CN112023049A

CN112023049A - New application of inhibitor of long-chain acyl coenzyme A synthetase 4 and medicine for treating cerebral ischemic stroke

Info

Publication number: CN112023049A
Application number: CN202010947278.5A
Authority: CN
Inventors: 庹清章; 雷鹏
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2020-12-04
Anticipated expiration: 2040-09-10
Also published as: CN112023049B

Abstract

The invention discloses a new application of an inhibitor of long-chain acyl coenzyme A synthetase 4 and a medicine for treating ischemic stroke, and relates to the technical field of ischemic stroke treatment. The invention discloses application of an inhibitor of long-chain acyl-CoA synthetase 4 in preparing a medicament for preventing or treating ischemic stroke. The research of the invention finds that long-chain acyl-coenzyme A synthetase 4(ACSL4) has a key role in ischemic stroke neuron damage, and the ACSL4 is used as a target point, and the ACSL4 inhibitor is used for verifying that the ACSL4 inhibitor can treat ischemic stroke in an MCAO model; the invention provides a new target and a new strategy for treating cerebral arterial thrombosis.

Description

New application of inhibitor of long-chain acyl coenzyme A synthetase 4 and medicine for treating cerebral ischemic stroke

Technical Field

The invention relates to the technical field of ischemic stroke treatment, in particular to a new application of an inhibitor of long-chain acyl-CoA synthetase 4 and a medicine for treating ischemic stroke.

Background

Vascular Cognitive Impairment (VCI) is estimated to account for 50% -70% of all people diagnosed with Cognitive Impairment. VCI emphasizes a continuous process, which refers to a class of syndromes ranging from mild cognitive impairment to dementia caused by risk factors of cerebrovascular disease (such as hypertension, diabetes, etc.), or cerebrovascular disease.

The incidence of cerebrovascular diseases of people in China is remarkably increased, and nearly half of the people in China can have VCI. Compared with another common cognitive disorder, namely Alzheimer's Disease (AD), the research on the pathogenesis of VCI is just started, effective prediction means such as pathological molecular images and peripheral markers are clinically lacked, the influence on the outcome of the cerebrovascular disease is still unclear, the intervention measures are limited to the intervention of traditional vascular risk factors and the evaluation of AD-related drugs, and effective treatment strategies and evidence-based evidence are lacked. Therefore, there is a need to develop relevant research on VCI and provide evidence-based evidence for creating new strategies for disease control.

Cerebral apoplexy (also called stroke) is a common acute cerebrovascular disease, which is a group of diseases causing brain tissue damage due to sudden rupture of cerebral vessels or failure of blood flow into the brain due to vessel occlusion, including ischemic and hemorrhagic stroke. The incidence rate of ischemic stroke is higher than that of hemorrhagic stroke, and accounts for about 80 percent of the total stroke. Occlusion or stenosis of internal carotid and vertebral arteries can cause ischemic stroke, patients are older than 40 years, more men than women, and even more serious patients can cause death. According to WHO statistics, stroke has become the second largest "death killer" worldwide. Along with the increasing aging degree of the population in China, the cerebral apoplexy becomes the first cause of death in China and is also the leading cause of disability of the elderly population. Stroke has the characteristics of high morbidity, high mortality and high disability rate. With the progress of modern medical level, if blood vessel dredging treatment is timely performed after stroke occurs, some people can completely recover, but because ischemic tissues are subjected to ischemia-reperfusion injury after blood flow is recovered, the functions of the tissues and organs cannot be recovered, but the dysfunction and structural injury of the tissues and organs are aggravated, and survivors who exceed 2/3 are left with disabled sequelae. However, the mechanism of occurrence of ischemia reperfusion injury is not completely elucidated at present, and an effective therapeutic target and related drugs are lacking.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a new application of an inhibitor of long-chain acyl-CoA synthetase 4 and a medicament for treating ischemic stroke. The research of the invention finds that long-chain acyl-coenzyme A synthetase 4(ACSL4) has a key role in ischemic stroke neuron damage, and by taking ACSL4 as a target point, the inhibitor of ACSL4 is used in an MCAO model to verify that the inhibitor of ACSL4 can treat ischemic stroke; the invention provides a new target and a new strategy for treating cerebral arterial thrombosis.

The invention is realized by the following steps:

the embodiment of the invention discovers that the expression profile of the peripheral blood mononuclear cell genome of the patient with the ischemic stroke is analyzed by combining the verification of related experiments: the iron death is a key pathway for the occurrence and development of cerebral apoplexy and vascular cognitive impairment, the key gene ACSL4 for regulating the iron death plays a key role in the process of ischemic cerebral apoplexy nerve injury, and further experiments show that the inhibition of ACSL4 can obviously improve the neurological score after ischemia reperfusion and reduce the cerebral infarction volume. The ACSL4 is shown to be a potential target for treating ischemic stroke.

