Medicine for treating cerebral hemorrhage and improving neuroprognosis of cerebral hemorrhage and application

Abstract

The invention provides a medicine for treating cerebral hemorrhage and improving neuroprognosis of cerebral hemorrhage and application, and particularly relates to application of a combined beta2 and beta3 receptor inhibitor in preparation of a medicine for preventing, treating or relieving cerebral hemorrhage-induced anemia and / or cerebral hemorrhage-induced stem progenitor cell change. The invention relates to application of a beta1 receptor inhibitor in preparation of a medicine for preventing, treating or relieving cerebral hemorrhage induced anemia, application of a beta2 receptor inhibitor in preparation of a medicine for preventing, treating or relieving cerebral hemorrhage induced stem progenitor cell change, and application of the beta1 receptor inhibitor, the beta2 receptor inhibitor and the beta3 receptor inhibitor in preparation of a medicine for improving cerebral hemorrhage neuroprognosis. The invention finds that cerebral hemorrhage-induced anemia and / or stem progenitor cell change can be reversed and cerebral hemorrhage nerve prognosis can be improved by administering an exogenous combination beta2 and beta3 receptor inhibitor, a beta2 receptor inhibitor and a beta3 receptor inhibitor. The invention discloses a novel application of a beta2 and beta3 receptor combined inhibitor, a beta2 receptor inhibitor and a beta3 receptor inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a medicine for treating cerebral hemorrhage and improving its neurological prognosis and its application. Background Art

[0002] Intracerebral hemorrhage (ICH) refers to bleeding caused by rupture of small arteries in the brain parenchyma due to non-traumatic reasons, which is characterized by high disability and mortality rates. After ICH occurs, the formation and expansion of hematoma will trigger a series of pathophysiological changes, including mechanical injury, secondary cerebral edema, inflammatory response and oxidative stress, which ultimately lead to neuronal damage and destruction of the blood-brain barrier. The pathological process of ICH is extremely complex, and there is currently no effective specific treatment, which makes its clinical management face great challenges. In addition to local brain tissue damage, ICH can also cause systemic reactions, among which abnormalities in the hematopoietic system are particularly prominent. Abnormal hematopoiesis in ICH patients is usually manifested as a decrease in red blood cells and hemoglobin, which leads to anemia. Anemia is a common complication and has been proven to be an important predictor of poor prognosis in ICH patients. The occurrence of anemia may aggravate brain hypoxia, affect the ability of brain tissue to repair, and further aggravate neurological damage. However, the specific mechanism of ICH-induced anemia is still unclear, and the role of anemia in the progression and long-term prognosis of ICH is still lacking in-depth research.

[0003] The autonomic nervous system (ANS) plays a vital role in maintaining the basic life functions of the body, including the sympathetic nervous system and the parasympathetic nervous system. Among them, the sympathetic nervous system plays an important role in stress response. It activates β receptors by releasing norepinephrine and epinephrine, thereby regulating multiple physiological processes such as cardiovascular function, metabolism, and immunity. Studies have shown that an overactivated sympathetic nervous system may aggravate cerebral vasoconstriction, increase blood-brain barrier permeability, and even affect systemic organ function. After intracerebral hemorrhage, dysfunction of the sympathetic nervous system may aggravate disease symptoms and even affect the course and prognosis of the disease. However, it is still unknown whether the dysfunction of the sympathetic nervous system is related to ICH-induced anemia and whether the sympathetic nervous system can affect bone marrow homeostasis and anemia after intracerebral hemorrhage by regulating β receptors. Further exploration of the potential neuro-hematopoietic regulatory mechanism will help reveal the pathological basis of ICH-related anemia and provide new therapeutic ideas for clinical intervention of ICH patients and improving the neurological prognosis of ICH. Summary of the invention

[0004] The purpose of the present invention is to provide the use of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor in the preparation of a drug for preventing, treating or alleviating anemia and / or stem cell changes induced by cerebral hemorrhage and the drug.

[0005] The present invention also provides the use of one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor in the preparation of a drug for improving the neurological prognosis of cerebral hemorrhage and the drug.

[0006] The present invention provides the following technical solutions: Use of a combination of β2 and β3 receptor inhibitors in the preparation of a drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage and / or changes in stem and progenitor cells induced by cerebral hemorrhage.

