Application of LAG3 agonist in preparation of medicine for removing hematoma after cerebral hemorrhage and application of LAG3 in regulation and control of hematoma removal after cerebral hemorrhage

Abstract

The invention relates to application of an LAG3 agonist in preparation of a medicine for removing hematoma after cerebral hemorrhage and application of LAG3 in regulation and control of removal of hematoma after cerebral hemorrhage, and belongs to the technical field of biological medicine. The LAG3 neutralizing antibody (C9B7W) is injected in an in-situ injection mode for the first time by utilizing a mouse ICH model, it is clear that LAG3 can regulate and control the phagocytic function of microglia after ICH and participate in hematoma removal, and it is revealed that LAG3 can serve as a potential molecular target for enhancing the phagocytic function of the microglia and promoting hematoma removal. Moreover, overexpression of Lag3 after ICH proves that the phagocytic function of microglial cells is enhanced, and hematoma removal is accelerated, which fully shows that the Lag3 agonist can be used for preparing the medicine for removing hematoma after cerebral hemorrhage. The experimental result of the invention makes it possible to apply LAG3 to prognostic treatment of ICH to promote the progress of endogenous hematoma removal and clinical research after ICH.

Description

Technical Field

[0001] The invention relates to application of a LAG3 agonist in preparing a drug for removing hematoma after cerebral hemorrhage and application of LAG3 in regulating the removal of hematoma after cerebral hemorrhage, and belongs to the technical field of biomedicine. Background Art

[0002] Intracerebral hemorrhage (ICH) refers to bleeding caused by rupture of small arteries in the brain parenchyma due to non-traumatic reasons. It is one of the subtypes of stroke and accounts for about 20-30% of all strokes in my country. Intracerebral hemorrhage is characterized by high morbidity, mortality and disability rates, and its incidence in my country has shown an increasing trend year by year. The space-occupying effect caused by hematoma after intracerebral hemorrhage and the secondary injury driven by hematoma components are the main causes of brain damage in patients. However, the current treatment methods for ICH are limited. Craniotomy not only has certain requirements for the volume of hematoma in patients, but also has no significant effect on the efficacy of surgery. Minimally invasive drainage has also been reported to be associated with a good prognosis only when the residual hematoma volume is less than 15mL, otherwise it cannot improve the prognosis of patients. Therefore, reducing the volume of hematoma after intracerebral hemorrhage through endogenous hematoma removal mechanism is a feasible research idea.

[0003] Studies have shown that microglia, as key innate immune cells, act as guardians of the brain. They begin to rapidly migrate to the injured area and proliferate 6 hours after cerebral hemorrhage. After cerebral hemorrhage, microglia protect surrounding cells by engulfing hematoma disintegration products, dead neurons, etc., and play a major role in hematoma clearance. At present, research on microglial clearance of hematoma after cerebral hemorrhage mainly focuses on scavenger receptors. Scavenger receptors are activated by binding to ligands on damaged red blood cells or free heme, prompting cells to ingest and engulf them. For example, after cerebral hemorrhage, red blood cells in the hematoma are dissolved to produce Hb (hemoglobin), and free Hb will attach to Hp (haptoglobin), and then the Hp-Hb complex can be internalized by phagocytes expressing CD163; CD47 changes from "don't eat me" to "eat me" signal to mediate the phagocytosis of macrophages. CD36 is a type II scavenger receptor expressed on the surface of macrophages. Cells that lack phagocytic ability can acquire phagocytic function after transfection with CD36. Studies have pointed out that microglia-derived interleukin-10 can regulate CD36 to accelerate hematoma clearance after intracerebral hemorrhage. At the same time, CD36 is also a key target of PPARγ-mediated downstream gene transcription. The activation of PPARγ can increase the expression of CD36, thereby promoting the phagocytic function of microglia. Other studies have shown that TLR (Toll-like receptor) may act as a regulator of microglial phagocytosis. TLR4 knockout leads to upregulation of CD36 around the hematoma, and inhibition of the TLR4 signaling pathway may promote the phagocytosis of microglia. Agonists of nuclear factorerythroid2-related factor 2 (Nrf2) can also increase the phagocytic ability of microglia and reduce the volume of hematomas. The above research content means that targeting phagocytosis-related targets on microglia is an important way to accelerate hematoma clearance, but there are still problems such as difficulty in converting therapeutic targets in clinical practice. This requires further clarification of the specific mechanism of hematoma clearance after intracerebral hemorrhage, and at the same time, finding innovative, effective and feasible therapeutic targets.

