Application of reagent for detecting complement factor in preparation of tinnitus diagnostic product

Abstract

The invention relates to the field of medicines, in particular to application of a reagent for detecting a complement factor in preparation of a tinnitus diagnostic product, the complement factor is selected from a complement component, a complement regulatory protein or a complement related chemotactic factor, the complement component is selected from C3, C5 and / or C1q, and tinnitus is chronic tinnitus. The peripheral blood can be used as a sample, the chronic tinnitus can be accurately diagnosed, theoretical and experimental bases are provided for clinical diagnosis of the chronic tinnitus, diagnosis and intervention can be carried out in the early stage of tinnitus, and further deterioration is prevented.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of reagents for detecting complement factors in the preparation of tinnitus diagnostic products. Background Technology

[0002] Tinnitus is the subjective auditory perception of the ear in the absence of an external sound source. Common tinnitus sounds include cicada chirping, ringing, buzzing, and humming. The rate of tinnitus patients seeking medical attention is very low, which can easily lead to disease progression and miss the optimal window for early intervention. Chronic tinnitus lasting longer than 6 months is often persistent and long-lasting, severely impacting the patient's physical and mental health. Many patients are overwhelmed by the tinnitus, often experiencing irritability, anxiety, depression, insomnia, and other psychosomatic disorders, as well as difficulty concentrating and lack of energy, significantly affecting their normal work. A small number of severe cases even have suicidal tendencies. Because the tinnitus of subjective tinnitus patients cannot be perceived by others, there is a significant lack of awareness of the harmful effects of tinnitus among family and social members. Tinnitus patients, due to a lack of understanding, often face strained family and social relationships. However, public awareness of tinnitus is generally insufficient, resulting in extremely low early diagnosis and intervention rates. Early diagnosis and exploration of simple and effective tinnitus intervention measures are essential for practicing, inheriting, and implementing the concept of proactive health.

[0003] Traditional tinnitus assessment includes history taking, specialist examination, audiological examination, psychoacoustic characterization of tinnitus, and severity assessment. When necessary, imaging studies such as CT / MRI and vascular ultrasound may be required to clarify the cause. A complete tinnitus diagnostic system requires a core team of professionals including ENT specialists, audiologists, radiologists, and technicians, as well as the necessary equipment in the hospital. Tinnitus assessment primarily relies on scales to help doctors develop individualized treatment plans. Commonly used tinnitus assessment scales include the Tinnitus Handicap Inventory (THI), the Visual Analogue Scale (VAS), and the Tinnitus Questionnaire (TQ). Currently, there is a lack of effective drug treatments for tinnitus; most clinical treatments focus on alleviating non-auditory symptoms associated with tinnitus. Various medications have been attempted to treat tinnitus, but none have achieved long-term efficacy. Furthermore, no medication has been approved by the US Food and Drug Administration or the European Medicines Agency for the treatment of tinnitus. The efficacy of traditional Chinese medicine and other treatments requires further evidence-based support. Psychological counseling and education are crucial components of tinnitus treatment. Based on this, cognitive behavioral therapy for tinnitus is currently the only method proven effective in alleviating tinnitus through large-scale randomized clinical trials. However, it is time-consuming and labor-intensive, making it unsuitable for application and promotion in my country, where tinnitus clinics are large and there is a shortage of specialized medical personnel.

[0004] With the development of neuroimaging, the central pathogenesis of tinnitus has become widely recognized. The development of tinnitus involves the peripheral auditory system, auditory centers, and non-auditory centers. Auditory deprivation, such as damage to inner and outer hair cells, leads to auditory center remodeling, initially exciting the primary auditory centers. Tinnitus is perceived when the sensory networks of the frontal lobe, parietal lobe, and limbic system are simultaneously activated. Further activation of the salience network reinforces the presence of tinnitus. Furthermore, after excitation of the emotional centers, negative emotions such as anxiety and depression will accompany tinnitus, severely affecting the patient's psychological state and mood. Once the hippocampus, parahippocampal gyrus, and amygdala are activated, the tinnitus and its associated negative emotional effects will persist. However, the key molecules and related mechanisms involved in this abnormal central neural activity remain unclear.

[0005] Although researchers have conducted extensive studies on the neural mechanisms of tinnitus, there is still a lack of laboratory-aided diagnostic evidence, and no relevant evidence from laboratory blood indicators to support the diagnosis of chronic subjective tinnitus. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a use of a reagent for detecting complement factors in the preparation of tinnitus diagnostic products, thereby solving the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention first provides the use of reagents for detecting complement factors or complement factor activation-related proteins in the preparation of tinnitus diagnostic products.

[0008] Preferably, the complement factor is selected from complement components, complement regulatory proteins, or complement-related chemokines.

[0009] Preferably, the complement component is selected from C2, C3, C5, C1q or complement membrane attack complex.

[0010] Preferably, the complement regulatory protein is selected from complement factor H (CFH), complement factor H-related protein 1 (CFHR1), CFI, IGHG4, CD46, CR1, CPB2, SERPING1, MBL2, or CPN2.

