CN114984221A

CN114984221A - Application of PP2A and activator thereof in pharmacy and marking of acute ischemic stroke

Info

Publication number: CN114984221A
Application number: CN202210579411.5A
Authority: CN
Inventors: 周煜东; 柳丹阳
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-09-02

Abstract

The invention discloses application of PP2A and an activator thereof in pharmacy and marking of acute ischemic stroke. Relates to the application of a protein phosphatase 2A activator in preparing a medicament for treating acute cerebral arterial thrombosis, the application of a PP2A activator as a biomarker and a target point of acute cerebral arterial thrombosis and the application in preparing an experimental reagent for inhibiting calcium overload of neuronal cells; the main components are the medicine of protein phosphatase 2A activator in acute cerebral arterial thrombosis, the medicine composition of the medicine for treating acute cerebral arterial thrombosis is combined with a pharmaceutically acceptable carrier to prepare a clinically acceptable preparation, and the application of the reagent for detecting the activity of PP2A enzyme in preparing the medicine for diagnosing acute cerebral arterial thrombosis is provided. The invention finds the application and principle of PP2A and an activator thereof in preventing and treating acute ischemic stroke, is used for preparing related medicaments, reagents and compositions, and is used for preparing diagnostic related reagents or kits.

Description

Application of PP2A and activator thereof in pharmacy and marking of acute ischemic stroke

Technical Field

The invention relates to application of a substance in the field of biological medicine, in particular to application of Protein phosphatase 2A (PP2A) and related substances thereof in pharmacy, marking and the like of acute ischemic stroke.

Background

The acute ischemic stroke refers to cerebral blood circulation disorder and cerebral blood perfusion decline caused by sudden attack, so as to cause ischemic injury caused by cerebral ischemia and anoxia, and comprises transient whole cerebral ischemic stroke caused by sudden cardiac arrest caused by reasons such as drowning, shock, heart failure and the like, and focal ischemic stroke caused by local cerebral embolism, and has the characteristics of high morbidity, high recurrence rate, high disability rate and high mortality rate. The acute ischemic stroke accounts for 69.6 percent of people suffering from stroke in China, has a trend of disease youthfulness, is one of main diseases harmful to public health, and therefore has great significance for preventing and treating the acute ischemic stroke.

According to the Chinese clinical management guideline for cerebrovascular diseases, acute ischemic stroke patients receive reperfusion therapy such as drugs and/or intravascular therapy in an urgent way, which is an effective method for improving clinical outcome. Reperfusion therapy protocols are however only applicable in cases that are in line with clinical treatment indications. More importantly, reperfusion therapy often causes ischemia reperfusion injury, further aggravates pathological damage of the nervous system, increases the death rate of patients, and even survivors often leave severe neurological and mental disorders such as paralysis, mental retardation and the like. Therefore, relieving the nerve injury accompanying and secondary to acute ischemic cerebral apoplexy reperfusion therapy is an urgent problem to be solved, and no effective means for relieving the ischemic reperfusion injury exists clinically at present.

The cellular calcium overload induced by cerebral ischemia-reperfusion injury is a core event causing neuronal cell death and also a key problem for curing acute ischemic stroke. Although the importance and significance of inhibiting the neuronal calcium overload on the treatment of acute ischemic stroke are widely accepted, the application of targeted inhibition of the neuronal calcium overload in the treatment of acute ischemic stroke at present has not been satisfactorily developed. In addition, the neuronal cell calcium overload is the result of the synergistic action of a plurality of calcium ion channel proteins and regulatory factors, and is closely related to the course development of the acute ischemic stroke, so that the accurate inhibition of the neuronal cell calcium overload at a proper time becomes a new strategy for preventing and treating the acute ischemic stroke.

Ischemia reperfusion causes calcium ion-calmodulin-dependent protein kinase II (Ca2+/calmodulin-dependent protein kinase II, CaMK II) to be activated by rapid autophosphorylation, so as to promote the permeation of glutamate receptors to calcium ions and aggravate calcium overload, and inhibition of phosphorylation of CaMK II can effectively inhibit calcium overload to protect neuronal cells. The phosphorylation state of CaMK II is dynamically and precisely regulated by protein phosphatase 2A (PP 2A). PP2A is one of the main performers for dephosphorylation in brain, and the application of PP2A has focused on the field of tumor therapy, for example, deletion or inactivation of PP2A promotes tumor development and metastasis; the PP2A activator can penetrate blood brain barrier, has strong in vivo antitumor activity, and has no obvious toxicity. However, the change rule of PP2A after acute ischemic stroke is not clear, and the action and mechanism of PP2A activator in the prevention and treatment process of acute ischemic stroke are not reported.

Disclosure of Invention

Aiming at the defects in the prior art, the first purpose of the invention is to provide the application of the protein phosphatase 2A (PP2A) activator in the treatment of acute ischemic stroke and the preparation of the medicine for treating acute ischemic stroke. The second purpose of the invention is to provide application of PP2A enzyme activity as a treatment target of acute ischemic stroke, and a medicament or a compound for enhancing PP2A enzyme activity can be used for treating acute ischemic stroke. The third purpose of the invention is to provide the application of the PP2A enzyme activity as a biological marker for diagnosing, evaluating the course progress of the early acute ischemic stroke and the clinical treatment effect.

The technical scheme of the invention is as follows:

the application of the protein phosphatase 2A comprises the following steps:

the application of the polypeptide as a biomarker and a target of acute ischemic stroke.

Secondly, the application of a protein phosphatase 2A activator:

the protein phosphatase 2A activator is a compound which directly or indirectly acts on the protein phosphatase 2A, improves the enzyme activity of the protein phosphatase and generates physiological action in the preparation of the medicine for treating acute cerebral arterial thrombosis.

