A method for constructing a phenylacetyl glutamine-induced sensorineural hearing loss animal model and application thereof

Abstract

This invention discloses a method for constructing an animal model of sensorineural hearing loss induced by phenylacetylglutamine and its application. The method includes: selecting 6-8 week old male C57BL / 6J mice, intraperitoneally injecting phenylacetylglutamine solution at a dose of 50 mg / kg body weight / day, once daily, for at least 4 months to obtain the sensorineural hearing loss animal model. This model is characterized by a significantly elevated auditory brainstem response threshold, loss of cochlear outer hair cells, and increased oxidative stress levels. This invention establishes for the first time an animal model of sensorineural hearing loss induced by the circulating metabolite phenylacetylglutamine, filling the technical gap in existing models that cannot simulate hearing loss mediated by metabolic abnormalities. The model construction method is simple to operate, has a high success rate, and good reproducibility, and can be used for research on the pathogenesis of sensorineural hearing loss and screening for preventive and therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing an animal model of sensorineural hearing loss induced by phenylacetylglutamine and its application. Background Technology

[0002] Sensorineural hearing loss (SNHL) is one of the most common sensory disorders worldwide. Its etiology is complex, involving multiple factors such as genetics, noise exposure, ototoxic drugs, aging, and metabolic abnormalities. In-depth research into the pathogenesis of SNHL, the search for new biomarkers and therapeutic targets, relies heavily on animal models that can accurately simulate the pathophysiological processes of human diseases.

[0003] Currently, researchers both domestically and internationally have established various animal models of non-SNHL, which mainly include the following categories: (1) Noise-induced hearing loss model: By exposing experimental animals to high-intensity broadband or narrowband noise, mechanical and metabolic damage to cochlear hair cells is directly caused, which in turn leads to hearing loss. This model is the most commonly used model for studying noise-induced hearing loss, but it cannot simulate hearing loss caused by other causes.

[0004] (2) Drug-induced hearing loss model: By injecting ototoxic drugs, such as aminoglycoside antibiotics like cisplatin and gentamicin, into the peritoneal cavity of animals, apoptosis and necrosis of cochlear hair cells are induced, resulting in an irreversible increase in the hearing threshold. This model is often used in research on the mechanisms of drug ototoxicity and protective strategies.

[0005] (3) Ischemia-reperfusion and radiation-induced model: Inner ear ischemia is simulated by surgical ligation of the labyrinthine artery, or inner ear tissue damage is induced by irradiation of the temporal bone region. This model is mainly used to study hearing loss caused by inner ear microcirculation disorders, but the surgical procedure is complex, has a high mortality rate, and poor reproducibility.

[0006] (4) Genetic engineering and aging models: Hereditary hearing loss models are constructed using naturally aging mice (such as the C57BL / 6J strain which naturally develops hearing loss at 12-18 months of age) or by overexpressing specific genes (such as the TZAP gene) (see the published text of Chinese Patent CN118599853A, published on September 6, 2024). These models are time-consuming and costly, and mainly reflect genetic or age factors.

[0007] However, all the existing models mentioned above share a common, unresolved technical limitation: they cannot simulate the pathophysiological process of sensorineural hearing loss mediated by abnormal circulating metabolites. In recent years, the concept of the "gut-inner ear axis" has been increasingly confirmed, and the influence of gut microbiota-dependent metabolites on inner ear function through blood circulation has become a research hotspot. For example, some studies have reported that the gut microbiota regulates age-related hearing loss through the metabolite 5-hydroxytryptophan (5-HTP) (see the study "Gut–Metabolome–Proteome Interactions in Age-Related Hearing Loss: Insights from Fecal Microbiota Transplantation and Multi-Omics Analyses" published in Advanced Science (2026), which first confirmed that the gut microbiota regulates age-related hearing loss through the metabolite 5-hydroxytryptophan (5-HTP)). However, this study involves 5-HTP, not the target molecule of this invention.

