Application of bilirubin in preparation of medicine for treating ischemic cerebrovascular disease

Abstract

The invention belongs to the field of medicine, and discloses application of bilirubin in preparation of medicine for treating ischemic cerebrovascular diseases, and the bilirubin can improve cognitive impairment caused by the ischemic cerebrovascular diseases; the severity of demyelination caused by ischemic cerebrovascular diseases is improved, the area of cerebral infarction is reduced, and the severity of neurological impairment caused by ischemic cerebrovascular diseases is relieved; inflammation, caused by ischemic cerebrovascular diseases, in blood and brain tissues is relieved, and activation of B cells and mediated immune response are inhibited; the oxidative stress of B cells is improved; the microglial cell activation caused by ischemic cerebrovascular diseases is relieved; the neuroinflammation reaction caused by ischemic cerebrovascular diseases is relieved. Good clinical application values are realized.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of bilirubin in preparing medicine for treating ischemic cerebrovascular disease. Background Art

[0002] Cerebrovascular disease is a general term for a class of diseases caused by cerebrovascular lesions that lead to brain dysfunction. It includes localized or diffuse brain dysfunction caused by various cerebrovascular lesions such as vascular occlusion or stenosis, vascular rupture, vascular malformation, vascular wall damage or permeability changes. Cerebrovascular diseases are mainly divided into ischemic cerebrovascular disease and hemorrhagic cerebrovascular disease, of which ischemic cerebrovascular disease accounts for about 85% of the total number of cerebrovascular diseases. Ischemic stroke caused by ischemic cerebrovascular disease is currently the second leading cause of death in humans.

[0003] The damage caused by ischemic cerebrovascular disease can widely affect the entire brain tissue. Its pathogenic mechanism and different diseased blood vessels can cause damage to brain tissue in different areas, thus causing different symptoms. The brain is mainly composed of white matter and gray matter. Gray matter is mainly composed of the cell bodies of neurons, while white matter is mainly composed of the myelin sheath wrapped around the outer layer of nerve fibers. In the context of ischemic cerebrovascular disease, the main manifestations of damage to the two are movement disorders, sensory disorders, balance disorders, cognitive disorders and other defects.

[0004] Bilirubin is a type of bile pigment and the main pigment in human bile. Bilirubin is the main metabolite of iron porphyrin compounds in the body. It is toxic and can cause irreversible damage to the brain and nervous system, but it also has antioxidant function and can inhibit the oxidation of linoleic acid and phospholipids. Bilirubin is an important basis for clinical diagnosis of jaundice and an important indicator of liver function.

[0005] Some studies have shown that serum bilirubin is related to lipid and lipoprotein oxidation and endothelial damage, which in turn promotes the formation of atherosclerotic plaques and arterial thrombosis. In recent years, more and more studies have shown that bilirubin is also a powerful intrinsic antioxidant. Bilirubin in serum has an antioxidant effect, which can reduce the degree of inflammation of atherosclerotic plaques, thereby reducing or delaying the formation of atherosclerotic plaques. However, the relationship between bilirubin and ischemic cerebrovascular disease has not been studied.

[0006] Therefore, exploring the application of bilirubin in ischemic cerebrovascular disease is crucial for alleviating neurological deficits, cognitive dysfunction and neuroinflammation caused by ischemic injury. Summary of the invention

[0007] The invention discloses the application of bilirubin in preparing a medicine for treating ischemic cerebrovascular disease, and has good clinical application value.

[0008] In order to achieve the above purpose, this application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of bilirubin in the preparation of a drug for treating ischemic cerebrovascular disease.

[0010] In the above technical solution, the ischemic cerebrovascular disease includes transient ischemic attack, different types of acute ischemic stroke, cerebral artery steal syndrome and chronic cerebral ischemia.

[0011] In the above technical solution, the bilirubin can improve cognitive dysfunction caused by ischemic cerebrovascular disease.

[0012] In the above technical solution, the bilirubin can improve the severity of demyelination caused by ischemic cerebrovascular disease, reduce the area of ​​cerebral infarction, and alleviate the severity of neurological deficits caused by ischemic cerebrovascular disease.

