Application of substances that inhibit lactation at the HSP90α-K408 site in astrocytes in epilepsy

Abstract

This invention provides the application of a substance that inhibits lactation at the HSP90α-K408 site in astrocytes in the treatment of epilepsy. The HSP90α-K408 site is the 408th amino acid position shown in SEQ ID NO:1. This invention targets HSP90α-K408 lactation in astrocytes as a therapeutic target for epilepsy and provides a polypeptide that inhibits HSP90α-K408 lactation in astrocytes. Mouse experiments have demonstrated the regulatory effect of this polypeptide on glutamate and glutamine levels, effectively controlling the number of neurons and contributing to the relief and treatment of epilepsy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of substances that inhibit lactation at the HSP90α-K408 site in astrocytes in epilepsy. Background Technology

[0002] Epilepsy is a chronic neurological disorder characterized by recurrent seizures caused by abnormal synchronous discharges of neurons in the brain. It ranks second globally in incidence among neurological diseases, after stroke. This disease not only severely impacts patients' physical, psychological, and cognitive functions but also imposes a heavy socioeconomic burden. Current clinical treatment primarily relies on antiepileptic drugs (AEDs), which inhibit abnormal neuronal discharges by regulating the activity of voltage-gated ion channels and neurotransmitter metabolic balance. However, existing drugs mostly target the terminal stages of seizures, failing to intervene in the core pathological process of the disease, leading to approximately 30% of patients eventually progressing to drug-resistant epilepsy. Developing new therapeutic targets and drugs for epilepsy remains a hot topic of ongoing interest for those skilled in the art. Summary of the Invention

[0003] This invention provides the application of a substance that inhibits lactation at the HSP90α-K408 site in astrocytes in epilepsy.

[0004] In a first aspect, the present invention provides the use of a substance that inhibits lactation at the HSP90α-K408 site in astrocytes in the preparation of a medicament for relieving and / or treating epilepsy, wherein the HSP90α-K408 site is the 408th amino acid position shown in SEQ ID NO:1.

[0005] As described above, the protein shown in SEQ ID NO:1 is the HSP90α protein (heat shock protein HSP90-alpha) derived from mice (Mus musculus), which consists of 733 amino acids. The lactation site is located at lysine (K) at position 408. The specific amino acid sequence shown in SEQ ID NO:1 is as follows:

[0006] MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPSKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRI KEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEEKEEKEEEKEKEEKESDDKPEIEDVGSDEEEEEKKDGDKKKKKKIKEKYIDQEELNKTKPIWTRNPDDI TNEEYGEFYKSLTNDWEEHLAVKHFSVEGQLEFRALLFVPRRAPFDLFENRKKKNNIKLYVRRVFIMDNCEELIPEYLNFIRGVVDSEDLPLNISREMLQQS K ILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSKNIKLGIHEDSQNRKKLSELLRYYTSASGDEMVSLKDYCTRMKENQKHIYFITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEYCVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFEN LCKIMKDILEKKVEKVVVSNRLVTSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHSIIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHANRIYRMIKLGLGIDEDDPTVDDTSAAVTEEMPPLEGDDDTSRMEEVD.

[0007] As described above, the amino acid sequence of the HSP90α protein derived from humans (Homo sapiens) differs from that of the HSP90α protein derived from mice, but the amino acid sequence near the lactation site is completely conserved.

[0008] As described above, the substance that inhibits lactation modification at the HSP90α-K408 site in astrocytes is an HSP90α-K408 blocking peptide, which is selected from at least one of the polypeptides whose amino acid sequence is shown in SEQ ID NO:2-6.

[0009] As described above, the amino acid sequence of the HSP90α K408 blocking peptide is SEQ ID NO:4.

[0010] As described above, substances that inhibit lactation at the HSP90α-K408 site in astrocytes are used in the preparation of drugs for the relief and / or treatment of epilepsy, embodied in at least one of A1)-A3):

[0011] A1) Reduce the concentration of glutamate and glutamate in hippocampal tissue;

[0012] A2) Increase the number of neurons in the CA1 and CA3 regions of the hippocampus;

[0013] A3) Reduce the frequency of spontaneous epileptic seizures.

[0014] As described above, the epilepsy refers to epilepsy in the subacute or chronic phase; the subacute phase refers to the first non-induced epileptic seizure occurring between 7 days and 3-6 months after the primary brain injury; the chronic phase refers to epileptic seizures occurring 3-6 months after the primary brain injury, or seizures that have occurred before this period and persist.

[0015] In a second aspect, the present invention provides a pharmaceutical composition comprising the substance described above that inhibits lactation at the HSP90α-K408 site in astrocytes and a pharmaceutically acceptable carrier.

[0016] In the pharmaceutical composition described above, the pharmaceutically acceptable carrier can be a substance that helps the substance to effectively enter cells and exert its effects, and the specific carrier can be determined based on factors such as the purpose of treatment, the stability of the substance, the required targeting, and the route of administration.

[0017] The pharmaceutical composition described above can be formulated into various dosage forms, including conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. In one specific embodiment, the pharmaceutical composition is an injection.

