CN115518161A

CN115518161A - Application of ZIP1 as epilepsy treatment target

Info

Publication number: CN115518161A
Application number: CN202211366856.1A
Authority: CN
Inventors: 李依泽; 丁冉; 张麟临; 于泳浩; 李元杰; 康佳敏; 宗琳玥; 乔丹
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2022-12-27
Anticipated expiration: 2042-11-02
Also published as: CN115518161B

Abstract

The invention discloses an application of ZIP1 as an epilepsy treatment target. The research finds that 4-AP-induced cerebral cortex epileptiform discharge is related to ZIP1 increase, the ZIP1 increase can cause reduction of GluA2 membrane protein expression, and ZIP1 knock-down can inhibit 4-AP-induced GluA2 membrane expression reduction. Thus, the present study demonstrates that ZIP1 is a critical epilepsy-related protein, and that inhibition of ZIP1 can reduce seizures and severity.

Description

Application of ZIP1 as epilepsy treatment target

Technical Field

The invention belongs to the field of biomedicine, and relates to application of ZIP1 as an epilepsy treatment target.

Background

Epilepsy is a clinically common central nervous system disease, which is a chronic brain disease with persistent epileptogenic tendency and is characterized by recurrent epileptic seizures. There are roughly five to sixty million patients worldwide, and this accounts for a large proportion of focal epilepsy, about 3500 thousands of patients. International union of epilepsy (ILAE) defined seizures in 2005 as transient seizure phenomena of signs and/or symptoms occurring due to abnormally excessive or synchronized neuronal activity in the brain, and this definition has been used up to now. The global burden of epilepsy is currently increased by 30% between 1990 and 2010, and in 2010 epilepsy patients have a disease burden higher than the sum of alzheimer's disease, dementia, multiple sclerosis and parkinson's disease, and almost ten years of time has passed, and there is apparently no trend toward a reduction in the burden of this disease. Depending on the brain region involved in the attack, various defects such as mental and cognitive disorders, autonomic nervous system disorders, and the like are exhibited, which seriously affect the life, work, and learning of the patient, and bring great pain and burden to the family and society of the patient.

The results of current studies indicate that approximately 60% of epileptic patients are caused by congenital causes, while around 40% are caused by developmental or acquired factors, such as stroke, traumatic brain injury, infection, tumors, and genetic defects. At present, the mechanism by which normal brain function progresses to seizure brain disease states is not well understood. In addition to factors such as sex and age, neurotransmitter release in the brain, neurotransmitter receptor function, ion channel function, connections between synapses, and neural circuits are important factors in the development of epilepsy. Epileptic seizures can cause damage to nervous tissues in the brain, and therefore, finding a key pathogenic mechanism of the disease has important academic value and clinical significance.

Disclosure of Invention

The present invention provides an inhibitor for preventing and/or treating epilepsy, comprising a substance inhibiting the activity of a ZIP1 protein, or a substance inhibiting or silencing the expression of a ZIP1 gene.

Preferably, the substance inhibiting the activity of the ZIP1 protein includes a substance inhibiting the synthesis of the ZIP1 protein or a substance promoting the degradation of the ZIP1 protein or a substance inhibiting the function of the ZIP1 protein.

Preferably, the substance inhibiting or silencing expression of the ZIP1 gene includes a substance interfering with expression of the ZIP1 gene or a substance knocking out the ZIP1 gene or a substance mutating the ZIP1 gene.

The substances include synthetic small molecules, chemical agents, antisense oligonucleotides, siRNA, miRNA, ribozymes, polypeptides, proteins; preferably, the polypeptide or protein includes hormones, cytokines, antibodies and fragments thereof.

The term "antisense oligonucleotide" refers to a short chain of nucleic acid (consisting of about 15 to 25 nucleotides) that has been chemically modified to have a base sequence complementary to a particular target sequence and which, upon entry into a cell, forms a duplex with the target sequence according to Watson-Crick base-complementary pairing rules.

