CN114028397A - Application of GSK126 serving as EZH2 inhibitor in preparation of medicine for preventing and/or treating cerebral arterial thrombosis - Google Patents
Application of GSK126 serving as EZH2 inhibitor in preparation of medicine for preventing and/or treating cerebral arterial thrombosis Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention discloses an application of GSK126 serving as an EZH2 inhibitor in preparation of a medicine for preventing and/or treating cerebral arterial thrombosis, and belongs to the field of biological medicines. According to the invention, the activity of EZH2 is inhibited by GSK126, so that the methylation level of rat hippocampal histone H3K27me3 can be reduced, the methylation level of hippocampal histone H3K27me3 can be prevented from being excessively increased after ischemic stroke occurs, and meanwhile, GSK126 is found to remarkably reduce neuron death of hippocampal CA1 area caused by ischemia and reduce the cerebral infarction area after ischemic stroke. Therefore, the GSK126 provided by the invention can be used for preventing and/or treating the ischemic stroke, provides a new drug choice for preventing and/or treating the ischemic stroke, and provides a new strategy and treatment mode for clinically treating the ischemic stroke.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of GSK126 serving as an EZH2 inhibitor in preparation of medicines for preventing and/or treating cerebral arterial thrombosis.
Background
Stroke is one of the three most fatal diseases in humans and is also the most common cause of disability. The latest statistical data show that: the proportion of people who may suffer stroke is up to one sixth. Stroke includes stroke and heart stroke, and stroke is also called stroke or cerebrovascular accident, and is an acute ischemic or hemorrhagic brain disease caused by circulatory disturbance of cerebral blood flow, so stroke is divided into ischemic stroke and hemorrhagic stroke.
At present, the treatment scheme for cerebral apoplexy is very limited, thrombolytic treatment medicines are generally used as main treatment means after the occurrence of the cerebral apoplexy, but the medicines capable of simultaneously preventing and treating the cerebral apoplexy are not reported. Moreover, the cause of direct neuronal damage, whether ischemic or hemorrhagic, is ischemic brain injury. After ischemia occurs, neuronal survival is determined by a balance of cell death and cytoprotective function that involves changes in the expression levels of hundreds of genes. H3K27me3 is a histone methylation form capable of widely inhibiting gene expression levels, while the H3K27me3 levels of the elderly are generally high. It is known that H3K27me3 levels are specifically mediated by the histone methylase EZH2 and affect the DNA methylation level at CG sites on adjacent DNA strands. EZH2 activity can be specifically inhibited by GSK126, and thereby reduce H3K27me3 levels. After the occurrence of stroke, the level of H3K27me3 in ischemic brain tissue is further increased, and whether the stroke can be prevented and treated by a mode that H3K27me3 level can be reversed by GSK-126 is unknown. Therefore, the deep research on the effect and the specific mechanism of the GSK-126 in the stroke is helpful for determining the activity level and the action mechanism of the EZH2 in the stroke, and provides a new strategy and thought for the treatment of the stroke.
Disclosure of Invention
The invention aims to provide a novel medical application of GSK126, discusses the activity level and action mechanism of EZH2 in cerebral apoplexy, and provides important data for further developing drugs for preventing and/or treating ischemic cerebral apoplexy.
In order to achieve the purpose, the invention adopts the following technical scheme:
provides the application of GSK126 serving as an EZH2 inhibitor in preparing medicines for preventing and/or treating cerebral arterial thrombosis.
The molecular formula of the GSK126 is C31H38N6O2The structural formula is as follows:
preferably, the medicament is used for reducing the death rate of hippocampal neuronal cells and reducing cerebral infarction area.
Preferably, the GSK126 is used in an amount of 200 mg/kg.
In another aspect, the present invention provides a pharmaceutical preparation for preventing and/or treating ischemic stroke, comprising (1) an effective amount of GSK126 as an active ingredient, and (2) optionally a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.
Animal experiments show that the methylation level of H3K27me3 in the hippocampus of a rat is obviously increased after ischemic stroke, and a massive death phenomenon occurs in neurons in the CA1 region of the hippocampus, which indicates that the methylation level of H3K27me3 is related to hippocampus death. According to the invention, the activity of EZH2 is inhibited by GSK126, so that the methylation level of rat hippocampal histone H3K27me3 can be reduced, the methylation level of hippocampal histone H3K27me3 can be prevented from being excessively increased after ischemic stroke occurs, and meanwhile, the GSK126 is found to remarkably reduce the neuron death of hippocampal CA1 area caused by cerebral ischemia and reduce the cerebral infarction area after ischemic stroke. Therefore, the GSK126 provided by the invention can be used for preventing and/or treating the ischemic stroke, provides a new drug choice for preventing and/or treating the ischemic stroke, and provides a new strategy and treatment mode for clinically treating the ischemic stroke.