[0018] Thirdly, the present invention provides a polypeptide selected from at least one polypeptide whose amino acid sequence is shown in SEQ ID NO:2-6.

[0019] Fourthly, the present invention provides the application of a polypeptide in inhibiting lactation at the HSP90α-K408 site in astrocytes.

[0020] Fifthly, the present invention provides a method for inhibiting lactation at the HSP90α-K408 site in astrocytes, wherein the above-mentioned polypeptide is contacted with astrocytes to inhibit lactation at the HSP90α-K408 site in astrocytes.

[0021] In a sixth aspect, the present invention provides a method for alleviating and / or treating epilepsy, comprising administering to a subject a therapeutically effective amount of any of the substances described above that inhibit lactation at the HSP90α-K408 site in astrocytes.

[0022] As described above, the subject refers to a subject suffering from epilepsy, whose clinical manifestations include loss of consciousness, generalized convulsions, foaming at the mouth, as well as sudden blank stares, pauses in movement (absence seizures), or involuntary twitching of parts of the body, abnormal sensations, etc.

[0023] As described above, the substance that inhibits lactation modification at the HSP90α-K408 site in astrocytes can be injected into the subject via intracerebrospinal fluid injection. This substance can target astrocytes in the hippocampus region through cerebrospinal fluid circulation.

[0024] In the method described above, the subject can be a mammal, which can be selected from bovine, equine, feline, canine, lagomorph, suidae, camel, rodent and primate animals, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, orangutans) and humans, preferably rats, mice and humans.

[0025] As described above, the dosage of the substance used to inhibit lactation at the HSP90α-K408 site in astrocytes is variable, depending on the method of administration, route of administration, individual age and / or weight, and the individual's condition, and is ultimately determined by the attending physician. Attached Figure Description

[0026] Figure 1 The results show the expression levels of EAAT2 protein in epileptic brain tissue. Specifically, a represents the results of Western blot analysis of EAAT2 protein monomers and multimers in perifocal tissue (PT) and epileptic focus tissue (EP) of epileptic patients; b represents the statistical analysis results of the corresponding grayscale values ​​in a, with β-actin used as an internal reference protein (n=5, paired t-test); c represents the results of Western blot analysis of EAAT2 protein in the hippocampus of mice in the Sham, KA 1d, KA 7d, and KA 28d groups; d represents the statistical analysis results of the corresponding grayscale values ​​in c, with β-actin used as an internal reference protein (n=3, one-way ANOVA).

[0027] Figure 2The results show the lactate content and lactation modification level in epileptic brain tissue. Specifically, a) lactate levels in EP and paired PT were detected by colorimetric method (n=15, paired t-test); b) lactate levels in hippocampal tissue of mice in the Sham, KA 1d, KA 7d, and KA 28d groups were detected by colorimetric method (n=6-7, one-way ANOVA); c) Western blot analysis of pan-lactation modification levels in EP and paired PT, with β-actin used as an internal reference protein (n=5); d) lactate levels in the Sham, KA 1d, KA 7d, and KA 28d groups. Western blot analysis results of pan-lactic acidification modification level in hippocampal tissue of group d mice; e is the gray value analysis result corresponding to d, β-actin is used as internal reference protein (n=3, one-way ANOVA); fg is a representative image of lactic acidification modification (Lactyl-K, green) and astrocyte (GFAP, red) localization in CA1 and CA3 regions of hippocampal tissue of co-labeled KA mice and Sham mice after immunofluorescence staining, scale bar=100 μm.

[0028] Figure 3 To identify lactation-modified proteins and sites in TLE mice using lactation modification proteomics; where a is the 4D-label-free quantitative proteomics detection procedure for lactation modification; and b is the differentially modified proteins and modification sites.

[0029] Figure 4 The results are from the lactation modification proteomics analysis; among them, a) shows the top 25 differentially expressed lactation modification proteins and their sites; b) shows the cellular functions and pathways enriched by GO-BP and KEGG enrichment analysis of differentially expressed proteins with increased lactation modification.

[0030] Figure 5The study aimed to investigate the K408 site lactation modification of HSP90α in astrocytes. Specifically, a) shows the HSP90α protein profile in the hippocampus of TLE mice; b) shows the HSP90α protein levels in five pairs of EP and corresponding PT tissues, with β-actin used as an internal control; c) shows the HSP90α protein levels in the hippocampus of mice in the Sham, KA 1d, KA 7d, and KA 28d groups, with β-actin used as an internal control; d) shows the pan-lactic acidification level in primary astrocytes after 24 hours with or without sodium lactate (10 μM or 20 μM), with β-actin used as an internal control; ef) shows the immunoprecipitation assay using HSP90α antibody or Lactyl-K antibody to detect the binding of HSP90α and Lactyl-K; g) shows the immunofluorescence staining assay to detect the co-localization level of HSP90α (green) and Lactyl-K (red) in primary astrocytes, with scale bar = 20. μm; h represents the conservation analysis of the HSP90α protein 408 site across different species; i represents the detection of Flag-HSP90α lactation modification level by immunoprecipitation using Flag antibody after overexpression of HSP90α-WT or HSP90α-K408R in astrocytes, with β-actin used as an internal control; j represents the specificity results of dot blot detection of anti-HSP90αK408lac polyclonal antibody; k represents the detection of HSP90αK408lac modification level in cells after treatment with solvent, 2-DG, or Oxamate for 12 hours, with β-actin used as an internal control.

