HEI-OC1 line were cultivated in 10-cm circular dishes for cell culture, using high-glucose medium (Gibco, 11965092, United States) supplemented with 10% fetal bovine serum (Gibco, A5670701, United States) and 0.1% ampicillin (Beyotime, ST008, China). The cell cultures were maintained at a temperature of 33°C in a controlled atmosphere with 10% CO2 concentration. Due to the unique properties of HEI-OC1 cells, their metabolic state is more stable under 33 °C culture conditions, which can better promote cell proliferation and reduce cell differentiation. Cells were passaged using a solution of 0.25% trypsin with EDTA (Gibco, 25200056, United States) when the cell population in the dish reached approximately 80%–90%.

Six mice were sterilized using 75% alcohol before undergoing dissecting to extract the cochlea. The isolated cochlea was then immersed in a 6 cm dissecting dish containing Hank’s Balanced Salt Solution (HBSS) (Beyotime, C0219, China). Using fine tweezers on a sterile surface, the spiral ligament, cochlear implant, and vascular structure were meticulously removed to isolate the cochlear basement membrane. This membrane was subsequently laid flat on a Cell-Tak-coated circular slide (Corning, 354,240, United States), which was then transferred to a four-hole dish with a six-cm diameter. The cochleas, numbering a total of twelve, were divided into separate experimental groups, control, BB-only, cisplatin-only, and BB combined with cisplatin groups. Each subgroup contained three basement membranes. These specimens were cultured in 3 mL of F12 high-glucose medium (Gibco, 11330–032, United States) along with B27 (Thermo Fisher, 17504044, United States), N2 (Thermo Fisher, 17502001, United States), and ampicillin, and were kept in a 5% carbon dioxide incubator at 37°C. After the attachment phase, Berberrubine (TargetMol, 15401–69–1, United States) was added to both the BB-only and the BB combined with cisplatin groups, achieving a final concentration of 5 μM and subsequently pre-incubated for 12 h. Subsequently, cisplatin was introduced to the groups cisplatin-only and BB combined with cisplatin groups, reaching a final concentration of 50 μM, after which the incubation continued for another 24 h.

Four-week-old male C57BL/6 mice were procured from Nanjing Qinglongshan Animal Co., Ltd., China. The animal studies conducted in strict compliance with the ethical standards of Southeast University, aligning with the national laws and regulations for animal experimentation. A total of twenty-four mice aged P28 were divided into four groups: control, BB gavage, cisplatin injury, and combined BB gavage with cisplatin injury. In the first week, the BB gavage groups and combined BB gavage with cisplatin injury groups received 20 mg/kg of BB (Yuanye, 15401–69–1, China) via gavage. The control and cisplatin injury groups were administered 0.9% saline orally. Moving into the second week, the gavage regimen continued for all groups, with the cisplatin-injured and combined BB gavage with cisplatin injury groups receiving an additional daily intraperitoneal injection of 4 mg/kg cisplatin. Similarly, the control group and the BB gavage group received injections of an equal amount of normal saline solution. Following the treatment period, the mice underwent ABR testing and hair cell counting.

The measurement of how alive the cells was done using the Cell Counting Kit-8 (CCK-8) (MCE, HY-K0301, United States). To begin with, the HEI-OC1 cells that were grown well were exposed to a 0.25% trypsin solution, and the enzymatic action was stopped by supplementing with an equivalent amount of growth medium. Subsequently, the cells were transferred to 15 mL centrifuge tubes for spinning at a speed of 1,000 rpm over a period of 5 min. The concentration of cells was determined with a cell counting chamber, followed by their seeding into 96-well plates at a density of 5,000 cells per well. Each well was categorized into different groups based on drug concentration, with 6 replicates wells in each group. Post drug treatment, CCK-8 reagent diluted in serum-free culture medium was added to each well and incubated for 1 h. A microplate reader was used to measure the absorbance at 450 nm, which was then utilized to compute the percentage of viable cells based on the obtained optical density readings.

