Use of lag3 agonists in the preparation of hematoma clearance drugs after cerebral hemorrhage and use of lag3 in regulating hematoma clearance after cerebral hemorrhage

Abstract

This invention relates to the application of LAG3 agonists in the preparation of drugs for hematoma clearance after intracerebral hemorrhage (ICH) and the application of LAG3 in regulating hematoma clearance after ICH, belonging to the field of biomedical technology. This invention utilizes a mouse ICH model to first inject a LAG3 neutralizing antibody (C9B7W) via orthotopic injection, clarifying that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance, revealing its potential molecular target for enhancing microglia phagocytic function and promoting hematoma clearance. Furthermore, overexpression of LAG3 after ICH is demonstrated. Lag3 The study confirmed that enhanced phagocytic function of microglia accelerated hematoma clearance, demonstrating that LAG3 agonists can be used as drugs for hematoma clearance after intracerebral hemorrhage. The experimental results of this invention make it possible to apply LAG3 to the prognostic treatment of ICH, thereby advancing basic and clinical research on endogenous hematoma clearance after ICH.

Description

Technical Field

[0001] This invention relates to the application of LAG3 agonists in the preparation of drugs for hematoma clearance after cerebral hemorrhage and the application of LAG3 in regulating hematoma clearance after cerebral hemorrhage, belonging to the field of biomedical technology. Background Technology

[0002] Intracerebral hemorrhage (ICH) refers to bleeding caused by the rupture of small arteries within the brain parenchyma, not due to trauma. It is a subtype of stroke, accounting for approximately 20-30% of all strokes in my country. ICH is characterized by high morbidity, mortality, and disability rates, and its incidence in my country is showing an increasing trend year by year. The mass effect caused by hematoma after ICH and secondary damage driven by hematoma components are the main causes of brain injury in patients. However, current treatment options for ICH are limited. Craniotomy not only has certain requirements regarding the volume of the hematoma, but its efficacy is also not significant. Minimally invasive drainage has also been reported to be associated with a good prognosis only when the residual hematoma volume is less than 15 mL; otherwise, it cannot improve the patient's prognosis. Therefore, reducing the hematoma volume after ICH through endogenous hematoma evacuation mechanisms is a feasible research approach.

[0003] Studies have shown that microglia, as key innate immune cells, act as guardians of the brain, rapidly migrating to and proliferating in the injured area as early as 6 hours after a brain hemorrhage. Following a brain hemorrhage, microglia protect surrounding cells by phagocytosing hematoma breakdown products and dead neurons, playing a crucial role in hematoma clearance. Current research on microglia's hematoma clearance after brain hemorrhage mainly focuses on scavenger receptors. These receptors are activated by binding to ligands on damaged red blood cells or free heme, prompting cells to take up and phagocytose them. For example, after a brain hemorrhage, the lysis of red blood cells in the hematoma produces hemoglobin (Hb). Free Hb attaches to haptoglobin (Hp), and the Hp-Hb complex can then be internalized by phagocytes expressing CD163. CD47 signals, shifting from a "don't eat me" to a "eat me" signal, mediate phagocytosis by macrophages. CD36, a type II scavenger receptor expressed on the surface of macrophages, allows cells lacking phagocytic capacity to acquire this function after transfection with CD36. Studies have shown that interleukin-10 derived from microglia can regulate CD36 to accelerate hematoma clearance after intracerebral hemorrhage. CD36 is also a key target for downstream gene transcription mediated by PPARγ; activation of PPARγ can increase CD36 expression, thereby promoting the phagocytic function of microglia. Other studies have indicated that TLRs (Toll-like receptors) may act as regulators of microglia phagocytosis; TLR4 knockout leads to upregulation of CD36 around the hematoma, and inhibition of the TLR4 signaling pathway may promote microglia phagocytosis. Agonists of nuclear factor erythroid 2-related factor 2 (Nrf2) can also increase the phagocytic capacity of microglia and reduce hematoma volume. These findings suggest that targeting phagocytosis-related targets on microglia is an important pathway to accelerate hematoma clearance. However, challenges remain in clinical practice regarding the conversion of therapeutic targets, necessitating further clarification of the specific mechanisms of hematoma clearance after intracerebral hemorrhage and the search for innovative, effective, and feasible therapeutic targets.

[0004] Lymphocyte Activation Gene 3 (LAG3) is an immune checkpoint primarily expressed on immune cells such as lymphocytes, microglia, and macrophages. Previous research on LAG3 has mainly focused on its role in regulating T lymphocyte function in peripheral tumors, influencing T lymphocyte immune responses by modulating T lymphocyte activation levels. For example, LAG3 exhibits a tumor immune escape mechanism similar to PD-1 (Programmed cell death protein 1). High expression of LAG3 on CD4+ and CD8+ T lymphocytes can cause T lymphocytes to lose their potent effector function, leading to tumor cell "escape"—these T lymphocytes are termed exhausted T lymphocytes. Conversely, low expression of LAG3 may result in reduced Treg cell inhibitory capacity, leading to excessive activation of immune cells. LAG3 and PD-1 synergistically inhibit anti-tumor immunity and autoimmunity. Studies have reported that co-blockade of LAG3 and PD-1 enhances the proliferation of tumor-infiltrating CD8+ T lymphocytes and cytokine production, thus LAG3 is considered a promising therapeutic target in cancer immunotherapy. Other studies have shown LAG3 expression on T lymphocytes in tumor models such as brain metastases and melanoma; in Parkinson's disease, LAG3 exacerbates neuronal α-synuclein endocytosis and deposition, while blocking LAG3 reduces neuronal α-synuclein deposition; in Alzheimer's disease, LAG3 has been reported to promote the transmission of pathological tau between neurons; furthermore, LAG3 has been reported to influence depressive-like behavior in unpredictable stimuli-induced depression models. However, while the regulation of peripheral T lymphocyte function by LAG3 after tumorigenesis is becoming increasingly clear, research on LAG3's effects on the central nervous system is limited. The role of LAG3 after cerebral hemorrhage and whether LAG3 regulates microglial function after cerebral hemorrhage remain unknown. Summary of the Invention

[0005] The first objective of this invention is to provide the application of LAG3 agonists in the preparation of hematoma evacuation drugs after cerebral hemorrhage, and to provide a novel phagocytosis-related target on microglia.

