Application of glutamate enzyme and related biological elements thereof in meniere disease diagnosis and treatment

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of glutamate enzyme and related biological elements thereof in meniere disease diagnosis and treatment. Specifically, the research finds that IL-1beta increases the glutamic acid level by up-regulating the expression of glutamate enzyme in HEI-OC1 cells. Treatment with a glutaminase inhibitor or an IL-1 [beta] receptor antagonist can alleviate auditory-vestibular dysfunction and reduce glutamic acid levels in endolymphatic hydrops mice. In a word, the research of the invention proves that the neurotransmitter release imbalance and immune response may cause the pathology of the Meniere's disease, possibly explains hearing loss and vertigo related to the Meniere's disease, and provides a new way for treatment intervention.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of glutaminase and related biological elements in the diagnosis and treatment of Meniere's disease. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Meniere's disease is a common and complex inner ear disease characterized by episodic vertigo, fluctuating sensorineural hearing loss, tinnitus, and / or aural fullness. Its pathological manifestation is endolymphatic hydrops, which may be caused by decreased reabsorption of endolymphatic fluid by the endolymphatic sac. Recent studies have shown that autoimmune inflammation, allergy, and immune response are associated with the occurrence and progression of Meniere's disease.

[0004] Previous studies on Meniere's disease have used a variety of methods to explain its pathological and molecular mechanisms. RNA sequencing studies of peripheral blood mononuclear cells have shown that Meniere's disease is associated with immune pathways, while transcriptome analysis of vestibular organs has shown a close association between Meniere's disease and neuropathy and autoimmune diseases. Proteomic studies of patients have found that overexpression of complement factor H in plasma and specific protein distribution in perilymph highlight the relationship between Meniere's disease and inflammatory diseases, energy metabolism, developmental disorders, and intercellular signaling. However, the use of plasma and perilymph in previous proteomic studies may not fully capture the changes in inner ear tissues. Because the inner ear structure is located deep in the temporal bone and is small in size, it is difficult to obtain and analyze, so there is limited understanding of the molecular changes in human inner ear diseases. Patients with Meniere's disease show varying degrees of vestibular dysfunction, and the exact location of the lesion and its molecular mechanism remain unclear.

[0005] Glutamate excitotoxicity is a pathological process that causes neuronal changes and damage through excessive release of glutamate and its analogs, which occurs in ischemia, Alzheimer's disease, and multiple sclerosis. Studies have shown that glutamate plays a key role in the occurrence and progression of cochlear synaptic damage. In addition, inflammation can affect glutamatergic neurotransmission and synaptic integrity. The inflammatory cytokine IL-17 enhances glutamate excitotoxicity by affecting the uptake of glutamate by astrocytes. In addition, glutamate can also induce upregulation of proinflammatory cytokines, further exacerbating excitotoxicity. However, the direct relationship between synaptic damage and Meniere's disease remains unclear. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide the application of glutaminase and its related biological elements in the diagnosis and treatment of Meniere's disease. Specifically, the present invention found through research that IL-1β increases glutamate levels by upregulating glutaminase expression in HEI-OC1 cells. Treatment with glutaminase inhibitors or IL-1β receptor antagonists can alleviate auditory-vestibular dysfunction in mice with endolymphatic hydrops and reduce their glutamate levels. In summary, the research of the present invention proves that imbalance in neurotransmitter release and immune response may lead to the pathology of Meniere's disease, which may explain the hearing loss and vertigo associated with the disease, and provide a new approach for diagnosis and treatment.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides the use of a substance for detecting the expression level of glutaminase and its related biological elements in the preparation of a product for screening, diagnosis, monitoring or prognosis of Meniere's disease.

[0009] Wherein, the glutamate-associated biological elements include IL-1β and glutamate.

[0010] Specifically, the present invention has found through research that the mRNA and protein levels of glutaminase in the vestibular organs of patients with Meniere's disease are significantly increased, and further research shows that IL-1β upregulates the expression of glutaminase in the inner ear, leading to increased glutamate levels, inducing neurodegeneration, thereby damaging the function of the auditory-vestibular system and mediating the occurrence of Meniere's disease. Therefore, the above-mentioned glutaminase and its related biological elements can be used as (auxiliary) diagnostic and prognostic markers for Meniere's disease.

[0011] The substances for detecting the expression level of glutaminase and its related biological elements may include reagents required for detecting the expression level of glutaminase encoding genes based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips and gene sequencing, reagents required for detecting the expression level of IL-1β based on immunoassay methods, and / or reagents required for detecting glutamate based on chromatography, mass spectrometry, spectroscopy, biological methods, etc.