To perform TUNEL staining, the 5 X equilibrium solution was first diluted to 1 X. Following dilution, the sample was placed in a four-well dish, and 1X equilibrium solution was added for equilibration for 30 min. Throughout this timeframe, the TUNEL reaction system (Beyotime, C1086, China) was prepared by combining ddH₂O, equilibrium solution, a fluorescent dye, and terminal deoxynucleotidyl transferase (TDT). After the equilibration process was finished, the balanced solution was removed, and 50 μL of the TUNEL reagent was added to each well of the 4-well dish. Following that step, the dish was placed in an incubator maintained at a temperature of 37°C for a duration of 1 hour, ensuring it was kept in the dark to prevent light exposure. Upon completion of the incubation period, the TUNEL staining solution was decanted, and DAPI staining solution (from Beyotime, C1002, China) was applied. Subsequently, the samples were subjected to a further incubation period of 1 hour at ambient temperature. Once the incubation was complete, the medium was aspirated off, and the samples underwent three successive rinses using PBS (Beyotime, C0221A, China) for 5 min per wash. The samples were subsequently sealed using nail polish and analyzed using a confocal microscope. Should immunofluorescence staining be necessary, the samples would undergo the appropriate immunofluorescence staining procedure.

For Annexin V-PI staining, initially the drug-treated cell samples underwent two washes with pre-cooled PBS. Subsequently, the Annexin V and PI Staining Solution, which was prepared using Binding Buffer (Beyotime, C1062S, China), was introduced to the cell samples. Using a pipette, Annexin V and PI Staining Solution was gently agitated to ensure thorough interaction with the cells. The cells were incubated for 10 min at ambient temperature. The setting was shielded from light. After incubation, the staining solution was aspirated or discarded and DAPI Staining Solution was applied for an additional 10-minute period. Following the DAPI staining procedure, the samples underwent three consecutive washes using PBS to remove any excess stain. To conclude the process, the samples were transferred onto microscope slides, and covered with coverslips, and then analyzed under a confocal scanning microscope.

To conduct ROS staining, had the culture medium removed. DCFH-DA (Beyotime, S0033M, China) was prepared by diluting it in a medium devoid of serum to a concentration ratio of 1:1,000. The diluted DCFH-DA solution was then applied to ensure full coverage of the cells. Following the preparation, The cells were placed in an incubator set to 37°C for 20 min. After the incubation, the cells underwent three washes with serum-free DMEM. This was followed by the removal of the staining solution and incubating the cells with DAPI staining for 10 min at room temperature. After the incubation, the cells underwent three rounds of washing using PBS. The samples were mounted onto slides with coverslips in place and then examined using a confocal microscope for detailed observation.

For MitoSOX staining, begin by removing the drug-treated samples from the culture medium. Prepare a MitoSOX staining solution (Beyotime, S0061S, China), dilute it to a concentration of 5 μM with HBSS. Apply the prepared MitoSOX solution to the samples, then place them in an incubator set to 37°C for a duration of 10 min, to ensure taking care to protect them from light exposure. Following incubation, carefully remove the staining solution and wash the samples three times with pre-warmed HBSS at 37°C, each wash lasting for 5 min. After the washing process, mount the cells onto slides and incubate them with DAPI staining solution at room temperature for 10 min, again protecting them from light. Subsequent washing of the samples should be done three times with PBS. To conclude, the cell samples onto microscope slides secure with coverslips, and then proceed to examine them under a confocal microscope. If immunofluorescence staining is necessary, follow the relevant immunofluorescence staining protocol.

To stain mitochondrial membrane potential, The working solution of JC-1 was diluted using ultra-pure water initially. Removal of the culture medium from the treated cell samples took place, followed by a single wash with PBS. Subsequently, the working solution of JC-1 (Beyotime, C2006, China) was combined with the culture medium and applied to the cell samples. The cells were then incubated at 37°C for 20 min. The JC-1 staining buffer was prepared using ultra-pure water and stored on ice. After the 20-minutes incubation, elimination of the staining solution and double washing of samples with the staining buffer took place. Lastly, the samples were observed using a confocal microscope.

HEI-OC1 cells, cochlear basement membrane, and cochlea samples underwent fixation in 4% paraformaldehyde. For the adult rat cochlea samples decalcification with EDTA solution was carried out prior to fixation. The cochlear basement membrane was then carefully dissected into three slices representing different turns (apical, middle, and basal) under a microscope post decalcification and fixation. Following the sample processing, the samples were permeabilized with PBST for 5 min, repeated three times. Subsequently, the samples were soaked with an immunofluorescence sealing solution for an hour. Once the blocking step was complete, the primary antibody specific for Myosin7a was introduced to the samples, which were then incubated at a temperature of 4°C in a refrigerator environment protected from light for an extended period overnight. On the following day, the primary antibody solution was aspirated off, the samples were washed extensively with PBST in three rounds of 5-minute intervals. Subsequent steps included the addition of the secondary antibody (goat anti-mouse AlexaFluor Plus 555) and DAPI, followed by an incubation period of 1 h at room temperature in the dark. Subsequently, the secondary antibody solution was eliminated, followed by a rigorous washing of the samples using PBST, which was done in triplicate for durations of 5 minutes per wash. When prepared, the samples were meticulously mounted onto DAKO-brand slides, topped with coverslips, and secured with a layer of clear nail polish to prevent leakage. The slides were left at room temperature for half an hour to allow the nail polish to dry before observation under a confocal microscope. The experiment utilized various primary and secondary antibodies as well as dyes such as goat anti-rabbit Alexa Fluor Plus 488, goat anti-rabbit Alexa Fluor Plus 555, goat anti-mouse AlexaFluor Plus 555 and phalloidin, in addition to DAPI staining solution. This meticulous process ensures the precise visualization and analysis of the cochlear structures and components in the samples, contributing valuable insights to the study of auditory system biology.

