Application of troxerutin in medicine for preventing and treating ototoxicity induced by cis-platinum

Abstract

The invention belongs to the field of medical application, and particularly relates to application of troxerutin in a medicine for preventing and treating ototoxicity induced by cis-platinum. The invention provides an application of troxerutin in a medicine for preventing and treating ototoxicity induced by cis-platinum, research finds that troxerutin can remarkably inhibit ROS accumulation, Fe overload and GPX4 reduction induced by cis-platinum, so that excessive accumulation of active oxygen and ferroptosis in hair cells are blocked, the auditory function is improved, troxerutin remarkably inhibits cell apoptosis induced by cis-platinum, and the troxerutin can be used for preventing and treating ototoxicity induced by cis-platinum. And a new way is provided for transformation application of traditional Chinese medicine micromolecules in ear toxicity prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical use, and more particularly to the use of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity. Background Technology

[0002] Sensorineural hearing loss (SNHL) is the most common type of permanent hearing impairment worldwide, and its main causes include drug toxicity, noise exposure, and aging.

[0003] Cisplatin is a commonly used chemotherapy drug in clinical practice, but its significant ototoxicity limits its application. Cisplatin can induce excessive accumulation of reactive oxygen species (ROS) in the stria vascularis and hair cells of the inner ear, leading to mitochondrial dysfunction, lipid peroxidation, and ferroptosis. Ferroptosis is an iron-dependent programmed cell death process characterized by Fe²⁺ accumulation, decreased GPX4, and increased ACSL4. Inhibiting ferroptosis can significantly reduce hair cell damage and represents a new generation of anti-otoxin strategies. Traditional antioxidants such as NAC can alleviate ROS, but their effects are singular and their stability is poor. Small molecules of flavonoids from traditional Chinese medicine have better protective potential due to their multi-target antioxidant, anti-inflammatory, and mitochondrial protective properties.

[0004] Troxerutin is a semi-synthetic flavonoid compound extracted from plants such as Sophora japonica, also known as vitamin P4 or trihydroxyethylrutin, and is an ethylated derivative of rutin. It is widely used clinically, primarily by enhancing vascular function and improving microcirculation, and plays a particularly important role in the treatment of vascular diseases and chronic venous insufficiency. Troxerutin possesses high free radical scavenging rate, good water solubility, and good biocompatibility. Currently, there are no reports on the role of troxerutin in preventing cisplatin-induced ototoxicity. Summary of the Invention

[0005] The purpose of this invention is to provide the application of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity. This invention has found that troxerutin can significantly inhibit cisplatin-induced ROS accumulation, Fe²⁺ overload, and GPX4 decrease, thereby blocking the excessive accumulation of reactive oxygen species and ferroptosis in hair cells and improving auditory function. Troxerutin also significantly inhibits cisplatin-induced apoptosis, providing a new approach for the translational application of small molecules of traditional Chinese medicine in the prevention and treatment of ototoxicity.

[0006] To address the aforementioned technical problems, the application of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity provided by this invention is achieved as follows: Application of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity.

[0007] Optionally, the troxerutin inhibits cisplatin-induced excessive ROS production.

[0008] Optionally, the troxerutin inhibits cisplatin-induced ferroptosis in hair cells.

[0009] Optionally, the troxerutin upregulates GPX4, downregulates ACSL4, reduces the expression of Caspase-3 and PARP, and blocks ferroptosis.

[0010] Optionally, the troxerutin inhibits cisplatin-induced apoptosis.

[0011] Optionally, the troxerutin reduces the expression of Caspase-3 and PARP, thereby inhibiting apoptosis.

[0012] Optionally, troxerutin can be prepared as an oral formulation, injection, sustained-release microsphere, or intratympanic injection formulation.

[0013] Optionally, troxerutin can be used in combination with antioxidants such as NAC, MitoQ, or NMN to form a compound formulation to enhance the protective effect.

[0014] Optionally, the concentration of troxerutin used is 5~20μM.

[0015] Optionally, the concentration of troxerutin used is 10 μM.

[0016] This invention relates to the application of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity. Cellular experiments using troxerutin pretreatment of cisplatin-damaged cells showed that troxerutin (Tro) significantly protected cisplatin-damaged cells. MitoSOX staining in mitochondrial ROS inhibition experiments revealed significantly enhanced mitochondrial ROS signaling in the cisplatin group, while a significant decrease was observed in the troxerutin-treated group (p<0.001), indicating that troxerutin possesses strong antioxidant capacity and can significantly inhibit cisplatin-induced ROS accumulation. Intracellular ROS detection experiments using CellROX assays showed that cisplatin significantly increased intracellular ROS, while the Tro group decreased ROS levels by approximately 50% (p<0.001). TUNEL assays demonstrating the apoptosis-inhibiting effect showed that cisplatin induced a large number of apoptotic cells, while Tro treatment reduced the proportion of apoptotic cells by 60%. Western blot analysis confirmed that Tro reduced the cleavage levels of Cleaved-Caspase-3 and PARP. In the ferroptosis mechanism experiment of this invention, Ferro Orange fluorescence detection showed that cisplatin significantly accumulated Fe²⁺, while Tro significantly restored iron homeostasis. GPX4 upregulation and ACSL4 downregulation confirmed that Tro exerts protective effects through the ferroptosis pathway. In the zebrafish model created in this invention, cisplatin reduced hair cells by 70% in Tg(brn3c:mGFP) zebrafish, while 10 μM Tro significantly restored hair cell number (p<0.05), verifying its in vivo auditory protective effect. In the mouse ABR detection experiment of this invention, after cisplatin modeling in BALB / c mice, administration of Tro (50 mg / kg, ip×7 d) showed an improvement in hearing threshold of more than 20 dB, suggesting that Tro restores auditory function in vivo.