[0008] Phenylacetylglutamine (PAGln) is an important gut microbiota-dependent metabolite that has been proven to be an independent risk factor for cardiovascular disease (Cell, 2020) and is closely related to cellular senescence. A search revealed existing reports on the use of PAGln to construct disease models. Chinese patent CN116491472B discloses the use of phenylacetylglutamine in constructing animal models of chronic wounds. This method involves administering PAGln and creating wounds on the animal's epidermis to obtain a chronic wound model with delayed healing. The target organ is the skin, and the research area is wound repair.

[0009] Chinese patent application CN120608012A discloses a phenylacetylglutamine / phenylacetylglycine-induced vascular smooth muscle cell senescence model, its establishment method, and its application. It falls under the category of cell models, targeting the cardiovascular system, and is not an in vivo auditory system model.

[0010] Although the applicant's previous clinical metabolomics studies found a correlation between PAGln levels in the plasma of patients with sudden SNHL and hearing loss (see the literature Untargeted plasma metabolite detection in sudden sensorineural hearing loss: identifying key metabolic signatures published in Frontiers in Molecular Biosciences, 2025, 12:1567064), prior to the filing date of this patent application, no literature or patent worldwide has disclosed an animal model for inducing sensorineural hearing loss in mammals by exogenous administration of PAGln, or a method for constructing such a model.

[0011] This technological gap directly leads to the following problems that cannot be solved: (1) It is impossible to directly verify at the level of live animals whether PAGln is an independent pathogenic factor causing SNHL; (2) There is a lack of suitable tools to study in depth the specific molecular pathways by which PAGln damages inner ear hair cells; (3) It is impossible to use this model to screen for preventive / therapeutic drugs that can target PAGln metabolism or block its effects.

[0012] Therefore, developing an animal model that is standardized in operation, phenotypically stable, and can specifically simulate inner ear damage caused by elevated circulating PAGln is of urgent and important significance for promoting basic research and clinical translation of metabolic hearing loss. Summary of the Invention

[0013] (a) The technical problem that the invention aims to solve The core technical problem that this invention aims to solve is to address the following deficiencies in existing technologies: (1) Existing animal models of sensorineural hearing loss cannot simulate the pathophysiological process of hearing loss mediated by abnormal circulating metabolites.

[0014] Existing noise-induced models, drug-induced models, ischemia-reperfusion and radiation-induced models, genetic engineering and aging models all induce hearing loss by directly damaging inner ear tissue or by utilizing the natural aging process. Their pathogenesis and core pathological mechanisms are completely unrelated to abnormal circulating metabolites and cannot reflect the true pathophysiological process of metabolic SNHL.

[0015] (2) There is a lack of animal models of sensorineural hearing loss induced by phenylacetylglutamine (PAGln) in the existing technology.

[0016] Although PAGln, as an important gut microbiota-dependent metabolite, has been proven to be an independent risk factor for cardiovascular disease and closely related to cellular senescence, and there are existing reports on the use of PAGln to construct chronic wound models and in vitro senescence models of vascular smooth muscle cells, as of the date of this application, no literature or patent worldwide has disclosed an animal model of sensorineural hearing loss induced in mammals by exogenous administration of PAGln.

[0017] This technological gap directly leads to: (a) the inability to verify at the live animal level whether PAGln is an independent pathogenic factor of SNHL; (b) the lack of suitable tools to study in depth the specific molecular mechanisms by which PAGln damages inner ear hair cells; and (c) the inability to use this model to screen for preventive / therapeutic drugs that target PAGln metabolism or block its effects.

[0018] (II) Objective of this invention Basic objective: To provide a method for constructing an animal model of PAGln-induced sensorineural hearing loss.

[0019] The primary objective of this invention is to provide a simple, highly successful, and reproducible method for constructing a PAGln-induced sensorineural hearing loss animal model. By optimizing the dosage, administration method, and administration cycle, a stable and reliable hearing loss phenotype and pathological changes can be obtained. Using this model, the effects of PAGln on inner ear microcirculation, oxidative stress, mitochondrial function, and apoptosis can be explored in depth, elucidating its pathogenic molecular basis.

[0020] Drug development objective: To provide a tool for screening drugs for the prevention and treatment of PAGln-related hearing loss.