[0011] Preferably, the complement-related chemokine is a platelet factor expressed on the surface of serum exosomes, selected from platelet factor 4 variant 1 (PF4V1).

[0012] Preferably, the tinnitus is chronic subjective tinnitus.

[0013] As described above, the use of the reagent for detecting complement factors in the preparation of tinnitus diagnostic products according to the present invention has the following beneficial effects: it provides relevant laboratory and molecular diagnostic evidence for the diagnosis of chronic subjective tinnitus, enabling more accurate and convenient diagnosis of chronic subjective tinnitus, allowing for early diagnosis and intervention to prevent further deterioration. Furthermore, the complement cascade reaction is an important pathway in the pathophysiological mechanism of chronic subjective tinnitus, and drugs based on complement factors have worthy of exploration in the drug treatment of chronic subjective tinnitus. Attached Figure Description

[0014] Figure 1 This chart shows the correlation analysis between clinical characteristics and blood parameters in patients with chronic subjective tinnitus. Purple indicates a positive correlation, and brown indicates a negative correlation. The correlation coefficients are labeled numerically.

[0015] Figure 2 The results show the identification of serum exosomes. A: Transmission electron microscopy results; B: Particle size analyzer results; C: Western blotting results.

[0016] Figure 3 A: Quality control of exosomal proteomics; B: PCA analysis; C: Correlation analysis of quality control samples.

[0017] Figure 4 Bubble chart showing the cellular component (CC) and molecular function (MF) analyses of differentially expressed proteins. A: Differentially expressed protein heatmap; B: Cellular component (CC) and molecular function (MF) analyses based on differentially expressed proteins.

[0018] Figure 5 The results are shown in the differential protein enrichment analysis, which shows significantly upregulated and significantly downregulated proteins.

[0019] Figure 6 The results of KEGG pathway enrichment analysis (purple) show proteins that are significantly upregulated (red) and downregulated (blue).

[0020] Figure 7 A: The top 10 significantly enriched pathways in the Reactome database; B: The names of differentially expressed proteins in the relevant pathways; C: ELISA validation of C3 and C5.

[0021] Figure 8 The results are displayed as WGCNA analysis results.

[0022] Figure 9 The results of GO-BP and Reactome enrichment analysis in WGCNA are shown.

[0023] Figure 10 The results are displayed as GSEA analysis results, where NES represents the standardized enrichment score.

[0024] Figure 11 Displays the names of differentially expressed proteins in the platelet activation pathway. Detailed Implementation

[0025] This article analyzes the proteomic characteristics of circulating serum exosomes in patients with chronic subjective tinnitus and healthy individuals based on DIA technology. It utilizes bioinformatics analysis to explore the relationship between exosomes and the pathogenesis of chronic tinnitus at the molecular level, and investigates the role and function of differentially expressed exosome proteins in the pathological process of tinnitus. This research lays the foundation for studying the mechanism of chronic tinnitus and developing personalized treatment methods.

[0026] This invention first provides the use of reagents for detecting complement factors or complement factor activation-related proteins in the preparation of tinnitus diagnostic products.

[0027] In some embodiments of the present invention, the complement factor activation-related proteins IGKC, IGLC2, IGHV2-5, IGHG3, IGHG2, IGLV7-46, or IGKVD-33 are used.

[0028] In some embodiments of the present invention, the complement factor is selected from complement components, complement regulatory proteins, or complement-related chemokines.

[0029] In some embodiments of the present invention, the complement component is selected from C2, C3, C5, C1q or the complement membrane attack complex (MAC), wherein the MAC is selected from C6, C7, C8A, C8B or C8G.

[0030] C3 is a key factor in both the classical and alternative pathways of the complement system. It is the most abundant complement component and plays a vital immune role. The complement system is an important humoral component of adaptive and innate immunity; when inappropriately activated, it can harm the host and lead to infectious, inflammatory, or autoimmune diseases.

[0031] Complement has been identified as a non-cytokine that plays a crucial role in coordinating platelet-mediated inflammation. The complement cascade is activated via classical, lectinic, or alternative pathways, which converge on the central molecule C3 of the complement system.

[0032] C1q is used to reflect the activation state of the classical complement pathway. MAC formation is closely related to membrane microparticles released by platelets and endothelial cells, which are precursors for thrombin formation.

[0033] The complement regulatory proteins are selected from complement factor H (CFH), complement factor H-related protein 1 (CFHR1), CFI, IGHG4, CD46, CR1, CPB2, SERPING1, MBL2, or CPN2.

[0034] Complement factor H (CFH) is a regulatory protein in the complement system. The main function of CFH is to act as a negative regulator of the complement activation pathway, controlling the complement cascade and preventing tissue damage caused by excessive activation of the complement system.

