Application of immediate early genes Fos and Egr2 in tinnitus disease model evaluation and intervention products

Abstract

The invention belongs to the technical field of biomedical products, and relates to application of immediate early genes Fos and Egr2 in tinnitus disease model evaluation and intervention products, the immediate early genes Fos and / or Egr2 are used as biomarkers for tinnitus maintenance, and tinnitus diseases are evaluated through detection of down-regulation of the immediate early genes Fos and Egr2 in an anterior claspite cortex ACC brain region when tinnitus occurs. Tinnitus is relieved by regulating and controlling early genes Fos and Egr2 in an AC brain region of the auditory cortex; according to the invention, diagnosis markers and treatment application are covered at the same time, a complete tinnitus disease assessment intervention system is formed, the bottleneck that traditional tinnitus research lacks objective biomarkers and targeted treatment means is overcome, and a solution is provided for clinical assessment and intervention of tinnitus through precise intervention at a gene level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical products, and specifically relates to the application of immediate premature genes Fos and Egr2 in tinnitus disease model assessment and intervention products. Background Art

[0002] Tinnitus is a meaningless and harmful sensation perceived in the absence of external sound or electrical stimulation. It often leads to psychological, psychiatric, and sleep disorders, placing a significant mental and economic burden on families and society. Tinnitus occurs in approximately one in six adults worldwide, affecting nearly one billion adults worldwide. For over 100 million people, tinnitus is a major problem that severely impacts their lives and work. Therefore, tinnitus has become a major global health issue that urgently needs to be addressed.

[0003] However, there are currently no effective diagnostic or treatment methods for tinnitus. The fundamental reasons are: the central mechanisms of tinnitus involve auditory and non-auditory networks, the interaction of which is not fully understood; the existence of a specific causative gene is not fully understood; and reliable, non-invasive, objective detection and assessment methods are lacking. Tinnitus research must first address issues that plague clinical practice. Tinnitus, a persistent clinical condition, troubles both patients and physicians alike. First, tinnitus has no physical signs, and clinicians lack objective means of evaluating it. They can only rely on subjective patient descriptions and psychophysical comparisons. Although functional brain imaging techniques such as fMRI and EEG can be used to investigate tinnitus, they are still far from clinical application. Second, tinnitus research provides a window into understanding the auditory system and even the mechanisms of brain function. Although tinnitus is a condition caused by plasticity disorders, research on it lags far behind similar conditions such as pain and depression, and tinnitus research urgently needs to be strengthened.

[0004] In terms of tinnitus intervention, first of all, in order to address tinnitus caused by the lack of sound signal input during the process of tinnitus signal recognition, appropriate sound therapy is given. According to the matching frequency and intensity of the tinnitus, music sound stimulation of specific frequency and intensity is selected to compensate for the lost auditory input and reduce the increase in spontaneous discharge caused by the reduced input. Alternatively, similar to the principle of tinnitus masking, rich environmental sounds are given to mask the tinnitus. However, the complexity and variability of tinnitus frequencies lead to poor treatment effects, and the possible noise damage effects are unclear. For patients with auditory system damage and whose tinnitus frequency happens to be at the hearing damage frequency, hearing aids can be given to compensate for the lost hearing information. However, hearing aids are more effective in compensating for low-frequency auditory information input. For patients with more common high-frequency tinnitus or whose tinnitus frequency is not consistent with the hearing damage frequency, hearing aids cannot effectively improve the existing tinnitus. And if the location of the auditory damage is postsynaptic, simply increasing the sound signal input will not improve the tinnitus. Secondly, transcranial magnetic stimulation (TMS) has been introduced into clinical practice to address the central cortical changes associated with tinnitus. The electromagnetic pulses delivered by TMS can, on the one hand, inhibit abnormal electrical activity in auditory-related neurons in tinnitus patients, reducing overexcitability in the auditory cortex; on the other hand, it can attenuate electrical activity in neurons in the cingulate gyrus of the limbic system. However, TMS may exacerbate tinnitus in evidence-based medicine, lacks long-term efficacy evidence, has unclear potential complications, and has yet to establish a definitive treatment model. Therefore, it is not recommended as a routine treatment. Furthermore, based on the theory of central plasticity in tinnitus, some researchers have attempted to alleviate tinnitus by administering high-intensity white noise or pure tone stimulation to tinnitus patients, attempting to modulate the plasticity of the auditory center through acoustic stimulation. However, whether this acoustic stimulation alters central plasticity positively or negatively, and whether it induces noise-induced damage, remains unknown. Furthermore, some researchers have explored vagus nerve electrical stimulation in animal models and volunteers, but this is still in the clinical trial stage. In addition, there are neurofeedback therapy, cognitive therapy, etc., but none of them have been confirmed to have a very definite therapeutic effect on improving the loudness of tinnitus.