[0013] In the above technical solution, the bilirubin can alleviate inflammation in the blood and brain tissue caused by ischemic cerebrovascular disease.

[0014] In the above technical solution, the bilirubin can improve the activation of B cells and the mediated immune response in ischemic cerebrovascular disease.

[0015] In the above technical solution, the bilirubin can alleviate the oxidative stress of B cells in ischemic cerebrovascular disease.

[0016] In the above technical solution, the bilirubin can alleviate the microglial activation caused by ischemic cerebrovascular disease.

[0017] In the above technical solution, the bilirubin can alleviate the neuroinflammatory response caused by ischemic cerebrovascular disease.

[0018] In a second aspect, the present invention provides a drug for treating ischemic cerebrovascular disease, which is composed of bilirubin at an effective dose, and can be a single component or a composition containing an effective dose of bilirubin, and the composition can include a pharmaceutically acceptable carrier.

[0019] The beneficial effects of the present invention are: the present invention creatively discovered a new application of bilirubin, namely, the application of bilirubin in the preparation of drugs for treating ischemic cerebrovascular disease. Bilirubin can improve cognitive dysfunction caused by ischemic cerebrovascular disease; improve the severity of demyelination caused by ischemic cerebrovascular disease, reduce the area of ​​cerebral infarction, and reduce the severity of neurological deficits caused by ischemic cerebrovascular disease; relieve inflammation in the blood and brain tissue caused by ischemic cerebrovascular disease, inhibit B cell activation and mediated immune response; inhibit B cell oxidative stress; relieve microglia activation caused by ischemic cerebrovascular disease; and relieve neuroinflammatory response caused by ischemic cerebrovascular disease. It has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a drug safety diagram for mice with ischemic cerebrovascular disease;

[0021] Figure 2 This is a statistical chart showing changes in neurological deficit scores in mice with ischemic cerebrovascular disease;

[0022] Figure 3 This is a statistical chart showing changes in learning and memory abilities in mice with ischemic cerebrovascular disease;

[0023] Figure 4 This is a statistical graph of the degree of white matter damage in mice with ischemic cerebrovascular disease;

[0024] Figure 5 This is a statistical chart of the changes in infarct area in mice with ischemic cerebrovascular disease;

[0025] Figure 6 This is a statistical diagram of the number of B cells in the peripheral blood of mice with ischemic cerebrovascular disease;

[0026] Figure 7 This is a statistical chart showing the changes in the number of B cells in the brain tissue of mice with ischemic cerebrovascular disease;

[0027] Figure 8 This is the GO enrichment analysis diagram of B cells in the peripheral blood of mice with ischemic cerebrovascular disease;

[0028] Fig. 9 This is the GO enrichment analysis diagram of B cells in the brain tissue of mice with ischemic cerebrovascular disease;

[0029] Fig.10 It is a statistical graph of the fluorescence intensity of MitoSOX in peripheral blood B cells of mice with ischemic cerebrovascular disease;

[0030] Fig.11 It is a statistical graph of the fluorescence intensity of MitoSOX, MitoSpy and ROS in human B cells cultured in vitro treated with bilirubin;

[0031] Fig.12 This is a statistical diagram of the activation degree of microglia in the brain tissue of mice with ischemic cerebrovascular disease;

[0032] Fig.13 This is a statistical chart of the proportion of cells positive for iNOS, CD16 / 32 and Iba1 in the brain tissue of mice with ischemic cerebrovascular disease;

[0033] Fig.14 This is the GSEA analysis of microglia in the brain tissue of mice with ischemic cerebrovascular disease;

[0034] Fig.15This is a statistical chart of the gene expression of TNFα, IL-1β, TGFβ, Arg-1, P2ry12 and Hexb in humanized microglia under the ischemia-hypoxia model. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0036] Ischemic cerebrovascular disease mouse model: The present invention uses middle cerebral artery occlusion (MCAO) surgery to establish a middle cerebral artery occlusion mouse model. Specifically, after the mouse is anesthetized with isoflurane, the skin is incised along the midline of the neck, the thyroid gland is peeled off, and the mouse trachea and the common carotid arteries on both sides are exposed. A silicone thread plug with an inner diameter of 0.18 mm is inserted into the internal carotid artery through the distal end of the right external carotid artery of the mouse, the incision is closed, the skin is sutured, and the cerebral blood flow is monitored. A decrease of 80% represents the successful establishment of the MCAO mouse model. 3 days and 28 days are selected as the observation time points for acute and chronic middle cerebral artery embolism injuries, respectively.

