CN115227692A - Application of reblatatin in preparation of medicine for treating chronic convulsion - Google Patents

Abstract

The invention discloses an application of Reblastatin in preparing a medicament for treating chronic convulsion. The research of the invention finds that after the Reblastatin is treated, the abnormal proliferation of hippocampal astrocytes of a mouse chronic convulsion model is effectively relieved, and proves that the Reblastatin can be used for preventing or treating chronic convulsion. The research result of the invention provides a new method for the clinical treatment of chronic convulsion.

Description

Application of reblatatin in preparation of medicine for treating chronic convulsion

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of Reblastatin in preparation of a medicine for treating chronic convulsion.

Background

Chronic convulsion is a chronic disease caused by abnormal discharge of neuron synchronization, and about 5000 thousands of people all over the world currently suffer from chronic convulsion. The incidence rate of chronic convulsion in China is 7 per thousand, about 600 million patients, and 40 million patients are newly added every year. Chronic convulsions have become one of the most common neurological diseases, and are ranked fifth in the disability-adjusted life year (DALY) statistical ranking. The chronic convulsion of the temporal lobe is the most common chronic convulsion which is difficult to treat by medicaments, and patients still cannot achieve satisfactory treatment effect after taking enough anti-chronic convulsion medicaments, and the chronic convulsion can be controlled only by a method of removing epileptogenic focus by operation. The medical and economic burden of the drug-refractory chronic convulsion on the society and families is far higher than that of the common chronic convulsion syndrome. Therefore, the pathogenesis of patients with intractable chronic convulsion such as chronic convulsion of temporal lobe is clarified, a novel effective anti-chronic convulsion drug target and a treatment method are developed, non-operative treatment selection is provided for the patients, and the treatment gap of the intractable chronic convulsion is filled.

Disclosure of Invention

The invention aims to provide a novel method for preventing or treating chronic convulsion, and the invention adopts the following technical scheme for realizing the aim:

the invention provides an application of a natural product in preparing a medicament for preventing or treating chronic convulsion, wherein the natural product is Reblastatin.

"prevention" as used herein means preventing the appearance of a disease or the recurrence of a disappeared disease in a subject at risk of the disease.

The term "treating" refers to ameliorating the disease or disorder referred to herein or the symptoms associated therewith to a significant extent.

In one embodiment, the Reblastatin can increase the expression level of Glt-1 in a cell.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide, mRNA or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into structures present and operating in a cell. Thus, according to the present invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of the transcribed polynucleotide, of the translated protein, or of the post-translationally modified protein should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of the protein (e.g., by proteolysis).

In a more preferred embodiment, the cells are primary astrocytes.

As one embodiment, the Reblastatin can relieve the abnormal proliferation of astrocytes in brain tissues.

As one implementation mode, the dosage form of the medicine comprises tablets, capsules, dropping pills, aerosols, pills, powder, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and freeze-dried powder injections.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating chronic convulsion, the pharmaceutical composition comprising reblatatin.

As an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier or excipient.

As an embodiment, the buffer includes Trizma, bicine, tricine, MOPS, MOPSO, MOBS, tris, hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, caconate (cacylate), CHES, DIPSO, EPPS, ethanolamine, glycine, HEPSO, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, TES, and TES

In one embodiment, the carrier comprises an antimicrobial agent, an isotonic agent, an antioxidant, a local anesthetic, a suspending agent, a dispersing agent, an emulsifying agent, a chelating agent, a thickening agent, or a solubilizing agent.

In one embodiment, the excipient comprises a carbohydrate, polymer, lipid, or mineral.

The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the reblatatin that produces a therapeutic effect. Typically, this amount will range from about 1% to about 90% of the active ingredient in 100%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%. The methods of making these compositions include the step of combining the reblatatin with a carrier and optionally one or more accessory ingredients. In general, pharmaceutical compositions can be prepared by uniformly and intimately bringing the reblatatin into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, sachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or lozenges (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or mouthwashes, each containing a predetermined amount of reblatatin as the active ingredient. The reblatatin may also be administered in the form of a honeyed pill, granule or paste.

The pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effect with respect to the condition to which it is administered. The precise dosage will depend on the activity of the compound, the mode of administration, the nature and severity of the condition, the age and weight of the patient and may require different dosages. Dosage administration can be carried out by a single administration in the form of individual dosage units (otherwise, several smaller dosage units) and also by multiple administrations of subdivided doses at specific time intervals.

The invention also provides a method of increasing glutamate uptake capacity of a cell for non-therapeutic purposes, said method comprising administering a reblatatin to the cell.

In one embodiment, the cells are primary astrocytes.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention discovers that the Reblastatin has obvious treatment effect on chronic convulsion for the first time, and the invention verifies that the abnormal proliferation of hippocampal astrocytes of a mouse chronic convulsion model is effectively relieved after the Reblastatin is treated by in vivo experiments. The invention provides a new method for clinical treatment of chronic convulsion, and has good application prospect.

Drawings

FIG. 1 shows the results of Western blotting for detecting Hsp90, hsp70, glt-1 and Actin levels;

FIG. 2 is a schematic view of the sample addition sequence of an Hsp90 protein labeling experiment;

FIG. 3 shows the results of the microcalorimetric electrophoresis experiment for detecting the affinity of Reblastatin (Z-R) to Hsp 90;

FIG. 4 is a result of a microcalorimetric electrophoresis experiment to detect the affinity of Geldadamycin (GA) to Hsp 90;

FIG. 5 is the result of a microcalorimetric electrophoresis experiment to detect the affinity of 17AAG to Hsp 90;

FIG. 6 shows the results of testing the ability of primary astrocytes treated with different drugs to take up glutamic acid;

FIG. 7 shows the results of 293T cytotoxicity assay;

FIG. 8 shows the results of HepG2 cytotoxicity assay;

FIG. 9 shows the Western blot detection of Glt-1 level of protein extracted from hippocampal tissue of C57 mouse injected with Z-R in abdominal cavity;

FIG. 10 is a statistical chart of Western blot detection of Glt-1 level of protein extracted from hippocampal tissue by C57 mouse intraperitoneal injection of Z-R;

FIG. 11 is an experimental result of Western blot detection of Glt-1 level of protein extracted from hippocampal tissues by intraperitoneal injection of Z-R in a C57 mouse chronic convulsion model;