The invention clarifies that iron death is a key path and a molecular mechanism for the occurrence and development of the stroke and the vascular cognitive disorder, provides a brand new theory for the pathogenesis of the stroke and the vascular cognitive disorder and lays a foundation for the development of related therapeutic drugs.

Based on this, in one aspect, the present invention provides the use of an inhibitor of long-chain acyl-coa synthetase 4 in the manufacture of a medicament for the prevention or treatment of ischemic stroke.

Based on the research results of the embodiments of the present invention, the inhibitor of long-chain acyl-CoA synthetase 4 has new applications, such as the preparation of drugs for preventing or treating ischemic stroke. The invention provides a new drug selection and strategy for treating ischemic stroke.

In alternative embodiments, the inhibitor is selected from the group consisting of triacin C (fatty acyl coa synthase inhibitor) and pioglitazone.

Based on the research results of the present invention, that is, the key role of long-chain acyl-CoA synthetase 4 in ischemic brain injury, those skilled in the art will easily think of using ACSL4 inhibitors other than Triacsin C and pioglitazone for treating ischemic stroke, which also falls within the scope of the present invention.

In another aspect, the present invention provides the use of an inhibitor of long chain acyl-coa synthetase 4 in the manufacture of a medicament for preventing or protecting neuronal cell damage following ischemic stroke.

The research result of the embodiment of the invention shows that the inhibitor of the long-chain acyl-CoA synthetase 4 can obviously improve the neurological score after ischemia-reperfusion, and the long-chain acyl-CoA synthetase 4 inhibitor can prevent or protect the neuronal cells from being damaged after ischemia-reperfusion. Therefore, the inhibitor of the long-chain acyl-CoA synthetase 4 can be used for preparing the medicine for preventing or protecting the damage of the neuron cells after the cerebral ischemic stroke.

In an alternative embodiment, the inhibitor is selected from the group consisting of triacin C and pioglitazone.

In another aspect, the invention provides the use of an inhibitor of long chain acyl-coa synthetase 4 in the manufacture of a medicament for reducing or reducing the volume of cerebral infarction following ischemic stroke.

The research result of the embodiment of the invention shows that the inhibitor of the long-chain acyl-coenzyme A synthetase 4 can obviously reduce the cerebral infarction volume after ischemia reperfusion, which indicates that the inhibitor of the long-chain acyl-coenzyme A synthetase 4 can be used for preparing the medicine for reducing or reducing the cerebral infarction volume after ischemic stroke.

In another aspect, the present invention provides a method for preparing a drug for preventing or treating ischemic stroke, comprising: the inhibitor of long-chain acyl coenzyme A synthetase 4 is used as the main raw material.

Based on the research results of the embodiments of the present invention, any method for producing or preparing a drug for preventing or treating ischemic stroke using an inhibitor of long-chain acyl-coa synthetase 4 as a main material is within the scope of the present invention. As for the usage amount of the inhibitor of the long-chain acyl-CoA synthetase 4 and other related auxiliary material types, and the dosage form of the drug, etc., those skilled in the art can reasonably select the inhibitor according to the actual administration object, administration route, curative effect, etc., so long as the drug takes the inhibitor of the long-chain acyl-CoA synthetase 4 as the main active ingredient, and the inhibitor belongs to the protection scope of the invention.

In an alternative embodiment, the medicament further comprises a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical agent for preventing or treating ischemic stroke, which targets long-chain acyl-coa synthetase 4 to inhibit its activity or expression.

Based on the results of the present invention, one skilled in the art can easily think of the treatment of ischemic stroke with long-chain acyl-coa synthetase 4 as a target or target, and the corresponding drug is easily obtained or screened by one skilled in the art based on the results of the present invention. These agents may be those which inhibit the activity of long chain acyl-coa synthetase 4, rendering it biologically non-functional; or inhibiting the expression of the long-chain acyl-CoA synthetase 4 gene, such as inhibiting transcription or translation, etc., and the medicines also take the long-chain acyl-CoA synthetase 4 as a target point and belong to the protection scope of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows iron death pathway-related genes that are significantly different in patients with ischemic stroke and age-matched normal populations.

FIG. 2 shows the changes of iron ions and lipid peroxides in the injured brain area of rodent model of ischemic stroke.

