Application of IL-6 antagonist in preparation of medicine for relieving anemia after cerebral hemorrhage and prognosis

Abstract

The invention relates to application of an IL-6 antagonist in preparation of drugs for relieving anemia after cerebral hemorrhage and prognosis, and belongs to the technical field of biological medicines. The invention clarifies that ICH patients have anemia, researches the influence of ICH on bone marrow (BM) erythropoiesis by using a striatum ICH model of 10-12 months old mice, and finds that the number of BM EMBIs of the mice after ICH is significantly reduced, and the ICH-induced anemia can be relieved and the bone marrow erythropoiesis of the ICH mice can be improved by using the IL-6 antagonist MR16-1 for treatment. The MR16-1 treatment not only improves the formation of BM EMBIs, but also increases EMBI macrophages. In addition, by means of IL-6 blocking, the neurological function defect after cerebral hemorrhage is effectively improved, and cerebral hematoma is reduced.

Description

Technical Field

[0001] The invention relates to application of an IL-6 antagonist in preparing a drug for alleviating anemia and prognosis 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 also called spontaneous cerebral hemorrhage, accounting for 20-30% of acute cerebrovascular diseases. About 80% of bleeding occurs in the cerebral hemispheres, and about 20% of bleeding occurs in the brainstem and cerebellum. According to the location of bleeding, it can be divided into 7 categories: basal ganglia hemorrhage, thalamic hemorrhage, lobar hemorrhage, brainstem hemorrhage, cerebellar hemorrhage and intraventricular hemorrhage; according to the cause, it can be divided into primary cerebral hemorrhage and secondary cerebral hemorrhage. Cerebral hemorrhage is a common and difficult to treat disease, with high disability and mortality rates. The one-month mortality rate of patients with cerebral hemorrhage is as high as 35-52%, and about 80% of surviving patients still have residual disabilities at the end of 6 months. It is one of the main causes of death and disability among residents.

[0003] The present invention has found through a large number of studies and clinical data analysis that, in addition to the currently commonly used evaluation indicators, anemia is also an important predictive factor for poor prognosis of acute cerebral hemorrhage. Although common anemia is caused by abnormal hematopoiesis, the molecular mechanism of ICH-induced anemia, that is, the correlation between anemia and ICH progression / prognosis, is largely unclear. It is worth noting that erythropoiesis occurs in erythroblast islands (EMBIs), which are composed of central macrophages surrounded by developing erythroid cells in the bone marrow (BM). In EMBIs, macrophages play a key role by secreting growth factors that enhance the survival and division of erythroblasts, provide iron for the production of hemoglobin, and phagocytose nuclei to avoid DNA-induced toxicity. These EMBIs promote the erythroid differentiation process from the late colony-forming unit erythroid (CFU-E) stage to reticulocytes. Despite the understanding of normal physiological regulation, the specific mechanism of anemia caused by EMBIs destruction after ICH has remained largely unexplored.

[0004] Increasing evidence indicates the role of activated innate immunity and inflammation in the pathogenesis of ICH. It has been reported that there is abnormal expression of multiple cytokines in ICH, and inflammatory diseases, although initially targeted to specific organs, have the ability to cause serious systemic health problems, including blood and bone marrow. This principle suggests that ICH that triggers systemic inflammation may lead to anemia or bone marrow dysfunction, which may aggravate the patient's prognosis. In order to describe the specific role of cytokines in the development of anemia after ICH, the expression levels of several key cytokines were analyzed in the early stage of this invention. The results of the analysis showed that interleukin-6 (IL-6) showed significant and unique expression in brain tissue, peripheral blood and bone marrow around the hemorrhage. IL-6 is a 26kD secreted protein that plays a vital role in immune response, especially through the gp130 signal transducer to induce B cells to produce antibodies and share functions with the IL-6 cytokine family. Despite the wide range of functions of IL-6, its impact on EMBIs has received little attention after ICH. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an IL-6 antagonist in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, thereby providing a new treatment approach for anemia caused by cerebral hemorrhage and improving prognosis.