FIG. 12 is a statistical chart of Western blot detection of Glt-1 level of protein extracted from hippocampal tissues by intraperitoneal injection of Z-R in a C57 mouse chronic convulsion model;

FIG. 13 is a brain electrical result chart of a C57 mouse chronic convulsion model;

FIG. 14 shows the frequency of chronic convulsions before and after intraperitoneal administration in Z-R and DMSO groups;

figure 15 is the frequency of chronic convulsive episodes following administration versus the pre-administration ratio for each mouse model of chronic convulsions;

fig. 16 is a result diagram of abnormal proliferation of astrocytes of hippocampus of a C57 mouse chronic convulsion model after Z-R treatment, wherein a diagram a shows the results of immunofluorescence labeling of protein marker GFAP specific for astrocytes of brain tissue of the C57 mouse chronic convulsion model, random selection of the region where the hippocampus is located to detect the fluorescence intensity thereof, comparison of the forms and amounts of astrocytes of hippocampus regions of each C57 mouse chronic convulsion model, a diagram B shows the fluorescence intensity of all selected hippocampus regions of the same treatment group, statistical analysis of the overall fluorescence intensity of the Z-R group and DMSO group, and comparison of the effects of different treatments on the forms and amounts of astrocytes of hippocampus of C57 mouse chronic convulsion model;

fig. 17 is a graph showing the results of abnormal proliferation of astrocytes in the hippocampal of the chronic convulsion model of C57 mice after Z-R treatment, in which a graph a shows the results of immunohistochemical labeling of astrocyte-specific expression protein marker GFAP in brain tissue of the chronic convulsion model of C57 mice, random selection of the region where the hippocampus is located to detect its fluorescence intensity, comparison of the forms and numbers of astrocytes in the hippocampal region of each chronic convulsion model of C57 mice, and a graph B shows the results of concentrating the fluorescence intensity of all selected hippocampal regions of the same treatment group, statistical analysis of the overall fluorescence intensity of the Z-R group and the DMSO group, and comparison of the influences of different treatments on the forms and numbers of astrocytes in the region of chronic convulsion model of C57 mice.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not noted in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Example 1 treatment of Low concentration Reblastatin (Z-R)/Geldamamycin (GA)/Tanesampycin (17-AAG) for 24h increased levels of Primary astrocytes Glt-1

1. Experimental Material

C57 suckling mouse (China, si Bei Fu, 1 day old), celLytic Cell Lysis Reagent lysate (US, sigma-aldrich, C2978-250 ML), 100X cocktail (China, bimake, B14001), beta-mercaptoethanol (US, sigma-aldrich, M3148-25 ML), 5X protein loading buffer, thermostatic metal bath (US, thermo Fisher Scientific, GP 10), glycerol (VWR Life Science, 0854-1L), tris (China, producer, A501492-0005), HCl (China, national drug group chemical Co., ltd., 10011008), SDS (US, sigma-aldrich, 74255-250G), bromophenol blue (US, sigma-aldrich, 114391-5G), electrophoresis apparatus (Chinese 3232n 32300), and mini electrophoresis apparatus (EPS 300), tanon, VE 180), transfer electrophoresis tank (Chinese, tanon, VE 186), stereomicroscope (Germany, ZEISS, 37081), thermostatic carbon dioxide incubator (U.S., thermo Fisher Scientific,150 i), fine forceps, ophthalmic scissors, 10CM petri dish, 12-well plate, 50ML centrifuge tube, 15ML centrifuge tube, pipette, syringe, DMEM (Chinese, zhongkemian, CM 15019), penicilin-Streptomycin (Chinese, zhongkemian, CC 004), PBS (Chinese, zhongkemian, CC 008), FBS (U.S., sigma-aldrich, F8687-500), poly-D-lysine ML (U.S., sigma-aldrich, P6407-5 MG), DMSO (U.S., sigma-aldrich, D2650-100 ML), pancreatin Trsin-0.25% (EDTA, zhongkemai morning, CC 012), geldamamycin (Chinese, selleck, S2713), tanespecin (Chinese, selectk, S1141), reblastatin (Chinese, synthesized by pharmaceutical Co., ltd.), conventional molecular weight markers for proteins (U.S., proteintetech, PL 00001), nitrocellulose membrane (U.S., PALL corporation, 66485), glt-1 (U.S., santa cruz, sc-365634), hsp90 (U.S., proteintech, 13171-1-AP), actin (U.S., CST, 4970S), hsp70 (U.S., proteintech, 3532 zxft 32-1-AP), goat Anti-Mouse IgG (H + dy L) Secondary IgG (H + dy L) Antibot (U.S., angrogen, 31430), anfun-conjuga Affinat 3532-S), goutu IgG (H + dy), C + Y (CTx @ S. Sub.S. Pat. 30), proteinoc-30G-30, K. Pat. No. 30G-30, S. 30, inc.;

formula of 5ml 5X protein loading buffer:

TABLE 1 5X protein loading buffer formulation

1M Tris-HCl (pH 6.8) 12.11g Tris-base was dissolved in 60ml ddH2O, after complete dissolution, pH =6.8 was adjusted with concentrated HCl, and then ddH2O was added to a constant volume of 100ml;

1.5M Tris-HCl (pH 8.8) 18.125g Tris-base was dissolved in 60ml ddH2O, after complete dissolution, pH =8.8 was adjusted with concentrated HCl, and then ddH2O was added to make volume 100ml;

weighing 144g of Glycine (Glycine) and 30g of Tris-base in 10 XRunningbuffer base solution (1000 ml system), dissolving in 800ml of ddH2O, and determining to dissolve to 1000ml after complete dissolution;

1 × Running buffer (electrophoretic solution): 895mL ddH2O was added with 100mL 10 × Running buffer (basic solution) and 5mL 20% SDS to prepare 1000mL electrophoretic solution;

1 × film transfer liquid: 100mL of 10 × Running buffer (base solution) and 150mL of methanol are added into 750mL of ddH2O to prepare 1000mL of membrane transferring solution;

weighing 12.1g Tris-Base and 87.6g NaCl in 10 xTBS (1000 ml system), dissolving in 800ml ddH2O, adjusting the pH to 7.6 after complete dissolution, and then fixing the volume to 1000ml;

100ml of 10 XTSS and 1ml of Tween-20 were added to 900ml of ddH2O, and the mixture was mixed well and used.