FIG. 3 shows the change of key iron death indicators in the injured brain region in rodent models of ischemic stroke.

FIG. 4. effect of iron death inducers on mice of ischemic stroke model.

FIG. 5 detection of iron death-related indicators following treatment of the neural cell line OGD.

FIG. 6 shows ACSL4 expression as a function of ischemia reperfusion time.

FIG. 7 shows the dynamic course of ACSL4 expression and neuronal cell death as a function of ischemia-reperfusion time.

FIG. 8 dynamic changes in ACSL4 expression and neuronal cell death following mouse ischemia reperfusion.

FIG. 9 shows the dynamic change of ACSL4 expression during OGD.

FIG. 10 Effect of ACSL4 knock-out or overexpression on cellular OGD models.

FIG. 11 is a graph showing the identification of ACSL4 knock-out or over-expression virus injected into CA3 region of mouse hippocampus.

FIG. 12 Effect of ACSL4 knock-out or overexpression on the mouse MCAO model.

FIG. 13 Effect of ACSL4 knockout on rat MCAO model.

Figure 14 effect of ACSL4 inhibitors on MCAO model mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The relevant experimental methods and detection methods referred to in the following examples are conventional in the art.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Analysis of peripheral blood mononuclear cell genome expression profile of ischemic stroke patient

Collecting peripheral blood of patients with ischemic stroke and age-matched normal people, separating and extracting peripheral blood mononuclear cells, and performing double-end sequencing based on an Illumina HiSeq technology sequencing platform.

By analyzing differential genes through STRING, we only screen relevant paths of down-regulated genes, further carry out cluster analysis on the down-regulated genes, and find three relevant paths, namely a Proteasome (Proteasome) path, an iron death (ferrotosis) path and a Huntington's disease (Huntington's disease) path, and find iron death path relevant genes which are remarkably different for patients with ischemic stroke and age-matched normal people (see figure 1). The blood brain barrier of a patient with ischemic stroke is damaged, and the change of genes in peripheral blood mononuclear cells can reflect the change condition of related genes of nerve cells in the brain, so that the nerve cells in the damaged brain area can regulate the death of the related nerve cells through an iron death pathway by supposing that the nerve cells in the damaged brain area can be killed in the development process of the ischemic stroke. Based on this, we will further explore and verify through cell and animal experiments in the subsequent examples.

Example 2

Iron death is the main mode of neuronal cell death in ischemic stroke

2.1 rodent model of ischemic stroke verification of neuronal iron death Process

To further explore and verify whether iron death is the main mode of ischemic stroke nerve cell death, we applied a rodent Middle cerebral artery infarction (MCAO) model to simulate the development process of ischemic stroke, and the results showed that the concentration of iron ions in the ischemic brain area was significantly increased compared with the non-ischemic area, and other metal ions did not significantly change (a in fig. 2); meanwhile, we detected the content of MDA (i.e. malondialdehyde, which is the end product of lipid peroxidation and represents the content of lipid peroxide) in the ischemic brain region, and the results showed that the content of lipid peroxide in the ischemic brain region was significantly increased (b, c in fig. 2), which further illustrates that nerve cells undergo an iron death process during ischemic stroke.

To further clarify that the ischemic brain injury process is mediated by iron death, we further examined changes in key biomarkers of the iron death pathway. The results show that the expression and activity of the key protein GPX4 (glutathione peroxidase 4) which regulates the iron death pathway are significantly reduced (a, b in figure 3); it was also found that the content of GSH (glutathione) was also significantly down-regulated (c, d in fig. 3); we observed cerebral cortical neurons in ischemic regions of rats using transmission electron microscopy and found that mitochondria in cerebral cortical neurons in ischemic regions showed significant contraction and increased mitochondrial membrane thickness, which are key morphological features of iron death of cells (fig. 3, e). From the above results, we conclude that iron death is the main mode of neuronal cell death during ischemic stroke.

To verify that iron death mediates ischemia reperfusion injury, we treated MCAO model mice with an iron death inducer, and found that the iron death inducer aggravates the degree of nerve injury, which is particularly marked by a significant increase in defect area (a, b in fig. 4) and a significant worsening of neurological symptoms (c in fig. 4); at the same time, the iron death inducer further increased the lipid peroxide content in the brain injury zone of MCAO model mice (d in fig. 4). The above results further indicate that iron death is the major mode of neuronal cell death during ischemic stroke.