[0006] In order to achieve the above-mentioned object, the technical scheme of the present invention for using the IL-6 antagonist in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage is:

[0007] Application of IL-6 antagonist in the preparation of drugs for alleviating anemia and prognosis after cerebral hemorrhage.

[0008] The beneficial effect of the above technical scheme is that the use of the IL-6 antagonist of the present invention in the preparation of drugs for alleviating anemia and prognosis after cerebral hemorrhage is a pioneering invention. The present invention uses the striatal ICH model of 10-12 month old mice to study the effect of ICH on BM erythropoiesis, and clarifies that anemia exists in ICH patients, and the number of BM EMBIs in mice after ICH is significantly reduced. Treatment with the IL-6 antagonist MR16-1 can alleviate ICH-induced anemia and improve bone marrow erythropoiesis in ICH mice. MR16-1 treatment not only improves the formation of BM EMBIs, but also increases EMBI macrophages. In addition, MR16-1 treatment effectively improves neurological deficits after cerebral hemorrhage.

[0009] This invention reveals for the first time the mechanism by which IL-6 induces terminal erythropoiesis disorder by acting on erythroid hematopoietic island macrophage disorder, thereby leading to anemia. This anemia mechanism involves deep-seated disorders in the erythroid cell microenvironment, and innovatively proposes that the pathological effect of IL-6 is located at the key node of erythroid hematopoiesis, thus providing a new idea for the treatment of anemia after cerebral hemorrhage.

[0010] Furthermore, the present invention proposes a unique perspective on the use of IL-6 antagonists in the treatment of anemia after cerebral hemorrhage, especially explaining the new mechanism of IL-6 causing anemia from the perspective of erythroid hematopoietic islands. This innovation not only breaks through the existing understanding of the role of IL-6 in chronic inflammation and bone marrow suppression, but also provides a unique and effective target for the treatment of anemia after cerebral hemorrhage, and therefore has significant innovation and application value.

[0011] As a further improvement, the drug for alleviating anemia after cerebral hemorrhage is a drug for enhancing the formation of erythrocyte islands in the bone marrow.

[0012] As a further improvement, the drug for enhancing the formation of bone marrow erythrocyte islands is a drug for increasing EMBI macrophages and reducing the number of granulocytes around the macrophages.

[0013] As a further improvement, the drug for alleviating anemia after cerebral hemorrhage is a drug for increasing peripheral blood red blood cells and hemoglobin.

[0014] As a further improvement, the drug for alleviating anemia after cerebral hemorrhage is a drug for enhancing bone marrow erythropoiesis.

[0015] As a further improvement, the drug for enhancing bone marrow erythropoiesis is a drug that reduces reticulocytes and increases the number of erythroid progenitor cells and terminal erythroblasts.

[0016] As a further improvement, the drug for alleviating prognosis after cerebral hemorrhage is a drug for alleviating neurological deficits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The peripheral blood red blood cell related indicators of clinical patients and D3 mice after cerebral hemorrhage in Example 1 of the present invention are abnormal (wherein, a is the comparison of red blood cells, hemoglobin, and white blood cells between clinical ICH patients and healthy people, b is the percentage of anemia in ICH patients, c is the mRS comparison between ICH patients and ICH anemia patients, d is the correlation analysis of hemoglobin and mRS in ICH anemia patients, e is a schematic diagram of the process of constructing a mouse ICH model, fg are blood routine white blood cell and red blood cell related indicators of mice on D1, D3, D7, and D14 after ICH, * represents P<0.05, *** represents P<0.001);