[0004] Lymphocyte Activation Gene 3 (LAG3) is an immune checkpoint that is mainly expressed on immune cells such as lymphocytes, microglia and macrophages. In the past, the focus of LAG3 research was mainly on regulating the function of T lymphocytes in the field of peripheral tumors. It affects the immune response of T lymphocytes by regulating the degree of activation of T lymphocytes. For example, LAG3 has a tumor immune escape mechanism similar to PD-1 (Programmed cell death protein 1, PD-1). The high expression of LAG3 on CD4+ and CD8+ T lymphocytes will cause T lymphocytes to lose their powerful effector function, thereby causing tumor cells to "escape". This type of T lymphocyte is called exhausted T lymphocyte; while low expression of LAG3 may lead to a decrease in the inhibitory ability of Treg cells and cause excessive activation of immune cells. LAG3 and PD-1 can synergize to inhibit anti-tumor immunity and autoimmunity. Studies have reported that the joint blockade of LAG3 and PD-1 can enhance the proliferation and cytokine production of tumor-infiltrating CD8+T lymphocytes, so LAG3 is also considered a promising therapeutic target in cancer immunotherapy. Other studies have shown that LAG3 expression was found on T lymphocytes in tumor models such as brain metastases and melanoma; in Parkinson's disease, LAG3 can aggravate the endocytic deposition of neuronal a-syn, and blocking LAG3 can reduce neuronal a-syn deposition; in Alzheimer's disease, LAG3 has also been reported to promote the transmission of pathological tau between neurons; in addition, LAG3 has also been reported to have an effect on depressive-like behavior in a depression model with unpredictable stimulation. However, so far, the regulation of peripheral T lymphocyte function by LAG3 after tumor occurrence has gradually become clear, but the research on LAG3 in the central nervous system is limited, and the role of LAG3 after cerebral hemorrhage and whether LAG3 regulates the function of microglia after cerebral hemorrhage are also unknown. Summary of the invention

[0005] The first purpose of the present invention is to provide the use of LAG3 agonist in the preparation of hematoma removal drugs after cerebral hemorrhage, and to provide a new phagocytosis-related target on microglia.

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

[0007] In order to achieve the above-mentioned object, the technical scheme of the application of LAG3 agonist in the preparation of a drug for removing hematoma after cerebral hemorrhage is:

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

[0009] The beneficial effect of the above technical scheme is that the application of the LAG3 agonist of the present invention in the preparation of a drug for hematoma removal after cerebral hemorrhage is a pioneering invention. The present invention first constructs a mouse striatal ICH model, and at the same time, in order to visualize the intensity and location of LAG3 in the brain, a neutralizing antibody (C9B7W) labeled with a CY5.5 fluorescent label is used to label LAG3. Then, the successfully labeled C9B7W and adeno-associated virus that specifically overexpresses Lag3 in microglia are respectively injected in situ into the mouse striatal ICH model. Through hematoma volume detection, hemoglobin content detection, immunofluorescence detection, flow cytometry detection, pHrodo-E.coli phagocytosis detection, etc., it is proved that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma removal, revealing that it can be used as a potential molecular target for enhancing the phagocytic function of microglia and promoting hematoma removal. In the future, it is expected that LAG3 will be applied to the prognosis treatment of ICH to promote the basic and clinical research progress on endogenous hematoma removal after ICH. This fully demonstrates that LAG3 agonists can be used as drugs for the removal of hematomas after cerebral hemorrhage.

[0010] As a further improvement, the drug for clearing hematoma after cerebral hemorrhage is a drug for reducing the volume of hematoma after cerebral hemorrhage.

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

[0012] As a further improvement, the drug for clearing hematoma after cerebral hemorrhage is a drug for improving 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 damage.

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

[0015] In order to achieve the above object, the technical solution of the application of LAG3 in regulating the removal of hematoma after cerebral hemorrhage in the present invention is:

[0016] Application of LAG3 in regulating hematoma clearance after cerebral hemorrhage, wherein the application is non-disease diagnosis and treatment.

[0017] The beneficial effect of the above technical solution is that: the present invention uses the mouse ICH model to inject LAG3 neutralizing antibody (C9B7W) for the first time by in situ injection, and clarifies that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance, revealing that it can be used as a potential molecular target for enhancing the phagocytic function of microglia and promoting hematoma clearance. In the future, it is expected that LAG3 will be applied to the prognosis treatment of ICH to promote the basic and clinical research progress on endogenous hematoma clearance after ICH. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the localization of perihematoma cells in which LAG3 is mainly expressed on microglia after cerebral hemorrhage in Example 1 of the present invention are shown (wherein, AC is the co-localization of LAG3 and IBA1, GFAP, and NeuN at D7 after ICH; D is the ratio analysis of the number of co-localized LAG3 and microglia at D7 after ICH (n=3); E is the co-localization map of LAG3 and microglia after ICH under a high-power microscope; F is the 3D map and Imaris modeling map of LAG3 and microglia after ICH, Scale bar=5μm);

