The study utilized C57BL/6 mice acquired from the SPF Animal Facility of Wenzhou Medical University (Wenzhou, China). For the purpose of creating animal models that replicate cisplatin-associated hearing damage, 7–8 weeks male mice weighing 18–20 g were randomly assigned to experimental and control groups. Only male mice were used in this study to minimize variability associated with sex-specific hormonal fluctuations. Age-matched adult male C57BL/6 J mice (postnatal week 7–8, 19.2 ± 0.8 g) underwent stratified randomization using a computer-generated sequence (GraphPad Prism 9.0), allocating subjects to experimental or control cohorts under SPF conditions. Baicalin (purity≥95%, Sigma-Aldrich, United States, 21967-41-9) was dissolved in a vehicle consisting of 10% DMSO, 40% PEG400(Polyethylene Glycol 400), and 50% sterile saline. Based on previous in vivo studies demonstrating the protective efficacy and acceptable safety profile of baicalin, a dose of 30 mg/kg was selected in the present study to evaluate its otoprotective effects against cisplatin-induced ototoxicity (Wen et al., 2023; Chong et al., 2025). Mice in the experimental groups received daily intraperitoneal injections of cisplatin (3 mg/kg, injection, Hengri), while those in the control group were administered 0.9% sterile saline or baicalin (30 mg/kg) for 7 consecutive days. To evaluate the otoprotective potential of baicalin against cisplatin-hearing loss, mice were intraperitoneally injected with baicalin (30 mg/kg), followed by an intraperitoneal administration of cisplatin (3 mg/kg) 2 h later once daily for 7 days. The baicalin dosage in our experiment was selected based on a prior study assessing chinese medicine formulas against age-related hearing loss (Shi et al., 2025). All animal procedures were conducted in compliance with protocols approved by the Animal Experimental Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Approval No. WYYY-AEC-YS-2023-0479), adhering to the ethical principles outlined in the Guidelines for the Welfare and Ethical Review of Experimental Animals (2018).

C57BL/6 mouse pups at postnatal day 3 (P3) were humanely euthanized in compliance with institutional animal care protocols. Temporal bones were rapidly excised and immersed in ice-cold Hanks’ Balanced Salt Solution (Hyclone, United States) to maintain tissue viability. Under sterile stereomicroscopic guidance, the cochlear duct was isolated through sequential removal of the bony capsule, lateral wall structures, and Reissner’s membrane. Isolated cochlear specimens were then affixed to CellTaK-coated glass coverslips (Fisher Scientific, PA) and maintained in a culture medium consisting of DMEM/F12 (Invitrogen, United States) enriched with 10% FBS (Gibco, United States), neurotrophic supplements (1% N2 and 2% B27, Invitrogen), and 50 mg/mL ampicillin (Sigma, United States). Cultures were incubated under standard physiological conditions (37 °C, 5% CO₂) for subsequent experimental procedures.

For cochlear explant experiments, baicalin was dissolved in DMSO and diluted in culture medium to the indicated final concentrations (final DMSO concentration <0.1%). After 24 h of stabilization in culture, explants were pretreated with baicalin (45 μM) for 2 h, followed by exposure to cisplatin (30 μM) for an additional 24 h in the continued presence or absence of baicalin. Control explants received an equivalent volume of vehicle.

Auditory brainstem response (ABR) thresholds in anesthetized mice were measured using 4–32 kHz clicks/tone bursts. Mice received xylazine-ketamine anesthesia (8 mg/kg and 50 mg/kg, i. m.) and were maintained on a heating pad (37 °C). Subdermal electrodes were placed at the vertex (active), ipsilateral mastoid (reference), and contralateral mastoid (ground). Acoustic stimuli (4–32 kHz) were delivered via calibrated transducers in a soundproof chamber, with intensity reduced from 90 dB SPL in 5 dB steps until waveform disappearance. ABR recordings were acquired using TDT System III hardware (Tucker-Davis Technologies, United States).

Distortion product otoacoustic emissions (DPOAEs) were recorded with two primary tones (f1 and f2, f2/f1 = 1.2, f2 set 10 dB lower than f1) presented at intensities decreasing from 80 to 20 dB SPL in 5-dB steps. Responses at the 2f1–f2 frequency were analyzed, and thresholds were defined as the lowest intensity at which the emission exceeded the surrounding noise floor by at least two standard deviations. Measurements were obtained at 4, 8, 16, 24, and 32 kHz using an acoustic probe connected to the TDT system.