[0017] The present invention has the following beneficial effects: 1. This invention reveals for the first time a novel application of troxerutin in the protection of auditory hair cells. The study found that troxerutin can significantly inhibit cisplatin-induced excessive ROS accumulation in cells, revealing the molecular mechanism by which troxerutin regulates ferroptosis through the ROS–Fe²⁺–GPX4 axis and blocks apoptosis by inhibiting Caspase-3 / PARP; troxerutin can prevent and treat cisplatin-induced ototoxicity through multiple targets and channels.

[0018] 2. This invention has discovered that troxerutin simultaneously inhibits apoptosis and ferroptosis, forming a synergistic protective network of "antioxidant-antiferroptosis". Experiments in zebrafish and mice have verified that troxerutin has a protective effect on hearing in zebrafish and can restore damaged hearing function in mice.

[0019] 3. Troxerutin has the advantage of being traceable to traditional Chinese medicine: it is derived from natural rutin, has high safety and stable pharmacokinetics; 4. Broad-spectrum applicability: It can be used for the prevention and treatment of drug-induced, noise-induced, and age-related hearing loss; 5. Strong potential for clinical translation: Troxerutin has a clinical safety basis, which can rapidly advance the research and development of hearing protection drugs. Attached Figure Description

[0020] Figure 1 This is a bar chart of the viability of HEI-OC1 cells treated with different concentrations of cisplatin according to Example 1 of the present invention; Figure 2 This is a bar chart illustrating the effect of troxerutin on the activity of cisplatin-treated HEI-OC1 cells according to Embodiment 2 of the present invention. Figure 3 This is a comparison chart of cell survival count and cell activity in the Con group, Cis group, and Tro+cis group set in Embodiment 2 of the present invention; where A is a comparison chart of cell survival count and B is a comparison chart of cell activity. Figure 4 This is a detection image of ROS levels in mitochondria in HEI-OC1 cells of the Con group, Cis group and Tro+cis group set in Embodiment 3 of the present invention. A is a comparison image of Mito SOX red fluorescent probe, B is a comparison image of cell viability, and C is a comparison image of fluorescence intensity. Figure 5 This is a cell ROX fluorescence staining detection image of the Con group, Cis group and Tro+cis group set in Embodiment 4 of the present invention to detect the intracellular ROS level. A is a picture of stained cells under a microscope and B is a comparison image of green fluorescence intensity. Figure 6 These are apoptosis images of the Con group, Cis group, and Tro+cis group set in Embodiment 5 of the present invention, where A is a TUNEL staining image, B is a TUNEL staining bar comparison image, and C is a detection image of apoptosis-related (Caspase-3 / PARP) protein expression in each group of cells by Western Blot method. Figure 7 This is a graph showing the intracellular Fe2+ content detection in the Con group, Cis group, and Tro+cis group set in Embodiment 7 of the present invention. A is a Ferro Orange fluorescence staining graph, B is a Ferro Orange fluorescence staining bar comparison graph, and C is a graph showing the expression levels of ferroptosis-related proteins (ACSL4 and GPX4) in each group of cells detected by Western Blot. Figure 8 This is an experimental diagram of zebrafish lateral line hair cell protection in Embodiment 9 of the present invention. A is a green fluorescence staining diagram of hair cells in the neurothalamus, B is a green fluorescence staining diagram of hair cells in the neurothalamus in the Tro (10 μM) group, Con group and Cis group, and C is a bar chart comparing the number of cells in the Tro (10 μM) group, Con group and Cis group. Figure 9 Example 10 of this invention: a line graph comparing the hearing thresholds of mice in the Con group, Cis group, and Tro+cis group. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Troxerutin is a semi-synthetic flavonoid compound extracted from plants such as Sophora japonica, also known as vitamin P4 or trihydroxyethylrutin. It is widely used clinically, primarily by enhancing vascular function and improving microcirculation, and plays a particularly important role in the treatment of vascular diseases and chronic venous insufficiency. As a classic vascular protectant, troxerutin has a clear efficacy in improving microcirculation and alleviating symptoms of venous diseases, and is especially suitable for the adjunctive treatment of chronic venous insufficiency and related microcirculatory disorders. The structural formula of troxerutin is as follows:

[0024] Example 1: Establishing a cisplatin-induced cell damage model 1.1 Cochlear hair cell culture Cell source and culture environment preparation: The mouse cochlear hair cell line HEI-OC1 used in this experiment was purchased from OriCell® by Cyagen (Guangzhou) Biotechnology Co., Ltd. The culture medium used was Dulbecco's Modified Eagle Medium (DMEM), a high-glucose medium suitable for HEI-OC1 cell growth, supplemented with 10% fetal bovine serum (FBS). All cells were placed in an incubator at 33 ℃ and 10% CO2 concentration.