[0007] Further, cerebral hemorrhage-induced anemia includes one or more of preventing, treating or alleviating cerebral hemorrhage-induced anemia; The changes in stem and progenitor cells induced by cerebral hemorrhage include one or more of the changes in the number of hematopoietic stem cells, the changes in the number of bone marrow megakaryocyte-erythroid progenitor cells, and the changes in the number of bone marrow granulocyte-macrophage progenitor cells.

[0008] The present invention also provides the use of the beta 2 receptor inhibitor in the preparation of a medicine for preventing, treating or alleviating anemia induced by cerebral hemorrhage.

[0009] Furthermore, anemia induced by cerebral hemorrhage includes one or more of a decrease in the number of red blood cells, a decrease in hemoglobin concentration, a decrease in erythroid progenitor cells, and a decrease in mature red blood cells.

[0010] The present invention also provides the use of a β3 receptor inhibitor in the preparation of a drug for preventing, treating or alleviating changes in stem and progenitor cells induced by cerebral hemorrhage.

[0011] Furthermore, the changes in stem and progenitor cells induced by cerebral hemorrhage include one or more of changes in the number of hematopoietic stem cells, changes in the number of bone marrow megakaryocyte-erythroid progenitor cells, and changes in the number of bone marrow granulocyte-macrophage progenitor cells.

[0012] The present invention also provides a drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage, which uses a combined β2 and β3 receptor inhibitor or a β2 receptor inhibitor as an active ingredient.

[0013] Furthermore, the drug also includes excipients; the combined β2 and β3 receptor inhibitor or the β2 receptor inhibitor and the excipients are made into a clinically acceptable preparation through a preparation process.

[0014] The present invention also provides a drug for preventing, treating or alleviating changes in stem and progenitor cells induced by cerebral hemorrhage, which uses a combined β2 and β3 receptor inhibitor or a β3 receptor inhibitor as an active ingredient.

[0015] Furthermore, the drug also includes excipients; the combined β2 and β3 receptor inhibitor or the β3 receptor inhibitor and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0016] The present invention also provides a drug for preventing, treating or alleviating drug-induced anemia and / or stem cell changes in cerebral hemorrhage, which uses one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor as active ingredients.

[0017] Furthermore, the drug has β2 receptor inhibitor and β3 receptor inhibitor as active ingredients.

[0018] Furthermore, the drug also includes excipients; one or more of the combined β2 and β3 receptor inhibitor, β2 receptor inhibitor, β3 receptor inhibitor and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0019] The present invention also provides the use of one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor in the preparation of a drug for improving the neurological prognosis of cerebral hemorrhage.

[0020] Furthermore, improving the neurological prognosis of cerebral hemorrhage includes reducing the hematoma volume and alleviating neurological deficits.

[0021] The present invention also provides a drug for improving the neurological prognosis of cerebral hemorrhage, which uses one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor as active ingredients.

[0022] It should be noted that, in the present invention, the combined β2 and β3 receptor inhibitor can be 6OHDA. The 6OHDA refers to hexahydroxydopamine, which is an autonomic nerve antagonist and can inhibit both β2 receptors and β3 receptors. The β2 receptor inhibitor can be ICI118551. The ICI118551 is a selective β2-adrenergic receptor antagonist with a CAS number of 1217094-53-5. The β3 receptor inhibitor can be SR59230A. The SR59230A is a selective β3-adrenergic receptor antagonist with a CAS number of 174689-39-5.

[0023] Bone marrow is the main organ for hematopoiesis, and the sympathetic nerves play an important role in regulating the differentiation and proliferation of hematopoietic stem / progenitor cells. Norepinephrine released by sympathetic nerves can act on β receptors on bone marrow stromal cells and hematopoietic cells to regulate the homeostasis of the hematopoietic microenvironment. Therefore, after ICH, sympathetic nerve dysfunction will affect bone marrow erythroid hematopoiesis through β receptors, leading to the occurrence of anemia. The present invention has found through research that combined β2 and β3 receptor inhibitors, β2 receptor inhibitors, and β3 receptor inhibitors can act on β receptors on bone marrow stromal cells and hematopoietic cells, thereby playing an important role in treating cerebral hemorrhage and improving the neurological prognosis of cerebral hemorrhage. Specifically, the present invention uses the mouse striatal ICH model to study the related effects of 6OHDA, ICI118551, and SR59230A on cerebral hemorrhage, and the following conclusions are obtained: (1) 6OHDA can be used to prevent, treat or alleviate anemia induced by cerebral hemorrhage, and can also be used to prevent, treat or alleviate changes in stem cells induced by cerebral hemorrhage. 6OHDA has a good inhibitory effect on the decrease in red blood cell count, hemoglobin concentration, erythroid progenitor cell decrease, and mature red blood cell decrease. It can help the recovery of anemia induced by cerebral hemorrhage, increase the RBC and HGB values, increase the number of early erythroid BFU-E and CFU-E, and Ter119 + At the same time, it also reverses the changes in the number of hematopoietic stem cells, the number of bone marrow megakaryocyte-erythroid progenitor cells, and the number of bone marrow granulocyte-macrophage progenitor cells, and can improve the lineage deviation of bone marrow cells induced by cerebral hemorrhage, reduce the number of HSCs, increase the number of MEP lineages, reduce the number of GMP lineages, and promote the recovery of stem progenitor cells.