[0031] Figure 6 This study investigated the regulation of EAAT2 protein stability by lactation modification at the HSP90α-K408 site in astrocytes. Specifically, ab represents the detection of EAAT2 protein levels and grayscale analysis after overexpression of HSP90α-WT or HSP90α-K408R in sh-HSP90α astrocytes (n=3, one-way ANOVA); c represents the extracellular glutamate concentration after treatment with 50 nM glutamate for 20, 40, 60, and 80 minutes following overexpression of HSP90α-WT or HSP90α-K408R in sh-HSP90α astrocytes (n=3, one-way ANOVA); and d represents the long-term glutamate uptake assay after treatment with 500 nM glutamate in sh-HSP90α astrocytes. Extracellular glutamate concentration after nM glutamate treatment for 20 minutes (n=3, one-way ANOVA); ef represents the expression level and gray value of EAAT2 protein after primary astrocytes overexpressing HSP90α-WT or HSP90α-K408R with or without sodium lactate, and after CHX treatment for 0, 4, 8 or 12 hours. β-actin was used as an internal control (n=3, t-test).

[0032] Figure 7 The HSP90α K408lac blocking peptide promotes hippocampal glutamate reuptake and attenuates the epileptic phenotype in mice; where a) is the design strategy for the HSP90α K408 blocking peptide; b) is the immunoprecipitation using Flag antibody and the detection of HSP90α using HSP90α K408lac antibody. The lactation inhibition capacity of different K408 blocking peptides; c shows the lactation inhibition capacity of different concentrations of blocking peptide K408-Peptide-3 detected by immunoprecipitation with Flag antibody and HSP90αK408lac antibody; d shows the lactation inhibition capacity of blocking peptide K408-Peptide-3 and mutant blocking peptide K408R-Peptide-3 detected by immunoprecipitation with Flag antibody and HSP90αK408lac antibody; e is a schematic diagram of the animal experiment process; f shows the number of spontaneous epileptic seizures in mice monitored by video electroencephalography from 35 to 42 days after modeling (n=7, one-way ANOVA); gh shows the number of neurons in the CA3 and CA1 regions of the hippocampus of mice sacrificed 42 days after modeling detected by Nissl staining (n=6, one-way ANOVA); i shows the concentration of glutamate (Glu) and glutamine (Gln) in the hippocampus of mice sacrificed 42 days after modeling detected by mass spectrometry (n=4, one-way ANOVA). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] The clinical samples used in the following examples were collected from epileptogenic foci located by stereotactic electroencephalography (SEEG) and surgically removed in the functional neurosurgery department of Beijing Tiantan Hospital, Capital Medical University, with the approval of the Ethics Committee and the signing of informed consent. The core epileptic discharge area tissue served as the experimental group, while peripheral tissue served as the control group. Specimens obtained during surgery were rinsed with saline to remove surface blood, and cut into two parts. One part was directly fixed in 4% paraformaldehyde at 4°C for paraffin sectioning. The other part was directly cryopreserved in liquid nitrogen for protein and RNA extraction.

[0036] In the following examples, the epilepsy animal model was established using the amygdala-targeted injection of kainic acid (KA). The specific procedure included: mice anesthetizing them with isoflurane gas and fixing them on a stereotaxic apparatus; the right amygdala was selected as the injection target (coordinates: 0.94 mm posterior to the anterior fontanelle, 2.75 mm lateral to the midline, depth 4.75 mm); 0.3 μL of KA solution (1 μg / μL, purchased from Sigma-Aldridge) was injected at a constant rate using a microinfusion pump; the sham-operated control group received an equal volume of physiological saline. Spontaneous recurrent epileptic seizures were recorded using a 24-hour video monitoring system, and behavioral scores were performed according to the Racine classification.

[0037] The protein immunoblotting procedure described in the following examples includes electrophoresis, transfer, blocking, antibody incubation, and signal detection. First, 20 μg of protein sample was mixed with 4× Sample Buffer to a 1× concentration. Lysis buffer was added to achieve a final concentration of 1-2 mg / mL. After denaturation at 95°C for 10 minutes, the sample was simultaneously loaded with a pre-stained protein marker for SDS-PAGE electrophoresis. The sample was run at a constant voltage of 80 V for 30 minutes to allow it to enter the separating gel, then switched to 120 V until bromophenol blue ran off the gel. Next, a pre-cooled, equilibrated NC membrane was aligned with the gel, and transfer was performed at a constant current of 200 mA for 1 hour at 4°C. After transfer, the membrane was blocked with 5% skim milk powder-TBST for 1 hour. After washing with TBST, it was incubated sequentially with primary antibody and HRP-labeled secondary antibody. After each antibody incubation, the membrane was thoroughly rinsed with TBST. Finally, the target protein signal was captured by dynamically adjusting the exposure time in the imaging system using an HRP chemiluminescent substrate reaction for 2 minutes.