Hair cell quantification was conducted following a modified method adapted from previous study (Zhang Q. et al., 2023). In summary, cochlear samples were immunostained with phalloidin to label HCs. After defining the regions of interest at low resolution (×20 magnification), at least two non-overlapping Z-stacks per turn were acquired using a ×40 objective. The lengths of selected cochlear segments were measured, and the total numbers of inner hair cells (IHCs) and outer hair cells (OHCs) in each segment were counted using ImageJ software. Hair cell loss was calculated for each 0.300 mm cochlear segment and normalized to cochlear length (0% = apex, 100% = base). Loss patterns of IHCs and OHCs were plotted along the tonotopic axis, and mean cochleograms were generated by averaging individual data using custom software.

HEI-OC1 cells were obtained from the UCLA Technology Development Group (Los Angeles, CA, United States). In the investigation of cisplatin-induced ototoxicity, HEI-OC1 cells served as the principal model owing to their consistent metabolic vulnerability profile with mouse cochlear cells (Kalinec et al., 1999). HEI-OC1 auditory cells were cultured under permissive conditions (33 °C, 5% CO₂) using DMEM (4.5 g/L glucose) supplemented with 10% FBS, 100 U/mL penicillin-streptomycin, and 50 μg/mL ampicillin (pH 7.4 adjusted with NaHCO₃). Cells were passaged at 80% confluence using 0.25% trypsin-EDTA.

For cell viability assessment, HEI-OC1 cells were seeded into 96-well plates at a density of 5 × 10³cells per well and allowed to adhere overnight. Cells were pretreated with baicalin (30, 45, or 60 μM) for 2 h, followed by co-incubation with cisplatin (30 μM) for 24 h at 37 °C/5% CO₂. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan, CK04) according to the manufacturer’s instructions. Briefly, 10 μL of CCK-8 reagent was added per well, plates were incubated at 37 °C for 2 h in the dark, and absorbance was read at 450 nm using a microplate reader (Bio-Rad, United States). In parallel, nuclear morphology was analyzed after 4% paraformaldehyde fixation and DAPI staining (1 μg/mL, 15 min) using fluorescence microscopy (Nikon Eclipse Ti2), with automated ImageJ quantification. All experiments used cells between passages 5-15.

Protein extracts from cochlear explants and HEI-OC1 cells were prepared using ice-cold RIPA buffer (Protein Biotech, China, PB180001) with protease inhibitors (Sigma). Lysates were centrifuged (12,000 × g, 10 min, 4 °C), and supernatants quantified via BCA assay (Beyotime, Chian, P0012). Equal protein aliquots underwent SDS-PAGE (12% gel) and PVDF membrane transfer (Millipore). Membranes were blocked with 5% skim milk/TBST (1 h, RT), then incubated with primary antibodies (CST: cleaved Caspase-3 1:500, p38 1:1000, p-p38 1:2000; Abcam: Bax/Bcl-2 1:500; ZSGB-BIO: β-actin 1:1000) in 3% milk/TBST at 4 °C overnight. After washing, membranes were incubated with the corresponding secondary antibodies (1:5000, 1 h, RT). Protein bands were visualized using ECL reagents (Millipore) and quantified with ImageJ software, normalizing target proteins to β-actin. Signals were detected by ECL (Millipore) and quantified using ImageJ.

HEI-OC1 cells were seeded in 6-well plates (1 × 10⁵/mL) and cultured at 37 °C/5% CO₂ for 24 h. Mitochondrial polarization status in cells was assessed using JC-1 (Beyotime Biotechnology, China, C2006) or tetramethylrhodamine methyl ester (TMRM, Invitrogen, United States, T668) probes. HEI-OC1 cells were incubated with 5 μg/mL JC-1 working solution at 37 °C for 20 min in the dark, followed by two washes with ice-cold JC-1 buffer (pH 7.4). Cells were then counterstained with DAPI (15 min, 25 °C) and immediately imaged by confocal microscopy. The ratio of red (aggregates) to green (monomers) fluorescence was quantified using ImageJ. For TMRM staining, HEI-OC1 cells were incubated with 15 nM TMRM in complete culture medium at 37 °C for 25 min in the dark. After incubation, cells were washed once with pre-warmed PBS and immediately subjected to confocal fluorescence imaging. Fluorescence intensity was quantified using ImageJ software.