[0006] The second objective of this invention is to provide the application of LAG3 in regulating hematoma clearance after cerebral hemorrhage, and to provide a new target that can effectively regulate the phagocytic capacity of microglia.

[0007] To achieve the above objectives, the technical solution for the application of LAG3 agonists in the preparation of hematoma evacuation drugs after cerebral hemorrhage in this invention is as follows:

[0008] Application of LAG3 agonists in the preparation of drugs for hematoma evacuation after cerebral hemorrhage.

[0009] The beneficial effects of the above technical solution are as follows: the application of the LAG3 agonist of this invention in the preparation of hematoma clearance drugs after intracerebral hemorrhage is a pioneering invention. This invention first constructs a mouse striatal ICH model. To visualize the intensity and localization of LAG3 in the brain, a neutralizing antibody against LAG3 (C9B7W) is labeled with a CY5.5 fluorescent tag. Then, the successfully labeled C9B7W and adeno-associated virus specifically overexpressing Lag3 in microglia are injected orally into the mouse striatal ICH model. Through hematoma volume detection, hemoglobin content detection, immunofluorescence detection, flow cytometry detection, and pHrodo-E. coli phagocytosis detection, it is demonstrated that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance, revealing its potential as a molecular target for enhancing microglia phagocytic function and promoting hematoma clearance. In the future, it is hoped that LAG3 can be applied to the prognostic treatment of ICH to promote basic and clinical research progress on endogenous hematoma clearance after ICH. This fully demonstrates that LAG3 agonists can be used as drugs for hematoma evacuation after cerebral hemorrhage.

[0010] As a further improvement, the hematoma-clearing drug after cerebral hemorrhage is a drug that reduces the volume of the hematoma after cerebral hemorrhage.

[0011] As a further improvement, the hematoma-clearing drug after cerebral hemorrhage is a drug that enhances the phagocytic capacity of microglia after cerebral hemorrhage.

[0012] As a further improvement, the hematoma-clearing drug after cerebral hemorrhage is a drug that improves motor function after cerebral hemorrhage.

[0013] As a further improvement, the drug for improving motor function after cerebral hemorrhage is a drug for improving neurological function damage.

[0014] As a further improvement, the drug for improving motor function after cerebral hemorrhage is a drug for improving limb and sensory impairments.

[0015] To achieve the above objectives, the technical solution for the application of LAG3 in regulating hematoma clearance after cerebral hemorrhage in this invention is as follows:

[0016] The application of LAG3 in regulating hematoma clearance after cerebral hemorrhage, wherein the application is not for disease diagnosis and treatment.

[0017] The beneficial effects of the above technical solution are as follows: This invention, by using a mouse ICH model, for the first time injecting LAG3 neutralizing antibody (C9B7W) in situ, clarifies that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance, revealing that it can serve as a potential molecular target for enhancing the phagocytic function of microglia and promoting hematoma clearance. In the future, it is expected that LAG3 can be applied to the prognostic treatment of ICH to promote the basic and clinical research progress on endogenous hematoma clearance after ICH. Attached Figure Description

[0018] Figure 1 This is a localization diagram of cells surrounding the hematoma after intracerebral hemorrhage, where LAG3 is mainly expressed on microglia in Example 1 of the present invention (where AC represents the co-localization of D7 LAG3 with IBA1, GFAP, and NeuN after ICH; D represents the proportion analysis of the number of co-localized D7 LAG3 and microglia after ICH (n=3); E is the co-localization diagram of LAG3 and microglia after ICH under high magnification; F is the 3D diagram and Imaris modeling diagram of LAG3 and microglia after ICH, scale bar=5μm).

[0019] Figure 2 This is a graph showing the results of LAG3 expression primarily on microglia rather than monocytes / macrophages after ICH in Example 1 of this invention (where A is a flow cytometry plot of microglia and monocytes / macrophages after ICH; B is the change in the number of microglia on D1, D3, D5, and D7 in the Sham and ICH groups (n=3-5, data from each group were subjected to Shapiro-Wilk test for normality, one-way ANOVA test was used, F=0.53, P=0.71, R...). 2=0.10, Tukey's multiple comparison test showed P>0.05 between groups; C is the mean fluorescence intensity of LAG3 on microglia in D1, D3, D5, and D7 of the Sham and ICH groups (n=3-5, data were subjected to Shapiro-Wilk test for normality, Brown-Forsythe ANOVA test, F=17.44, P<0.001, Games-Howell multiple comparison test showed *P<0.05D1 vs D5, ***P<0.001Sham vs D5); D is the change in the number of monocytes and macrophages in D1, D3, D5, and D7 of the Sham and ICH groups (n=3-5, data were subjected to Shapiro-Wilk test for normality, Brown-Forsythe ANOVA test, F=15.82, P<0.001, Games-Howell multiple comparison test showed ***P<0.001Sham vs D5). D3, **P<0.01D1 vs D3, **P<0.01D3 vs D7); E represents the mean fluorescence intensity of LAG3 on D1, D3, D5, and D7 monocytes / macrophages in the Sham and ICH groups (n=3-5, data from each group were subjected to the Shapiro-Wilk test for normality, Brown-Forsythe ANOVA test was used, F=3.77, P=0.05, Games-Howell multiple comparison test showed P>0.05 between groups); F is a comparison of the peak diagrams of the mean fluorescence intensity of LAG3 on unstained LAG3, D5 microglia, and monocytes / macrophages after ICH).