[0025] Cell passage culture: Observe cell growth regularly using an inverted microscope. Passage the cells when they reach 80% confluence. Discard the old culture medium from the culture dish, and gently wash the cells 2-3 times with pre-warmed PBS to remove residual culture medium and metabolites. Then, add an appropriate amount of trypsin solution for digestion. Next, gently pipette the cell suspension to form a homogeneous single-cell suspension. Finally, according to the passage ratio, take an appropriate amount of cell suspension and seed it into a new culture dish, add fresh DMEM medium containing 10% FBS, and return it to the incubator for continued culture.

[0026] 1.2. HEI-OC1 cells were exposed to different concentrations of cisplatin (0–50 μM) for 24 h. Cell seeding and pre-culture: HEI-OC1 cells were slowly and evenly added to each well of a 96-well plate at a density of 10,000 cells per well using a pipette, avoiding uneven cell distribution or the formation of air bubbles. After seeding, the 96-well plate was placed in an incubator and cultured for 24 hours to allow the cells to fully adhere to the culture environment.

[0027] Cisplatin treatment and culture: When cells reach the logarithmic growth phase, carefully aspirate the old culture medium from the culture dish and gently wash the cells 2-3 times with sterile PBS. Divide the cells into an untreated control group and groups treated with different concentrations (0–50 μM) for 24 h. According to the experimental design, slowly and evenly add the appropriate concentration of cisplatin solution to each group of cells. Return the treated cell culture dishes to a 33 °C, 10% CO2 incubator and continue culturing for 24 h. Microscopic observation: After cisplatin treatment, observe cell growth using an inverted microscope, including cell morphology, adhesion and aggregation, and characteristics of apoptosis or necrosis. Adjust the microscope magnification and focus during observation to obtain clear images, and record and compare detailed data.

[0028] 1.3. CCK-8 assay for cell viability Cell viability was measured and evaluated using a cell counting kit (Cell Counting Kit 8, CCK8, VH785, Dojindo) 24 h after cisplatin treatment.

[0029] Carefully aspirate the culture medium from the culture plate. Add an appropriate amount of serum-free culture medium and CCK-8 reagent to each well according to the CCK-8 kit instructions, precisely controlling the reagent dosage. Incubate the culture plate at 37 ℃ for 2 h to allow the CCK-8 reagent to react with the mitochondrial dehydrogenase of live cells, generating a water-soluble orange-yellow product. After incubation, measure the absorbance (optical density, OD) of each well at a wavelength of 450 nm using a microplate reader, and calculate the cell viability of different treatment groups.

[0030] The results are as follows Figure 1The bar chart shows HEI-OC1 cells treated with different concentrations of cisplatin. CCK8 assay results indicated that, compared to the untreated control group, cell viability was significantly reduced in all cisplatin-treated groups in a dose-dependent manner (p < 0.0001). The decrease in cell viability was particularly pronounced and statistically significant when the cisplatin concentration reached 30 μM or higher. Based on these experimental data, 30 μM was ultimately selected as the standard cisplatin concentration for subsequent in vitro cell experiments.

[0031] Example 2: Protective effect of troxerutin (Tro) against cisplatin-induced HEI-OC1 cell damage. First, HEI-OC1 cells were pretreated with Tro at different concentration gradients (0, 1, 5, 10, 20, and 50 μM) for 2 h, followed by treatment with 30 μM cisplatin and the aforementioned different concentrations of Tro for 24 h. Cell viability was then assessed using the CCK-8 assay. Results are shown below. Figure 2 The results showed that, compared with cells treated with cisplatin alone, Tro concentration of 10 μM had the most significant protective effect on hair cells (p < 0.0001). Therefore, 10 μM Tro pretreatment for 2 h was selected as the optimal treatment condition for this study.

[0032] Subsequently, HEI-OC1 cells were divided into: a control group (Con), a cisplatin-damaged group (Cis), a group pretreated with 10 μM Troxerutin for 2 h, and a group co-treated with 30 μM cisplatin (Tro+cis). Cells in each group were cultured for 24 h, and the number of surviving cells in each group was counted. Figure 3 A), and the viability of cells in each group was detected using the CCK8 assay. Figure 3 B). The experimental results all showed that, compared with the control group, the number of cells in the 30 μM cisplatin treatment group was significantly reduced (p < 0.0001). More importantly, compared with the 30 μM cisplatin treatment group, the number of viable cells in the groups pretreated with 10 μM Tro for 2 h and co-treated with 30 μM cisplatin was significantly increased (p < 0.0001). Based on the above results, it can be inferred that Tro can effectively protect hair cells and reduce the damage to cells caused by cisplatin ototoxicity.

[0033] Example 3: Detection of mitochondrial ROS levels by Mito SOX staining To further investigate whether Tro has the effect of inhibiting cisplatin-induced ROS accumulation, this study used the highly specific ROS indicator Mito SOX red fluorescent detection reagent to detect the ROS levels in each group of cells and mitochondria, thereby revealing the potential mechanism of Tro in regulating cellular oxidative stress.