[0012] The products include but are not limited to primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting the expression level of glutaminase and its related biological elements.

[0013] A second aspect of the present invention provides a system for screening, diagnosing, monitoring or prognosing Meniere's disease, the system comprising:

[0014] An acquisition module is configured to: acquire the expression level of glutamate and its related biological elements in the test sample of the subject;

[0015] The evaluation module is configured to evaluate and judge the disease condition of the subject according to the subject's glutaminase and related biological elements obtained by the acquisition module.

[0016] Wherein, the glutamate-associated biological elements include IL-1β and glutamate.

[0017] The sample to be tested can be an inner ear tissue sample or peripheral blood of a subject.

[0018] The third aspect of the present invention provides the use of glutaminase and related biological elements as targets in the preparation and / or screening of drugs for Meniere's disease.

[0019] Specifically, the effects of a candidate drug on glutaminase and related biological elements before and after use can be used to determine whether the candidate drug can be used to prevent or treat Meniere's disease.

[0020] Specifically, the method for screening drugs for preventing or treating Meniere's disease includes:

[0021] a) treating a system expressing and / or containing glutamate and its related biological elements with a candidate substance; setting up a parallel control without treatment with the candidate substance;

[0022] b) After completing step a), detecting the expression level of the glutamate enzyme and its related biological elements in the system; if the expression level of the glutamate enzyme and its related biological elements in the system treated with the candidate substance is significantly downregulated compared with the parallel control, the candidate substance can be used as a candidate drug for preventing or treating Meniere's disease.

[0023] Wherein, the glutamate-associated biological elements include IL-1β and glutamate.

[0024] The system may be a cell system, a solution system, a tissue system, an organ system or an animal system, and is not specifically limited herein.

[0025] Wherein, the cells in the cell system may be HEI-OC1 cells;

[0026] The organ in the organ system may be the ear, which is an important organ of hearing and balance and consists of three parts: the outer ear, the middle ear and the inner ear. Among them, the inner ear is the main organ of hearing and balance, including the vestibule, the cochlea and the semicircular canals. It is also the focus of the present invention.

[0027] Animal embodiments include humans and non-human animals, wherein the non-human animals are mammals, such as rats, mice, guinea pigs, rabbits, pigs, dogs, monkeys, gorillas, etc., which are not specifically limited here.

[0028] The fourth aspect of the present invention provides the use of a substance for inhibiting the expression of glutaminase and its related biological elements or reducing their activity in any one or more of the following:

[0029] a) preparing products that reduce cochlear synaptic damage;

[0030] b) Products to alleviate endolymphatic hydrops-mediated auditory-vestibular dysfunction;

[0031] c) Products for the prevention and / or treatment of Meniere's disease.

[0032] Among them, the substances that inhibit glutaminase and its related biological elements can include but are not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, antagonists, siRNA (SEQ ID NO.1-2), shRNA against glutaminase and its related biological elements, substances for implementing lentiviral infection or gene knockout, antibodies, etc., and are not specifically limited here.

[0033] In a specific embodiment of the present invention, the substance that inhibits the expression of glutaminase and its related biological elements or reduces their activity includes glutaminase inhibitor CB-839 (CAS: 1439399-58-2) and interleukin-1β receptor inhibitor anakinra (CAS: 143090-92-0).

[0034] The product can be a drug or an experimental reagent for non-medical use, and the experimental reagent can be used for basic research. For example, the product can be used to regulate the relevant phenotypes of experimental animals for endolymphatic hydrops, thereby preparing relevant animal models, thereby providing convenient conditions for the mechanism research of diseases such as Meniere's disease.

[0035] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0036] The inactive pharmaceutical ingredients may be carriers, excipients, diluents, etc. commonly used in pharmacy. Meanwhile, the inactive pharmaceutical ingredients such as carriers, excipients, diluents, etc. that may be included are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0037] The medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be applied optionally via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0038] The drug administration subjects can be humans and non-human mammals, such as rats, mice, guinea pigs, rabbits, pigs, dogs, monkeys, gorillas, etc.