[0020] Figure 3 The images show the results of the effective action of LAG3 neutralizing antibody on microglia after in situ injection in Example 1 of this invention (where A is a comparison of the absorbance of C9B7W and C9B7W-CY5.5; B is in vivo imaging and statistical diagrams at different time points after in situ injection of C9B7W-CY5.5, with ICH+C9B7W on the far left and ICH+C9B7W-CY5.5 on the right; C is brain slice imaging and statistical diagrams at different time points after in situ injection of C9B7W-CY5.5, with ICH+C9B7W on the far left and ICH+C9B7W-CY5.5 on the right; D is the fluorescence co-localization map of IBA1 and C9B7W-CY5.5 after ICH).

[0021] Figure 4The graph shows the results of slowed hematoma clearance after LAG3 function blockade in Example 1 of this invention (where A is a comparison and statistical graph of hematoma volume between the C9B7W group and the IgG group after ICH at D3 and D5, scale bar = 10 mm (D3 n = 3, D5 n = 13-15, data of each group are subject to Shapiro-Wilk test for normality, two-way ANOVA test, Bonferroni multiple comparison test shows **P < 0.01 D5 IgG vs C9B7W); B is a comparison and statistical graph of hemoglobin content between the C9B7W group and the IgG group (n = 8-13, data of each group are subject to Shapiro-Wilk test). The normality test was performed (F=2.47, P=0.23; unpaired t-test was used, t=2.11, *P<0.05); C is a comparison of the average fluorescence intensity of CD68 in the C9B7W and IgG groups after immunofluorescence; D is a statistical graph of the average fluorescence intensity of CD68 in the D5C9B7W and IgG groups after immunofluorescence ICH (n=4, the data of each group were subject to the Shapiro-Wilk test for normality, F=2.21, P=0.53; unpaired t-test was used, t=2.88, *P<0.05); E is a comparison of the peak diagrams of the average fluorescence intensity of CD68 in the C9B7W and IgG groups by flow cytometry; F is a statistical graph of the average fluorescence intensity of CD68 in the C9B7W and IgG groups by flow cytometry (n=7, the data of each group were not subject to the Shapiro-Wilk test for normality, and the Mann-Whitney test was used). test*P<0.05); G is a comparison of the average fluorescence intensity peaks of LAMP1 in the C9B7W group and the IgG group by flow cytometry; H is a flow cytometry statistical plot of the average fluorescence intensity of LAMP1 in the C9B7W group and the IgG group (n=3-4, data are subject to Shapiro-Wilk test for normality, F=1.99, P=0.70, unpaired t-test is used, t=1.17, P=0.30);

[0022] Figure 5The figures show the results of reducing hematoma volume after ICH in Example 1 of this invention by overexpressing Lag3 on microglia (where A is the comparison and statistics of hematoma volume between the Lag3 overexpression group and the control group, scale bar = 10 mm (n = 5-10, data of each group were subjected to Shapiro-Wilk test for normality, F = 2.63, P = 0.21, unpaired t-test was used, t = 2.57, *P < 0.05); B is the statistics of hematoma volume and hemoglobin content between the Lag3 overexpression group and the control group (n = 5-10, data of each group were subjected to Shapiro-Wilk test for normality, F = 1.47, P = 0.58, unpaired t-test was used, t = 3.00, *P < 0.05); C is the statistics of microglia number between the Lag3 overexpression group and the control group (n = 3-5, data of each group were subjected to Shapiro-Wilk test for normality, F = 1.47, P = 0.58, unpaired t-test was used, t = 3.00, *P < 0.05); C is the statistics of microglia number between the Lag3 overexpression group and the control group (n = 3-5, data of each group were subjected to Shapiro-Wilk test for normality). The test showed normality (F = 1.1, P > 0.05), and an unpaired t-test was used (t = 0.35, P > 0.05). D represents the average fluorescence intensity of CD68 in the Lag3 overexpression group and the control group (n = 3–5, and the data for each group follow the Shapiro-Wilk standard). The normality test for the test results was performed (F = 4.78, P = 0.36; unpaired t-test was used, t = 1.09, P = 0.32); E represents the average fluorescence intensity of LAMP1 in the Lag3 overexpression group and the control group (n = 3-5; data for each group were subject to the Shapiro-Wilk test for normality, F = 3.11, P = 0.52; unpaired t-test was used, t = 0.17, P > 0.05); F represents the pHrodo+ microglia in the Lag3 overexpression group and the control group (n = 7; data for each group were subject to the normality test, F = 1.38, P = 0.71; unpaired t-test was used, t = 1.49, P = 0.16).

[0023] Figure 6This invention relates to the effect of LAG3 function blockade or overexpression on motor function in mice after ICH in Example 1 of this invention (where A is the NDS score of mice in the IgG group and C9B7W group (n=10-14, data of each group were subjected to Shapiro-Wilk test for normality, F=1.31, P=0.70, unpaired t-test was used, t=4.11, ***P<0.001); B is the suspension test time of mice in the IgG group and C9B7W group (n=9-12, data of each group were subjected to Shapiro-Wilk test for normality, F=1.50, P=0.52, unpaired t-test was used, t=3.17, **P<0.01); C is the number of slips of the contralateral limb / total steps (%) of mice in the grid test of mice in the IgG group and C9B7W group (n=9-12, data of each group were subjected to Shapiro-Wilk test for normality, F=1.50, P=0.52, unpaired t-test was used, t=3.17, **P<0.01); C is the grid test number of slips of the contralateral limb / total steps (%) of mice in the IgG group and C9B7W group (n=9-12, data of each group were subjected to Shapiro-Wilk test for normality). The normality test for the test was performed (F = 1.48, P = 0.59; unpaired t-test was used, t = 2.32, *P < 0.05); D represents the NDS scores of mice in the control group and the Lag3 overexpression group (n = 5-11, data from each group did not conform to the Shapiro-Wilk test for normality, Mann-Whitney test was used, *P < 0.05); E represents the duration of suspension test in the control group and the Lag3 overexpression group (n = 5-11, data from each group conformed to the Shapiro-Wilk test for normality, F = 6.74, P = 0.08; unpaired t-test was used, t = 2.16, *P < 0.05); F represents the percentage of slippage of the contralateral limb / total steps in the grid test in the control group and the Lag3 overexpression group (n = 5-11, data from each group conformed to the Shapiro-Wilk test for normality, F = 6.74, P = 0.08, unpaired t-test was used, t = 2.16, *P < 0.05); F represents the percentage of slippage of the contralateral limb / total steps in the grid test in the control group and the Lag3 overexpression group (n = 5-11, data from each group conformed to the Shapiro-Wilk test for normality). Normality test, F = 2.19, P = 0.29; unpaired t-test, t = 2.41, *P < 0.05). Detailed Implementation