[0037] Bilirubin administration: The present invention selects chemically synthesized bilirubin, the solvent is 0.5% sodium carboxymethyl cellulose, the bilirubin concentration is 1.67 mg / ml, and the administration is carried out by gavage. Specifically, the mice after MCAO surgery are gavaged for 30 consecutive days. The mice in the control group are gavaged with an equal volume of 0.5% sodium carboxymethyl cellulose solution.

[0038] Example 1 Bilirubin has drug safety

[0039] Mice after MCAO were gavaged with bilirubin for 30 consecutive days, and mice in the control group were gavaged with an equal volume of sodium carboxymethylcellulose solution. 1 ml of peripheral blood was collected from mice using a blood collection tube without anticoagulant, left to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm / min for 15 min in a 4°C centrifuge. The upper serum was collected and frozen in an ultra-low temperature refrigerator at -80°C. An automatic biochemical analyzer was used to detect liver and kidney function indicators of mice, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), total bile acid (TBA), urea nitrogen (BUN), creatinine (CREA) and uric acid (UA).

[0040] Figure 1The results showed that the liver and kidney function indicators such as alanine aminotransferase, aspartate aminotransferase, total bile acid, urea nitrogen, creatinine and uric acid in the mice in the oral bilirubin group were not significantly abnormal compared with those in the control group, indicating that oral bilirubin is safe. However, the total bilirubin level of the mice in the oral bilirubin group was slightly increased within the normal range compared with the mice in the control group (P<0.05).

[0041] Example 2 Bilirubin can effectively improve neurological deficits in MCAO mice

[0042] Modified Neurological Severity Scores (mNSS) is a behavioral experiment used to evaluate the symptoms of neurological deficits in animals, including motor function, sensory function and reflexes. In this example, the mNSS scores of mice were tested at five time points: before surgery, 3 days, 7 days, 14 days, 21 days and 28 days after MCAO, to evaluate the severity of their neurological deficits.

[0043] Figure 2 The results showed that compared with the control mice, the mice in the oral bilirubin group had lower mNSS scores, showing significant differences at 28 days after MCAO surgery, indicating that oral bilirubin helps to repair neurological function in mice with chronic ischemic cerebrovascular disease.

[0044] The above results indicate that oral bilirubin treatment in mice with ischemic cerebrovascular disease can effectively improve the symptoms of neurological deficits caused by it.

[0045] Example 3 Bilirubin improves cognitive dysfunction caused by ischemic cerebrovascular disease

[0046] The novel object recognition experiment is a behavioral experiment that uses the characteristic of mice that they like to explore new objects to test their learning and memory abilities. The first day of the experiment is the adaptation day, and the mice are placed in a 40×40×40 cm cube open field to adapt for 10 minutes. The next day is the test day, and two cubes are placed in a fixed area to allow the mice to get familiar with them for 10 minutes. After 1 hour, one of the cubes is replaced with a sphere, and the exploration time of the mice on the new object is observed. The learning and memory abilities of the mice are evaluated based on the novel object exploration index (new object head exploration time / new object head exploration time + old object head exploration time).

[0047] Figure 3 The results showed that compared with the MCAO mice in the control group, the mice in the bilirubin-treated group explored the new objects for a longer time, indicating that the learning and memory abilities of mice with ischemic cerebrovascular disease were improved after administration of bilirubin.

[0048] The above results show that the administration of bilirubin in ischemic cerebrovascular disease can effectively improve the cognitive dysfunction caused by it.