[0039] Beneficial technical effects of one or more of the above technical solutions:

[0040] The above technical scheme uses mass spectrometry to analyze the changes in protein features in the vestibular end organs (VO) of patients with Meniere's disease. Differential protein functional enrichment analysis showed that differentially expressed proteins in the vestibular end organs were related to synaptic processes. In addition, compared with the control group, the expression level of glutaminase in the vestibular terminal organs of patients with Meniere's disease was increased. Further studies have shown that IL-1β increases glutamate levels by upregulating glutaminase expression in HEI-OC1 cells. Treatment with glutaminase inhibitors or IL-1β receptor antagonists can alleviate auditory-vestibular dysfunction and reduce glutamate levels in mice with endolymphatic hydrops. These results indicate that imbalance in neurotransmitter release and abnormal immune response may lead to the pathology of Meniere's disease, explain the hearing loss and vertigo attacks associated with the disease, and provide new therapeutic targets for its treatment, so it has good potential for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0042] Figure 1 Different proteins in VO were identified by quantitative proteomics analysis in the embodiment of the present invention. Among them, a is the result of quantitative principal component analysis of VO protein in VS and MD patients. b is the volcano map of all proteins identified in VO samples. c is the distribution histogram of DAPs in VO. d is the heat map of DAPs and their subcellular localization.

[0043] Figure 2 : Functional enrichment analysis of DAPs in VO in the embodiment of the present invention. Wherein, a is a bubble diagram of VO biological process. b is a bubble diagram of VO KEGG. c is the normalized enrichment score of the selected representative and significantly enriched gene sets in VSVO and MDVO samples.

[0044] Figure 3 : DAPs related to synaptic vesicle cycle in VSVO and MDVO in the embodiment of the present invention. Wherein, a is a protein-protein interaction network. b is a heat map showing DAPs related to synaptic vesicle cycle. c is qRT-PCR detection of GLS expression in VO of VS and MD patients. d is a representative image of GLS immunohistochemical staining in VO of VS and MD patients.

[0045] Figure 4 Anakinra reduces inner ear glutamate levels by downregulating GLS in the embodiments of the present invention. Wherein, a is HEI-OC1 cells stimulated with IL-1β for 24 hours, and qRT-PCR is used to detect the expression of Gls gene. B is HEI-OC1 cells stimulated with IL-1β for 24 hours, and western blotting is used to detect the expression of GLS protein. C is the glutamate level of HEI-OC1 cells stimulated with IL-1β for 24 hours. D is HEI-OC1 cells treated with or without siGls, and qRT-PCR is used to detect Gls expression. E is Western blotting to detect the expression of GLS in HEI-OC1 cells transfected or not transfected with siGls. F is the glutamate level of HEI-OC1 cells stimulated with IL-1β and / or siGls for 24 hours. G is the serum and inner ear glutamate levels of mice that were not treated or pretreated with anakinra and lipopolysaccharide or an equal amount of saline. H is the ultrastructure of VO shown by transmission electron microscopy. I is the expression of Gls in the inner ear of mice detected by qRT-PCR.

[0046] Figure 5 Inhibiting GLS in the embodiments of the present invention can alleviate the phenotype of EH mice and reduce the level of inner ear glutamate. Among them, a is a mouse given lipopolysaccharide for 3 consecutive days, and the GLS inhibitor CB-839 was given at the same time, and the analysis was performed on the 5th day. B is the representative wave and threshold of ABR. C is the ABR threshold of pure tone. D is the representative wave of VEMP, P1-N1 peak amplitude, and P1 and N1 peak latency of VEMP under 100dB nHL. E is the serum and inner ear glutamate levels of mice. F is the transmission electron microscopy observation of the ultrastructure of mouse VO. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0048] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0049] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0050] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] Example

[0052] Materials and Methods

[0053] Patients and tissue samples

[0054] Patients with Meniere's disease were recruited according to the 2015 diagnostic criteria for Meniere's disease. From December 2018 to November 2023, a total of 16 patients with Meniere's disease aged 46 to 74 years (mean age, 58.8 ± 6.2 years) and 14 patients with vestibular schwannoma (VS) aged 45 to 74 years (mean age, 54.9 ± 8.2 years) were enrolled in Shandong Provincial Otolaryngology Hospital. Written informed consent was obtained from all participants. This study protocol was conducted in accordance with the principles of human research in the Declaration of Helsinki, as revised in 2013, and was approved by the Ethics Committee of Shandong Provincial Otolaryngology Hospital (Ethics Review Number: XYK-20180603).