[0038] In the following examples, continuous variables that conform to a normal distribution are expressed as mean ± standard deviation. The independent samples t-test is used for comparisons between two groups, and analysis of variance (ANOVA) is used for comparisons among multiple groups. P A value <0.05 was considered statistically significant. SPSS 22.0 and GraphPad Prism 7 statistical graphing software were primarily used for data analysis, processing, and graphing.

[0039] Example 1: Downregulation of glutamate transporter-excitatory amino acid transporter 2 (EAAT2) expression in epileptic brain tissue

[0040] Five pairs of perifocal tissue (PT) and epileptic focus (EP) samples were collected from epilepsy patients. The content of EAAT2 protein in the samples was detected using Western blotting analysis. Results are as follows: Figure 1 As shown in Figure ab, it can be seen that, compared with PT, the expression levels of both EAAT2 protein monomers (cytoplasm) and multimers (cell membrane) are significantly decreased in EP.

[0041] Hippocampal tissue was harvested from mice with KA temporal lobe epilepsy (KA group) on days 1, 7, and 28 after KA injection. Hippocampal tissue from the control group (Sham group) was used as a control. The same Western blot analysis method was used to detect the EAAT2 content in the samples. Results are as follows: Figure 1 As shown in the middle CD, it can be seen that, compared with the Sham group, the expression level of EAAT2 in the hippocampus of KA group mice did not change significantly in the acute phase (KA 1d), but gradually decreased in the subacute phase (KA 7d) and chronic phase (KA 28d).

[0042] The above results indicate that EAAT2 expression is specifically downregulated in epileptic brain tissue, and mainly occurs in the subacute and chronic phases.

[0043] Example 2: Lactic acid content and lactation modification level were significantly upregulated in epileptic brain tissue.

[0044] 1. EP samples and corresponding PT tissue samples were collected from 15 pairs of epilepsy patients, and the lactate level in the tissues was detected using a colorimetric method. Specific steps included: collecting samples and rinsing the tissues with pre-cooled PBS buffer. The tissues were placed in an ice bath, and 4-6 times the volume of the detection reagent buffer was added. The brain tissues were homogenized thoroughly at 4°C and 6000 rpm, repeating the homogenization process 10-15 times. The tissues were centrifuged at 4°C and 16000 rpm for 5 minutes, and the supernatant was collected. A standard curve was prepared using the lactate standard provided in the lactate detection kit (abcam, Cat. No. ab65330). The reaction system was prepared by adding 50 μl of reaction mixture to each standard well and sample well, and 50 μl of background control mixture to each background well. After mixing and incubating in the dark at room temperature for 30 min, the absorbance was measured at 570 nm using a microplate reader. A standard curve was constructed based on the serially diluted standards, and the lactate content of the samples was calculated. Results are shown below. Figure 2 As shown in Figure a, the lactate level in EP was significantly higher than that in PT.

[0045] 2. Using the same detection method, the changes in lactate content in the hippocampus of KA-induced temporal lobe epilepsy mice were verified, and the results are as follows: Figure 2 As shown in Figure b, lactate levels rise during the subacute phase and tend to return to normal levels during the chronic phase.

[0046] 3. Western blot analysis was performed to detect the level of lactation modification in brain tissue of epilepsy patients and hippocampal tissue of epilepsy mouse models using a pan-lactic acidification modification antibody. Results are as follows: Figure 2 As shown in the results, the pan-lactic acidification level in the EP tissue of epilepsy patients was significantly higher than that in the PT tissue. In the mouse model, the pan-lactic acidification level in the hippocampus at KA 1 d, KA 7 d, and KA 28 d was significantly higher than that in the Sham group.

[0047] 4. Identify the types of lactated cells using immunofluorescence assays. Specifically: Brain tissue was fixed in 4% paraformaldehyde solution at 4°C for 24 hours, then subjected to a gradient sucrose solution of 20% and 30%, embedded in OCT, sectioned using a cryostat, 25 μm thick, mounted on poly-L-lysine-treated slides, and stored at -80°C for later use. Immunofluorescence staining: Frozen sections were thawed at room temperature for 30 minutes, washed three times with PBS for 5 minutes each time, and then permeabilized in PBS containing 0.1% Triton X-100 for 10 minutes, followed by three washes with PBS for 5 minutes each time. Gently shake the slide several times manually to remove all liquid. Prepare a 4% sheep serum blocking solution with PBS and block at room temperature for 30 minutes. Remove all liquid, do not wash, add primary antibody, and incubate at 37°C for 1 hour or overnight at 4°C. Wash three times with PBS for 5 minutes each time (for double-labeled staining, prepare a primary antibody mixture according to the dilution factor). After removing the primary antibody, wash the cells three times with PBS. Add fluorescein-labeled secondary antibody, protect from light, incubate at 37°C for 60 minutes, and rinse with 0.01M PBS for 5 minutes each time, three times. Mount with anti-quenching mounting medium and store at 4°C, protected from light. Observe and photograph under a fluorescence microscope. Results are as follows. Figure 2 As shown in fg, staining results of the CA1 and CA3 regions of the hippocampus indicate that lactation modification mainly occurs in neurons and astrocytes, and the level of lactation modification in astrocytes is significantly increased after epilepsy modeling.