[0019] Figure 2 The results of Example 1 of the present invention show that LAG3 is mainly expressed in microglia rather than mononuclear macrophages after ICH (wherein, A is a flow cytometry diagram of microglia and mononuclear macrophages after ICH; B is the change in the number of microglia at D1, D3, D5, and D7 in the Sham group and the ICH group (n=3-5, each group of data is subject to the Shapiro-Wilk test normality test, using one-way ANOVA test, F=0.53, P=0.71, R 2=0.10, Tukey's multiple comparison test showed P>0.05 among the groups); C is the average fluorescence intensity of LAG3 on microglia at D1, D3, D5, and D7 in the Sham group and ICH group (n=3-5, the data of each group were subject to the Shapiro-Wilk test normality test, Brown-Forsythe ANOVA test was used, 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 mononuclear macrophages at D1, D3, D5, and D7 in the Sham group and ICH group (n=3-5, the data of each group were subject to the Shapiro-Wilk test normality test, Brown-Forsythe ANOVA test was used, F=15.82, P<0.001, Games-Howell multiple comparison test showed ***P<0.001Sham vs D3, **P<0.01D1 vs D3, **P<0.01D3 vs D7); E is the average fluorescence intensity of LAG3 on mononuclear macrophages at D1, D3, D5, and D7 in the Sham group and ICH group (n=3-5, the data of each group were subject to the Shapiro-Wilk test normality test, Brown-Forsythe ANOVAtest was used, F=3.77, P=0.05, and the Games-Howell multiple comparison test showed that P>0.05 among the groups); F is the peak graph comparison of the average fluorescence intensity of LAG3 on microglia and mononuclear macrophages without LAG3 staining at D5 after ICH);

[0020] Figure 3 The results of in situ injection of LAG3 neutralizing antibodies in Example 1 of the present invention effectively acting on microglia are shown in Figure 1 (wherein A is a comparison of the absorbance of C9B7W and C9B7W-CY5.5; B is a live imaging diagram and statistical diagram 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 a brain slice imaging diagram and statistical diagram 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 a fluorescence co-localization diagram of IBA1 and C9B7W-CY5.5 after ICH);

[0021] Figure 4The results of the slowing down of hematoma clearance after LAG3 function blocking in Example 1 of the present invention are shown in Figure 1 (wherein, A is a comparison and statistical diagram of hematoma volume between the C9B7W group and the IgG group at D3 and D5 after ICH, Scale bar = 10 mm (D3 n = 3, D5 n = 13-15, each group of data is subject to the Shapiro-Wilk test normality test, using two-way ANOVA test, Bonferroni multiple comparison test shows **P < 0.01D5 IgG vs C9B7W); B is a statistical diagram of hemoglobin content comparison between the C9B7W group and the IgG group (n = 8-13, each group of data is subject to the Shapiro-Wilk test normality test. test normality test, F=2.47, P=0.23, unpaired t-test, t=2.11, *P<0.05); C is a comparison of the mean fluorescence intensity of CD68 in the C9B7W group and the IgG group by immunofluorescence; D is a statistical graph of the mean fluorescence intensity of CD68 in the C9B7W group and the IgG group on D5 after ICH by immunofluorescence (n=4, the data of each group obeyed the Shapiro-Wilk test normality test, F=2.21, P=0.53, unpaired t-test, t=2.88, *P<0.05); E is a comparison of the peak graph of the mean fluorescence intensity of CD68 in the C9B7W group and the IgG group by flow cytometry; F is a statistical graph of the mean fluorescence intensity of CD68 in the C9B7W group and the IgG group by flow cytometry (n=7, the data of each group did not obey the Shapiro-Wilk test normality test, and the Mann Whitney test*P<0.05); G is the flow cytometry comparison of the LAMP1 mean fluorescence intensity peak graph of the C9B7W group and the IgG group; H is the flow cytometry statistical graph of the LAMP1 mean fluorescence intensity of the C9B7W group and the IgG group (n=3-4, data were subjected to the Shapiro-Wilk test normality test, F=1.99, P=0.70, unpaired t-test was used, t=1.17, P=0.30);

[0022] Figure 5The result diagram of overexpression of Lag3 on microglia in Example 1 of the present invention to reduce the hematoma volume after ICH (wherein, A is the comparison and statistics of hematoma volume between the Lag3 overexpression group and the control group, Scale bar = 10mm (n = 5-10, each group of data is subject to the Shapiro-Wilk test normality test, F = 2.63, P = 0.21, unpaired t-test, 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, each group of data is subject to the Shapiro-Wilk test normality test, F = 1.47, P = 0.58, unpaired t-test, t = 3.00, *P < 0.05); C is the statistics of the number of microglia in the Lag3 overexpression group and the control group (n = 3-5, each group of data is subject to the Shapiro-Wilk test normality test, F = 1.47, P = 0.58, unpaired t-test, t = 3.00, *P < 0.05). Normality test, F=1.1, P>0.05, unpaired t-test, t=0.35, P>0.05; D is the mean fluorescence intensity of CD68 in the Lag3 overexpression group and the control group (n=3-5, data of each group were subjected to Shapiro-Wilk test normality test, F=4.78, P=0.36, unpaired t-test, t=1.09, P=0.32); E is the statistics of the average fluorescence intensity of LAMP1 in the Lag3 overexpression group and the control group (n=3-5, the data of each group obeyed the Shapiro-Wilktest normality test, F=3.11, P=0.52, unpaired t-test, t=0.17, P>0.05); F is the statistics of pHrodo+ microglia in the Lag3 overexpression group and the control group (n=7, the data of each group obeyed the normality test, F=1.38, P=0.71, unpaired t-test, t=1.49, P=0.16);