[0035] Complement factor H-related protein 1 (CFHR1) is a complement regulatory protein of the complement system. As a component of the complement factor H protein family, CFHR1, along with complement component 3, complement component 5, factor H (CFH), and factor I (CFI), plays an important regulatory role in the activation of the complement alternative pathway.

[0036] In some embodiments of the present invention, the complement-related chemokine is a platelet factor expressed on the surface of exosomes in serum.

[0037] The proteomics study of this invention revealed a significant upregulation of platelet factor 4 variant 1 (PF4V1). Figure 8The chemokine PF4V1, encoded by a homologous non-allelic gene of PF4, can bind to the CXCR3 receptor and is released during platelet aggregation, exhibiting chemotactic anti-angiogenic function. Therefore, it can be concluded that platelet activation and complement-mediated inflammation are involved in the mechanism of tinnitus.

[0038] In some embodiments of the present invention, the tinnitus is chronic tinnitus; preferably, the tinnitus is chronic subjective tinnitus.

[0039] Most patients with chronic subjective tinnitus have grade II-III tinnitus disorder; most patients with chronic tinnitus accompanied by hearing loss exhibit high-frequency hearing loss.

[0040] In some embodiments of the present invention, the reagent for detecting complement factors is a reagent for detecting free complement factors in serum or a reagent for detecting complement factors encapsulated by exosomes in serum.

[0041] Free complement factors in serum are complement components that exist in serum in a soluble form.

[0042] In some embodiments of the present invention, the reagent for detecting complement factor activation-related proteins is a reagent for detecting free complement factor activation-related proteins in serum or a reagent for detecting complement factor activation-related proteins encapsulated by exosomes in serum.

[0043] This invention reveals that complement components are significantly elevated in the blood circulation of patients with chronic tinnitus. Specifically, complement components are highly expressed in serum-derived exosomes from patients with chronic subjective tinnitus. Therefore, it is hypothesized that exosomes carry complement factors and participate in the occurrence and development of tinnitus by mediating the interaction between neurons and microglia.

[0044] Exosomes are nanoscale extracellular microvesicles rich in proteins and other bioactive molecules. Besides playing a crucial role in intercellular signal transduction, their excellent biocompatibility allows them to cross the blood-brain barrier and participate in the pathological processes of central nervous system diseases, thus making them potential diagnostic markers. As endogenous nanoscale vesicles, exosomes can cross the blood-brain barrier and the stria vascularis-labyrinthine barrier, and mediate long-distance protein transport or inter-cell transport via paracrine signaling. Therefore, exosomes also hold great promise as therapeutic targets for diseases.

[0045] In some embodiments of the present invention, reagents for detecting complement factors and reagents for detecting platelet activation are used together to prepare tinnitus diagnostic products.

[0046] In some embodiments of the present invention, the reagent for detecting platelet activation is selected from substances used to detect one or more of the following indicators: mean platelet volume, platelet distribution width, or platelet activation-related proteins in exosomes. Preferably, the platelet activation-related proteins are selected from one or more of the following: A1BG, HRG, LGALS3BP, ORM1, A2M, VTI1A, CLEC3B, APOH, SERPINA3, TF, GNG2, CD9, MPIG6B, SERPINA1, PLG, SERPI NG1, AHSG, APOA1, KNG1, SERPINA4, LY6G6F, PLA2G4A, LAMP2, FGA, ANXA5, ABCC4, VAV1, GNAI3, FLNA, CLEC1B, RASGRP2 , YWHAZ, VCL, GNAI2, WDR1, GP9, CALM1, ACTN4, RAB27B, MAPK1, CFL1, GNB4, PLEK, CD36, RHOA, TUBA4A, FCER1G, GNB1 (such as Figure 11 ).

[0047] Platelets express complement receptors on their surface. Complement components can bind to these receptors, triggering platelet activation and aggregation.

[0048] In the aforementioned tinnitus diagnostic product, the active ingredient that performs the function may be solely a reagent for detecting complement factors, or it may include a reagent for detecting platelet activation, or it may contain other substances. That is, the reagent for detecting complement factors is the sole active ingredient or one of the active ingredients of the product. The product can be used in combination with other products or alone.

[0049] The product can be a single-component substance or a multi-component substance.

[0050] The product may be in the form of small chemical molecules, proteins, peptides, lipids, carbohydrates, nucleic acids, oligonucleotides, ribozymes, aptamers, cofactors, ligands, ligand mimetics, receptors, enzymes, kinases, phosphatases, cytokines, growth factors, metal ions, chelates, antisense nucleic acids, repressive RNA, microRNA, small interfering RNA (siRNA), antibodies or their antigen-binding moieties, antibody mimics, antagonists, blockers, or inhibitors, or other forms suitable for detecting complement factors.

[0051] The product may be a complement assay kit, an exosome extraction kit, or a serum processing reagent, etc.

[0052] The product can be a commercially available reagent, such as a commercially available exosome extraction kit using the MagCapture kit (Wako, Japan).

[0053] There are no special restrictions on the form of the product; it can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0054] The product is primarily designed for blood samples.