Each experimental condition was assessed through a minimum of three independent repetitions. Results are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons among groups in GraphPad Prism 10.0 and SPSS 20.0. A p-value <0.05 was considered statistically significant.

Previous study has shown baicalin alleviated gentamicin-triggered organ of Corti hair cell toxicity (Zhang and Yu, 2019), so we introduced baicalin in our study to determine whether it had protective effects on auditory cells against cisplatin. To assess baicalin’s protective efficacy in vivo, C57BL/6 mice were treated with baicalin at a dosage of 30 mg/kg body weight and subsequently injected with 3 mg/kg cisplatin 2 h later for 7 days, based on earlier studies (Wang et al., 2023; Guo et al., 2024) (Figure 1a). Six saline-treated animals without cisplatin or baicalin administration formed the control group. To evaluate the otoprotective efficacy of baicalin, auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) thresholds were systematically quantified across a range of frequencies (4, 8, 16, 24, 32 kHz) in murine subjects. As shown in Figure 1b, cisplatin administration resulted in a pronounced elevation of DPOAE thresholds at all tested frequencies compared with the control group (p < 0.001). Notably, the degree of impairment was more pronounced at higher frequencies (16–32 kHz), reflecting the basal turn vulnerability of cochlear hair cells to cisplatin injury. In contrast, animals co-treated with cisplatin and baicalin displayed significantly reduced DPOAE threshold shifts (p < 0.001 vs. cisplatin alone), indicating that baicalin effectively preserved OHC function. Similarly, ABR threshold shifts (Figure 1c) were markedly elevated following cisplatin exposure (p < 0.001 vs. control), but were substantially attenuated in the cisplatin + baicalin group (p < 0.001 vs. cisplatin), suggesting that baicalin protected against both hair cell dysfunction and auditory pathway impairment.

To corroborate these functional observations, we next examined the structural integrity of cochlear hair cells. Immunofluorescence staining for Myosin7a and phalloidin (Figure 1d) revealed that in control and baicalin-alone groups, the three rows of OHCs and one row of IHCs were well preserved throughout all cochlear turns. In contrast, cisplatin treatment induced extensive OHC loss, particularly in the middle and basal turns, while IHCs remained largely intact. Notably, baicalin co-treatment markedly reduced OHC loss and preserved the characteristic V-shaped stereocilia arrangement, especially in the basal turn. Quantitative analysis (Figure 1e) confirmed these findings: IHC counts showed no significant differences among groups at any cochlear turn, whereas OHC numbers were dramatically reduced by cisplatin (p < 0.001 vs. control). Co-administration of baicalin significantly preserved OHC survival at the apical, middle, and basal turns (p < 0.001 vs. cisplatin), whereas baicalin alone had no effect on hair cell counts.

Encouraged by the in vivo evidence of baicalin’s protective role against cisplatin-induced hearing loss, we next sought to validate its protective effects at the cellular level. To this end, we employed HEI-OC1 auditory cells to investigate the underlying cellular responses and dose-dependent cytoprotective efficacy of baicalin against cisplatin-induced ototoxicity. HEI-OC1 auditory cells were pretreated with a concentration gradient of baicalin (30, 45, 60 μM) for 2 h, subsequently sustained in co-exposure to 30 μM cisplatin for an additional 24-h period (Figure 2a). Immunostaining findings revealed that HEI-OC1 subjected to cisplatin exhibited a gradual recovery in cell count reduction and nucleus deformation with the rising concentrations of baicalin (Figure 2b). Quantitative CCK-8 analysis indicated that dose-dependent viability of HEI-OC1 cells was significantly increased to 63.17% ± 2.06% following the administration of 30 μM baicalin compared with the control group, and it was further enhanced to 76.81% ± 2.20% at concentrations of 45 μM, 74.83% ± 2.09% at 60μM (Figure 2c). Based on dose-response optimization, a 24-h pretreatment with 45 μM baicalin was established as the standard protocol for subsequent HEI-OC1 cellular investigations.

