Application of bilirubin in preparation of medicine for treating vascular cognitive impairment

Abstract

The invention belongs to the field of medicines, and discloses application of bilirubin in preparation of a medicine for treating vascular cognitive impairment. Bilirubin can improve the working, learning and memory ability after cerebrovascular diseases; the severity of myelination after cerebrovascular diseases is improved; the inflammatory response in blood caused by cerebrovascular diseases is relieved, the activation of B cells and the mediated immune response are inhibited, and the peripheral inflammation is inhibited; the oxidative stress of B cells is improved; mitigating activation of microglial cells; the inflammatory response of microglial cells is obviously inhibited, and the inflammatory injury of a nervous system is improved; 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 a drug for treating vascular cognitive dysfunction. Background Art

[0002] Vascular dementia (VaD) refers to dementia mainly caused by cerebrovascular disease or impaired cerebral blood flow, and belongs to the category of vascular cognitive impairment (VCI). Vascular cognitive impairment includes all cognitive impairments with cerebrovascular disease or impaired cerebral blood flow, mainly manifested by demyelinating white matter damage caused by chronic hypoperfusion. With the aggravation of aging, the prevalence of senile dementia in my country has increased significantly. Epidemiological studies have shown that the prevalence of vascular cognitive dysfunction in the elderly over 65 years old in my country is 1.1% to 3.0%, and the annual incidence rate is 5 to 9 / 1000 people. Among the elderly population over 65 years old, the overall prevalence of mild cognitive impairment is 20.8%, of which 42.0% is caused by cerebrovascular disease and vascular risk factors; and among all dementia cases, vascular cognitive dysfunction accounts for about 20%, which is the most common type of dementia except Alzheimer's disease, and has caused serious social and medical burdens. With the gradual acceleration of the aging process of my country's population and the increase in the number of patients with cerebrovascular diseases, the incidence of vascular cognitive dysfunction in the middle-aged and elderly population is also gradually increasing, which has brought a heavy burden to families and society. In addition, due to the complexity of the injury process, there is currently no effective clinical treatment plan. The treatment of vascular cognitive dysfunction is a new area that urgently needs to be studied.

[0003] Studies have reported that neuroinflammation plays an important role in the pathogenesis of vascular cognitive impairment. The brain's response to vascular injury involves an acute and long-term inflammatory process, characterized by rapid activation of microglia, production of proinflammatory cytokines, and entry of various types of leukocytes (including lymphocytes, neutrophils, and monocytes) into ischemic brain tissue, which together lead to vascular cognitive dysfunction. Neuroinflammation can also lead to damage to oligodendrocytes, which in turn causes damage to myelin and lesions in white matter, ultimately exacerbating cognitive dysfunction. By regulating neuroinflammatory responses, it is expected that effective therapeutic strategies will be developed to improve cognitive function and quality of life in patients with VCI.

[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 suggested that bilirubin is associated with lipid and lipoprotein oxidation and endothelial damage, which in turn promotes vascular atherosclerosis and thrombosis. However, in recent years, more and more studies have shown that bilirubin is also a powerful intrinsic antioxidant, and bilirubin in serum has an antioxidant effect. However, the relationship between bilirubin and vascular cognitive dysfunction has not been studied.

[0006] Therefore, exploring the application of bilirubin in vascular cognitive dysfunction is crucial for alleviating cognitive dysfunction caused by white matter damage and alleviating neuroinflammation. Summary of the invention

[0007] The invention discloses the application of bilirubin in preparing a medicine for treating vascular cognitive dysfunction, 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 vascular cognitive dysfunction.

[0010] In the above technical solution, the bilirubin can improve the working and learning memory abilities after cerebrovascular disease.

[0011] In the above technical solution, the bilirubin can reduce the severity of demyelinating damage after cerebrovascular disease.

[0012] In the above technical solution, the bilirubin can alleviate the inflammatory response caused by cerebrovascular disease.

