Method for relieving noise-induced nerve injury and application thereof

Abstract

The invention belongs to the field of life science and drug screening and evaluation, and particularly relates to a method for relieving noise-induced nerve injury by screening drugs. Comprising the following steps: (1) constructing a sound wave device; (2) establishing a sound wave induced nerve injury model; (3) exploring a sound wave induced nerve injury mechanism; and (4) screening compounds. The nerve injury of the vertebrate caused by sound waves is relieved by screening drugs, and an experimental basis is provided for further revealing a nerve injury mechanism caused by sound waves such as noise and research and development of drugs for preventing and treating noise-induced nerve injury.

Description

Technical Field

[0001] The invention belongs to the field of life science and drug screening and evaluation, and specifically relates to a method for alleviating macular noise-induced nerve damage by screening drugs and an application thereof. Background Art

[0002] Noise exposure (NE) is the most common cause of hearing loss in adults worldwide. Noise exposure causes oxidative stress, increases reactive oxygen species (ROS) levels, and leads to hair cell damage, which in turn causes noise-induced hearing loss (NIHL) (ST JOHN JA, KEY B. HuC-eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones [J]. Journal of molecular histology, 2012, 43 (6): 615-23.). Unlike functional deafness, the main pathological basis of NIHL is the loss of sensory hair cells, and this hearing loss is permanent because mammalian hair cells cannot regenerate. Although some studies in the past few decades have focused on alleviating noise-induced hair cell damage by interfering with ROS levels, there is no clinically relevant intervention target to date, and no FDA-approved drugs have been provided to reduce the severity of NIHL. This is partly due to the complexity of the sensory hair cell death pathway in response to various factors and the uncertainty of potential molecular targets. Therefore, it is crucial to further explore the key targets in the pathogenesis of NIHL.

[0003] Due to their high genetic homology with humans, zebrafish are common organisms in neuroscience research. As a new vertebrate model organism between cells and mammals, zebrafish have been widely used in various biomedical research fields, including developmental biology, toxicology, and genetic and behavioral research. The auditory organ of zebrafish is mainly composed of the inner ear, Weber's organ and lateral line system. The hearing range of juvenile and adult zebrafish is between 100 and 4000 Hz (the human hearing range is 20 Hz to 20 kHz) (Defourny J, Aghaie A, Perfettini I, et al. Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage. Proc Natl Acad Sci USA, 2019, 116(16): 8010-8017.; Wang J, Song Q, Yu D, et al. Ontogenetic development of the auditory sensory organ in zebrafish (Danio rerio): changes in hearing sensitivity and related morphology. Sci Rep, 2015, 5: 15943.; Breitzler L, Lau IH, Fonseca PJ, et al. Noise-induced hearing loss in zebrafish: investigating structural and functional inner ear damage and In summary, zebrafish have a well-developed and sensitive hearing system, which can be used to study the central nervous system effects of various auditory and vibration stimuli, and have great potential in the field of hearing research. At present, some models have been developed to study the main effects of noise exposure on zebrafish behavior, including individual behavioral changes and group behavioral changes.For example, juvenile zebrafish exposed to noise for 18 hours showed hyperkinesis and enhanced startle reactivity (Higgs DM, Rollo AK, Souza MJ, et al. Development of form and function in peripheral auditory structures of the zebrafish (Danio rerio). Journal of the Acoustical Society of America, 2003, 113 (2): 1145-1154.). Shafiei Sabet et al. found that noise exposure also affects the foraging behavior of zebrafish and causes escape behavior of zebrafish as the noise level increases (Bhandiwad AA, Raible DW, Rubel EW, et al. Noise-Induced Hypersensitization of the Acoustic Startle Response in Larval Zebrafish. Journal of the Association for Research in Otolaryngology, 2018, 19 (6): 741-752.).

[0004] Transient receptor potential vanilloid (TRPV) channels are cation-gated channels that serve as detectors for various sensory modalities, such as pain, heat, cold, and taste (Shafiei Sabet S, Van Dooren D, Slabbekoorn H. Son et lumière: Sound and light effects on spatial distribution and swimming behavior in captive zebrafish. Environmental Pollution, 2016, 212: 480-488.; Ishibashi T, Takumida M, Akagi N, et al. Expression of transient receptor potential vanilloid (TRPV) 1, 2, 3, and 4 in mouse inner ear [J]. Acta Otolaryngol, 2008, 128(12): 1286-1293. Studies have found that TRPV channels contribute to normal cochlear physiological functions, such as hair cell mechanoelectric transduction channels and chain nerve function, to a certain extent. However, to date, the exact mechanism of action of TRPV channels on normal mammalian cochlear physiology is still unclear. For example, whether TRPV channels exist in the cochlea as homopolymers or heteropolymers, and how the above forms are regulated and function under normal conditions and environmental stress conditions. And the role of TRPV channels expressed on immune cells in mediating or regulating noise- and drug-induced hearing loss. TRPV1 is the first member of the TRPV family to be discovered and cloned, and is also one of the most widely studied and most concerned non-selective cation channel receptors. TRPV1 is mainly expressed in nociceptive sensory neurons and is mainly involved in the conduction of nociceptive signals and pain modulation in vivo. A better understanding of the function and regulation mode of TRPV1 channels in the cochlea may help develop new treatments for hearing loss.

[0005] References

[0006] [1]Park H C,Kim C H,Bae Y K,et al.Analysis of upstream elements inthe HuC promoter leads to the establishment of transgenic zebrafish withfluorescent neurons.[J].Developmental Biology,2000,227(2):279-293.

[0007] [2]ST JOHN J A,KEY B.HuC-eGFP mosaic labelling of neurons inzebrafish enables in vivo live cell imaging of growth cones[J].Journal ofmolecular histology,2012,43(6):615-23.

[0008] [3]Defourny J,Aghaie A,Perfettini I,et al.Pejvakin-mediated pexophagyprotects auditory hair cells against noise-induced damage.Proc Natl Acad SciU S A,2019,116(16):8010-8017.