2. Experimental methods

Isolation culture of Primary astrocytes

Intercellular preparation 45min before the experiment: DMEM complete Petri dish, pipette, 15ml centrifuge tube, syringe, double distilled water (sterilized above with UV), PBS, DMEM complete Medium (incubated above 37 ℃), penicilin-Streptomyces, PDL

Intercellular preparation work 15min before the experiment: by H ₂ An O1.

The method comprises the following steps of arranging a test bed, preparing ice, and numbering 123 three 50ml centrifuge tubes, wherein 75% ethanol is added into 1, 2 and 3 pieces of PBS, 10ml culture dishes, a stereomicroscope and an alcohol spray cleaning wiping tool are filled in the centrifuge tubes, and the dissecting tool needs to be sterilized by an alcohol lamp;

soaking a suckling mouse in an alcohol centrifuge tube for disinfection, immersing the mouse in PBS (phosphate buffer solution) in the tube 2 for killing the mouse, washing the tube 3 with PBS once, and transferring the washed tube into a culture dish containing PBS;

cutting off the head of the rat from the neck, cutting off the skull along the middle bone seam, and pulling the head to take out the brain;

under a stereomicroscope, dividing brain tissues into a left hemisphere and a right hemisphere, pinching off a sniffing ball, stripping off a blood membrane from the position, turning over the blood membrane to obtain a hippocampus, stripping off the brain stem tissues such as the hippocampus and the like, only remaining cerebral cortex, transferring the brain stem tissues into a mixed double-resistant PBS centrifugal tube, and stripping off a plurality of brain tissues to be treated together;

entering cells, washing each vessel coated with PDL with a culture medium, pouring most of original PBS, suspending the residual PBS in cerebral cortex, pouring the residual PBS into the culture vessel, sucking off the redundant PBS, adding new PBS, washing for 3 times, and sucking up the PBS as much as possible in the last time;

adding 2ml of culture medium, repeatedly blowing and sucking with a gun head for about 50 times to break the brain tissue until no obvious blocky structure exists, then changing to an injector, and repeatedly blowing and sucking for about 20 times until the color of the solution is uniform;

transferring the cell suspension into a 15ml centrifuge tube, and centrifuging at 800rpm for 5min to settle the cells to the bottom of the tube;

discarding the supernatant medium, adding 2ml of the medium again, sucking the cells evenly, adding 10ml of DMEM containing 20% FBS into each dish, and placing the cells into each dish according to 3 brain tissues/dish to coat the dishes with PDL;

shaking in the shape of a cross or a 8 to uniformly distribute the cells in the culture dish, putting the culture dish into an incubator, and shaking the cells again to obtain the cell culture dish;

observing the growth condition of the cells after 1d, washing for 3 times by PBS, and changing the liquid;

blowing off surface dead neurons after 3d, washing with PBS for 3 times, and changing the solution;

culturing until the cells grow over a 12-hole plate;

compound treatment of astrocytes

Taking primary astrocytes growing in a dish of 10cm, removing the culture medium, and washing with PBS for 3 times;

taking 1ml of pancreatin digestive cells, digesting for 3min at 37 ℃, and shaking up once every 1min;

removing pancreatin, adding 2ml of complete culture medium and blowing down cells;

centrifuging at 800rpm for 3min;

discarding the supernatant, taking 3ml of culture medium to suspend the cells, and inoculating the cells into a 12-well plate according to 1:3;

culturing until the cells grow full of 12-well plates, and treating the cells with Geldanamycin (GA), 17AAG and Reblastatin (Z-R) at final concentrations of 2, 4, 8, 16, 32, 50, 200, 1000, 5000 and 20000nmol/L for 24h;

cell lysis and protein extraction for western blot

Washing astrocytes in each well with PBS 3 times;

adding 120ul of cocktail-containing cell lysate into each hole, and shaking in an ice bath until cells are fully lysed;

centrifuging the cell lysate for 10min at 12000rpm at 4 ℃;

after centrifugation, 80ul of supernatant was taken from each sample, and 20ul of 5X protein loading buffer was added;

heating at 95 deg.C for denaturation for 5min;

preparing 10% of separation glue and 5% of concentrated glue;

adding 10ul of fully and uniformly mixed protein samples into each hole, and setting 160V constant voltage for electrophoresis for 60min;

setting a constant voltage of 156V for electrophoresis for 60min;

setting 300mA constant current to flow into the membrane for 100min;

cutting the nitrocellulose membrane according to the target protein strip after the membrane conversion is finished;

adding milk powder into TBST to prepare 5% confining liquid, and incubating for 60min;

the primary antibody was prepared with the blocking solution and incubated overnight in a refrigerator at 4 ℃ as follows:

TABLE 2 Primary antibody formulation

Taking out the nitrocellulose membrane, and washing for 3 × 5min by TBST;

secondary antibody (1;

washing with TBST for 3 × 5min, and adsorbing TBST on the dry film for color development;

preparing a luminescent liquid;

incubating the luminous liquid for 1min;

photosensitive developing of the photosensitive film.

3. Results of the experiment

As shown in figure 1, isolated and cultured milk mouse astrocytes, 2, 4, 8, 16, 32, 50, 200, 1000, 5000, 20000nmol/L Geldamamycin, 17AAG, reblastatin treatment of astrocytes for 24h, cell lysis to extract protein, western blotting to detect Hsp90, hsp70, glt-1, actin level. A larger range of concentrations of Geldamamycin, 17AAG, IPI-504, reblastatin increased primary astrocyte Glt-1 levels.

Example 2Z-R has significantly greater affinity for Hsp90 than GA/17-AAG

1. Experimental materials

His-Tag Hsp90 (abcam, ab48801, USA), monolith Capillaries (Nanotemper, mo-K022, germany), his-Tag Labeling Kit RED-tris-NTA 2nd Generation (Nanotemper, mo-L018, germany),

PCR Tube Strips (America, MERK, AXYPCR 0208C), monolith (Nanotemper, monolith. TM. 115, germany), tycho NT.6 (Nanotemper, TY-002, germany), DMSO (Sigma-aldrich, D2650-100 ML), PBS-T (Ready-to-use), ddH ₂ O, 1.5ml EP tube, geldaramycin (Chinese, selleck, S2713), tanespecycin (Chinese, selelck, S1141), reblastatin (Chinese, synthesized by drugs);

2. experimental methods

Hsp90 protein labeling assay

Add 8.0mL ddH2O to a5 XPBS-T vial to dilute to 1 XPPBS-T;

add 25u L PBS-T to dilute the dye to 5u M;

2 μ L of dye (5 μ M) was mixed with 198 μ L of PBS-T to give 200 μ L of dye (50 nM);

prepare 30. Mu.L of His-tagged protein (4. Mu.M, diluted with PBS-T);

adding 10 mu L of PBS-T into the No. 2-16 PCR tubes respectively;

add 20. Mu.L of His-tagged protein (4. Mu.M) to PCR tube # 1;

and (3) taking 10 mu L of ligand (His-tagged protein) from the No. 1 tube, adding into the No. 2 tube, repeatedly blowing and beating by using a pipette, uniformly mixing, taking 10 mu L of ligand, adding into the No. 3 tube, uniformly mixing, and then completing the dilution of the No. 4-16 tube according to the same method. Finally discard the excess 10 μ L Ligand from tube 16; the loading sequence is shown in FIG. 2.