[0055] The ampulla and cystic maculae were collected during labyrinthectomy. For liquid chromatography-tandem mass spectrometry (LC-MS / MS), the samples were homogenized in lysis buffer (8 M urea, 1% protease inhibitor cocktail, Calbiochem) and then sonicated on ice. After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was collected and the protein concentration was measured using a BCA kit (Shanghai Biotech Co., Ltd.) according to the manufacturer's instructions. For frozen sections, the collected vestibular tissue samples were immediately frozen with liquid nitrogen and stored at -80 °C until use.

[0056] LC-MS / MS

[0057] For LC-MS / MS, the protein solution was digested with trypsin and dissolved in 0.1% formic acid before loading onto a reversed-phase analytical column. Subsequently, elution was performed on an EASY-nLC 1000 ultra-high performance liquid chromatography system with a gradient mobile phase B (0.1% formic acid) from 6% to 23% in 26 min, from 23% to 35% in 8 min, to 80% in 3 min, and then maintained at 80% for the last 3 min at a flow rate of 400 nL / min. The resulting peptides entered the capillary source and were then analyzed by liquid chromatography-tandem mass spectrometry in a tims-TOF Pro (Bruker, Karlsruhe, Germany). The electrospray voltage was 1.4 kV. Precursors and fragments were analyzed using an orbitrap, and the secondary mass spectrometry scan range was from 100 to 1700 m / z. Ten fragment spectra were acquired per cycle, and the dynamic exclusion time was set to 24 s. The automatic gain control (AGC) was 5E4. The fixed first mass number was set to 100 m / z.

[0058] Protein identification and quantification

[0059] Proteins were identified in the Uniport database using the MaxQuant search engine (version 1.6.6.0). For the search parameters, the digestion method was set to trypsin / P, the number of missed cleavage sites was four, and the mass tolerance for the first and main searches was 70 ppm. The fragment mass tolerance was 0.04 Da. For fixed modifications, carbamidomethylation on cysteine ​​was selected; for variable modifications, acetylation modification and oxidation of methionine were selected. Other conditions were as follows: false discovery rate (FDR) <1%, peptide spectrum match (PSM) <1%, and the minimum score of modified peptides >40. Protein quantification was based on the median number of unique peptides of the protein. The difference in protein abundance between the two groups was compared by calculating the relative expression ratio of the target protein in the sample to the reference protein. Finally, the median ratio of the protein was used to normalize the peptide ratio.

[0060] Bioinformatics analysis

[0061] To analyze the identified differentially expressed proteins, Gene Ontology (GO) (InterProScan, version 5.14-53.0) terms were used, including cellular components, molecular functions, and biological processes. InterproScan (version 5.14-53.0) was used to annotate the functional descriptions of the identified protein domains. Protein pathways were annotated using the KEGG database (KAAS, version 2.0; KEGGMapper, version 2.5). Protein subcellular localization was predicted using Wolfpsort (version 0.2). For functional enrichment analysis of GO, pathways, and protein domains, the enrichment of target proteins was tested using a two-sided Fisher's exact test, and a corrected p-value < 0.05 was considered significant. Protein clustering analysis was performed using the ComplexHeatmap R package (R, version 3.4). The filtered P matrix was transformed using -log10(P value). Enrichment-based clustering was visualized using the R package heatmap (version 2.03). Protein-protein interactions (PPIs) based on the STRING database were visualized using the R package networkD3 (version 0.4). Genes in this network were ranked according to degree centrality values ​​using Cytoscape (version 3.10.0). Gene set enrichment analysis (GSEA) was based on the algorithm developed by the Broad Institute (https: / / www.gsea-msigdb.org / gsea / index.jsp). For single-sample GSEA (ssGSEA), the relative quantitative values ​​of vestibular organ proteins and the KEGG annotation data of this project were used as input and ssGSEA2.0 was used.

[0062] (https: / / github.com / broadinstitute / ssGSEA2.0) was used for analysis.

[0063] Animal model establishment and intervention

[0064] Wild-type male C57BL / 6 mice (6-8 weeks old, 17-25 g in body weight) were purchased from the Animal Center of Shandong University and housed in a temperature-controlled room (20-22°C) with a 12 / 12 h light / dark cycle and free access to food and drinking water. All study protocols were approved by the Animal Care Committee of Shandong University and complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. C57BL / 6 mice were subjected to the LPS-induced endolymphatic hydrops (EH) model. Briefly, LPS in saline (2 mg / kg, Sigma) was injected postauricularly (pa) for three days, while the control group was injected postauricularly with an equal amount of 0.9% saline. To evaluate the role of interleukin-1β in LPS-induced endolymphatic hydrops, mice were intraperitoneally (ip) injected with the interleukin-1 receptor antagonist anakinra (10 mg / kg, HY-108841, MCE) or an equal amount of saline 30 min before LPS stimulation. To evaluate the effects of glutaminase inhibition, C57BL / 6 mice were injected postauricularly with LPS and simultaneously with intraperitoneal (ip) injection of the glutaminase inhibitor CB-839 (HY-12248, MCE, 28 mg / kg). In all cases, hearing and vestibular function were assessed on day 5 after the first LPS injection. Auditory function assessment (auditory brainstem response, ABR)