[0024] Existing research indicates that microglia play a crucial role in hematoma clearance after intracerebral hemorrhage (ICH) by phagocytosing hematoma breakdown products and dead neurons to protect surrounding cells. Numerous studies have shown that targeting phagocytosis-related targets on microglia is an important pathway to accelerate hematoma clearance; however, challenges remain in clinical practice regarding the conversion of therapeutic targets. This necessitates further clarification of the specific mechanisms of hematoma clearance after ICH and the search for innovative, effective, and feasible therapeutic targets. Based on this, this invention provides the application of LAG3 agonists in the preparation of drugs for hematoma clearance after intracerebral hemorrhage.

[0025] This invention utilizes a mouse ICH model to inject a LAG3 neutralizing antibody (C9B7W) in situ for the first time, clarifying that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance. It reveals that LAG3 can serve as a potential molecular target for enhancing microglia phagocytic function and promoting hematoma clearance. In the future, it is hoped that LAG3 can be applied to the prognostic treatment of ICH to promote the basic and clinical research progress on endogenous hematoma clearance after ICH.

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; however, these embodiments are merely examples and do not constitute any limitation on the scope of the present invention. Modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent manufacturers.

[0027] Statistical methods:

[0028] The flow cytometry results of this invention were analyzed using FlowJo10 software. All statistical analyses were performed in GraphPad Prism9. For each group of data, the Shapiro-Wilk test for normality was performed first. For comparisons between two groups, if they conformed to a normal distribution and had homogeneous variances, an unpaired t-test was used; if they conformed to a normal distribution but had unequal variances, an unpaired t-test with Welch correction was used; if they did not conform to a normal distribution, the non-parametric Mann-Whitney test was used. Statistical results are expressed as mean ± standard deviation (mean ± SD). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference.

[0029] Specific embodiments of the application of the LAG3 agonist in the preparation of hematoma evacuation drugs after cerebral hemorrhage and the application of LAG3 in regulating hematoma evacuation after cerebral hemorrhage:

[0030] Example 1: In situ injection of LAG3 neutralizing antibody effectively targets microglia.

[0031] In this embodiment, a mouse striatal ICH model was first constructed. Immunofluorescence was used to observe the cellular localization of LAG3 around the hematoma, and flow cytometry was used to detect the changes in LAG3 expression on microglia on D1, D3, D5, and D7 after ICH.

[0032] Second, LAG3 function was blocked using a LAG3 neutralizing antibody (C9B7W), and C9B7W was labeled with a fluorescent tag CY5.5. In vivo imaging technology was used to observe the effect of a single in situ injection of C9B7W-CY5.5 in the mouse brain after ICH.

[0033] Third, after in situ injection of C9B7W, the hematoma volume and hemoglobin content of the C9B7W group and the IgG group on day 5 after ICH were compared. Immunofluorescence and flow cytometry were used to detect the expression of CD68 and LAMP1 in microglia and their phagocytosis of pHrodo-labeled E. coli. Adeno-associated virus that specifically overexpresses Lag3 on microglia was used to observe the phagocytic function of microglia and hematoma clearance on day 5 after ICH.

[0034] The specific steps are as follows:

[0035] All experimental animals were 2-3 month old male C57BL / 6 mice, weighing approximately 22-30g. The animals had free access to food and water, and all experiments strictly adhered to relevant national regulations. In this embodiment, animals were randomly assigned to groups using a website (http: / / www.randomizer.org): the Sham group (injected with saline), the ICH group (injected with 0.5μL saline containing 0.075U type VII collagenase), the IgG group (injected with 1μL PBS containing 5μg of IgG isotype control + 0.5μL saline containing 0.075U type VII collagenase), the C9B7W group (injected with 1μL PBS containing 5μg of anti-mouse LAG-3 neutralizing antibody + 0.5μL saline containing 0.075U type VII collagenase), and the C9B7W-CY5.5 group (injected with 1μL PBS containing 5μg of anti-mouse LAG-3 neutralizing antibody (with the CY5.5 tag)). PBS + 0.5 μL saline containing 0.075 U type VII collagenase, Control group (injected with negative control adeno-associated virus + 0.5 μL saline containing 0.075 U type VII collagenase) and Lag3 overexpression group (injected with adeno-associated virus specifically overexpressing Lag3 on microglia + 0.5 μL saline containing 0.075 U type VII collagenase).

[0036] 1. Construction of the mouse striatal ICH model

[0037] (1) Anesthesia: Mice were placed in a gas anesthesia device and inhaled 2.5% isoflurane to induce deep anesthesia. In subsequent experiments, 1.5% isoflurane was used to maintain the anesthesia of mice.

[0038] (2) Fixation: Fix the mouse's incisors to the incisor hook of the stereotaxic instrument, push one ear rod into the mouse's external auditory canal so that the mouse's head is in the center, then push the other ear rod into the external auditory canal on the other side, press against the skull and tighten the knob to adjust the height and keep it horizontal.