Add 10. Mu.L of each dye (50 nM) into 1-16 wells and mix well with a pipette;

incubation for 30 minutes at room temperature;

detection on the machine after sucking the sample with a capillary, the instrument set to 40% LED/excitation power and medium MST power (set to 40% MST power in NT.Control software);

kd was calculated by Kd fitting model using mo.control or mo.affinity Analysis software.

Hsp90 and Z-R/GA/17-AAG interaction detection

Preparing PBST: add 8mL ddH2O to 5 XPBS-T vial to dilute to 1 XPBST;

preparing PBST containing 2% DMSO according to experimental requirements;

adding 25uL PBST to dilute the dye into 5uM, and diluting 5uM RED protein dye mother liquor into 100nM RED protein dye according to the experiment requirement;

the 11.49um Hsp90 protein mother liquor is diluted to 200nM Hsp90 protein according to the experimental requirement:

uniformly mixing the prepared 100nM RED protein dye and 200nM Hsp90 protein in equal volume, and incubating at room temperature for 30min;

the sample was centrifuged at 14 000g at 4 ℃ for 15min and the supernatant was taken to a new EP tube;

diluting Z-R/GA/17-AAG according to the experiment requirement;

preparing 2 ul PBST + DMSO (2%) buffer diluted Z-R/GA/17-AAG, and adding the Z-R/GA/17-AAG into the corresponding No. 1 PCR tube respectively;

in each set of experiments, 10ul PBST + DMSO (2%) buffer is added into each of the other 15 PCR tubes, gradient dilution is sequentially completed, and 10ul is discarded in the last tube;

preparation of 1800ul PBST diluted Hsp90-RED (20 nM);

in each set of experiment, 10ul Hsp90-RED was added to each of 16 PCR tubes and mixed well;

the sample was drawn up using a capillary and tested on the machine.

3. Results of the experiment

As shown in FIGS. 3-5, microcalorimetric results showed that Reblastatin (Z-R) has much higher affinity for Hsp90 than Geldanamycin (GA) and 17AAG.

The Kd between Reblastatin and Hsp90 was 2.3X 10-9mol/L, between Geldamycin and Hsp90 was 2.4X 10-7mol/L, and between 17AAG and Hsp90 was 2.0X 10-4mol/L in PBST +2% DMSO environmental system as determined by microcalorimetric electrophoresis.

Example 3Z-R significantly better than GA/17-AAG in improving the uptake of glutamate by primary astrocytes

1. Experimental Material

C57 suckling mice (china, si Bei Fu, 1 day old), glutamic acid detection kit (usa, cell biolabs INC, STA-674), costar assay plate (usa, corning, 3925), electrothermal thermostated incubator (china, shanghai-heng science instruments ltd., DHP), stereomicroscope (germany, ZEISS, 37081), thermostated carbon dioxide incubator (usa, thermo Fisher Scientific,150 i), fine tweezers, ophthalmic scissors, 10CM petri dishes, 12 well plates, 50ML centrifuge tubes, 15ML centrifuge tubes, syringes, DMEM (china, zhongkechen, CM 15019), penillilin-strepomycin (china, zhongkemeichen, CC 004), FBS (US, sigma-aldrich, F8687-500 ML), poly-D-lysine hydrate (US, sigma-aldrich, P6407-5 MG), DMEM without glutamate (China, mach Cork., CM 15018), pancreatin Trypsin-EDTA0.25% (China, mach Cork., CC 012), 10CM dish, multifunctional microplate reader FlexStation3 (US, molecular Devices 86783), L-Glutamic acid (US, sigma-aldrich, G8415-100G), geldamamycin (China, serlleck, S2713), taneslimycin (China, sel, select, S1141), reblastatin (Chinese, synthesized by drugs), DMSO (US, sigma-aldrich, D50-100 ML);

2. experimental method

Separating and culturing primary astrocytes by the same method as the experiment I;

adding Z-R/GA/17-AAG according to a final concentration of 100nM for treating for 36h when the primary astrocytes overgrow each hole of the 12-hole plate;

changing the culture medium to DMEM without glutamate, and treating for 12h to reduce the intracellular glutamate level;

weighing L-glutamic acid, dissolving in ddH ₂ Preparing mother liquor;

changing new DMEM without glutamate, adding glutamic acid according to a final concentration of 20uM, and uniformly mixing;

collecting cell culture supernatant at 20min, 40min, and 80 min;

centrifuging at 4 deg.C and 10 000rpm for 5min, removing insoluble particles, and diluting the supernatant by 100 times for use;

preparing a detection reagent:

1X assay buffer: diluting 10X buffer solution with deionized water, and stirring or vortexing until the solution is uniform;

TABLE 3 Reaction Mix formulations

Add 50ul of sample and blank to Costar assay plate;

adding 50ul Reaction Mix to each well, mixing well, incubating at 37 deg.C in the dark for 30min;

reading the plate, exciting light at 550nm and emitting light at 590nm;

the glutamate concentration of each sample was calculated from the standard.

3. Results of the experiment

As shown in FIG. 6, 100nmol/L Z-R treatment significantly improved the glutamate uptake capacity of primary astrocytes.

The DMSO, the 100 nmol/L17 AAG and the 100nmol/L ReBLASTIn are respectively used for treating astrocytes for 36h, the culture is carried out for 12h in a glutamate-free medium, the astrocytes are cultured in20 uM glutamate treatment solution, and cell culture supernatant is taken at 20min, 40min and 80min to detect the glutamate concentration.