[0024] (2) ICI118551 can be used to prevent, treat or alleviate anemia induced by cerebral hemorrhage, but it does not reverse the changes in stem and progenitor cells induced by cerebral hemorrhage.

[0025] (3) SR59230A can be used to prevent, treat or alleviate the changes in stem cells induced by cerebral hemorrhage, but it cannot reverse the anemia induced by cerebral hemorrhage.

[0026] In summary, the sympathetic nervous system plays an important role in bone marrow homeostasis and hematopoietic homeostasis after ICH. Targeted inhibition of sympathetic nervous system activity by 6OHDA or inhibition of β2 receptors in bone marrow-derived monocytes by ICI118551 can reverse anemia induced by ICH and improve the prognosis of ICH. Targeted inhibition of β3 receptors by SR59230A can reverse the changes in stem and progenitor cells induced by ICH.

[0027] (4) 6OHDA, ICI118551, and SR59230A can improve the neurological prognosis of cerebral hemorrhage, significantly reduce the hematoma volume of cerebral hemorrhage, and alleviate the neurological deficits of cerebral hemorrhage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 For healthy control individuals and IC H Comparison chart of the patient's red blood cell count (RBC), hemoglobin concentration (HGB), and white blood cell count (WBC); Figure 2 This is a statistical chart of the proportion of anemia in ICH patients; Figure 3 The mRS comparison chart of ICH patients (ICH) and ICH patients with anemia (ICH+anemia); Figure 4 This is a correlation analysis chart between hemoglobin concentration (HGB) and mRS in ICH patients; Figure 5 Schematic diagram of constructing mouse ICH model; Figure 6 This is the change chart of white blood cell count (WBC) in ICH mice; Figure 7 The graphs are the changes of red blood cell index (RBC), hemoglobin concentration (HGB), hematocrit (HCT) and reticulocyte count (Retic) in ICH mice; Figure 8 This is a comparison of bone marrow norepinephrine expression between the sham operation group (Sham) and the intracerebral hemorrhage group (ICH); Fig. 9 This is a schematic diagram of the 6OHDA administration mode; Fig.10 This is a comparison of the IL6 content in serum and bone marrow supernatant of the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.11 Comparison of red blood cell index (RBC) and hemoglobin concentration (HGB) in the blood of mice in the PBS solvent group (Veh) and 6OHDA group (6OHDA); Fig.12 This is a comparison chart of the reticulocyte count (Retic) in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.13Comparison of BFU-E in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.14 Comparison of CFU-E in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.15 Ter119 in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA) + Cell number comparison chart; Fig.16 This is a comparison of the number of erythroid progenitor cells in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.17 This is a comparison of the number of blood island EBI in the blood of mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.18 The blood island display and statistical graph of Giemsa staining of PBS solvent group (Veh) and 6OHDA group (6OHDA); Fig.19 This is a comparison chart of the number of hematopoietic lineages in the mouse bone marrow of the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig. 20 Comparison of the number of neutrophils and LY6C monocytes, hematoma volume and neurological deficit score between mice in the PBS solvent group (Veh) and the 6OHDA group (6OHDA); Fig.21 This is a schematic diagram of the ICI118551 administration mode; Fig. 22 This is a comparison chart of the serum IL6 content in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.23 This is a comparison of the IL6 content in the bone marrow supernatant of the mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.24 This is a comparison chart of the red blood cell index (RBC), hemoglobin concentration (HGB) and reticulocyte count (Retic) in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.25 Comparison of BFU-E of early progenitor cells in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.26Comparison of CFU-E of early progenitor cells in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig. 27 Ter119 in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551) + Cell number comparison chart; Fig.28 This is a comparison of the number of erythroid progenitor cells in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.29 This is a comparison of the number of blood islands in the blood of mice