[0034] HEI-OC1 cells were divided into: control group (Con), cisplatin-damaged group (Cis), group pretreated with 10 μM Tro for 2 h and group co-treated with 30 μM cisplatin (Tro+cis). Cells from each group were cultured at 33 °C in 10% CO2 for 24 h. Then, 1–2 mL of 5 μM Mito SOX staining reagent (M36008, Invitrogen™) was applied to the cells. The cells were then incubated at 33 °C in 10% CO2 for 30 min. After staining, the culture medium containing Mito SOX was carefully removed. The cells were then washed three times with pre-cooled PBS to room temperature. After washing, 4% PFA solution was added to fix the cells for 15 min. After fixation, the cells were gently washed three times again with PBS. An appropriate amount of DAPI staining solution (D523, Dojindo) was evenly added to the cells, and staining was performed for 10 min. The cells were then gently washed three times with PBS. Cells were examined under a confocal microscope within 2 h after staining to ensure accurate experimental data were obtained within the optimal timeframe when the cell staining state was relatively stable and the fluorescence signal had not significantly decayed.

[0035] The levels of ROS in mitochondria of HEI-OC1 cells from different treatment groups were thoroughly investigated using a 5 μM Mito SOX red fluorescent probe. The results are as follows: Figure 4 A. Simultaneously affects cell viability ( Figure 4 B) and fluorescence intensity ( Figure 4 C) Further comparative analysis was conducted. Cell viability results were consistent with the above. Compared with the control group, the number of cells in the cisplatin-treated group was significantly reduced (p < 0.0001). Compared with the cisplatin-treated group, the number of cells surviving after Tro pretreatment and co-treatment with cisplatin was significantly increased (p < 0.0001). Fluorescence intensity analysis showed that, compared with the control group, the red fluorescence of Mito SOX in the cisplatin group was significantly enhanced (p < 0.0001). However, after Tro pretreatment, the red fluorescence of Mito SOX was significantly weakened compared with the cisplatin group (p < 0.001). This result indicates that Tro pretreatment can effectively reduce cisplatin-induced ROS accumulation in mitochondria, protecting mitochondrial function from oxidative stress damage.

[0036] Example 4: Cell ROX fluorescence staining to detect intracellular ROS levels Experimental grouping: HEI-OC1 cells were divided into: control group (Con), cisplatin-damaged group (Cis), 10 μM Tro pretreatment for 2 h and 30 μM cisplatin co-treatment group (Tro+cis). Cells in each group were cultured at 33 °C and 10% CO2 for 24 h. Accurately measure an appropriate amount of Cell ROX reagent (C10444, Life Technologies Corporation) and add it to each group of cells according to the experimental design requirements to achieve a final concentration of 5 μM. Then, incubate the cell culture dishes at 33 ℃ in a 10% CO2 incubator for 30 min. After incubation, carefully aspirate the culture medium containing Cell ROX reagent. Slowly add pre-cooled PBS along the wall of the culture dish, gently agitate to cover the cell surface, and then aspirate. Repeat this process three times. Fix the cells with 4% PFA solution for 15 min. After fixation, wash the cells three times with PBS to remove residual formaldehyde solution. Then, add an appropriate amount of DAPI staining solution (D523, Dojindo) and stain in the dark for 10 min. After staining, wash the cells three times with PBS. Observe the cells using a confocal microscope, and record and analyze the images in detail.

[0037] After treating each group of cells, they were incubated with 5 μM Cell ROX green fluorescent dye, and then the stained cells were observed using a confocal microscope. Figure 5 A). The results showed that, compared with the control group, the green fluorescence intensity of Cell ROX in HEI-OC1 cells treated with cisplatin was significantly enhanced (p < 0.0001), while when cells were pretreated with Tro and then co-treated with cisplatin, the green fluorescence intensity of Cell ROX was significantly weakened compared with the cisplatin group (p < 0.0001). Figure 5 B). This phenomenon indicates that cisplatin can induce a significant increase in intracellular ROS levels, thereby exacerbating cellular oxidative stress. Tro pretreatment can inhibit cisplatin-induced intracellular ROS production and play a protective role in mitigating cellular oxidative stress damage.

[0038] Example 5: TUNEL assay for detecting cell apoptosis To investigate the potential mechanism by which Tro plays a role in cisplatin-induced damage to HEI-OC1 cells, this study examined the effects of Tro treatment, cisplatin treatment, and their combinations on apoptosis.

[0039] 5.1 HEI-OC1 cells were divided into: control group (Con), cisplatin-damaged group (Cis), group pretreated with 10 μM Tro for 2 h, and group co-treated with 30 μM cisplatin (Tro+cis). Cells in each group were cultured at 33 °C and 10% CO2 for 24 h. Subsequently, TUNEL staining was used to detect apoptosis in each group.

[0040] TUNEL staining technique for detecting cell apoptosis: Add an appropriate amount of 4% PFA to each group of cells and fix at room temperature for 30 min. After fixation, wash the cells with pre-cooled PBS to prepare for the next permeabilization process. Dilute 2 mg / mL proteinase K with PBS at a ratio of 1:100 to prepare a working solution with a final concentration of 20 μg / mL. Add the solution slowly and evenly using a pipette, avoiding air bubbles and ensuring coverage of the entire sample area. Incubate at room temperature for 5 min to allow proteinase K to permeate the cell membrane and nuclear membrane to a suitable extent, facilitating the subsequent TUNEL assay solution (MA0223, Meilunbio) to enter the cells and specifically bind to the ends of apoptotic DNA breaks.