Example 4Z-R treatment 293T/HepG2 is less cytotoxic than/GA/17-AAG

1. Experimental Material

Constant temperature carbon dioxide incubator (U.S., thermo Fisher Scientific,150 i), trypsin-EDTA0.25% (china, chinese michael, CC 012), penicilin-Streptomycin (china, chinese michael, CC 004), FBS (U.S., sigma-aldrich, F8687-500 ML), PBS (chinese, chinese michael, CC 008), DMSO (U.S., sigma-aldrich, D2650-100 ML), geldanamycin (china, selleck, S2713), tanespimycin (china, seleleck, S1141), reblatatin (china, synthesized by drugs), 293T, hepG, 96-well plate, multichannel adjustable pipettor (china, darongxing, YE203AM 0135463), cell counting kit-8 (us, MCE, HY-K0301), multifunctional microplate reader SynergyH1 (us, biotek, 86780);

2. experimental methods

293T and HepG2 culture and plating

293T and HepG2 are cultured, and cells in logarithmic growth phase are taken and washed for 3 times by PBS;

0.25% pancreatin digest cells, 10% fbs DMEM medium to terminate digestion;

centrifuging at 800rpm for 3min to pellet the cells, 10% FBS DMEM medium to resuspend the cells and adjust the cell concentration to 3x 10 ⁴ Per ml;

cells were seeded into 96-well plates at 100ul cell suspension per well, 5% CO at 37 ℃ ₂ Culturing;

cell activity detection by Z-R/GA/17-AAG treatment 293T and HepG2

Culturing 293T and HepG224h;

preparing compound mother liquor with the concentration of 20000uM, 8000uM, 4000uM, 2000uM, 400uM, 200uM, 40uM, 20uM, 4uM and 2uM by DMSO;

diluting the mother liquor 400 times, and dissolving in 10% FBS DMEM complete medium to obtain 50000nM, 20000nM, 10000nM, 5000nM, 1000nM, 500nM, 100nM, 50nM, 10nuM, 5nM compound working solution medium and DMSO solvent only medium;

discarding culture medium inoculated with 293T and HepG296 pore plates, adding 100ul of compound working solution culture medium into each pore, and setting 3 multiple pores;

37℃5％CO ₂ culturing for 24h, 48h and 72h;

CCK-8 detection of Z-R/GA/17-AAG treatment on activity of each cell

Respectively detecting 293T and HepG2 treated by Z-R/GA/17-AAG for 24h, 48h and 72h;

adding 10ul CCK-8 into each 100ul culture medium to prepare a CCK-8 detection solution;

discarding the culture medium in 293T and HepG224h 96-well plates, and adding 100 mu L of CCK-8-containing detection solution into each well;

incubating for 2h in an incubator at 37 ℃;

setting 450nm excitation light for the multifunctional microplate reader SynergyH1 to measure the absorbance of each cell culture well;

group data were counted and compound treatment groups IC calculated using Prism ₅₀ A numerical value;

3. results of the experiment

HepG2 cytotoxicity assay

As shown in FIG. 7, 293T cytotoxicity test preliminarily verifies that 293T cells are treated under the same condition for 24h, and Z-R cytotoxicity is weaker than 17AAG and GA; the treatment time was 48h, Z-R was less cytotoxic than GA; the treatment time was 72h and Z-R was less cytotoxic than GA.

As shown in FIG. 8, the HepG2 cytotoxicity test preliminarily verified that the HepG2 cells were treated under the same conditions, 24h was treated, and the toxicity of Z-R was weaker than 17AAG; when the treatment is carried out for 48h, the toxicity of Z-R is weaker than that of 17AAG and GA; 72h, the toxicity of Z-R is weaker than that of 17AAG and GA.

Example 5Z-R treatment elevated Normal C57 mouse Hippocampus tissue Glt-1 levels

1. Experimental Material

C57 mice (China, si Bei Fu, 8 weeks old), syringes, reblastatin (China, produced by pharmaceuticals), ultrasonic cell disruptor (China, ningbo New Ganoderma Biotech GmbH, JY 92-IIN), du's homogenizer (USA, sigma-aldrich, P1110-1 EA), beta-mercaptoethanol (USA, sigma-aldrich, M3148-25 ML), 5X protein loadbuffer, thermostatic metal bath (USA, thermo Fis Scientific, GP 10), glycerol (VWR Life Science, 0854-1L), chinese (China, manufacturer, A501-0005), HCl (China, pharmaceutical group Chemicals, inc, 10011008), SDS (USA, sigma-aldrich, 74255G-250G), bromophenol blue (USA, sigma, 24 zxft 4324-435), SDS-electrophoresis (SDS-3245), DMSO-3745, taxzone-3745), and Taxzone electrophoresis (Taxzone-32 );

2. experimental methods

C57 mouse intraperitoneal injection Z-R

C57 mice were acclimated for one week;

weighing each mouse, and calculating the dosage of each mouse compound according to the weight and the dosage of the administration agent, wherein the dosage of the administration agent is designed to be 2, 10 and 20mg/kg;

dissolving Z-R in DMSO to prepare injection for each mouse, wherein the total volume of the injection is 50ul, the injection is continuously performed for 3 times, the intraperitoneal injection is performed every time at intervals of 1 day, and the hippocampal tissue is taken on the 6 th day;

protein extracted from C57 mouse hippocampal tissue

After the administration, mouse hippocampal tissue was taken and 4% SDS lysate was prepared;

TABLE 4% SDS lysate preparation

Preparing 1.5ml EP tubes, 350ul 4% SDS lysate per tube;

taking a mouse brain tissue;

preparing a Du's homogenizer, and pre-grinding the waste brain tissue;

adding Hippocampus tissue into homogenizer, adding 350ul PBS, and lightly grinding to reduce foam generation;

the ground homogenate was taken out and placed in a pre-prepared EP tube,

carrying out ultrasound by an ultrasonic cell crusher, wherein the ultrasonic power is 7%, the ultrasound is carried out for 5s, the time is suspended for 2s, the ultrasound is repeated for 2 times, and the temperature is reduced by ice bath;

centrifuging at 13000rpm for 10min at room temperature;

transferring the centrifuged supernatant into a new EP tube;

taking a used amount of tissue lysate, adding 5X protein loadingbuffer, and preparing by the same method as experiment I;

heating at 95 deg.C for denaturation for 5min;

western blot for detecting the level of hippocampal tissue Glt-1 of each mouse

Western blot is the same as experiment one;

TABLE 5 antibody preparation

3. Results of the experiment

As shown in FIGS. 9-10, western blot was performed to detect the level of Glt-1 in protein extracted from hippocampal tissue obtained by intraperitoneal injection of Z-R in C57 mice.