[0048] Example 3: Lactic acidification modification proteomics identification of lactation-modified proteins and sites in TLE mice

[0049] like Figure 3As shown in Figure a, to identify differentially expressed lactation-modified proteins and specific modification sites in epileptic brain tissue during epilepsy, this embodiment performed omics screening on KA temporal lobe epilepsy mice and sham-operated mice. Video EEG behavioral monitoring was performed from day 35 to day 42 post-modeling. The three TLE mice with the highest number of seizures and three Sham mice were selected, and hippocampal tissue was extracted and pooled to reduce inter-group differences. 4D-label-free quantitative proteomics detection of lactation modifications was then performed. Figure 3 As shown in Figure b, the modalomics study identified 188 lactation-modified proteins, of which 181 showed increased lactation levels and 7 showed decreased lactation levels; a total of 319 lactation modification sites were identified. Figure 4 As shown in Figure a, the 25 proteins with the highest differences in lactation modification levels are displayed (Log). 2 FC > 1.7, P < 0.05). Functional enrichment analysis was performed on proteins with increased lactation modification levels, such as... Figure 4 As shown in Figure b, functional enrichment analysis revealed that lactation modification is closely related to functions such as cell signaling, amino acid metabolism pathways, and energy metabolism pathways. Based on literature review, enrichment analysis, and preliminary experimental results, heat shock protein 90α (HSP90α) was selected. 2 (FC=2.1) to conduct subsequent experiments.

[0050] Example 4: Lactation modification at the K408 site in HSP90α cells of astrocytes

[0051] 1. After thawing the hippocampal tissue of a mouse model of epilepsy frozen at -80℃, add lysis buffer containing 8 M urea, 1% protease inhibitor, 3 μM TSA, and 50 mM NAM at a ratio of 1:4. After sonicating to disrupt the cells, centrifuge at 12,000 g for 10 minutes at 4℃ and collect the supernatant. Protein concentration is determined by the BCA method. Then, take an equal volume of protein sample and enrich the protein by precipitation with 20% trichloroacetic acid (TCA) at 4℃ for 2 hours. Centrifuge at 4500 g for 5 minutes and discard the supernatant. Wash 2-3 times with pre-cooled acetone to remove impurities, vacuum dry, reconstitute with 200 mM TEAB solution, and sonicate. Add trypsin at a ratio of 1:50 and incubate overnight at 37℃. Perform subsequent reduction with 5 mM DTT at 56℃ for 30 minutes and 11... alkylation with mM iodoacetamide at room temperature in the dark for 15 minutes; finally, the peptide was dissolved in mobile phase A (0.1% formic acid + 2% acetonitrile aqueous solution), and gradient separation was performed using a NanoElute ultra-high performance liquid chromatography system. After ionization by a Capillary ion source, the ion was introduced into a timsTOF Pro mass spectrometer. The precursor ion and secondary fragment spectra were acquired using PASEF mode. Each primary spectrum triggered 10 secondary scans of charge states 0-5, and the dynamic exclusion time was set to 30 seconds to optimize data coverage. The results are as follows: Figure 5 As shown in Figure a, lactation modification was observed at the HSP90α-K408 site, and the modification level was elevated in epileptic tissue.

[0052] 2. The levels of HSP90α protein in the brain tissue of epilepsy patients and mouse models of epilepsy were detected, such as... Figure 5 As shown in Figure bc, epilepsy does not affect the expression level of HSP90α protein.

[0053] 3. To further validate the proteomics data, primary cortical astrocytes were extracted from suckling mice, cultured, and treated with sodium lactate. Western blot analysis results are shown below. Figure 5 As shown in Figure d, the results indicate that 24 hours of sodium lactate treatment led to an increase in pan-lactation levels with increasing sodium lactate concentration.

[0054] 4. Total protein was extracted from brain tissue or cells using IP lysis buffer. The extract was washed with pre-cooled PBS and a 50% Protein A / G agarose bead suspension was prepared. 100 μl of agarose beads were added to 1 ml of the total protein solution, and the mixture was incubated at 4°C for 30 minutes to pre-remove non-specific binding. After centrifugation, the supernatant was collected for BCA quantification. Samples were aliquoted according to the results: 30 μg of protein from the Input group was mixed with IP lysis buffer and 5×Sample buffer to prepare a 20 μl system; 500-1000 μg of protein from the IP group was mixed with lysis buffer to prepare a 200 μl system. Then, the target primary antibody and IgG negative control antibody were added according to the manufacturer's instructions, and the mixture was gently vortexed overnight at 4°C. Finally, 60-100 μl of agarose beads were added, and the mixture was incubated at 4°C for 12 hours to capture the complex. After collecting the agarose bead-antigen-antibody complex by centrifugation, the mixture was washed three times with pre-cooled PBS to remove free impurities. The complex was then resuspended in 2×Sample buffer and boiled at 95°C for 5 minutes to dissociate it. The supernatant was then collected after centrifugation and stored at -80°C. Subsequent Western blot analysis was performed to detect the target protein interaction. The results are as follows: Figure 5 As shown in the figure, Co-IP detection revealed that HSP90α protein can be lactated, and the lactation level increases with increasing sodium lactate concentration.