[0005] Therefore, existing medical technologies still lack a deep understanding of the neural mechanisms of tinnitus, which further limits the follow-up of tinnitus intervention measures. Currently, there is still a lack of specific biomarkers for detecting tinnitus, and there is no drug or recognized intervention method for tinnitus. Summary of the Invention

[0006] The present invention aims to use Fos and Egr2 immediate premature genes as a combined clinical history, audiological assessment, and tinnitus assessment to objectively detect tinnitus, and to integrate the regulation of the upstream modification target H3K27me3 of Fos and Egr2 immediate premature genes into the preparation of a comprehensive tinnitus intervention method.

[0007] The present invention provides the following technical solutions:

[0008] Application of immediate-early-acting genes Fos and Egr2 in the evaluation of tinnitus disease models.

[0009] Preferably, the immediate-early-break genes Fos and / or Egr2 are used as biomarkers for the maintenance of tinnitus. By detecting the downregulation of the immediate-early-break genes Fos and Egr2 in the anterior cingulate cortex (ACC) brain region when tinnitus occurs, it can be used as a clinical assessment of tinnitus disease and a reference for disease prevention guidance and treatment decision-making.

[0010] Preferably, the applications include: audiological assessment, tinnitus assessment, and objective tinnitus detection.

[0011] Preferably, the application includes: a blood detection kit, which rapidly detects the biomarker by RT-qPCR.

[0012] The present invention also discloses the application of immediate early genes Fos and Egr2 in tinnitus disease intervention products.

[0013] Preferably, the application alleviates tinnitus by regulating the immediate-early genes Fos and / or Egr2 in the auditory cortex (AC) brain region.

[0014] Preferably, the application regulates tinnitus by regulating IEGs neutral early genes Fos and / or Egr2 through H3K27me3; downregulation of IEGs neutral early genes Fos and Egr2 is related to H3K27me3 modification, and H3K27me3 modification negatively regulates IEGs. H3K27me3 regulates IEGs epigenetic modification in tinnitus models, which can be used as a means of non-invasively regulating tinnitus at the epigenetic level compared to local gene regulation in brain regions.

[0015] Preferably, the tinnitus intervention products include: drugs, medical devices, biological products, and rehabilitation products. Drugs include chemical drugs, biopharmaceuticals, and traditional Chinese medicines that intervene in the disease process through pharmacological effects. Medical devices include therapeutic equipment, diagnostic and treatment instruments, and the like that assist with treatment through physical or technical means. Biological products include vaccines and therapeutic stem cells that utilize biotechnology to intervene in the disease. Rehabilitation products include sound therapy equipment and training devices for functional recovery or symptom management.

[0016] The beneficial effects of the present invention are:

[0017] 1. This invention covers both diagnostic markers (Fos / Egr2 expression level detection) and therapeutic applications (AC / ACC region gene regulation), forming a comprehensive tinnitus disease assessment and intervention system. This overcomes the bottleneck of traditional tinnitus research, which lacks objective biomarkers and targeted treatments. Through precise intervention at the genetic level, it provides a solution for the clinical assessment and intervention of tinnitus.

[0018] 2. The present invention discloses that biomarkers can be rapidly detected by RT-qPCR (sensitivity reaches p < 0.01), and the salicylic acid model verifies the universality of drug-induced tinnitus, laying the foundation for the development of blood detection kits and drug screening platforms.