Western blot results show that 2 and 10mg/kg dosage of Z-R can obviously improve Glt-1 level of C57 mouse hippocampal tissue, and 25mg/kg dosage can reduce Glt-1 level of C57 mouse hippocampal tissue.

Example 6Z-R treatment elevated levels of Hippocampus tissue Glt-1 in the model of chronic convulsions in C57 mice

1. Experimental Material

C57 mice (China, st Bei Fu, 8 weeks old), kainic Acid (US, sigma-aldrich,420318-10 MG), brain stereotaxic apparatus (China, rewold's Life technologies, E03275-003), chloral hydrate (China, bio, A600288-0250), PBS (China, zhongke, CC 008), reblastatin (China, synthesized by drugs), syringe, reblastatin (China, synthesized by drugs), ultrasonic cell disruptor (China, ningbo Xinzhi Biotechnology GmbH, JY 92-IIN), du's homogenizer (US, sigma-aldrich, P1110-1 EA), beta-mercaptoethanol (sigma-aldrich, M3148-25 ML), 5X protein lobuffer, thermostated metal bath (US, thermo Fisher Scientific, GP 10), glycerol (VWR Life Science, 0854-1L), tris (chinese, sheng, a 501492-0005), HCl (chinese, national group chemical agents limited, 10011008), SDS (us, sigma-aldrich, 74255-250G), bromophenol blue (us, sigma-aldrich, 114391-5G), electrophoresis apparatus (chinese, tanon), mini vertical electrophoresis tank (china, tanon, VE 180), transfer electrophoresis tank (chinese, tanon, VE 186), 3536-1 (us, proteintech, 15-1-AP), action (us, proteintech, 60008-1-Ig) DMSO (us, sigma-aldrich, D2650-100 ML);

2. experimental methods

Preparation of model for chronic convulsion of mouse C57 caused by hippocampal injection of Kainic acid

C57 mice were acclimated for one week;

5% chloral hydrate is prepared by PBS, intraperitoneal injection is carried out according to 1g of chloral hydrate with the weight of 0.5ug, and the mice are anesthetized;

adding PBS, and preparing Kainic acid according to 1 ug/ul;

after the mice are completely anesthetized, the mice are fixed in a brain stereotaxic instrument and are preserved, 230nl of Kainic acid is injected into the right hippocampus of each mouse, the anterior fontanel of the mouse is taken as the origin, and the injection coordinates are (X-0.2cm, Y-0.2cm, Z-0.18 cm);

after suturing, the mice are placed at 37 ℃ for rewarming, are collected after the mice recover to be clear-headed, and are raised for 4 weeks to build a chronic convulsion mouse model;

c57 chronic convulsion mouse intraperitoneal injection Z-R

Weighing each chronic convulsion mouse, and calculating the dosage of each mouse compound according to the weight and the dosage of the drug, wherein the dosage of the drug is designed to be 0.5, 1, 2, 5 and 10mg/kg;

dissolving Z-R in DMSO to prepare injection for each mouse, wherein the total volume of the injection is 50ul, the injection is continuously carried out for 3 times, the intraperitoneal injection is carried out every time at intervals of 1 day, and the hippocampal tissue is taken on the 6 th day;

protein extracted from hippocampal tissue of C57 chronic convulsion mouse

Experimental procedure is as in example 5

Western blot detection of the level of hippocampal tissue Glt-1 of each chronic convulsion mouse

Experimental procedure is as in example 5

3. Results of the experiment

As shown in figures 11-12, western blot was performed to detect the level of Glt-1 by injecting Z-R into the abdominal cavity of a chronic convulsion model in C57 mice to extract protein from hippocampal tissue.

Western blot results show that 1, 2 and 5mg/kg Z-R can obviously improve the level of Glt-1 of hippocampal tissue of a C57 mouse, and 10mg/kg Z-R has no obvious influence on the level of Glt-1 of hippocampal tissue of a chronic convulsion model of the C57 mouse.

Example 7 C57 Chronic convulsion mouse model intraperitoneal injection of Z-R remarkably inhibits chronic convulsion attack

1. Experimental materials

C57 mice (china, si Bei Fu, 8 weeks old), kainic Acid (usa, sigma-aldrich,420318-10 MG), brain stereotaxic apparatus (china, rewarded life science, E03275-003), chloral hydrate (china, industrial, a 600288-0250), PBS (china, chinese maymon, CC 008), reblatatin (china, pharmaceutical synthesis), syringe, reblatatin (china, pharmaceutical synthesis), DMSO (usa, sigma-aldrich, D2650-100 ML), electroencephalogram amplifier (china, guangzhou edi electronics limited, china-201E), denture base resin (china, shanghai new century dental materials limited, type II class I), dental resin (2500, shanghai new dental materials limited, type II self-setting powder), medical disposable needle electrode (rich medical needle twisted wire, west ampere, T-13/NE, 0.13/13);

2. experimental methods

Preparation of model for C57 mouse chronic convulsion caused by hippocampal injection of Kainic acid

The preparation of the C57 mouse chronic convulsion model is the same as that of experiment six;

c57 mouse chronic convulsion model installation electrode cap

A 5% chloral hydrate anesthesia mouse chronic convulsion model;

fixing the anesthetized mouse chronic convulsion model in a brain stereotaxic instrument, preparing skin, cutting head skin at the top of the skull, cutting a circular area with the diameter of about 0.6cm, removing the skin and corresponding subcutaneous tissues of the corresponding area of the skull, and fully exposing the skull;

drilling holes with the anterior fontanel of the mouse chronic convulsion model as the origin and coordinates of (X-0.2cm, Y-0.2 cm), (X +0.2cm, Y + 0.2cm) and installing electrodes;

a bone cement fixation electrode;

rewarming at 37 ℃ until the mouse chronic convulsion model recovers clear-headed, and detecting the electroencephalogram after the mouse chronic convulsion model is recovered and raised for 2 weeks;

electroencephalogram for detecting C57 mouse chronic convulsion model

Connecting the brain electrode of the C57 mouse chronic convulsion model with electroencephalogram amplifier equipment through a medical disposable needle electrode stranded wire;

setting leads and continuously recording mouse electroencephalogram signals;

continuously recording for 2 weeks, counting chronic convulsion attack situations of mice, randomly dividing a chronic convulsion model of the mice with the frequency of chronic convulsion attack not less than 0.5 times/day into 2 groups, injecting Z-R into the abdominal cavity of the first group, and injecting solvent DMSO into the abdominal cavity of the second group as a control;

according to the previous experimental result, preparing Z-R according to the administration dose of 4mg/kg, dissolving the Z-R in DMSO to prepare the injection for each mouse, wherein the total administration volume is 50ul, carrying out intraperitoneal injection for 1 time every 1 day, and continuously administering for 21 days;

the frequency change of the chronic convulsion attack of each group of mice before and after administration is counted.