[0021] This invention provides a tool for screening drugs that can prevent and treat sensorineural hearing loss by modulating PAGln levels or blocking its effects. Using this model, the efficacy of different types of drugs can be evaluated, providing new drug candidates for clinical treatment.

[0022] Further objective: To provide a reference technical paradigm for studying the relationship between other circulating metabolites and hearing loss.

[0023] The ultimate goal of this invention is to provide a universal technical paradigm for constructing animal models of sensorineural hearing loss induced by other circulating metabolites. The method established in this invention can be extended to study the relationship between trimethylamine oxide, short-chain fatty acids, 5-hydroxytryptophan, and other gut microbiota metabolites, as well as other circulating metabolites, and hearing loss, thereby promoting the development of the entire field of metabolic hearing loss research.

[0024] Based on the above-mentioned technical problems to be solved, the present invention aims to provide the following five technical solutions: In a first aspect, the present invention provides a method for constructing an animal model of sensorineural hearing loss induced by phenylacetylglutamine, comprising the following steps: (1) Selection and preparation of experimental animals: Male C57BL / 6J mice aged 6-8 weeks and weighing 18-25g were selected as model animals. The mice were housed in an SPF-grade animal room with a temperature of 22±2℃, humidity of 50±10%, and alternating light and dark for 12 hours. They were allowed free access to water and food and were kept in an acclimatization environment for 7 days.

[0025] (2) Preparation of PAGln solution: Accurately weigh phenylacetylglutamine powder with a purity of ≥98%, dissolve it in 0.9% sterile physiological saline, prepare a PAGln solution with a concentration of 5 mg / mL, vortex thoroughly until completely dissolved, filter through a 0.22 μm sterile filter membrane for sterilization, and store at 4℃ for later use.

[0026] (3) Continuous intraperitoneal injection: After acclimatization, mice were randomly divided into a model group and a control group, with at least 6 mice in each group. Mice in the model group were injected intraperitoneally with the PAGln solution prepared in step (2) at a dose of 50 mg / kg body weight / day, once a day; mice in the control group were injected intraperitoneally with an equal volume of 0.9% saline. The administration was continued for at least 4 months.

[0027] (4) Validation of hearing function in the model: The hearing threshold of mice was detected by auditory brainstem response (ABR) before drug administration and at 28 days, 2 months, 3 months and 4 months after drug administration. After anesthetizing the mice with 4% chloral hydrate via intraperitoneal injection, the recording electrode was inserted subcutaneously on the top of the skull, the reference electrode was inserted subcutaneously behind the test ear, and the grounding electrode was inserted subcutaneously behind the contralateral ear. Short clicks and short pure tone stimuli at 4kHz, 8kHz, 16kHz and 24kHz were given, respectively. The stimulation intensity started at 90 dB SPL and decreased in 5 dB SPL increments. The lowest stimulation intensity that elicited a repeatable ABR waveform was recorded, which was the hearing threshold at that frequency.

[0028] (5) Histopathological verification of the model: After the ABR test, mouse cochlear tissue was taken, fixed with 4% paraformaldehyde and decalcified with 10% EDTA, the cochlear basilar membrane was separated, and hair cells were labeled with Myosin VIIa immunofluorescence staining. The number of hair cells was observed and counted under a laser confocal microscope. At the same time, the level of reactive oxygen species in the cochlear tissue was detected by nitrotetrazole blue (NBT) in situ staining.

[0029] Through the above steps, an animal model of sensorineural hearing loss was obtained.

[0030] Secondly, the present invention provides an animal model of sensorineural hearing loss constructed by the above method.

[0031] The model has the following characteristics: the ABR threshold of mice in the model group was significantly higher than that in the control group at click, 4kHz, 8kHz, 16kHz and 24kHz frequencies (≥20 dB SPL), there was obvious loss of hair cells in the outer hair cell membrane of the cochlear basilar membrane, and the level of oxidative stress in the cochlear tissue was significantly increased.

[0032] Thirdly, the present invention provides the application of the animal model in studying the pathogenesis of sensorineural hearing loss.

[0033] This model can be used to explore the effects of PAGln on inner ear microcirculation, oxidative stress, mitochondrial function, and apoptosis, and to elucidate the molecular basis of its pathogenesis.