Generally, the protein phosphatase 2A activator is used for resisting tumor and preparing antitumor drugs, and the invention particularly finds that the protein phosphatase 2A activator can be used for treating acute ischemic stroke, and further provides the application of preparing the drugs for treating acute ischemic stroke.

The application in preparing the medicine for treating acute ischemic stroke.

The drug specifically aims at enhancing the enzymatic activity of protein phosphatase 2A so as to play a role in treating acute ischemic stroke.

The acute ischemic stroke is transient global cerebral ischemia or cerebral ischemia caused by sudden cardiac arrest due to drowning, shock and other reasons, and focal stroke caused by cerebral embolism.

The acute ischemic stroke is mainly characterized by cerebral edema, cerebral infarction, neuronal apoptosis, motor function and cognitive dysfunction and the like.

The protein phosphatase 2A activator is one or the mixture of DT061 and iHAP 1;

DT061 is N- [ (1R,2R,3S) -2-hydroxy-3-benzoxazine-10-cyclohexyl ] -4- (trifluoromethoxy) benzenesulfonamide, CAS No.1809427-19-7, the molecular formula is as follows:

iHAP1 is 2-chloro-10- (4-methoxybenzoyl) -10H-phenothiazine, CAS No.105925-39-1, of the formula:

thirdly, the application of a protein phosphatase 2A activator is characterized in that:

the protein phosphatase 2A activator is applied to the preparation of an experimental reagent for inhibiting the overload of neuronal calcium.

The mechanism of action of the protein phosphatase 2A activator is to inhibit neuronal cell calcium overload. The protein phosphatase 2A activator can be used for treating acute ischemic stroke by inhibiting calcium overload of neuronal cells.

The protein phosphatase 2A activator can enhance enzyme activity of protein phosphatase 2A in neuron cell, and promote calcium ion-calmodulin dependent protein kinase II (Ca) ²⁺ The CALMODULIN-dependent protein kinase II, CaMK II) dephosphorylates, promotes the migration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, AMPA) receptor GluA1 subunit to a postsynaptic membrane, reduces the permeability to calcium ions and inhibits the calcium overload of neuronal cells.

Fourthly, a medicine for acute ischemic stroke, the main component of which comprises a protein phosphatase 2A activator.

And fifthly, a medicine for acute cerebral arterial thrombosis: the medicine is used for treating acute ischemic stroke. The drug targets protein phosphatase 2A in activated neuronal cells.

Preferably, the medicine is an oral medicine or a gastric perfusion medicine.

The drug increases the enzymatic activity of protein phosphatase 2A in neuronal cells without affecting the amount of protein expression of protein phosphatase 2A.

The medicine can inhibit calcium overload of neuronal cells by enhancing enzyme activity of protein phosphatase 2A to treat acute ischemic stroke.

The medicine targets protein phosphatase 2A in neuronal cells, enhances the enzyme activity of the protein phosphatase 2A, promotes the dephosphorylation of CaMKII, inhibits the permeation of AMPA receptors to calcium ions, relieves the calcium overload of the neuronal cells, and plays a role in treating acute ischemic stroke.

The treatment of the medicament includes reducing at least one of cerebral edema, cerebral infarction, neuronal apoptosis, motor function and cognitive dysfunction after acute ischemic stroke.

Sixthly, a pharmaceutical composition, which comprises the acute ischemic stroke medicament and a clinical acute ischemic stroke treatment medicament.

The medicine for clinically treating acute ischemic stroke comprises recombinant tissue plasminogen activator, urokinase and tenecteplase.

The therapeutic effect of the pharmaceutical composition includes reducing at least one of cerebral edema, cerebral infarction, neuronal apoptosis, motor function and cognitive dysfunction after ischemic stroke.

Seventhly, a pharmaceutical preparation, which comprises the medicine or the pharmaceutical composition for acute cerebral arterial thrombosis and is a clinically acceptable preparation prepared by combining the medicine or the pharmaceutical composition with a pharmaceutically acceptable carrier.

The preparation comprises tablets, capsules, granules, injections, pills, powders and various microparticle administration systems.

The application of one reagent in preparing a preparation or a kit for diagnosing acute ischemic stroke is characterized in that the reagent is used for detecting the activity of PP2A enzyme and is used for detecting the unit enzyme activity change of PP 2A.

The detection object of the reagent is a neuron cell.

The enzyme activity is the ratio of the total PP2A enzyme activity of the sample to the concentration of PP2A protein.

The reagent is especially used for detecting and diagnosing the condition of the early course of acute ischemic stroke, and the early course of the disease is 0.5 to 8 hours after the whole cerebral ischemia reperfusion.

The expression of PP2A is unchanged in early stage after acute cerebral ischemic stroke, but the activity of PP2A enzyme presents the change characteristics of ascending before descending, the activity of PP2A enzyme is reduced, the damage of neurons is aggravated, and the activity of PP2A enzyme in the neurons is detected to judge the progress of the disease course or the clinical treatment effect.

In specific implementation, the PP2A activator is introduced into cells, and the activity of PP2A enzyme in neuronal cells is used as a detection object to detect and obtain the effects of treating and preventing acute ischemic stroke of the PP2A activator.

The invention relates to application of a PP2A activator in pharmacy and marking of acute ischemic stroke. The invention finds that the PP2A activator has good treatment effect in a transient whole-brain ischemic stroke model of a mouse and a focal ischemic stroke model of the mouse by improving the activity of the PP2A enzyme, and particularly shows that the PP2A activator can relieve cerebral edema, cerebral infarction, neuronal apoptosis, motor function and cognitive dysfunction caused by acute ischemic stroke, the action mechanism of the activator is that calcium overload of neuronal cells is inhibited by enhancing the activity of the PP2A enzyme, so that the PP2A enzyme activity can be used as a treatment target, and the medicament or compound for enhancing the activity can be used for treating acute ischemic stroke. Further, after the acute ischemic stroke, the activity of PP2A enzyme of the cerebral cortex of the mouse dynamically changes along with time, and the activity can be used for judging and evaluating the course development and the drug treatment effect of the acute ischemic stroke. Based on the method, the invention provides the application of the PP2A activator in pharmacy and marking of acute ischemic stroke, and the application of PP2A enzyme activity as an acute ischemic stroke treatment target and a biomarker.