In the present invention, "complementary" means that two nucleotides can be paired under hybridization conditions, for example, the relationship between adenine (A) and thymine (T) or uracil (U), and the relationship between cytosine (C) and guanine (G).

The term "ribozyme" refers to an RNA molecule that functions to catalyze a specific biochemical reaction.

The term "siRNA" refers to a ribonucleic acid (RNA) capable of inhibiting the expression of a target gene, including a region of a sense RNA fragment and a region of an antisense RNA fragment.

The term "miRNA" refers to a ribonucleic acid (RNA) molecule of about 21 to 23 nucleotides, widely found in eukaryotes, which regulates the expression of other genes.

In the present invention, the antisense oligonucleotide, ribozyme, siRNA or miRNA may be designed to target a gene of interest or regulatory sequence thereof, such as a gene whose expression is desired to be inhibited or its regulatory sequence, in order to inhibit or reduce its expression. The gene or its regulatory sequence targeted may be any gene or its regulatory sequence for which it is desired to inhibit or reduce its expression, such as those from pathogens or involved in cancer formation and development, particularly targeting ZIP1. The antisense oligonucleotide, ribozyme, siRNA or miRNA of the present invention can be designed according to conventional methods.

The conventional design method of siRNA can be referred to the published data of company websites such as Reynoldsa, et al, nature Biotechnology, 2004, vol.22: 326-330, amhion, qiagen, etc. The conventional design method of miRNA can be referred to in literature (Lo HL, etc., gene therapy, 2007, 14: 1503-1512), the method for selecting target sequence is similar to the design method of siRNA, for example, the designed sense strand containing target sequence and corresponding antisense strand can be replaced on pri-microRNA, so that the constructed miRNA can prevent the expression of mRNA containing target sequence. The conventional design of ribozymes can be found in the literature (HaseloffJ et al, nature, 1988, volume 334: 585-591), for example, by placing nucleotide sequences complementary to the sequence around the target sequence before and after the conserved core sequence of the ribozyme (e.g., hammerhead structure) so that the constructed ribozyme can cleave nucleic acid containing the target sequence at the target sequence. Conventional design methods for antisense oligonucleotides are described in the literature (Matveeva OV et al, nucleic acids research, 2003, vol.31: pages 4989-4994).

The term "sense strand" refers to a nucleotide strand having the same sequence as the coding strand of a gene.

The term "antisense strand" refers to a strand of an siRNA that comprises a region that is completely or substantially complementary to a target sequence. The term "complementary region" refers to a region of the antisense strand that is completely or substantially complementary to a target mRNA sequence. In the case where the complementary region is not fully complementary to the target sequence, the mismatch may be located in an internal or terminal region of the molecule. Typically, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2 or 1 nucleotide of the 5 'and/or 3' end. The portion of the antisense strand most sensitive to mismatches is referred to as the "seed region".

The antisense oligonucleotide, ribozyme, siRNA or miRNA of the present invention includes a modification product of chemically modifying a constituent part of the phosphate backbone and/or ribose and/or base constituting the antisense oligonucleotide, ribozyme, siRNA or miRNA, and the modification method is known in the art, and a modification suitable for the present invention may be selected from the group consisting of: locked Nucleic Acids (LNA), unlocked Nucleic Acids (UNA), 2 '-methoxyethyl, 2' -O-alkyl, 2 '-O-methyl, 2' -O-allyl, 2 '-C-allyl, 2' -fluoro, 2 '-deoxy, 2' -hydroxy, phosphate backbone, fluorescent probes, ligand modifications, or combinations thereof.

The ZIP1 gene knockout substance further comprises a gene editing tool for knocking out a ZIP1 gene.

Preferably, the gene editing tools include Cre-lox recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR/Cas9 technology.

Preferably, the gene editing tool is CRISPR/Cas9 technology.