3. Results of the experiment

FIG. 13 is the brain electricity of the C57 mouse chronic convulsion model.

Wherein, the upper diagram: in the normal state, the C57 mouse chronic convulsion model electroencephalogram can show sparse sharp waves and spike waves;

the following figures: the C57 mouse chronic convulsion model electroencephalogram under the chronic convulsion attack state has a large amount of sharp waves and spike waves which are densely appeared, and the electroencephalogram activity after the attack is finished is obviously inhibited.

As shown in figures 14-15, electroencephalogram results show that the frequency of chronic convulsion attacks of mice can be remarkably reduced by intraperitoneal injection of the Z-R with the dose of 4 mg/kg.

FIG. 14: the frequency of chronic convulsion before and after the Z-R group and the DMSO group are subjected to intraperitoneal injection is changed, the frequency of chronic convulsion after the Z-R group is subjected to intraperitoneal injection is reduced to 52.4 percent of the baseline level, and the frequency of chronic convulsion after the DMSO group is subjected to intraperitoneal injection is increased to 153 percent of the baseline level;

FIG. 15 is a schematic view of: after the chronic convulsion model of each mouse is administrated, the frequency of chronic convulsion attacks is obviously reduced compared with that of a DMSO group after the chronic convulsion model is administrated compared with that before the chronic convulsion model is administrated, the ratio of the Z-R group is mostly lower than 1, and the ratio of the DMSO group is mostly higher than 1,Z-R group.

Example 8 C57 mice Chronic convulsion model intraperitoneal injection of Z-R to alleviate astrocyte dysplasia in brain tissue

1. Experimental Material

Paraffin microtomes (RM 2235 cwEU, leica fiber systems ltd., germany), cold plates of modular tissue embedding systems (EG 1150C, shanghai leica instruments ltd., germany), baking sheet instruments (HI 1220, shanghai leica instruments ltd., germany), water bath devices (HI 1210, shanghai leica instruments ltd., germany), adhesive slides (china, santa, 188105), microscope cover slips (china, santa, 10212450C), environmentally friendly transparent dewaxing fluids (china, china sequoyiki bridge, ZLI-9315), absolute ethanol (china, tianjin seiyuan chemical reagents ltd., usa), GFAP (Cell signaling technology, 3670S), autoclaves (china, supperl GmbH, inc., AS 20-4.0-90), a multifunctional induction cooker (China, america, EF 197), a wet box, citric acid (China, national group chemical reagent, inc., 10007118), sodium citrate (USA, sigma-aldrich, S1804-500G), super PAP Pen (China, china fir bridge, ZLI-9305), bovine serum album (USA, sigma-aldrich, A1933-100G), triton X-100, a universal two-step method kit (Mouse/rabbit enhanced polymer method detection system) (China, china fir bridge, PV-9000), GFAP (US, cell signaling technology, 3670S), donkey anti-dynamic Mouse IgG (H + L) HigCross-Adsored absorbent company, adsorry ^TM 488 (Invitrogen, A-21202, USA), DAPI (sigma-aldrich, D9542-1MG, USA), leica DM 6B Upper Microscope (Leica,DM 6B), an anti-fluorescence attenuation block tablet (china, solibao, S2100), a DAB color reagent kit (china, sequoia jinqiao, ZLI-9019), and a primary anti-diluent (china, solarbio, a 1810);

2. experimental methods

Brain tissue section of C57 mouse chronic convulsion model

Chloral hydrate 5% was anesthetized in C57 mouse chronic convulsions model for 21 consecutive days after administration;

fixing the mouse on an operating table, opening the thoracic cavity of the mouse, and cutting the right auricle;

perfusing about 20ml of PBS from the left ventricle to clear blood from the circulation of the mice;

injecting about 20ml of 4-th PFA from the left ventricle, preliminarily fixing the whole body tissues and organs of the mouse;

dissecting mouse cranium, taking out intact brain tissue, cutting off cerebellum and olfactory bulb, placing the rest brain tissue in 4% PFA solution, and fixing in rotary shaking table for 48 hr;

transferring the brain tissue of the mouse to a 15% sucrose solution, and placing the mouse brain tissue in a rotary shaking table for dehydration for 24 hours;

transferring the brain tissue of the mouse to a 30% sucrose solution, and placing the mouse brain tissue in a rotary shaking table for dehydration for 24 hours;

embedding mouse brain tissue by paraffin;

embedding a mouse brain tissue slice, cutting out classical hippocampal tissue, continuously slicing according to the thickness of 5ul, drying the slices, and storing at normal temperature;

immunofluorescence staining of C57 mouse chronic convulsion model brain tissue section

Taking a paraffin section of brain tissue of a C57 mouse chronic convulsion model, and heating at 60 ℃ for 10min to dissolve the paraffin of the tissue section;

completely immersing the tissue slices in the dewaxing solution, and slowly shaking for dewaxing for 10min;

repeatedly dewaxing the tissue slices once;

completely immersing the tissue slices in 100% ethanol, and slowly shaking for rehydration for 10min;

100% ethanol is rehydrated once again;

completely immersing the tissue slices in 75% ethanol, and slowly shaking for rehydration for 5min;

completely immersing the tissue slices in 50% ethanol, and slowly shaking for rehydration for 5min;

completely immersing the tissue slices in single distilled water, and slowly shaking for rehydration for 5min;

the PBS completely submerges the tissue slices, and slowly shakes for 5min;

antigen retrieval, weighing 0.38g of citric acid and 2.4g of sodium citrate, dissolving in 1L Shan Zhengshui, placing in an autoclave, heating with an electromagnetic oven on strong fire until gas is emitted, heating with slow fire for 3min, discharging gas, and cooling to room temperature;

transferring the slices to PBS for soaking for 5min;