Mice were administered an isobarbital solution at a dosage rate of 10 mg/kg of body weight, rendered unconscious and then placed on a warming pad maintained at a temperature of 37°C. Judging the effectiveness of anesthesia by finger pinching reflex, if there is no response when pinching the toes of mice, it indicates that anesthesia is complete. Following anesthesia, ABR thresholds were recorded using a TDT system (Tucker Davies Technologies, Gainesville, FL, United States) at frequencies of 4, 8, 12, 16, 24, and 32 kHz.

The HEI-OC1 cells were distributed into two groups and cultured in six-well plates, designating three wells per group for the experiment. One of the groups served as the control, while the other was subjected to BB treatment. Once the cells had adhered, the experimental group received BB at a concentration of 5 μM, which was maintained for a period of 24 h. Following the treatment, RNA was harvested from the cells in each well and then proceeded to RNA sequencing, conducted by Lianchuan Biotechnology Co., Ltd., China. following successful RNA quality control checks.

Total RNA was extracted from lysed cells and reverse-transcribed into cDNA using a cDNA synthesis kit (Vazyme, RC112-01, China). The qRT-PCR reaction was set up with primers and a qRT-PCR master mix (Vazyme, Q511-02, China), The qRT-PCR program started with an initial denaturation at 95°C for 5 min, then 40 cycles of alternating between 95°C for 10 s and 60°C for 30 s. Following the amplification cycles, a melting curve analysis was conducted, which included steps at 95°C for 15 s, 60°C for a minute, and a final step at 95°C for another 15 s. The collected data were analyzed through the comparative Ct (cycle threshold) method, utilizing GAPDH as a housekeeping gene for normalization.

The data are expressed as mean values with their accompanying standard deviations (SD). For data that follows a normal distribution, a Student’s t-test was employed to assess the differences between two-group comparisons, while analysis of variance (ANOVA) followed by the Dunnett’s test was used for the comparison of three or more groups. For data that does not conform to a normal distribution, we use logarithmic transformation to transform it into data that conforms to a normal distribution, and then use the above statistical methods for analysis. Statistical analyses were conducted using Microsoft Excel and GraphPad Prism 9, with results considered statistically significant for P < 0.05.

A range of cisplatin concentrations, spanning from 0 to 100 μM in increments of 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM and 100 μM was applied to HEI-OC1 cells for exposure. The study’s findings indicated that cisplatin exhibited a lethal impact on approximately 50% of the HEI-OC1 cells within the concentration range of 20–30 μM (Figure 1B). Therefore, we chose 30 μM cisplatin for further investigation. To establish the optimal concentration of BB for treating HEI-OC1 cells before cisplatin treatment, varying doses of BB (1, 2, 5, 10, 20, 50, and 100 μM) were tested. The CCK-8 assay showed no toxic effects at cisplatin concentrations from 1 to 20 μM (Figure 1C). When HEI-OC1 cells were pre-incubated with BB concentrations ranging from 1 to 30 μM for 12 h, followed by concurrent treatment with 30 μM cisplatin and BB for an additional 24 h, it was found that a 5 μM of BB was effective in mitigating the damage caused by cisplatin to the cells (Figure 1D). Additionally, in explants culture models, pre-treatment with 5 μM BB alleviated the hair cell damage caused by cisplatin (Figures 1E–H). In summary, BB alleviates cisplatin-induced cell injury, enhancing cell survival in the exposure of cisplatin.

