Application of MIF inhibitor in preparation of drugs for regulating and controlling central B lymphocytes and neuroinflammation

Abstract

The invention discloses an application of an MIF inhibitor in preparation of drugs for regulating and controlling central B lymphocytes and neuroinflammation, the MIF inhibitor reduces generation of proinflammatory cytokines by inhibiting oxidative stress, differentiation and chemotactic effects of B lymphocytes and mediated immune response of B lymphocytes, so that neuroinflammation is relieved, and vascular cognitive dysfunction is improved. The invention provides a new strategy and potential medicine for the treatment of the vascular cognitive impairment.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of a MIF inhibitor in the preparation of drugs for regulating central B lymphocytes and neuroinflammation. Background Art

[0002] Vascular dementia (VaD) refers to dementia mainly caused by cerebrovascular disease or impaired cerebral blood flow. It belongs to the category of vascular cognitive impairment (VCI), which includes all cognitive impairments caused by cerebrovascular disease or impaired cerebral blood flow. It mainly manifests as demyelinating white matter damage caused by chronic hypoperfusion. 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. There is currently no effective clinical treatment plan, and the treatment of vascular cognitive dysfunction is a new field that needs to be studied urgently.

[0003] Studies have reported that neuroinflammation plays an important role in the pathogenesis of vascular cognitive dysfunction. The brain's response to vascular injury includes an acute and long-term inflammatory process, characterized by the rapid activation of microglia, the production of proinflammatory cytokines, and the entry of various types of white blood cells (including lymphocytes, neutrophils, and monocytes) into ischemic brain tissue. These mechanisms together lead to vascular cognitive dysfunction. As an important component of lymphocytes, B lymphocytes have immunoregulatory functions and participate in regulating the functions of macrophages, dendritic cells, natural killer cells and other cells by producing cytokines. In addition, B lymphocytes can also differentiate into plasma cells under antigen stimulation and with the assistance of Th cells, produce high-affinity antibodies, and exercise humoral immune functions. After the initial immune response, some high-affinity cells differentiate into memory B cells and continue to participate in the immune process in the body.

[0004] Macrophage migration inhibitory factor (MIF), as a cytokine secreted by T cells, participates in innate and adaptive immune responses. In addition to immune cells, MIF is secreted by a variety of non-immune cells, including hematopoietic cells, endothelial cells, and neurons. It is worth noting that MIF is involved in regulating the functions of various components of the nervous system, such as neurons, glial cells, and endothelial cells. It induces neuroinflammation through chemokine-like effects and causes cell death through nuclease activity. However, its role in vascular cognitive dysfunction and its effects and mechanisms on B lymphocytes remain to be explored.

[0005] Therefore, there is an urgent need to develop an inhibitor that can effectively regulate B lymphocytes as a potential therapeutic drug to improve neuroinflammation mediated by vascular cognitive impairment. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide an application of an MIF inhibitor in the preparation of a drug for regulating central B lymphocytes and neuroinflammation. The MIF inhibitor of the present invention can inhibit the activity of MIF, effectively reduce the activation of central B lymphocytes and neuroinflammatory responses, and thus treat or prevent related nervous system diseases.

[0007] To achieve the above object, the technical solution designed by the present invention is as follows:

[0008] The present invention provides an application of an MIF inhibitor in the preparation of a drug for regulating central B lymphocytes.

[0009] The present invention also provides an application of an MIF inhibitor in the preparation of a drug for treating or improving neuroinflammation-related diseases.

[0010] Further, the neuroinflammation-related diseases are vascular cognitive impairment, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, autoimmune encephalitis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, etc.

[0011] The present invention also provides an application of an MIF inhibitor in the preparation of a drug for improving vascular cognitive impairment.

[0012] Further, the MIF inhibitor regulates the function of B lymphocytes or reduces neuroinflammation to improve vascular cognitive impairment.

[0013] The above MIF inhibitor reduces neuroinflammation by inhibiting oxidative stress, differentiation, chemotaxis of B lymphocytes and the immune response mediated by them.

[0014] Still further, the MIF inhibitor is ISO-1.

[0015] The present invention also provides a drug for improving vascular cognitive impairment, and the drug contains an MIF inhibitor.

[0016] Further, the MIF inhibitor is ISO-1.

[0017] Further, the drug further includes excipients, and the excipients are DMSO, PEG300, Tween-80 and physiological saline.

[0018] Furthermore, the drug is an injection or other dosage form.