[0039] (3) Expose the anterior and posterior fontanelles: Disinfect the mouse head with iodine solution, and use a scalpel blade to make a 2cm incision along the sagittal axis of the mouse head to expose the anterior and posterior fontanelles;

[0040] (4) Leveling and positioning: With the anterior fontanelle as the origin, adjust the values ​​of the X, Y and Z axes to 0. Move the glass electrode forward and backward and left and right respectively to observe the change in distance between the electrode and the surface of the skull. That is, the error of the Z axis is less than 0.2mm. After leveling, move the glass electrode back to the anterior fontanelle and then move it to the target coordinates (0.8mm in front of the anterior fontanelle, 2.1mm to the left, and 3.3mm deep).

[0041] (5) Drilling: After marking the coordinates with a pencil, drill holes at the marked locations until a hollowed-out feel is achieved;

[0042] (6) Injection: Use a glass electrode to draw the injection solution, fix the glass electrode to the drilling point, and adjust the Z-axis to 0 when the electrode and the drilling plane are at the same height. Start injection after the electrode reaches a depth of 3.3 mm, and keep the speed at 0.1 μL / min.

[0043] (7) Stop the needle: In order to avoid the solution sticking to the glass electrode wall, stop the needle for 2 minutes after the injection is completed, lift it 0.2 mm, and then stop the needle for 6 minutes.

[0044] (8) Lifting and suturing: After the needle is stopped, slowly lift the glass electrode and suture the mouse head skin after it leaves the skull surface.

[0045] 2. Neutralizing antibody and virus injection

[0046] (1) C9B7W and IgG injection

[0047] Take 5 μg of C9B7W or IgG with a concentration of 8.57 mg / mL, and adjust the volume to 1 μL with PBS buffer at pH 7. Inject it in situ 0.8 mm anterior to the anterior fontanelle and 2.1 mm lateral to the left side 2 h after ICH modeling, to a depth of 3.3 mm.

[0048] (2) Adeno-associated virus injection

[0049] Twenty-one days before ICH modeling, 1 μL of adeno-associated virus (pAAV-F4 / 80p-MCS-EGFP-3Flag-SV40-PolyA) that specifically overexpresses LAG3 in microglia or negative control virus was injected into the anterior fontanelle at a depth of 3.3 mm, 2.5 mm to the left.

[0050] 3. Fluorescent label CY5.5 marked C9B7W

[0051] Add 0.1 mg of CY5.5 powder to a centrifuge tube containing 1 mL of DMSO, wrap with aluminum foil to protect from light, and shake to ensure complete dissolution. Dilute 8.57 mg / mL C9B7W 10 times to 0.857 mg / mL and add to the CY5.5 solution. Stir overnight at 4°C. Filter unbound CY5.5 using an ultrafiltration centrifuge tube and aliquot at -80°C. Add 2 μL of C9B7W and C9B7W-CY5.5 solutions to the sample cell of a UV spectrophotometer and measure their absorbance. Compare the absorbance peaks to determine if labeling was successful.

[0052] 4. Live imaging

[0053] Two hours after establishing the mouse striatal ICH model, C9B7W-CY5.5 was injected orally. The fluorescence intensity of C9B7W-CY5.5 around the hematoma was measured at 1, 3, 5, and 7 days post-ICH. The small animal in vivo imaging system was initialized for 30 minutes before the experiment. Mice were anesthetized with isoflurane, and the scalp was cut to fully expose the skull. Anesthetized mice were placed in the in vivo imaging chamber for fluorescence imaging detection. Subsequent image processing and analysis were performed using the accompanying software.

[0054] 5. Behavioral testing

[0055] Blinding test: In this embodiment, all behavioral tests were conducted using the blinding rule. The mice were grouped and labeled by an experimenter who was completely unaware of the experimental grouping, or the following behavioral tests were evaluated directly by an experimenter who was unaware of the experimental grouping.

[0056] (1) Neurological deficit score

[0057] Under standard indoor conditions, mice were placed in the behavioral laboratory for 1 hour to acclimatize before undergoing the tests listed in the table below. The tests included 6 items, each with 5 levels, ranging from 4 points to 0 points, as detailed in Table 1. Mice with a total score below 4 points or above 20 points were disqualified.

[0058] Table 1 Neurological deficit scores

[0059]

[0060]

[0061] (2) Suspension test

[0062] Mice were placed in a behavioral laboratory for 1 hour to acclimatize before the experiment. The experimental setup consisted of an iron frame with a 1mm thick, 55cm long iron wire placed on top at a height of 50cm. A soft object was placed below to prevent injury to the mice. During the experiment, the mice were required to support their suspended weight using their forelimbs. To prevent the mice from using all four limbs, their hind limbs were taped or similar materials. The time each mouse spent on the iron wire was recorded, and the average of three experiments for each mouse was taken.

[0063] (3) Grid testing

[0064] Under standard indoor conditions, mice were placed in a behavioral laboratory for 1 hour to acclimatize before the experiment. Mice were placed on a 35cm×30cm×40cm wire mesh and walked for 3 minutes. A camera was used to record the video from below the mesh. The total number of steps and the number of slips on the contralateral hind limb of the mouse within 3 minutes were counted. The percentage of slips was calculated as (number of slips / total steps) × 100%.

[0065] 6. Material selection for grouting

[0066] Mice were deeply anesthetized with 2.5% isoflurane. After deep anesthesia, the mice were fixed to a stage, and the chest cavity was opened with scissors to fully expose the heart. A perfusion needle was inserted into the left apex of the heart, and the right atrial appendage was simultaneously cut open. Approximately 50 mL of 37°C 0.9% NaCl was infused into the left ventricle, and perfusion was stopped when the liver turned completely white. Then, paraformaldehyde at 4°C was used for perfusion, and perfusion was stopped when the mouse's muscles became rigid and the neck stiffened. The skull was dissected, and the intact brain tissue was removed. Note: If fresh tissue is required, perfusion with paraformaldehyde is unnecessary.