The invention has the advantages that:

1. the invention discovers that the PP2A activator shows good therapeutic action in various acute ischemic cerebral apoplexy models, and can relieve model animal modeling cerebral edema, cerebral infarction, neuronal cell apoptosis, motor function and cognitive dysfunction by inhibiting calcium overload of neuronal cells, so the PP2A activator can be used for preparing the medicine for preventing and treating acute ischemic cerebral apoplexy.

2. The invention also discovers that the enhancement of the enzyme activity of PP2A in the neuron cells can inhibit calcium overload caused by acute ischemic stroke, and the calcium overload is one of the core pathological mechanisms of the acute ischemic stroke. Therefore, the activity of the PP2A enzyme can be used as a therapeutic target for acute ischemic stroke. The PP2A enzyme activity is negatively correlated with pathological characteristics such as neuronal apoptosis and cognitive disorder caused by acute ischemic stroke, and the medicine or compound for enhancing the PP2A enzyme activity in neuronal cells can inhibit calcium overload, so that the medicine or compound can be used for treating acute ischemic stroke.

3. The invention discovers the change characteristics of the PP2A enzyme activity and the expression level in the early stage of acute ischemic stroke, namely the characteristics that the PP2A enzyme activity is firstly increased in reactivity and then decreased in the early stage of an in-vivo and in-vitro acute ischemic stroke model. Whereas the PP2A protein level was not significantly changed. Therefore, the activity of the PP2A enzyme can be used as a biomarker for the development of the course of acute ischemic stroke and the treatment effect, and a substance for detecting the activity of the PP2A enzyme can be used for preparing a reagent or a kit for diagnosing and evaluating the development of the course of disease and the treatment effect.

Drawings

FIG. 1: the experimental flow chart of example 1 is shown. Wherein a is an experimental process of treating by using a PP2A activator after the mouse transient whole cerebral ischemic stroke is modeled by Bilateral Common Carotid Artery Occlusion (BCCAO), wherein a PP2A antagonist administration group is arranged for further proving the treatment effect of the PP2A activator; b is the experimental process of using PP2A activator to treat the mouse after the mouse focal ischemic stroke, namely the Middle Cerebral Artery Occlusion (MCAO) is modeled.

FIG. 2: a is the change in activity of PP2A in the hippocampal region of mice 4 hours after DT061 and iHAP1 administration (n-6 in each group, mean ± sd, one-way anova,. p < 0.001); b is the expression of the apoptosis marker protein Cleaved-caspase-3 in CA1 region of hippocampus of mice 4 hours after DT061 and iHAP1 are administrated; c is the relative quantitative statistics of clean-caspase-3 (Control, BCCAO and LB-100, n 11, DT061, n 14, LB-100+ DT061, n 15, iHAP1, n 16, mean ± standard deviation, one-way variance analysis, × p <0.001, × p <0.01, × p < 0.05).

FIG. 3: a is an open field experiment schematic diagram; b is the locomotor speed of the mice 7 days after treatment with or without DT061 or iHAP1 (Control group, n-16, BCCAO group, n-18, DT061 and LB-100 group, n-12, iHAP1 group, n-14, LB-100+ DT061 group, n-13, mean ± standard deviation, one-way anova, ns, no significant difference); c is the distance of movement of the mouse (data analysis as with b); c is a representative movement trajectory of the mouse, and the inner black box is the central region of the experimental device (dashed region in a).

FIG. 4: a is a Y maze experimental schematic diagram; b is spontaneous alternation response rate of mice 7 days after treatment with or without DT061 or iHAP1 (Control group, n-12, BCCAO, DT061 and LB-100 group, n-11, iHAP1 group, n-13, LB-100+ DT061 group, n-10, mean ± standard deviation, one-way anova, × p <0.001, × p < 0.05); c is the total number of arm entries in the mouse (data analysis is the same as b, where ns, no significant difference).

FIG. 5: a is a new object identification experiment schematic diagram; b is the preferential index for the mice to explore new objects 7 days after treatment with or without DT061 or iHAP1 (Control and BCCAO groups, n 10, DT061 groups, n 11, iHAP1 and LB-100+ DT061 groups, n 9, LB-100 groups, n 8, mean ± standard deviation, one-way anova, <0.01, < 0.05); and c is a representative motion track, wherein a black line represents the exploration track of the mouse on the object, and small squares at the upper left and lower right represent the old object and the new object respectively (a is a track diagram).

FIG. 6: a is expression of mouse hippocampal CA1 zone neuronal marker NeuN 7 days after treatment with or without DT061 or iHAP1 (Control group, n-9, BCCAO and DT061 group, n-17, iHAP1 group, n-13, LB-100 group, n-15, LB-100+ DT061 group, n-19, mean ± standard deviation, one-way anova, <0.001, < 0.05); b is a NeuN immunostaining pattern.

FIG. 7: a is brain water content of mice 4 hours after treatment with or without DT061 (n 6 per group, mean ± sd,. monobasic analysis,. p <0.001,. p < 0.01); b is a representative MAP2 immunostaining pattern, with infarct areas within the white dashed box; c is the volume of mouse brain infarcts (n-6 in each group, mean ± sd, unpaired bilateral t-test,. p < 0.01).

FIG. 8: a is a pole climbing experiment, a cylinder experiment and a corner experiment which are carried out 1 day after the mice are treated by DT 061; b is the total time taken for the mice to climb the pole (Control and DT061 group, n 12, MCAO group, n 10, mean ± sd, one-way anova,. p <0.001,. p < 0.01); c is the difference in the number of bilateral forelimbs used by the mice in the cylinder experiment (data analysis is the same as b); d is the number of right turns of the mouse in the corner experiment (data analysis same as b).