[0019] 3. The present invention discloses that the synchronous downregulation of immediate-early genes such as Fos / Egr2 in the auditory cortex (AC) and anterior cingulate cortex (ACC) is a core molecular event in the occurrence of tinnitus, providing a specific biomarker combination (Fos+Egr2) for tinnitus diagnosis.

[0020] 4. The present invention discloses that the Fos gene has region-specific functions in the central nervous system: overexpression in the ACC region can induce tinnitus-like behavior (establishing a new central tinnitus model), while overexpression in the AC region can alleviate symptoms, achieving bidirectional regulation of the same target.

[0021] 5. The present invention discloses a Fos / Egr2 targeted expression technology based on AAV vectors, which significantly improves the gap startle ratio in the noise-induced tinnitus model, providing a new intervention approach for clinical gene therapy.

[0022] 6. The present invention verified the direct association between Fos / Egr2 gene downregulation and tinnitus through two independent modeling methods: noise exposure (116dB SPL narrow-band noise monaural stimulation) and intraperitoneal injection of sodium salicylate (250mg / kg). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 FIG is a diagram showing the establishment of a noise-induced tinnitus mouse model (n=30) for the application of the immediate-early genes Fos and Egr2 in tinnitus disease model assessment and intervention products of the present invention;

[0024] Figure 2 FIG is a diagram of the establishment of a salicylic acid-induced tinnitus mouse model of the present invention (n=30);

[0025] Figure 3 This is a diagram showing the gene expression pattern in the noise-induced tinnitus mouse AC of the present invention;

[0026] Figure 4 This is a diagram showing the gene expression pattern in the ACC of noise-induced tinnitus mice of the present invention;

[0027] Figure 5 This is a graph showing changes in AC and ACC neutral early genes and startle reflex in mice during different time periods of sodium salicylate modeling;

[0028] Figure 6 This is a diagram of tinnitus-like behavior induced by overexpression of FOS in the ACC region of wild-type mice of the present invention;

[0029] Figure 7 This is a diagram showing that overexpression of FOS / EGR2 in tinnitus mice AC can alleviate symptoms;

[0030] Figure 8 This is a graph showing changes in H3K27me3 protein in the AC / ACC of mice on day 7 of noise-induced tinnitus;

[0031] Figure 9 This figure shows increased expression of IEGs (Fos and Egr families) and decreased H3K27me3 protein in the AC / ACC of mice on the first day of noise-induced tinnitus. DETAILED DESCRIPTION

[0032] The following will provide a clear and complete description of the relevant technologies in the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example

[0034] Findings related to the immediate premature genes Fos and Egr2 and tinnitus intervention, such as Figures 1 to 9 As shown, the details are as follows:

[0035] 1. Behavioral verification of the startle reflex in tinnitus models and noise and sodium salicylate modeling:

[0036] Acoustic startle reflex (ASR): Gap pre-pulse inhibition of the acoustic startle (GPIAS) is a reflex that is suppressed when a silent gap is inserted into the background noise preceding a startle stimulus. Tinnitus mice are unable to perceive gaps due to persistent tinnitus. The paradigm utilizes white noise (WN) and narrowband sounds with a bandwidth of 1 kHz and center frequencies of 8, 12, 16, and 20 kHz as background sounds at a sound pressure level of 70 dB SPL. A 50 ms gap is introduced 130 ms before the startle stimulus. Gap detection is assessed using the gap startle ratio (GPR), which is the ratio of the peak-to-peak amplitude of the startle waveform in trials with a gap to the peak-to-peak amplitude of the startle waveform in trials without a gap. Noise-exposed mice are considered tinnitus if their GPIAS startle ratio increases by more than 0.3 at at least one tested frequency. Prepulse inhibition (PPI) is performed in quiet. A 50-ms non-startle sound (prestimulus) at 70 dB SPL, the same background sound used in gap detection testing, is presented 130 ms before the startle stimulus. Prestimulus inhibition is assessed using the PPI startle ratio, defined as the ratio of the peak-to-peak startle waveform in trials with prestimulus to the peak-to-peak startle waveform in trials with startle alone. A normal prestimulus ratio is less than 0.9; frequencies with prestimulus ratios greater than 0.9 are excluded.