CRISPR/Cas9 is a technology for specific DNA modification of targeted genes by RNA-guided nuclease Cas9 protein. The principle of the technology is that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, and the complex guides nuclease Cas9 protein to cut double-stranded DNA at a sequence target site paired with the crRNA, so that the genome DNA sequence is edited.

The present invention provides a pharmaceutical composition for preventing or treating epilepsy, comprising the aforementioned inhibitor.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable" is meant a non-toxic material that does not detract from the active ingredient. Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, a. Rgennaro eds., mack Publishing Company (1990) and handbook of Pharmaceutical excipients, 3 rd edition, a.kibbe eds., pharmaceutical Press (2000)).

Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate and the like; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, and soybean polysaccharide.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may further include any one of glycine, cysteine and glutamic acid. Saccharides include monosaccharides such as glucose, mannose, galactose, fructose, and the like; sugar alcohols such as mannitol, cellosolve, xylitol, and the like; disaccharides such as sucrose, maltose, lactose, and the like; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, etc. and their derivatives. The cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium hydroxymethylcellulose. Surfactants include ionic or non-ionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and the corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium, potassium, calcium, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylene diamine tetracetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir device. Oral administration or injection administration is preferred. The pharmaceutical composition of the invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The agents of the invention may also be used in combination with other agents for the treatment of epilepsy, and other therapeutic compounds may be administered simultaneously with the principal active ingredient (e.g., an inhibitor of ZIP 1), even in the same composition. The other therapeutic compounds may also be administered separately, in a separate composition or in a different dosage form than the primary active ingredient. A partial dose of the main ingredient (e.g., an inhibitor of ZIP 1) may be administered concurrently with other therapeutic compounds, while other doses may be administered separately.

The invention also provides the application of the inhibitor or the pharmaceutical composition in preparing a medicament for preventing or treating epilepsy.

Furthermore, the dosage forms of the medicine comprise tablets, capsules, granules, pills, dripping pills, syrups, powders, suppositories, drops, emulsions, injections, solutions or suspensions.

The invention also provides an application of ZIP1 in preparation of an epilepsia animal model.

In one embodiment, the animal model is prepared by promoting the expression level of ZIP1 in an animal by, but not limited to, introducing ZIP1 into the genome of the animal using an expression vector to overexpress ZIP1. The expression vector comprises a plasmid vector, a virus vector, a cosmid vector, a Bacterial Artificial Chromosome (BAC), a Yeast Artificial Chromosome (YAC) or other non-plasmid vectors.

The animals of the present invention include mammals, birds, and fishes.

Further, the animal is a mammal. Mammals include, but are not limited to, livestock, swine, cattle, sheep, goats, chickens, rabbits, fish, zebrafish, dogs, rats, cats. In one embodiment of the present invention, the mammal is a mouse.

Preferably, the animal model is prepared by modifying the expression level of ZIP1 in an animal.

Preferably, the animal is a mouse.

The present invention also provides a method for screening a candidate drug for the prevention and/or treatment of epilepsy, the method comprising:

(1) Treating a system expressing or containing ZIP1 with a test substance;

(2) Detecting the expression level of ZIP1 in said system;

(3) Selecting an agent that downregulates the expression level of ZIP1 as a candidate drug.

Further, the system includes a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

In an embodiment of the invention, the method of screening for a candidate drug for the treatment of epilepsy further comprises: the candidate drug obtained in the above step is further tested for its effect of treating epilepsy, and if the test compound has a significant effect of treating epilepsy, the candidate drug is indicated as a candidate drug for treating epilepsy.

The invention also provides application of the ZIP1 in screening candidate drugs for preventing and/or treating epilepsy.