[0009] [4]Wang J,Song Q,Yu D,et al.Ontogenetic development of the auditorysensory organ in zebrafish(Danio rerio):changes in hearing sensitivity andrelated morphology.Sci Rep,2015,5:15943.

[0010] [5]Breitzler L,Lau I H,Fonseca P J,et al.Noise-induced hearing lossin zebrafish:investigating structural and functional inner ear damage andrecovery.Hearing Research,2020,391:107952.

[0011] [6]Higgs D M,Rollo AK,Souza M J,et al.Development of form andfunction in peripheral auditory structures of the zebrafish(Danio rerio).Journal of the Acoustical Society of America,2003,113(2):1145-1154.

[0012] [7]Bhandiwad AA,Raible D W,Rubel E W,et al.Noise-InducedHypersensitization of the Acoustic Startle Response in LarvalZebrafish.Journal of the Association for Research in Otolaryngology,2018,19(6):741-752.

[0013] [8]ShafieiSabet S,Van Dooren D,Slabbekoorn H.Son et lumière:Sound andlight effects on spatial distribution and swimming behavior in captivezebrafish.Environmental Pollution,2016,212:480-488.

[0014] [9]Ishibashi T,Takumida M,Akagi N,et al.Expression of transientreceptor potential vanilloid(TRPV)1,2,3,and 4in mouse inner ear[J].ActaOtolaryngol,2008,128(12):1286-1293.

[0015]

[10] SOLANKI K, RAJPOOT S, BEZSONOV EE, et al. The expanding roles of neuronal nitric oxide synthase(NOS1)[J]. PeerJ, 2022,10:e13651. Summary of the invention

[0016] The present invention aims to provide an application of screening and obtaining drugs to alleviate the nerve damage of zebrafish caused by sound waves.

[0017] The present invention first provides a method for establishing a sound wave induced nerve injury model in a simulated closed environment, comprising the following steps:

[0018] (1) Select a vertebrate or cell (e.g., transgenic zebrafish, gene knockout mouse or human cell line, and the corresponding wild-type vertebrate cell can be used as a control), place it in a culture vessel (e.g., a microplate), and place it in an incubator;

[0019] (2) acoustic wave stimulation treatment: using acoustic waves to continuously stimulate the selected vertebrate cells;

[0020] (3) Quantitative analysis by fluorescence microscopy and RT-PCR analysis: The transgenic vertebrate cells were photographed and saved under a fluorescence microscope, and then image processing software was used to analyze the images and calculate their fluorescence intensity; RT-PCR was used to analyze the expression levels of the elavl3, pax6a, bcl2l11 and / or ncama1 genes;

[0021] (4) Result evaluation: Based on the gene expression level, a sound wave-induced vertebrate nerve injury model has been established.

[0022] Preferably, the transgenic zebrafish are HuC (elavl3)-GFP transgenic zebrafish and wild-type zebrafish 3-6 days (preferably 4 days) after fertilization;

[0023] The incubator is a constant temperature incubator;

[0024] The sound wave is 800-1200 Hz, 100-160 dB, preferably 1000 Hz, 130 dB;

[0025] The continuous stimulation time is 10-14 hours, preferably 12 hours;

[0026] The image processing software is imageJ software.

[0027] In one embodiment, the device for the acoustic wave stimulation treatment is as follows:

[0028] Use Cool Edit Pro software to generate a waveform of a certain frequency, and the computer volume controls the intensity of the sound wave;

[0029] The sound wave signal is transmitted to a speaker fixed in the incubator via Bluetooth. The speaker is fixed at the center of the top wall of the incubator, which is equivalent to a circular piston radiator on a rigid baffle, with a radiation angle downward.

[0030] A zebrafish culture vessel and a measuring microphone are placed directly below the speaker to receive sound wave signals; specifically, the measuring microphone is a standard microphone with a frequency response of ±0.1dB in the frequency band of 10Hz to 10000Hz and a flat response curve;

[0031] The distance between the standard microphone and the speaker is the same as the distance between the zebrafish culture device and the speaker;

[0032] The standard microphone is connected to the microphone preamplifier and the preamplifier power supply box to convert the sound signal into an electrical signal, and the strength and frequency of the sound can be observed in real time on the oscilloscope.

[0033] In a preferred embodiment, in the result judgment of step (4), a T test is used to calculate the error and significance through three sets of parallel data, and then the calculation results are statistically processed. According to the statistical processing results that the expression levels of elavl3, pax6a, bcl2l11 and / or ncama1 genes are statistically different from those of the control group, it is judged that a zebrafish sound wave-induced nerve injury model has been established.

[0034] Specifically, the internal dimensions of the constant temperature incubator are 41×36×36 cm, the dimensions of the speaker are 6.3×6.3×8.2 cm, the diameter of the sound radiation surface is 4.5 cm, the distance between the radiation surface and the culture dish is 15.5 cm, and the acoustic sensitivity of the measurement microphone used is M=46.23 mV / Pa.

[0035] The present invention provides a use of the acoustic wave induced nerve injury model obtained by the establishment method, wherein the model is used to evaluate or screen drugs that reduce nerve injury caused by acoustic wave stimulation. For example, noise-induced nerve injury can lead to noise-induced hearing loss or noise-induced deafness, hearing noise-induced damage and other diseases, and thus these drugs can be used to treat these diseases.