[0037] Noise modeling: Normal hearing is essential for detecting sound gaps, so in order to prevent complete hearing loss, monaural noise exposure was implemented. Before the experiment began, the animals were anesthetized with 1% to 1.5% isoflurane and soundproofed with foam earplugs in the non-modeled ear. They were then placed in an acoustically shielded room equipped with a sound transmission system. Narrowband noise with a center frequency of 16 kHz and a carrier frequency of 1 kHz was given 10 cm away from the exposed ear of the mouse and played at a sound pressure level of 116 dB SPL for 45 minutes. Figure 1 As shown in the figure, the ASR test of tinnitus mice after noise modeling showed that the startle ratio to GPIAS at frequencies of 8, 12, and 16 kHz increased significantly ( Figure 1 (D) shows that the tinnitus frequency is mainly at these frequencies. The hearing of tinnitus mice and non-tinnitus mice is reduced at 8, 12, and 24 kHz, and the hearing of tinnitus mice is also reduced at 32 kHz.

[0038] Sodium salicylate modeling: GPIAS and PPI were measured before and 1 hour after intraperitoneal injection of sodium salicylate (250 mg / kg) in mice. The average values ​​of GPIAS and PPI before and after injection were calculated once a day for 3 consecutive days. The method of evaluating tinnitus in mice by ASR was the same as that of noise-exposed tinnitus mice. Figure 2 As shown, the ASR test showed a significant increase in the startle ratio to GPIAS at 16 kHz ( Figure 2 C), indicating that the tinnitus frequency is mainly at these frequencies.

[0039] 2. Fos and Egr2 genes were downregulated in the auditory cortex (AC) and anterior cingulate cortex (ACC) of noise- and salicylic acid-induced tinnitus mice:

[0040] To study the changes in gene expression in the AC of noise-induced tinnitus mice, AC brain tissues of noise-induced tinnitus mice were extracted and analyzed using RNA-seq ( Figure 3 A). Downregulated genes include IEGs (Fos, Egr2) that regulate synaptic plasticity. We verified the expression of these genes in AC by RT-qPCR and found that it was consistent with the RNA-seq results ( Figure 3 C). To reveal the biological processes associated with tinnitus, we performed GO enrichment analysis using DAVID. Similarly, we extracted brain tissue from the ACC of noise-induced tinnitus mice and analyzed gene expression in the ACC using RNA-seq ( Figure 4 A). Similar to the gene expression in AC, we found that the top of the downregulated genes were IEGs (Fos, Egr1, Egr3, Egr4, Nr4a1, Npas4, Homer1) that regulate synaptic plasticity and late response genes (Bdnf) ( Figure 4 B). Upregulated genes include extracellular matrix-related genes (Col1a2, Col5a2, Col9a3, Col13a1, Ltbp2). Also, at 2 hours and 12 hours after salicylic acid modeling in the tinnitus stage, IEGs genes such as Fos and Egr2 were downregulated in the AC and ACC brain regions. However, 24 hours after salicylic acid modeling (non-tinnitus stage), Fos and Egr2 genes were upregulated ( Figure 5 CF).

[0041] Therefore, we verified in two tinnitus models that Fos and Egr2 genes were downregulated when tinnitus occurred. These two immediate-early genes can serve as biomarkers for the maintenance of tinnitus.

[0042] 3. Overexpression of Fos in the ACC of wild-type mice can induce tinnitus, while overexpression of Fos or Egr2 genes in the AC brain region of tinnitus mice can alleviate tinnitus:

[0043] Mouse brain regions overexpressing Fos and Egr2 were treated with viral microinjection. Animals were anesthetized with isoflurane and placed in a stereotaxic adapter. After skin removal, the skull was exposed. A craniotomy was performed above the target brain region using a dental drill. The coordinates of the target regions were determined based on previous literature: the AC region (anteroposterior axis: -4.2 mm, left-right axis: ±2.65 mm, superior-inferior axis: -2.65 mm) and the ACC region (anteroposterior axis: +0.55 mm, left-right axis: ±0.3 mm, superior-inferior axis: -0.9 mm). Using a RWD viral microinjector, 200 nl of AVV viral vectors encoding Fos and Egr2, as well as a control virus, were injected into the auditory cortex contralateral to the model ear or the bilateral anterior cingulate cortex at a rate of 50 nl / min. The microinjector remained at the injection site for an additional 10 minutes before being slowly withdrawn to prevent spillage.

