Application of MLK3 inhibitor in preparation of medicine for treating ischemic cerebrovascular disease
By using MLK3 inhibitors, the problems of white matter damage and cognitive dysfunction caused by ischemic cerebrovascular disease are solved, and effective treatment of ischemic cerebrovascular disease is achieved, which has good clinical application value.
Patent Information
- Application Number
- CN202411866489.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-04-18
AI Technical Summary
Ischemic cerebrovascular disease leads to cerebral white matter damage and cognitive dysfunction, and there is no effective treatment method in the prior art.
MLK3 inhibitors, including URMC-099, MLK-IN-1, CEP-1347, CS640, MLK-IN-2, and CEP1347-VHL-02, are used as drugs for the treatment of ischemic cerebrovascular diseases to improve cognitive dysfunction and alleviate white matter damage.
MLK3 inhibitors can improve cognitive dysfunction caused by ischemic cerebrovascular disease, relieve white matter damage, reduce mitochondrial damage and neuroinflammatory response of nerve cells, and relieve oxidative stress and ferrous death in microglia.
Smart Images
Figure CN119838013A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine, and in particular relates to the use of an MLK3 inhibitor in preparing a 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. Ischemic cerebrovascular disease has a high incidence, high recurrence rate, high mortality and disability rate, and a heavy disease burden, and is a major threat to people's health.
[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 myelin sheaths wrapped around the outer layer of nerve fibers. In the context of ischemic cerebrovascular disease, the clinical manifestations of white matter damage vary greatly: periventricular white matter damage is mostly cap-shaped, thin line-shaped or halo-shaped. Smaller cap-shaped or dot-shaped ones may be asymptomatic and progress slowly; deep white matter damage is mostly dot-shaped, sheet-shaped or large-scale fusion-shaped. The lesions progress rapidly and can produce multiple clinical manifestations such as cognitive dysfunction, abnormal emotional fluctuations, unstable gait, and urinary incontinence, which brings serious socioeconomic and family problems to daily life.
[0004] Mixed lineage protein kinase 3 (MLK3) is a member of the serine / threonine kinase MLK family and plays an important role in the proliferation, migration and invasion of cancer cells. However, there are no reports on the use of MLK3 inhibitors in the treatment of ischemic cerebrovascular disease.
[0005] Therefore, the development of specific inhibitors can serve as a potential therapeutic drug to improve white matter damage caused by ischemic cerebrovascular disease. Summary of the invention
[0006] The invention discloses the use of an MLK3 inhibitor in preparing a medicine for treating ischemic cerebrovascular disease, which has good clinical application value.
[0007] In order to achieve the above purpose, this application adopts the following technical solutions:
[0008] In a first aspect, the present invention provides use of an MLK3 inhibitor in the preparation of a medicament for treating ischemic cerebrovascular disease.
[0009] 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.
[0010] In the above technical scheme, the MLK3 inhibitors include URMC-099, MLK-IN-1, CEP-1347, CS640, MLK-IN-2, and CEP1347-VHL-02.
[0011] In the above technical solution, the MLK3 inhibitor can improve cognitive dysfunction caused by ischemic cerebrovascular disease.
[0012] In the above technical solution, the MLK3 inhibitor can alleviate white matter damage in ischemic cerebrovascular disease.
[0013] In the above technical solution, the MLK3 inhibitor can alleviate mitochondrial damage of nerve cells in ischemic cerebrovascular disease.
[0014] In the above technical solution, the MLK3 inhibitor can alleviate the neuroinflammatory response in ischemic cerebrovascular disease.
[0015] In the above technical solution, the MLK3 inhibitor can alleviate the oxidative stress and ferroptosis of microglia in ischemic cerebrovascular disease.
[0016] In a second aspect, the present invention provides a drug for treating ischemic cerebrovascular disease, which is composed of an MLK3 inhibitor at an effective dose, and can be a single component or a composition comprising an effective dose of the MLK3 inhibitor, wherein the composition can include a pharmaceutically acceptable carrier.