in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.30 The blood island display and statistical graph of Giemsa staining of PBS solvent group (Veh) and β2-AR inhibitor group (ICI118551); Fig.31 This is a comparison chart of the number of hematopoietic lineages in the mouse bone marrow of the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.32 This is a comparison of the number of mouse neutrophils and LY6C monocytes in the PBS solvent group (Veh) and the β2-AR inhibitor group (ICI118551); Fig.33 This is a schematic diagram of the drug administration mode of SR59230A; Fig.34 This is a comparison chart of the red blood cell index (RBC), hemoglobin concentration (HGB) and reticulocyte count (Retic) in the blood of mice in the sham operation group (Sham), intracerebral hemorrhage group (ICH+PBS), intracerebral hemorrhage + β3-adrenergic receptor inhibitor group (ICH+SR59230A), intracerebral hemorrhage + IL6 antagonist group (ICH+an-tilL6), and intracerebral hemorrhage + β3-adrenergic receptor inhibitor + IL6 antagonist group (ICH+SR59230A+an-tilL6); Fig.35 Ter119 in the mouse bone marrow of the sham operation group (Sham), ICH group (ICH+PBS), ICH+β3-adrenergic receptor inhibitor group (ICH+SR59230A), ICH+IL6 antagonist group (ICH+an-tilL6), and ICH+β3-adrenergic receptor inhibitor+IL6 antagonist group (ICH+SR59230A+an-tilL6) + Cell comparison chart; Fig.36This is a comparison of the number of HSC cells in the mouse bone marrow of the sham operation group (Sham), intracerebral hemorrhage group (ICH+PBS), intracerebral hemorrhage + β3-adrenergic receptor inhibitor group (ICH+SR59230A), intracerebral hemorrhage + IL6 antagonist group (ICH+an-tilL6), and intracerebral hemorrhage + β3-adrenergic receptor inhibitor + IL6 antagonist group (ICH+SR59230A+an-tilL6); Fig.37 This is a comparison of the number of GMP cells in the mouse bone marrow of the sham operation group (Sham), intracerebral hemorrhage group (ICH+PBS), intracerebral hemorrhage + β3-adrenergic receptor inhibitor group (ICH+SR59230A), intracerebral hemorrhage + IL6 antagonist group (ICH+an-tilL6), and intracerebral hemorrhage + β3-adrenergic receptor inhibitor + IL6 antagonist group (ICH+SR59230A+an-tilL6); Fig.38 This is a comparison of the number of bone marrow MEP cells in mice in the sham operation group (Sham), intracerebral hemorrhage group (ICH+PBS), intracerebral hemorrhage + β3-adrenergic receptor inhibitor group (ICH+SR59230A), intracerebral hemorrhage + IL6 antagonist group (ICH+an-tilL6), and intracerebral hemorrhage + β3-adrenergic receptor inhibitor + IL6 antagonist group (ICH+SR59230A+an-tilL6). DETAILED DESCRIPTION

[0030] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. In the embodiments of the present invention, if specific conditions are not specified, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially, and the raw materials of different manufacturers and models do not affect the implementation of the technical solution of the present invention and the realization of the technical effect. Example 1

[0031] The drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage and / or changes in stem and progenitor cells induced by cerebral hemorrhage of this embodiment has 6OHDA as an active ingredient and also includes auxiliary materials; the 6OHDA and the auxiliary materials are prepared into a clinically acceptable preparation through a preparation process.

[0032] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0033] The drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage and / or changes in stem and progenitor cells induced by cerebral hemorrhage described in this embodiment is obtained by the following method: 6OHDA was taken and dissolved in water so that the concentration of 6OHDA was 12.5 mg / ml to obtain an injection.

[0034] It should be noted that the concentration of the injection in this embodiment is not unique, and those skilled in the art can adjust it within a reasonable range as needed. Example 2

[0035] The drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage of this embodiment has ICI118551 as an active ingredient and also includes excipients; the ICI118551 and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0036] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0037] The drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage described in this embodiment is obtained by the following method: ICI118551 was taken and dissolved in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) to a concentration of 0.5 mg / ml to obtain an injection.