[0018] Figure 2The ICH in Example 2 of the present invention leads to a decrease in EMBI macrophages and an increase in granulocytes in EMBIs (wherein, a is the gating strategy and quantitative statistics for detecting blood island EMBIs macrophages by flow cytometry, b is the flow peak graph of the classic macrophage functional markers Mertk, Vcam1, CD163, and MHC-II, c is a schematic diagram of blood island cell enrichment, df is the ratio of EMBIs and CD11b around blood islands after ICH detected by flow cytometry, gi is a comparison of red blood cells and granulocytes around blood islands enriched after cerebral hemorrhage shown by Giemsa staining, * represents P<0.05, and ** represents P<0.01);

[0019] Figure 3 The increased IL-6 level in BM after ICH and the enhanced IL-6R activity in EMBI macrophages in Example 3 of the present invention (wherein, AB represents the enhanced inflammatory response after ICH, C represents the expression of IL6 in bone marrow supernatant, serum and brain tissue after ICH in QAM-INF-1 analysis, * represents P<0.05, *** represents P<0.001);

[0020] Figure 4 IL-6 antagonism in Example 4 of the present invention is beneficial to erythropoiesis rather than granulopoiesis after ICH (wherein, a is the use of MR16-1 and the construction of the ICH model, b is the reduction of IL6 in the bone marrow supernatant after IL6 antagonism, c is the reduction of Hepcidin in both the bone marrow supernatant and serum after IL6 antagonism, d is the analysis of red blood cell related indicators in the IL6 antagonism group, eh is the analysis of BFU-E and CFU-E in the IL6 antagonism group, i is the Ter119 in the bone marrow of the IL6 antagonism group + Quantitative analysis, jl is the quantitative analysis of EMBIs in the bone marrow and the quantitative analysis of granulocytes in EMBIs in the IL6 antagonist group, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001);

[0021] Figure 5 The neurological prognosis of IL-6 knockout mice after ICH in Example 5 of the present invention was significantly improved (wherein A is the neurological deficit score, B is the hanging wire experiment, * represents P<0.05, and ** represents P<0.01). DETAILED DESCRIPTION

[0022] The present invention has found through a large amount of research and clinical data analysis that anemia is associated with increased disability and severity after cerebral hemorrhage. Cerebral hemorrhage can cause anemia, but the molecular mechanism of cerebral hemorrhage-induced anemia is unclear, and there is no relevant effective drug for treating anemia caused by cerebral hemorrhage. Based on this, the present invention provides the use of IL-6 antagonists in the preparation of drugs for alleviating anemia and prognosis after cerebral hemorrhage.

[0023] The present invention firstly found through data query and research that anemia caused by chemotherapy and radiotherapy and common anemia are usually due to bone marrow suppression, inhibition of erythropoiesis or immune hemolytic anemia caused by drugs. The core mechanism of this type of anemia lies in the damage to hematopoietic stem cells in the bone marrow during radiotherapy or chemotherapy, or the immune response induced by drugs, which leads to a decrease in hemoglobin and red blood cells. Therefore, the role of some inflammatory factors in this context is mainly focused on indirect anemia caused by chronic inflammatory response.

[0024] However, the present invention has discovered through a large number of studies and experiments that anemia caused by cerebral hemorrhage is due to abnormalities in the brain-bone marrow axis and specific physiological changes caused by inflammatory responses after hemorrhage, which is completely different from anemia caused by existing chemotherapy and radiotherapy and common anemia. The research of the present invention reveals for the first time that IL-6 induces terminal erythropoiesis disorders by acting on erythroid hematopoietic island macrophage disorders, thereby leading to anemia. This anemia mechanism is not just the conventional inflammatory anemia caused by hepcidin, etc., but involves deep-seated disorders of the erythroid cell microenvironment. This mechanism innovatively proposes that the pathological effect of IL-6 is located at the key node of erythroid hematopoiesis, thus providing a new idea for the treatment of anemia after cerebral hemorrhage.

[0025] 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.