[0065] ABR responses were measured using the TDT system 3 (Tucker-Davis Technologies, USA) with short pure tone stimulation at 4, 8, 12, 16, 24 and 32 kHz, with 1024 stimulation repetitions per recording in a soundproof room. Briefly, mice were anesthetized by intraperitoneal injection of a mixture of xylazine (10 mg / kg) and ketamine (100 mg / kg). Needle electrodes were inserted into the subcutaneous tissue of the top of the head (recording electrode), the mastoid region under the ear of the ipsilateral ear (reference electrode), and the back (ground electrode). The sound intensity started at 90 dB sound pressure level (SPL) and then decreased by 5 dB to the hearing threshold. The ABR threshold was determined at each frequency, which refers to the minimum sound pressure level at which a reliable ABR record is produced and one or more distinguishable waves can be clearly identified by visual inspection. Repeat this process for low sound pressure levels close to the threshold to ensure waveform consistency.

[0066] Vestibular evoked myogenic potential (VEMP)

[0067] After anesthesia, the neck of the mouse was hyperextended and stabilized with a hanging wire fixed behind the front teeth. The mouse was maintained in a prone position with the head elevated and the legs freely extended. During the recording, a platinum needle electrode was inserted into the neck extensor muscle and the reference electrode was placed in the midline of the cervical occipital region. The ground electrode was then repositioned. VEMP testing was performed on each animal at a stimulus intensity of 100 dB normal hearing level (nHL). The response threshold was the lowest threshold at which the waveform appeared, and its reproducibility was verified by continuous runs (>3 times). Finally, the latency and amplitude of the negative and positive peaks were measured.

[0068] Vestibular ocular reflex (VOR)

[0069] The mice were fixed in a non-invasive animal fixation device. The horizontal eye position signals were recorded using a vestibular function test system based on binocular video oculography (VOG) provided by Professor Chen Fangyi's team from the Southern University of Science and Technology. ) was mounted on a translation stage at a 45° angle to the anteroposterior axis of the mouse. Illumination for video recording was achieved using two near-infrared light-emitting diode lamps (940 nm, Chuangdaxin, China) attached to the camera. The eye tracker tracked the region of interest (ROI) at a rate of 60 frames per second. The ROI containing the pupil in each frame was automatically selected using a template matching method. Subsequently, ellipse fitting was applied to determine the pupil center and to extract the horizontal component from the eye movement recordings. Calibration was performed to convert the acquired translation distance into eye rotation angles. After calibration, a series of linear accelerations were recorded. To measure the vestibulo-ocular reflex response, horizontal rotations were recorded at a rate of 20° / s (0.2, 0.5, 0.8, 1.0, 1.6, 3.2, and 5.0 Hz). The exported eye position data were Fourier transformed using MATLAB 2016b software to obtain the amplitude data of the eye movements. The vestibulo-ocular reflex gain was calculated as the ratio of the response amplitude to the stimulus amplitude.

[0070] Cell lines, cell culture, and treatment

[0071] HEI-OC1 cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco, USA) at 33°C in a humidified incubator containing 10% carbon dioxide. In the experiment, HEI-OC1 cells were stimulated with interleukin-1β (10, 25, 50 ng / ml, HY-P7073A, MCE) for 24 hours. A pair of small interfering RNAs (siRNAs) targeting the mouse glutaminase (GLS) gene was used to silence the expression of GLS in HEI-OC1 cells. The sequences of the GLS siRNAs used are as follows: sense strand, 5′-GAGGGAAGGUUGCUGAUUATT-3′, SEQ ID NO.1; antisense strand, 5′-UAAUCAGCAACCUUCCCUCTT-3′, SEQID NO.2. A non-targeting scrambled siRNA was used as a negative control treatment (Jima Gene, Shanghai, China). Briefly, siRNA and Lipofectamine RNAiMAX (Invitrogen, USA) were added to Opti-MEM (Invitrogen). The siRNA and Lipofectamine RNAiMAX solutions were then mixed and incubated at room temperature. An equal volume of the mixture was added to a culture plate and cultured for 48 hours.