[0019] Advantages of the present invention:

[0020] The MIF inhibitor of the present invention reduces the production of pro-inflammatory cytokines by inhibiting the oxidative stress, differentiation, chemotaxis and immune responses mediated by B lymphocytes, thereby reducing neuroinflammation and improving vascular cognitive impairment. The present invention provides a new strategy and potential drugs for the treatment of vascular cognitive impairment. Description of the Drawings

[0021] Figure 1 It is a statistical chart of ROS and GSH / GSSG ratio;

[0022] Figure 2 It is a statistical chart of IgD-IgM-B cells, IgD-IgM+B cells, IgD+IgM-B, CD138+B cells;

[0023] Figure 3 A is a B cell GO enrichment analysis chart of MIF group and Vehicle group;

[0024] Figure 3 B is a B cell GO enrichment analysis chart of cytokines+MIF group and cytokines+Vehicle group;

[0025] Figure 4 It is a statistical chart of B cell chemotaxis index;

[0026] Figure 5 It is a statistical chart of B cell number;

[0027] Figure 6 It is a statistical chart of the learning and memory ability of mice;

[0028] Figure 7 It is a statistical chart of the changes in working memory and reference memory of mice;

[0029] Figure 8 It is a statistical chart of the white matter injury degree score of mice. Detailed Embodiments

[0030] The present invention will be further described in detail below with reference to specific embodiments for the understanding of those skilled in the art.

[0031] Example 1 MIF exacerbates oxidative stress in B cells

[0032] Oxidative stress (Reactive Oxygen Species, ROS) ROS is generated from oxygen molecules during normal cellular processes or in response to harmful exogenous factors. ROS plays a central role in redox regulation and cell signaling, but excessive ROS production can trigger oxidative stress. ROS includes superoxide (O2·-), hydrogen peroxide (H2O2), and hydroxyl radical (OH·). The ratio of GSH (reduced glutathione) to GSSG (oxidized glutathione) is an important indicator of the cellular redox state. Under normal conditions, most of the glutathione (GSH) in cells exists in the reduced form, while the proportion of oxidized glutathione (GSSG) is relatively small. Changes in this ratio can reflect the level of oxidative stress within the cell. Specifically, the lower the ratio of GSH to GSSG, the higher the degree of oxidation of the cell.

[0033] In this example, mouse B cells were extracted in vitro and cultured. The specific steps are as follows:

[0034] Under sterile conditions, the spleens of 8-week-old normal mice were placed in a six-well plate containing an appropriate amount of cell culture medium. The spleen tissues were ground into a single cell suspension, and erythrocyte lysate was added to treat the cell suspension. After terminating the erythrocyte lysis, the supernatant was removed by centrifugation, leaving the leukocyte layer. The cells were washed with PBS to remove residual lysate and debris. The cell pellet was collected by centrifugation and then resuspended in an appropriate amount of culture medium. Mouse primary B cells were further obtained by magnetic bead sorting and then cultured. At the same time, cytokines were used to activate the B cells. Specifically, anti-CD40 (10 ug / ml), anti-IgM (10 ug / ml), and R848 (0.05 uM) were used to treat the B cells simultaneously and cultured until they were in good condition.

[0035] In this example, MIF protein (MedChemExpress, HY-P7388) was used to treat well-cultured mouse primary B cells and cytokine-activated B cells. Specifically, an in vitro intervention was performed at a concentration of 100 ng / ml for 24 h. Then, B cells from each group were collected, and the expression level of ROS in B cells was measured by flow cytometry. The supernatants of B cells from each group were collected, and a GSH and GSSG detection kit (purchased from Beyotime) was used to measure the contents of GSH and GSSG to reflect the level of oxidative stress in B cells under different treatment conditions.

[0036] The grouping is as follows:

[0037] Vehicle group: Control group B cells

[0038] MIF group: B cells intervened with MIF

[0039] cytokines + Vehicle group: Activated B cells

[0040] Cytokines + MIF group: B cells activated by MIF

[0041] Such as Figure 1 shown: Compared with the ROS expression level in activated B cells, after MIF intervened in activated B cells, the expression level of ROS increased significantly ( Figure 1 left figure). Moreover, compared with GSH / GSSG in activated B cells, after MIF intervened in activated B cells, GSH / GSSG decreased significantly ( Figure 1 right figure). It is worth noting that although there is no significant difference in the ROS expression level between the MIF group and the Vehicle group, the GSH / GSSG ratio is significantly lower in the MIF group than in the Vehicle group. This indicates that after MIF intervenes in B cells in vitro, the oxidative stress of B cells is enhanced, thus indicating that MIF can be used as a potential therapeutic target for regulating the oxidative stress of B cells.