[0041] Prepare an appropriate amount of TUNEL assay solution to ensure its activity and effectiveness. Protect the solution from light during preparation. For example, using a 50 μL system, accurately measure 5 μL of TdT Enzyme (10×) and 45 μL of FITC-12-dUTP Labeling Mix using a high-precision pipette under water bath conditions, and slowly mix them thoroughly. Avoid generating air bubbles and minimize light exposure during mixing. After preparation, store in an ice-water bath protected from light until ready for use.

[0042] Using a pipette, slowly and evenly add 50 μL of TUNEL assay solution to the pretreated sample. Ensure the assay solution completely covers the cell sample area. Incubate the sample with the added TUNEL assay solution at 33 °C in a 10% CO2 incubator for 60 min in the dark. After incubation, wash the sample three times with pre-cooled PBS. After washing, mount the sample with an anti-fluorescence attenuation mounting medium, avoiding air bubbles, and then gently cover with a coverslip, ensuring a uniform and tight mount. Observe the mounted sample under a confocal microscope. Set the excitation (Ex) wavelength to 450-500 nm and the emission (Em) wavelength to 515-565 nm.

[0043] 5.2 Experimental Results: The results are as follows Figure 6 A and Figure 6 B showed that, compared with the control group, the number of TUNEL-positive cells was significantly increased after cisplatin treatment (p < 0.0001), indicating that cisplatin strongly induces apoptosis. In stark contrast, the percentage of TUNEL-positive cells in the Tro pretreatment group and the combination therapy group was significantly reduced compared with the cisplatin-only experimental group (p < 0.01). This finding suggests that Tro plays a positive and effective role in inhibiting cisplatin-induced apoptosis.

[0044] Example 6: Western Blot Experiment To further explore the intrinsic apoptosis pathway involved in cisplatin-induced cell death, Western blotting was used to analyze the expression levels of apoptosis-related proteins PARP, Caspase-3, and Cleaved-Caspase-3.

[0045] 6.1 Cell Lysis and Protein Extraction: Remove cells from the cell culture incubator and aspirate the culture medium. Gently wash three times with pre-cooled PBS; add protease and phosphatase inhibitors to the lysis buffer according to the instructions, and mix thoroughly; add 10... 6 Add 0.1 mL of lysis buffer to each cell, evenly covering the cell surface. Scrape the cells off with a cell scraper under ice-water bath conditions to induce lysis. Transfer the cell lysis mixture to a pre-chilled centrifuge tube and incubate at 4 °C on a shaker for 30 min to fully release intracellular proteins. After incubation, centrifuge the sample at 12000 rpm for 20 min at 4 °C. Transfer the supernatant to a new pre-chilled centrifuge tube and place the tube in an ice-water bath. The supernatant obtained at this point is the desired protein sample.

[0046] 6.2 Protein Quantification: Standard Curve Construction: Using bovine serum albumin (BSA) as the standard, prepare a series of BSA standard solutions with different concentrations (e.g., 0, 25, 50, 100, 200, and 400 μg / mL). A blank control was also set up, with only lysis buffer added. Bicinchoninic Acid (BCA) Assay: Following the instructions of the BCA protein quantification kit, add an appropriate amount of BCA working solution to the standard solution and protein sample, mix well, and incubate at 37 ℃ for 30-60 min to allow the protein to fully react with the reagent. After incubation, cool to room temperature and measure the OD value of each tube at 562 nm using a microplate reader. Protein Concentration Calculation: Based on the OD value of the protein sample, find the corresponding protein concentration on the standard curve. Record the concentration of each protein sample for subsequent adjustment of the SDS-PAGE loading amount.

[0047] 6.3 SDS-PAGE Electrophoresis: Select appropriate separating and stacking gels according to the experimental purpose and protein molecular weight, and follow the instructions of the SDS-PAGE gel preparation kit. When preparing the separating gel, mix all reagents. After the separating gel solidifies, pour off the supernatant, blot dry with filter paper, then pour in the stacking gel, insert the comb, and allow it to solidify. Sample preparation: Calculate the loading volume based on the protein quantification results, ensuring a loading amount of 20 μg. Add an appropriate amount of loading buffer, mix well, and heat in a boiling water bath for 5-10 min to denature the protein. Briefly centrifuge and set aside. Sample loading and electrophoresis: Add the prepared protein sample and protein molecular weight standard sequentially to the wells, avoiding air bubbles. Turn on the power and perform constant voltage or constant current electrophoresis. During the stacking gel stage, concentrate the protein at 80 V. After the sample enters the separating gel, increase the voltage to 120 V until the bromophenol blue indicator reaches the bottom of the gel.