[0038] The 0.5% bovine serum albumin (BSA) means that every 100 ml of solution contains 0.5 g of bovine serum albumin (BSA); the phosphate buffered saline is a suitable concentration for injection, for example, it can be a phosphate buffer with a concentration of 0.01-0.1 mol / L and a pH value of 7.2-7.4, which will not be described in detail below.

[0039] It should be noted that the concentration of the injection in this embodiment is not unique, and those skilled in the art can adjust it within a reasonable range as needed. Example 3

[0040] The drug for preventing, treating or alleviating changes in stem and progenitor cells induced by cerebral hemorrhage in this embodiment has SR59230A as an active ingredient and also includes excipients; the SR59230A and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0041] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0042] The drug for preventing, treating or alleviating the changes in stem and progenitor cells induced by cerebral hemorrhage described in this embodiment is obtained by the following method: SR59230A was taken and dissolved in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) to a concentration of 0.5 mg / ml to obtain an injection.

[0043] It should be noted that the concentration of the injection in this embodiment is not unique, and those skilled in the art can adjust it within a reasonable range as needed. Example 4

[0044] The drug for improving the neurological prognosis of cerebral hemorrhage of this embodiment has 6OHDA as an active ingredient and also includes auxiliary materials; the 6OHDA and the auxiliary materials are prepared into a clinically acceptable preparation through a preparation process.

[0045] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0046] The drug for improving the neurological prognosis of cerebral hemorrhage in this example is prepared by the same method as in Example 1. Example 5

[0047] The drug for improving the neurological prognosis of cerebral hemorrhage in this embodiment has ICI118551 as an active ingredient and also includes excipients. The ICI118551 and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0048] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0049] The drug for improving the neurological prognosis of cerebral hemorrhage in this example is prepared by the same method as in Example 2. Example 6

[0050] The drug for improving the neurological prognosis of cerebral hemorrhage of this embodiment has SR59230A as an active ingredient and also includes excipients; the SR59230A and the excipients are prepared into a clinically acceptable preparation through a preparation process.

[0051] The drug in this embodiment is an injection, and those skilled in the art can select the excipients according to actual conditions, which may be osmotic pressure regulators, stabilizers, antioxidants, buffers, etc., or other clinically acceptable pharmaceutical excipients.

[0052] The drug for improving the neurological prognosis of cerebral hemorrhage in this example is prepared by the same method as in Example 3. Example 7

[0053] Below, the following experiments are carried out to verify the technical effects of the present invention: 1. Construction of mouse intracerebral hemorrhage model

[0054] This experiment used 12-month-old WTC57BL / 6 mice to make the model. The intracerebral hemorrhage group (ICH) used type VIIs collagenase to stereotaxically inject into the left caudate nucleus of mice to make the intracerebral hemorrhage model. The injection dose was 0.5µL saline containing 0.075U collagenase. The injection site was 0.6mm in front of the anterior fontanelle and 2.0mm on the left side. The needle insertion depth was 2.9mm. The injection time was 5min, and the needle was retained for 10min. The sham group (Sham) was injected with an equal amount of 0.9% sterile saline.

[0055] The body temperature of mice was monitored and maintained within the normal range. Mice that died within 24 hours after surgery or mice with a neurological deficit score of less than 4 points were excluded from the experiment, and other mice were included in the experiment.

[0056] Among them, the neurological function deficit score was tested in a blind manner, and the mice were grouped and numbered by an experimenter who was completely unaware of the experimental groups, or the neurological function deficit score was directly performed by an experimenter who was unaware of the experimental groups.

[0057] When the indoor environment meets the standards, place the mice in the behavioral laboratory for 1 hour before acclimatization and then perform the tests in the table below. The test includes 6 items, each with 5 levels, with the highest score being 4 and the lowest score being 0.

[0058] Table 1 Neurological deficit scores

[0059] 2. Preparation of single cell suspension from mouse bone marrow and spleen First, after the mice were anesthetized, they were killed by cervical dislocation, and the femur, tibia and humerus were separated. Buffer I buffer (1×PBS+2%FBS+1%EDTA) was aspirated using a 1mL syringe to repeatedly rinse the mouse bone marrow cavity, and the cells were repeatedly blown up and down with a 1mL pipette to form a single-cell suspension. The bone marrow single-cell suspension was filtered with a 70µM cell filter before subsequent detection.