[0023] Figure 6The effect of LAG3 function blocking or overexpression on the motor function of mice after ICH in Example 1 of the present invention (wherein, A is the NDS score of mice in the IgG group and C9B7W group (n=10-14, each group of data is subject to the Shapiro-Wilk test normality test, F=1.31, P=0.70, unpaired t-test, t=4.11, ***P<0.001); B is the persistence time of mice in the IgG group and C9B7W group in the hanging wire experiment (n=9-12, each group of data is subject to the Shapiro-Wilk test normality test, F=1.50, P=0.52, unpaired t-test, t=3.17, **P<0.01); C is the number of slips / total steps (%) of the contralateral limbs of mice in the IgG group and C9B7W group in the grid test (n=9-12, each group of data is subject to the Shapiro-Wilk test normality test, F=1.50, P=0.52, unpaired t-test, t=3.17, **P<0.01). The results of the Shapiro-Wilk test showed that the normality of the control group and the Lag3 overexpression group was 1.48, 0.59, and the unpaired t-test was used, t=2.32, *P<0.05); D was the NDS score of the control group and the Lag3 overexpression group (n=5-11, the data of each group did not obey the Shapiro-Wilk test normality test, and the Mann-Whitney test was used *P<0.05); E was the persistence time of the control group and the Lag3 overexpression group in the hanging wire experiment (n=5-11, the data of each group obeyed the Shapiro-Wilk test normality test, F=6.74, P=0.08, and the unpaired t-test was used, t=2.16, *P<0.05); F was the number of slips / total steps of the contralateral limbs of the mice in the grid test (n=5-11, the data of each group obeyed the Shapiro-Wilk test normality test) Normality test, F=2.19, P=0.29, unpaired t-test, t=2.41, *P<0.05)). DETAILED DESCRIPTION

[0024] Studies of the prior art have shown that microglia after ICH protect surrounding cells by engulfing hematoma disintegration products, dead neurons, etc., and play a major role in hematoma removal. A large number of studies have shown that targeting phagocytosis-related targets on microglia is an important way to accelerate hematoma removal, but there are still problems such as difficulty in converting therapeutic targets in clinical practice. This requires further clarification of the specific mechanism of hematoma removal after ICH, and at the same time finding innovative, effective and feasible therapeutic targets. Based on this, the present invention provides the use of LAG3 agonists in the preparation of hematoma removal drugs after cerebral hemorrhage.

[0025] The present invention utilizes the mouse ICH model to inject LAG3 neutralizing antibody (C9B7W) for the first time by in situ injection, and clarifies that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma clearance, revealing that it can be used as a potential molecular target for enhancing the phagocytic function of microglia and promoting hematoma clearance. In the future, it is expected that LAG3 will be applied to the prognosis treatment of ICH to promote the basic and clinical research progress on endogenous hematoma clearance after ICH.

[0026] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation on the scope of the present invention. The details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention. The test methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0027] Statistical methods:

[0028] The flow cytometry test results of the present invention were analyzed on FlowJo10 software. All statistical analyses in the article were analyzed in GraphPad Prism9. Each group of data was first subjected to the Shapiro-Wilk test normality test. The two groups were compared. If they conformed to the normal distribution and the variance was equal, the parametric test unpaired t-test was used; if they obeyed the normal distribution and the variance was uneven, the unpaired t-test and Welch correction were used. If they did not obey the normal distribution, the non-parametric test Mann-Whitney test was used. The statistical results of the data are expressed as mean ± standard deviation (mean ± SD). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** P < 0.0001, and ns indicates no statistical difference.

[0029] Specific examples of the use of the LAG3 agonist of the present invention in preparing a drug for removing hematoma after cerebral hemorrhage and the use of LAG3 in regulating the removal of hematoma after cerebral hemorrhage:

[0030] Example 1 In situ injection of LAG3 neutralizing antibodies effectively acts on microglia

[0031] In this example, a mouse striatal ICH model was first constructed, and the cellular localization of LAG3 around the hematoma was observed by immunofluorescence, and the expression changes of LAG3 on microglia at D1, D3, D5, and D7 after ICH were detected by flow cytometry.

[0032] Second, a LAG3 neutralizing antibody (C9B7W) was used to block LAG3 function, and C9B7W was labeled with the 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 D5 after ICH were compared, and the expression of microglial CD68 and LAMP1 and the phagocytosis of pHrodo-labeled Escherichia coli (E. coli) were detected by immunofluorescence and flow cytometry. Adeno-associated virus that specifically overexpressed Lag3 on microglia was used to observe the phagocytic function of microglia and hematoma clearance on D5 after ICH.