[0034] Fourthly, the present invention provides the application of the animal model in screening drugs for the prevention or treatment of sensorineural hearing loss.

[0035] The drugs mentioned include, but are not limited to: drugs that reduce PAGln levels in the body (such as certain antibiotics, probiotics, enzyme inhibitors), drugs that promote PAGln excretion, drugs that block the effects of PAGln (such as β2-adrenergic receptor antagonists), and drugs that improve PAGln-induced oxidative stress and mitochondrial dysfunction.

[0036] Fifthly, the present invention provides the use of phenylacetylglutamine in the preparation of reagents or kits for constructing animal models of sensorineural hearing loss.

[0037] Compared with the prior art, the technical solution provided by the present invention has the following significant and unexpected beneficial effects: 1. The PAGln-induced SNHL animal model was established and validated for the first time, filling a technological gap.

[0038] This invention is the first in the world to demonstrate that exogenous administration of a single circulating metabolite, PAGln, can directly induce sensorineural hearing loss in mammals. The model constructed in this invention is the world's first in vivo animal model of PAGln-induced hearing loss, successfully filling the technological gap in existing SNHL models that cannot reflect the pathophysiological process of "metabolic abnormality-inner ear damage," and providing the first operable in vivo experimental platform for research on the "gut-inner ear axis" and metabolic hearing loss.

[0039] 2. The model building method is extremely simple to operate, has a high success rate, good repeatability, and low cost.

[0040] Compared to existing ischemic models requiring complex surgery (with mortality rates as high as 30%-50%) or noise / radiation models requiring expensive equipment, this invention uses only a conventional intraperitoneal injection method, requiring no special surgical skills or large-scale specialized equipment. It can be successfully carried out in any laboratory with basic animal experimental conditions. Through the optimized parameter combination (specific dosage and cycle) of this invention, the model construction success rate is over 90%, the animal mortality rate is less than 5%, and the ABR threshold variation between different batches of experiments is small, ensuring high reproducibility and low cost in scientific research.

[0041] 3. The model phenotype is stable, and the pathological features are highly consistent with clinical findings.

[0042] The model of this invention exhibits a significant and stable increase in the auditory brainstem response (ABR) threshold in the high-frequency region (8kHz-24kHz). Histological verification shows that PAGln primarily leads to the loss of outer hair cells in the cochlear basilar membrane, while the inner hair cells are relatively well preserved. This pathological feature is highly consistent with the pathological characteristics of sensorineural hearing loss (with preferential damage to outer hair cells) observed in patients with metabolic disorders such as hypertension, hyperlipidemia, and diabetes, demonstrating the excellent clinical relevance of this model.

[0043] 4. It provides an irreplaceable tool for screening therapeutic drugs targeting the PAGln pathway.

[0044] Based on the model constructed in this invention, researchers can intuitively evaluate the efficacy of candidate drugs in vivo. Specifically, it can be used to screen: drugs that reduce PAGln levels in the body (such as specific antibiotics and probiotic preparations), antagonists that block the binding of PAGln to receptors (such as β2-adrenergic receptors), and protective agents that alleviate PAGln-induced oxidative stress damage. This opens up a completely new drug screening pathway for developing novel treatment strategies for metabolic hearing loss.

[0045] 5. It provides a technical paradigm that can be referenced for other circulating metabolite-related hearing loss models.

[0046] The technical route of "exogenous intraperitoneal injection of a single circulating metabolite - auditory function and morphological assessment" established in this invention is not only applicable to PAGln, but can also be directly applied to the study of the relationship between other gut microbiota metabolites such as trimethylamine oxide (TMAO) and short-chain fatty acids and hearing loss. It has extremely high scientific research demonstration value and promotion significance. Attached Figure Description