Drawings

FIG. 1 shows a schematic diagram of the operation of the uterine embryo electroporation experiment; wherein, A: displaying an experimental flow chart of embryo electroporation experiment transfection exogenous AMPA receptor, and marking key operation time points; e15 represents the embryo electroporation experiment performed on the day 15 of pregnancy, P0 represents the birth date of the mouse, and P60-70 (red solid line) represents the two-photon imaging experiment period; b: a schematic diagram of a method for marking a somatosensory cortex in an embryo electroporation transfection receptor experiment; left panel, marking plasmid DNA injected into lateral ventricle; the middle diagram shows the crown view of the placement position of the electric rotating plate-shaped positive and negative electrodes; the right side figure shows the craniocerebral view of the placement position of the electric rotating plate-shaped positive and negative electrodes;

FIG. 2 shows a two-photon microscope live animal targeted fluorescence labeling patch clamp recording result chart; wherein, A: a schematic diagram of a two-photon microscope living animal targeting whole-cell patch clamp; the figure shows a water mirror (40X 0.8 NA), a recording electrode (containing a fluorescent dye Alexa 5940.1mM), fluorescence labeled neurons located at the 2 nd and 3 rd layers of the cortex, a recording incubator, and the like; b: the process is recorded by a two-photon living animal targeted fluorescence labeling neuron patch clamp. The upper two figures and the lower two figures respectively show the imaging effect when the electrode is far away from and presses the same neuron under the laser wavelengths of 910nm and 800 nm; it can be clearly shown that at 800nm wavelength, the electrodes and neuronal cell bodies are recorded, while at 910nm wavelength, the electrodes are not clearly shown;

FIG. 3 is a graph showing the results of testing protein expression of different groups of ZIP1 and GluA2 on membranes by ELISA method; wherein, A: expressing ZIP1 protein; b: gluA2 protein expression; n =10; * P <0.001, compared to group C; $ P <0.001, compared to group C after 4-AP administration; one-wayANOVA;

FIG. 4 is a graph showing the results of 4-AP injection induced cortical neuron damage, showing the range of administration of the injection electrode and the location of the monitoring electrode under the superficial cerebral cortex; wherein, A: the administration range of the injection electrode under the cerebral superficial cortex and the position of the monitoring electrode; b: reconstructing a neuron structure by using the z-stack image;

FIG. 5 is a graph showing the results of 4-AP injection in whole cell mode and in cell contact mode to induce epileptic-related action potential firing; wherein, A: (ii) a whole cell structure; b: and recording the adsorbed cells.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references. 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 commercially available. It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE ZIP1 correlation study with epilepsy

1. Experimental groups

Male C57BL6 mice, 30, 1 month old, were purchased from the experimental animals center of the military medical science institute of the people's liberation army, china. The random number table method was used to divide into 3 groups (n = 10):

control plasmid +4-AP (group C): in an embryonic E15 electroporation experiment, CRISPR/Cas9 control plasmid (SANTACRUZ, sc-418922, usa) was injected using a microinjector, right ventricle 1 μ 1, right cerebral cortex injected 4-AP induced epilepsy 6 weeks after birth;

ZIP KO +4-AP (group Z): in the embryo E15 electroporation experiment, the ZIP1CRISPR/Cas9 KO plasmid (SANTACRUZ, sc-424394, USA) was injected with a micro-syringe, the right ventricle was 1 μ 1, and the right cerebral cortex was injected with 4-AP to induce epilepsy at 8 weeks after birth.

2. Experimental methods and procedures

1. Uterine embryo electroporation

Uterine embryo electroporation is a DNA transfection technique that drives negatively charged DNA transiently through the cell membrane into the cell by electrical impulses. The experimental procedure was as follows:

(1) Uterine embryo electroporation experimental animals were housed by laboratory experienced laboratory personnel and female mice were observed daily for the presence of vaginal emboli. When the female mouse is judged to be possibly pregnant, the female mouse is singly raised. The day of observation of the mouse vaginal embolus was defined as day 0 of the embryo (E0). The time for the embryo electroporation experiment was E15, see FIG. 1.