[0034] The specific operations are as follows:

[0035] All experimental animals used 2-3 month old C57BL / 6 male mice, weighing about 22-30 g, and the experimental animals had free access to food and water. All experiments strictly complied with relevant national regulations. In this example, the animals were randomly divided into Sham group (injected with normal saline) and ICH group (injected with 0.5 μL normal saline containing 0.075 U type VII collagenase), IgG group (injected with 1 μL PBS containing 5 μg IgG isotype control + 0.5 μL normal saline containing 0.075 U type VII collagenase), C9B7W group (injected with 1 μL PBS containing 5 μg anti-mouse LAG-3 neutralizing antibody + 0.5 μL normal saline containing 0.075 U type VII collagenase) and C9B7W-CY5.5 group (injected with 1 μL PBS containing 5 μg anti-mouse LAG-3 neutralizing antibody (containing CY5.5 label) PBS + 0.075U type VII collagenase in 0.5μL normal saline), Control group (injection of negative control adeno-associated virus + 0.075U type VII collagenase in 0.5μL normal saline) and overexpression Lag3 group (Over-expression group, injection of adeno-associated virus that specifically overexpresses Lag3 on microglia + 0.075U type VII collagenase in 0.5μL normal saline).

[0036] 1. Construction of mouse striatal ICH model

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

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

[0039] (3) Exposure of the anterior and posterior bregma: Use iodine to disinfect the mouse head, and use a surgical blade to make a 2 cm incision along the sagittal axis of the mouse head to expose the anterior and posterior bregma;

[0040] (4) Leveling and positioning: The glass electrode is set at the bregma as the origin, and the values ​​of the X, Y, and Z axes are all adjusted to 0. The electrode is translated forward and backward and left and right to observe the change in the distance from the electrode to the skull surface. That is, the size error of the Z axis is less than 0.2 mm. After leveling, the glass electrode is moved back to the bregma, and then to the target coordinates (0.8 mm in front of the bregma, 2.1 mm to the left, and 3.3 mm deep);

[0041] (5) Drilling: Mark the coordinates with a pencil and drill holes at the marked locations until a hollow feeling is felt;

[0042] (6) Injection: Use a glass electrode to extract the injection solution, fix the glass electrode to the drill hole, adjust the Z axis to 0 when the electrode and the drill hole plane are at the same height, and start injection after the electrode reaches a depth of 3.3 mm, and maintain the speed at 0.1 μL / min;

[0043] (7) Stop the needle: To prevent the solution from sticking to the glass electrode wall, stop the needle for 2 minutes after the injection is completed, lift it up 0.2 mm, and then stop the needle for another 6 minutes;

[0044] (8) Lifting and suturing: After the needle is stopped, slowly lift the glass electrode and suture the mouse's 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, dilute it to 1 μL with PBS buffer at pH 7, and inject it in situ 2 hours after ICH modeling at the coordinates 0.8 mm in front of the anterior fontanelle, 2.1 mm to the left, and a depth of 3.3 mm.

[0048] (2) Adeno-associated virus injection

[0049] 21 days before ICH modeling, 1 μL of microglia-specific adeno-associated virus overexpressing LAG3 (pAAV-F4 / 80p-MCS-EGFP-3Flag-SV40-PolyA) or negative control virus was injected 0.8 mm in front of the anterior bregma, 2.5 mm to the left, and 3.3 mm deep.

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

[0051] Take 0.1 mg of CY5.5 powder and add it to a centrifuge tube containing 1 mL of DMSO. Wrap it with tin foil and shake it in the dark to fully dissolve it. Dilute 8.57 mg / mL C9B7W 10 times to 0.857 mg / mL, add it to the CY5.5 solution, and stir it at 4°C overnight. Use an ultrafiltration centrifuge tube to filter the unbound CY5.5 and store it in aliquots at -80°C. Take 2 μL of C9B7W and C9B7W-CY5.5 solutions respectively and add them to the sample slot of the UV spectrophotometer to detect their respective absorbances. Determine whether the labeling is successful by comparing the absorbance peaks.

[0052] 4. In vivo imaging

[0053] C9B7W-CY5.5 was injected in situ 2 hours after the establishment of the mouse striatal ICH model, and the fluorescence intensity of C9B7W-CY5.5 around the hematoma was detected 1, 3, 5, and 7 days after ICH. The small animal in vivo imaging system was turned on and initialized for 30 minutes before the experiment. The mice were anesthetized with isoflurane, and the scalp was cut to fully expose the skull. The anesthetized mice were placed in the in vivo imaging dark box for fluorescence imaging detection, and the image processing application equipment supporting software was used for image processing and analysis.

[0054] 5. Behavioral testing

[0055] Blind testing: The behavioral tests in this example were all performed in a blind manner, with an experimenter who was completely unaware of the experimental groupings grouping the mice or directly evaluating the following behavioral tests by an experimenter who was unaware of the experimental groupings.

[0056] (1) Neurological deficit score

[0057] When the indoor environment meets the standards, the mice were placed in the behavioral laboratory for 1 hour of pre-adaptation and then the following tests were performed in sequence. The test includes 6 items, each with 5 levels, with the highest score being 4 points and the lowest score being 0 points, as shown in Table 1. ICH mice with a total score of less than 4 points and a score of more than 20 points will be eliminated.