[0060] 3. Hematopoietic stem cell testing Bone marrow cells (about 6×10 7Biotin-Ter119, Biotin-B220, Biotin-CD3e, Biotin-Gr1, and Biotin-CD11b antibodies were added to each cell culture medium, mixed well, and incubated on a horizontal shaker for 30 minutes. The cells were washed once with Buffer I buffer, centrifuged at 300 g, and the supernatant was removed. The cells were resuspended in Buffer I buffer containing Biotinbeads and incubated for 15 minutes. The cells were resuspended by centrifugation, and the cell suspension was placed on a magnetic rack to enrich Lin cells. The enriched Lin cells (about 2×10 5 After centrifugation, the corresponding flow cytometry antibodies were added to detect hematopoietic stem cells (HSC), hematopoietic progenitor cells (MPP), myeloid progenitor cells (CMP), lymphoid progenitor cells (CMP), megakaryocyte erythroid progenitor cells (MEP) and granulocyte monocytic progenitor cells (GMP).

[0061] 4. EBI Enrichment The specific solution used for enriching EBI according to the serum gradient density method is shown in Table 2. Take the mouse femur and tibia, and quickly flush out the bone marrow with 500μL of 0% density gradient solution, gently blow twice with a 1mL gun, and then filter with a 70μm cell filter, and take 10μL of cells for counting. The bone marrow cell solution containing blood islands is adjusted to a volume of 5mL with 0% density gradient solution, and slowly added to the tube containing the layered density solution with a pipette, and placed on the 0% gradient. Leave at room temperature for 30min without collision. Aspirate and discard the 0% and 1.5% density gradient layers, and collect the 3% density layer in a 15mL centrifuge tube. Take out an appropriate volume of 16% paraformaldehyde solution and add it directly to the 3% density layer solution, so that the final concentration of PFA is 4%, and gently shake it on a shaker at room temperature for 20min to fix the blood islands. Add 5mL PBS + 0.5% BSA, centrifuge at 450g for 5 minutes at room temperature, discard the supernatant, add 5mL PBS + 0.5% BSA again, and centrifuge under the same conditions. The antibodies used for mouse bone marrow flow staining are: F4 / 80 (5µg / 106cells), Ter119 (0.5µg / 106cells) and Hoechst33342 (5µM). Flow cytometry was performed using a BD flow cytometer and the data were analyzed and processed using Flow Jo software.

[0062] Table 2 Density gradient solution

[0063] 5. Giemsa staining Place 200 µL of cell suspension in a cell centrifuge to obtain adherent cell clusters, fix with methanol for 2 min, wash off excess methanol with water, prepare with Giemsa stain and diluent (1:10), stain for 15 min, wash with water, dry and seal.

[0064] 6. Quantitative analysis of Cytospins Take 0.1×10 6 Enriched blood island cells were collected, resuspended in 200 µL PBS, placed in a spinner (Shandon Cytospin, Thermo scientific), centrifuged at 280 rpm for 10 min, and then the spinner was taken out and dried for Wright-Giemsa staining. The number of EBI and the number of nucleated red blood cells and granulocytes surrounding them were quantitatively analyzed.

[0065] 7. Drug treatment In the 6OHDA group (6OHDA), the injection of Example 1 was administered to mice two days before the cerebral hemorrhage model was established. The injection was administered once a day, with each intraperitoneal injection of 100 mg / kg. The mice were killed on the third day after the cerebral hemorrhage model was established. The β2-AR inhibitor group (ICI118551) was intraperitoneally administered to mice with the injection of Example 2 at a dose of 2 mg / kg per mouse on the day of cerebral hemorrhage modeling; In the cerebral hemorrhage + β3-adrenergic receptor inhibitor group (ICH + SR59230A), the injection of Example 3 was intraperitoneally administered to mice at a dose of 60 mg / kg per mouse on the day of cerebral hemorrhage modeling.

[0066] The cerebral hemorrhage + β3-adrenergic receptor inhibitor + IL6 antagonist group (ICH + SR59230A + an-tilL6) was intraperitoneally administered with IL6 antagonist at a dose of 8 mg / kg per mouse one week before and on the day of cerebral hemorrhage modeling, and the injection in Example 3 was intraperitoneally administered with 60 mg / kg per mouse on the day of cerebral hemorrhage modeling.

[0067] In the ICH+an-tilL6 group, IL6 antagonist was intraperitoneally administered once a week before and on the day of ICH modeling at a dose of 8 mg / kg per mouse.