(2) Before the experiment, all surgical instruments are sterilized under high pressure, and the experiment operation is ensured to be carried out in an aseptic environment.

(3) The pregnant mouse is anesthetized and induced by isoflurane (1-1.2%), the head and four pregnant mice are fixed on a heating blanket, the anal temperature of the pregnant mouse is maintained between 37 ℃ and 38 ℃, and the breathing frequency is 90-120 times per minute. The abdomen was depilated, the skin was disinfected, the uterine horn was cut open at approximately 1cm of the abdominal wall, and the embryos were carefully lifted out of the abdominal cavity.

(4) A micro syringe pump, 1. Mu.l of plasmid fluid containing Fast Green was injected into the right ventricle. A pulsed voltage 35V for E15 (50ms on,950ms off, 1HZ) was applied using a 3mm tweezer electrode attached to an electroporator (CUY 21EDIT, π Protech) facing the somatosensory cortex, see FIG. 1.

(5) At the end of the uterine electroporation experiment, the animal was conscious and the embryo was carefully returned to the abdominal cavity and the abdominal wall opening was sutured. After the experiment was completed, pregnant rats were continued to be individually bred and provided with sufficient food and water. After birth, mice were re-tested for two-photon imaging until adulthood (8-10 weeks).

2. Two-photon microscope observation live animal fluorescence labeled GluA2 receptor

The receptor observation experiment adopted is a NikonA1RMP two-photon microscope system, a MaiTai deep two-photon infrared femtosecond laser (Spectral Physics company, wavelength range: 690-1040nm, pulse width 70fs, pulse frequency 80 MHz). High speed resonance scanners (resonants scanners) scan and four channel NDD detectors detect the fluorescent signals. The microscope imaging software was Nikon NIS-Element C. When imaging a GluA2 receptor, the imaging is carried out by adopting the parameters of 910nm laser wavelength, 40 multiplied by 0.8NA water mirror, 512 multiplied by 512 pixels, 30 frames per second, the field of view is 70 mu m multiplied by 70 mu m, and the laser power range is adjusted to be 20-35mW according to the imaging depth. The dosing experiments were recorded for 30-60 seconds each time. Acute phase of change, interval 20s imaging. Late chronic changes were imaged 5min apart. And after the experiment is finished, adjusting the laser intensity according to the imaging depth to perform three-dimensional imaging on the neurons.

3. Electrophysiological recording of neurons from fluorescently labeled AMPA receptor GluA2 subunits in living animals

Craniotomy window surgery

Living mouse two-photon guided patch clamp recording

(1) The electrode is immersed and a positive pressure of 100-200mbar is applied. The two-photon guided lower electrode into the cortex, scanning speed 30 frames per second, pixel 512 x 512. The recording of non-fluorescently labeled neurons was performed as before.

(2) In the recording of AMPA receptor-labeled neurons, the laser wavelength was first adjusted to 910nm to clearly show the fluorescently labeled receptors and cell structures (SEP-GluA 2 and dsRed 2).

(3) After the position of the neuron cell is determined, the laser wavelength is adjusted to 800nm, and the tip of a recording electrode (Alexa 594) is clearly displayed. The laser wavelength was modulated at any time according to the cell body position and the electrode position, see fig. 2. Pressing the electrode against the cell, sucking the cell by negative pressure to reach G omega resistance, removing the negative pressure, and switching to a current clamp or a voltage clamp to record an electric signal.

(4) When a spontaneous postsynaptic current is recorded (sEPSC), the cell voltage is clamped at-70 mv, i.e., the Cl-reversal potential. When spontaneous postsynaptic inhibitory currents (sIPSC) were recorded, the cell voltage was clamped at 0mv, the reversal potential of excitatory synaptic current.

(5) Electrophysiological recordings were filtered using an Axonpatch 200B patch-clamp amplifier and Digidata1550B digital-to-analog converter (Molecular Devices, foster City, calif., USA) with a data acquisition frequency of 20kHz and 10kHz.