[0055] 5. After embedding the tissue sample in paraffin, section it to a thickness of 4-6 μm. Fix with 4% paraformaldehyde solution at room temperature for 15-30 minutes, then permeabilize with PBS solution containing 0.1% Triton X-100 to enhance cell membrane permeability. Next, block with 10% normal serum matched to the species from which the secondary antibody is derived for 30 minutes at room temperature to eliminate non-specific binding. Then, add the working solution of the primary antibody against the target antigen and incubate overnight in a humidified chamber at 4°C. The next day, thoroughly wash the sections with PBS buffer, add the corresponding species-labeled working solution of the secondary antibody, and incubate at room temperature in the dark for 1 hour. After washing again with PBS, mount with mounting medium containing DAPI. Finally, observe and acquire fluorescence images at a specific excitation wavelength under a fluorescence microscope. Figure 5 As shown in Figure g, immunofluorescence assays confirmed that HSP90α co-localizes with Lactyl-K in the cytoplasm of primary astrocytes.

[0056] 6. To clarify the HSP90α lactation modification site, the amino acid sequence of HSP90α protein in hippocampal tissues of different species was analyzed. The results are as follows: Figure 5 As shown in h, the HSP90α 408 site is highly conserved among different species.

[0057] 7. Using a mouse cDNA library as a template, the coding region of the HSP90α gene was amplified by PCR and cloned into the pcDNA3.1(+) eukaryotic expression vector to construct the HSP90α wild-type plasmid. Based on this, using overlap extension PCR or a site-directed mutagenesis kit, primers containing the K408R point mutation were designed using the wild-type plasmid as a template to replace the codon encoding lysine (K) at position 408 with the codon encoding arginine (R). After PCR amplification and DpnI digestion of the template, the obtained mutant fragment was transformed into competent cells and amplified. Finally, the correctness of the plasmid sequence was verified by DNA sequencing. The plasmid and empty vector were transfected using Lipofectamine 3000 (Invitrogen), and cells were collected 48 hours after transfection for subsequent analysis. Cells were grown at a rate of 3 × 10⁶ cells / year. 5 Cells were seeded per well in 6-well plates. When cell confluence reached 30%-50%, infection was performed: 1 ml of complete culture medium containing 40 μl Hitrans GP infection enhancer and 20 μl LV was added to each well. After 12-16 hours of infection, the medium was replaced with fresh serum. HSP90α wild-type plasmid and K408R mutant plasmid were constructed and transfected into astrocytes. Co-IP analysis was performed, and the results are as follows: Figure 5 As shown in Figure i, mutation at site 408 can significantly reduce the level of HSP90α lactation modification.

[0058] 8. Construct antibodies against the HSP90α-K408 lactation modification site, including:

[0059] (1) Synthesized polypeptide sequences for antigen preparation. Two modified polypeptide sequences (modified polypeptide A and modified polypeptide B) were designed and synthesized, and their immunogenicity and specificity were ensured. At the same time, a modified control polypeptide and a negative unmodified polypeptide were designed for antibody detection.

[0060] Modified polypeptide A: LQQS-(Lactyl)K-ILKVIRKN; Antigen region: 404-416aa;

[0061] Modified polypeptide B: QQS-(Lactyl)K-ILKVIRK antigen region: 405-415aa;

[0062] Modified control peptide C: LQQSKIL-(Lactyl)K-VIRKN; Antigen region: 404-416aa;

[0063] Unmodified peptide: LQQSKLKVIRKN Antigen region: 404-416aa.

[0064] (2) Animal immunization

[0065] The peptides were KLH conjugated. Two New Zealand White rabbits were immunized with each peptide, for a total of four rabbits. The immunization method involved injecting the antigen subcutaneously at two points on both shoulders and intramuscularly at two points on both hind legs. A total of six immunizations were performed, with blood samples collected four times after the fourth immunization.

[0066] (3) Serum screening and purification

[0067] The antibody titer in serum was determined using ELISA results, and Western blotting was performed on the serum. A serum titer (OD450 > 1.0) of 1:10,000 (after the 3rd or 4th immunization) was considered satisfactory and ready for further purification. Based on the combined ELISA and WB results, serum from 2-4 rabbits was selected for two-step affinity purification of Protein A and the antigenic peptide.

[0068] (4) ELISA detection of anti-HSP90α peptide (K408lac)

[0069]

[0070] The results showed that the recognition ability of at least one modified peptide was no less than that of a 1:50K dilution.

[0071] (5) Immunospot detection of anti-HSP90α peptide (K408lac)

[0072] Modified and unmodified peptides at different doses (e.g., 4 ng, 16 ng) were immobilized on a solid membrane, incubated with anti-HSP90α peptide (K408lac) antibody (1:2000), followed by the addition of enzyme-labeled secondary antibody and chemiluminescent substrate, and the binding of peptides and antibodies was detected.

[0073] Using the glucose metabolism pathway inhibitors 2-DG and Oxamate, it was demonstrated that intracellular lactate levels can regulate lactation at the HSP90α-K408 site. Figure 5 (jk).