BSA was dissolved in PBS to prepare 5% and 1% BSA solutions, and 0.1% Triton X-100 was added;

wiping the area around the glass slide where the brain tissue is located, and forming a pen-drawing circle;

blocking, and puncturing, 5% BSA blocking containing 0.1% Triton X-100 for 30min;

preparing a GFAP antibody by using an anti-dilution solution, and incubating overnight;

taking out the slices, spin-drying the primary antibody, and washing for 3x5min by PBST;

1% BSA to prepare a fluorescent secondary antibody, and incubating for 60min at normal temperature;

1% BSA diluted DAPI, stained nuclei for 5min;

PBST is washed for 3x5min, the anti-fluorescence attenuation mounting agent is mounted for mounting, and the Leica DM 6B is placed in a fluorescence microscope for shooting statistics;

the statistical method comprises the following steps: a plurality of parts of the hippocampal region of the tissue section are randomly framed and quantified by applying a self-contained image quantification function of Leica Application Suite X software, and statistical analysis is carried out on a quantitative result.

Immunohistochemical staining of brain tissue section of C57 mouse chronic convulsion model

Taking a paraffin section of brain tissue of a C57 mouse chronic convulsion model, and heating at 60 ℃ for 10min to dissolve the paraffin of the tissue section;

completely immersing the tissue slices in the dewaxing solution, and slowly shaking for dewaxing for 10min;

repeatedly dewaxing the tissue slices once;

completely immersing the tissue slices in 100% ethanol, and slowly shaking for rehydration for 10min;

repeating the 100% ethanol rehydration once;

completely immersing the tissue slices in 75% ethanol, and slowly shaking for rehydration for 5min;

completely immersing the tissue slices in 50% ethanol, and slowly shaking for rehydration for 5min;

completely immersing the tissue slices in single distilled water, and slowly shaking for rehydration for 5min;

the PBS completely submerges the tissue slices, and slowly shakes for 5min;

antigen retrieval, weighing 0.38g of citric acid and 2.4g of sodium citrate, dissolving in 1L Shan Zhengshui, placing in an autoclave, heating with an electromagnetic oven on strong fire until gas is emitted, heating with slow fire for 3min, discharging gas, and cooling to room temperature;

transferring the slices to PBS for soaking for 5min;

wiping the area around the glass slide where the brain tissue is located, and forming a pen-drawing circle;

repairing endogenous peroxidase blocker antigen for 30min, and washing with PBST for 5min;

blocking, perforating, blocking with 5% of Triton X-100 by 0.1% BSA for 30min;

primary anti-diluent 1;

taking out the slices, spin-drying the primary antibody, and washing for 3x5min by PBST;

incubating the reaction enhancing solution at room temperature for 20min;

PBST washing for 3x5min;

incubating the enhanced enzyme goat anti-mouse/rabbit IgG polymer for 20min at room temperature;

PBST for 3x5min;

DAB color development is carried out for 2min and 20s;

PBST is washed for 3x5min, the anti-fluorescence attenuation mounting agent is mounted for mounting, and the Leica DM 6B is placed in a fluorescence microscope for shooting statistics;

the statistical method comprises the following steps: a plurality of parts of the hippocampal region of the tissue section are randomly framed and quantified by applying a self-contained image quantification function of Leica Application Suite X software, and statistical analysis is carried out on a quantitative result.

3. Results of the experiment

As shown in FIG. 16, the immunofluorescent-labeled GFAP results indicated that abnormal proliferation of hippocampal astrocytes was effectively alleviated in the chronic convulsion model of C57 mice after Z-R treatment.

FIG. 16A: immunofluorescence marks C57 mouse chronic convulsion model brain tissue astrocyte specific expression protein marker GFAP, random frame selection of the region of the hippocampus is carried out to detect the fluorescence intensity, and the form and the number of astrocytes in the hippocampus region of each C57 mouse chronic convulsion model are compared.

FIG. 16B: and (3) concentrating fluorescence intensities of all the selected hippocampal regions in the same treatment group, performing statistical analysis on the overall fluorescence intensities of the Z-R group and the DMSO group, and comparing influences of different treatments on the form and quantity of astrocytes in the hippocampal regions of the C57 mouse chronic convulsion model.

As shown in fig. 17, immunohistochemical labeling GFAP results showed effective relief of hippocampal astrocyte hyperplasia in the chronic convulsive model of C57 mice after Z-R treatment.

FIG. 17A: immunohistochemistry marks the brain tissue astrocyte specific expression protein marker GFAP of the C57 mouse chronic convulsion model, randomly selecting the region where the hippocampus is located to detect the fluorescence intensity, and comparing the form and the number of the astrocytes in the hippocampus region of each C57 mouse chronic convulsion model.

FIG. 17B: and (3) concentrating the fluorescence intensity of all the frame-selected hippocampal regions of the same treatment group, performing statistical analysis on the overall fluorescence intensity of the Z-R group and the DMSO group, and comparing the influences of different treatments on the form and the quantity of astrocytes in the hippocampal regions of the C57 mouse chronic convulsion model.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. The application of a natural product in preparing a medicament for preventing or treating chronic convulsion is characterized in that the natural product is reblatatin.

2. The use of claim 1, wherein the reblatatin is capable of increasing the expression level of Glt-1 in a cell, preferably a primary astrocyte.

3. The use according to claim 1, wherein the reblatatin is capable of alleviating abnormal proliferation of astrocytes in brain tissue.

4. The use of claim 1, wherein the pharmaceutical dosage form comprises tablet, capsule, drop pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, and lyophilized powder for injection.

5. A pharmaceutical composition for preventing or treating chronic convulsions, wherein the pharmaceutical composition comprises a reblatatin.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable buffer, carrier or excipient.

7. The pharmaceutical composition of claim 6, wherein the buffer comprises Trizma, bicine, tricine, MOPS, MOPSO, MOBS, tris, hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, caconate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolalactasic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

8. The pharmaceutical composition of claim 6, wherein the carrier comprises an antimicrobial agent, an isotonic agent, an antioxidant, a local anesthetic, a suspending agent, a dispersing agent, an emulsifying agent, a chelating agent, a thickening agent, or a solubilizing agent.

9. The pharmaceutical composition of claim 6, wherein the excipient comprises a carbohydrate, a polymer, a lipid, or a mineral.

10. A method for increasing glutamate uptake capacity of a cell for non-therapeutic purposes, said method comprising administering a reblatatin to a cell, preferably said cell is a primary astrocyte.

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