[0036] The present invention further provides a method for evaluating or screening nerve injury drugs using the acoustic wave-induced nerve injury model obtained by the establishment method, characterized in that it comprises the following steps:

[0037] (1) Compound treatment: Take the test substance, for example, select a potential antagonist against TRPV1 and / or NOS1, and add it to the culture medium of zebrafish larvae in the zebrafish sound wave induced nerve injury model established according to any one of claims 1 to 4 (specifically, the final concentration is 10 -2 mM to 10 -4 mM, preferably 10 -3 mM);

[0038] (2) Fluorescence microscopy quantitative analysis and RT-PCR analysis

[0039] The transgenic zebrafish were photographed and saved under a fluorescence microscope, and then image processing software was used to analyze the images and calculate their fluorescence intensity; RT-PCR was used to analyze the expression levels of trpv1, chga, atf4a, and aifm1 and their effects on cell apoptosis;

[0040] (3) Result processing: Statistical processing is performed on the calculation results. Based on the statistical processing results, the efficacy of the test compound is evaluated or screened. If the efficacy of the compound after use is statistically significantly different from that before use, it indicates that the compound is effective.

[0041] Specifically, the antagonist in step (3) is vanilloid receptor antagonist 1 or pentamidine isethionate.

[0042] The present invention further provides the use of the compound screened based on the method, or the antagonist of TRPV1 or the antagonist of NOS1 in the preparation of a drug for reducing sound damage to vertebrate nerves; preferably, the sound wave is 800-1200HZ, 100-160dB, preferably 1000HZ, 130dB.

[0043] Specifically, the TRPV1 antagonist is vanilloid receptor antagonist 1, and the NOS1 antagonist is pentamidine isethionate.

[0044] The present invention is applied to alleviate the nerve damage of zebrafish caused by sound waves by interfering with the expression of TRPV1. Specifically, wild-type and transgenic zebrafish with fluorescent marker proteins introduced, such as HuC (elavl3)-GFP transgenic zebrafish models, are used to inhibit TRPV1 and NOS1, and RT-PCR, confocal fluorescence microscopy imaging and other methods are used to observe and verify their effects on nerve damage. Through repeated comparison and observation, it is found that zebrafish inhibiting TRPV1 and NOS1 by small molecule antagonists have significantly alleviated nerve damage compared with wild zebrafish. It is characterized in that the nerve damage marker gene elavl3 is significantly downregulated after sound stimulation, and the small molecule drugs Vanilloid receptor antagonist 1 and Pentamidine isethionate can alleviate this phenomenon, and the small molecule drugs Vanilloid receptor antagonist 1 and Pentamidine isethionate can significantly alleviate zebrafish cell apoptosis caused by sound waves.

[0045] The present invention provides a signal pathway in which TRPV1 and NOS1 respond to sound waves and regulate the death of zebrafish larvae's neurons, wherein the pathway includes 11 genes, namely, trpv1, trpv4, trpa1a, hmox1a, rtn4a, nos1, chga, atf4a, ailfm1, hspa4, and ubc (Ishibashi T, Takumida M, Akagi N, et al. Expression of transient receptor potential vanilloid (TRPV) 1, 2, 3, and 4 in mouse inner ear [J]. Acta Otolaryngol, 2008, 128 (12): 1286-1293.; SOLANKI K, RAJPOOT S, BEZSONOV EE, et al. The expanding roles of neuronal nitric oxide synthase (NOS1) [J]. Peer J, 2022, 10: e13651.).

[0046] The present invention provides drugs for alleviating nerve damage caused by sound waves in animals such as zebrafish, mice or humans, such as vanilloid receptor antagonist 1 and pentamidine isethionate, which have specific antagonistic effects on TRPV1 and NOS1, respectively.

[0047] The present invention achieves inhibition of TRPV1 and NOS1 through small molecule antagonists vanilloid receptor antagonist 1 and pentamidine isethionate, alleviates the nerve damage of animals such as zebrafish, mice or human cells caused by sound waves, and provides an experimental basis for further revealing the mechanism of noise-induced nerve damage in animals and for research on drugs for preventing and treating nerve damage caused by sound waves (such as noise). BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 . Schematic diagram of the acoustic stimulation experimental device and the expression of genes related to zebrafish nerve damage after acoustic stimulation. Figure 1 Figure A shows the computer, constant temperature incubator, Bluetooth speaker, standard microphone, microphone preamplifier, preamplifier power supply box and oscilloscope in the sound wave stimulation experimental device. Figure 1 Figure B shows the specific conditions for the acoustic stimulation experiment on zebrafish, including the frequency, intensity and duration of the acoustic stimulation. Figure 1 C and D show the fluorescence intensity of zebrafish (elavl3:EGFP) observed using a confocal fluorescence microscope. Figure 1 Middle E shows the use of immunohistochemistry to analyze liver damage in zebrafish induced by acoustic stimulation.

[0049] Figure 2 .Results of sound-wave-induced neural injury in zebrafish larvae. Figure 2 A and B show the fluorescence intensity of zebrafish (elavl3:EGFP) observed using a confocal fluorescence microscope. Figure 2 Center C shows the use of immunohistochemistry to analyze liver damage in zebrafish induced by acoustic stimulation. Figure 2 Center C shows the relative mRNA expression of zebrafish nerve injury-related genes (elavl3, pax6a, bcl2l11, ncama1) after acoustic stimulation analyzed by RT-PCR.

[0050] Figure 3 .Transcriptome epigenetics reveals the functional regulation of zebrafish under acoustic stimulation. Figure 3The cluster heat map in A shows the differential gene expression between the experimental group and the control group (without acoustic stimulation) after 6 hours and 12 hours of acoustic stimulation. The vertical axis is the normalized FPKM expression value of the differentially expressed genes. Different colors represent differences in gene expression levels, with high expression in red and low expression in blue. The closer the color distribution of the two samples, the more similar the expression patterns. Figure 3 Middle B is a volcano map of differential gene expression between the experimental group stimulated by sound wave for 6 hours and 12 hours and the control group (without sound wave stimulation). Red dots represent significantly up-regulated genes, green dots represent significantly down-regulated genes, and blue dots represent genes with no differential expression. Figure 3 Middle C shows the number of DEGs after sonic stimulation of zebrafish for 6 hours and 12 hours. Figure 3 In D, the Venn diagram shows the overlapping DEGs between different comparison groups.