[0055] In some embodiments of the present invention, the product is a drug, and the drug further includes a carrier or excipients.

[0056] The "carrier or excipient" should be compatible with the active ingredient, meaning it can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0057] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0059] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0060] The following examples first investigated and analyzed the clinical characteristics and peripheral blood immune-related indicators of patients with chronic subjective tinnitus at the tinnitus clinic of the Eye, Ear, Nose and Throat Hospital affiliated with Fudan University. A cross-sectional study was conducted to observe changes in some blood indicators related to chronic tinnitus, and to preliminarily explore peripheral blood factors associated with chronic tinnitus. Based on this, proteomic analysis of peripheral blood serum exosomes from the above-mentioned representative population revealed that peripheral blood platelet-related indicators and complement levels were significantly elevated in patients with chronic subjective tinnitus compared to the control group. Differentially expressed serum exosome proteins were associated with complement cascade reactions and platelet activation processes, leading to the following conclusions:

[0061] 1. Patients with chronic subjective tinnitus exhibit higher levels of platelet activity in their peripheral blood;

[0062] 2. The complement cascade is an important pathway in the pathophysiological mechanism of chronic subjective tinnitus;

[0063] 3. The differentially expressed proteins from exosomes in the tinnitus group were functionally enriched in the complement activation pathway and the platelet activation pathway.

[0064] Given the close relationship between complement and platelets, the results of this study suggest that patients with chronic tinnitus may have a chronic inflammatory state in the central microcirculation or a tendency to thrombosis.

[0065] Example 1: Clinical characteristics of patients with chronic subjective tinnitus

[0066] This study was approved by the Ethics Committee of the Eye, Ear, Nose and Throat Hospital affiliated with Fudan University (No.: 2017048-1). The inclusion / exclusion criteria are as follows:

[0067] Inclusion criteria:

[0068] 1) Age 18-70;

[0069] 2) The chief complaint is unilateral or bilateral non-pulsatile tinnitus that has lasted for more than 6 months;

[0070] 3) The patient participates voluntarily and gives informed consent.

[0071] Exclusion criteria:

[0072] 1) There are known potential causes of tinnitus, such as middle ear diseases, acoustic neuroma, Meniere's disease, etc.

[0073] 2) Recently infected, or have taken antibiotics within the past 30 days;

[0074] 3) Pregnant or breastfeeding women;

[0075] 4) Comorbid other systemic diseases, such as poorly controlled hypertension, diabetes, kidney disease, tumors, etc.;

[0076] 5) The patient has a serious mental illness that affects the questionnaire response and assessment.

[0077] This study included 200 patients with chronic subjective tinnitus who visited the Tinnitus Clinic of the Eye, Ear, Nose and Throat Hospital affiliated with Fudan University in Shanghai from September 2021 to December 2022. Before enrollment, all patients underwent relevant ENT examinations, including otoscopy, acoustic impedance, pure tone audiometry, and temporal bone CT. Subsequently, they completed a tinnitus questionnaire using the Fudan Tinnitus RS.

[0078] Statistical methods

[0079] The Kolmogorov-Smirnov test is used to assess whether continuous variables conform to a normal distribution. Normally distributed continuous data are described by the mean [standard deviation] (mean [SD]); non-normally distributed continuous data are described by the median [interquartile range] (median [IQR]). Categorical variables are expressed as frequency or percentage (%).

[0080] This study included 200 patients with chronic subjective tinnitus, of whom 105 were male and 95 were female (Table 1). The age distribution was 41.00 [33.00, 52.00] years, and the median duration of illness was 18 months. 88 patients had unilateral tinnitus, while the remaining 112 patients had bilateral tinnitus.

[0081] Table 1. Basic characteristics of 200 patients with chronic subjective tinnitus.

[0082]

[0083]

[0084] Hearing status of enrolled patients

[0085] Of the patients included, 43.1% did not have hearing loss, while the majority of the other group of patients with hearing loss presented with high-frequency hearing loss (Table 2).

[0086] Table 2. Audiological examination results of 200 patients with chronic subjective tinnitus

[0087]

[0088] Example 2: Changes in blood cell count characteristics in patients with chronic subjective tinnitus

[0089] Materials and methods

[0090] Control group recruitment:

[0091] In Example 1, peripheral venous blood samples were collected from all patients with chronic subjective tinnitus for routine blood tests and complement measurements. Simultaneously, a control group matched for age and sex was recruited from healthy individuals, and blood samples were collected from this group for reference.

[0092] Blood parameters were measured in the morning after participants had fasted for 6-8 hours, by collecting peripheral blood samples via venous puncture at the elbow. The obtained blood samples were used for complete blood count and routine serum complement level testing.

[0093] Collect a blood sample in an EDTA tube. Measure complete blood count (CBC) parameters using a Sysmex XN-A1 automated blood counting system (Kobe, Japan) within 0.5 hours of blood collection.