2.2 cellular oxygen sugar deprivation experiments to verify the cellular iron death Process

In order to further explore whether iron death is the main death mode of Oxygen-sugar deficient cells, a cell OGD (Oxygen-glucose depletion) model is applied to simulate the Oxygen-sugar deficiency process of cells. The results show that the survival rate of the nerve cell line N27 cells is remarkably reduced after OGD2 hours and 18 hours of reperfusion (a, b in figure 5); detecting the content of intracellular lipid peroxides by using a flow cytometer in combination with a cell lipid peroxide reagent BODIPY581/591C11, and finding that the intracellular lipid peroxides are obviously increased (C in figure 5); after cell lysis, a significant increase in lipid peroxides was also observed using another lipid peroxide detection reagent MDA (fig. 5 d); meanwhile, it was found that the fluorescence of intracellular DCF (indicating ROS content) was significantly enhanced after OGD treatment (e in FIG. 5), and the ROS content detected by microplate reader was significantly increased (f in FIG. 5). We also observed the morphological changes of mitochondria within cells after OGD treatment using transmission electron microscopy, and the results showed significant intramitochondrial shrinkage and increased mitochondrial membrane thickness (g in fig. 5), which are key morphological features of iron death in cells.

From the above results, we conclude that iron death is the primary mode of oxygen deprivation cell death, which will further validate from the cellular level that ischemia reperfusion injury is mediated by iron death.

Example 3

ACSL4 is a key gene for regulating pig death of nerve cells of cerebral arterial thrombosis

3.1 significant downregulation of ACSL4 during cerebral ischemia-reperfusion injury

The gene ACSL4 which is changed remarkably in the hippocampal tissue of the MCAO model mouse is screened by applying a transcriptome sequencing technology and combining a proteomic analysis technology, which shows that ACSL4 is remarkably reduced in the ischemia-reperfusion injury process, but as a regulatory gene which is critical to iron death, the expression of the gene can promote the occurrence of iron death, and the reduction is caused in the process. To explain this phenomenon, we examined the expression of ACSL4 as a function of the ischemia-reperfusion time by immunoblotting, and the results showed (fig. 6) that the expression of ACSL4 in the hippocampal tissues of the ischemic side of MCAO mice and rats decreased with the prolongation of the ischemia time, indicating that there was a depletion process of ACSL4 as a key enzyme for the regulation of iron death during cerebral ischemia-reperfusion.

To further clarify that consumption of ACSL4 was not due to cell death, we examined the dynamic course of ACSL4 expression and neuronal cell death using immunofluorescence staining and showed (fig. 7) that 3 hours of ischemia-reperfusion did not result in neuronal cell death, while a significant decrease in ACSL4 expression occurred, indicating that down-regulation of ACSL4 was not due to neuronal cell death and that down-regulation of ACSL4 caused neuronal cell death due to ischemia-reperfusion.

We further validated the above conclusions in the mouse MCAO model, first we observed that as the ischemia reperfusion time increased, the area of the mouse brain defect gradually increased, and the neuronal number and ACSL4 protein expression were stained by NeuN and ACSL4, respectively, and found that ACSL4 was down-regulated prior to the decrease in neuronal number, further suggesting that ACSL4 mediated neuronal cell death during ischemia reperfusion (fig. 8).

3.2 significant downregulation of ACSL4 during OGD in cells

We further verified the dynamic expression of ACSL4 as a function of OGD time at the cellular level, and the results showed that the expression of ACSL4 decreased correspondingly as OGD time increased (fig. 9), indicating that ACSL4 plays a key role in the cell death process caused by OGD.

Example 4

ACSL4 knock-out is effective in reducing ischemia reperfusion injury

4.1ACSL4 knockout reduces OGD-induced neuronal cell death

We use CRISPR-Cas9 gene editing technology and AAV transfection technology to realize the knockout and over-expression of N27 cell ACSL4, and the result shows that the knockout and over-expression efficiency can be both (a in FIG. 10). To elucidate that ACSL4 regulates cell death by the iron death pathway, we treated ACSL knockout and overexpressing cells with the iron death inducers Erastin and RSL3 (b, c in fig. 10), and the results showed that the knockout of ACSL4 was effective in inhibiting cell death by the iron death inducers, whereas the overexpression of ACSL4 was effective in emphasizing cell death by the iron death inducers, indicating that ACSL4 mediates cell death by the iron death pathway; to elucidate that cell death caused by OGD is also mediated by ACSL4, we treated an ACSL4 knockout and overexpression cell line with OGD and then examined the cell activity by CCK8, and the results showed that ((d in fig. 10) the ACSL4 knockout effectively reversed cell death caused by OGD, whereas the ACSL4 overexpression cell line died more severely after OGD treatment, indicating that ACSL4 mediates the occurrence of cellular iron death caused by OGD.