[0049] Example 4 Bilirubin reduces the severity of brain damage in ischemic cerebrovascular disease

[0050] (1) Bilirubin can improve the severity of demyelination in MCAO mice

[0051] Demyelination of the central nervous system is one of the important pathological characteristics of ischemic cerebrovascular disease. Mouse brain tissue was obtained and sliced ​​for Luxol Fast Blue (LFB) staining. LFB staining is a staining method that shows the morphological structure and pathological changes of nerve myelin sheaths. It can reflect the severity of brain white matter damage by evaluating the area of ​​corpus callosum demyelination.

[0052] Specifically, the frozen sections were warmed to room temperature and then washed with phosphate buffer, tap water and ultrapure water for 5 minutes each, followed by gradient dehydration in 75%-95%-100% alcohol, and stained in 0.1% LFB dye at 60°C for 6-8 hours. After staining, the sections were taken out and placed at room temperature, and then repeatedly separated under a microscope using 0.05% lithium carbonate differentiation solution and 75% alcohol until the myelin sheath was stained blue and the background was almost colorless. At this point, intact myelinated fibers were stained blue, while demyelinated fibers were colorless. According to previous research methods, the percentage of the corpus callosum demyelination area to the total white matter area was calculated.

[0053] The results show that Figure 4 ), the mice in the control group had severe white matter damage 28 days after MCAO modeling. After oral administration of bilirubin, the degree of demyelination in the mice was significantly alleviated, which indicates that oral bilirubin can significantly improve the severity of demyelination caused by ischemic cerebrovascular disease, thereby exerting its protective effect on white matter structure.

[0054] (2) Bilirubin can reduce the infarct size in MCAO mice

[0055] MAP2 (microtubule-associated protein 2), as a cytoskeletal protein, is mainly expressed in neurons and performs many functions related to nerve growth and development, such as stabilizing microtubules, regulating the transport of organelles in axons and dendrites, and anchoring regulatory proteins in the signal transduction process. Therefore, it plays an important role in neuronal growth, synaptic plasticity, and neuronal apoptosis. By counting the expression of MAP2 protein in neurons, we can understand the damage of neurons and then reflect the severity of cerebral infarction in mice. Specifically, frozen sections of brain tissue of MCAO model mice were taken for immunofluorescence staining, and the area of ​​MAP2 expression loss in brain tissue was evaluated to reflect the situation of cerebral infarction in ischemic cerebrovascular disease.

[0056] Figure 5 The results showed that after MCAO modeling, obvious infarction areas were visible in the mouse brain slices, and the infarction area was reduced after oral administration of bilirubin. This indicates that bilirubin can reduce the infarction area of ​​mice with ischemic cerebrovascular disease and promote nerve repair.

[0057] In conclusion, oral administration of bilirubin can effectively reduce the severity of brain damage after ischemic cerebrovascular disease in mice and promote their neural repair.

[0058] Example 5 Bilirubin relieves inflammation in the blood and brain tissue caused by ischemic cerebrovascular disease

[0059] As an important component of lymphocytes, B cells have immune regulatory functions and participate in regulating macrophages, dendritic cells, natural killer cells, etc. by producing cytokines. In addition, B cells can also differentiate into plasma cells under antigen stimulation and Th cell assistance, produce high-affinity antibodies, and exercise humoral immune functions. After the initial immunization, some high-affinity cells differentiate into memory B cells and continue to participate in the immune process in the body. Previous studies have shown that B cells play an important role in ischemic cerebrovascular disease and can cause immune abnormalities.

[0060] (1) Bilirubin can alleviate inflammation in peripheral blood caused by ischemic cerebrovascular disease

[0061] Flow cytometry is a high-throughput, rapid, and accurate technique that can be used for immunophenotyping, signal transduction analysis, and interpretation of cell population heterogeneity. Specifically, at 30 days after MCAO surgery in the bilirubin gavage group and the control group, the mice were anesthetized, peripheral blood was collected, peripheral blood immune cell markers were selected for flow cytometry analysis, and corresponding antibody staining was performed. After incubation at 4°C for 30 minutes, red blood cells were lysed, and absolute counting microspheres were finally added. The resulting cell suspension was filtered through a 40um filter into the flow tube, ready for loading onto the machine to analyze changes in immune cells in mouse peripheral blood.