[0047] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 This is an overall flowchart of the method for constructing an animal model of sensorineural hearing loss induced by PAGln according to the present invention; Figure 2 This is a graph showing the changes in ABR thresholds at various frequencies in mice after 28 days of continuous intraperitoneal injection of different doses of PAGln according to the present invention. Figure 3 This is a graph showing the changes in ABR threshold in mice after continuous intraperitoneal injection of 50 mg / kg PAGln for different time periods according to the present invention; Figure 4 These are immunofluorescence staining images (Myosin VIIa staining) of hair cells in the cochlear basement membrane of mice in the control group and the 50 mg / kg PAGln group of this invention, where (A) is the control group and (B) is the experimental group; Figure 5 This is a comparison of oxidative stress indices in the cochlear tissue of mice in the control group and the 50 mg / kg PAGln group of the present invention, wherein (A) is the control group and (B) is the experimental group. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Example 1 See Figures 1 to 5 As shown, this invention provides a method for constructing an animal model of sensorineural hearing loss induced by phenylacetylglutamine (PAGln). The animal model constructed by this invention is simple to operate, has a high success rate (>90%), and good reproducibility. It can accurately simulate the pathophysiological process of sensorineural hearing loss in humans caused by metabolic abnormalities and gut microbiota metabolic disorders, providing a key tool for the study of the pathogenesis of this type of disease and the screening of therapeutic drugs.

[0051] To more clearly illustrate the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings: Detailed steps of the animal model construction method based on Figure 1: Figure 1 illustrates the complete process of constructing the animal model of the present invention, including five core steps: preparation of experimental animals, preparation of PAGln solution, continuous intraperitoneal injection administration, model functional verification, and model histological verification. The specific implementation methods of each step are as follows: (I) Step S1: Selection and preparation of experimental animals: Executor: Experimental operators; Target of execution: non-human mammals; Triggering condition: 7 days before the start of the experiment; Perform the following actions: 1. Select male C57BL / 6J mice aged 6-8 weeks and weighing 18-25g. This strain of mice has a clear genetic background and stable hearing, and is the most commonly used experimental animal in hearing research. 2. Mice were housed in an SPF-grade animal room with a temperature of 22±2℃, humidity of 50±10%, and 12 hours of light / 12 hours of darkness; 3. Provide free access to water and food, allow for 7 days of acclimatization, and exclude individuals with hearing abnormalities; Operating environment parameters: SPF-grade animal room, temperature 22±2℃, humidity 50±10%; Purpose of this procedure: To ensure the health and genetic uniformity of laboratory animals and reduce experimental errors; End condition: Mice showed no abnormal behavior after 7 days of acclimatization feeding; Results: Healthy mice that meet the experimental requirements were obtained.

[0052] (ii) Step S2: Preparation of PAGln solution: Executor: Experimental operators; Subject matter: Phenylacetylglutamine powder, 0.9% physiological saline; Triggering condition: within 24 hours prior to each administration; Perform the following actions: 1. Accurately weigh phenylacetylglutamine powder with a purity ≥98%. 2. Dissolve in 0.9% sterile physiological saline to prepare a 5 mg / mL PAGln solution. 3. Vortex thoroughly until completely dissolved, then filter through a 0.22μm sterile membrane for sterilization. 4. Aliquot into 5mL sterile EP tubes and store at 4°C. Shelf life is 7 days. Operating environment parameters: Aseptic operation inside a biosafety cabinet; Function of this step: To prepare a sterile, accurately concentrated PAGln drug delivery solution; End condition: Obtain a filtered and sterilized PAGln solution; Results: PAGln solution that can be directly used for intraperitoneal injection.

[0053] (iii) Step S3: Continuous intraperitoneal injection for drug administration; Executor: Experimental operators; Execution object: Healthy mice obtained in step S1; Triggering condition: Day 1 after the end of acclimatization period; Perform the following actions: 1. Mice were randomly divided into a model group and a control group, with at least 6 mice in each group; 2. Mice in the model group were intraperitoneally injected with PAGln solution at a dose of 50 mg / kg body weight / day, once daily in the afternoon; 3. Control group mice were intraperitoneally injected with an equal volume of 0.9% physiological saline; 4. Administer the drug continuously for 7-42 days, recording the mice's weight, diet, and activity levels daily; Operating environment parameters: Animal room, room temperature; Action of the procedure: By administering PAGln exogenously, it simulates the pathophysiological state of elevated concentrations of circulating metabolites in the body, thereby inducing hearing loss; Termination condition: Completion of the predetermined dosing cycle; Results: Mice showed dose-dependent and time-dependent sensorineural hearing loss.