[0058] Table 1 Neurological deficit scores

[0059]

[0060]

[0061] (2) Hanging wire experiment

[0062] The mice were placed in the behavioral laboratory to adapt for 1 hour before the experiment. The experimental device was an iron frame with a 1mm thick and 55cm long iron wire placed on the top, 50cm high, and a soft object placed below to prevent the mice from getting hurt. In the experiment, the mice needed to support their own suspended weight with both upper limbs. In order to prevent the mice from using their limbs, their lower limbs were glued with tape or other items. The time each mouse persisted on the iron wire was recorded, and the average of three experiments was taken for each mouse.

[0063] (3) Grid test

[0064] When the indoor environment meets the standard, the mice were placed in the behavioral laboratory for 1 hour of pre-adaptation before the experiment. The mice were placed on a 35cm×30cm×40cm steel wire grid and walked for 3 minutes. A camera was used to shoot and record the video under the grid. The total number of steps and the number of slips of the lower limbs on the side opposite to the injury of the mice within 3 minutes were counted. The percentage of mice slipping = (number of slips / total number of steps) × 100%.

[0065] 6. Infusion

[0066] Use 2.5% isoflurane to induce deep anesthesia in mice. When the mice are deeply anesthetized, fix the limbs of the mice on the stage, open the chest cavity with scissors, and fully expose the heart. Insert the perfusion needle into the left apex of the heart and cut the right atrial appendage at the same time. Infuse 37℃ 0.9% NaCl (about 50mL) from the left ventricle, and stop perfusion when the liver turns completely white. Then use 4℃ paraformaldehyde for perfusion. When the muscles of the mouse are stiff and the neck is stiff, stop perfusion. Peel off the skull and remove the intact brain tissue. Note: If fresh tissue is needed, there is no need to use paraformaldehyde for perfusion.

[0067] 7. Hematoma volume measurement

[0068] The method of perfusion sampling is shown in step 6. The mice were sampled on D5 after ICH modeling. The fresh intact brain tissue was cut into 1 mm thick coronal brain slices, and the front and back of each brain tissue were photographed with a measuring ruler. Image J software was used to analyze the hematoma volume, hematoma volume = the sum of the hemorrhage volume of each brain tissue (mm3), and the hemorrhage volume of each brain tissue = (front hemorrhage area × 1mm + back hemorrhage area × 1mm) / 2).

[0069] 8. Hemoglobin content detection

[0070] The perfusion sampling method is shown in step 6. The mice were sampled on D5 after ICH modeling. The hemoglobin content of brain tissue was quantified using a hemoglobin detection kit (Sigma). The center of the hematoma was collected with 500 mL of ice PBS, the tissue was homogenized for 1 min, and centrifuged at 16000g for 15 min. 50 μL of the supernatant was transferred to a flat-bottomed 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] The mice were sampled on D1, D3, D5 and D7 after ICH modeling. Fresh mouse perihematoma tissue was obtained and the tissue was prepared into a single cell suspension using a mild mouse brain tissue enzymatic dissociation kit. An efficient debris removal reagent was used for high-speed centrifugation to remove impurities, and then a red blood cell lysis reagent was used to remove residual red blood cells to obtain the final single cell suspension. Take the cell sample to be tested for 10 6 , add 50 μL of flow cytometry antibodies diluted in the corresponding proportion. Use a micropipette to gently blow and mix, and incubate at 4 ° C for 30 minutes. After centrifugation and discarding the supernatant, add 1 mL of cold PBS and centrifuge and wash twice to remove unbound excess antibody components. Finally, add 300 μL of cold PBS to the cells, blow and mix, store at 4 ° C in the dark, detect on the machine according to the operating procedure, and then use Flowjo flow analysis software for data analysis. The cell flow gating strategy involved in this embodiment includes microglia (7AAD-LY6G-CD45intCD11b+), monocytes and macrophages (7AAD-LY6G-CD45highCD11b+), T lymphocytes (7AAD-CD45+CD3+), and also includes detection of indicators such as CD68 and LAMP1 on microglia.

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

[0074] Mice were sampled on D5 after ICH modeling, and fresh mouse perihematoma tissue was obtained. The tissue was prepared into a single-cell suspension using a mild mouse brain tissue enzymatic dissociation kit. An efficient debris removal reagent was used for high-speed centrifugation to remove impurities, and then a red blood cell lysis reagent was used to remove residual red blood cells to obtain the final single-cell suspension. pHrodo is an acid-dependent dye, and the fluorescence of the E. coli labeled with it will increase as the pH value decreases from the neutral cytoplasmic pH to the acidic pH value of the endosomal body. 50μg / mL pHrodo-E. coli was added to the single-cell suspension, incubated at 37°C for 2h, and then incubated with microglia-related flow cytometry antibodies for detection on the machine.

[0075] 11. Immunofluorescence

[0076] (1) Infusion sampling

[0077] The perfusion sampling method is shown in 6. The mice were sampled on D5 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 fixation, the brain tissue was first precipitated in 20% sucrose for 24 hours and then in 30% sucrose for 48 hours.

