New application of F-box protein 48 inhibitor and medicine for treating ischemic cerebrovascular disease

Abstract

The invention discloses a new application of an inhibitor of F-box protein 48 and a medicine for treating ischemic cerebrovascular diseases, and relates to the field of biological medicine, research finds that the F-box protein 48 has a key effect in neuronal damage of cerebral arterial thrombosis, and the inhibitor of the F-box protein 48 can prevent or treat cerebral arterial thrombosis, such as cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis, cerebral arterial thrombosis and cerebral arterial thrombosis. The invention provides a new application of the F-Box protein 48 inhibitor, and provides a new target and strategy for treatment of cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a new use of an inhibitor of F-box protein 48 and a drug for treating ischemic cerebrovascular disease. Background Art

[0002] Vascular cognitive impairment is a clinical stroke or subclinical vascular brain injury caused by cerebrovascular lesions and their risk factors. It involves a clinical syndrome with impairment of at least one cognitive domain, ranging from mild cognitive impairment to dementia, and also includes varying degrees of cognitive impairment caused by mixed pathologies such as Alzheimer's disease. Stroke, also known as stroke, is a common disease in neurology. It is mostly caused by sudden rupture of cerebral blood vessels or blood vessel blockage that prevents blood from flowing into the brain, causing brain tissue damage. It is characterized by high morbidity, high mortality, and high disability rate. In recent years, due to the increase in life and work pressure, the incidence of stroke has increased year by year and tends to be younger. Stroke includes ischemic stroke and hemorrhagic stroke. Among them, the incidence of ischemic stroke is higher, accounting for about 75% to 90% of the total number of strokes. Ischemic stroke often occurs in a quiet state, and is manifested by dysphagia, slurred articulation, crooked corners of the mouth, dysphagia, hemiplegia, sensory impairment, and in severe cases, incontinence. Hemorrhagic stroke often occurs in an active state, and is manifested by headache, nausea, projectile vomiting, hemiplegia, sensory impairment, and in severe cases, impaired consciousness and coma.

[0003] At present, the exact cause and pathological process of this disease are still unclear. The mechanism of brain damage caused by ischemic stroke is relatively complex. It is currently believed to be related to the cascade of neural damage, mainly including glutamate excitotoxicity, oxidative stress injury and immune inflammatory injury. However, since there is no unified answer to the mechanism of ischemic stroke, the direction of the cure for stroke is unclear, and existing drug treatments have problems such as poor therapeutic effects and poor prognosis. There is a lack of drugs that can treat or improve stroke. At the same time, stroke is an important cause of vascular cognitive impairment, so intervention treatment of stroke is also an important issue in solving vascular cognitive impairment.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of an inhibitor of F-box protein 48 and a drug for treating ischemic cerebrovascular disease.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides the use of an FBox protein 48 inhibitor in the preparation of a drug for preventing, treating or assisting in the treatment of stroke.

[0008] In a second aspect, an embodiment of the present invention provides a method for screening drugs for preventing, treating or assisting in the treatment of stroke, comprising: using an FBox protein 48 inhibitor as a candidate drug to screen out drugs that can prevent, treat or assist in the treatment of stroke.

[0009] In a third aspect, an embodiment of the present invention provides a drug for preventing, treating or assisting in the treatment of stroke, wherein the active ingredients include: an FBox protein 48 inhibitor and other drugs for preventing or treating stroke.

[0010] The present invention has the following beneficial effects:

[0011] The present invention has found that F-Box protein 48 (Fbxo48) plays a key role in neuronal damage in ischemic stroke, and inhibitors of Fbxo48 can prevent or treat ischemic stroke. The present invention provides a new use of F-Box protein 48 inhibitors and provides a new target and strategy for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 To investigate the effects of adding or not adding BC1618 on the neuronal cells in the glucose-oxygen deprivation-reperfusion model (HT22 cell model);

[0014] Figure 2 The effect of adding or not adding BC1618 on neuronal cells in the mouse MCAO model; A is the result of TTC staining, where the white area is the infarct area; B is the statistics of the infarct area; C is the neurological score after 0 hr and 24 hr of cerebral ischemia-reperfusion;

[0015] Figure 3 For histochemical staining, the brain tissue of mice after cerebral ischemia-reperfusion was sectioned and stained with Nissl to mark the morphology of neurons. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] In a first aspect, an embodiment of the present invention provides the use of an FBox protein 48 inhibitor in the preparation of a drug for preventing, treating or assisting in the treatment of stroke.