[0054] (iv) Step S4: Verification of the model's hearing function: Executor: Experimental operators; Target: Mice after drug administration; Triggering conditions: 1 day before administration and on days 7, 14, 21, 28, 35, and 42 after administration; Perform the following actions: 1. Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate at a dose of 0.01 mL / g body weight; 2. After anesthetizing, place the mouse in a soundproof and shielded room, insert the recording electrode subcutaneously at the top of the skull, insert the reference electrode subcutaneously behind the test ear, and insert the grounding electrode subcutaneously behind the contralateral ear. 3. Using an auditory brainstem response (ABR) tester, short click sounds and short pure tone stimuli at 4kHz, 8kHz, 16kHz, 24kHz, and 32kHz were administered respectively; 4. Starting with a stimulus intensity of 90 dB SPL, decrease the intensity in increments of 5 dB SPL. Record the lowest stimulus intensity that elicits a repeatable ABR waveform, which is the hearing threshold for that frequency.

[0055] (V) Step S5: Verification of the model's hearing function: Executor: Laboratory operators Target: Mice after drug administration Triggering conditions: 1 day before administration and at 7, 14, 21, 28, 2 months, 3 months, and 4 months after administration. Perform the following actions: 1. Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate at a dose of 0.01 mL / g body weight; 2. After anesthetizing, place the mouse in a soundproof and shielded room, insert the recording electrode subcutaneously at the top of the skull, insert the reference electrode subcutaneously behind the test ear, and insert the grounding electrode subcutaneously behind the contralateral ear. 3. Using an auditory brainstem response (ABR) tester, short click sounds and short pure tone stimuli at 4kHz, 8kHz, 16kHz, and 24kHz were administered respectively; 4. Starting with a stimulus intensity of 90 dB SPL, decrease the intensity in increments of 5 dB SPL. Record the lowest stimulus intensity that elicits a repeatable ABR waveform, which is the hearing threshold for that frequency.

[0056] Operating environment parameters: soundproof and shielded room, temperature 25±2℃; Purpose of this step: To objectively assess the hearing function of mice and verify whether the model has been successfully constructed; Termination condition: Complete hearing threshold testing for all frequencies; Results: ABR threshold data for mice at various frequencies were obtained. The ABR threshold of mice in the model group was significantly higher than that of the control group (≥20 dB SPL).

[0057] (vi) Step S6: Histopathological and molecular biological validation of the model: Executor: Experimental operators; Subject of the test: Mice after hearing testing; Triggering condition: After the last hearing test; Perform the following actions: 1. Mice were euthanized by cervical dislocation, and both cochleas were quickly removed; 2. Fix with 4% paraformaldehyde at 4℃ for 24 hours, then decalcify with 10% EDTA solution at 4℃ for 72 hours, changing the decalcification solution daily; 3. The cochlear basilar membrane was separated under a dissecting microscope and divided into three parts: the apical turn, the mid-turn, and the basal turn; 4. Hair cells were labeled with Myosin VIIa immunofluorescence staining, and the number of hair cells was observed and counted under a laser confocal microscope; 5. A portion of cochlear tissue was taken, and the level of reactive oxygen species generated in the cochlear tissue was detected by in situ staining with Nitro Blue Tetrazolium (NBT).

[0058] Operating environment parameters: laboratory, room temperature; Steps and their purpose: To verify the pathological changes in the model at the histological and molecular biological levels and to preliminarily clarify its pathophysiological mechanisms; Termination criteria: Completion of all histological and molecular biological tests; Results: The number of outer hair cells in the cochlea of ​​mice in the model group was significantly reduced, and the level of oxidative stress was significantly increased.

[0059] Example 2 Based on Example 1, this example provides a variety of technical solutions.