[0072] Immunohistochemical analysis

[0073] Inner ear specimens were obtained from patients with Meniere's disease and fixed with 4% paraformaldehyde overnight at 4°C. The specimens were dehydrated with phosphate buffer containing 15%, 20%, and 30% sucrose in sequence and then embedded in OCT compound (Tissue-Tek, USA). Specimens with a thickness of 5 μm were sliced ​​using a cryostat (Leica CM 1850, Germany) and stored at −80°C. The expression of glutaminase in the vestibular organs was evaluated using a DAB detection kit (streptavidin-biotin) (SP-9000-D; Zhongshan Jinqiao, Beijing, China). Briefly, the sections were treated with 0.3% hydrogen peroxide for 30 minutes to inhibit endogenous peroxidase activity. To prevent nonspecific binding, the sections were incubated with 10% normal goat serum for 20 minutes. The sections were then incubated with anti-glutaminase antibody (1:200, sc-74430; USA) at 4°C overnight. After washing with PBS, the sections were incubated with biotinylated anti-rabbit or anti-mouse IgG for 30 minutes, and then incubated with DAB complex for 1 minute. The sections were counterstained with hematoxylin, dehydrated, and mounted with neutral gum (G8590; Solebol, China). Images were acquired using a Leica microscope.

[0074] Enzyme-linked immunosorbent assay (ELISA)

[0075] Mouse cochleae were obtained, immediately snap-frozen in liquid nitrogen, and stored at -80°C. Subsequently, cochlear tissue was placed in lysis buffer and homogenized. The homogenate was then centrifuged and the supernatant was collected for ELISA analysis. Glutamate concentrations in serum and cochleae were determined using paired antibodies and standard recombinant mouse glutamate (ab83389; Abcam, USA) according to the manufacturer's instructions. Signals were detected at 450 nm using a microplate reader (Bio-Rad).

[0076] Transmission Electron Microscopy (TEM)

[0077] Mouse cochleae were obtained, fixed immediately with 3% glutaraldehyde fixative and 1% osmium acid, dehydrated, infiltrated, and embedded in Epon 812. Subsequently, ultrathin radial sections were stained with lead citrate and uranyl acetate and then observed under a transmission electron microscope (JEOL-1200EX, Japan Electronics).

[0078] Real-time quantitative PCR (qRT-PCR)

[0079] Total RNA was extracted using an RNA extraction kit (RNeasy Mini QIAcube Kit, QIAGEN, Germany). The relative expression level of RNA was measured by qRT-PCR using an Eppendorf AG 22331PCR instrument (Eppendorf, Germany). The qRT-PCR reaction mixture included 2×SYBR Green Premix EX Taq (RR42LR; Takara, Japan), cDNA template, forward and reverse primers, and deionized water. The qRT-PCR parameters included an initial denaturation step at 95°C, followed by 40 cycles of denaturation at 95°C, annealing at 60°C, and finally extension at 72°C. Melting curve analysis was used to confirm the specificity of each PCR. GAPDH was used to normalize the target mRNA expression. The comparative Ct method was used to determine the fold change of each gene.

[0080] Western blotting

[0081] Total protein from HEI-OC1 cells was extracted using ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail. Equal amounts of protein samples were heat denatured at 99°C for 10 minutes and then separated by 10% SDS-PAGE gel electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked in 5% skim milk for 1 hour and then incubated with anti-glutaminase antibodies (1:100, sc-74430; Santa Cruz) and anti-GAPDH antibodies (1:8000, ab8245; Abcam). The next day, the membranes were incubated with HRP-conjugated secondary antibodies (1:10000) for 1 hour. Finally, protein signals were visualized using an ECL kit (Millipore). All blocking, incubation, and washing steps were performed using Tris-buffered saline containing 0.05% Tween 20.

[0082] Statistical analysis

[0083] In addition to bioinformatics analysis, data were statistically analyzed using SPSS 21.0 statistical software (SPSS). Bioinformatics-related statistical software and methods are described in the bioinformatics analysis section. Quantitative data are presented as mean ± standard error of the mean (SEM) and analyzed by t-test or one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

[0084] result

[0085] Protein identification and differentially abundant protein (DAP) screening in the human vestibular organ

[0086] To determine the differences in VO between Meniere's disease and vestibular schwannoma samples, we performed quantitative proteomic analysis. First, unsupervised principal component analysis (PCA) was performed, and the score plot showed that the Meniere's disease group could be distinguished from the vestibular schwannoma group ( Figure 1 a). Quantitative analysis of individual proteins revealed 119 differentially abundant proteins (66 upregulated and 63 downregulated) in the vestibular organs, with a difference of more than 1.5-fold compared with the vestibular schwannoma group (p<0.05; Figure 1 b, c). The up-regulated and down-regulated proteins and their subcellular localization are presented in the form of heat maps. Figure 1 e. These results provide a proteomic map of the vestibular apparatus in patients with Meniere's disease and vestibular schwannoma.