Drawings
FIG. 1 is a graph showing the results of Western blot analysis of hippocampal cells before and after ischemia in rats, respectively, before and after GSK126 treatment (I/R); GSK126 indicates GSK126 pretreatment, I/R indicates four artery ligation whole brain ischemia reperfusion; "-" indicates that the treatment was not performed, "+" indicates that the treatment was performed;
FIG. 2 is a statistical graph (I/R) of the methylation level of H3K27me3 before and after GSK126 treatment of hippocampal cells of the brain before and after rat ischemia; wherein, Control is a Control group rat brain hippocampus sample which is not operated and administered, GSK126 is a group rat brain hippocampus sample which is pretreated by GSK126, I/R is a four-artery ligation whole brain ischemia reperfusion group rat brain hippocampus sample, and GSK126+ I/R is a four-artery ligation whole brain ischemia reperfusion group rat brain hippocampus sample after GSK126 pretreatment; data are expressed as Mean ± SEM, # p <0.001, # p <0.01, # p < 0.05;
fig. 3 is a graph of niemann's staining before and after treatment of hippocampal neuronal cells with GSK126 before and after rat ischemia: (A) nissl staining of rat hippocampal neuronal cells without ischemia and treatment with GSK126 injection (Control group); (B) nissl staining pattern of rat hippocampal neuronal cells treated with GSK126 alone (GSK126 group); (C) nissl staining pattern of ischemia reperfusion treated rat hippocampal neuronal cells only (group I/R); (D) nissl staining patterns of rat hippocampal neuronal cells treated with both ischemia reperfusion and pre-injection of GSK126 (I/R + GSK126 group); the small picture in each group of pictures is the amplification effect of the hippocampal CA1 region, and the scale bar is 1: 200 of a carrier;
FIG. 4 is a statistical chart of the cell numbers of hippocampal neurons before and after rat ischemia treatment by GSK126, respectively; wherein, Control is a Control group rat brain hippocampus sample which is not operated and administered, GSK126 is a group rat brain hippocampus sample which is pretreated by GSK126, I/R is a four-artery ligation whole brain ischemia reperfusion group rat brain hippocampus sample, and GSK126+ I/R is a four-artery ligation whole brain ischemia reperfusion group rat brain hippocampus sample after GSK126 pretreatment; data are expressed as Mean ± SEM, # p <0.01, # p <0.05, ns indicates no significant difference;
FIG. 5 is a graph showing the results of Western blot analysis of hippocampal cells before and after rat ischemia treatment with GSK126, respectively (MCAO); GSK126 represents GSK126 pretreatment, and MCAO represents rat cerebral artery embolism modeling; "-" indicates that the treatment was not performed, "+" indicates that the treatment was performed;
FIG. 6 is a statistical chart of the methylation level of H3K27me3 (MCAO) before and after GSK126 treatment of hippocampal cells of the brain before and after rat ischemia; wherein, Control is a Control rat brain hippocampus sample without operation and administration, GSK126 is a rat brain hippocampus sample pretreated by GSK126, MCAO is a rat brain hippocampus sample of a rat brain middle artery embolism modeling group, and GSK126 and MCAO are rat brain hippocampus samples of the rat brain middle artery embolism modeling group pretreated by the GSK 126; data are expressed as Mean ± SEM, # p <0.001, # p <0.01, # p < 0.05.
Fig. 7 is a TTC staining pattern of rats modeled by MCAO before and after treatment with GSK126, respectively: TTC staining pattern (Control group) after modeling of cerebral MCAO of rats not injected with GSK 126; TTC staining pattern after brain MCAO modeling of rats injected with GSK126 (GSK126 group); wherein the white areas show areas of cerebral infarction;
FIG. 8 is a statistical graph of infarct size of rats modeled by MCAO before and after treatment with GSK126, wherein Control is a group modeled by MCAO without GSK126 pretreatment, and GSK126 is a group modeled by MCAO and preprocessed by GSK 126; data are expressed as Mean ± SEM,. xp < 0.05.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
Unless otherwise indicated, reagents, methods and equipment used in the present invention are conventional in the art, and materials of kits used in the following examples are commercially available.
Example 1
Provides the application of GSK126 serving as an EZH2 inhibitor in preparing medicines for preventing and/or treating cerebral arterial thrombosis. The medicine is used for reducing the death rate of hippocampal neuron cells and reducing cerebral infarction area.
The specific experimental process is as follows:
experimental animals: male SD rats were purchased from the laboratory animal center of Xuzhou medical university under license number SCXK (Su) 2015-. Rats are placed under the standard conditions of humidity of 50 +/-10% and temperature of 23 +/-2 ℃ for adapting to survival for 12 hours in each day and night. Rats can drink and eat freely. All animal management and treatment protocols were approved by the animal ethics committee of xu nationality medical university. All experiments were performed as recommended by the ethical guidelines for managing and using animal behavior.