[0080] (3) Slicing, pasting and baking

[0081] After the cerebellum was removed, the brain tissue was placed on a round tray with the olfactory bulb facing upward. After embedding, the brain tissue was first rapidly cooled at -80°C for 10 minutes, then placed at -20°C for 15 minutes, and then fixed in the card slot of the freezing slicer, the temperature was set to -20°C, and the unnecessary tissue was quickly cut off first, and the slices were slowly sliced ​​after the lesions were seen, ensuring that the brain slices were symmetrical, with a slice thickness of 25μm, and the slicing was completed until the lesions disappeared. After slicing, the brain slices were rinsed 3 times with 0.01M PBS, and after washing, they were carefully attached to the slide with a small brush, and then placed in an oven for drying.

[0082] (4) Dyeing

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

[0084] 2) Blocking: Each slide was blocked with 200 μL of blocking solution (10% fetal bovine serum and 0.1% Triton-X 100 in PBS) in a wet box at room temperature for 2 h.

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

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

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

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

[0089] 7) Sealing and detection: Use absorbent paper to absorb the liquid and seal the slides with sealing solution containing anti-fluorescence quenching agent.

[0090] 8) Filming: Use a confocal fluorescence microscope for filming. Select 3-4 brain slices from each brain tissue. Take a picture of each brain slice around the hematoma. The number of filming fields should be greater than 4. Then use Image J software to statistically analyze the number of immunofluorescent cells or the number of co-labeled cells.

[0091] 12. Experimental Results

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

[0093] LAG3 and IBA1 (microglia and monocytes and macrophages), GFAP (astrocytes), and NeuN (neurons) were double-labeled by immunofluorescence experiments ( Figure 1 AC) to observe the localization of LAG3 in cells around the hematoma after ICH. The results showed that about 64.33% of IBA1+ cells (microglia and mononuclear macrophages) around the hematoma after ICH expressed LAG3 ( Figure 1 D), while astrocytes and neurons do not express LAG3 ( Figure 1 BC). In addition, 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), and 3D modeling of microglia and LAG3 also confirmed this ( Figure 1 F).

[0094] Since IBA1 cannot distinguish microglia from peripheral infiltrating mononuclear macrophages well, this example uses flow cytometry to distinguish microglia from peripheral infiltrating mononuclear macrophages and observes the expression of LAG3 on them. First, the main cell clusters of the tissue surrounding the hematoma after ICH were circled, and after the de-adhesion step, the 7AAD- living cells were circled, and after removing the LY6G+ granulocytes, microglia (CD45intCD11b+) and peripheral infiltrating mononuclear macrophages (CD45highCD11b+) ( 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 with the Sham group, the expression of LAG3 on microglia decreased at D1 after ICH, and then gradually increased, and significantly increased at 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 infiltrating monocytes and macrophages increased significantly on D3 after ICH, and 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 the expression level of LAG3 in microglia is higher than that in monocytes and macrophages ( Figure 2 F).

[0095] (2) In situ injection of LAG3 neutralizing antibodies effectively acts on microglia (specific experimental results are shown in Figure 3 shown):

[0096] After ICH, LAG3 neutralizing antibody (anti-mouse LAG-3, C9B7W) was injected in situ into the mouse striatum to observe the phenotypic changes of ICH. The antibody can recognize the D2 structural threshold of LAG3 and bind to it to block the function of LAG3. In order to visualize the intensity and localization of C9B7W in the brain, C9B7W was labeled with CY5.5 fluorescent label, and then the absorbance of C9B7W and C9B7W-CY5.5 was detected to determine whether the fluorescent label was successfully labeled. The results of the ultraviolet spectrophotometer showed that compared with C9B7W, C9B7W-CY5.5 had a significant absorption peak at 673nm, indicating that the CY5.5 fluorescent label had been successfully labeled with C9B7W ( Figure 3 A).

[0097] After a single in situ injection of C9B7W-CY5.5 after ICH, the fluorescence intensity of C9B7W-CY5.5 in the mouse brain at different time points was detected using in vivo imaging technology. Compared with the C9B7W group, there was obvious red fluorescence in the mouse brain and brain slices at D1, D3, D5, and D7 after in situ injection of C9B7W-CY5.5, and the fluorescence intensity was the highest on D3 after ICH, and then gradually decreased, but there was still obvious fluorescence expression on D5 after ICH ( Figure 3 BC).

[0098] Immunofluorescence was then used to further observe the co-localization of C9B7W-CY5.5 and IBA1. The results showed that C9B7W-CY5.5 and IBA1 co-localized at D5 after ICH ( Figure 3 D), indicating that C9B7W can act on microglia after ICH to block the function of LAG3.