[0026] The preparation process of ICH models (cerebral hemorrhage models) used in the embodiments of the present invention is as follows:

[0027] The present invention uses WTC57BL / 6 12-month-old mice to prepare the model. Collagenase hemorrhage model: The cerebral hemorrhage model is prepared by stereotaxic injection of type VIIs collagenase in the left caudate nucleus. The injection dose is 0.5 μL of normal saline containing 0.075U collagenase, the injection site is 0.6mm in front of the anterior fontanelle, 2.0mm in the front left side, the needle insertion depth is 2.9mm, the injection time is 5min, and the needle is retained for 10min. The sham operation group (Sham) was injected with 0.9% equal amount of sterile saline. The body temperature changes of the mice were monitored and maintained within the normal range. Mice that died within 24h after surgery or mice with a neurological deficit score of <4 points were excluded from the experiment, and other mice were included in the experiment.

[0028] The process of preparing mouse bone marrow and spleen single cell suspension in the embodiment of the present invention is as follows:

[0029] First, after the mice were anesthetized, they were killed by cervical dislocation, and the femur, tibia and humerus were separated. Buffer I (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 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.

[0030] The process of behavioral detection in the embodiment of the present invention is as follows:

[0031] Blind testing: All behavioral tests were performed in a blind manner, with an experimenter who was completely unaware of the experimental groups marking the mice into groups or directly evaluating the following behavioral tests.

[0032] 1) Neurological deficit score

[0033] When the indoor environment meets the standards, the mice were placed in the behavioral laboratory for 1 hour of pre-adaptation and then tested in Table 1. The test includes 6 items, each with 5 levels, with the highest score being 4 and the lowest score being 0. ICH mice with a total score of less than 4 points or more than 20 points will be eliminated.

[0034] Table 1 Neurological deficit scores

[0035]

[0036] 2) Hanging wire experiment

[0037] When the indoor environment meets the standards, the mice are placed in the behavioral laboratory to adapt for 1 hour in advance before the experiment. The experimental device is 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 need to use both upper limbs to support their suspended weight. In order to prevent the mice from using their limbs, their lower limbs should be glued with tape or other items. The time each mouse persists on the iron wire is recorded, and the average value of three experiments is taken for each mouse.

[0038] The process of EMBI enrichment in the embodiment of the present invention is as follows:

[0039] According to the serum gradient density method, the specific solution preparation method for enriching EMBI 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 the blood island is adjusted to a volume of 5mL with a 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 a suitable volume of 16% paraformaldehyde solution and add it directly to the 3% density layer solution to make the final concentration of PFA 4%, and gently shake it on a shaker at room temperature for 20min to fix the blood island. Add 5 mL PBS + 0.5% BSA, centrifuge at 450 g for 5 min at room temperature, discard the supernatant, add 5 mL PBS + 0.5% BSA again, and centrifuge under the same conditions. The antibody used for mouse bone marrow flow cytometry staining is: F4 / 80 (5 μg / 10 6 cells), Ter119 (0.5 μg / 10 6 cells) and Hoechst33342 (5 μM). Flow cytometry was performed using a BD flow cytometer, and the data were analyzed and processed using Flow Jo software.

[0040] Table 2 Preparation method of density gradient solution

[0041]

[0042] The process of Giemsa staining in the embodiment of the present invention is as follows:

[0043] Place 200 μL of cell suspension in a cell centrifuge to obtain adherent cell clusters, fix with methanol for 2 minutes, wash off excess methanol with water, prepare with Giemsa stain and diluent (1:10), stain for 15 minutes, wash with water, dry and seal.

[0044] The process of quantitative analysis of Cytospins in the embodiment of the present invention is as follows:

[0045] Take 0.1×10 6 The enriched blood island cells were resuspended in 200 μL PBS, placed in the EMBI in a spin slice machine (Shandon Cytospin, Thermo scientific), centrifuged at 280 rpm for 10 min, and the spin slices were taken out and dried for Wright-Giemsa staining. The number of EMBI and the number of nucleated red blood cells and granulocytes surrounding them were quantitatively analyzed.