[0067] 7. Hematoma volume measurement

[0068] The perfusion sampling method is described in step 6. Mice were sampled on day 5 after ICH modeling. Fresh, intact brain tissue was cut into 1mm thick coronal brain slices. The front and back of each brain tissue were photographed with a measuring ruler. ImageJ software was used to analyze the hematoma volume. Hematoma volume = the sum of the hemorrhage volume of each brain tissue slice (mm3). The hemorrhage volume of each brain tissue slice = (the hemorrhage area on the front × 1mm + the hemorrhage area on the back × 1mm) / 2).

[0069] 8. Hemoglobin content detection

[0070] The perfusion sampling method is described in step 6. Mice were subjected to ICH modeling on day 5. The hemoglobin content of the brain tissue was quantified using a hemoglobin assay kit (Sigma). The hematoma center was collected with 500 mL of ice-cold PBS, homogenized for 1 min, centrifuged at 16000 g for 15 min, and 50 μL of the supernatant was transferred to a flat-bottomed plate of a 96-well plate. After incubation with the reaction reagent for 5 min, the absorbance was measured at 400 nm. The hemoglobin content was calculated using a standard curve.

[0071] 9. Flow cytometry

[0072] Mice were subjected to ICH modeling on days 1, 3, 5, and 7. Fresh tissue surrounding the hematoma was collected and prepared into single-cell suspensions using a mild mouse brain tissue enzymatic digestion kit. High-speed centrifugation with a high-efficiency defraction reagent was used to remove impurities, followed by removal of residual red blood cells using a erythrocyte lysis reagent to obtain the final single-cell suspension. 10 cells were taken as the sample for testing. 6 Add 50 μL of flow cytometry antibody diluted to the appropriate ratio to each cell. Gently mix with a micropipette and incubate at 4°C for 30 min. After centrifugation and discarding the supernatant, add 1 mL of cold PBS and wash twice by centrifugation to remove unbound excess antibody components. Finally, add 300 μL of cold PBS to the cells, mix well, and store at 4°C protected from light. Perform flow cytometry analysis according to the procedure, and then analyze the data using Flowjo flow cytometry software. The cell gating strategy involved in this example includes microglia (7AAD-LY6G-CD45intCD11b+), monocytes / macrophages (7AAD-LY6G-CD45highCD11b+), and T lymphocytes (7AAD-CD45+CD3+). In addition, it also includes the detection of indicators such as CD68 and LAMP1 on microglia.

[0073] 10. pHrodo-E. coli phagocytosis detection

[0074] Mice were carcass-positive on day 5 after ICH modeling. Fresh tissue surrounding the hematoma was collected and prepared into a single-cell suspension using a mild mouse brain tissue digestion kit. High-speed centrifugation with a high-efficiency defraction reagent was used to remove impurities, followed by erythrocyte lysis reagent to remove residual red blood cells, yielding the final single-cell suspension. pHrodo is an acid-dependent dye; the fluorescence of E. coli labeled with pHrodo increases as the pH decreases from the neutral cytoplasmic pH to the acidic pH of the endosome. 50 μg / mL pHrodo-E. coli was added to the single-cell suspension and incubated at 37°C for 2 hours, followed by incubation with a flow cytometry antibody targeting microglia for analysis.

[0075] 11. Immunofluorescence

[0076] (1) Grinding material sourcing

[0077] The perfusion sampling method is described in 6. Mice were sampled on day 5 after ICH modeling. The intact brain tissue was fixed in 4% paraformaldehyde for 24 hours and stored at 4°C.

[0078] (2) Gradient dehydration

[0079] After the brain tissue was fixed, it was first immersed in 20% sucrose for 24 hours, and then immersed in 30% sucrose for 48 hours.

[0080] (3) Slicing, mounting, and baking of slices

[0081] After cerebellar resection, the brain tissue was placed on a circular tray with the olfactory bulb facing upwards. After embedding, the brain tissue was first rapidly cooled at -80°C for 10 minutes, then thawed at -20°C for 15 minutes. The brain tissue was then fixed in the cryostat slot at -20°C. Unwanted tissue was quickly removed, and once the lesion was visible, sections were slowly cut, ensuring symmetry. Section thickness was 25 μm, and cutting continued until the lesion disappeared. After sectioning, the brain slices were rinsed three times with 0.01M PBS. After rinsing, the slices were carefully mounted onto glass slides using a small brush and then dried in an oven.

[0082] (4) Staining

[0083] 1) Washing: Wash the brain slices three times with 0.01M PBS, 5 minutes each time.

[0084] 2) Blocking: Use 200 μL of blocking solution (10% fetal bovine serum and 0.1% Triton-X 100 in PBS) for one slide and block at room temperature for 2 h in a humidified chamber.

[0085] 3) Incubation with primary antibody: Discard the blocking solution, add the primary antibody and antibody dilution solution (0.01M PBS) to the slide in a certain ratio, and incubate overnight at 4°C.

[0086] 4) Washing: Wash brain slices three times with 0.01M PBS, 5 minutes each time.

[0087] 5) Incubation with secondary antibody: Add fluorescent secondary antibody to the brain slice in the dark and incubate at room temperature for 2 hours;

[0088] 6) Washing: Wash brain slices three times with 0.01M PBS, 5 minutes each time.

[0089] 7) Sealing and testing: Blot dry the liquid with absorbent paper and seal the slide with a sealing solution containing an anti-fluorescence quencher.

[0090] 8) Imaging: Confocal fluorescence microscopy was used for imaging. 3-4 brain slices were selected from each brain tissue. Each brain slice was photographed around the hematoma. The number of fields of view was greater than 4. ImageJ software was then used to statistically analyze the number of immunofluorescence cells or the number of co-labeled cells.

[0091] 12. Experimental Results

[0092] (1) LAG3 was expressed on microglia after ICH, and its expression was significantly increased on microglia 5 days after ICH (specific experimental results are as follows). Figure 1 , 2 (As shown).