Reagent: GSK126 was purchased from bioengineering, inc.
Antibody: H3K27me3 and Histone H3 were both available from Abcam, USA.
First, GSK126 reduces ischemia-induced neuronal death in CA1 region of hippocampus cerebri by reducing histone H3K27me3 methylation level and inhibiting H3K27me3 elevation
(1) Establishment and administration of rat four-artery ligation cerebral ischemia reperfusion model (I/R)
Rats were first administered a 200mg/kg/day dose of GSK126 by lateral ventricle injection for seven consecutive days. Electrocoagulation of vertebral arteries on two sides of the rat on the seventh day, surgical ligation of carotid artery for 15min on the eighth day to cause cerebral ischemia model, releasing carotid artery after 15min for blood reperfusion, and taking brain tissue of the rat according to the time required by the study to carry out the next study.
(2) Detection of histone H3K27me3 methylation level by Western blotting method
Histone H3K27me3 methylation level assay material was taken from rat brain hippocampus CA1 region tissue 24H after I/R model surgery, hippocampus tissue was immediately lysed with RIPA lysate, protein was separated by 10% SDS-PAGE gel electrophoresis after centrifugation to remove precipitates, protein isolate was transferred to Nitrocellulose (NC) membrane, and 3% BSA blocked for 1H. And (3) incubating the NC membrane with the corresponding primary antibody at 4 ℃ overnight, eluting the next day, adding the corresponding secondary antibody, and incubating for 1h at room temperature. Antibodies used included H3K27me3, Histone H3. And (4) ECL developing, scanning by a fluorescence imaging system to form a graph, and expressing the relative expression content of the target protein by the ratio of optical density.
(3) Neisser's staining method for detecting survival condition of rat hippocampal neuron
Tissues for observation of neural cell survival were obtained from rat brain tissue 7 days after surgery. Taking out brain, slicing with paraffin, dewaxing to water, and dewatering with gradient alcohol. Repeatedly washing with distilled water, placing in a warm box, and dyeing with tar violet for 30 s. Eluting with distilled water, dehydrating in ethanol, treating with xylene, and sealing with neutral gum.
(4) Results of the experiment
The results of western blot experiments are shown in fig. 1 and fig. 2, with Histone H3 as an internal reference, and the ratio of H3K27me3 to Histone H3 as a reference value of the methylation level of H3K27me3, it was found that GSK126 pretreatment can effectively reduce the level of rat hippocampus H3K27me3 (Control vs. GSK 126: 0.6035 ± 0.0088vs.0.3244 ± 0.0788, p <0.05), and that the methylation level of H3K27me3 caused by global cerebral ischemia can be effectively inhibited (Control vs. I/R: 0.6035 ± 0.0088vs.1.505 ± 0.0874, p <0.001) (I/R vs. GSK126+ I/R: 1.505 ± 0.0874vs.0.5697 ± 0.1104, p <0.01), and the difference has statistical significance (p < 0.05). The results suggest that GSK126 pretreatment can effectively inhibit the elevated level of methylation of H3K27me3 in brain tissue after global cerebral ischemia.
The results of the Neisseria staining are shown in FIGS. 3 and 4, and FIG. 3 shows the normal survival of neurons in purple. Based on the number of neurons in CA1 area of hippocampal in Control (Control) rats, the pretreatment of GSK126 did not affect the survival number of neurons in CA1 area of hippocampus (FIG. 4, Control vs. GSK126: 84 + -8.021 vs.82 + -5.200), and the neurons in CA1 area of hippocampus of rats suffered from massive death after global cerebral ischemia (FIG. 4, Control vs. I/R:84 + -8.021 vs.37 + -5.196, p <0.01), and the pretreatment of GSK126 could effectively save the death of hippocampal neurons after global cerebral ischemia (FIG. 4, I/R vs. GSK126+ I/R: 37 + -5.196 vs.68.67 + -6.888, p < 0.05). The results suggest that GSK126 pretreatment can effectively rescue hippocampal CA1 area neurons from dying after stroke.
II, GSK126 reduces cerebral infarction area after ischemia by reducing histone H3K27me3 methylation level and inhibiting H3K27me3 from rising
(1) Establishment and administration of Middle artery occlusion model (Middle artery occlusion/reperfusion, MCAO) in rat
Rats were first given a 200mg/kg/day dose of GSK126 by lateral ventricle injection for seven consecutive days. The common carotid artery, the external carotid artery and the internal carotid artery are separated bluntly after the rat is anesthetized. The common carotid artery and the external carotid artery are ligated, the internal carotid artery is clamped by an artery clamp, and a small opening is cut at the common carotid artery to insert a prepared fish line.