[0046] The process of OAM-INF-1 inflammation chip detection in the embodiment of the present invention is as follows:

[0047] Collect bone marrow supernatant from sham-operated and cerebral hemorrhage aged mice. After surgery, take the slide chip out of the box, balance at room temperature for 20-30 minutes, open the packaging bag, remove the seal, and then place the chip in a vacuum dryer or dry at room temperature for 1-2 hours. Gradient dilution of the standard sample for standard curve detection. Add 10μL of sample diluent to each well, incubate on a shaker at room temperature for 1 hour, and block the quantitative antibody chip. Draw out the buffer in each well, add 100L of standard solution and diluted sample to the well (bone marrow supernatant and brain tissue are loaded at 500μg / mL, and serum is diluted 2 times for loading), and incubate on a shaker at 4℃ overnight. Wash the slide with the ThermoScientific Well wash Versa chip washer, which is divided into two steps. First, wash with 1× wash solution I, 250μL of 1× wash solution I per well, wash 10 times, shake for 10s each time, and select high shaking intensity. Dilute 20× wash solution I with deionized water. Then switch to 1× Wash Solution II channel for washing, 250μL of 1× Wash Solution I per well, wash 6 times, shake for 10s each time, choose high shaking intensity, and dilute 20× Wash Solution II with deionized water. Incubate the detection antibody mixture, centrifuge the detection antibody mixture tube, then add 1.4mL of sample diluent, mix well and centrifuge again quickly. Add 80μL of detection antibody to each well and incubate on a shaker at 37℃ for 2h. Centrifuge the Cy3-streptavidin tube, then add 1.4mL of sample diluent, mix well and centrifuge again quickly. Add 80μL of Cy3-streptavidin to each well, wrap the slide with aluminum foil to incubate in the dark, and incubate on a shaker at 37℃ for 1 hour. Remove the slide frame, be careful not to touch the side of the slide printed with antibodies with your hands. Use a laser scanner such as InnoScan300 Microarray Scanner to scan the signal, using Cy3 or green channel (excitation frequency -532nm).

[0048] The process of clinical data collection in the embodiment of the present invention is as follows:

[0049] The blood routine data of patients with acute basal ganglia cerebral hemorrhage were collected as the research group, and the prognostic indicators such as cerebral edema and hematoma reflected by CT and MRI imaging were collected, and correlation analysis was performed using SPSS software. The inclusion criteria for patients with cerebral hemorrhage were as follows: age 50-70 years, meeting the diagnostic criteria for hypertensive cerebral hemorrhage (Chinese Stroke Association StrokeCouncil Guideline, 2020, doi:10.1136 / svn-2020-000433), no concurrent infection, tumor and autoimmune disease, and no use of drugs that affect immune function. The inclusion criteria for patients in the control group were the same as above (except for the diagnostic criteria).

[0050] Specific examples of the use of the IL-6 antagonist of the present invention in the preparation of drugs for alleviating anemia and prognosis after cerebral hemorrhage:

[0051] The present invention uses a 12-month-old mouse striatal ICH model to study the effect of ICH on bone marrow (BM) erythropoiesis, and clarifies that ICH patients have anemia, and the number of BM EMBIs in mice after ICH is significantly reduced. Treatment with the IL-6 antagonist MR16-1 can alleviate ICH-induced anemia and improve bone marrow erythropoiesis in ICH mice. MR16-1 treatment not only improves the formation of BMEMBI, but also increases EMBI macrophages. In addition, MR16-1 treatment effectively improves neurological deficits after cerebral hemorrhage and reduces cerebral hematomas. Administration of recombinant IL-6 to wild-type mice leads to reduced levels of red blood cells and hemoglobin, while inhibiting erythropoiesis in BM. The specific implementation operations are as follows:

[0052] Example 1 Anemia is associated with increased disability and severity after intracerebral hemorrhage