[0074] Example 5: Lactation modification at the HSP90α-K408 site in astrocytes regulates the stability of EAAT2 protein.

[0075] 1. Literature review shows that the HSP90 family is closely related to the stability of EAAT2 protein. To verify whether lactation modification at the HSP90α-K408 site can regulate the stability of excitatory amino acid transporter 2 (EAAT2) protein, HSP90α knockdown astrocytes were first constructed to exclude the influence of endogenous HSP90α. Specifically: LV3 (H1 / GFP & Puro) lentiviral vectors were used to construct shRNA lentiviruses targeting mouse HSP90α (shHSP90) and negative control lentiviruses (shNC), respectively. The target sequence for shHSP90α was GGTCGTGGAACAAAGGTTATC (SEQ ID NO:8), and the negative control shNC used a scrambled sequence TTCTCCGAACGTGTCACGT (SEQ ID NO:9) that showed no significant homology to the mouse genome, as verified by BLAST. After annealing the above oligonucleotide chains to form double strands, they were cloned into LV3 vectors using BamHI and EcoRI restriction endonuclease sites. After transformation, amplification, and sequencing verification, the correct recombinant plasmid, along with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G, was co-transfected into 293T cells for viral packaging. The supernatant was collected, filtered, and concentrated by ultracentrifugation to obtain high-titer lentiviral particles. Subsequently, under a multiplicity of infection (MOI) of 5, the obtained lentiviral particles were combined with 8... μg / mL of polybrene was added to cultured mouse astrocytes. After 24 hours of infection, the medium was replaced with complete medium. After 48 hours of further culture, the medium was replaced with medium containing 2 μg / mL puromycin for continuous screening. The medium was changed every 2-3 days until all untransfected cells in the control group died. The surviving cells at this point were the stable cell lines that successfully integrated shRNA. Finally, GFP expression was observed by fluorescence microscopy and the knockdown efficiency of the target protein was verified by Western blotting to confirm successful construction.

[0076] Subsequently, HSP90α-WT and HSP90α-K408R proteins were overexpressed in HSP90α-knockdown astrocytes, and the cells were treated with sodium lactate. The results showed that HSP90α lactation significantly inhibited the expression of EAAT2 protein, and mutation at the K408 site could rescue this effect. Figure 6 (ab).

[0077] 2. To investigate whether HSP90α lactation can affect the ability of astrocytes to take up glutamate by regulating EAAT2 protein, short-term and long-term glutamate uptake assays were performed in vitro.

[0078] Astrocytes transfected with plasmids were counted and seeded into 6-well plates. The astrocytes in the 6-well plates were then placed in Opti-MEM medium containing 2% fetal bovine serum for 12 hours to induce glutamate depletion. After removing the medium, the plates were washed twice with uptake buffer (1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 10 mM d-glucose, 122 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO₄, 25 mM Hepes, pH 7.4). Immediately afterwards, 2 ml of glutamate-containing uptake buffer was added to each well. The glutamate concentration for short-term uptake experiments was 500 nM, and for long-term uptake experiments, it was 20 μM. At each time point, 100 μl of supernatant was collected and centrifuged at 3500 rpm for 10 minutes. Extracellular glutamate concentration was determined using a glutamate / glutamate oxidase assay kit according to the manufacturer's instructions. Data are presented as bar charts or decreasing curves of extracellular glutamate concentration.

[0079] The results showed that HSP90α lactation inhibited astrocyte uptake of extracellular glutamate, while inhibiting lactation by the K408 mutation could salvage this function. Figure 6 medium cd).

[0080] 3. HSP90α can bind to the 20S proteasome subunit, assisting its chaperone protein in completing proteasome degradation and maintaining normal cellular physiological functions. We found that inhibiting the lactation modification of HSP90α at the K408 site significantly prolongs the half-life of EAAT2 protein and maintains the stability of EAAT2 protein in astrocytes. Figure 6 in ef).

[0081] Example 6: HSP90α K408 blocking peptide inhibits glutamate accumulation in the hippocampus of epileptic mice and weakens the epileptic phenotype.

[0082] 1. Based on the modification site of HSP90α, five blocking peptides were designed and named K408-Peptide-1# to K408-Peptide-5#, respectively, and the transcellular sequence HLYVSPWGG was linked to their front end.

[0083] The HSP90α K408lac blocking peptide sequence is shown below, synthesized by Nanjing Baode Biotechnology Co., Ltd.

[0084] K408-Peptide-1#:HLYVSPWGGISREMLQQSKILK (SEQ ID NO:2);

[0085] K408-Peptide-2#: HLYVSPWGGQQSKILKVIRKNL (SEQ ID NO:3);

[0086] K408-Peptide-3#:HLYVSPWGGEMLQQSKILKVIR (SEQ ID NO:4);

[0087] K408-Peptide-4#:HLYVSPWGGREMLQQSKILKVI (SEQ ID NO:5);

[0088] K408-Peptide-5#: HLYVSPWGGMLQQSKILKVIRK (SEQ ID NO:6);

[0089] K408R-Peptide-3#: HLYVSPWGGEMLQQSRILKVIR (SEQ ID NO:7);.