[0094] Complement assays were performed using blood collection tubes containing a coagulant, followed by centrifugation at 1318 × g for 10 minutes. The Roche Diagnostics 8000 biochemical analyzer (Mannheim, Germany) measured the levels of C3, C4, C1q and total complement activity (CH50) in serum within 3 hours after blood collection, and performed the determination using immunoturbidimetry according to the instructions.

[0095] Statistical methods:

[0096] Statistical analysis was performed using R Version 4.1.2. The Kolmogorov-Smirnov test was used to assess whether continuous variables conformed to a normal distribution. For normally distributed continuous data, the mean [standard deviation] (mean [SD]) was used for comparisons between groups using the independent samples t-test; for non-normally distributed continuous data, the median [interquartile range] (median [IQR]) was used for comparisons between groups using the Wilcox rank-sum test (Mann-Whitney U test). Categorical variables were expressed as frequencies or percentages (%), and comparisons between groups were performed using the χ² test; Spearman correlation coefficients (r) were used to determine the correlation between parameters and were represented by a heatmap; p < 0.05 was considered statistically significant.

[0097] 2.1 Peripheral blood cell count in patients with chronic tinnitus and the control group

[0098] As shown in Table 3, the erythrocyte count, hematocrit, mean erythrocyte volume, and mean erythrocyte hemoglobin concentration in the tinnitus group were significantly higher than those in the control group, with statistically significant differences (P values ​​of 0.002, <0.001, 0.003, and <0.001, respectively). Furthermore, the monocyte and eosinophil counts in the tinnitus group were also significantly higher than those in the control group (P values ​​of 0.001 and 0.011, respectively). However, there were no significant P values ​​for other indicators between the two groups. The erythrocyte count, hematocrit, mean erythrocyte volume, and mean erythrocyte hemoglobin concentration values ​​in the tinnitus group are used in routine blood tests to define erythrocyte size and hemoglobin content. Mean erythrocyte volume determines erythrocyte size, while mean erythrocyte hemoglobin concentration represents the amount of hemoglobin per unit volume and reflects the mean hemoglobin concentration. The structural characteristics of erythrocytes are affected by the pathophysiology of many diseases, potentially impairing microcirculatory blood flow.

[0099] Table 3 Comparison of blood cell parameters between the two groups

[0100]

[0101] 2.2 Peripheral blood platelet count in patients with chronic tinnitus and the control group

[0102] The comparison of platelet-related parameters between the control group and the tinnitus group (Table 4) showed that the mean platelet volume (MPV) and platelet distribution width (PDW) were significantly higher in the tinnitus group than in the control group (P<0.05). This indicates that tinnitus patients may have a larger mean platelet volume and a wider platelet distribution width. MPV assesses platelet size, while PDW represents changes in platelet size distribution. Increased levels of these parameters indicate that a large number of platelets have been released from the bone marrow into the bloodstream; therefore, MPV and PDW can be considered indicators of platelet activation.

[0103] Table 4 Comparison of platelet-related parameters between the two groups

[0104]

[0105] 2.3 Peripheral blood complement levels in patients with chronic tinnitus and the control group

[0106] The complement levels in the control group and the tinnitus group are shown in Table 5. The results showed that compared with the control group, the C3 level in the tinnitus group was significantly higher (P<0.001), while the C4 level showed no significant difference (P=0.076). Furthermore, the C1Q level in the tinnitus group was slightly higher than that in the control group (P=0.037), and the serum total complement activity level was slightly lower than that in the control group (P=0.046). These results suggest that changes in C3 levels may play a major role in the pathogenesis of tinnitus, while changes in C4 may not have a significant impact on its development. These results may contribute to a deeper understanding of the pathogenesis of tinnitus and have certain guiding significance in clinical practice.

[0107] Table 9. Comparison of serum complement levels between the two groups.

[0108]

[0109] 2.4 Correlation analysis between clinical characteristics and blood indicators in patients with chronic subjective tinnitus

[0110] Spearman correlation analysis using heatmaps ( Figure 1 The results showed that complement C3 and C1Q levels were significantly positively correlated; platelet parameter PDW was significantly positively correlated with MPV and P-LCR levels, and MPV was significantly positively correlated with P-LCR levels; THI score was significantly positively correlated with AIS score and VAS score; meanwhile, THI score, VAS score, and unilateral / bilateral tinnitus were not significantly correlated with complement or platelet-related blood parameters.

[0111] Example 3: Serum exosomal proteomics analysis of patients with chronic subjective tinnitus

[0112] Methods and steps:

[0113] 3.1 Extraction and identification of serum exosomes

[0114] Exosome extraction in this experiment was performed using the MagCapture kit (Wako, Japan). The principle is based on the purification method using Tim4 magnetic beads to bind to phosphatidylserine (PS) on the surface of exosomes. The extraction method was performed according to the manufacturer's instructions. This method can conveniently and efficiently extract high-purity PS-positive exosomes from membrane surfaces. The principle involves using a pH-neutral metal chelating reagent to elute the captured exosomes from the magnetic beads, obtaining intact exosomes for various downstream experiments, including electron microscopy analysis, nanoparticle tracking analysis, and component (protein, lipid, nucleic acid, etc.) analysis. Healthy individuals and patients with chronic subjective tinnitus exhibiting representative characteristics of the cross-sectional study analyzed in Example 2 were recruited. 4 ml of fasting venous blood was collected, and 1 ml of serum was extracted into centrifuge tubes. Exosomes were extracted from the serum according to the kit's instructions.