To further validate these findings at the level of primary tissue cultures, we next investigated the protective role of baicalin in cisplatin-exposed cochlear hair cells (HCs). After baicalin pretreatment, cochleae were exposed to cisplatin with or without continued baicalin to evaluate differences in HC survival. Spatiotemporal patterns of hair cell degeneration were quantitatively mapped through immunocytochemistry and cochleogram analysis, with specific focus on quantifying IHCs and OHCs cell loss gradients across experimental cohorts. Consistent with the quantitative mapping in Figures 2d–f, cisplatin administration induced a tonotopic gradient of hair cell degeneration, with the loss of OHCs being more severe than that of IHCs, which aligns with prior research indicating that cisplatin disproportionately affects OHCs (Bu et al., 2022; Gibaja et al., 2022). Baicalin alone did not cause measurable HC loss. Importantly, co-treatment with baicalin significantly attenuated cisplatin-induced HC degeneration. The protective effect was evident across all cochlear regions but was particularly pronounced in the mid-basal and basal turns. Moreover, baicalin preserved IHCs more effectively in the basal segment, where cisplatin-induced loss was otherwise prominent. These findings indicate that baicalin confers significant protection against cisplatin ototoxicity, with preferential preservation of OHCs along the tonotopic axis and a stronger effect in the basal cochlea.

To systematically evaluate the cytoprotective efficacy of baicalin on injury induced by cisplatin, the Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was utilized to detect apoptotic cells, which provides a sensitive and reliable indication of DNA fragmentation as a hallmark of programmed cell death. As demonstrated in the representative fluorescence micrographs of Figures 3a,b, cisplatin-induced apoptotic damage in cochlear hair cells (HCs) was characterized by the co-localization of TUNEL-positive nuclei (red fluorescence) with preserved phalloidin immunostaining (green fluorescence). Notably, the cisplatin + baicalin group exhibited a markedly lower proportion of TUNEL-positive cells (27.35% ± 2.63%) in comparison to the cisplatin group (53.12% ± 2.18%), indicating that baicalin effectively attenuated cisplatin-triggered apoptotic cell death in the cochlea. Consistently, TUNEL results in HEI-OC1 cells further corroborated these findings, revealing that a high percentage of TUNEL-positive cells (61.02% ± 1.98%) was detected in the cisplatin group, whereas a significantly reduced percentage of TUNEL-positive cells (18.56% ± 2.23%) was observed in the cisplatin + baicalin group (Figures 3c,d). These in vitro results collectively highlight a reproducible anti-apoptotic effect of baicalin across different experimental models.

Moreover, mechanistic interrogation of apoptotic signaling pathways was performed through protein-level profiling of core apoptosis-regulatory genes. Compared to the cisplatin-only group, HEI-OC1 cells co-treated with cisplatin and baicalin for 24 h exhibited a significant downregulation of cleaved Caspase-3 and the pro-apoptotic protein Bax, accompanied by a pronounced upregulation of the anti-apoptotic protein Bcl-2 (p < 0.001) (Figures 3e,f). This molecular expression profile suggests that baicalin inhibits the intrinsic apoptotic cascade, thereby enhancing cell survival.

ROS have been demonstrated to exhibit a strong correlation with the mechanism of HC injury caused by ototoxic medications (Thakur et al., 2024; Maniaci et al., 2024; Mirzaei et al., 2021), and excessive mitochondrial ROS production is widely recognized as a key initiator of oxidative stress–mediated cellular apoptosis. To investigate the association between baicalin and oxidative strain in auditory cells, Mito-SOX Red was employed to assess mitochondrial reactive oxygen species (ROS) buildup in HEI-OC1 cell line and cochlear explants following the different treatments. HEI-OC1 cells were incubated with baicalin before the co-treatment with cisplatin for 24 h, thereby enabling us to evaluate the potential preventive effect of baicalin on cisplatin-triggered oxidative injury. As shown in Figure 4a, the control group and the baicalin-only group exhibited virtually no MitoSox-Red–positive cells, suggesting that baicalin alone does not induce mitochondrial oxidative stress and that basal ROS levels in auditory cells remain low under physiological conditions. The relative fluorescence intensity of Mito-SOX Red exhibited a marked upregulation after 24 h of cisplatin exposure compared to the uninjured controls (p < 0.001), with this escalation being significantly attenuated by baicalin pretreatment (p < 0.01 vs. cisplatin alone) (Figures 4a,c), indicating that baicalin is able to effectively suppress cisplatin-induced mitochondrial ROS overproduction in auditory cells.