[0087] Functional analysis of differentially abundant proteins in the vestibular organ

[0088] To understand the characteristics and functions of the proteins identified in the vestibular organs, the differentially abundant proteins between vestibular schwannoma and Meniere's disease samples were annotated by subcellular localization, GO, and KEGG pathways. GO enrichment results showed that synaptic processes, including secretion, exocytosis, and neurotransmitter transport, were significantly affected ( Figure 2 a). In addition, biological processes related to immune responses, such as neutrophil activation and cytokine signaling, were also significant ( Figure 2 a). In the KEGG database, GABAergic synapses were significantly enriched, and most of the enriched proteins were involved in complement and coagulation cascade pathways ( Figure 2 b).

[0089] Consistent with the GO and KEGG results, synaptic vesicle recycling was also significantly enriched in PPI analysis ( Figure 3 a), supporting its potential role in the pathogenesis of Meniere's disease. GSEA showed consistent positive enrichment of GO terms related to synaptic vesicle recycling and IL-17 signaling pathway ( Figure 2 c). The heat map further showed that synaptic-related proteins were significantly altered in Meniere's disease ( Figure 3 b). Glutaminase (GLS) is a key enzyme in the glutamate biosynthesis pathway, catalyzing the hydrolysis of glutamine to produce glutamate and participating in the pathogenesis of various neurological diseases. Compared with the control group, the mRNA and protein levels of glutaminase in MDVO were significantly increased ( Figure 3 c, d). Taken together, these results suggest that synapse-associated transmitter release plays an important role in the pathology of Meniere's disease.

[0090] Anakinra alleviates the phenotype of mice with endolymphatic hydrops and reduces glutamate levels in the inner ear by downregulating glutaminase

[0091] Interleukin-1β (IL-1β) has been shown to affect glutamate release, uptake, and receptor expression, thereby affecting synaptic transmission and neuronal excitability. Our study suggests that IL-1β plays a key role in the pathogenesis of Meniere's disease, with glutaminase levels significantly increased in MDVO compared with controls ( Figure 3 c, d). Therefore, we first verified that IL-1β regulates glutamate synthesis through glutaminase. As shown in the figure, in HEI-OC1 cells, IL-1β promoted the expression of glutaminase mRNA and protein ( Figure 4 a, b), and increased glutamate levels in culture supernatant and cells ( Figure 4 c). In contrast, downregulation of glutaminase using small interfering RNA (siRNA) suppressed the increase in glutamate levels induced by IL-1β ( Figure 4df). In addition, anakinra significantly reversed the LPS-induced increase in glutamate in the inner ear and serum of mice ( Figure 4 g), Transmission electron microscopy (TEM) sections showed that the ribbon synapses in vestibular hair cells of mice treated with lipopolysaccharide had abnormal structures, the presynaptic ribbon structures were extremely swollen, and the ribbon structures were "free floating" in the cytoplasm, away from the active sites. In addition, it was found that anakinra inhibited synaptic damage ( Figure 4 h), and reversed the LPS-induced upregulation of glutaminase in the cochlea ( Figure 4 i) Taken together, these findings collectively suggest that IL-1β upregulates glutaminase expression in the inner ear, leading to elevated glutamate levels that impair the function of the auditory-vestibular system.

[0092] Inhibition of glutaminase alleviates the phenotype of mice with endolymphatic hydrops and reduces glutamate levels in the inner ear

[0093] Subsequently, mice were treated with the glutaminase inhibitor CB-839 to elucidate the effect of glutaminase on endolymphatic hydrops mice ( Figure 5 a). The results showed that the ABR thresholds of LPS mice were significantly higher compared with the saline group, and CB-839 treatment significantly reversed these effects ( Figure 5 b, c). Regarding vestibular function, LPS mice showed prolonged latencies of P1 and N1 and decreased P1-N1 amplitude compared with control mice ( Figure 5 d). CB-839 treatment can reduce the latency of N1 ( Figure 5 d). In addition, CB-839 significantly reversed the LPS-induced increase in glutamate in the inner ear and serum of mice ( Figure 5 e), and reduced synaptic damage as shown in TEM sections ( Figure 5 f). Taken together, these data suggest that CB-839 attenuates the impairment of auditory-vestibular function and reduces glutamate levels in the LPS-induced endolymphatic hydrops mouse model.