[0017] The beneficial effect of the present invention is that the present invention creatively discovers the use of MLK3 inhibitors in the preparation of drugs for treating ischemic cerebrovascular disease. MLK3 inhibitors improve cognitive dysfunction caused by ischemic cerebrovascular disease, relieve white matter damage in ischemic cerebrovascular disease, relieve mitochondrial damage of nerve cells in ischemic cerebrovascular disease, relieve neuroinflammatory response in ischemic cerebrovascular disease, mitochondrial damage, oxidative stress and ferroptosis of microglia in ischemic cerebrovascular disease, and have good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a statistical chart of changes in working memory in mice.
[0019] Figure 2 This is a statistical chart of white matter damage degree scores.
[0020] Figure 3This is the G ratio statistical chart.
[0021] Figure 4 This is a statistical diagram of the degree of mitochondrial vacuolization.
[0022] Figure 5 is a statistical diagram of the degree of microglial activation.
[0023] Figure 6 is the statistical diagram of 8-OHdG fluorescence intensity,
[0024] Figure 7 is the statistical diagram of 4-HNE fluorescence intensity,
[0025] Figure 8 Fluorescence intensity statistics of mitoSOX and FerroOrgance DETAILED DESCRIPTION
[0026] 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.
[0027] Ischemic cerebrovascular disease mouse model: The present invention uses bilateral carotid artery stenosis (BCAS) surgery to establish an ischemic cerebrovascular disease 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. January was selected as the observation time point for ischemic cerebrovascular disease.
[0028] Example 1 MLK3 inhibitor improves cognitive dysfunction caused by ischemic cerebrovascular disease
[0029] 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.
[0030] URMC-099 is a specific inhibitor of MLK3. It has brain penetration. Specifically, it is administered by intraperitoneal injection.
[0031] Sham group: normal mice were given an equal volume of PBS solution by intraperitoneal injection for 1 month;
[0032] BCAS+URMC-099 group: The mice after BCAS surgery were injected once a day, 10 mg / Kg each time, for 1 month.
[0033] 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 to drive them to explore the maze. After each arm was numbered and food was placed, the mice were placed in the middle of the platform, the timing was started, and the number of each entry into the arm was recorded. The experiment ended when each arm was entered. After 7 days of training, the records 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.
[0034] Figure 1 The results showed that the number of repeated visits of mice in the BCAS group increased compared with that of mice in the Sham group, indicating that the working memory of mice after BCAS surgery was impaired. At the same time, the revisit errors of mice in the BCAS+URMC-099 group were significantly reduced compared with those in the BCAS group, indicating that the working memory of BCAS mice given MLK3 inhibitors was restored. This shows that ischemic cerebrovascular disease mainly impairs the working memory of mice. The above results show that the application of MLK3 inhibitors can improve the working memory of mice with ischemic cerebrovascular disease, thereby improving the cognitive dysfunction caused by ischemic cerebrovascular disease.
[0035] Example 2 MLK3 inhibitors alleviate white matter damage in ischemic cerebrovascular disease
[0036] 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. Mouse brain tissue was obtained and sliced for LuxolFast Blue (LFB) staining.
[0037] 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 with 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 research methods, the degree of myelin loss was scored and counted according to grade 0: normal; grade 1: nerve fiber disorder; grade 2: visible vacuoles; grade 3: myelinated fibers disappeared.
[0038] Figure 2The results showed that compared with the sham operation group, the white matter injury score of BCAS mice was significantly increased after modeling. However, compared with the BCAS group, the white matter injury score of the BCAS+URMC-099 group was significantly reduced. This suggests that the application of MLK3 inhibitors can help alleviate white matter damage in ischemic cerebrovascular disease.
[0039] G ratio is further used to evaluate the degree of demyelination and thus the degree of white matter damage in ischemic cerebrovascular disease.
[0040] G ratio refers to the axon diameter / (axon + myelin diameter). Under normal circumstances, its value ranges from 0.5 to 0.7. The larger the G ratio, the thinner the myelin sheath, indicating the possibility of demyelination.
[0041] Specifically, the myelin in the brain slices was fixed and stained with osmium acid, and then the axon diameter of the nerve fiber and the diameter of the total nerve fiber (axon + myelin sheath) were measured using a microscope. The G ratio was calculated according to the formula to evaluate white matter damage in ischemic cerebrovascular disease.