[0053] Considering the clinical incidence of anemia in patients with intracerebral hemorrhage (ICH), this example first analyzed the red blood cell (RBCs) count, hemoglobin concentration (HGB) and white blood cell (WBCs) count of ICH patients (n=111). The results showed that compared with healthy control individuals, the red blood cell count and hemoglobin level of ICH patients were significantly decreased, while the white blood cell count in peripheral blood increased ( Figure 1 The analysis results showed that approximately 35.1% (39 of 111) of ICH patients showed anemia ( Figure 1 In addition, the mRS scores of ICH patients with anemia at 3 months were significantly higher than those of ICH patients without anemia ( Figure 1 c), and there was a negative correlation between the patient's hemoglobin level and mRS score ( Figure 1 (shown in d).

[0054] Subsequently, this example verified these clinical results in mice, and established a collagenase-induced striatal ICH model in 12-month-old mice ( Figure 1 Compared with control mice, ICH mice induced significant anemia on day 3 ( Figure 1 g), red blood cell indices (such as red blood cell count, hemoglobin level, red blood cell standards) decreased significantly, and reticulocyte count and white blood cell count increased ( Figure 1 f and g).

[0055] In summary, the results of Example 1 indicate that cerebral hemorrhage can lead to anemia, and anemia is associated with more severe ICH progression and poorer patient prognosis.

[0056] Note: mRS score (modified Rankin Scale) is a clinical scale for evaluating the functional status and degree of disability of patients with neurological diseases. It is widely used in the prognosis assessment of cerebrovascular diseases (such as stroke). mRS score is a concise assessment tool with a score range of 0 to 6. The higher the score, the more severe the patient's functional impairment and disability.

[0057] Example 2: EMBI macrophages are damaged after intracerebral hemorrhage, and EMBI favors granulocyte production rather than erythrocyte production

[0058] In the context of erythropoiesis, macrophages interact closely with early erythroblasts, providing a special microenvironment called erythroid islands (EMBIs). In order to evaluate whether ICH-induced erythropoiesis inhibition is related to EMBIs, this example used 12-month-old EpoR-tdTomato-Cre mice (Zhang, Huan et al. "EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissuemacrophages and hematopoietic cells." Blood vol.138, 20(2021): 1986-1997. doi: 10.1182 / blood.2021011410) to perform ICH and found that the number of BM EMBI macrophages was significantly impaired in ICH mice ( Figure 2 Notably, ICH mice did not show a significant decrease in classical macrophage functional markers, including Mertk, Vcam1, CD163, and MHC-II ( Figure 2 (as shown in b).

[0059] Previous research results have shown that EMBIs central macrophages are adapted to optimize the production of erythrocytes or neutrophils. In order to further investigate the proportion of CD11b-expressing cells associated with EMBIs in BM after ICH, this example first improved the method of isolating natural EMBIs from mouse BM by density gradient sedimentation ( Figure 2 The results of the analysis showed that the number of EMBIs decreased, while the CD11b+ cells representing granulocytes increased significantly, indicating that the microenvironment had changed and that EMBIs after ICH favored granulocyte production rather than erythropoiesis ( Figure 2 (shown in df).

[0060] In addition, Giemsa staining of EMBI also showed that the granulocyte precursors around EMBI increased significantly and the developing erythroid cells decreased after ICH ( Figure 2 (as shown in gi).

[0061] In summary, the results of Example 2 indicate that ICH leads to a decrease in EMBI macrophages and an increase in EMBI granulocytes, and this imbalance may subsequently impair erythropoiesis after ICH.

[0062] Example 3 IL-6 receptor IL-6R is preferentially expressed on EMBI macrophages

[0063] Studies have shown that inflammation plays a crucial role in secondary brain damage after intracerebral hemorrhage, and inflammatory cytokines may play a role in this process by affecting the lineage specificity of pluripotent stem cells and EMBI. Therefore, the present invention assumes that inflammatory cytokines may contribute to the inhibition of erythropoiesis observed in the ICH mouse model. To investigate this, this example uses the QAM-INF-1 chip (a tool for quantitatively measuring 40 mouse cytokines) to measure inflammatory factors in peripheral blood and bone marrow fluid. The results are shown in Figure 2. Figure 3 shown.

