Application of ERK5 inhibitor in preparation of medicine for treating ischemic cerebrovascular disease

Abstract

The invention discloses an application of an ERK5 inhibitor in preparation of a medicine for treating ischemic cerebrovascular disease, the ERK5 inhibitor improves cognitive impairment caused by ischemic cerebrovascular disease, alleviates white matter damage in ischemic cerebrovascular disease, alleviates mitochondrial damage of nerve cells in ischemic cerebrovascular disease, and improves the curative effect of ischemic cerebrovascular disease. The neuroinflammatory response in the ischemic cerebrovascular disease and the mitochondrial injury, oxidative stress and ferroptosis of small glial cells in the ischemic cerebrovascular disease are relieved, and the medicine has a good clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of brain injury treatment, and in particular to the use of an ERK5 inhibitor in the preparation of 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, and China is the country with the highest lifetime risk of stroke in the world. Ischemic cerebrovascular disease has a high incidence rate, high recurrence rate, high mortality and disability rate, and a heavy disease burden. It is a major threat to the health of the Chinese people and has now risen to the first place in the cause of death among Chinese citizens.

[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] The extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway is associated with increased cell proliferation, migration, survival, and angiogenesis. Previous studies on ERK5 have focused on its role in tumors. According to statistics, the gene MAPK7 that edits ERK5 was found to be amplified in 50% of liver cancer patients, and ERK5 was highly expressed in breast cancer and prostate cancer. At the same time, in vitro experimental results showed that ERK5 inhibitors can effectively inhibit the growth and spread of tumor cells. Therefore, ERK5 inhibitors are considered to be one of the most promising methods for cancer treatment. At present, some ERK5 inhibitors have been studied in the preclinical and clinical trial stages, including AX-15836, XMD8-92, etc. However, there are no reports on the use of ERK5 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 various types of diseases. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of an ERK5 inhibitor (the ERK5 inhibitor is XMD8-92) in the preparation of a drug for treating ischemic cerebrovascular disease. The ERK5 inhibitor and the drug of the present invention can improve white matter damage caused by various types of diseases as antagonists.

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

[0008] The present invention provides an application of an ERK5 inhibitor in preparing a medicine for treating ischemic cerebrovascular disease. The ERK5 inhibitor is XMD8-92.

[0009] Furthermore, the ischemic cerebrovascular disease includes transient ischemic attack, different types of acute ischemic stroke, cerebral artery steal syndrome and chronic cerebral ischemia.

[0010] The present invention also provides a medicine for treating ischemic cerebrovascular disease, characterized in that the medicine contains an effective amount of ERK5 inhibitor.

[0011] Furthermore, the medicine also contains pharmaceutically acceptable adjuvants.

[0012] Beneficial effects of the present invention:

[0013] The present invention creatively discovered the use of ERK5 inhibitors in the preparation of drugs for treating ischemic cerebrovascular disease. ERK5 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

[0014] Figure 1 This is a statistical chart of changes in working memory in mice.

[0015] Figure 2 This is a statistical chart of white matter damage degree scores.

[0016] Figure 3 This is the G ratio statistical chart.

[0017] Figure 4 This is a statistical diagram of the degree of mitochondrial vacuolization.

[0018] Figure 5 is a statistical diagram of the degree of microglial activation.

[0019] Figure 6 is the statistical diagram of 8-OHdG fluorescence intensity,

[0020] Figure 7 Statistical graph of 4-HNE fluorescence intensity. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0022] Example 1 ERK5 inhibitor improves cognitive dysfunction caused by ischemic cerebrovascular disease

[0023] In this example, bilateral carotid artery stenosis (BCAS) surgery was used to establish a mouse model of white matter injury. The specific steps are as follows:

[0024] a. After the mice were anesthetized with isoflurane, the skin was incised along the midline of the neck, the thyroid gland was peeled off, and the trachea and bilateral common carotid arteries of the mice were exposed;

[0025] b. 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 was wound around the bilateral common carotid arteries of the mice, the incision was closed, the skin was sutured, and the cerebral blood flow was monitored to decrease by 40%, which represented the successful establishment of the BCAS mouse model (hereinafter referred to as the BCAS group). One month was selected as the observation time point for white matter damage.

[0026] XMD8-92 is an effective ERK5 inhibitor (purchased from MedChemExpress; catalog name: HY-14443, inhibitor purity is 99.67%). The specific method of intragastric administration is as follows:

[0027] Sham group: normal mice were given an equal volume of PBS solution by intragastric gavage for 1 month;

[0028] BCAS+XMD8-92 group: The mice after BCAS surgery were gavaged once every three days, 25 mg / Kg each time, for one month.

[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] 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.

[0031] like 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, compared with the BCAS group, the revisit errors of mice in the BCAS+XMD8-92 group were significantly reduced, indicating that the working memory of BCAS mice given ERK5 inhibitors was restored. This shows that ischemic cerebrovascular disease impaired the working memory of mice. The above results show that the application of ERK5 inhibitors can improve the working memory of mice with ischemic cerebrovascular disease, thereby improving the cognitive dysfunction caused by ischemic cerebrovascular disease.

[0032] Example 2 ERK5 inhibitors alleviate white matter damage in ischemic leukoencephalopathy

[0033] 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 area of ​​myelin loss in the corpus callosum. Take mouse brain tissue and slice it for LuxolFast Blue (LFB) staining. The specific steps are as follows:

[0034] a. 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% to 95% to 100% alcohol, and stain them in 0.1% LFB dye at 60°C for 6 to 8 hours.