[0115] The extracted exosomes were analyzed for concentration and particle size distribution using Zetaview (Particle Metrix GmbH) to determine the actual content and purity of the samples. Data were analyzed using ZetaView 8.04.02 software. Exosome morphology was observed using a Tecnai G2 F20 TWIN transmission electron microscope from FEI. Western blot analysis was performed to detect exosome marker proteins, specifically Alix (CST:2171), a protein related to exosome synthesis, and CD63 (Santa Cruz:sc5275), a four-transmembrane protein.

[0116] 3.2 Serum exosomal proteomics based on DIA quantitative technology

[0117] 1) Protein extraction and peptide enzymatic hydrolysis:

[0118] a) Sample testing: Mix an appropriate amount of protein and extract 20 μg of protein for electrophoresis testing.

[0119] b) Enzymatic digestion: All samples were digested in solution with trypsin. Dithiothreitol was added and reacted for 2 h; iodoacetamide was added and shaken for 30 min; NH4HCO3 buffer was added; 2 μg of intracellular protease Lys-C was added and stored at 37 °C for 4 h; then 2 μg of Trypsin was added and stored for 16 h; desalted and lyophilized.

[0120] c) Reconstitution: 0.1% formaldehyde aqueous solution (FA).

[0121] d) Peptide concentration detection: Peptide concentration was determined at OD280 using a spectrophotometer.

[0122] e) Peptide separation: Take 100 μg of low-abundance peptides and fractionate them using the HPRP method, collecting all components. f) Mass spectrometry detection: Take 2 μg of peptides and incorporate an appropriate amount of iRT standard peptides.

[0123] 2) Data-dependent acquisition (DDA) mass spectrometry detection:

[0124]

[0125]

[0126] 3) DIA Analysis

[0127]

[0128] 4) Data Analysis

[0129] ①DDA data:

[0130] Import the Spectronaut software. Use the human_uniprot database.

[0131] Enzyme: trypsin;

[0132] Maximum deletion cleavage site: 1;

[0133] Fixed modification: Carbamidomethyl(C);

[0134] Dynamic modification settings: Oxidation(M) and Acetyl(Protein N-term);

[0135] The FDR of proteins identified by database retrieval is <1%.

[0136] ②DIA data:

[0137] Import the Spectronaut software. Use the human_uniprot database.

[0138] Residence time prediction type: Dynamic iRT; MS2 layer interference correction enabled;

[0139] Set cross-run normalization; FDR < 1%.

[0140] 3.3 Differential protein analysis and biological function analysis of exosomes

[0141] 1) Quality control (QC)

[0142] First, the quantitative information of the target protein set was normalized (to the (-1,1) interval). Then, the Complexheatmap R package (R Version 4.1.2) was used to classify both sample and protein expression levels simultaneously. Quality control samples were inserted into the sample queue to monitor and evaluate the stability of the system and the reliability of the data. Further QC assessments were performed using the coefficient of variation (CVs), principal component analysis (PCA), and Pearson correlation coefficient. Generally, lower CVs indicate higher clustering tendency in PCA, and correlation coefficients closer to 1 indicate more stable experimental configurations. PCA was used as an unsupervised multivariate data analysis method using the R packages Factoextra and FactoMineR.

[0143] 2) Identification of differentially expressed proteins (DEPs)

[0144] The quantitative proteomic identification methods were first presented in tabular form, listing the number of identified and quantified peptides. Missing data were estimated using chain equations applied with the RandomForest package. Next, key screening criteria for DEPs were a p-value <0.05 based on an independent samples t-test and a reduced change value greater than 1.5 or less than 0.67. DEPs were screened using the R package ComplexHeatmap.

[0145] 3) Functional enrichment analysis

[0146] To predict biological function, we focused on enrichment analyses of all DEPs using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome. GO functional annotation aimed to elucidate the underlying biological processes (BP), molecular functions (MF), and cellular components (CC) associated with proteins. CC and MF analyses were performed on all DEPs using the Annotated, Visualized, and Synthetic Discovery Database (DAVID). The top seven terms for each category were visualized using the R package ggplot2. BP analyses were performed separately on upregulated and downregulated DEPs using the ClusterProfiler v.4.2 package in Bioconductor. ClusterProfiler was used to assign each protein to the KEGG database, revealing the potential roles of upregulated and downregulated proteins in the network. Furthermore, the enrichPathway feature in the "ReactomePA" package of Bioconductor was used to further analyze the DEPs of interest.