[0042] Example 2 MIF promotes the differentiation of B cells towards maturity

[0043] The brain's response to vascular injury involves an acute and chronic inflammatory process. During the chronic inflammatory process, mature B cells, on the one hand, continuously produce autoantibodies to attack cerebrovascular endothelial cells or nerve tissues, resulting in vascular damage and blood-brain barrier disruption; on the other hand, by continuously secreting pro-inflammatory cytokines (such as IL-6, TNF-α, IL-1β), they exacerbate vascular inflammation and neuroinflammation, thus maintaining a chronic low-grade inflammatory state and promoting neuronal damage and cognitive decline.

[0044] Extract B cells and intervene as the above treatment method, and group them as follows:

[0045] Cytokines + Vehicle group: Activated B cells,

[0046] Cytokines + MIF group: B cells activated by MIF;

[0047] Then use IgD-BV780, IgM-BV421 and CD138-PE antibodies to label B cells in each group, use a flow cytometer to detect the labeled cells, and identify and count B cell subsets through different fluorescence signals. Among them, IgD is a marker of naive B cells, IgM is a marker of mature B cells, and CD138 is a marker of plasma cells.

[0048] Such as Figure 2As shown: Compared with the cytokines + Vehicle group, the IgD-IgM-B cells in the cytokines + MIF group increased, the IgD-IgM+B cells increased, the IgD+IgM-B cells decreased, and the CD138+B cells increased. This shows that: After MIF intervened in activated B cells, the number of mature B cells and plasma cells increased.

[0049] In summary, it can be seen that: After MIF intervened in B cells in vitro, it drove the differentiation and maturation of B cells. This shows that: MIF can be used as a potential therapeutic target for regulating B cell differentiation.

[0050] Example 3 MIF enhanced the immune response mediated by B cells

[0051] Extract B cells and perform intervention and grouping according to the above treatment method. Then, collect the RNA of the four groups of B cells for transcriptome analysis and draw a GO enrichment analysis map.

[0052] As Figure 3 Shown in A: When the MIF group was compared with the Vehicle group, it was found that after MIF intervened in B cells, the pathways related to adaptive immune response, the pathways promoting immune response, the pathways related to cytokine production, and the pathways related to inflammatory response in B cells were up-regulated. While the pathways related to lipid metabolism, the pathways maintaining cell homeostasis, and the pathways inhibiting signal transduction were all down-regulated.

[0053] Similarly, as Figure 3 Shown in B: When the cytokines + MIF group was compared with the cytokines + Vehicle group, it was found that after MIF intervened in activated B cells, the immune and inflammatory related pathways in B cells were up-regulated, while the metabolism related pathways were down-regulated.

[0054] In summary, it is shown that: After intervening in B cells with MIF, the regulation of the immune response by B cells after activation is enhanced, and at the same time, the metabolism and homeostasis of cells are affected, thereby enhancing the immune-inflammatory response mediated by B cells, suggesting that MIF can be used as a potential therapeutic target for the immune response mediated by B cells.

[0055] Example 4 MIF enhanced the chemotaxis of B cells

[0056] The chemotaxis of B cells refers to the process by which B cells migrate to specific tissues (such as cerebrovascular or brain parenchyma) under the guidance of chemokines. In vascular cognitive impairment, the blood-brain barrier is damaged, and the expression of chemokines (such as CXCL12, CXCL13) increases, attracting B cells to migrate from the periphery to the brain. Infiltrating B cells can differentiate into plasma cells, produce autoantibodies, or exacerbate the inflammatory response by secreting pro-inflammatory cytokines (such as IL-6, TNF-α), thereby leading to vascular damage and neurodegeneration.

[0057] Extract B cells using the above treatment method. In this example, mouse primary B cells cultured well were treated with MIF protein (extracted from the supernatant of microglia after OGD for 12 h). The specific steps are as follows:

[0058] Intervene in vitro at a concentration of 100 ng / ml for 24 h. The treatment method and grouping of cytokines are as described above. Then, select an appropriate chemokine and create a concentration gradient in the culture plate. Collect B cells from each group for chemotaxis experiments to measure the number of migrated cells and calculate the chemotaxis index.