FIG. 9: a is a representative movement track of a mouse in an open field experiment; b is the distance traveled by the mice 7 days after treatment with or without DT061 (Control group, n-12, MCAO group, n-17, DT061 group, n-16, mean ± standard deviation, one-way anova, ns, no significant difference); c is the speed of movement of the mice (data analysis as in b).

FIG. 10: a is spontaneous alternation response rate in Y maze experiments in mice 7 days after treatment with or without DT061 (Control group, n-20, MCAO and DT061 group, n-11, mean ± standard deviation, one-way anova,. p < 0.01); b is the total arm entry number of the mice (data analysis is the same as b, where ns, has no significant difference).

FIG. 11: a is a representative trajectory of a mouse in a new object discrimination experiment; b is the preferential index for the new object exploration by mice 7 days after treatment with or without DT061 (Control and MCAO panel, n 21, DT061 panel, n 14, mean ± sd, one-way anova,. p < 0.05).

FIG. 12: the experimental flow chart of example 2 is shown. Wherein, a is a process for investigating the treatment effect and action mechanism of DT061 or iHAP1 in a primary mouse cortical neuron OGD model, and the action of a PP2A activator is verified by using a PP2A antagonist; b is the primary mouse cortical neuron infected by the lentivirus loaded with the CAMK II alpha-T286 site autophosphorylation or dephosphorylation mutation plasmid, is used for verifying the treatment effect and action mechanism of DT061 or iHAP1, and verifies the action of a PP2A activator by using a PP2A antagonist; and c is the plasmid information map of lentivirus loading.

FIG. 13: a is the effect of DT061 or iHAP1 treatment on the enzyme activity of primary neuronal cells PP2A (n ═ 5 in each group, mean ± sd, × p <0.001, × p < 0.05); b is primary neuronal cell viability following DT061 or iHAP1 treatment (n-5 per group, mean ± standard deviation, one-way anova,. p <0.001,. p <0.01,. p < 0.05); c is the activity of Lactate Dehydrogenase (LDH) in the medium after DT061 or iHAP1 treatment (n ═ 6 in each group, mean ± sd, one-way anova,. p <0.001,. p <0.01,. p < 0.05).

FIG. 14: a is the effect of DT061 or iHAP1 treatment on the expression of p-CaMK II and CaMK II in primary neurons modelled by OGD; b is the ratio of p-cammkii to cammkii after DT061 or iHAP1 treatment (n-5 in each group, mean ± sd,. p <0.001,. p <0.01,. p <0.05 for one-way variance analysis); c expression of glu a1 on cell membranes (using glu a1 antibody that specifically recognizes transmembrane fragments) after DT061 or iHAP1 treatment (n-18 in each group, mean ± sd, one-way anova,. p <0.001,. p < 0.01); d is a representative graph of GluA1 expression.

FIG. 15 is a schematic view of: a is primary neuronal cell viability after treatment with DT061 or iHAP1 after lentivirus infection (no significant difference in n-5, mean ± sd, one-way anova, × p <0.001, × p <0.05, ns per group); b is the activity of LDH in the medium after DT061 or iHAP1 treatment (n ═ 6 per group, assay as in a).

FIG. 16: a is the expression of p-CaMK II and CaMK II after treatment with DT061 or iHAP1 of primary neurons infected with lentivirus; b is the ratio of p-CaMK ii to CaMK ii after DT061 or iHAP1 treatment of lentivirus infected primary neurons (n-4 in each group, mean ± sd, one-way anova,. p <0.001,. p <0.01, ns, no significant difference); c expression of glu a1 on primary neuronal cell membranes infected with lentivirus after DT061 or iHAP1 treatment (n-18 in each group, mean ± sd,. p <0.001,. p <0.01, ns, no significant difference); d is a representative graph of lentivirus (mGFP) and GluA1 expression.

FIG. 17: the experimental flow chart of example 3 is shown. a represents the detection of PP2A protein expression and enzyme activity, and the expression of clear-caspase-3 at different time points after the BCCAO of mice.

FIG. 18 is a schematic view of: a is a clean-caspase-3 immunostaining pattern of mouse cerebral cortex at different time after BCCAO; b is relative quantitative statistics of clean-caspase-3 (NC group, n-14, 1h, n-13, 2h, n-16, 4h, n-19, 8h, n-20, mean ± sd, unpaired independent sample two-sided t-test with NC group, p <0.001, p < 0.01).

FIG. 19: a is the activity of PP2A enzyme of the cerebral cortex of the mouse at different time after BCCAO (n is 4 in each group, the average value is +/-standard deviation, and unpaired independent sample double-side t test is carried out on the n and the NC groups respectively, and p is less than 0.01); b is the protein expression of PP2A in the immunoblot experiment; c is semi-quantitative statistics of PP2A protein expression in immunoblot experiments (each group n is 5, mean ± standard deviation, unpaired independent sample bilateral t-test with NC group, respectively, ns, no significant difference); d is the expression of PP2A protein in enzyme-linked immunoassay (data analysis as in c).

Detailed Description

The invention is further described with reference to the accompanying drawings and the detailed description.