[0062] like Figure 6 AB results showed that compared with the control group, the B cells in the peripheral blood of mice in the bilirubin gavage group were significantly reduced (p < 0.0001), while there was no significant difference in CD8+T cells, neutrophils, monocytes, macrophages, NK cells and NKT cells; the absolute count of B cells in the peripheral blood showed that compared with the control group, the B cell count in the peripheral blood of mice in the bilirubin gavage group was significantly reduced (p < 0.001). This shows that bilirubin can effectively reduce the aggregation of B cells in the peripheral blood of mice after MCAO, thereby reducing the inflammatory response in the peripheral blood.

[0063] At the same time, Ficoll lymphocyte separation medium was used to separate mouse peripheral blood mononuclear cells (PBMC). The CD45-positive immune cells were flow-sorted and single-cell sequencing was performed. Single-cell clustering analysis was also performed, and a UMAP map was drawn for the immune cell composition of mouse peripheral blood, and the single-cell transcriptome characteristics of mouse B cells were obtained by clustering. By comparing the control group and the bilirubin gavage group, the results Figure 6 C shows that the proportion of B cells in the peripheral blood of mice in the bilirubin gavage group was significantly reduced. This further indicates that bilirubin can effectively reduce B cells in the peripheral blood of mice after MCAO and alleviate inflammatory responses.

[0064] (2) Bilirubin relieves inflammation in brain tissue caused by ischemic cerebrovascular disease

[0065] Specifically, frozen sections of the mouse brains of the bilirubin gavage group and the control group at 30 days after MCAO surgery were taken, and immunofluorescence staining was performed using B220 (B cell marker) to detect the number of B cells in the brain tissue. Specifically, the frozen sections were rewarmed at room temperature and washed with phosphate buffer for 5 minutes. Then, the membrane was broken at room temperature for 15 minutes with Triton X-100 immunofluorescence permeabilization solution. Subsequently, the specimen was blocked at room temperature for 15 minutes with immunofluorescence rapid blocking solution. After the antibody was diluted and mixed with the primary antibody diluent, 10 μL of diluent was added to each sample, incubated at 4°C for 12 hours, and then incubated with the secondary antibody for one hour in the dark at room temperature. Finally, the density of B220-positive B cells in the infarct was observed and counted using a fluorescence microscope.

[0066] result Figure 7 A shows that B cells accumulate in large numbers in the brain infarction foci of mice in the MCAO control group, indicating that B cells enter the brain tissue infarction foci and accumulate after ischemic injury, and participate in the pathogenesis of ischemic cerebrovascular disease. Compared with the mice in the control group, the density of B cells in the brain infarction foci of mice in the bilirubin gavage group was greatly reduced, indicating that bilirubin can effectively reduce the infiltration of B cells in the brain of mice after MCAO. At the same time, the brains of mice in the bilirubin gavage group and the control group at 30 days after MCAO surgery were taken, the lesion side tissues were separated under a microscope, and then prepared into single cell suspensions and subjected to single cell analysis. The results showed that compared with the control group, the infiltration of B cells in the brain tissue of mice in the bilirubin gavage group was reduced ( Figure 7 B). This suggests that bilirubin can effectively reduce the infiltration of B cells in the brain of mice after MCAO, thereby alleviating the inflammatory response in the brain tissue.

[0067] Example 6 Bilirubin improves B cell activation and mediated immune response in ischemic cerebrovascular disease

[0068] GO pathway enrichment analysis of mouse peripheral blood B cells indicated that after bilirubin administration, B cell activation, immune response activation, and oxidative stress response decreased, while the regulation of oxidative phosphorylation and the ability to regulate metabolism and energy homeostasis increased ( Figure 8 ). At the same time, GO pathway enrichment analysis of B cells in brain tissue also suggests that after bilirubin administration, B cell function changes and tends to be more stable in metabolism ( Fig. 9 ). This suggests that bilirubin can effectively improve the activation of B cells after MCAO, making them more stable, thereby alleviating the immune response.