[0093] LAG3 and IBA1 (microglia and monocytes / macrophages), GFAP (astrocytes), and NeuN (neurons) were double-labeled using immunofluorescence assays. Figure 1 AC was used to observe the cellular localization of LAG3 around the hematoma after ICH. The results showed that approximately 64.33% of IBA1+ cells (microglia and monocytes / macrophages) around the hematoma after ICH expressed LAG3. Figure 1 D), while astrocytes and neurons do not express LAG3 ( Figure 1 BC). Furthermore, under high magnification, it can be clearly observed that LAG3 is mainly expressed on the cell bodies and processes of microglia, but not in the nucleus. Figure 1 E), 3D modeling of microglia and LAG3 also confirms this. Figure 1 F).

[0094] Since IBA1 cannot effectively distinguish between microglia and peripherally infiltrating monocytes / macrophages, this embodiment uses flow cytometry to differentiate between microglia and peripherally infiltrating monocytes / macrophages and observe the expression of LAG3 on them. First, the principal cell clusters of the tissue surrounding the hematoma after ICH were de-adhesed. After a de-adhesion step, 7AAD- viable cells were de-selected. After removing LY6G+ granulocytes, microglia (CD45intCD11b+) and peripherally infiltrating monocytes / macrophages (CD45highCD11b+) were obtained. Figure 2 A). The results showed that the number of microglia did not change significantly after ICH (Sham 8.6±2.62% vs D1 9.99±1.97% vs D3 8.82±2.34% vs D5 8.06±2.12% vs D7 9.58±2.46%). Figure 2 B), however, compared to the Sham group, LAG3 expression on microglia showed a decreasing trend on D1 after ICH, followed by a gradual increase, and a significant increase on D5 (Sham 26.68±2.74 vs D1 18.02±9.08 vs D3 31.9±5.08 vs D5 40.74±3.55 vs D7 44.23±3.50). Figure 2C). The number of monocytes / macrophages infiltrating D3 significantly increased after ICH, then gradually decreased (Sham 1.38±0.99% vs D1 6.00±2.63% vs D3 16.44±2.65% vs D5 13.09±6.76% vs D7 2.40±2.18%). Figure 2 D). Although the expression trend of LAG3 in monocytes and macrophages was consistent with that in microglia (Sham 22.7±5.73 vs D1 19.48±7.74 vs D3 24.36±5.33 vs D5 28.6±2.48 vs D7 37.2±9.72), Figure 2 E), but LAG3 expression levels are higher in microglia than in monocytes / macrophages (E). Figure 2 F).

[0095] (2) Oral injection of LAG3 neutralizing antibody effectively targets microglia (specific experimental results are as follows). Figure 3 As shown):

[0096] After ICH (intra-hemispheric encephalopathy), an anti-mouse LAG3 antibody (C9B7W) was injected orally into the striatum of mice to observe phenotypic changes following ICH. This antibody recognizes and binds to the D2 structural threshold of LAG3, thus blocking LAG3 function. To visualize the intensity and localization of C9B7W in the brain, C9B7W was labeled with a CY5.5 fluorescent tag. The absorbance of C9B7W and C9B7W-CY5.5 was then measured to determine whether the fluorescent tagging was successful. UV spectrophotometry results showed that C9B7W-CY5.5 had a significant absorption peak at 673 nm compared to C9B7W, indicating that the CY5.5 fluorescent tag had been successfully applied to C9B7W. Figure 3 A).

[0097] A single orthotopic injection of C9B7W-CY5.5 was administered after ICH (in vivo imaging). The fluorescence intensity of C9B7W-CY5.5 in the mouse brain at different time points was detected using in vivo imaging. Compared with the C9B7W group, significant red fluorescence was observed in the brains and brain slices of mice on days 1, 3, 5, and 7 after orthotopic injection of C9B7W-CY5.5. The fluorescence intensity was highest on day 3 after ICH, gradually decreasing thereafter, but significant fluorescence expression was still observed on day 5 after ICH. Figure 3 BC).

[0098] Subsequently, immunofluorescence was used to further observe the co-localization of C9B7W-CY5.5 and IBA1. The results showed that C9B7W-CY5.5 and IBA1 co-localized on day 5 after ICH. Figure 3 D) indicates that C9B7W can act on microglia after ICH to block LAG3.

[0099] (3) LAG3 function blockade slows down hematoma clearance (specific experimental results are as follows) Figure 4 As shown):

[0100] First, the effect of LAG3 function blockade on hematoma clearance after ICH was observed. Results showed no difference in hematoma volume between the IgG group and the C9B7W group on day 3 after ICH (IgG group: 10.37±0.40 mm). 3 vs C9B7W group 9.08±2.35mm 3 , Figure 4 A) indicates that there was no difference in the initial hemorrhage volume between the two groups. However, the hematoma volume in the C9B7W group on day 5 after ICH was higher than that in the IgG group (IgG group 3.07±1.54 mm). 3 vs C9B7W group 5.86±2.58mm 3 , Figure 4 A) indicates that hematoma clearance was delayed in the C9B7W group, and the higher hemoglobin content in the C9B7W group compared to the IgG group also confirms this (IgG group 9.25±2.90 mg / dL vs C9B7W group 13.07±4.57 mg / dL). Figure 4 B).

[0101] Second, both immunofluorescence and flow cytometry results showed that CD68 expression was reduced in microglia of the C9B7W group compared to the IgG group (immunofluorescence: IgG group 50.65±9.98 vs C9B7W group 33.35±6.72; flow cytometry: IgG group 34.89±6.50 vs C9B7W group 25.84±10.36). Figure 4 In CF (C9B7W), LAMP1 expression showed a decreasing trend but no significant difference (IgG group 273±67.67 vs C9B7W group 196.8±95.45). Figure 4 The results (GH) indicate that LAG3 function blockade reduces the phagocytic function of microglia after ICH, thus affecting hematoma clearance after ICH.