4. Preparation of living animal cortical receptor level drug-induced epilepsy model and epilepsy inhibition experiment

The experimental steps are as follows:

(1) The dosing electrode is drawn with a resistance of 4-6M omega. Extracellular fluid containing 4-AP (2 mM) and GABA (50 mM) was flushed into the electrode. 4-AP (2 mM) electrodes with the fluorescent indicator Alexa594 were placed on the surface of the sensory cortex of the animal body with a craniotomy under the guidance of a two-photon microscope (800 nm). GABA (50 mM) electrodes with the same fluorescent indicator Alexa594 were placed on the cortical surface at a horizontal distance of 100-200 μm.

(2) By adjusting the laser wavelength to 910nm, clearly shown fluorescently labeled AMPA (SEP-GluA 1) receptor and neuronal structures (dsRed 2) are found below the cortical surface. The two administration electrodes are slowly moved toward the target dendrite by the micromanipulator at the same time. Avoid the electrode to be blocked by stroma or cells, influence the administration. And (3) after the electrode and the dendrite are positioned on the same plane (the distance is 30-50 mu m), recording the basic fluorescence data of receptor imaging, and determining the receptor expression and the morphological structure of the dendrite.

(3) The drug delivery electrode contained 4-AP and was given a positive pressure of 30-50mpa to blow out the drug while imaging data was recorded.

(4) One minute after dosing, the experimental group blew the intra-electrode drug containing the inhibitory neurotransmitter GABA (50 mM) at the same positive pressure level. The change of the fluorescence of the receptor and the morphological structure of the dendrite are continuously observed. The control group was given extracellular fluid without GABA.

5、ELISA

After the final two-photon imaging and patch clamp experiment, the mice are sacrificed, and the expression of the ZIP1 and GluR2 proteins of the right cerebral cortex is measured by ELISA method by taking the L4-5 dorsal root ganglia. The brain cortex tissue is added with precooled tissue protein lysate and ground into tissue homogenate. And (3) centrifuging the homogenate for 5min at 4 ℃,12000rpm, wherein the centrifugation radius is 10cm, and obtaining the supernatant, namely the total protein of the spinal cord tissue. Membrane proteins were extracted using a membrane protein extraction kit (Thermo, USA) according to the instructions. The expression of ZIP1 and GluR2 proteins was determined experimentally using Mouse GRIA2/GLUR2 (Sandwich ELISA) ELISAKit (LSBio, LS-F8852, USA) and Mouse Slc39a1/Zinc transporter ZIP1 ELISAKit (EUAab, E15589m, china) according to the instructions.

6. Statistical analysis

Using SPSS 18.0 statistical software, normally distributed data as mean + -standard deviation

Showing that the measurement data comparison of random block design adopts one-factor analysis of variance, the measurement data comparison of repeated measurement design adopts the analysis of variance of repeated measurement design, P<A difference of 0.05 is statistically significant.

3. Results

FIG. 3 shows that 4-AP administration resulted in increased ZIP1 expression in cell membranes, whereas ZIP1 knockdown resulted in a significant decrease in ZIP1 protein expression. In group C, administration of 4-AP reduced the membrane protein expression of AMPAR-GluA2, while administration of ZIP1 knock-down reduced the 4-AP-induced reduction in GluA2 membrane protein expression.

Under two-photon imaging, another injection electrode filled with 4-AP and Alexa594 was inserted into the cortex to induce epileptiform events with only a few neurons. When the injection electrode reaches the same focal point as the neuron repaired or attached to the cell, a gentle hit of about 40 mbar releases the internal solution. Under two-photon time delay imaging, a neuron structure is reconstructed by using z-stack images every 15 min. Surprisingly, it was found that the local injection of 4-AP was significant for neuronal damage, especially after 15 min. These results indicate that acute focal 4-AP injection can rapidly induce epileptiform events and neuronal damage at the single cell scale, and the results are shown in figure 4.