The examples of the invention are as follows:

first, experiment method

1. Drug configurations and dosing regimens.

Animal experiments, DT061 is prepared into 100mg/mL mother solution by DMSO, diluted by normal saline and administered by gastric lavage according to the dosage of 1 mg/kg; the iHAP1 was gavaged with 0.5mg/kg of 10% DMSO + 90% corn oil as the solvent, and two kinds of solvents were gavaged at random in the same number of mice in the control group. In addition, BCCAO experiment set PP2A inhibitor-administered group confirmed the therapeutic effect of DT061, LB-100 was formulated at 1. mu.g/mL with artificial cerebrospinal fluid (aCSF), 1. mu.L (0.5. mu.L per side) was administered to each mouse via cannula for 1 hour, then mouse DT061(1mg/kg) was administered via gavage and BCCAO molding was performed, and the remaining groups of mice were injected with an equal volume of aCSF via cannula. In the cell experiment, DT061 and iHAP1 were prepared in DMSO as 100. mu.M and 50. mu.M mother solutions, respectively, and then diluted in neuronal medium to final concentrations of 1. mu.M and 0.5. mu.M, respectively, and LB-100 was prepared in neuronal medium to final concentration of 3.5. mu.M. After LB-100 treated primary neurons for 1 hour, DT061 or iHAP1 was added while OGD molding was performed, and the drug was continuously present during and 30 minutes after molding.

2. Mouse acute whole brain ischemic stroke is a Bilateral Common Coronary Artery Occlusion (BCCAO) model. Mice were induced and maintained under anesthesia with 3% and 1.5% isoflurane, respectively. The mouse was fixed in the supine position, the electronic thermometer probe was inserted into the anus of the mouse 1 cm, and the body temperature was maintained at 38.0 + -0.2 deg.C using a heating pad and heating lamps. Removing hair from anterior cervical region of mouse, cutting skin of mouse at 1.5 cm in front of neck, separating bilateral common carotid artery, clamping with artery clamp, and releasing the artery clamp after 10 min ischemia to allow blood to flow again. Finally, the tissue site is restored, and the wound is sutured and sterilized. After the mice were awakened, they were returned to normal breeding environment and provided with water and food. Mice in the sham (Control) group underwent the procedures described above except for occluding the blood vessel.

3. Mouse focal ischemic stroke is a model of Middle Cerebral Artery Occlusion (MCAO). Anesthesia, preoperative preparation and postoperative care were as in method 2. The difference is that the external carotid artery and the internal carotid artery of the mouse are separated, an 8-0 nylon thread plug is inserted into the internal carotid artery from the external carotid artery on the right side for about 0.9 cm until the initial part of the middle cerebral artery, and after 60 minutes, the thread plug is taken out to restore the blood flow to be reperfused.

4. The bilateral hippocampal CA1 region sleeves were embedded in the mice. Mice were induced and maintained under anesthesia with 3% and 1.5% isoflurane, respectively. The mouse is fixed on a brain stereotaxic apparatus in a supine position, hairs on the surface of a skull are removed, the skull epidermis is cut, and attached mucous membrane tissues are wiped off by dry cotton. The height of the brain tissue is adjusted to ensure the left and right level and the front and back level to be consistent. Bilateral hippocampal CA1 regions (ML ═ 1.15, AP ═ 1.83, DV ═ 1.45) and 2-3 fixation points distant from the suture were located on the surface of the skull by means of stereomicroscope according to mouse brain atlas, and carefully drilled, where the miniscrew was screwed in. The double cannula was then drilled through the CA1 area on both sides and slowly lowered into the target nucleus. After the surface of the mouse skull is dried, the inserting core needle and the screw are lightly fixed on the surface of the mouse skull by medical dental cement, after the dental cement is solidified, the mouse is taken down, and after the mouse recovers for 7-10 days, an experiment is carried out.

5. And (4) performing immunofluorescence staining. Mice were deeply anesthetized with isoflurane, their four limbs were fixed, the heart was exposed, blood was flushed with normal saline through the right atrium, then perfused with 4% paraformaldehyde solution, and the brains were carefully removed and fixed in 4% paraformaldehyde solution. Dehydrating with 30% sucrose, embedding brain tissue in the frozen sliced tablet, placing in a constant-cooling box, quickly freezing for 30 min, and then obtaining a frozen slice of the coronary brain tissue with the size of 30 μm of the target brain area. Frozen sections were blocked with 10% normal goat serum for 2 hours at room temperature, and clear-caspase-3, MAP2 or NeuN primary antibody was added as needed and incubated overnight in a refrigerator at 4 ℃. The next day, sections were washed, and Alexa-546 labeled secondary antibody was incubated at room temperature for 1.5 hours, and after another wash, mounted with an anti-fluorescence quencher. And (4) taking a fluorescence photo by using a laser confocal microscope, counting the fluorescence staining intensity by ImageJ, and correcting the fluorescence staining intensity by using the average fluorescence intensity of a control group.

6. The serine/threonine co-immunoprecipitation kit detects the activity of the PP2A enzyme. Mouse cerebral cortex or cultured primary neuronal cells were collected in 1.5mL EP tubes, added with 300. mu.L of a binding buffer containing protease and phosphatase inhibitors (50mM Tris, 150mM NaCl, 0.5% Triton X-100, pH 7.5), sonicated, placed on ice, allowed to stand for 10 minutes, then centrifuged at 14,000 Xg for 10 minutes at 4 ℃, and the supernatant was collected for protein quantification and the total protein concentration of each sample was adjusted to be uniform. 50 μ L of magnetic beads (100 μ g) were placed in a 1.5mL EP tube, washed twice with buffer, added to a final concentration of 4 μ g of PP2A (05-421, Sigma-Aldrich) primary antibody, mixed well at room temperature, and after 15 minutes the antibody was removed and the beads washed. Adding 200 μ L protein extract, and mixing at room temperature for 1 hr to combine antigen and antibody. Washing the magnetic bead-antibody-antigen complex three times by using buffer solution, and finally uniformly transferring the complex into two new EP tubes, wherein one tube is added with an enzyme substrate to detect the enzyme activity of PP2A, and the other tube is used for detecting the protein concentration of PP2A by using an enzyme-linked immunosorbent assay, and the final result is represented as:

7. the water content of the brain of the mouse is detected as encephaledema. Mouse meridian CO ₂ After the death of the anesthesia, brain tissue is taken, the injured lateral half brain is separated, and the wet weight and the dry weight after drying for 48 hours at 100 ℃ are respectively weighed, and the cerebral edema calculation formula is as follows:

8. cerebral infarction area test. Collecting 6 brain tissue sections at the same position at the same interval, obtaining MAP2 fluorescence staining pictures according to the method 5, counting the positive staining area of MAP2 on the non-infarction side by ImageJ, subtracting the positive staining area of MAP2 on the infarction side to obtain the infarction area, and obtaining the infarction volume according to the 6 brain tissue sections.