[0102] (4) Overexpression of Lag3 on microglia reduces hematoma volume after ICH (specific experimental results are shown in the figure). Figure 5 As shown):

[0103] Twenty-one days before ICH, AAV-Lag3-F4 / 80-EGFP was injected to overexpress Lag3 on microglia to construct an ICH model. Results showed that the hematoma volume in the Lag3-overexpressing group was significantly lower on day 5 after ICH than in the control group (control group 0.96±0.43 mm). 3 vs. Lag3 overexpression group: 0.51 ± 0.27 mm 3 , Figure 5 A), and the hemoglobin level in the Lag3 overexpression group was also lower than that in the control group (control group 13.94±3.32 mg / dL vs Lag3 overexpression group 9.13±2.74 mg / dL). Figure 5 B) indicates that overexpression of Lag3 on microglia can accelerate hematoma clearance after ICH.

[0104] Furthermore, the Lag3 overexpression group did not exhibit an increased number of microglia (control group 10.88±3.10% vs. Lag3 overexpression group 10.06±3.26%). Figure 5 C), CD68 expression on microglia (control group 29.13±4.81 vs Lag3 overexpression group 36.34±10.52), Figure 5 D) and the number of E. coli phagocytosed (control group 87.6±3.15% vs Lag3 overexpression group 90.33±3.69%). Figure 5 F) all showed an increasing trend, but LAMP1 expression remained basically unchanged. Figure 5 D). This indicates that the phagocytic function of microglia overexpressing Lag3 is enhanced after ICH.

[0105] (5) LAG3 function blockade aggravated motor function deficits in mice after ICH, while overexpression of Lag3 in microglia improved motor function in mice after ICH (specific experimental results are as follows). Figure 6 As shown):

[0106] ICH significantly impairs the neurological function of mice, accompanied by limb and sensory disturbances. Neurological deficit scores are used to assess the degree of neurological deficit in mice. The suspension test observes the mice's motor function, including grip strength and balance, while the grid test assesses the mice's sensorimotor coordination.

[0107] The results showed that, compared with the IgG group, the D5 C9B7W group mice after ICH had more severe neurological deficits (IgG group 5.2±1.55 points vs C9B7W group 8.07±1.77 points). Figure 6 A) The persistence time in the suspension test was shortened (IgG group 30.27±10.06s vs C9B7W group 17.64±8.23s). Figure 6 B) In the grid test, the percentage of slippage on the contralateral lower limb was also higher in the IgG group than in the IgG group (IgG group 25.5±10.01% vs C9B7W group 37.08±12.18%). Figure 6 C).

[0108] Compared with the control group, the neurological deficit score of ICH mice was reduced after overexpression of Lag3 in microglia (control group 9.4±2.61 points vs. Lag3 overexpression group 6.73±1.35 points). Figure 6 D), the duration of the suspension test increased (control group 9.34±2.99s vs Lag3 overexpression group 17.19±7.77s). Figure 6 E) The percentage of slippage of the contralateral lower limb in the grid test was reduced (30.8±12.95% in the control group vs. 17.64±8.74% in the Lag3 overexpression group). Figure 6 F).

[0109] In summary, this invention first constructed a mouse striatal ICH model. To visualize the intensity and localization of LAG3 in the brain, a neutralizing antibody against LAG3 (C9B7W) was labeled with a CY5.5 fluorescent tag. Then, the successfully labeled C9B7W and an adeno-associated virus specifically overexpressing Lag3 in microglia were injected orally into the mouse striatal ICH model. Hematoma volume, hemoglobin content, immunofluorescence, flow cytometry, and pHrodo-E. coli phagocytosis assays demonstrated that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance. This reveals LAG3 as a potential molecular target for enhancing microglia phagocytic function and promoting hematoma clearance. Future research on LAG3 for the prognostic treatment of ICH is expected to advance basic and clinical research on endogenous hematoma clearance after ICH. This fully demonstrates that LAG3 agonists can be used as drugs for hematoma clearance after intracerebral hemorrhage.

[0110] Furthermore, the inventors of this invention analyzed the single-cell transcriptome of microglia after ICH in published articles, and the results showed that the expression trend of Lag3 was highly consistent with that of phagocytosis-related genes (Lamp1, Trem2 and Il10), which also proves that the LAG3 discovered in this invention can regulate the phagocytic function of microglia after ICH.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of LAG3 agonists in the preparation of drugs for hematoma evacuation after cerebral hemorrhage, characterized in that: The LAG3 agonist is an adeno-associated virus that specifically overexpresses Lag3 on microglia.

2. The application of the LAG3 agonist according to claim 1 in the preparation of a hematoma evacuation drug after cerebral hemorrhage, characterized in that: The hematoma-clearing drug mentioned above is a drug that reduces the volume of the hematoma after cerebral hemorrhage.

3. The application of the LAG3 agonist according to claim 1 in the preparation of a hematoma evacuation drug after cerebral hemorrhage, characterized in that: The hematoma-clearing drug mentioned above is a drug that enhances the phagocytic ability of microglia after cerebral hemorrhage.

4. The application of the LAG3 agonist according to claim 1 in the preparation of a hematoma evacuation drug after cerebral hemorrhage, characterized in that: The hematoma-clearing drug mentioned above is a drug that improves motor function after cerebral hemorrhage.

5. The application of the LAG3 agonist according to claim 4 in the preparation of a hematoma evacuation drug after cerebral hemorrhage, characterized in that: The drug mentioned for improving motor function after cerebral hemorrhage is a drug for improving neurological function damage.

6. The application of the LAG3 agonist according to claim 4 in the preparation of a hematoma evacuation drug after cerebral hemorrhage, characterized in that: The drugs mentioned for improving motor function after cerebral hemorrhage are those for improving limb and sensory impairments.

Images