[0147] 4) Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA)

[0148] The WGCNA algorithm is a commonly used algorithm for constructing weighted co-expression networks. Using the `Eigengenes` package in R, Pearson correlation was used to determine the correlation between each module and the trait. Eigengene-based connections were used to assess the correlation between modules and gene expression values. Generally, the closer a module's membership is to +1 or -1, the higher the correlation between the module and the trait. Key co-expression modules were then selected, and the differentially expressed proteins corresponding to the selected modules were used for further analysis. GSEA was performed on total proteins using the `fgsea` package in R, and GOBP and KEGG pathway enrichment analyses were performed. GSEA provides mechanistic insights into the gene lists generated by genome-scale experiments. Proteins were sorted according to folding changes, and GSEA was performed and visualized using the `Cluster Profiler` package in R.

[0149] result:

[0150] 1. Identification of serum exosomes

[0151] TEM and NTA revealed the detailed morphology and particle size distribution of exosomes from serum samples. TEM showed that the exosomes possessed a classic cup-shaped structure with a double membrane, with a diameter of 40-150 nm. NTA showed that the mean diameters of serum exosomes in 97.1% of the chronic tinnitus group and 98.6% of the control group were 105.3 nm and 108.7 nm, respectively. Western blot analysis confirmed the presence of exosome-specific marker proteins; exosomes in both groups were positive for CD63 and ALIX, but negative for Calnexin. Figure 2 ).

[0152] 2. Quality control and screening of differentially expressed proteins in exosomes

[0153] We used quality control samples to determine overall data quality and the reproducibility of the analysis. The CV of the quality control samples was approximately 20%. Figure 3 A) indicates that there were only slight fluctuations and satisfactory stability between samples during the testing process. In the PCA plot, the quality control samples showed a high degree of aggregation (…). Figure 3 B) demonstrates the reasonable stability and high accuracy of the detection system. Significant correlations exist among the various quality control samples. Figure 3 C) confirms the high stability of the testing process and the reliable data repeatability.

[0154] 3. Functional enrichment analysis of differentially expressed proteins in exosomals

[0155] Next, differentially expressed proteins were analyzed. Based on the screening criteria for differentially expressed proteins: a fold change greater than 1.5 or less than 0.67, and a p-value <0.05, a total of 332 significantly upregulated and 118 significantly downregulated differentially expressed proteins were identified from serum exosomes of chronic tinnitus. Figure 4 A), serving as the basis for subsequent bioinformatics analysis. A bubble diagram based on differentially expressed protein cellular components (CC) and molecular functions (MF) analysis (…). Figure 4 B) presented the top 7 enriched terms, with the most prominent term in the CC category being "exosomes," which confirms that most of the differentially expressed exosomal proteins we screened are located in exosomes.

[0156] Next, bio-pathway BP enrichment analysis was performed on the differentially regulated proteins. R language was used to analyze significantly upregulated and significantly downregulated differentially regulated proteins, and representative enrichment terms are listed below. Figure 5 The significant terms among the upregulated differentially expressed proteins indicate that chronic tinnitus exosomes are involved in various immune-related functions, including humoral immune responses (GO:0006959), adaptive immune responses based on somatic reorganization of immunoglobulin superfamily structures (GO:0002460), leukocyte-mediated immunity (GO:0002443), complement activation (GO:0006956), and activation of immune responses (GO:0002253). For downregulated differentially expressed proteins, BP terms revolve around wound healing (GO:0042060), platelet activation (GO:0030168), platelet aggregation (GO:0070527), blood coagulation (GO:0007596), positive regulation of supramolecular fibrous tissue (GO:1902905), actin filament tissue (GO:0007015), homocellular-cell adhesion (GO:0034109), regulation of cell component size (GO:0032535), actin polymerization or depolymerization (GO:0008154), and regulation of actin filament tissue (GO:0110053).

[0157] In KEGG pathway enrichment analysis, it is worth noting that significant pathways are often intertwined based on certain key proteins, because a single protein can participate in multiple signaling pathways. Figure 6 The images show proteins corresponding to functionally associated KEGG pathways, such as C3, C5, C6, C7, C8A, C8B, and / or C8G. Two significantly enriched KEGG pathways identified by upregulated differentially expressed proteins are the complement and coagulation cascade (hsa04610) and the proteasome pathway (hsa03050).

[0158] Figure 7The bar chart in Figure A shows the top 10 significantly enriched pathways in the Reactome database and their corresponding definitions. The complement cascade (R-HSA-166658) pathway was significantly enriched. Furthermore, among all significantly enriched pathways, some differentially expressed proteins were found to be enriched in the sound sensory processing pathway (R-HSA-9659379). Among the proteins involved in the sound sensory pathway, CAPZB and ACTB are associated with the differentiation and morphology of inner hair cells related to auditory function. Figure 7 Section B lists proteins associated with the complement cascade and auditory processing. Key complement factors C3 and C5 are significantly upregulated in tinnitus patients within the complement cascade pathway. The major regulators of the complement pathway, complement factor H (CFH) and complement factor H-associated protein 1 (CFHR1), were also screened as differentially upregulated proteins. The components of the complement cascade (C3, C5) were subsequently validated at the protein level using ELISA. Figure 7 C). Therefore, complement factors are specific for diagnosing chronic subjective tinnitus.