[0068] In the cerebral hemorrhage control group (ICH+PBS), mice were injected with phosphate buffered saline (PBS) for two consecutive days, once a day, with each intraperitoneal injection of 100 mg / kg, two days before the cerebral hemorrhage model was established.

[0069] The PBS solvent group (Veh) was intraperitoneally injected with phosphate buffered saline (PBS) into 12-month-old WTC57BL / 6 mice, and the injection dose was the same as the dose of the control group. For example, when the PBS solvent group (Veh) was compared with the 6OHDA group (6OHDA), the administration dose of the PBS solvent group (Veh) was the same as that of the 6OHDA group (6OHDA), and so on.

[0070] Experimental Results Based on the above experiments, the following conclusions are obtained: 1. Anemia is associated with increased disability and severity after intracerebral hemorrhage Clinically, patients with intracerebral hemorrhage (ICH) have a certain risk of anemia. To understand the incidence of anemia in ICH patients, routine blood tests were performed on 115 patients with ICH, and the red blood cell count, hemoglobin concentration, and white blood cell count of ICH patients (n=115) were analyzed.

[0071] The research results show that if Figure 1 As shown in Figure 2, compared with healthy controls, the red blood cell count and hemoglobin level of ICH patients (ICHpatient) were significantly decreased, while the white blood cell count in peripheral blood increased. Figure 2 As shown, approximately 33.9% (39 of 115) of ICH patients showed anemia.

[0072] In addition, the mRS score (Modified-Rankin-Scale) was performed on patients with cerebral hemorrhage who had anemia at 3 months to evaluate the patient's neurological recovery status. Figure 3 As shown in Figure 2, the mRS score was significantly higher than that of ICH patients without anemia, and there was a negative correlation between the hemoglobin level and the mRS score. Figure 4 shown.

[0073] The above clinical results were also verified in mice, such as Figure 5 As shown in Figure 2, a collagenase-induced striatal ICH model was established in 12-month-old mice. Figure 6-7 As shown, ICH mice induced obvious anemia on the third day, with a significant decrease in red blood cell indices (including red blood cell count, hemoglobin level, and hematocrit), and an increase in reticulocyte count and white blood cell count.

[0074] Conclusions: Anemia is associated with increased disability and severity after ICH, ICH can cause anemia, and anemia is associated with more severe ICH progression and worse patient outcomes.

[0075] 2. 6OHDA treatment alleviates ICH-induced anemia and improves bone marrow erythropoiesis in ICH mice; 6OHDA, ICI118551, and SR59230A improve neurological prognosis in intracerebral hemorrhage like Figure 8 As shown, the sympathetic nervous system changes after cerebral hemorrhage, and the secretion of norepinephrine in the bone marrow supernatant increases, which may be related to the disorder of the bone marrow hematopoietic system after cerebral hemorrhage.

[0076] like Fig. 9 As shown in Figure 2, 6OHDA was intraperitoneally administered two days before the cerebral hemorrhage model was established. Figure 10-12 As shown, 6OHDA administration significantly reduced the IL6 content in serum and bone marrow, increased the counts of RBC and HGB in the blood of mice, and reduced the counts of Retic.

[0077] In addition, 6OHDA administration increased the early and terminal differentiation of BFU-E, CFU-E and Ter119 in ICH mice. + The number has increased significantly ( Figure 13-15 ), while the hematopoietic islands ( Figure 16-17 ) and erythroid progenitor cells surrounding blood islands ( Fig.18 ) also increased. 6OHDA treatment also improved the lineage bias of mouse bone marrow cells, with an increase in the number of MEP lineages and a decrease in the number of GMP lineages ( Fig.19 ), the number of Ly6C monocytes decreased, the volume of hematoma decreased, and the neurological deficit improved ( Fig. 20 ), which is beneficial to the development of the red blood cell, improves anemia after cerebral hemorrhage, and improves the neurological prognosis of cerebral hemorrhage.

[0078] It can be seen that 6OHDA can treat and alleviate ICH-induced anemia, improve bone marrow erythropoiesis in ICH mice, and improve the neurological prognosis of cerebral hemorrhage.

[0079] In addition, 6OHDA inhibits the expression or function of β2 and β3 adrenergic receptors at the same time, affecting norepinephrine-mediated signal transduction. Therefore, β2 receptor inhibitor ICI118551 and β3 receptor inhibitor SR59230A can also improve neurological prognosis after intracerebral hemorrhage by regulating neurotransmitter pathways, reducing inflammatory responses and improving neural plasticity.