[0059] As Figure 4 shown: Compared with the Vehicle group, the chemotaxis index of B cells in the MIF group was significantly increased; compared with the B cells in the cytokines + Vehicle group, the chemotaxis index of B cells in the cytokines + MIF group was significantly increased. It should be noted that there was no significant difference in the chemotaxis index of B cells between the Vehicle group and the cytokines + Vehicle group. This indicates that in vitro MIF intervention on B cells enhanced the chemotaxis of B cells, and this enhanced chemotaxis was independent of the functional state of B cells. Thus, it shows that MIF can be used as a potential therapeutic target for regulating the chemotactic function of B cells.

[0060] Example 5 MIF inhibitor alleviates the chemotaxis of meningeal B cells in vascular cognitive impairment

[0061] In this example, a mouse model of vascular cognitive impairment was established by bilateral common carotid artery stenosis (BCAS) surgery. The specific steps are as follows:

[0062] After anesthetizing the mice with isoflurane, cut the skin along the midline of the neck, dissect the thyroid gland, and expose the trachea and bilateral common carotid arteries of the mice. Wind a microspring with an inner diameter of 0.18 mm, a pitch of 0.50 mm, and a total length of 2.5 mm around the bilateral common carotid arteries of the mice, close the incision, suture the skin, and monitor a 40% decrease in cerebral blood flow, indicating successful establishment of the BCAS mouse model. Select 30 days as the observation time point for vascular cognitive impairment.

[0063] ISO-1 is a commonly used MIF inhibitor (MedChemExpress, HY-16692, inhibitor purity is 99.94%), and it is administered by intraperitoneal injection as follows:

[0064] BCAS + Vehicle group: Mice after BCAS surgery were intraperitoneally injected with an equal volume of PBS solution for 3 days.

[0065] BCAS + ISO-1 group: Mice after BCAS surgery were intraperitoneally injected with 35 mg / kg of ISO-1 solution for 3 consecutive days.

[0066] Flow cytometry is a high-throughput, rapid, and accurate technique that can be used for immunophenotyping analysis, signal transduction analysis, and interpretation of cell population heterogeneity. Specifically, the meningeal tissue was dissected from the mouse skull and placed in 1 ml of 1640 medium. 20 μl of 5% collagenase II and 10 μl of 10 mg / ml DNase were added, and the mixture was incubated in an incubator at 37°C for 45 min, with pipetting every 15 min until the tissue was completely dissociated. 1 ml of 1640 medium containing 20% serum was added to terminate the digestion. The mixture was centrifuged at 500 g for 5 min, the supernatant was discarded, 100 μl of 1:1000 Fvs700 was added, and the mixture was incubated at 4°C for 10 min. 1 ml of 2% FACS was added, and the mixture was centrifuged at 500 g for 5 min. The supernatant was discarded, 100 μl of 1:100 Fc block was added to block for 10 min, and then mouse meningeal B cells were labeled by CD19-APC and B220-FITC double-label flow cytometry and incubated at 4°C for 30 minutes. The obtained cell suspension was filtered through a 40-μm filter into a flow tube and prepared for analysis on the machine.

[0067] As Figure 5 Shown: Compared with the BCAS + Vehicle group, the number of meningeal B cells in the BCAS + ISO-1 group was significantly reduced. This indicates that: Application of MIF inhibitors helps to alleviate the chemotaxis of meningeal B cells caused by vascular cognitive impairment, thereby alleviating neuroinflammation.

[0068] Example 6 MIF inhibitor alleviates cognitive impairment caused by vascular dementia

[0069] (1) MIF inhibitor improves the learning and memory ability of BCAS mice

[0070] The novel object recognition experiment is a behavioral experiment that uses the characteristics of mice's preference for exploring novel objects to detect their learning and memory abilities. The specific steps are as follows:

[0071] Mice were treated and grouped as described above.

[0072] On the first day of the experiment, which was the adaptation day, the mice were placed in a 40×40×40 cm cube open field for 10 min to adapt. The next day was the test day. Two cubes were placed in a fixed area for the mice to become familiar with for 10 min. One hour later, one of the cubes was replaced with a sphere, and the exploration time of the mice for the novel object was observed. The learning and memory ability of the mice was evaluated according to the novel object exploration index (novel object head exploration time / novel object head exploration time + old object head exploration time).

[0073] As Figure 6Results showed that compared with the BCAS+Vehicle group, mice in the BCAS+ISO-1 group spent more time exploring new objects, indicating that the learning and memory abilities of BCAS mice were improved after the application of the MIF inhibitor.