[0013] In the above technical solution, the bilirubin can improve the activation of B cells and the mediated immune response in cerebrovascular disease and inhibit peripheral inflammation.

[0014] In the above technical solution, the bilirubin can alleviate the oxidative stress of B cells in cerebrovascular disease.

[0015] In the above technical solution, the bilirubin can alleviate the microglial activation caused by cerebrovascular disease.

[0016] In the above technical solution, the bilirubin can alleviate the neuroinflammatory response caused by cerebrovascular disease.

[0017] In a second aspect, the present invention provides a drug for treating vascular cognitive dysfunction, 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.

[0018] 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 vascular cognitive dysfunction. Bilirubin can improve working and learning memory ability after cerebrovascular disease; improve the severity of demyelination after cerebrovascular disease; relieve inflammatory response in the blood caused by cerebrovascular disease, inhibit B cell activation and mediated immune response, inhibit peripheral inflammation; improve B cell oxidative stress; reduce microglia activation; significantly inhibit microglia inflammatory response, improve nervous system inflammatory damage; and have good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a drug safety map for mice;

[0020] Figure 2 This is a statistical chart showing changes in working memory and reference memory in mice;

[0021] Figure 3 This is a statistical chart showing changes in the learning and memory abilities of mice;

[0022] Figure 4 This is a statistical graph of the degree of white matter damage in mice;

[0023] Figure 5 This is a statistical diagram of the number of B cells in the peripheral blood of mice;

[0024] Figure 6 This is the GO enrichment analysis diagram of B cells in mouse peripheral blood;

[0025] Figure 7 It is a statistical graph of the fluorescence intensity of MitoSOX, MitoSpy and ROS in human B cells cultured in vitro treated with bilirubin;

[0026] Figure 8 This is a statistical diagram of the activation degree of microglia in mouse brain tissue;

[0027] Fig. 9 This is a statistical chart of the proportion of cells positive for CD16 / 32, iNOS and Iba1 in mouse brain tissue;

[0028] Fig.10 This is the GSEA analysis of microglia in mouse brain tissue;

[0029] Fig.11 This 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

[0030] 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.

[0031] Vascular cognitive dysfunction mouse model: The present invention uses bilateral carotid artery stenosis surgery to establish a vascular cognitive dysfunction 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 bilateral common carotid arteries are exposed. A micro spring with an inner diameter of 0.18 mm, a pitch of 0.50 mm, and a total length of 2.5 mm is wrapped around the bilateral common carotid arteries of the mouse, the incision is closed, the skin is sutured, and the cerebral blood flow is monitored to decrease by 40%, representing the successful establishment of the BCAS mouse model. Day 30 is selected as the observation time point for cognitive dysfunction.

[0032] 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, mice after BCAS 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.

[0033] Example 1 Bilirubin has drug safety

[0034] Mice after BCAS 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 in a 4°C centrifuge for 15 minutes. 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).

[0035] Figure 1 The 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<0.01).

[0036] Example 2 Bilirubin improves working and learning memory abilities after cerebrovascular disease

[0037] (1) Bilirubin can improve the working memory ability of mice after cerebrovascular disease

[0038] The eight-arm maze consists of eight identical arms arranged radially at equal angles and a common platform in the middle. It is a behavioral device used to detect the cognitive function of mice. It is usually used to evaluate changes in cognitive function of mice under the influence of drugs or brain damage.

[0039] Specifically, mice were fasted and deprived of water for 6-8h before the experiment to induce their foraging behavior in the experiment. Food was placed in the eight arms of the maze, thereby driving them to explore the maze. After each arm was numbered and food was put in, the mice were placed in the middle of the platform, timing was started, and the number of each entry into the arm was recorded. The experiment ended after each arm had been entered. After 7 days of training, the record of the first eight times the mice entered the arm and the number of times the mice repeatedly visited the arm during the entire experiment were all working memory errors. After three days, only bait was placed in the fixed arms 2, 4, 6, and 8, and mice were put in. The number of repeated visits of the mice during the process of eating the food in the four arms was recorded, and repeated three times a day for a total of 9 times, which was recorded as reference memory errors.