[0080] 3. Treatment with the β2 receptor inhibitor ICI118551 alleviates ICH-induced anemia but does not reverse changes in stem and progenitor cells Sympathetic nerves activate β receptors by releasing norepinephrine and regulate various physiological processes. Both β2 and β3 receptors are part of the sympathetic nervous system. By inhibiting β receptors with ICI118551, we can observe changes in anemia and bone marrow hematopoietic stem cell homeostasis after cerebral hemorrhage. Fig.21As shown in Figure 2, on the day of modeling, the β2 inhibitor ICI118551 was intraperitoneally administered to mice. Figure 22-24 As shown, ICI118551 administration also significantly reduced the IL6 content in serum and bone marrow, increased the counts of RBC and HGB in the blood of mice, and reduced the counts of Retic.

[0081] In addition, ICI118551 administration also increased early and terminal differentiation in ICH mice, such as Figure 25-27 As shown, BFU-E, CFU-E and Ter119 + The number has increased significantly. Figure 28-30 As shown in Figure 3, the number of hematopoietic islands and erythroid progenitor cells surrounding the blood islands also increased. Figure 31-32 As shown, consistent with 6OHDA treatment, ICI118551 administration also improved the lineage bias of mouse bone marrow cells, reduced the number of Ly6C monocytes, favored erythroid development, and improved anemia after intracerebral hemorrhage.

[0082] β3 receptor inhibitor SR59230A treatment alleviates ICH-induced changes in stem cells but cannot reverse ICH-induced anemia like Fig.33 As shown, on the day of modeling, the β3 inhibitor SR59230A was intraperitoneally administered to mice. Fig.34 As shown in Figure 2, SR59230A administration did not change the counts of RBC, HGB, and Retic in the blood of ICH mice. Fig.35 As shown, SR59230A administration can improve the lineage skewing of bone marrow cells in ICH mice, and the number of HSCs is reduced ( Fig.36 ), increasing the number of MEP lineages ( Fig.38 ), reduce the number of GMP lineages ( Fig.37 ).

[0083] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. Use of a combination of β2 and β3 receptor inhibitors in the preparation of a drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage and / or changes in stem and progenitor cells induced by cerebral hemorrhage.

2. The use according to claim 1, characterized in that: Anemia induced by intracerebral hemorrhage includes one or more of a decrease in the number of red blood cells, a decrease in hemoglobin concentration, a decrease in erythroid progenitor cells, and a decrease in mature red blood cells; The changes in stem and progenitor cells induced by cerebral hemorrhage include one or more of the changes in the number of hematopoietic stem cells, the changes in the number of bone marrow megakaryocyte-erythroid progenitor cells, and the changes in the number of bone marrow granulocyte-macrophage progenitor cells.

3. Use of β2 receptor inhibitors in the preparation of drugs for preventing, treating or alleviating anemia induced by cerebral hemorrhage.

4. The use according to claim 3, characterized in that: Anemia induced by cerebral hemorrhage includes one or more of a decrease in the number of red blood cells, a decrease in hemoglobin concentration, a decrease in erythroid progenitor cells, and a decrease in mature red blood cells.

5. Use of β3 receptor inhibitors in the preparation of drugs for preventing, treating or alleviating changes in stem and progenitor cells induced by cerebral hemorrhage.

6. The use according to claim 5, characterized in that: The changes in stem and progenitor cells induced by cerebral hemorrhage include one or more of the changes in the number of hematopoietic stem cells, the changes in the number of bone marrow megakaryocyte-erythroid progenitor cells, and the changes in the number of bone marrow granulocyte-macrophage progenitor cells.

7. A drug for preventing, treating or alleviating anemia induced by cerebral hemorrhage and / or changes in stem and progenitor cells induced by cerebral hemorrhage, characterized in that: The active ingredient is one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor.

8. Use of one or more of β2 and β3 receptor inhibitors, β2 receptor inhibitors, and β3 receptor inhibitors in the preparation of drugs for improving the neurological prognosis of cerebral hemorrhage.

9. The use according to claim 8, characterized in that: The improvement of neurological prognosis of cerebral hemorrhage includes reducing hematoma volume and alleviating neurological deficits.

10. A drug for improving the neurological prognosis of cerebral hemorrhage, characterized in that: The active ingredient is one or more of a combined β2 and β3 receptor inhibitor, a β2 receptor inhibitor, and a β3 receptor inhibitor.

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