply anesthetized. After perfusion, the brain tissue was removed from the skull and placed in a glass dish containing a small amount of pre-chilled saline. Under a stereomicroscope, the brain tissue was carefully dissected using the optic chiasm, third ventricle, and pituitary stalk as anatomical landmarks. Hypothalamic tissue was removed using microtweezers and placed into pre-labeled centrifuge tubes. Brain tissue washing and lysis: Hypothalamic tissue was washed three times with PBS to remove residual blood. 500 μL of radioimmunoprecipitation assay lysis buffer (RIPA) containing protease and phosphatase inhibitors was added to each centrifuge tube. The inhibitor-to-lysis buffer ratio was 1:100 (1 volume of protease and phosphatase inhibitors per 100 volumes of RIPA buffer). The brain tissue was thoroughly ground using a frozen tissue homogenizer and lysed at 4°C for 30 minutes. The lysed samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein Extraction and Processing: After centrifugation, carefully aspirate the supernatant, transfer it to new, labeled centrifuge tubes, and determine protein concentration according to the BCA kit (Beyotime, China) instructions. Dilute the sample to the appropriate concentration using PBS. Add 5× loading buffer at a 4:1 volume ratio and mix thoroughly to obtain a mixture. Heat denature the mixture in a 100°C metal bath for 5 minutes, then immediately cool it in an ice-water mixture, centrifuge it, and aliquot it for storage at -20°C.

[0046] 11. Protein electrophoresis

[0047] Separate and detect protein samples using SDS-polyacrylamide gel (SDS-PAGE) electrophoresis: Prepare an SDS-PAGE gel. Load the tissue protein sample into the sample loading wells of the stacking gel. Simultaneously, add a protein molecular weight marker to the appropriate wells as a molecular weight reference. Loading buffer is added to the unused wells on both sides to prevent edge effects. Place the gel plate in the electrophoresis tank and add running buffer (Tris-glycine-SDS buffer). Run the gel at a constant voltage of 80 V until the indicator (bromophenol blue) rises above the stacking gel. Then, adjust the voltage to 120 V and continue running until the target protein bands are clearly resolved. After electrophoresis, carefully remove the gel and excise the portion of the gel corresponding to the target band using a gel knife. A polyvinylidene fluoride (PVDF) membrane (Millipore, USA) is activated with methanol and placed in a transfer apparatus along with the excised gel. After transfer, the PVDF membrane is blocked with freshly prepared 5% fetal bovine serum (BSA) for 1 hour at room temperature to block nonspecific binding. After blocking, the PVDF membrane was incubated with the corresponding primary antibody overnight at 4°C. The next day, the membrane was washed three times with TBST (Tris-buffered saline containing 0.1 wt% Tween-20) for 5 minutes each. The membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. After incubation, the membrane was washed again with TBST three times for 5 minutes each. The membrane was stored at 4°C and visualized using electrochemiluminescence (ECL).

[0048] 12. RNA Extraction and Fluorescence Quantitative PCR Detection

[0049] RNA from hypothalamic tissue was extracted using the TRIzol method (Invitrogen), and 1 μg of RNA was reverse transcribed using ABScript III RTMaster Mix. qPCR was performed using ABclonal SYBR Green Mix on a QuantStudio 5. Glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) as internal reference, 2 –ΔΔCt All operations were performed under RNase-free conditions to ensure RNA integrity.

[0050] The primers used in this experiment are shown in Table 1 below:

[0051] Table 1

[0052]

[0053] 13. Microcannula Implantation and Drug Administration into the Lateral Ventricle

[0054] Three days before surgery, C57 mice were placed in the experimental environment and allowed to acclimate. Under 1% isoflurane anesthesia, a craniotomy was performed on a stereotaxic apparatus (Rayward). A cannula was implanted in the right lateral ventricle (0.30 mm posterior to bregma, 1.00 mm lateral to the midline, and 0.25 mm subdural) and secured with dental cement. Following surgery, the mice were observed to ensure normal function. One week after surgery, 1 µL of artificial cerebrospinal fluid (aCSF) or AZA1 (100 µg / mouse) was infused via a polyurethane (PU) tubing connected to a microinjection pump.

[0055] 2. Experimental Results Analysis

[0056] 1. Increased hypothalamic and serum inflammatory factors after sepsis

[0057] Cecal puncture and ligation (CLP) is the gold standard for animal models of sepsis, effectively simulating the clinical pathological features of patients following intraperitoneal infection, including systemic inflammation, immune dysfunction, and multi-organ damage. The CLP model, which utilizes mid-cecal ligation, induces a moderate systemic inflammatory response and has a higher survival rate (approximately 50%), reducing experimental failures due to high mortality and improving experimental reproducibility.

[0058] Detection of proinflammatory factor proteins and mRNA in hypothalamic tissue after CLP ( Figure 1 AD), and serum inflammatory factor ELISA detection ( Figure 1 EG). From Figure 1 It can be seen that the inflammatory factors TNF-a, IL-1β, and IL-6 in the hypothalamus and serum increased at different points, indicating that the development of sepsis is not only accompanied by an increase in systemic inflammatory factors, but also by hypothalamic neuroinflammation.

[0059] 2. Immune response of hypothalamic microglia after sepsis

[0060] 24 hours after CLP modeling, microglia in the hypothalamic arcuate nucleus (ARC) showed obvious aggregation and activation. Specifically, microglia activation was most obvious in the median eminence-arcuate nucleus complex (ME-ARC) region lacking the blood-brain barrier, while microglia in the blood-brain barrier-intact region where the ARC gradually extended into the brain parenchyma did not show obvious activation ( Figure 2 Middle AB). After CLP, ARC microglia showed shortened branches, roughness, and amebic changes ( Figure 2 The results indicate that the immune response process of hypothalamic microglia activation and migration occurs after sepsis.