[0040] Figure 2 AB showed that the mice in the bilirubin gavage group had significantly lower revisit errors than the control group, and were more inclined to choose food from different arms in the first eight times, indicating that the working memory of BCAS mice gavaged with bilirubin was significantly improved. At this time, there was no statistical significance in the reference memory test results among the three groups of mice ( Figure 2 C), which indicates that chronic cerebrovascular hypoperfusion in mice mainly impaired the working memory of mice, and there was no significant change in reference memory. The above results indicate that bilirubin can improve the working memory ability of mice after cerebrovascular disease.

[0041] (2) Bilirubin can improve the learning and memory ability of mice after cerebrovascular disease

[0042] 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).

[0043] Figure 3The results showed that compared with the BCAS mice in the control group, the mice in the bilirubin gavage group spent longer exploring new objects, indicating that in the context of cerebrovascular disease, the learning and memory abilities of mice were improved after administration of bilirubin.

[0044] The above results show that oral bilirubin can effectively improve cognitive dysfunction after cerebrovascular disease.

[0045] Example 3 Bilirubin reduces the severity of demyelinating damage after cerebrovascular disease

[0046] Vascular cognitive dysfunction is a vascular demyelinating disease caused by chronic ischemia and hypoxia, and its main pathological change is the loss of myelin in the central nervous system. 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 white matter damage by evaluating the degree of myelin loss in the corpus callosum.

[0047] Specifically, the frozen sections were rewarmed at room temperature and washed with phosphate buffer, tap water and ultrapure water for 5 minutes each, then dehydrated in 75% to 95% to 100% alcohol in a gradient manner, and placed in 0.1% LFB dye for staining at 60°C for 6 to 8 hours. After staining, the sections were taken out and placed at room temperature, and then the sections were repeatedly color-separated under a microscope using 0.05% lithium carbonate differentiation solution and 75% alcohol until the myelin sheath was dyed blue and the background was almost colorless. At this time, the intact myelinated fibers were dyed blue, while the lost myelinated fibers were colorless. According to previous studies, the degree of white matter damage caused by ischemia is divided into 4 levels, namely normal (0 points), myelin fiber disorder (1 point), obvious vacuoles (2 points), and myelin fiber disappearance (3 points).

[0048] The results show that Figure 4 ), the control group mice had severe white matter damage 30 days after BCAS modeling, and the degree of myelin damage in mice was significantly alleviated after oral administration of bilirubin, which indicates that the application of bilirubin can significantly reduce the severity of demyelination after cerebrovascular disease, thereby playing a protective role in brain white matter structure.

[0049] Example 4 Bilirubin relieves inflammatory response in the blood caused by cerebrovascular disease.

[0050] 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 body's immune process. Previous studies have shown that B cells play an important role in cerebrovascular disease and can cause immune abnormalities.

[0051] 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 BCAS surgery in the bilirubin gavage group and the control group, mice were anesthetized by inhalation of isoflurane. Peripheral blood was collected from mice, and peripheral blood immune cell markers were selected for flow cytometry analysis. Corresponding antibody staining was performed, and red blood cells were lysed after incubation at 4°C for 30 minutes. The resulting cell suspension was filtered through a 40um filter into a flow tube, ready for loading onto the machine to analyze changes in immune cells in mouse peripheral blood.

[0052] like Figure 5 The 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 < 0.01), while there was no significant difference in CD8+T cells, neutrophils, monocytes, macrophages, NK cells and NKT cells. This shows that bilirubin can effectively reduce the aggregation of B cells in the peripheral blood of mice after BCAS, thereby alleviating the inflammatory response in the peripheral blood.