By creating mouse models to induce hearing loss with cisplatin (Figure 2A), Our investigation delved into the potential of BB to safeguard against hearing impairment triggered by cisplatin. The ABR results revealed severe hearing impairment in the cisplatin group with significantly elevated thresholds. Conversely, the group treated with BB showed a significant decrease in hearing impairment caused by cisplatin across the tested frequencies, which included 4, 8, 12, 16, and 24 kHz (Figure 2B). The immunofluorescence examination revealed a significant depletion of hair cells across the cochlea apex, midsection, and base in the basilar membrane area. Furthermore, pretreatment with BB mitigated this damage and restored hair cell counts, underscoring its role in safeguarding cochlear hair cells (Figures 2C–F). In conclusion, BB exhibits a protective effect against hearing loss caused by cisplatin in mice, highlighting its potential as a therapeutic agent for mitigating the ototoxic effects of cisplatin.

We utilized TUNEL and Cleaved Caspase-3 staining to evaluate apoptosis, pretreated HEI-OC1 cells with BB for 12 h before exposing them to cisplatin for 24 h. Compared to the control group, the group treated with cisplatin showed a significant elevation in indicators of apoptosis, such as TUNEL positivity and levels of Cleaved Caspase-3. In contrast, the group pretreated with BB showed a notable decrease in these apoptotic markers (Figures 3A–D). Additionally, the use of Annexin V-PI staining provided an additional method to evaluate the extent of apoptosis. Under the same treatment conditions, cisplatin exposure resulted in higher apoptosis rates than the control group. However, the addition of BB substantially reduced the apoptotic markers (Figures 3E–G). In summary, BB significantly mitigates cisplatin-induced apoptosis in HEI-OC1 cells.

In our study, cochlear explants underwent TUNEL and Cleaved Caspase-3 staining. BB was introduced at a concentration of 5 µM for 12 h, followed by cisplatin exposure for 24 h. Immunofluorescence analysis revealed that, in the cisplatin-treated group, the count of dual positive cells for TUNEL/myosin 7a and Cleaved Caspase-3/myosin 7a was elevated. Conversely, BB treatment mitigated hair cell damage, substantially reducing the levels of TUNEL and Cleaved Caspase-3, indicative of apoptosis (Figures 4A–D). In conclusion, BB significantly reduced hair cell apoptosis induced by cisplatin in the cochlear basement membrane in cochlear explants.

A significant accumulation of ROS in the mitochondrial compartment is crucial for the onset of hearing loss caused by cisplatin. To investigate ROS levels in HEI-OC1 cells, we utilized DCFH, MitoSOX staining, and JC-1 analysis to assess mitochondrial membrane potential. Following treatment with BB and cisplatin, the cells were stained with DCFH, MitoSOX, and JC-1. Following cisplatin treatment, a noticeable rise in the number of cells staining positively for DCFH, MitoSOX, and JC-1 indicators was detected when compared with the untreated control group. Notably, pre-treatment with BB significantly decreased the number of cells showing positivity for DCFH, MitoSOX, and JC-1 in HEI-OC1 cells (Figures 5A–G). Furthermore, the expression of MitoSOX in cochlear explants was evaluated in a similar fashion. In line with the cell-based results, cisplatin administration led to an elevation in ROS levels in cochlear hair cells, while BB pre-treatment mitigated ROS production (Figures 5H–I). In summary, the findings demonstrate that BB successfully diminishes the production of ROS triggered by cisplatin in both the HEI-OC1 cell line and cochlear explant cultures.

To further investigate how BB antagonizes cisplatin-induced ototoxicity, an RNA sequencing analysis was conducted on HEI-OC1 cells following treatment with BB. The data indicated that treatment with BB led to the upregulation of 226 genes and the downregulation of 65 genes, in contrast to the gene expression levels observed in the control group (Figures 6A–C). Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified that one of the major pathways affected by BB treatment is the promotion of folate synthesis (Figure 6D). Following up with Gene Set Enrichment Analysis (GSEA), it was found that the pathway associated with folate biosynthesis was significantly enriched in the group treated with BB when compared to the untreated control group (Figure 6E). Notably, genes involved in folate biosynthesis and metabolism such as Ccnd2, Reln, Pgf, Mylk3, Ppplr12c, and Thbsl were significantly altered in the BB treatment group. Real-time fluorescence quantitative PCR further validated the changes in mRNA expression related to folate biosynthesis in the BB treatment group compared to the control group (Figure 6F). The expression of Ccnd2, Reln, Pgf, and Ppplr12 genes increased, while the expression of Mylk3 and Thbsl genes decreased. The overall effect of these changes is to promote folate synthesis. The collective results imply that Promoting folate synthesis may be one of the mechanisms by which berberine antagonizes cisplatin ototoxicity.