[0042] Figure 3 The results showed that compared with the Sham group, the G ratio of BCAS mice was significantly increased after modeling. However, compared with the BCAS group, the G ratio of the BCAS+URMC-099 group was significantly reduced. This suggests that the use of MLK3 inhibitors can help alleviate the degree of demyelination in ischemic cerebrovascular disease, thereby helping to alleviate white matter damage.
[0043] Example 3 MLK3 inhibitor alleviates mitochondrial damage in neurons in ischemic cerebrovascular disease
[0044] Mitochondria are the main site for cells to generate ATP. They are important organelles that promote cell energy conversion and participate in many important life activities such as cell aging, apoptosis, signal transduction, and electrolyte balance. Studies have shown that mitochondrial damage plays an important role in the pathogenesis of ischemic cerebrovascular disease.
[0045] Specifically, the morphological examination of mitochondria in nerve cells was performed by transmission electron microscopy. Brain tissue less than 1 mm3 was taken, fixed with 2.5% glutaraldehyde for 2 hours, rinsed with phosphate buffer 3 times, 15 minutes each time; then fixed with 1% osmium acid fixative for 2 hours, rinsed with phosphate buffer 3 times, 15 minutes each time. Then, dehydrated with gradient ethanol at 4°C, treated with pure acetone + embedding solution (2:1) at room temperature for 4 hours, pure acetone + embedding solution (1:2) at room temperature overnight, treated with pure embedding solution at 37°C for 3 hours, sliced with an ultrathin slicer, with a thickness of about 50 nm, double stained with 3% uranyl acetate-lead citrate, observed under a transmission electron microscope, filmed, and the degree of mitochondrial vacuolization was evaluated.
[0046] Figure 4 The results showed that compared with the Sham group, the degree of mitochondrial vacuolization in BCAS mice was significantly increased after modeling. However, compared with the BCAS group, the mitochondrial vacuolization in the BCAS+URMC-099 group was significantly reduced. This suggests that the use of MLK3 inhibitors can help alleviate mitochondrial damage in ischemic cerebrovascular disease.
[0047] Example 4 MLK3 inhibitors alleviate neuroinflammatory responses in ischemic cerebrovascular disease
[0048] Neuroinflammatory response is considered to be an important pathological mechanism for causing white matter lesions. Neuroinflammation is a basic immune response, which is manifested by enhanced glial cell activation, proinflammatory cytokine secretion, blood-brain barrier permeability and peripheral leukocyte invasion, thereby protecting the body from endogenous and exogenous damage. Microglia are innate immune cells of the central nervous system, and their activation can mediate neuroinflammatory response. The specific manifestations of microglial activation are increased density, cell body area and roundness.
[0049] Immunofluorescence staining was used to perform stereological analysis of microglia (Iba-1) in the damaged white matter area of mice to evaluate the activation of microglia. Figure 5 As shown in the figure, compared with the Sham group, the density of microglia in the white matter area of BCAS mice increased significantly after modeling, the cell body area increased, and the roundness also increased significantly, indicating that the microglia in the ischemic cerebrovascular disease injury area were significantly activated. However, the aggregation of microglia in the white matter area of BCAS mice in the MLK3-URMC-099 group was alleviated, the cell body area decreased, and the roundness also decreased significantly ( Figure 5 ), indicating that microglial activation was significantly reduced after the application of MLK3 inhibitors. This suggests that in the context of ischemic cerebrovascular disease, MLK3 inhibitors can reduce microglial activation and reduce neuroinflammation, thereby benefiting the alleviation of microglial-induced neural damage in ischemic cerebrovascular disease.
[0050] Example 5 MLK3 inhibitors alleviate oxidative stress in microglia in ischemic cerebrovascular disease
[0051] 8-Hydroxydeoxyguanosine (8-OHdG) is a ROS-induced DNA purine residue modification, which is a sensitive indicator of oxidative DNA damage and a common biomarker of oxidative stress. Immunofluorescence staining of 8-OHdG and Iba-1 mouse brain tissue sections was performed to detect the oxidative stress level of microglia.