[0051] Figure 4 .The top 20 upstream regulatory signals determined based on KEGG enrichment and GO analysis, including gene functions, signaling pathways, transcription factors, ion channels, and enzymes. Figure 4 Middle E shows the activation levels of key regulatory factors of TRPV ion channels and NO signaling pathways determined by KEGG enrichment and GO analysis after 12 hours of acoustic stimulation of zebrafish. Solid arrows = direct interaction, dotted arrows = indirect interaction. The shadow part is other pathways in the neural stimulation response, and the color-highlighted part is the TRPV and NOS1-related pathways.

[0052] Figure 5 .The possible pathways by which TRPV1 and NOS1 activation lead to neuronal cell death, protein phosphorylation, and neuronal apoptosis are demonstrated. Figure 5 In center A, solid line = confirmed signaling pathway, dashed line = unconfirmed signaling pathway, arrow = positive feedback regulation, T-shaped head = negative feedback regulation; Figure 5 Figure 2B shows the results of RT-PCR analysis of the mRNA expression levels of genes related to TRPV ion channels and NO signaling pathways (trpv1, trpv4, trpa1a, hmox1a, rtn4a, nos1, chga, atf4a, ailfm1, hspa4) in zebrafish nerve damage induced by sound wave stimulation.

[0053] Figure 6 .The effect of inhibiting trpv1 expression on zebrafish nerve damage under acoustic stimulation was analyzed by RT-PCR and confocal fluorescence microscopy. Figure 6 Center A shows the inhibitory effect of TRPV1 expression using morpholino interference technology. Figure 6B and C show the changes in fluorescence expression of zebrafish before and after acoustic stimulation after inhibiting trpv1 expression in zebrafish larvae (elavl3:GFP). The fluorescence intensity represents the expression level of elavl3. Figure 6 D, E, F, and G in the middle show the expression of genes related to the TRPV1 pathway before and after sound wave stimulation after inhibiting trpv1 expression in zebrafish larvae (elavl3:GFP).

[0054] Figure 7 Zebrafish were treated with TRPV1 and NOS1 antagonists Vanilloid receptor antagonist 1 and pentamidine isethionate (working concentration was 10 -3 mM), and the effects of the above small molecule compounds on zebrafish nerve damage induced by sound wave stimulation were analyzed by RT-PCR and confocal fluorescence microscopy. Figure 7 A in the middle is a schematic diagram of the experimental process and the targets of the two drugs. Figure 7 B and D show the fluorescence expression of zebrafish larvae (elavl3:GFP) in the drug-treated group and the control group after sound wave stimulation. The fluorescence intensity represents the expression level of elavl3. Figure 7 C and E show the effects of two drugs on zebrafish cell apoptosis with and without sound wave stimulation.

[0055] Figure 8 It was demonstrated that TRPV1 is a key target for noise-induced neural damage in mice, and Trpv1 gene knockout significantly reduced neural damage caused by sound wave exposure in mice.

[0056] Fig. 9 Western Blot detection and immunohistochemical analysis demonstrated the role of TRPV1 in neural injury in mice and human cells.

[0057] Fig.10 Mice were treated with TRPV1 antagonist Vanilloid receptor antagonist 1, and the effects of the above-mentioned small molecule compounds on sound wave stimulation-induced nerve damage in mice were analyzed using open field tests and HE staining. DETAILED DESCRIPTION

[0058] The present invention is described in more detail below in conjunction with specific embodiments, which is for a better understanding of the present invention but does not constitute a limitation of the present invention.

[0059] Example 1. Acoustic Wave Stimulation Experimental Device

[0060] In order to provide a suitable environment for the growth of zebrafish, the acoustic stimulation experiment was carried out in a closed constant temperature incubator. The sound-generating device is a loudspeaker, and the real-time detection equipment is composed of a measurement microphone and an oscilloscope. The overall situation of the experimental device is as follows Figure 1 shown.

[0061] Cool Edit Pro software generates sound waves of a certain frequency, and the computer volume controls the intensity of the sound waves, which are transmitted to the speaker (Philips BT100 Bluetooth speaker) via Bluetooth. The speaker is fixed at the top center of the incubator, which can be approximated as a circular piston radiator on a rigid baffle, with a downward radiation angle. The zebrafish culture container and measurement microphone are placed below the speaker to receive sound signals. The measurement microphone device used in the experiment is a standard microphone (Hangzhou Aihua Instrument Co., Ltd., AWA14423 model), with a frequency response of ±0.1dB in the frequency band of 10Hz to 10000Hz, and a flat response curve, so the effect of sound reception and measurement devices on sound amplification can be ignored. When placing, ensure that the distance between the standard microphone and the speaker is the same as the distance between the zebrafish culture device and the speaker, so it is considered that the sound pressure at the standard microphone measurement point is the sound pressure at the zebrafish culture location. The standard microphone was connected to the microphone preamplifier and preamplifier power supply box (Hangzhou Aihua Instrument Co., Ltd.) to convert the acoustic signal into an electrical signal, and the intensity and frequency of the sound were observed in real time on an oscilloscope (Tektronix Technology Co., Ltd., model TBS1102X). Figure 1 As shown in A.

[0062] Example 2: Experiment on sound wave stimulation of zebrafish

[0063] 1) Basic properties of sound waves and data conversion

[0064] Sound is a pressure wave generated by the mechanical vibration of an object and propagates in a medium. Frequency and sound pressure are important properties that describe sound. The speed of vibration is the essence of the frequency of sound waves. A sound wave signal with periodic vibration and a unique period is a sine wave, also known as a single-frequency signal. The reciprocal of the period is the frequency f of the single-frequency signal, and the unit is Hertz (Hz). Based on the theory of Fourier transform, any sound wave can be decomposed into a superposition of sine waves, that is, it reflects the characteristics of a discrete line spectrum or a continuous spectrum in the frequency domain.