[0060] (a) The effect of different doses of PAGln on the model effect (refer to Figure 2) Appendix Figure 2 The changes in ABR threshold in mice at click, 4kHz, 8kHz, 16kHz and 24kHz frequencies were shown after 28 days of continuous intraperitoneal injection of different doses of PAGln (50mg / kg, 100mg / kg and 150mg / kg).

[0061] As can be seen from Figure 2: 1. Dose dependence: PAGln-induced hearing loss did not show a significant dose dependence, and the ABR thresholds of mice at various frequencies did not increase with increasing dosage; 2. Frequency dependence: PAGln has a more significant effect on high-frequency hearing, with the ABR threshold increase at 8kHz, 16kHz, and 24kHz being significantly greater than that at low and mid frequencies; 3. Optimal dosage: 50 mg / kg body weight / day is the optimal dosage of this invention. After continuous administration for 4 months, the ABR threshold of mice at various frequencies increased by 35-45 dB SPL compared with the control group, and the animal mortality rate was <5%, with a model success rate of >90%.

[0062] Experimental results: As attached Figure 2As shown, all three doses of PAGln induced an increase in ABR thresholds at all frequencies in mice, with more pronounced high-frequency hearing loss. The 50 mg / kg dose group consistently induced significant hearing loss, while the 100 mg / kg and 150 mg / kg dose groups showed no statistically significant difference in ABR threshold increase compared to the 50 mg / kg group (P>0.05). Furthermore, mice in the 100 mg / kg and 150 mg / kg dose groups experienced some degree of weight loss and reduced activity, while mice in the 50 mg / kg dose group maintained good general condition and showed no significant systemic toxicity. Therefore, 50 mg / kg body weight / day is the optimal dosage according to this invention.

[0063] (ii) The effect of different drug administration times on the model effect (refer to Figure 3) Figure 3 shows the changes in ABR thresholds at various frequencies in mice after continuous intraperitoneal injection of 50 mg / kg PAGln for different durations (28 days, 2 months, 3 months, and 4 months).

[0064] As can be seen from Figure 3: 1. Time dependence: PAGln-induced hearing loss is significantly time-dependent; as the duration of administration increases, the ABR threshold in mice gradually rises. 2. Optimal dosing cycle: Continuous dosing for 4 months is the optimal dosing cycle of this invention. At this time, the model is stable, the pathological changes are obvious, and the experimental period is moderate.

[0065] Experimental results: As attached Figure 3 As shown, with prolonged administration, the ABR thresholds at various frequencies in the model group mice gradually increased, exhibiting a clear time-dependent effect. Significant hearing loss was observed in the mice after 28 days of administration; the hearing loss worsened further at 2 and 3 months; and after 4 months, the increase in ABR thresholds stabilized, reaching a plateau. Considering both model stability and experimental duration, a continuous administration period of 4 months is the optimal administration period for this invention.

[0066] (III) Pathological changes in the model tissue as shown in Figure 4 Figure 4 shows the immunofluorescence staining of hair cells at the base of the cochlear basilar membrane in mice of the control group and the 50 mg / kg PAGln group (Myosin VIIa staining, green fluorescent labeling of hair cells).

[0067] As can be seen from Figure 4: 1. In the control group, the hair cells of the cochlear basilar membrane were neatly arranged, in normal number, and without obvious loss; 2. Significant loss of external hair cells in the cochlear basilar membrane of mice in the PAGln group; 3. The number of inner hair cells in the PAGln group mice was not significantly reduced, which is consistent with the pathological characteristics of clinical metabolic hearing loss, which mainly damages outer hair cells.

[0068] A mouse model of sensorineural hearing loss induced by PAGln was constructed following the steps in Example 1. During the administration period, some model mice were given the candidate drug to be screened (e.g., the β2-adrenergic receptor antagonist ICI 118,551, at a dose of 1 mg / kg / day, intraperitoneally). A model control group (treated with PAGln only) and a blank control group (treated with saline only) were also established. The ABR threshold of mice in each group was measured before administration and at 2 and 4 months after administration. After administration, cochlea was harvested for hair cell counting and oxidative stress level assessment.