9. Behavioral experiments in mice. Examination of the mouse motor ability: the pole climbing experiment is used for inspecting the balance adjustment capability and limb coordination capability of a mouse, an experimental device is a vertical long rod with a rough surface, the length of the long rod is 50 cm, the diameter of the long rod is 8 cm, the vertical long rod is erected in a cage where the mouse lives, the head of the mouse is placed upwards at a position close to the top end, the time of the mouse in starting turning and climbing down along the pole is recorded, if pole climbing cannot be completed, 120 seconds are recorded, and the average value is obtained after 3 times of testing of each mouse. Cylinder experiment, consider the asymmetric use of the limbs of a mouse, which is a transparent plexiglass cylinder (15 × 20 cm), place the mouse into the cylinder and record the number of times the mouse stands in contact with the cylinder wall using the left (L), right (R) and bilateral (B) forelimbs within 5 minutes. Mice normally tend to use bilateral upper limb touches to climb the cylindrical wall to explore behavior, but the rate of mice using non-injured lateral upper limbs is significantly increased after MCAO modelling. And (3) performing corner experiments, inspecting the symmetric sensory and motor defects of the mice, wherein the experimental device consists of two walls with an included angle of 30 degrees, placing the mice at the tips of the two walls, and recording the times that the mice turn to the side, which is not damaged, of the body from the corner in 10 experiments. Open field experiments, reflecting the locomotor ability of the mice, the experimental setup was a square box (40 × 40 cm) with a middle area of 20 × 20 cm drawn in the middle. The mice were placed in the middle area and allowed to run freely for 5 minutes, and the distance and speed of movement were counted. The cognitive function of the mice was investigated: each group of mice was tested for cognitive function on the premise that there was no difference in distance and speed of movement in the open field experiment. The Y maze inspects the motor ability and the working memory of the mouse, and the experimental device is composed of three equal-length arms to form a 'Y' shape, namely A, B, C three arms. Placing a mouse at the tail end of the arm A, recording the sequence of the mouse entering the three arms and the total times of arm entering within 5 minutes, continuously entering the three different arms to be correct alternating reverse (alternation), and calculating the spontaneous alternating reaction rate when the total times of arm entering of each group of mice are not different, wherein the formula is as follows:

the experimental device is a square box (40 multiplied by 40 cm), after the mouse adapts to the environment for 5 minutes, two completely identical objects which are not seen by the animal are placed at the position equidistant from the edge, the time for the mouse to search the two objects within 5 minutes is recorded, then one object is changed into a new object which has different colors, shapes and materials but the same size, after 1 hour, the mouse is placed again, and the time for the mouse to search the two objects (T) is recorded _{Old age} 、T _New ) The Preferential index (Preferential index) of the mouse for the new object exploration is calculated by the formula:

10. and (3) culturing primary cortical neurons. C57BL/6J mice were anesthetized with ether for 18 days of pregnancy, the mother mice were quickly sacrificed and the fetal mice were removed, sterilized with alcohol and placed in Duchen buffer. Clean skin layers were separated under a dissecting scope and transferred to a 15mL centrifuge tube. 0.25% pancreatin was added and digested in a thermostatic water bath at 37 ℃ for 12 minutes. The digestion was stopped by adding 10% serum, centrifuged at 1,000 Xg for 5 minutes at room temperature, and the cell pellet was gently resuspended in neuronal culture. Neuronal cells were seeded into petri dishes or well plates previously coated with polylysine, with half a day of replacement with fresh medium every 4-5 days. Neurons were cultured in vitro for 10 days before use in the experiment.

11. Primary cortical neuron oxygen-glucose-deprivation (OGD) model. Sucking out the original neuron cell culture solution in the culture dish or the pore plate, replacing the original neuron cell culture solution with the sugar-free serum-free culture solution subjected to the anoxic treatment, placing the culture solution into a Billups-Rothenberg cell anoxic chamber, culturing the cell culture solution in an incubator with 95% N2-5% CO2 and 37 ℃ for 30 minutes, and then recovering to a normal culture medium and culture conditions of 95% air and 5% CO 2.

12. Cell viability. Cell counting kit (CCK8) can be reduced by dehydrogenase in mitochondria to generate water-soluble orange-yellow formazan products, the color depth after reaction is in direct proportion to Cell proliferation and in inverse proportion to cytotoxicity, and the quantity of living cells is indirectly reflected. Primary neuronal cells were cultured in 96-well plates, and at the appropriate time after molding or dosing, the original medium was removed, 100 μ L of medium containing 10% CCK8 was added to each well, and a blank set without cells was set. The well plate was incubated in an incubator for 2 hours, and the absorbance value (A) at a wavelength of 450 nm was measured using a microplate reader. The cell viability calculation formula is:

13. lactate Dehydrogenase (LDH) activity in cell culture medium. LDH is a stable cytoplasmic enzyme that normally cannot permeate the cell membrane. When cells are damaged the cell membrane leaks and LDH is released outside the cell. Detection of LDH enzyme activity in cell culture medium was used to assess the extent of cell damage. In the experiment, a standard curve is made by using a standard substance provided by the kit, and a positive control hole is arranged. Cell supernatants were harvested, centrifuged at 1,000 Xg for 5 minutes at room temperature to remove suspended cells, and 0.05mL of cell supernatant was added per well followed by 50. mu.L of assay buffer containing 4% LDH substrate. The absorbance at the wavelength of 450 nm was measured with a microplate reader at 5 minute intervals until the maximum absorbance approached the maximum of the standard curve. The concentration of LDH (C nmol) was calculated from the standard curve, and the test time interval (. DELTA.T), the activity of LDH in cell culture medium (LDH activity) was calculated according to the formula:

14. western blot experiment. After anesthetizing with CO2, mice were rapidly decapitated to harvest brains, cerebral cortex was separated, primary cultured cortical neurons were harvested, and cell masses were obtained by centrifugation at 1,000 Xg for 5 minutes. For Western blotting, a RIPA lysate containing Cocktail, a protease inhibitor, was added at a ratio of 1:10(10mL/mg) to prepare a homogenate on ice, which was centrifuged at 12,000 Xg for 20 minutes at 4 ℃. Taking 10 mu L of supernatant, carrying out protein quantification according to the BCA kit instruction, diluting the sample to the same concentration, adding a loading buffer solution, and carrying out boiling water bath for 5 minutes to fully denature the protein. Naturally cooling and storing in a refrigerator at-80 deg.C. 4-12% SDS-PAGE gel was placed in an electrophoresis tank, and after adding running buffer, 30. mu.g of protein was added to each well, followed by electrophoresis at 160V. The wet transfer method transfers the proteins on the gel to a PVDF membrane. PVDF membrane with 5% skimmed milk powder-PBS closed liquid temperature after 2 hours of closed incubation primary antibody, placed in 4 degrees C refrigerator overnight 7-12 hours. Horseradish peroxidase-labeled secondary antibody was incubated at room temperature for 1.5 hours. And (3) after the PVDF membrane is cleaned again, adding ECL luminous liquid, and developing by using a gel imaging system method. The results were analyzed using ImageJ software.

15. Immunocytochemistry experiments. Primary neuronal cells for immunocytochemical staining experiments were cultured on polylysine coated slides. The slide was washed once with PBS and blocked with 10% normal donkey serum for 2 hours at room temperature. GluA1(MAB2263, Millipore, which specifically recognizes the GluA1 transmembrane fragment) was incubated for 1 hour at room temperature. The primary antibody was washed gently with PBS and Alexa-546 labeled secondary antibody was incubated for 1 hour at room temperature in the dark. And (3) lightly washing the secondary antibody in the dark, sealing the secondary antibody by using a DAPI sealing agent, taking a fluorescence photo by using a laser confocal microscope, analyzing the fluorescence intensity of the image by using ImageJ software, and correcting the fluorescence intensity by using the mean value of the fluorescence intensity of a control group.

16. And (5) virus infection experiments. The 5 th day of primary neuron cell culture is the appropriate infection time. One half of the original medium was aspirated and stored aseptically, and then lentivirus was added at a multiplicity of infection of 10. Lentivirus infection for 8-12 hours, discard the medium, then add rapidly the other half of the original medium collected before infection, which was preheated. The virus-containing medium can not be directly discarded, and the inactivation operation should be carried out first. After further culturing for 5 days, the infection condition of the empty vector lentivirus infection group only expressing mGFP is observed under a fluorescence microscope, and a protein sample is collected for subsequent experiments.

17. Enzyme-linked immunosorbent assay. The method comprises the steps of preparing a standard curve by using a PP2A standard substance matched with a PP2A specificity enzyme-linked immunosorbent assay kit, adding a sample with unknown concentration into a micropore ELISA plate, incubating with a biotin-labeled antibody, adding avidin-labeled horseradish peroxidase, incubating and washing, adding a chromogenic substrate, reading the absorbance of the sample by using an ELISA reader, and detecting the protein concentration of PP2A in the sample by using the standard curve.

Second, experimental results

DT061 and iHAP1 have therapeutic effects in BCCAO model mice (FIGS. 1-6).

The experimental design is shown in figure 1.

The results of detecting the activity of the PP2A enzyme and the apoptosis of neurons are shown in figure 2, and DT061 and iHAP1 after being administrated for 4 hours remarkably enhance the activity of the PP2A enzyme in the hippocampal region of a mouse and reduce the expression of a hippocampal CA1 region apoptosis marker protein clear-caspase-3 caused by BCCAO molding. The above-described effects of DT061 were blocked by the inhibitor LB-100 of PP2A, and LB-100 administration significantly increased the expression of cleared-caspase-3 following BCCAO.

Open field experiments are shown in fig. 3, where it can be seen that the locomotor ability of the mice was not changed at day 7 after BCCAO modelling.

Y maze experiment As shown in FIG. 4, it can be seen that DT061 and iHAP1 treatments significantly improved working memory impairment due to BCCAO modeling, and this effect of DT061 was blocked by LB-100.

Experiments on new object discrimination as shown in fig. 5, it can be seen that DT061 and iHAP1 treatments significantly improved image discrimination memory impairment due to BCCAO modeling, and this effect of DT061 was blocked by LB-100.

Immunofluorescence experiment detection of neuron marker NeuN is shown in figure 6, and it can be seen that BCCAO causes a large amount of loss of neuron cells in the CA1 region of the hippocampus compared with control mice, DT061 and iHAP1 treatment can remarkably improve the loss phenomenon of neuron cells in the CA1 region of the hippocampus of mice, and the effect of DT061 is blocked by LB-100.

DT061 had therapeutic effects in MCAO model mice (FIGS. 7-11).

The detection of the water content and the cerebral infarction area of the mouse brain is shown in figure 7, and the figure shows that 4 hours after MCAO molding, the mouse brain tissue has obvious cerebral edema and cerebral infarction, and the DT061 treatment can obviously inhibit the cerebral edema and cerebral infarction caused by MCAO.

The behavioral experiment for detecting the motor ability and the cognitive ability of the mouse is shown in figure 8, and the results show that the motor ability of the mouse is damaged when a pole climbing experiment, a cylinder experiment and a corner experiment are carried out 1 day after MCAO (micro channel occlusion) modeling, and DT061 treatment can obviously improve the mouse balance regulation and limb coordination imbalance, symmetric sensory motor defect and limb motor asymmetry caused by MCAO.