[0018] F-Box protein 48, also known as FBXO48 or Fbxo48, is involved in the SCF-dependent proteasomal ubiquitin-dependent protein catabolism process. It is part of the SCF ubiquitin ligase complex.

[0019] In some embodiments, the stroke comprises ischemic stroke.

[0020] In some embodiments, the prevention, treatment or adjunctive treatment of stroke includes: prevention, treatment or adjunctive treatment of neuronal cell damage in ischemic stroke.

[0021] In some embodiments, the prevention, treatment or adjunctive treatment of stroke includes: prevention, treatment or adjunctive treatment of cerebral infarction in ischemic stroke.

[0022] In some embodiments, the prevention, treatment or auxiliary treatment of cerebral infarction in ischemic stroke includes inhibiting cerebral infarction in ischemic stroke or reducing the volume of cerebral infarction in ischemic stroke.

[0023] It has been verified that the inhibitor of FBXO48 can significantly improve the neurological scores after ischemia-reperfusion and reduce the volume of cerebral infarction, indicating that FBXO48 is a key protein in MCAO-induced ischemic brain damage.

[0024] In some embodiments, the FBox protein 48 inhibitor includes: an agent that inhibits the activity and / or expression level of FBox protein 48.

[0025] The FBox protein 48 inhibitor can inhibit the activity of FBox protein 48, causing it to lose its normal biological function; it can also inhibit the expression of the FBox protein 48 gene, such as inhibiting transcription and / or inhibiting translation. As long as FBox protein 48 is used as a target, it falls within the protection scope of the present application.

[0026] In some embodiments, the expression level includes protein expression level and / or gene expression level. The agent that inhibits the expression level may include a gene editing agent.

[0027] In some embodiments, the FBox protein 48 inhibitor comprises BC1618.

[0028] It should be noted that the use of other Fbxo48 inhibitors except BC1618 for the treatment of ischemic stroke also falls within the scope of protection of the present invention.

[0029] On the other hand, an embodiment of the present invention provides a method for screening drugs for preventing, treating or assisting in the treatment of stroke, comprising: using FBox protein 48 as a target and an FBox protein 48 inhibitor as a candidate drug to screen out drugs that can prevent, treat or assist in the treatment of stroke.

[0030] In some embodiments, the FBox protein 48 inhibitor includes: an agent that inhibits the activity and / or expression level of FBox protein 48.

[0031] In some embodiments, the expression level includes protein expression level and / or gene expression level. The agent that inhibits the expression level may include a gene editing agent.

[0032] In some embodiments, the FBox protein 48 inhibitor comprises BC1618.

[0033] BC1618 is an orally active Fbxo48 inhibitor that stimulates Ampk signaling (prevents activated pAmpkα from being degraded by Fbxo48-mediated proteasomes). BC1618 promotes mitochondrial fission, promotes autophagy, and improves hepatic insulin sensitivity.

[0034] In addition, an embodiment of the present invention further provides a drug for preventing, treating or assisting in the treatment of stroke, wherein the active ingredients thereof include: an FBox protein 48 inhibitor and other drugs for preventing or treating stroke.

[0035] The "drug" and "drug composition" herein can be equivalently replaced. The drug includes not only the active ingredient of the present invention but also a pharmaceutically acceptable carrier.

[0036] Optionally, the carrier includes, but is not limited to, diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorption carriers, etc.

[0037] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0038] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0039] Example 1

[0040] In vitro, the HT22 neuronal cell line was used to simulate the pathological process of ischemic stroke through glucose-oxygen deprivation-reperfusion experiment to explore the protective effect of FBXO48 inhibition on neuronal damage in this process.

[0041] Experimental process:

[0042] The cells were plated in 96-well plates at a 50% seeding density. After 24 hours, the culture medium was replaced with sugar-free medium and placed in a three-gas incubator with 5% carbon dioxide and 95% nitrogen for 2 hours to deprive the cells of sugar and oxygen. The cells were then replaced with normal culture medium and culture conditions for 18 hours, which was reperfusion. After reperfusion, the cell survival rate was detected by MTT.

[0043] The experimental and control groups were treated by pretreatment. In the experimental group, BC1618 was added to the pre-warmed culture medium at a final concentration of 2 μM. In the control group, an equal amount of DMSO was added. The pre-prepared experimental and control culture medium was replaced 4 hours before the sugar-free culture medium was replaced, and the treatment was carried out for 4 hours. After the completion of glucose-oxygen deprivation, the pre-prepared experimental and control culture medium was used for reperfusion culture for 18 hours.