[0069] The efficacy of candidate drugs in preventing or treating PAGln-induced sensorineural hearing loss was evaluated by comparing changes in ABR threshold, hair cell survival rate, and oxidative stress levels between the drug-treated group and the model control group. If the candidate drug significantly inhibits the increase in ABR threshold, reduces the loss of outer hair cells, and lowers the level of oxidative stress in cochlear tissue, it indicates that the drug has the potential to prevent or treat PAGln-related sensorineural hearing loss.

[0070] (iv) Model molecular biological changes (see Figure 5) Figure 5 shows a comparison of oxidative stress indices in the cochlear tissue of mice in the control group and the 150 mg / kg PAGln group.

[0071] As can be seen from Figure 5: 1. Compared with the control group, the NBT content in the cochlear tissue of mice in the PAGln group was significantly increased, and the blue-purple color was more obvious and profound; 2. These results indicate that oxidative stress is one of the important pathological mechanisms by which PAGln induces hearing loss.

[0072] A mouse model of sensorineural hearing loss induced by PAGln was constructed following the steps in Example 1. During the administration period, some model mice were given the candidate drug to be screened (e.g., the β2-adrenergic receptor antagonist ICI 118,551, at a dose of 1 mg / kg / day, intraperitoneally). A model control group (treated with PAGln only) and a blank control group (treated with saline only) were also established. The ABR threshold of mice in each group was measured before administration and at 2 and 4 months after administration. After administration, cochlea was harvested for hair cell counting and oxidative stress level assessment.

[0073] The efficacy of candidate drugs in preventing or treating PAGln-induced sensorineural hearing loss was evaluated by comparing changes in ABR threshold, hair cell survival rate, and oxidative stress levels between the drug-treated group and the model control group. If the candidate drug significantly inhibits the increase in ABR threshold, reduces the loss of outer hair cells, and lowers the level of oxidative stress in cochlear tissue, it indicates that the drug has the potential to prevent or treat PAGln-related sensorineural hearing loss.

[0074] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for constructing an animal model of sensorineural hearing loss induced by phenylacetylglutamine and its application, characterized in that: Includes the following steps: (1) Preparation of experimental animals: Male C57BL / 6J mice aged 6-8 weeks and weighing 18-25g were selected and placed in an SPF-grade animal room for 7 days of acclimatization, and individuals with hearing abnormalities were removed. (2) Preparation of phenylacetylglutamine solution: Phenylacetylglutamine powder with a purity of ≥98% was dissolved in 0.9% sterile physiological saline to prepare a solution of 3.33~5mg / mL. After sterilization by filtration through a 0.22μm sterile filter membrane, it was stored at 4℃. (3) Drug treatment: Mice were randomly divided into a model group and a control group. The model group was injected intraperitoneally with 50-150 mg / kg body weight of phenylacetylglutamine solution daily, while the control group was injected intraperitoneally with an equal volume of 0.9% sterile saline daily for 7-120 days. (4) Model validation: The hearing threshold of mice was detected by auditory brainstem response, and the cochlear tissue pathology and molecular biological tests were combined to confirm the acquisition of an animal model of sensorineural hearing loss.

2. The construction method according to claim 1, characterized in that, In step (3), the dosage of phenylacetylglutamine is 50 mg / kg body weight / day.

3. The construction method according to claim 1, characterized in that, The continuous dosing period in step (3) is 4 months.

4. The construction method according to claim 1, characterized in that, The stimulation frequencies for auditory brainstem response testing in step (4) include click short sounds, 4kHz, 8kHz, 16kHz, and 24kHz short pure tones.

5. The construction method according to claim 1, characterized in that, In step (4), the cochlear tissue pathology was performed by using Myosin VIIa immunofluorescence staining to label cochlear hair cells.

6. The construction method according to claim 1, characterized in that, In step (4), the molecular biological detection is to detect the oxidative stress index ROS in cochlear tissue.

7. An animal model of sensorineural hearing loss induced by phenylacetylglutamine, characterized in that, The animal model is prepared by the construction method according to any one of claims 1 to 6.

8. The application of the animal model of claim 7 in the study of the pathogenesis of sensorineural hearing loss.

9. The use of the animal model of claim 7 in screening drugs for the prevention or treatment of sensorineural hearing loss.

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