[0048] 6.4 Transfer: Prepare transfer materials: Cut polyvinylidene fluoride (PVDF) membrane and filter paper to the same size as the gel. Immerse the PVDF membrane in methanol for 1-2 min, then transfer it to transfer buffer for equilibration for 10-15 min. Simultaneously, immerse the filter paper in transfer buffer. Following the transfer apparatus instructions, place the sponge pad, 4 layers of filter paper, gel, PVDF membrane, 4 layers of filter paper, and sponge pad in sequence on the transfer clamp, ensuring no air bubbles between layers. Place the assembled clamp into the transfer tank, correctly connect the electrodes (usually the gel is near the negative electrode, and the PVDF membrane is near the positive electrode), and add an appropriate amount of transfer buffer. Turn on the power and transfer the membrane in an ice bath. Select transfer conditions based on the protein molecular weight: for small proteins, use a constant current of 200-300 mA for 1 h; for large proteins, extend the time or increase the current. After transfer, turn off the power, carefully remove the PVDF membrane, and mark the front and back sides and the standard position of the protein molecular weight to identify the target protein band.

[0049] 6.5 Blocking and Antibody Incubation: Place the PVDF membrane in blocking buffer containing 5% skim milk, i.e., Tris-buffered saline or phosphate-buffered saline with Tween-20 (TBST), and gently incubate at 37 °C on a shaker for 1 h. Primary antibody incubation: Gently wash three times with TBST buffer, 5-10 min each time. Then place the membrane in an incubation chamber containing diluted primary antibodies. The primary antibody dilution ratios are as follows: PARP (1:1000, Proteintech), Caspase-3 (1:1000, Proteintech), Cleaved-Caspase-3 (1:1000, Invitrogen), ACSL4 (1:1000, Invitrogen), GPX4 (1:1000, Abcam), Tubulin (1:1000, Abcam), and GAPDH (1:1000, Abcam). Incubate overnight on a shaker at 4 ℃ to allow the primary antibody to fully bind to the target protein. Secondary antibody incubation: The next day, wash the PVDF membrane three times with TBST buffer, 10-15 min each time. After washing, place it in the appropriate secondary antibody incubation solution according to the manufacturer's instructions. Incubate on a shaker at 37 ℃ for 2 h. After incubation, wash 3-5 times with TBST buffer, 10-15 min each time, to remove unbound secondary antibody.

[0050] 6.6 Color Development and Result Analysis: Color Development: Immediately and evenly drop the color developing solution onto the PVDF membrane, ensuring complete coverage. Place the membrane in a dark chamber for 1-5 minutes to react; Exposure and Imaging: Place the dark chamber in a chemiluminescence imager and select an appropriate exposure time based on the luminescence intensity. Multiple exposures can be performed to obtain images of different intensities to achieve optimal strip clarity and contrast. Save the image file after exposure.

[0051] 6.7 Results Analysis: Statistical analysis of protein bands was performed using ImageJ software.

[0052] Figure 6 The results showed that the expression levels of several apoptosis-related proteins, including PARP and Caspase-3, were increased in the Cis group. Compared with the Cis group, the expression levels of PARP and Caspase-3 were decreased in the Tro+cis group. This indicates that Tro can inhibit cisplatin-induced apoptosis. These results suggest that Tro has the ability to attenuate cisplatin-induced apoptosis in HEI-OC1 cells.

[0053] Example 7: Intracellular Fe 2+ Content detection 7.1 Estimating intracellular Fe by detecting the fluorescence intensity of Ferro Orange 2+ content.

[0054] Prepare 1 μM Ferro Orange working solution (F374, Dojindo); select HEI-OC1 cells in the logarithmic growth phase and in good condition, and slowly and evenly seed an appropriate amount of cell suspension into a fluorescent culture dish using a pipette, and incubate overnight at 33°C in a 10% CO2 incubator; remove the culture dish after overnight culture, discard the supernatant, and wash the cells 3 times with PBS; HEI-OC1 cells were divided into four groups: control group (Con), cisplatin-damaged group (Cis), group pretreated with 10 μM Tro for 2 h, and group co-treated with 30 μM cisplatin (Tro+cis). All groups were cultured at 33 °C in 10% CO2 for 24 h. The culture medium was removed from each group, and the cells were washed three times with pre-cooled PBS. A suitable amount of 1 μM Ferro Orange working solution was added to the cell culture dish, ensuring complete coverage of the cell layer. After addition, the cells were incubated at 33 °C in 10% CO2 for 30 min. After incubation, the cell culture dish was quickly placed on the stage of a laser confocal microscope. Cells were observed and fluorescence images were acquired. The Ex wavelength was set to 561 nm, and the Em wavelength to 570-620 nm.

[0055] 7.2 Result Description: Detection of intracellular Fe using a 1 μM Ferro Orange fluorescent probe 2+ Horizontal. Ferro Orange fluorescence staining in each group was observed using a confocal microscope, and the results are as follows: Figure 7 As shown in A and B, compared with the control group, cisplatin-induced HEI-OC1 cells had higher intracellular Fe content. 2+ The content was significantly increased, as evidenced by a significantly higher Ferro Orange fluorescence signal compared to normal control cells (p < 0.0001). This phenomenon confirms that cisplatin can induce intracellular Fe... 2+ Increased levels. Further research revealed that, compared to the cisplatin group, the Tro+cis co-treatment group had significantly higher intracellular Fe levels. 2+ The content was significantly reduced (p < 0.0001). This indicates that Tro intervened in ferroptosis-related signaling pathways at the cellular level, reducing the iron-dependent iron deficiency. 2+ The mediated oxidative stress and cell damage further confirm that Tro plays an important protective role in resisting cisplatin-induced ferroptosis.