[0065] Another most commonly used physical quantity for sound wave representation is sound pressure, which is used to measure the strength of sound. The atmospheric pressure will change when disturbed by sound waves. Therefore, the difference between the pressure when there is a sound field in the medium and the pressure when there is no sound field is called the sound pressure of the sound field in the medium. The basic unit is Pascal (Pa). Sound pressure can be measured by a microphone. The greater the sound pressure input to the microphone, the greater the output electrical signal. The impedance relationship between input and output can be expressed by acoustic sensitivity, which is determined by the material and manufacturing process of the microphone and can be obtained through measurement. Therefore, after knowing the sensitivity of the microphone used and the output voltage, the output sound pressure can be obtained as shown in Formula 1.

[0066]

[0067] Where P is the measured sound pressure; V pp is the peak-to-peak voltage output by the transmitter. In this experiment, it is approximately V pp is the peak-to-peak value of the voltage displayed by the oscilloscope; M is the sensitivity of the measurement microphone used.

[0068] In acoustics, the effective value of sound pressure can be expressed by sound pressure level (SPL), as shown in Formula 2.

[0069]

[0070] Where, L p is the sound pressure level in dB; P is the effective value of the sound pressure to be expressed; P ref is a reference value; according to the general international standard, in air acoustics, P ref =20μPa, in hydroacoustics, P ref =1μPa. Therefore, it can be estimated that the sound pressure level of the sound source in seawater and in air, which produces the same sound pressure, differs by about 26dB.

[0071] 2) Setting of acoustic stimulation experiment parameters

[0072] In this experiment, a continuous sine wave with a frequency of 1kHz was used as the radiated sound signal to stimulate zebrafish. The specific parameters were set as sampling rate 48000, stereo channel, sampling accuracy 16 bits, fundamental frequency 1000Hz, modulation frequency 0Hz, sound pressure level 130dB, sine wave type. The internal dimensions of the constant temperature incubator were 41×36×36cm, the dimensions of the speaker were 6.3×6.3×8.2cm, the diameter of the sound radiation surface was 4.5cm, the distance between the radiation surface and the culture dish was 15.5cm, and the acoustic sensitivity of the measurement microphone used was M=46.23mV / Pa.

[0073] Example 3: Sound wave induced neural damage in zebrafish larvae

[0074] HuC (elavl3)-GFP transgenic zebrafish (Park HC, Kim CH, Bae YK, et al. Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. [J]. Developmental Biology, 2000, 227 (2): 279-293.) larvae (4 dpf) were treated with phenylthiourea (PTU, 0.0045%) to block pigment formation and exposed to Figure 1 The zebrafish larvae were stimulated with the acoustic wave device (1000 Hz, 130 dB SPL) for 12 hours, and the expression of the gene elavl3 was detected using a laser confocal microscope. Compared with the control group, the fluorescence level of zebrafish larvae in the acoustic wave stimulation group was significantly reduced (P<0.01), and the expression level of elavl3 was significantly downregulated ( Figure 2 A and B in the middle). elavl3 plays an important role in neuronal development and individual behavior and is an early neuronal marker. The expression of other genes related to nerve damage was further detected by RT-PCR ( Figure 2 D), it was found that in addition to elavl3, the expression of pax6a was also downregulated (P<0.001). As a brain marker gene, pax6a is widely expressed in various parts of the brain. It plays an important role in the cortical division of the central nervous system and the differentiation of neural stem cells (NSCs) into different neurons. It may also be related to the dorsal-ventral polarization of the spinal cord mediated by spinal cord induction signals. The mRNA levels of ncam1a, a gene that plays an important role in the development of the zebrafish nervous system, and bcl2l11, which is involved in the apoptosis pathway, were upregulated (P<0.01). Since the Bcl-2 family is a key regulator of apoptosis, its anti-apoptotic and pro-apoptotic members synergistically play an important role in controlling hepatocyte apoptosis in healthy livers. Therefore, the effect of sound waves on the liver tissue of zebrafish larvae was investigated. Immunohistochemistry results showed that compared with the control group, the overall structure of zebrafish liver tissue was abnormal after sound wave stimulation, the structure of tissue hepatocytes was loose, a large number of hepatocytes were obviously edematous, hepatocytes were slightly fatty, and tissue blood vessels were congested and dilated ( Figure 2 Middle C). This indicates that acoustic stimulation not only damages the nerves of zebrafish larvae, but also damages their livers.

[0075] Example 4: Transcriptome landscape analysis reveals functional regulation of zebrafish larvae in response to acoustic stimulation