(2) Detection of histone H3K27me3 methylation level by Western blotting method
Histone H3K27me3 methylation level detection material rat brain hippocampus CA1 region tissue 24H after MCAO model operation is taken, hippocampus tissue is immediately cracked by RIPA lysate, protein is separated by 10% SDS-PAGE gel electrophoresis after sediment is removed by centrifugation, the separated protein is transferred to a Nitrocellulose (NC) membrane, and 3% BSA is blocked for 1H. And (3) incubating the NC membrane with the corresponding primary antibody at 4 ℃ overnight, eluting the next day, adding the corresponding secondary antibody, and incubating for 1h at room temperature. Antibodies used included H3K27me3, Histone H3. And (4) ECL developing, scanning by a fluorescence imaging system to form a graph, and expressing the relative expression content of the target protein by the ratio of optical density.
(3) Detection of cerebral infarction area of rat by TTC staining method
Taking brain, quickly freezing in refrigerator at-20 deg.C for 20min, and slicing. And placing the slices in TTC, covering with tin foil paper, and placing in a 37 ℃ incubator for 15-30 min. Effect after dyeing: white is subcortical infarction.
(4) Results of the experiment
As shown in fig. 5 and fig. 6, with Histone H3 as an internal reference, and the ratio of H3K27me3 to Histone H3 as a reference value of the methylation level of H3K27me3, it was found that GSK126 pretreatment can effectively reduce the level of H3K27me3 in rat hippocampus (Control vs. GSK 126: 1.109 ± 0.096vs.0.7935 ± 0.0309, p <0.05), and that ischemia-induced cerebral ischemia (MCAO) can effectively inhibit the methylation level of H3K27me3 in ischemia-reperfusion region (Control vs. MCAO: 1.109 ± 0.096vs.2.026 ± 0.1553, p <0.01) (MCAO vs. GSK126+ MCAO: 2.026 ± 0.1553vs. 1.1. ± 0.0882, p < 0.456), and that the difference is less than 0.05 (p < 0.05). The results suggest that GSK126 pretreatment can effectively inhibit the increase of the methylation level of H3K27me3 in the ischemic brain tissue after the ischemic brain.
TTC staining results are shown in fig. 7 and 8, and white areas shown in fig. 7 are infarcted brain tissues, and each group of infarcted middle cerebral artery (right side) showed a cerebral tissue infarct compared to non-infarcted middle cerebral artery (left side) brain tissues, but it can be seen from fig. 8 that GSK126 pretreatment was effective in reducing the cerebral infarct area caused by MCAO (control vs. GSK 126: 0.73 ± 0.013vs.0.46 ± 0.074, p < 0.05).
The animal experiments show that the GSK126 can inhibit the activity of EZH2, so that the methylation level of rat hippocampal histone H3K27me3 can be reduced, the methylation level of hippocampal histone H3K27me3 can be prevented from being excessively increased after ischemic stroke occurs, and meanwhile, the GSK126 can obviously relieve the neuron death of a hippocampal CA1 area caused by ischemia and reduce the cerebral infarction area after ischemic stroke. Shows that GSK126 can be used as an EZH2 inhibitor in the preparation of medicines for preventing and/or treating cerebral arterial thrombosis.
Claims (5)
- Application of GSK126 serving as an EZH2 inhibitor in preparation of medicines for preventing and/or treating cerebral arterial thrombosis.
- 2. Use of GSK126 as EZH2 inhibitor for the preparation of a medicament for the prevention and/or treatment of stroke, according to claim 1, for reducing the death rate of hippocampal neuronal cells and reducing the cerebral infarct size.
- 3. The use of GSK126 as an EZH2 inhibitor for the preparation of a medicament for the prevention and/or treatment of stroke, as claimed in claim 1, wherein GSK126 is used in an amount of 200 mg/kg.
- 4. A pharmaceutical preparation for preventing and/or treating ischemic stroke, comprising (1) an effective amount of GSK126 as an active ingredient, and (2) optionally a pharmaceutically acceptable carrier.
- 5. The pharmaceutical preparation for preventing and/or treating ischemic stroke according to claim 3, wherein the pharmaceutical dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup or injection.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160340674A1 (en) * | 2014-02-10 | 2016-11-24 | University Of Rochester | Compositions and Methods to Inhibit EZH2 for the Treatment of Cardiovascular Diseases |
| CN107105651A (en) * | 2014-11-06 | 2017-08-29 | 达纳-法伯癌症研究所股份有限公司 | Ezh2 inhibitor and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20160340674A1 (en) * | 2014-02-10 | 2016-11-24 | University Of Rochester | Compositions and Methods to Inhibit EZH2 for the Treatment of Cardiovascular Diseases |
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