[0056] In addition, the expression levels of ferroptosis-related proteins (ACSL4 and GPX4) in each group of cells were detected by Western blotting. Figure 7C. The study found that ACSL4 expression levels were significantly increased in the cisplatin group, while GPX4 expression levels were decreased. Compared to the cisplatin group, the Tro combination group showed significantly increased GPX4 expression levels and significantly decreased ACSL4 expression levels. These results indicate that Tro possesses the ability to alleviate cisplatin-induced ferroptosis in HEI-OC1 cells. This finding provides experimental evidence for a deeper understanding of the mechanism by which Tro acts in resisting cisplatin ototoxicity and protecting hair cells from ferroptosis.

[0057] Example 8: Zebrafish rearing Zebrafish, as a newly emerging vertebrate model organism in recent years, has attracted much attention in the field of biological research. Zebrafish lateral line hair cells, both structurally and functionally, are remarkably similar to mammalian inner ear hair cells, both possessing the ability to sense mechanical stimuli and convert them into electrical signals. This characteristic makes zebrafish-based research results highly valuable for translation, providing important references for a deeper understanding of the physiological and pathological processes of the mammalian inner ear. This study used a Tg(brn3c:mGFP) transgenic zebrafish model to investigate the protective effect of Tro against cisplatin-induced hair cell damage. The hair cells specifically express green fluorescent protein (GFP), providing a direct labeling system for observing hair cell morphology and survival in vivo. Quantitative analysis using GFP immunofluorescence staining directly assessed the degree of protection against cisplatin-induced hair cell damage by Tro pretreatment.

[0058] The Tg(brn3c:mGFP)s356t transgenic zebrafish used in this experiment were provided by the Zebrafish Laboratory of Peking University. The entire animal experimental protocol strictly followed relevant national and international regulations and requirements for animal experiments, and had been formally approved by the Animal Ethics Committee of Peking University, ensuring that animal welfare was fully respected and protected throughout the entire experimental process, and that ethical standards were strictly followed.

[0059] In zebrafish husbandry and management, an advanced fully automated zebrafish rearing system was selected to create a stable and suitable living environment for the zebrafish. This system precisely and strictly controls various key environmental parameters: the temperature is consistently maintained at 28 ± 0.5 ℃, the pH value is stably maintained at 7.0, and the conductivity is set at 500 μS. These regulated environmental conditions are highly compatible with the environment required for zebrafish's natural growth, contributing to their healthy growth and development. Simultaneously, the rearing environment follows a 14 h / 10 h light-dark cycle, simulating the natural diurnal rhythm to ensure that the zebrafish's normal physiological rhythms are not disturbed. Furthermore, following a scientifically sound feeding plan, the zebrafish are fed twice daily at regular intervals to ensure they receive sufficient and balanced nutrients.

[0060] Zebrafish embryos were obtained by collecting them using the natural spawning characteristics of adult zebrafish. On day 5 post-fertilization (dpf), morphologically normal and vigorous juveniles were carefully selected from successfully hatched embryos according to strict selection criteria. These rigorously selected juveniles will serve as high-quality experimental subjects for subsequent research, laying a solid foundation for ensuring the reliability and accuracy of experimental results.

[0061] Example 9: Quantitative Analysis of Zebrafish Lateral Line Hair Cell Count Using GFP Fluorescence Staining 9.1 A 24-well plate was used to conduct the test drug treatment experiment on zebrafish larvae at 5 dpf post-fertilization. GFP green fluorescent antibody reagent (1:1000, A11122, Invitrogen) was used to specifically stain each group of zebrafish larvae, and the staining results were quantitatively analyzed to count the number of hair cells. The zebrafish were divided into three groups: control group (Con), cisplatin-treated group (Cis), and group pretreated with Tro for 2 h followed by co-treatment with cisplatin (Tro+cis). After treatment, GFP green fluorescent reagent was used to stain the lateral line hair cells of each group of zebrafish, and the stained hair cells were observed and quantitatively analyzed.

[0062] 9.2 Description of Results In the experiment, zebrafish with a growth factor of 5 dpf were selected and divided into: a control group, a cisplatin-treated group, a group pretreated with Tro (1 μM, 5 μM, 10 μM) for 2 h, and a group co-treated with cisplatin. After 1 h of treatment in each group, GFP green fluorescence staining was performed on the hair cells in the neurothalamus of each group. The results showed that compared with the control group, the number of hair cells in the zebrafish treated with cisplatin was significantly reduced; while compared with the cisplatin group, a Tro concentration of 10 μM could significantly reduce the cisplatin-induced loss of hair cells. Figure 8 A). The zebrafish were then further divided into: a control group, a cisplatin-treated group, a group pretreated with Tro (10 μM) for 2 h, and a group co-treated with cisplatin. The number of hair cells was observed and quantified. Figure 8 (B, C). The results showed that cisplatin reduced hair cells by nearly 70%, while 10 μMTro significantly restored the number of hair cells (p < 0.05), indicating that Tro has a protective effect against cisplatin-induced hair cell damage in zebrafish.

[0063] Example 10: Mouse ABR Detection To investigate whether Tro (Tr) has a protective effect against cisplatin-induced hearing loss in mice, a cisplatin-induced hearing loss model was established in mice. BALB / c mice aged 28 days were selected, and the hearing loss model was successfully established by intravenous injection of cisplatin via the tail vein. ABR (Auditory Breast Rate) tests were then performed to assess the hearing status of the mice.