[0044] Experimental results:

[0045] The results showed that the use of BC1618 could significantly reduce the cell death of HT22 neuronal cell line after glucose-oxygen deprivation and reperfusion ( Figure 1 ).

[0046] Example 2

[0047] Verify the key role of FBXO48 in neuronal damage in ischemic stroke.

[0048] Experimental methods:

[0049] In this example, FBXO48 inhibitor BC1618 was administered intranasally to inhibit the activity of FBXO48 in mouse brain tissue, and the control group was treated with a solvent. The specific steps are as follows:

[0050] 5mg BC1618 powder was dissolved in 60μL DMSO, followed by the addition of 100μL tween-80, 200μL PEG-400 and 640μL saline. The final BC1618 treatment concentration was 5mg / mL, and each mouse was given 20μL per day. The control group was given an equal amount of solvent. The drug was administered for 3 consecutive days, and then the rats underwent MCAO surgery to cause artificial blockage of the middle cerebral artery. After 60min of blockage, the blockage was removed and reperfused for 18hr. Neurological scoring was performed, and then the complete brain tissue was surgically removed, frozen at -80℃ for 4min, and then the brain tissue was evenly cut into five pieces and stained in preheated TTC dye. The number of parallel repetitions was 5.

[0051] Experimental results:

[0052] The results showed that mice with suppressed FBXO48 activity were significantly protected from MCAO-induced functional impairment ( Figure 2 Middle A), cerebral infarction volume measured by TTC staining ( Figure 2 A, B) and neurological function scores ( Figure 2 Middle C) shows the functional impairment induced by MCAO. These results show the key role of FBXO48 in neuronal death in ischemic stroke.

[0053] Example 3

[0054] Verification: FBXO48 inhibitor can rescue neuronal damage in mice after cerebral ischemia-reperfusion.

[0055] Experimental methods:

[0056] The mouse brain tissue mentioned in Example 2 was paraffin sectioned and stained with Nissl to determine the state of neuronal cells. The results are shown in Figure 3 .

[0057] Experimental results:

[0058] The results showed that the neurons of mice shrank significantly and had abnormal morphology after cerebral ischemia-reperfusion, indicating severe neuronal damage after cerebral ischemia-reperfusion. The use of BC1618 can alleviate such neuronal abnormalities and rescue the damage of mouse neurons after cerebral ischemia-reperfusion ( Figure 3 ).

[0059] The above data fully demonstrate that FBXO48 is a key protein in MCAO-induced ischemic brain injury and FBXO48 is a therapeutic target for ischemic stroke.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of FBox protein 48 inhibitors in the preparation of drugs for the prevention, treatment or auxiliary treatment of stroke.

2. The use according to claim 1, characterized in that: The stroke includes ischemic stroke.

3. The use according to claim 1, characterized in that: The prevention, treatment or auxiliary treatment of stroke includes: prevention, treatment or auxiliary treatment of neuronal cell damage in ischemic stroke.

4. The use according to claim 1, characterized in that: The prevention, treatment or auxiliary treatment of stroke includes: prevention, treatment or auxiliary treatment of cerebral infarction of ischemic stroke; Optionally, the prevention, treatment or auxiliary treatment of cerebral infarction caused by ischemic stroke includes inhibiting cerebral infarction caused by ischemic stroke or reducing the volume of cerebral infarction caused by ischemic stroke.

5. The use according to claim 1, characterized in that: The FBox protein 48 inhibitor includes: an agent that inhibits the activity and / or expression level of FBox protein 48; Optionally, the expression level includes protein expression level and / or gene expression level.

6. The use according to claim 5, characterized in that: The FBox protein 48 inhibitors include BC1618.

7. A method for screening drugs for preventing, treating or assisting in the treatment of stroke, characterized in that: It includes: FBox protein 48 inhibitors are used as candidate drugs to screen out drugs that can prevent, treat or assist in the treatment of stroke.

8. The screening method according to claim 7, characterized in that The FBox protein 48 inhibitor includes: an agent that inhibits the activity and / or expression level of FBox protein 48.

9. The screening method according to claim 8, characterized in that The expression level includes protein expression level and / or mRNA expression level.

10. A drug for preventing, treating or assisting in treating stroke, characterized in that: Its active ingredients include: FBox protein 48 inhibitors and other drugs used to prevent or treat stroke.

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