[0069] Example 7 Bilirubin relieves oxidative stress of B cells in ischemic cerebrovascular disease

[0070] MitoSOX is a commonly used cell fluorescent probe and a living cell permeable dye. It can be used to detect changes in intracellular reactive oxygen species (ROS) levels. The MitoSpy probe fluorescently labels mitochondria by specifically targeting the membrane and can be used as an indicator of cell health. Reactive oxygen species (ROS) refers to the general term for oxygen-containing free radicals and peroxides that are easily formed and are related to oxygen metabolism in the body, including superoxide radical anions (O2·-), hydrogen peroxide (H2O2), hydroxyl radicals (OH·), etc., which can reflect the degree of oxidative stress.

[0071] Specifically, at 30 days after MCAO surgery in the bilirubin gavage group and the control group, the mice were anesthetized by inhalation of isoflurane. Peripheral blood was collected from the mice, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll lymphocyte separation medium. The PBMCs were blocked with Fc-Block and labeled with CD19-APC flow cytometry antibodies, incubated at 4°C for 30 minutes, and the resulting cell suspension was filtered through a 40um filter into a flow tube, and flow cytometry was used to detect the fluorescence intensity of MitoSOX in B cells in each group.

[0072] like Fig.10 The results showed that the mean fluorescence intensity of MitoSOX in the bilirubin gavage group was significantly reduced compared with that in the control group, suggesting that oral bilirubin can help alleviate mitochondrial damage of B cells in ischemic cerebrovascular disease.

[0073] Specifically, peripheral blood was collected from normal subjects, and peripheral blood mononuclear cells (PBMC) were separated using Ficoll lymphocyte separation medium. The PBMCs were further sorted using magnetic beads to obtain primary human B cells and then cultured. Next, DMSO was used to dissolve chemically synthesized bilirubin, and primary human B cells were treated in vitro at a concentration of 20 nM for 24 hours. Then, flow cytometry was used to detect the fluorescence intensity of MitoSOX, MitoSpy and ROS in each group of B cells. Fig.11The results showed that the mean fluorescence intensity of MitoSOX, MitoSpy and ROS in B cells in the bilirubin gavage group was significantly reduced compared with the control group. This indicates that bilirubin helps to alleviate mitochondrial damage and oxidative stress of B cells in ischemic cerebrovascular disease, thereby helping to alleviate neuroinflammation.

[0074] Example 8 Bilirubin alleviates microglial activation caused by ischemic cerebrovascular disease

[0075] Microglia, as innate immune cells of the central nervous system, play an important role in immune surveillance, host defense and brain tissue repair. Studies have shown that in the core and surrounding areas of ischemic cerebrovascular disease lesions, activated microglia release proinflammatory factors and chemokines, leading to an inflammatory microenvironment, causing cell damage, immune cell infiltration, aggravating the permeability of the blood-brain barrier, and inhibiting myelin regeneration and repair.

[0076] The specific manifestations of microglial activation are increased density, firmness, and roundness. Fig.12 As shown in the figure, the aggregation of microglia in the infarct edge area of ​​MCAO mice in the bilirubin gavage group was reduced, the cell area increased, and the firmness and roundness decreased significantly, indicating that microglial activation was significantly reduced after bilirubin gavage. This shows that in the context of ischemic cerebrovascular disease damage, oral bilirubin can effectively reduce the activation of microglia, significantly inhibit the inflammatory response of microglia, and improve inflammatory damage to the nervous system.

[0077] Example 9 Bilirubin relieves inflammatory response caused by ischemic cerebrovascular disease

[0078] (1) Bilirubin reduces the expression of pro-inflammatory markers in the infarct area of ​​ischemic cerebrovascular disease

[0079] Studies have shown that microglia can regulate neuroinflammation in ischemic cerebrovascular disease. With the activation of microglia, the expression levels of pro-inflammatory markers such as microglial CD16 / 32 and iNOS will be enhanced. Frozen sections of brain tissue of MCAO model mice were taken for immunofluorescence staining. The ratio of microglial inflammation-related markers and microglial markers (Iba1) double positive in the corpus callosum was evaluated to reflect the level of neuroinflammation in ischemic cerebrovascular disease.