[0044] Wild-type mice overexpressing Fos in the ACC region developed tinnitus-like behavior one week after the overexpression, but the tinnitus-like behavior (startle reflex) disappeared after three weeks of overexpression (see Figure 6 The results of a study in which noise-induced tinnitus mice were microinjected with viruses in the AC and ACC brain regions for three consecutive weeks indicated that the tinnitus behavior was improved after local overexpression of Fos virus in the AC brain region of tinnitus mice (see Figure 7 ).

[0045] Overexpressing Fos in the ACC of wild-type mice induces tinnitus, while overexpressing Fos and Egr2 in the AC of tinnitus mice alleviates tinnitus. Thus, tinnitus can be alleviated by regulating immediate-early genes in the AC, while a central tinnitus model can be induced by regulating Fos in the ACC.

[0046] 4. H3K27me3 dynamically regulates Fos and Egr2 immediate early genes during the onset (1 day after noise modeling) and maintenance (7 days after noise modeling) of tinnitus:

[0047] Downregulation of Fos and Egr2 in IEGs is associated with H3K27me3 modification. In this embodiment, 7 days after noise-induced tinnitus in mice, the expression of IEGs such as Fos and Egr2 decreased, and H3K27me3 protein in the AC and ACC brain regions increased significantly (as shown below). Figure 8 As shown in Figure 2 ), one day after noise modeling, the expression of IEGs such as Fos and Egr2 increased in the AC and ACC brain regions of tinnitus mice, while the H3K27me3 protein decreased (as shown in Figure 2 ). Figure 9 This study validated the negative regulation of IEGs by H3K27me3 modification. H3K27me3 regulation of IEG epigenetic modifications in tinnitus models could serve as a noninvasive epigenetic approach to regulate tinnitus, compared to localized gene regulation in the brain.

[0048] In summary, the present invention covers both diagnostic markers and therapeutic applications, forming a complete tinnitus disease assessment and intervention system. This overcomes the bottleneck of traditional tinnitus research, which lacks objective biomarkers and targeted treatment methods. Through precise intervention at the genetic level, it provides a solution for the clinical assessment and intervention of tinnitus. Therefore, the present invention has broad application prospects in the field of tinnitus disease assessment and intervention.

[0049] It should be emphasized that the above are only preferred embodiments of the present invention and do not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of the immediate premature genes Fos and Egr2 in the evaluation of tinnitus disease models.

2. The use of the immediate early gene Fos, Egr2 in the evaluation of tinnitus disease model according to claim 1, characterized in that: The immediate early gene Fos and / or Egr2 serves as a biomarker for the maintenance of tinnitus.

3. The use of the immediate early gene Fos, Egr2 in the evaluation of tinnitus disease model according to claim 2, characterized in that: The applications include: audiological assessment, tinnitus assessment, and objective tinnitus detection.

4. The use of the immediate early gene Fos, Egr2 in the evaluation of tinnitus disease model according to claim 2, characterized in that: The application includes: a blood detection kit, which quickly detects the biomarker through RT-qPCR.

5. Application of the immediate premature genes Fos and Egr2 in tinnitus disease intervention products.

6. The use of the immediate early gene Fos, Egr2 in a tinnitus disease intervention product according to claim 5, characterized in that: The application alleviates tinnitus by regulating the immediate early genes Fos and / or Egr2 in the auditory cortex AC brain region.

7. The use of the immediate early gene Fos, Egr2 in a tinnitus disease intervention product according to claim 5, characterized in that: The application regulates tinnitus by regulating IEGs neutral early genes Fos and / or Egr2 through H3K27me3.

8. The use of the immediate early gene Fos, Egr2 in a tinnitus disease intervention product according to claim 5, characterized in that: The tinnitus disease intervention products include: drugs, medical devices, biological products, and rehabilitation products.

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