[0076] RNA sequencing (RNA-seq) was used to study the changes in the transcriptional profile of zebrafish under acoustic stimulation. Wild-type zebrafish larvae (4dpf) were continuously exposed to sound waves (1000HZ, 130dB SPL) for 12h, collected at different time points (0h, 6h, 12h), and quantitatively analyzed using RNA-seq to quantify gene expression and observe the dynamic response of zebrafish larvae to external acoustic stimulation. The threshold value |log2(Fold Change)|>1 and the corrected p value (padj)<0.05 were used to identify differentially expressed genes (DEGs) relative to the control group. We collected zebrafish at different time points (6h, 12h) to observe the dynamic response of zebrafish larvae to external acoustic stimulation, and drew a cluster heat map to show the differentially expressed genes (DEGs) at specified time points after acoustic stimulation ( Figure 3 A). A total of 3717 DEGs were identified at 6 hours postinfection (6hpi) and 12hpi, including 2052 at 6hpi and 1665 at 12hpi. The detailed list of EDGs for all control groups is shown in Supplementary Data Table 1. The volcano plot visually shows the differential gene distribution between the acoustic stimulation group and the control group, with 941 and 740 up-regulated genes and 1111 and 925 down-regulated genes identified at 6hpi and 12hpi, respectively ( Figure 3 In addition, we classified the DEGs at different time points after acoustic stimulation, as shown in the Venn diagram ( Figure 3 (D). To gain a deeper understanding of the potential gene regulatory network in response to acoustic stimulation, we performed KEGG enrichment and Gene Ontology (GO) analysis on the upregulated DEGs at 6hpi and 12hpi, and retrieved annotations using the GO database. The top 20 pathologies, signaling pathways, transcription factors, ion channels, and enzymes were enriched, respectively. Different colors represent the activation status of the corresponding signaling pathways, with activated colors in red and inhibited colors in blue ( Figure 4 AD). It was found that genes related to sensory organ development, connective tissue cell development, head development, cell process formation, cell sensitivity, etc. were significantly down-regulated, while genes related to body death, nerve cell death, etc. were significantly up-regulated ( Figure 4 We then performed pathway enrichment analysis to explore functional pathways that may be associated with the mechanism of acoustic wave exposure. We found that the neurovascular coupling signaling pathway, synaptic long-term depression, calcium signaling, and endocannabinoid neuron synaptic pathway were activated ( Figure 4In the middle B), some regulatory factors (RB1, PIAS1, ZBTB20, ATF4, HIF1, NPM1) were significantly differentially regulated. Among them, the regulatory factors RB1, PIAS1, and ZBTB20 related to neurological disorders were upregulated at 6-12 hpi, while the factors ATF4, HIF1, and NPM1 related to normal physiological activities of nerves were downregulated at 6-12 hpi ( Figure 4 C), the expression levels of mechanosensitive ion channel TRPV4 and prostaglandin endoperoxide synthase 2 (PTGS2) were significantly reduced, and the expression of PTGS2 and other related enzymes was downregulated at 6-12 hpi, which may have led to the downregulation of neurovascular coupling signaling pathways ( Figure 4 (middle D).

[0077] Example 5: TRPV channels and NO signaling pathways are involved in the regulation of sound waves on the nerves of zebrafish larvae

[0078] By determining the activation levels of TRPV ion channels and NO signaling pathways at 12 hours, core analysis and pathway analysis were performed based on the mRNA folding changes in the RNA-seq experiment, and the above two regulatory factors and their target genes were reconstructed. Figure 4 The gene regulatory network (GRN) in E. The GRN results confirmed that the two genes were associated under nerve stimulation conditions, which may lead to the activation of TRPV4. The activated TRPV4 promoted a severe neuronal injury response, in which highly activated downstream regulatory factors were associated with neuronal cell death and neuronal apoptosis. Based on the above results, we speculated that TRPV may be involved in the signaling pathway of sound wave regulation of zebrafish nerve injury ( Figure 5 (A) Sound wave stimulation may inhibit the zebrafish TRPV4 ion channel, downregulate the expression of Fos, activate the TRPV1 channel, upregulate AIFM1, and enhance the Ca2+ in the cochlear tissue. 2+ Influx of NOS1 upregulates the expression of NOS1, promotes the synthesis of NO, and thus increases the release of NO, which ultimately leads to neuronal cell death, protein phosphorylation, and neuronal apoptosis. To verify the above speculation, we used RT-PCR to detect the expression of genes in this pathway under the condition of sound wave stimulation. The results showed that after 12 hours of 1000Hz sound wave stimulation, the expression of trpv1, trpv4, trpa1a, hmox1a, rtn4a, nos1, chga, atf4a, ailfm1, and hspa4 were all upregulated.

[0079] To further verify the involvement of TRPV1 in the regulation of sound waves on the nerves of zebrafish larvae and confirm the accuracy and reliability of transcriptome data, morpholino interference technology was used to inhibit the expression of TRPV1 in zebrafish ( Figure 6(A) The fluorescence expression of zebrafish larvae was analyzed by laser confocal microscopy. The fluorescence intensity represents the expression level of elavl3. The results showed that the level of sound-induced nerve damage in zebrafish was significantly reduced after inhibiting trpv1 expression ( Figure 6 B and C). The expression of TRPV1 pathway-related genes (trpv1, chga, atf4a, aifm1) after sound wave stimulation was analyzed by RT-PCR, and it was found that the expression of the above genes was downregulated after trpv1 inhibition, further verifying that Figure 5 TRPV1 in A is involved in the signaling pathway of sound wave regulation of zebrafish larvae's nerves ( Figure 6 (D, E, F, G).

[0080] Example 6: Inhibition of TRPV1 and NOS1 activation can alleviate sound stimulation-induced neural damage

[0081] In previous studies, we speculated that sound wave stimulation may activate TRPV1 channels and promote the expression of NOS1. Therefore, we selected Vanilloid receptor antagonist 1 and Pentamidine isethionate, antagonists of TRPV1 and NOS1, in order to screen out small molecule compounds that can alleviate nerve damage induced by sound wave stimulation and verify the present invention. -3 mM) was added to the culture medium of zebrafish larvae, and the fluorescent zebrafish larvae (elavl3:GFP) were continuously stimulated with sound waves (1000HZ, 130dB) for 12 hours ( Figure 7 A), fluorescence confocal microscopy results showed that the nerve injury marker gene elavl3 was significantly downregulated after sound wave stimulation, and small molecule drugs Vanilloid receptor antagonist 1 and Pentamidine isethionate can alleviate this phenomenon ( Figure 7 B, D), and small molecule drugs Vanilloid receptor antagonist 1 and Pentamidine isethionate can significantly alleviate zebrafish cell apoptosis caused by sound wave stimulation ( Figure 7 (C, E) (P < 0.001).

[0082] Example 7: Trpv1 gene knockout significantly reduces nerve damage caused by sound wave exposure

[0083] In this study, CRISPR-Cas9 technology was used to construct a Trpv1 gene knockout (Trpv1 - / -) mice and HEK293T cell line with TRPV1 gene knockout (Trpv1- / -) were used to investigate the role of TRPV1 in sound-induced nerve injury and cell apoptosis.