[0074] (2) The MIF inhibitor improves the working memory of BCAS mice

[0075] 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 and is usually used to evaluate the changes in cognitive function in mice under the influence of drugs or brain injury. The specific steps are as follows:

[0076] Before the experiment, mice were fasted and water-deprived for 6-8 h to induce their foraging behavior in the experiment. Food was placed in the eight arms of the maze to drive them to explore the maze. After each arm was numbered and food was placed in it, the mice were placed in the middle of the platform, and the timer was started. The number of the arm entered each time was recorded. The experiment ended after each arm had been entered. After 7 days of training, the records of the first eight times the mice entered the arms and the number of times the mice repeatedly visited the arms during the entire experiment were working memory errors. In the following three days, food was only placed in the fixed arms 2, 4, 6, and 8. The mice were put in, and the number of repeated visits during the process of the mice eating the food in the four arms was recorded. This was repeated three times a day for a total of 9 times, which was recorded as the reference memory error.

[0077] As Figure 7 A-B showed that compared with the BCAS+Vehicle group, the revisit errors of mice in the BCAS+ISO-1 group were significantly reduced, and they were more inclined to choose the food in different arms in the first eight times, indicating that the working memory of BCAS mice given the MIF inhibitor was improved. At this time, there was no statistical significance in the reference memory test results between the two groups of mice ( Figure 7 C), which indicated that vascular cognitive impairment mainly damaged the working memory of mice, and there was no obvious change in the reference memory.

[0078] The above results indicated that the application of the MIF inhibitor could improve the working memory of mice with vascular cognitive impairment, thereby improving cognitive impairment.

[0079] Example 7 The MIF inhibitor alleviates myelin loss caused by vascular cognitive impairment

[0080] Mice were treated and grouped as described above. Luxol Fast Blue (LFB) staining was used to evaluate the severity of white matter damage. LFB staining is a staining method for showing the morphological structure and pathological changes of nerve myelin, and the severity of white matter damage can be reflected by evaluating the area of myelin loss in the corpus callosum. Mouse brain tissues were taken and sectioned for LFB staining. The specific steps are as follows:

[0081] After rewarming the frozen sections at room temperature, wash them with phosphate buffer, tap water, and ultrapure water for 5 minutes each. Then, dehydrate them in a gradient of 75% - 95% - 100% alcohol, and place them in 0.1% LFB dye for staining at 60°C for 6 - 8 hours. After staining, take out the sections and place them at room temperature. Then, under the microscope, repeatedly destain the sections with 0.05% lithium carbonate destaining solution and 75% alcohol until the myelin sheaths are stained blue and the background is nearly colorless. At this time, the intact myelin sheath fibers are stained blue, while the lost myelin sheath fibers are colorless. According to the previous research method, score and count the degree of myelin sheath loss according to grade 0: normal; grade 1: disordered nerve fibers; grade 2: visible vacuole formation; grade 3: disappearance of myelin sheath fibers.

[0082] As Figure 8 The results showed that: compared with the BCAS + Vehicle group, the white matter damage score in the BCAS + ISO-1 group was significantly reduced. It can be seen from this that using MIF inhibitors helps to alleviate myelin sheath loss caused by vascular cognitive impairment.

[0083] Other parts not described in detail are prior art. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a MIF inhibitor in the preparation of a drug for regulating central B lymphocytes.

2. Use of a MIF inhibitor in the preparation of a drug for treating or improving a neuroinflammation-related disease.

3. The application according to claim 2, wherein: The neuroinflammation-related diseases are vascular cognitive impairment, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, autoimmune encephalitis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, etc.

4. Use of a MIF inhibitor in the preparation of a drug for improving vascular cognitive impairment.

5. The application according to claim 4, characterized in that: The MIF inhibitor regulates the function of B lymphocytes or reduces neuroinflammation to improve vascular cognitive impairment.

6. The application according to any one of claims 1 to 5, characterized in that: The MIF inhibitor is ISO-1.

7. A drug for improving vascular cognitive impairment, characterized in that: The drug contains a MIF inhibitor.

8. The drug according to claim 7, wherein: The MIF inhibitor is ISO-1.

9. The medicament according to claim 7 or 8, characterized in that: The drug further comprises excipients, and the excipients are DMSO, PEG300, Tween-80, and physiological saline.

10. The drug according to claim 9, characterized in that: The drug is an injection or other dosage form.

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