[0094] Meniere's disease is one of the most common diseases in the field of otolaryngology. Previous proteomic studies have used plasma and perilymph samples from patients with Meniere's disease to investigate molecular changes associated with the disease. However, these studies have limitations in revealing changes in inner ear tissues. In this study, we analyzed the proteomic patterns of vestibular organs in patients with Meniere's disease and vestibular schwannoma, revealing the following: 1) There were 119 differentially abundant proteins in the vestibular organs of Meniere's disease compared with the vestibular schwannoma group, and these differentially abundant proteins were closely related to synapse-related processes; 2) Interleukin-1β increased glutamate levels by promoting the expression of glutaminase in HEI-OC1 cells; 3) The glutaminase inhibitor CB-839 or the interleukin-1β receptor antagonist anakinra alleviated the phenotype of mice with endolymphatic hydrops and reduced their glutamate levels. Therefore, our data provide comprehensive insights into the overall molecular landscape of Meniere's disease and suggest that synaptic vesicle recycling and dysregulated immune responses are involved in Meniere's disease and partially explain hearing loss and vertigo attacks.

[0095] Matters not covered by the present invention are known technologies.

[0096] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of substances for detecting the expression level of glutaminase and its related biological elements in the preparation of products for screening, diagnosis, monitoring or prognosis of Meniere's disease; wherein: The glutamate-associated biological element comprises IL-1β and glutamate.

2. The use according to claim 1, characterized in that: The substances for detecting the expression level of glutaminase and its related biological elements include reagents required for detecting the expression level of glutaminase encoding gene based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing, reagents required for detecting the expression level of IL-1β based on immunoassay method, and / or reagents required for detecting glutamate based on chromatography, mass spectrometry, spectroscopy and biological method; The products include primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting the expression levels of glutaminase and its related biological elements.

3. A system for screening, diagnosis, monitoring or prognosis of Meniere's disease, characterized in that: The system comprises: An acquisition module is configured to: acquire the expression level of glutamate and its related biological elements in the test sample of the subject; An evaluation module, which is configured to: evaluate and judge the disease condition of the subject according to the subject's glutaminase and related biological elements obtained by the acquisition module; Wherein, the glutamate-associated biological elements include IL-1β and glutamate.

4. The use according to claim 3, characterized in that: The sample to be tested includes an inner ear tissue sample of a subject.

5. Application of glutaminase and its related biological elements as targets in the preparation and / or screening of drugs for Meniere's disease; wherein: The glutamate-associated biological element comprises IL-1β and glutamate.

6. The use according to claim 5, characterized in that Methods for screening drugs to prevent or treat Meniere's disease include: a) treating a system expressing and / or containing glutamate and its related biological elements with a candidate substance; setting up a parallel control without treatment with the candidate substance; b) After completing step a), detecting the expression level of the glutamate enzyme and its related biological elements in the system; if the expression level of the glutamate enzyme and its related biological elements in the system treated with the candidate substance is significantly downregulated compared with the parallel control, the candidate substance can be used as a candidate drug for preventing or treating Meniere's disease.

7. The use according to claim 6, characterized in that The system is a cell system, a solution system, a tissue system, an organ system or an animal system.

8. Use of a substance that inhibits the expression of glutaminase and its related biological elements or reduces their activity in any one or more of the following, wherein the glutaminase-related biological elements include IL-1β and glutamate: a) preparing products for inhibiting cochlear synaptic damage; b) Products to alleviate endolymphatic hydrops-mediated auditory-vestibular dysfunction; c) Products for the prevention and / or treatment of Meniere's disease.

9. The use according to claim 8, characterized in that The substances that inhibit glutaminase and its related biological elements include but are not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, antagonists, siRNA (SEQ ID NO.1-2), shRNA, substances for implementing lentiviral infection or gene knockout, and antibodies against glutaminase and its related biological elements.

10. The use according to claim 9, characterized in that: The substances that inhibit the expression of glutaminase and related biological elements or reduce their activity include glutaminase inhibitor CB-839 and interleukin-1β receptor inhibitor anakinra.

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