To provide molecular evidence for baicalin’s pharmacological inhibition of cisplatin-triggered ROS overproduction, MitoSOX Red mitochondrial superoxide staining with fluorescence intensity quantification was systematically performed in cultured hair cells (Figure 4b), thereby providing additional experimental evidence at the tissue level. The relative fluorescence intensity of MitoSOX Red in baicalin-cotreat group decreased substantially compared to the cisplatin-only group (p < 0.01) (Figures 4b,d), further confirming that baicalin markedly alleviates mitochondrial ROS accumulation within cochlear hair cells. Together, these findings strongly suggest that baicalin confers protection against cisplatin-induced oxidative stress both in auditory cell lines and in cochlear explants, thereby supporting its potential as a pharmacological agent for mitigating ototoxic injury.

Mitochondria are widely recognized as the principal generators of intracellular ROS and concurrently the organelle is most susceptible to ROS-triggered injury (Willems et al., 2015; Chakrabarty and Chandel, 2021), thereby serving as a central target for oxidative damage–mediated cellular dysfunction. To assess the primary indicator of mitochondrial dysfunction (Bazhin et al., 2020), we evaluated differences in mitochondrial membrane potential (ΔΨm) between baicalin-treated and untreated HEI-OC1 cells following cisplatin-induced damage using dual fluorescence detection with TMRM and JC-1 probes, both of which are established and complementary methods for monitoring mitochondrial polarization status. As depicted in Figures 5a,c, JC-1 probe exhibited characteristic mitochondrial aggregation with dominant red fluorescence in control HEI-OC1 cells, consistent with preserved transmembrane electrochemical polarity and intact ΔΨm. In contrast, cisplatin-exposed cells demonstrated a marked fluorescence shift towards green spectral dominance, reflecting JC-1 monomer predominance, which quantitatively confirmed ΔΨm collapse through fluorometric analysis (p < 0.001 vs. control). However, the JC-1 red/green fluorescence ratio notably increased in HEI-OC1 cells co-treated with baicalin (p < 0.001 vs. cisplatin alone), indicating partial restoration of membrane potential and suggesting that baicalin significantly counteracts cisplatin-induced mitochondrial depolarization.

Furthermore, TMRM staining showed a similar trend and served as an additional validation of ΔΨm changes. As shown in Figures 5b,d, the substantial reductions of fluorescence intensity of TMRM in the cisplatin-impaired cells also indicated the loss of ΔΨm (p < 0.001 vs. control), while baicalin treatment significantly restored TMRM fluorescence intensity (p < 0.01 vs. cisplatin alone). Collectively, these findings provide convergent evidence from two independent assays demonstrating that baicalin effectively preserves mitochondrial membrane potential and mitigates cisplatin-induced mitochondrial dysfunction in auditory cells.

Given the established correlation between oxidative stress-induced apoptosis and P38 MAPK activation (Zhang Q. et al., 2023; Debatti et al., 2017; Niemietz and Brown, 2023), we interrogated this signaling axis to determine its mechanistic contribution to ROS accumulation in HEI-OC1 cells. To this end, we performed pharmacological modulation of p38 signaling using both agonists and inhibitors to clarify its role in baicalin-mediated protection against cisplatin ototoxicity. MitoSOX™ Red staining revealed that cisplatin treatment caused a significant increase in mitochondrial ROS production compared to the control cells (p < 0.001), whereas baicalin co-treatment markedly suppressed this overproduction (p < 0.001 vs. cisplatin). Notably, co-administration of the p38 agonist anisomycin largely abolished the antioxidant effect of baicalin, resulting in ROS levels comparable to those observed in the cisplatin-only group, while inhibition of p38 signaling by SB203580 recapitulated the ROS-suppressive effect comparable to baicalin treatment alone (Figures 6a,c). TUNEL staining further confirmed that cisplatin exposure led to substantial apoptotic cell death (p < 0.001 vs. control), which was significantly alleviated by baicalin co-treatment (p < 0.001 vs. cisplatin). Similar to ROS results, anisomycin abrogated the anti-apoptotic effect of baicalin, whereas SB203580 treatment markedly reduced TUNEL-positive cells (Figures 6b,d).

At the molecular level, Western blot analysis revealed a marked increase in p-p38 protein levels following cisplatin treatment, accompanied by a reduction in p38 expression. Baicalin treatment effectively reduced p-p38 expression, an effect diminished by anisomycin but mimicked by SB203580 (Figures 6e,f). Collectively, the results demonstrate that baicalin attenuates cisplatin-induced mitochondrial ROS accumulation and apoptosis in auditory cells, at least in part, through inhibition of p38 MAPK activation.