4.2ACSL4 knockout reduces ischemia reperfusion injury

In order to further discuss and verify the action and mechanism of ACSL4 in ischemic stroke nerve injury, CRISPR-Cas9 gene editing technology and AAV transfection technology are applied to construct ACSL4 gene knockout and over-expression viruses, the relevant viruses are injected into a mouse hippocampal CA3 region (figure 11) through mouse brain stereotaxic localization, and the identification result shows that the knockout or over-expression efficiency of ACSL4 in the hippocampal CA3 region meets the experimental requirements.

After 30 days of virus injection, an MCAO model is constructed for the mice, and the result shows that the over-expression of ACSL4 can obviously increase the cerebral infarction area of the mice induced by MCAO (a in figure 12), the neurological symptoms of the mice are obviously aggravated (b in figure 12), and the movement coordination capacity of the mice is obviously reduced (c and d in figure 12); compared with the ACSL4 over-expression mouse, the ACSL4 knockout mouse shows the diametrically opposite symptoms, and the ACSL4 knockout can obviously reduce the cerebral infarct size of the MCAO-induced mouse (e in fig. 12), relieve the neurological function symptoms of the mouse (f in fig. 12), and simultaneously obviously enhance the motor coordination capacity of the mouse (g, h in fig. 12). The results show that ACSL4 is a key protease for regulating ischemia-reperfusion injury, and can be used as a potential target for treating ischemic stroke.

We further verified the above conclusions using rats, also the ACSL4 knock-out virus, by stereotactic injection of the virus into the rat cortex, and after 30 days the MCAO model was constructed in rats, which showed that the ACSL4 knock-out did not affect the cerebral cortex perfusion in the MCAO model rats (a, b in fig. 13), but the neurological symptoms of the rats were significantly reduced (c in fig. 13) and the cerebral infarct size was significantly reduced (d in fig. 13). The results show that the ACSL4 knockout can reverse MCAO-induced rat ischemic brain injury, and the ACSL4 can be used as a potential target for treating ischemic stroke.

4.3ACSL4 inhibitors are effective in reducing ischemia reperfusion injury

In order to verify the intervention effect of ACSL4 as a target for treating ischemic stroke, an ACSL family inhibitor, namely, Triacin C is used for treating MCAO model mice, and the result shows that the Triacin C can obviously relieve the nerve function symptoms of the MCAO model mice (a in figure 14) and obviously reduce the cerebral infarction area (b in figure 14). The results further show that the inhibition of ACSL4 can reverse MCAO-induced ischemic brain injury, and ACSL4 can be used as a potential target for treating ischemic stroke.

The invention firstly provides a key path for the occurrence and development of the cerebral apoplexy and the vascular cognitive disorder, and clarifies the molecular mechanism of the iron death involved in the cerebral apoplexy and the vascular cognitive disorder; finding out the key gene ACSL4 for regulating iron death in the nerve injury process of ischemic stroke. The gene is used as a target point, and the ACSL4 inhibitor is used for treating or improving the effect of ischemic stroke.

By combining the experimental results, the invention provides a new target point, namely ACSL4, for the treatment of cerebral arterial thrombosis, and can play a role in treating cerebral arterial thrombosis by inhibiting ACSL 4.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an inhibitor of long chain acyl-coa synthetase 4 in the manufacture of a medicament for the prevention or treatment of ischemic stroke.

2. The use according to claim 1, wherein the inhibitor is selected from the group consisting of triacin C and pioglitazone.

3. Use of an inhibitor of long chain acyl-coa synthetase 4 in the manufacture of a medicament for preventing or protecting neuronal cell damage following ischemic stroke.

4. The use according to claim 3, wherein the inhibitor is selected from the group consisting of Triacsin C and pioglitazone.

5. Use of an inhibitor of long chain acyl-coa synthetase 4 in the manufacture of a medicament for reducing or reducing the volume of cerebral infarction following ischemic stroke.

6. The use according to claim 5, wherein the inhibitor is selected from the group consisting of Triacsin C and pioglitazone.

7. A method for preparing a medicament for preventing or treating ischemic stroke, comprising: the inhibitor of long-chain acyl coenzyme A synthetase 4 is used as the main raw material.

8. The method of claim 7, wherein the inhibitor is selected from the group consisting of Triacsin C and pioglitazone.

9. The method of claim 7 or 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

10.A drug for preventing or treating ischemic stroke, which is characterized by inhibiting the activity or expression of long-chain acyl-CoA synthetase 4 as a target.