[0061] 3. Arhgap24 regulates hypothalamic microglial immune responses after sepsis

[0062] The dynamic remodeling of cell morphology is highly dependent on the activation of Rho family small G proteins Rac1 and Cdc42, and Arhgap24, as an important GTPase activating protein (GAP), plays a key negative feedback regulatory role by promoting the transition of Rac1 / Cdc42 to the GDP-bound state. Immunofluorescence staining of the arcuate nucleus confirmed the increased expression of Arhgap24 on microglia ( Figure 3 Western blotting of the hypothalamus showed that Arhgap24 protein was significantly increased 1 day after CLP ( Figure 3 Chinese BC).

[0063] To further clarify the changes of Arhgap24 in microglia induced by inflammation, the BV2 cell line was stimulated with lipopolysaccharide (LPS) to simulate the inflammatory environment in vitro. Immunofluorescence staining and protein immunoblotting experiments showed that the expression of Arhgap24 protein in microglia increased significantly after LPS treatment ( Figure 4 AC).

[0064] 4. AZA1 regulates the immune response of hypothalamic microglia in sepsis through the Arhgap24-Rac1 / Cdc42 signaling axis and improves prognosis.

[0065] In order to determine the optimal concentration of AZA1, different concentrations of AZA1 (0.1 μM-10 μM) were used for stimulation and CCK-8 assay was performed to determine the half-maximal inhibitory concentration (IC 50 ), the test results are shown in Figure 5 The data are presented as mean ± SEM, and the IC 50 The results showed that AZA1 could significantly inhibit the viability of BV2 cells at a low micromolar level.

[0066] Figure 6 Figure 2 shows the results of AZA1 blocking pseudopodia formation in BV2 cells; A: Representative images of F-actin (red) and DAPI (blue) immunofluorescence staining of BV2 cells in the control group, under AZA1 stimulation, LPS stimulation, and combined stimulation; BC: Statistical results of the length and number of pseudopodia of BV2 cells in each group; *: P < 0.05; **: P < 0.01.

[0067] Figure 7 Figure 2 shows the results of AZA1 inhibiting BV2 cell proliferation; A: representative images of BrdU (green) and DAPI (blue) immunofluorescence staining of BV2 cells in the control group, under AZA1 stimulation, LPS stimulation, and combined stimulation; B: statistical results of BrdU-positive cells in BV2 cells in each group; *: P < 0.05; **: P < 0.01.

[0068] from Figure 6 As shown in Figures AC, in the control group, BV2 cells had a small number of pseudopodia, but after LPS stimulation, the number of filopodia increased significantly. After AZA1 treatment, the number and length of cell pseudopodia decreased significantly, both in the control group and under LPS combined treatment conditions. At the same time, AZA1 treatment significantly slowed the proliferation rate of BV2 cells. The BrdU proliferation assay showed that both AZA1 treatment alone and combined with LPS treatment significantly reduced the BrdU positive rate ( Figure 7 Middle AB), indicating that the Rac1 / Cdc42 inhibitor AZA1 not only inhibits cell motility and structural remodeling, but also effectively weakens the proliferation activity of microglia.

[0069] Figure 8 Figure 1 shows that AZA1 improves the immune response of hypothalamic microglia in sepsis and improves prognosis; A: Representative images of immunofluorescence staining of Iba1 (green) and DAPI (blue) in the arcuate nucleus of mice treated with sham surgery + artificial cerebrospinal fluid (aCSF), sham surgery + AZA1 administration, CLP + artificial cerebrospinal fluid, and CLP + AZA1 administration; B: Statistical results of microglial cell density in the arcuate nucleus of the four groups of mice; CG: morphological analysis results of microglial cells in each group; H: Statistical results of survival of mice in each group; I: Hypothalamic inflammatory factors in each group Il1b mRNA quantitative results graph; J: Body weight change curve of each group of mice; K: Clinical score results of each group on day 1 of modeling; LN: Statistical results of serum pro-inflammatory factor levels of each group on day 1 of modeling; ns: no statistical difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0070] After CLP, AZA1 treatment significantly reduced CLP-induced microglial migration to the ARC region and overactivation ( Figure 8 Daily intracerebroventricular (ICV) injection of AZA1 significantly improved the survival rate of CLP mice ( Figure 8 Middle H) and clinical scores ( Figure 8 K), and inhibit hypothalamic inflammatory factors Il1b mRNA expression ( Figure 8 Inhibits the levels of systemic inflammatory factors IL-1β, IL-6, and TNF-α ( Figure 8 AZA1 treatment had no significant effect on the body weight of mice ( Figure 8 J). This indicates that in the CLP model, AZA1 inhibits Rac1 / Cdc42, reduces neuroinflammation by limiting the pathological activation and excessive migration of microglia, and improves systemic inflammation and clinical prognosis.

[0071] In summary, the application of AZA1 provided by the present invention in the preparation of a drug for treating sepsis and the drug, the present invention AZA1 can mediate the homeostasis of hypothalamic microglia through the Arhgap24-Rac1 / Cdc42 signaling axis, regulate the immune response of sepsis, thereby promoting the recovery of sepsis and improving the prognosis, providing a potential new strategy for central nervous system immune regulation for the treatment of patients with systemic sepsis.

[0072] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Use of AZA1 in the preparation of a medicament for treating sepsis, wherein the structural formula of AZA1 is as follows: .

2. The use according to claim 1, characterized in that AZA1 mediates hypothalamic microglial homeostasis through the Arhgap24-Rac1 / Cdc42 signaling axis, regulates the immune response to sepsis, promotes recovery from sepsis, and improves prognosis.

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