[0159] 4. WGCNA and GSEA analysis of exosome proteomics

[0160] WGCNA and GSEA conducted further investigations. In WGCNA, by setting the soft threshold to 8, 10 co-expression modules were obtained, of which 6 modules showed a significant correlation with tinnitus. Figure 8 A). The most relevant protein cluster ( Figure 8 B) was selected as the primary analysis due to its superior co-expression performance. Within this protein cluster, 175 out of 344 proteins showed significant differential changes, as illustrated in the ring heatmap. Figure 8 C).

[0161] In addition, the top 15 pathways in GO-BP and Reactome pathway enrichment analyses were screened to predict the function of these key proteins. Figure 9 Complement activation, humoral immune response, and platelet aggregation were significantly enriched.

[0162] In the GSEA analysis, the most representative pathways from the GO-BP and KEGG databases are shown below. Figure 10 Exosomal proteins associated with complement coagulation cascade, complement replacement activation, and immune response are generally upregulated.

[0163] The changes in complement factors obtained in this embodiment are consistent with the changes obtained through blood cell characteristics in Example 2, which proves that complement factors are specific for diagnosing chronic subjective tinnitus.

[0164] Example 4: Specificity and sensitivity of complement factor combined with platelet markers in diagnosing tinnitus.

[0165] Peripheral venous blood samples were collected from 237 age- and sex-matched healthy individuals and 200 patients clinically diagnosed with chronic subjective tinnitus for the determination of complement C3, C5, MPV, and PDW. The measurement methods were the same as in Example 2.

[0166] The results showed that, compared with healthy individuals serving as negative controls, 200 patients with chronic subjective tinnitus exhibited significant differences in serum complement C1 and C3, as well as hematological indicators such as MPV and PDW. A multivariate logistic regression model was established using these indicators to effectively detect chronic subjective tinnitus. The method was as follows: the optimal model prediction threshold was determined by analyzing the area under the receiver operating characteristic curve (ROC). Model validation was performed on the entire population in this embodiment, yielding true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN). Using the following formula, the model demonstrated good specificity (0.625) and sensitivity (0.654):

[0167] Specificity = TN / (TN+FP) × 100%

[0168] Sensitivity = TP / (TP+FN)×100%.

[0169] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of reagents for detecting complement factors or complement factor activation-related proteins in the preparation of tinnitus diagnostic products.

2. The use according to claim 1, characterized in that, The complement factor is selected from complement components, complement regulatory proteins, or complement-related chemokines; and / or, the complement factor activates related proteins IGKC, IGLC2, IGHV2-5, IGHG3, IGHG2, IGLV7-46, or IGKVD-33.

3. The use according to claim 2, characterized in that, The complement component is selected from C2, C3, C5, C1q or complement membrane attack complex; preferably, the complement membrane attack complex is selected from C6, C7, C8A, C8B and / or C8G.

4. The use according to claim 2, characterized in that, The complement regulatory protein is selected from complement factor H, complement factor H-related protein 1, CFI, IGHG4, CD46, CR1, CPB2, SERPING1, MBL2, or CPN2.

5. The use according to claim 2, characterized in that, The complement-related chemokine is a platelet factor expressed on the surface of exosomes; preferably, the complement-related chemokine is platelet factor 4 variant 1.

6. The use according to claim 1, characterized in that, The tinnitus described is chronic tinnitus.

7. The use according to claim 1, characterized in that, The tinnitus described is chronic subjective tinnitus.

8. The use according to claim 1, characterized in that, The reagent used for detecting complement factors is selected from reagents used for detecting free complement factors in serum or reagents used for detecting complement factors in exosomes.

9. The use according to claim 1, characterized in that, The tinnitus diagnostic product also includes reagents for detecting platelet activation.

10. The use according to claim 9, characterized in that, The reagent used to detect platelet activation is selected from substances used to detect one or more of the following indicators: mean platelet volume, platelet distribution width, or platelet activation-related proteins in exosomes. Preferably, the platelet activation-related proteins are selected from one or more of the following: A1BG, HRG, LGALS3BP, ORM1, A2M, VTI1A, CLEC3B, APOH, SERPINA3, TF, GNG2, CD9, MPIG6B, SERPINA1, PLG, SERPING1, A HSG, APOA1, KNG1, SERPINA4, LY6G6F, PLA2G4A, LAMP2, FGA, ANXA5, ABCC4, VAV1, GNAI3, FLNA, CLEC1B, RASGRP2, Y WHAZ, VCL, GNAI2, WDR1, GP9, CALM1, ACTN4, RAB27B, MAPK1, CFL1, GNB4, PLEK, CD36, RHOA, TUBA4A, FCER1G, GNB1.

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