[0052] Figure 6The results showed that compared with the sham group, the fluorescence intensity of 8-OHdG was significantly increased after BCAS mouse modeling. However, compared with the BCAS group, the fluorescence intensity of 8-OHdG in the BCAS+URMC-099 group was significantly reduced. This indicates that the use of MLK3 inhibitors can help alleviate the oxidative stress of microglia in ischemic cerebrovascular disease.
[0053] Example 6 MLK3 inhibitor alleviates ferroptosis of microglia in ischemic cerebrovascular disease
[0054] 4-Hydroxynonenal (4-HNE) is an important lipid peroxidation marker of ferroptosis and accumulates in large quantities during ferroptosis. Immunofluorescence staining of brain tissue sections of mice with 4-HNE and Iba-1 was performed to detect the level of ferroptosis in microglia.
[0055] Figure 7 The results showed that compared with the sham group, the fluorescence intensity of 4-HNE was significantly increased after BCAS mouse modeling. However, compared with the BCAS group, the fluorescence intensity of 4-HNE in the BCAS+URMC-099 group was significantly reduced. This indicates that MLK3 inhibitors can help alleviate the iron death of microglia in ischemic cerebrovascular disease.
[0056] Example 7 MLK3 inhibitors alleviate mitochondrial damage, oxidative stress and ferroptosis of microglia in ischemic cerebrovascular disease
[0057] MLK3 inhibitors were further added in vitro to observe the changes in microglia in ischemic cerebrovascular disease.
[0058] Ischemic cerebrovascular disease causes damage to the white matter of the brain, and the distribution of white matter in the brain is different, and the clinical manifestations vary greatly. Based on this, an in vitro model of white matter damage was constructed. Specifically, the brain tissue of the newborn rat was peeled off, chopped and digested, and cultured in a 37°C incubator containing 5% CO2 for 14 days. Primary microglia were collected and stimulated with exogenous myelin to simulate the white matter damage environment in vitro. That is:
[0059] Control group: primary microglia without any treatment
[0060] Myelin group: primary microglia stimulated with exogenous myelin 10 μg / ml
[0061] Myelin+URMC-099 group: After stimulating primary microglia with exogenous myelin 10 μg / ml, primary microglia were treated with URMC-099, 100 nM, for 24 h.
[0062] 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. FerroOrange is a ferrous ion probe that can be used to detect changes in intracellular ferrous iron levels.
[0063] Immunofluorescence staining of mouse brain tissue sections with mitoSOX, FerroOrgance and Iba-1 was performed to detect the oxidative stress and ferroptosis levels of microglia.
[0064] Figure 8 The results showed that compared with the control group and the myelin group, the mean fluorescence intensity of mitoSOX and FerroOrgance in the myelin+URMC-099 group was significantly reduced, suggesting that the application of MLK3 inhibitors can help alleviate the oxidative stress and ferroptosis of microglia in ischemic cerebrovascular disease.
[0065] 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 MLK3 inhibitors in the preparation of drugs for treating 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 MLK3 inhibitors include URMC-099, MLK-IN-1, CEP-1347, CS640, MLK-IN-2, and CEP1347-VHL-02.
4. The application according to claim 1, characterized in that: The MLK3 inhibitor can improve cognitive dysfunction caused by ischemic cerebrovascular disease.
5. The application according to claim 1, characterized in that: The MLK3 inhibitor can alleviate white matter damage in ischemic cerebrovascular disease.
6. The use according to claim 1, characterized in that: The MLK3 inhibitor can alleviate mitochondrial damage of nerve cells in ischemic cerebrovascular disease.
7. The use according to claim 1, characterized in that: The MLK3 inhibitor can alleviate the neuroinflammatory response in ischemic cerebrovascular disease.
8. The use according to claim 1, characterized in that: The MLK3 inhibitor can alleviate mitochondrial damage, oxidative stress and ferroptosis of microglia in ischemic cerebrovascular disease.
9. A drug for treating ischemic cerebrovascular disease, characterized in that: The composition is composed of an MLK3 inhibitor at an effective dose, and can be a single component or a composition comprising an effective dose of the MLK3 inhibitor, wherein the composition can include a pharmaceutically acceptable carrier.