[0064] The results of principal component analysis (PCA) of RNA-Seq data of blood island macrophages sorted after ICH showed that the data of the control group and the experimental group had good separation and consistency ( Figure 3 The results of RNA-Seq in the control group and the ICH group showed that GO analysis was based on the enrichment of differentially expressed genes in the two groups into inflammation-related pathways, indicating that the inflammatory response after ICH changed significantly ( Figure 3 The IL-6 concentrations in the peripheral blood and bone marrow fluid of ICH mice were significantly higher than those in the control group ( Figure 3 (as shown in C).

[0065] Example 4 IL-6 antagonist alleviates anemia after cerebral hemorrhage

[0066] This example investigates the effects of one week of treatment with MR16-1 (an IL-6 antagonist, administered at a dose of 8 mg / kg) on ​​the survival of mice after ICH. Figure 4 Treatment with MR16-1 in mice after ICH resulted in lower levels of IL-6 and hepcidin in BM supernatant and serum at day 3 compared with treatment with isotype control ( Figure 4 (as shown in bc).

[0067] Interestingly, MR16-1 treatment not only alleviated ICH-induced anemia but also enhanced bone marrow erythropoiesis in mice, as evidenced by increased peripheral blood red blood cell and hemoglobin levels, decreased reticulocyte counts, and increased numbers of erythroid progenitors and terminal erythroblasts ( Figure 4 MR16-1 treatment also enhanced the formation of BM EMBIs, increased EMBI macrophages, and decreased the number of granulocytes surrounding these macrophages ( Figure 4 (as shown in Figure 1).

[0068] In summary, the results of Example 4 indicate that IL-6 antagonism reverses anemia, at least to some extent, by restoring BM-EMBI balance after ICH, thereby favoring erythropoiesis rather than granulopoiesis.

[0069] Example 5 Neuromotor function of IL-6 knockout mice significantly improved after ICH

[0070] This example uses IL6 knockout mice to compare the changes in neuromotor function between WT mice and IL6 knockout mice after cerebral hemorrhage. Compared with WT mice, the neurological deficits of IL6 knockout mice on D3 after cerebral hemorrhage were significantly reduced ( Figure 5 The upper limbs persisted longer in the hanging wire test ( Figure 5 (shown in B).

[0071] 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 IL-6 antagonists in the preparation of drugs for alleviating anemia and prognosis after cerebral hemorrhage.

2. The use of the IL-6 antagonist according to claim 1 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for alleviating anemia after cerebral hemorrhage is a drug for enhancing the formation of erythrocyte islands in the bone marrow.

3. The use of the IL-6 antagonist according to claim 2 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for enhancing the formation of bone marrow erythrocyte islands is a drug for increasing EMBI macrophages and reducing the number of granulocytes around the macrophages.

4. The use of the IL-6 antagonist according to claim 1 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for alleviating anemia after cerebral hemorrhage is a drug for increasing peripheral blood red blood cells and hemoglobin.

5. The use of the IL-6 antagonist according to claim 4 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for alleviating anemia after cerebral hemorrhage is a drug for enhancing bone marrow erythropoiesis.

6. Use of the IL-6 antagonist according to claim 5 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for enhancing bone marrow erythropoiesis is a drug for reducing reticulocytes and increasing the number of erythroid progenitor cells and terminal erythroblasts.

7. Use of the IL-6 antagonist according to claim 1 in the preparation of a drug for alleviating anemia and prognosis after cerebral hemorrhage, characterized in that: The drug for alleviating prognosis after cerebral hemorrhage is a drug for alleviating neurological deficits.

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