[0053] Example 5 Bilirubin improves B cell activation and mediated immune response in cerebrovascular disease and inhibits peripheral inflammation

[0054] At the same time, Ficoll lymphocyte separation fluid was used to separate mouse peripheral blood mononuclear cells (PBMC). For the PBMCs obtained, 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. 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 ability to regulate oxidative phosphorylation and metabolism and energy homeostasis increased ( Figure 6 ). This suggests that bilirubin can effectively improve the activation of B cells after cerebrovascular disease, making them more stable, thereby alleviating the immune response and inhibiting peripheral inflammation.

[0055] Example 6 Bilirubin relieves oxidative stress of B cells in cerebrovascular disease

[0056] 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.

[0057] 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 separated using magnetic beads to obtain primary human B cells and then cultured. Chemically synthesized bilirubin was dissolved in DMSO, and human primary 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. Figure 7 The 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 oral bilirubin can help alleviate mitochondrial damage and oxidative stress of B cells in cerebrovascular disease, thereby helping to alleviate neuroinflammation.

[0058] Example 7 Bilirubin alleviates microglial activation caused by cerebrovascular disease

[0059] 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 areas of chronic hypoperfusion lesions, activated microglia release proinflammatory factors and chemokines, leading to an inflammatory microenvironment, causing cell damage, immune cell infiltration, increased blood-brain barrier permeability, and inhibition of myelin regeneration and repair. Cerebrovascular disease can also cause microglia activation. The specific manifestations of microglial activation are increased density, cell body area, firmness, and roundness.

[0060] like Figure 8 As shown in the figure, compared with the control group mice, the aggregation of microglia in the white matter region of BCAS mice in the bilirubin group was reduced, the cell body area was reduced, and the firmness and roundness were also significantly reduced, indicating that microglial activation was significantly reduced after bilirubin administration. This shows that bilirubin can effectively reduce microglial activation after cerebrovascular disease, thereby alleviating neuroinflammation.

[0061] Example 8 Bilirubin relieves neuroinflammatory response caused by cerebrovascular disease

[0062] (1) Bilirubin reduces the expression of pro-inflammatory markers in brain tissue after cerebral hemorrhage

[0063] Studies have shown that during the activation of microglia, the expression levels of inflammatory markers such as CD16 / 32 and iNOS are enhanced. The brain tissue of BCAS model mice was obtained, sliced, and immunofluorescence staining was performed to evaluate the ratio of microglial inflammation-related markers and microglial markers (Iba1) in the corpus callosum to reflect the level of neuroinflammation after cerebrovascular disease.

[0064] like Fig. 9 As shown, it can be observed that the proportion of CD16 / 32 and iNOS double-positive cells and Iba1 in the white matter area of ​​mice treated with bilirubin was reduced.

[0065] At the same time, single-cell transcriptome sequencing was performed on cells in the white matter of brain tissue, and the transcriptome characteristics of microglia in brain tissue were identified through UMAP cluster analysis. The GSEA gene set enrichment analysis of microglia also suggested that after bilirubin administration, microglia genes showed significant enrichment for chemotaxis and cytokine-mediated neuroinflammatory response downregulation ( Fig.10 ). This suggests that bilirubin can significantly inhibit microglia-mediated inflammatory responses, thereby improving inflammatory damage to the nervous system.

[0066] (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

[0067] 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. Then, the sugar-containing DMEM 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 after cerebrovascular disease.

[0068] like Fig.11 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 after cerebrovascular disease, thereby improving inflammatory damage to the nervous system.

[0069] 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. Application of bilirubin in the preparation of drugs for the treatment of vascular cognitive dysfunction.

2. The application according to claim 1, characterized in that: The bilirubin can improve working and learning memory abilities after cerebrovascular disease.

3. The application according to claim 1, characterized in that: The bilirubin can reduce the severity of demyelinating damage after cerebrovascular disease.

4. The application according to claim 1, characterized in that: The bilirubin can alleviate the inflammatory response in the blood caused by cerebrovascular disease.

5. The application according to claim 1, characterized in that: The bilirubin can improve the activation of B cells and the mediated immune response in cerebrovascular disease and inhibit peripheral inflammation.

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

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

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

9. A drug for treating vascular cognitive dysfunction, 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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