[0099] (3) Hematoma clearance slowed down after LAG3 function blockade (specific experimental results are shown in Figure 4 shown):

[0100] First, the effect of LAG3 functional blockade on hematoma clearance after ICH was observed. The results showed that there was no difference in hematoma volume between the IgG group and the C9B7W group on D3 after ICH (IgG group 10.37±0.40mm 3 vs C9B7W group 9.08±2.35mm 3 , Figure 4 A), indicating that there was no difference in the initial bleeding volume between the two groups. However, the hematoma volume of the C9B7W group on D5 after ICH was higher than that of the IgG group (3.07±1.54mm in the IgG group). 3 vs C9B7W group 5.86±2.58mm 3 , Figure 4 A), indicating that the hematoma clearance in the C9B7W group was delayed, which was also verified by the higher hemoglobin content in the C9B7W group than in the IgG group (IgG group 9.25±2.90mg / dL vs C9B7W group 13.07±4.57mg / dL, Figure 4 B).

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

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

[0103] AAV-Lag3-F4 / 80-EGFP was injected 21 days before ICH to overexpress Lag3 on microglia to construct an ICH model. The results showed that the hematoma volume of the Lag3 overexpression group on D5 after ICH was significantly lower than that of the control group (0.96±0.43mm in the control group). 3 vs. Lag3 overexpression group: 0.51±0.27mm 3 , Figure 5 A), and the hemoglobin content of the Lag3 overexpression group was also lower than that of the control group (control group 13.94±3.32mg / dL vs Lag3 overexpression group 9.13±2.74mg / dL, Figure 5 B), indicating that overexpression of Lag3 on microglia can accelerate hematoma clearance after ICH.

[0104] Moreover, the Lag3 overexpression group did not show the phenotype of increased 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 phagocytosed E. coli (control group 87.6±3.15% vs Lag3 overexpression group 90.33±3.69%, Figure 5 F) showed an increasing trend, but the expression of LAMP1 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 aggravates the motor function deficits of mice after ICH, while overexpression of microglial Lag3 improves the motor function of mice after ICH (specific experimental results are shown in Figure 6 shown):

[0106] The neurological function of mice after ICH will be significantly reduced, accompanied by limb disorders and sensory disorders, etc. The neurological deficit score is used to determine the degree of neurological deficit in mice, the hanging wire experiment is used to observe the movement of mice including grip strength and balance, etc., and the grid test is used to determine the sensory-motor coordination ability of mice.

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

[0108] Compared with the control group, the neurological deficit score of ICH mice after overexpression of microglial Lag3 was reduced (9.4±2.61 points in the control group vs 6.73±1.35 points in the Lag3 overexpression group, Figure 6 D), the persistence time of the hanging wire experiment increased (9.34±2.99s in the control group vs 17.19±7.77s in the Lag3 overexpression group, Figure 6 E), the percentage of slipping of the lower limb on the opposite side of the injury in the grid test was reduced (control group 30.8±12.95% vs Lag3 overexpression group 17.64±8.74%, Figure 6 F).

[0109] In summary, the present invention first constructed a mouse striatal ICH model, and at the same time, in order to visualize the intensity and location of LAG3 in the brain, a neutralizing antibody (C9B7W) labeled with a CY5.5 fluorescent label was used to label LAG3. Then, the successfully labeled C9B7W and the adeno-associated virus that specifically overexpresses Lag3 in microglia were respectively injected in situ into the mouse striatal ICH model. Through hematoma volume detection, hemoglobin content detection, immunofluorescence detection, flow cytometry detection, pHrodo-E.coli phagocytosis detection, etc., it was proved that LAG3 can regulate the phagocytic function of microglia after ICH and participate in hematoma removal, revealing that it can be used as a potential molecular target for enhancing the phagocytic function of microglia and promoting hematoma removal. In the future, it is expected that LAG3 will be applied to the prognosis and treatment of ICH to promote the basic and clinical research progress on the removal of endogenous hematomas after ICH. This fully demonstrates that the agonist of LAG3 can be used as a drug for preparing hematoma removal after cerebral hemorrhage.

[0110] Furthermore, the inventors of the present 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 phagocytosis-related genes (Lamp1, Trem2 and Il10), which also proved on the other hand that LAG3 discovered in the present invention can regulate the phagocytic function of microglia after ICH.

[0111] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. Application of LAG3 agonists in the preparation of drugs for removing hematoma after cerebral hemorrhage.

2. Use of the LAG3 agonist according to claim 1 in the preparation of a drug for removing hematoma after cerebral hemorrhage, characterized in that: The drug for removing hematoma after cerebral hemorrhage is a drug for reducing the volume of hematoma after cerebral hemorrhage.

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

4. Use of the LAG3 agonist according to claim 1 in the preparation of a drug for removing hematoma after cerebral hemorrhage, characterized in that: The drug for removing hematoma after cerebral hemorrhage is a drug for improving motor function after cerebral hemorrhage.

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

6. Use of the LAG3 agonist according to claim 4 in the preparation of a drug for removing hematoma after cerebral hemorrhage, characterized in that: The drug for improving motor function after cerebral hemorrhage is a drug for improving limb disorders and sensory disorders.

7. Application of LAG3 in regulating hematoma clearance after cerebral hemorrhage, wherein the application is non-disease diagnosis and treatment.

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