[0090] 2. After transfecting HSP90α wild-type plasmid with HEK293T for 24 hours, cells were treated with lactate, and then a blocking peptide (25 μg / mL) was added 2 hours later. The results showed that K408-Peptide-1# to K408-Peptide-5# could inhibit the lactation level at the K408 site of HSP90α, with K408-Peptide-3# showing the best inhibitory effect. K408-Peptide-3# was used for subsequent experiments. Figure 7 (b)

[0091] In addition, solutions of the blocking peptide K408-Peptide-3# were prepared at concentrations of 0, 5, 10, 25, and 50 μg / mL, and their inhibitory effect on HSP90α lactation was analyzed. The results are as follows: Figure 7 As shown in Figure c, gradient treatment with the blocking peptide K408-Peptide-3 also indicates that this inhibitory effect depends on the concentration of the blocking peptide. Furthermore, mutating K in K408-Peptide-3 to R, i.e., K408R-Peptide-3, eliminates its blocking effect.

[0092] 3. Verify the antiepileptic ability of K408-Peptide-3#, such as... Figure 7As shown in Figure e, K408-Peptide-3# and K408R-Peptide-3# were injected into the lateral ventricle 30 minutes before KA injection, and a supplemental injection of the blocking peptide was given on the third day after KA injection. Intraventricular injection of the blocking peptide intervention: The blocking peptide was injected into the ventricle at a rate of 0.4 μl / min (relative to the anterior fontanelle: AP=0.46 mm, ML=1.0 mm, DV=-2.5 mm), with an injection volume of 5 μL (1 mmol / L), 30 minutes before KA injection. The control group received an equal volume of sterile PBS. Behavioral monitoring of mice was performed, and the results are shown below. Figure 7 As shown in Figure f, K408-Peptide-3# significantly inhibited the number of spontaneous seizures in mice, while K408R-Peptide-3 had no significant therapeutic effect compared to the control group.

[0093] Mouse brain tissue, after perfusion fixation, was embedded in paraffin and sectioned to a thickness of 5-8 μm. The sections were then dewaxed to water and stained with 0.5%-1% tar purple or toluidine blue at 50-60℃ for 20-40 minutes. After staining, the sections were briefly washed with distilled water and then successively immersed in 70% ethanol and 95% ethanol for differentiation. This process required microscopic control until the background was nearly colorless and Nissl bodies in the neuronal cytoplasm were clearly visible as deep blue or purple patches. The sections were then rapidly dehydrated with anhydrous ethanol, cleared with xylene, and finally mounted with neutral resin. The structural integrity and the distribution and density of Nissl bodies in neurons in the CA1 and CA3 regions of the hippocampus were observed under an optical microscope. Results are as follows: Figure 7 As shown in the figure, the number of neurons in the CA1 and CA3 regions of mice in the K408-Peptide-3 group was significantly increased.

[0094] Mass spectrometry analysis was performed on extracted mouse hippocampal tissue, and the results are as follows: Figure 7 As shown in Figure i, the concentrations of glutamate and its downstream metabolite glutamine in the hippocampus of mice in the K408-Peptide-3 group were significantly decreased, while the K408R-Peptide-3 treatment group did not affect the concentrations of glutamate and its downstream metabolite glutamine in the hippocampus.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a substance that inhibits lactation at the HSP90α-K408 site in astrocytes in the preparation of a drug for relieving and / or treating epilepsy, wherein the HSP90α-K408 site is the 408th amino acid position shown in SEQ ID NO:1; The substance that inhibits lactation at the HSP90α-K408 site in astrocytes is an HSP90α-K408 blocking peptide, which is selected from at least one of the polypeptides whose amino acid sequence is shown in SEQ ID NO:2-6.

2. The application according to claim 1, characterized in that, The amino acid sequence of the HSP90α K408 blocking peptide is SEQ ID NO:

4.

3. The application according to any one of claims 1-2, characterized in that, Substances that inhibit lactation at the HSP90α-K408 site in astrocytes are present in at least one of A1)-A3) for the preparation of drugs that alleviate and / or treat epilepsy: A1) Reduce the concentration of glutamate and glutamate in hippocampal tissue; A2) Increase the number of neurons in the CA1 and CA3 regions of the hippocampus; A3) Reduce the frequency of spontaneous epileptic seizures.

4. The application according to any one of claims 1-2, characterized in that, The epilepsy referred to is either in the subacute or chronic phase.

5. A pharmaceutical composition, characterized in that, The invention includes a substance that inhibits lactation at the HSP90α-K408 site in astrocytes and a pharmaceutically acceptable carrier; the substance that inhibits lactation at the HSP90α-K408 site in astrocytes is an HSP90α-K408 blocking peptide, wherein the HSP90α-K408 blocking peptide is selected from at least one of the polypeptides whose amino acid sequence is shown in SEQ ID NO:2-6.

6. The pharmaceutical composition according to claim 5, characterized in that, The amino acid sequence of the HSP90α K408 blocking peptide is SEQ ID NO:

4.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that, The pharmaceutical composition is an injectable preparation.

8. A polypeptide, characterized in that, The polypeptide is selected from at least one polypeptide whose amino acid sequence is shown in SEQ ID NO:2-6.

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