[0035] b. After staining, take out the slices and place them at room temperature. Then, use 0.05% lithium carbonate differentiation solution and 75% alcohol to repeatedly separate the slices under a microscope until the myelin sheath is dyed blue and the background is almost colorless. At this time, the intact myelinated fibers are dyed blue, while the myelinated fibers are colorless. According to previous research methods, the statistical rules are as follows:

[0036] Level 0: Normal;

[0037] Grade 1: Nerve fiber disorder;

[0038] Grade 2: cavitation is visible;

[0039] Grade 4: Myelin fibers disappear

[0040] The degree of demyelination was scored and counted according to the above rules.

[0041] like Figure 2 The results showed that compared with the Sham 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+XMD8-92 group was significantly reduced. Therefore, the application of ERK5 inhibitors can help alleviate white matter damage in ischemic cerebrovascular disease.

[0042] G ratio is further used to evaluate the degree of demyelination and thus the degree of white matter damage in ischemic cerebrovascular disease.

[0043] 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.

[0044] 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.

[0045] like 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+XMD8-92 group was significantly reduced. This suggests that the use of ERK5 inhibitors can help alleviate the degree of demyelination in ischemic cerebrovascular disease, thereby helping to alleviate white matter damage.

[0046] Example 3 ERK5 inhibitor alleviates mitochondrial damage in neurons during ischemic cerebrovascular disease

[0047] 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.

[0048] The morphological examination of mitochondria in nerve cells was performed using transmission electron microscopy. The specific steps are as follows:

[0049] Take brain tissue less than 1mm3, fix it with 2.5% glutaraldehyde for 2 hours, rinse it with phosphate buffer 3 times, 15 minutes each time; then fix it with 1% osmium acid fixative for 2 hours, rinse it with phosphate buffer 3 times, 15 minutes each time. Then dehydrate it with gradient ethanol at 4℃, treat it with pure acetone + embedding solution (2:1) at room temperature for 4 hours, pure acetone + embedding solution (1:2) at room temperature overnight, treat it with pure embedding solution at 37℃ for 3 hours, use ultrathin slicer to slice it, the thickness is about 50nm, use 3% uranyl acetate-lead citrate double staining, observe it under transmission electron microscope, take pictures, and evaluate the degree of mitochondrial vacuolization.

[0050] like Figure 4 The results showed that compared with the Sham group, the degree of mitochondrial vacuolation in BCAS mice was significantly increased after modeling. However, compared with the BCAS group, the mitochondrial vacuolation in the BCAS+XMD8-92 group was significantly reduced. This suggests that the use of ERK5 inhibitors can help alleviate mitochondrial damage in ischemic cerebrovascular disease.

[0051] Example 4 ERK5 inhibitor alleviates neuroinflammatory response in ischemic cerebrovascular disease

[0052] 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.

[0053] Immunofluorescence staining was used to perform stereological analysis of microglia (Iba-1) in the injured area of ​​ischemic cerebrovascular disease in mice to evaluate the activation of microglia.

[0054] like Figure 5 The results showed that compared with the Sham group, the density of microglia in the ischemic cerebrovascular disease white matter area of ​​BCAS mice was significantly increased, the cell body area was enlarged, and the roundness was also significantly increased after ischemic cerebrovascular disease injury, indicating that microglia were significantly activated after ischemic cerebrovascular disease injury. However, the aggregation of microglia in the ischemic cerebrovascular disease injury area of ​​BCAS mice in the XMD8-92 group was reduced, the cell body area was reduced, and the roundness was also significantly decreased ( Figure 5 ), indicating that microglial activation was significantly alleviated after application of ERK5 inhibitor.

[0055] It can be seen from this that in the context of ischemic cerebrovascular disease injury, ERK5 inhibitors can reduce the activation of microglia and alleviate neuroinflammation, thereby benefiting the alleviation of neural damage caused by microglia in ischemic cerebrovascular disease.

[0056] Example 5 ERK5 inhibitors help alleviate oxidative stress in microglia in ischemic cerebrovascular disease

[0057] 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.

[0058] like Figure 6 The results showed that compared with the Sham group, the fluorescence intensity of 8-OHdG was significantly increased after BCAS mice were modeled. However, compared with the BCAS group, the fluorescence intensity of 8-OHdG in the BCAS+XMD8-92 group was significantly reduced. This indicates that ERK5 inhibitors can help alleviate the oxidative stress of microglia in ischemic leukoencephalopathy.

[0059] Example 6 ERK5 inhibitors help alleviate ferroptosis of microglia in ischemic leukoencephalopathy

[0060] 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.

[0061] like 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+XMD8-92 group was significantly reduced. This indicates that ERK5 inhibitors can help alleviate the iron death of microglia in ischemic leukoencephalopathy.

[0062] Although the above embodiments have been described 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 creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of an ERK5 inhibitor in the preparation of a drug for treating ischemic cerebrovascular disease, characterized in that: The ERK5 inhibitor is XMD8-92.

2. The use 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. A drug for treating ischemic cerebrovascular disease, characterized in that: The medicament comprises an effective amount of an ERK5 inhibitor.

4. The drug according to claim 3, characterized in that: The medicine further comprises a pharmaceutically acceptable adjuvant.

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