[0084] The WT and Trpv1 knockout genes (Trpv1 - / - ) mice, exposed to Figure 8 The sound wave device shown in A (1000HZ, 130dB SPL) was used for 6 hours. Figure 8 Middle B shows the movement trajectory of WT mice after treatment with different drugs: the upper figure is the control group, the lower figure is the sound wave group; the left side is the group without drug treatment; the middle is the normal saline (control) treatment group, which was given an intrathecal injection of 0.9% NaCl; the right side is the group given an intrathecal injection of the TRPV1 antagonist vanilloid receptor antagonist 1. Figure 8 C shows the expression of WT and Trpv1 in an open field experiment. - / - Activity levels of mice under different drug treatments (n=6). Figure 8 D shows WT and Trpv1 - / - The number of times mice stood up in the open field test (n=6).

[0085] Figure 8 Middle E shows the struggling time of WT and Trpv1- / - mice in the sound wave exposure group and the control group (n=6) in the tail suspension test (TST) to evaluate the degree of depression in mice. The experimental results showed that the struggling time of WT mice in the sound wave treatment group was significantly reduced compared with the control group (p=0.0036). In contrast, the struggling time of Trpv1- / - mice after sound wave stimulation did not change significantly compared with the control group mice.

[0086] The movement trajectory of mice showed that the range of motion of WT mice in the sound wave exposure group in the central area was significantly reduced compared with that of mice in the control group ( Figure 8 Middle B), activity level (p = 0.0064) ( Figure 8 C), and the number of standing times (p = 0.0405) ( Figure 8 These findings reflect typical anxiety behaviors. In contrast, Trpv1 - / - The central range of motion, activity level, and number of standing times of mice under acoustic stimulation were not significantly different from those of control mice. The OFT results showed that acoustic exposure caused abnormal changes in the emotions and behaviors of mice and inhibited their autonomous and exploratory behaviors. This indicates that acoustic stimulation may increase the severity of depression in mice, while knocking out TRPV1 may have a protective effect.

[0087] Figure 8Middle F shows the results of hematoxylin-eosin staining (HE staining) of brain tissue of WT mice treated with different drugs in the sound wave stimulation group and the control group (bar scale: 100μm). The results showed that WT mice exposed to sound waves showed obvious neurological damage compared with the control group mice (no sound wave stimulation). The neurons were sparsely arranged and disordered, the cell bodies shrank or dissolved, the morphology was irregular, and the neurons degenerated. However, compared with WT mice exposed to sound waves, the Trpv1 - / - The mice showed orderly arrangement of neurons, clear and regular cell body structure, no obvious degeneration, and significant relief of nerve damage.

[0088] Fig. 9 A shows the expression of Trpv1 in wild-type (WT) mice and Trpv1 knockout ( - / - The expression levels of TRPV1, CHGA, and ATF4 in mice before and after sound wave stimulation were investigated. The results showed that the expression levels of TRPV1, CHGA, and ATF4 were upregulated in mice after 12 hours of sound wave stimulation, which was consistent with the findings in zebrafish. In addition, in wild-type (WT) and Trpv1 knockout (Trpv1 - / - ) In HEK293T cells, the expression levels of PERK, eIF2α, ATF4, CHOP and TRPV1 proteins before and after acoustic stimulation were detected by Western Blot. The results showed that in the TRPV1 overexpression group, the expression levels of the above proteins were significantly higher than those in the TRPV1 knockout group, such as Fig. 9 As shown in B.

[0089] For WT and Trpv1 - / - Immunohistochemical analysis was performed on the brain tissues of mice in the acoustic wave stimulation group and the control group (bar: 20 μm) (n=3). Fig. 9 Center C shows the number of positive cells in immunohistochemical analysis (n=3). In the immunohistochemical analysis, specific antibodies for PERK, eIF2α, ATF4, and CHOP were used, and the expression of these proteins in the brain tissue of Trpv1- / - mice and WT mice after acoustic stimulation was detected. The results showed that the positive staining of PERK, eIF2α, ATF4, and CHOP was significantly enhanced after WT mice were exposed to acoustic waves, while only weak staining was shown in WT mice without acoustic stimulation. These findings suggest that TRPV1 can be activated by acoustic stimulation, which may lead to calcium imbalance in mouse and human cells, activate endoplasmic reticulum stress, and lead to cell apoptosis, thereby inducing neural damage and neurobehavioral abnormalities.

[0090] Example 8: Trpv1 antagonists effectively reduce nerve damage caused by sound wave exposure

[0091] To verify the effectiveness of the TRPV1 antagonist vanilloid receptor antagonist 1, the TRPV1 antagonist was injected into the lateral ventricle of mice and exposed to a sound wave device (1000HZ, 130dB SPL) for 6 hours to further verify whether drug inhibition of TRPV1 can also alleviate neurobehavioral abnormalities and neural damage in mice caused by sound waves.

[0092] Fig.10 A in the middle shows the movement trajectory of WT mice after treatment with different drugs: the upper figure is the control group, the lower figure is the sound wave group; the left side is the group without drug treatment; the middle is the normal saline (control) treatment group, which was given an intrathecal injection of 0.9% NaCl; the right side is the group given an intrathecal injection of the TRPV1 antagonist vanilloid receptor antagonist 1. Fig.10 In the open field experiment, WT and Trpv1 - / - Activity levels of mice under different drug treatments (n=6).

[0093] Fig.10 Center C shows WT and Trpv1 - / - The number of standing postures of mice in the open field test (n=6). OFT results showed that compared with the blank control group and the saline control group, mice treated with TRPV1 antagonists showed significantly reduced neurobehavioral effects after sound wave exposure, as shown by an increase in the range of motion in the central area ( Fig.10 A), a significant increase in activity level ( Fig.10 B) and a significant increase in the number of standing postures ( Fig.10 Middle C).