The results in FIG. 5 show that the frequency of action potentials given to group C after 4-AP increases rapidly in both whole cell and cell contact modes (on A: whole cell structure, 1.5. + -. 0.13Hz before 4-AP, 5.1. + -. 0.73Hz after 4-AP, n =6 on B: adsorbed cell record, 1.7. + -. 0.15Hz, 5.2. + -. 0.63Hz after 4-AP, n = 7). The frequency of the action potential given to the group Z after 4-AP also increases rapidly. However, compared to group C, the knock-down of ZIP1 resulted in a significant decrease in the action potential frequency for group Z (under A: whole cell structure, 1.6. + -. 0.24Hz before 4-AP, 3.2. + -. 0.47Hz after 4-AP, n =8 under B: adsorbed cell record, 1.8. + -. 0.22Hz, 3.4. + -. 0.59Hz after 4-AP, n = 6).

In summary, a decrease in GluA2 is associated with cerebral cortical epileptiform discharges. The research finds that 4-AP-induced cerebral cortex epileptiform discharge is related to ZIP1 increase, the ZIP1 increase can cause reduction of GluA2 membrane protein expression, and ZIP1 knock-down can inhibit 4-AP-induced GluA2 membrane expression reduction. Thus, the present study demonstrates that ZIP1 is a critical epilepsy-related protein, and that inhibition of ZIP1 can reduce seizures and severity.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims

1. An inhibitor for preventing and/or treating epilepsy, comprising a substance inhibiting the activity of ZIP1 protein, or a substance inhibiting or silencing the expression of ZIP1 gene; preferably, the substance inhibiting the activity of the ZIP1 protein comprises a substance inhibiting the synthesis of the ZIP1 protein or a substance promoting the degradation of the ZIP1 protein or a substance inhibiting the function of the ZIP1 protein; preferably, the substance inhibiting or silencing expression of the ZIP1 gene includes a substance interfering with expression of the ZIP1 gene or a substance knocking out the ZIP1 gene or a substance mutating the ZIP1 gene.

2. The inhibitor of claim 1, wherein the substance comprises a synthetic small molecule, chemical agent, antisense oligonucleotide, siRNA, miRNA, ribozyme, polypeptide, protein; preferably, the polypeptide or protein includes hormones, cytokines, antibodies and fragments thereof.

3. The inhibitor according to claim 1, wherein the substance knocking out the ZIP1 gene further comprises a gene editing tool for knocking out the ZIP1 gene; preferably, the gene editing tools include Cre-lox recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR/Cas9 technology; preferably, the gene editing tool is CRISPR/Cas9 technology.

4. A pharmaceutical composition for preventing or treating epilepsy, comprising the inhibitor of any one of claims 1-3, preferably further comprising a pharmaceutically acceptable carrier.

5. Use of the inhibitor of any one of claims 1-3 or the pharmaceutical composition of claim 4 for the manufacture of a medicament for the prevention or treatment of epilepsy.

6. The use as claimed in claim 5, wherein the medicament is in the form of tablets, capsules, granules, pills, drops, syrups, powders, suppositories, drops, emulsions, injections, solutions or suspensions.

Application of ZIP1 in preparation of an epilepsy animal model; preferably, the animal model is prepared by modifying the expression level of ZIP1 in an animal; preferably, the animal is a mouse.

8. A method of screening for a candidate agent for the prevention and/or treatment of epilepsy, comprising:

(1) Treating a system expressing or containing ZIP1 with a test substance;

(2) Detecting the expression level of ZIP1 in said system;

(3) Selecting a substance capable of down-regulating the expression level of ZIP1 as a candidate drug.

9. The method of claim 8, wherein the system comprises a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Use of ZIP1 for screening a candidate drug for the prevention and/or treatment of epilepsy.