Open field experiments are shown in figure 9, where it can be seen that the locomotor activity of the mice did not change on day 7 after MCAO modelling.

The Y maze experiment is shown in fig. 10, where it can be seen that DT061 treatment significantly improved working memory impairment due to MCAO modelling.

The new object identification experiment is shown in fig. 11, and it can be seen that DT061 treatment can significantly improve image identification memory impairment caused by MCAO modeling.

DT061 and iHAP1 have therapeutic effects on primary neuronal cells modeled by OGD (FIGS. 12-16).

The experimental design is shown in fig. 12.

The results of the detection of the enzyme activity, cell viability and LDH viability in cell culture medium of PP2A are shown in fig. 13, where it can be seen that DT061 and iHAP1 treatment significantly enhanced the enzyme activity of PP2A in primary cortical neuronal cells and reduced the decrease in cell viability and the enhancement of LDH viability in culture medium caused by OGD molding. The above-mentioned effects of DT061 are blocked by LB-100, and the administration of LB-100 significantly inhibits the enzymatic activity of PP2A and aggravates cell damage.

Results of detecting CaMKII phosphorylation by a protein immunoblotting experiment and detecting GluA1 level by an immunocytochemistry experiment are shown in FIG. 14, and it can be seen that pT286-CaMKII phosphorylation level and GluA1 distribution on cell membranes are significantly increased compared with a control group after primary neurons are subjected to OGD modeling. DT061 and iHAP1 treatment significantly inhibited pT286-CaMKII phosphorylation levels and GluA1 distribution on cell membranes. The above-mentioned effects of DT061 were blocked by LB-100 and LB-100 administration significantly increased the level of phosphorylation of pT286-CaMKII and the distribution of GluA1 on the cell membrane after OGD modeling.

The results of cell viability assay and LDH viability in cell culture medium are shown in FIG. 15, which shows that after OGD modeling of primary neuronal cells infected with lentivirus, cell viability decreased and LDH viability in culture medium increased after infection with CaMKII autophosphorylated T286D lentivirus, at which time DT061 and iHAP1 lost their therapeutic effect. While the cell viability of the CaMKII-infected dephosphorylated T286A lentivirus was increased, the LDH viability in the culture medium was decreased, and the LB-100 treatment did not aggravate the cell damage.

Results of protein immunoblotting experiment detection of CaMKII phosphorylation and immunocytochemistry experiment detection of GluA1 level are shown in FIG. 16, and it can be seen that after primary neuron cells infected with lentivirus are OGD molded, pT286-CaMKII phosphorylation level of neurons infected with CaMKII autophosphorylation T286D lentivirus and GluA1 level on cell membranes are both significantly increased, and DT061 and iHAP1 also cannot play a role. While the phosphorylation level of pT286-CaMKII of neurons infected with CaMKII dephosphorylated T286A lentivirus and the level of GluA1 on the cell membrane were both significantly reduced, while LB-100 treatment did not affect the phosphorylation level of pT286-CaMKII and the level of GluA1 on the cell membrane.

Characterization of PP2A protein expression and enzymatic Activity after acute ischemic brain tissue (FIGS. 17-19)

The experimental design is shown in figure 17.

Immunofluorescent staining experiments are shown in FIG. 18, which shows that within 8 hours after BCCAO modeling, the expression of mouse cerebral cortex clear-caspase-3 is gradually increased along with time.

The experiment for detecting the activity of PP2A enzyme and the expression of PP2A protein is shown in figure 19, which shows that the reactivity of the activity of PP2A enzyme in the cerebral cortex of a mouse is increased within 8 hours after BCCAO molding, and the reactivity reaches the peak value within 2 hours and then is reduced. While the protein expression level of PP2A was unchanged.

Claims

1. The application of protein phosphatase 2A is characterized in that: the application of the polypeptide as a biomarker and a target of acute ischemic stroke.

2. The application of a protein phosphatase 2A activator is characterized in that: the protein phosphatase 2A activator is a compound which directly or indirectly acts on the protein phosphatase 2A, improves the enzyme activity of the protein phosphatase and generates physiological action in the preparation of the medicine for treating acute cerebral arterial thrombosis.

3. The use of a protein phosphatase 2A activator according to claim 2, wherein: the application in preparing the medicine for treating acute ischemic stroke.

4. The use of a protein phosphatase 2A activator according to claim 2, wherein:

the protein phosphatase 2A activator is one or the mixture of DT061 and iHAP 1; DT061 is N- [ (1R,2R,3S) -2-hydroxy-3-benzoxazine-10-cyclohexyl ] -4- (trifluoromethoxy) benzenesulfonamide, CAS No.1809427-19-7, the molecular formula is as follows:

5. the application of a protein phosphatase 2A activator is characterized in that: the protein phosphatase 2A activator is applied to the preparation of an experimental reagent for inhibiting the overload of neuronal calcium.

6. A medicine for acute cerebral arterial thrombosis is characterized in that: the main component comprises a protein phosphatase 2A activator.

7. The medicine for acute ischemic stroke according to claim 5, characterized in that: the drug targets protein phosphatase 2A in activated neuronal cells.

8. A pharmaceutical composition comprising the pharmaceutical agent of any one of claims 1 to 5 and a therapeutic agent for clinical acute ischemic stroke.

9. A pharmaceutical formulation characterized by: comprises the medicament as defined in any one of claims 1 to 6 or the pharmaceutical composition as defined in claim 7, and is a clinically acceptable preparation prepared by combining the medicament or the pharmaceutical composition with a pharmaceutically acceptable carrier.

10. Use of a reagent, characterized in that: the application of the reagent in preparing a preparation or a kit for diagnosing acute ischemic stroke is that the reagent detects the activity of PP2A enzyme.