[0080] like Fig.13 As shown in the figure, it can be observed that the proportion of pro-inflammatory markers iNOS, CD16 / 32 and Iba1 double positive cells in the infarct area of ​​mice treated with bilirubin decreased. At the same time, the GSEA gene set enrichment analysis of microglia in brain tissue also suggested that after bilirubin administration, the genes of microglia showed significant enrichment of down-regulation of neuroinflammatory response and immune response pathways ( Fig.14). This suggests that bilirubin can significantly inhibit microglia-mediated inflammatory responses, thereby improving inflammatory damage to the nervous system.

[0081] (2) In vitro B cell co-culture, the expression of pro-inflammatory markers of HMC3 was reduced, while the expression of anti-inflammatory and homeostatic markers was increased

[0082] Specifically, after obtaining human primary B cells cultured to a good state as described above, DMSO was used to dissolve chemically synthesized bilirubin, and human primary B cells were pretreated in vitro at a concentration of 20 nM for 24 hours. At the same time, HMC3 cells (humanized microglial cell line) were added with phosphate solution PBS in vitro and cultured for 4 hours in a three-gas incubator at 37°C, 0.3% O2, and 5% CO2. Subsequently, the sugar-containing DMEM culture medium was replaced and placed in a 37°C, 74% N2, 21% O2, 5% CO2 cell culture incubator for reoxygenation culture for 24 hours, and co-cultured with control group B cells and bilirubin-treated group B cells. RNA of microglia was extracted, and qPCR was used to detect the expression of inflammation-related markers and homeostatic markers in microglia to reflect the level of neuroinflammation in ischemic cerebrovascular disease.

[0083] like Fig.15 As shown, compared with microglia co-cultured with B cells in the control group, the expression of pro-inflammatory markers (TNFα, IL-1β) in microglia co-cultured with B cells treated with bilirubin was reduced, while the expression of anti-inflammatory markers (TGFβ, Arg-1) and homeostatic markers (P2ry12, Hexb) was increased. This indicates that bilirubin can significantly inhibit the microglia-mediated neuroinflammatory response in ischemic cerebrovascular disease, thereby improving inflammatory damage to the nervous system.

[0084] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. The application of bilirubin in the preparation of drugs for the treatment of ischemic cerebrovascular disease.

2. The application according to claim 1, characterized in that: The ischemic cerebrovascular disease includes transient ischemic attack, different types of acute ischemic stroke, cerebral artery steal syndrome and chronic cerebral ischemia.

3. The application according to claim 1, characterized in that: The bilirubin can improve cognitive dysfunction caused by ischemic cerebrovascular disease.

4. The application according to claim 1, characterized in that: The bilirubin can improve the severity of demyelination caused by ischemic cerebrovascular disease, reduce the area of ​​cerebral infarction, and alleviate the severity of neurological deficits caused by ischemic cerebrovascular disease.

5. The application according to claim 1, characterized in that: The bilirubin can alleviate inflammation in the blood and brain tissue caused by ischemic cerebrovascular disease.

6. The use according to claim 1, characterized in that: The bilirubin can improve the activation of B cells and the mediated immune response in ischemic cerebrovascular disease.

7. The use according to claim 1, characterized in that: The bilirubin can alleviate the oxidative stress of B cells in ischemic cerebrovascular disease.

8. The use according to claim 1, characterized in that: The bilirubin can alleviate the microglial activation caused by ischemic cerebrovascular disease.

9. The use according to claim 1, characterized in that: The bilirubin can alleviate the neuroinflammatory response caused by ischemic cerebrovascular disease.

10. A drug for treating ischemic cerebrovascular disease, characterized in that: The composition is composed of bilirubin at an effective dose, and can be a single component or a composition containing an effective dose of bilirubin, wherein the composition can include a pharmaceutically acceptable carrier.

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