[0064] 10.1 Laboratory Animals This experiment used healthy, 4-week-old BALB / c mice of similar weight and normal development as experimental subjects, sourced from Top Biotech (Guangdong, China). All animal experiments conducted at the First Affiliated Hospital of Shenzhen University strictly adhered to the protocols established by the institution's Animal Care and Use Committee. Before the start of the experiment, the experimental protocol underwent rigorous review and approval by the committee, fully protecting the rights of the experimental animals. The selected BALB / c mice were randomly divided into several groups to ensure the reliability and scientific rigor of the experimental results.

[0065] 10.2ABR audiometry In a soundproof and interference-free professional experimental environment, standardized equipment and procedures were used to perform Auditory Brainstem Response (ABR) threshold testing on all mice. ABR testing, as a widely used and sensitive technique for assessing auditory function, accurately reflects the functional state of different neural nuclei and synaptic connections in the auditory conduction pathway. First, tribromoethanol was used as an anesthetic, administered via intraperitoneal injection at a dose of 0.2 mL / 10 g to ensure the mice remained quiet and physiologically stable during the testing process. Once the mice were adequately anesthetized, they were gently and steadily placed on a constant-temperature heating pad to maintain stable body temperature and avoid affecting the results. Subsequently, a multi-channel neurophysiological signal acquisition and analysis system was used to measure the ABR hearing threshold of the mice. During measurement, six specific frequencies—8, 12, 16, 24, and 32 kHz—were selected sequentially to determine the mouse hearing threshold. A specific stimulation paradigm was used for each frequency: short sounds were used as stimulation signals, with the stimulation intensity gradually decreasing from a high level, such as 90 dB, until no obvious ABR waveform could be detected. At each stimulus intensity, stimulation was repeated multiple times (e.g., 500 times) to ensure sufficient reliability and stability of the acquired ABR signals. Using specialized data acquisition software and systems, the electrophysiological response signals generated by the mouse brainstem after sound stimulation were recorded synchronously. The acquired ABR signals underwent precise analysis and processing, including filtering, amplification, and averaging, to improve the signal-to-noise ratio and clearly identify ABR waveform characteristics. Based on parameters such as the presence, latency, and amplitude of the ABR waveform, the minimum stimulus intensity at which mice could produce a recognizable ABR response at each frequency was determined, i.e., the hearing threshold at that frequency. Detailed recording of hearing threshold data at each frequency provided crucial and accurate experimental evidence for analyzing changes in hearing function in mice under different treatment groups and exploring the effects of drugs or experimental factors on the mouse auditory system.

[0066] 10.3 Result Description: In the auditory function assessment, mice were divided into three groups: a control group (Con), a cisplatin group (Cis), and a Tro+cisplatin group (Tro+Cis). Placed in a specific acoustically shielded environment, mice in each group were subjected to sound stimuli of different frequencies and intensities. Throughout the testing process, changes in the mice's ABR threshold were precisely recorded and compared. The results are as follows: Figure 9Statistical analysis revealed that cisplatin treatment significantly increased the hearing threshold in mice, indicating that cisplatin can induce hearing loss in mice. Compared with the cisplatin group, mice pretreated with Tro showed significantly reduced hearing thresholds at 24 kHz (p < 0.05) and 32 kHz (p < 0.01) frequencies. Tro administration resulted in an ABR (Audio-Resonance Scale) improvement of over 20 dB in hearing thresholds, suggesting that Tro restores auditory function in vivo. This result indicates that Tro has a protective effect against cisplatin-induced hearing loss. These findings demonstrate that Tro can inhibit cisplatin-induced hearing and hair cell damage in vivo, providing valuable reference for research on the prevention and treatment of hearing loss.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of troxerutin in the prevention and treatment of cisplatin-induced ototoxicity.

2. The application of troxerutin according to claim 1 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The troxerutin mentioned above inhibits cisplatin-induced excessive ROS production.

3. The application of troxerutin according to claim 2 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The troxerutin mentioned above inhibits cisplatin-induced ferroptosis in hair cells.

4. The application of troxerutin according to claim 3 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The troxerutin mentioned above upregulates GPX4 and downregulates ACSL4, thus blocking ferroptosis.

5. The application of troxerutin according to claim 2 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The troxerutin mentioned above inhibits cisplatin-induced apoptosis.

6. The application of troxerutin according to claim 5 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The troxerutin reduces the expression of Caspase-3 and PARP, and inhibits apoptosis.

7. The application of troxerutin according to claim 1 in the prevention and treatment of cisplatin-induced ototoxicity can be prepared as an oral formulation, injection, sustained-release microspheres, or intratympanic injection formulation.

8. The use of troxerutin according to claim 1 or 7 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, Troxerutin can be used in combination with antioxidants such as NAC, MitoQ, or NMN to form compound preparations to enhance the protective effect.

9. The application of troxerutin according to claim 1 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The concentration of troxerutin used is 5~20μM.

10. The application of troxerutin according to claim 9 in the prevention and treatment of cisplatin-induced ototoxicity, characterized in that, The concentration of troxerutin used is 10 μM.

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