[0094] Fig.10 D shows the recording of WT and Trpv1 in the tail suspension experiment - / - The struggling time of mice in the sound wave exposure group and the control group (n=6). The results showed that after sound wave exposure, the struggling time of mice in the blank control group and the saline control group was significantly reduced; however, the struggling time of mice in the antagonist treatment group was not significantly different from that of mice in the control group that were not exposed to sound waves.

[0095] Fig.10Middle E shows the results of hematoxylin-eosin staining of brain tissue of WT mice treated with different drugs in the sound wave stimulation group and the control group (bar scale: 100μm). The results showed that the blank control group and saline control group mice exposed to sound waves showed significant neurological damage. In contrast, the degree of brain damage in mice treated with TRPV1 antagonists after sound wave exposure was significantly reduced. The neurons were arranged in an orderly manner, with clear morphology and no obvious degeneration. This shows that TRPV1 plays a key role in the process of neural damage and abnormal neurological function in mice caused by sound waves. It was verified that the use of antagonists (vanilloid receptor antagonist 1) to inhibit TRPV1 can effectively reduce the adverse neurological effects caused by sound wave exposure.

Claims

1. A method for establishing a sound wave induced nerve injury model in a simulated environment, characterized in that: The steps include: (1) Animal selection: vertebrates or cells (e.g. transgenic zebrafish, knockout mice or human cell lines); (2) Acoustic wave stimulation treatment: using sound waves (such as noise) to continuously stimulate the selected animals or cells; (3) Fluorescence microscopy quantitative analysis and RT-PCR analysis: The transgenic vertebrate cells were photographed and saved under a fluorescence microscope, and then image processing software was used to analyze the images and calculate their fluorescence intensity; RT-PCR analysis elavl3 , pax6a , bcl2l11 and / or ncama1 Gene expression levels; (4) Result judgment: Based on the gene expression level, a sound wave-induced vertebrate nerve injury model has been established.

2. The method according to claim 1, characterized in that: The transgenic zebrafish are selected from HuC ( elavl3 )-GFP transgenic zebrafish and wild-type zebrafish; The cells are placed in an incubator, specifically the incubator is a constant temperature incubator; The sound wave is 800-1200 Hz, 100-160 dB, preferably 1000 Hz, 130 dB; The continuous stimulation time is 10-14 hours, preferably 12 hours; The image processing software is imageJ software.

3. The establishment method according to claim 1, characterized in that: The equipment for the acoustic wave stimulation treatment is as follows: Use Cool Edit Pro software to generate a waveform of a certain frequency, and the computer volume controls the intensity of the sound wave; The sound wave signal is transmitted to a speaker fixed in the incubator via Bluetooth. The speaker is fixed at the center of the top wall of the incubator, which is equivalent to a circular piston radiator on a rigid baffle, with a radiation angle downward. A zebrafish culture vessel and a measuring microphone are placed directly below the speaker to receive sound wave signals; specifically, the measuring microphone is a standard microphone with a frequency response of ±0.1dB in the frequency band of 10Hz to 10000Hz and a flat response curve; The distance between the standard microphone and the speaker is the same as the distance between the zebrafish culture device and the speaker; The standard microphone is connected to the microphone preamplifier and the preamplifier power supply box to convert the sound signal into an electrical signal, and the strength and frequency of the sound can be observed in real time on the oscilloscope.

4. The establishment method according to claim 1, characterized in that: In step (4), the T test is used to calculate the error and significance of three sets of parallel data, and then the calculation results are statistically processed. elavl3 , pax6a , bcl2l11 and / or ncama1 The gene expression level was statistically different from that of the control group. The statistical processing results showed that the zebrafish sound wave-induced nerve injury model had been established.

5. The establishment method according to claim 3, characterized in that: The inner dimensions of the constant temperature incubator are 41×36×36 cm, the dimensions of the speaker are 6.3×6.3×8.2 cm, the diameter of the sound radiation surface is 4.5 cm, the distance between the radiation surface and the culture dish is 15.5 cm, and the acoustic sensitivity of the measurement microphone used is M = 46.23 mV / Pa.

6. Use of a zebrafish acoustic wave-induced nerve injury model obtained by the establishment method according to any one of claims 1 to 5, characterized in that: The model is used to evaluate or screen drugs that reduce nerve damage caused by acoustic stimulation in zebrafish.

7. A method for evaluating or screening nerve injury drugs using the fish acoustic wave induced nerve injury model obtained by the establishment method according to any one of claims 1 to 5, characterized in that: The steps include: (1) Compound treatment: Take the test substance, for example, select a potential antagonist against TRPV1 and / or NOS1, and add it to the sound wave induced nerve injury model established by the method according to any one of claims 1 to 4, for example, for transgenic zebrafish, add it to the culture medium of its juvenile fish (specifically, the final concentration is 10 -2 mM to 10 -4 mM, preferably 10 -3 mM); (2) Fluorescence microscopy quantitative analysis and RT-PCR analysis The acoustic wave-induced nerve injury model was photographed and saved under a fluorescence microscope, and then image processing software was used to analyze the image and calculate its fluorescence intensity; RT-PCR analysis trpv1,chga,atf4a,aifm1 The expression level of β-catenin and its effect on cell apoptosis; (3) Result processing: The calculation results are statistically processed, and the efficacy of the test compound is evaluated or screened based on the statistical processing results. If the efficacy of the compound after use is statistically significantly different from that before use, it means that the compound is effective.

8. The method according to claim 7, characterized in that The antagonist in step (3) is vanilloid receptor antagonist No. 1 or pentamidine isethionate.

9. Use of the compound screened by the method according to claim 7 or 8, a TRPV1 antagonist or a NOS1 antagonist in the preparation of a drug for reducing nerve damage caused by sound waves in vertebrates; Preferably, the sound wave is 800-1200 Hz, 100-160 dB, preferably 1000 Hz, 130 dB.

10. The use according to claim 9, characterized in that The TRPV1 antagonist is grass receptor antagonist No. 1, and the NOS1 antagonist is pentamidine isethionate.

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