A total of 48 BALB/c mice (3 days) were purchased from the Animal Experimental Center, Jinzhou Medical University, China. A total of 48 BALB/c mice (20-22 g, 4 weeks, male and female) with normal Preyer's reflexes were purchased from the Animal Experimental Center, Dalian Medical University, China [license no. SCXK (Liao) 2008-0002]. All animal studies complied with the regulations for the Management of Laboratory Animals published by the Ministry of Science and Technology of the People's Republic of China, and all animal protocols were approved by the Animal Care and Use Committee of Jinzhou Medical University. All mice were fed a standard commercial diet and housed at an ambient temperature of 20-24°C with a relative humidity of 50±5% under a 12/12 h light-dark cycle in a specific pathogen-free facility during the experiment. All animals were allowed 1 week to acclimate before treatment and then were randomly divided into four groups (n=12 in each group) and treated for five days as follows: i) Normal control group, where the mice received physiological saline [0.5 ml/100 g, intra-peritoneally (i.p)]; ii) UA group, where the mice received UA (purity ≥98%; cat. no. N1823, APExBIO Technology LLC; 80 mg/kg/day, i.p); iii) CDDP group, where the mice received CDDP (Sigma-Aldrich; Merck KGaA; 4.5 mg/kg/day, i.p,) and iv) UA plus CDDP (UA+CDDP) group, where the mice received UA (80 mg/kg/day, i.p) followed by CDDP (4.5 mg/kg/day, i.p) 30 min later each day (Fig. 1A).

During the treatment period, the weight of the experimental animals was measured every day (Fig. S1). Although CDDP is toxic, the survival rate of mice is 100% during the administration (33).

For the analysis of the auditory threshold, ABR was recorded 1 day before and 5 days after related drug treatment with tone bursts of 8, 12 and 24 kHz using the Smart EP & OAE auditory evoked potential recording system (Intelligent Hearing Systems). The mice were anesthetized using pentobarbital sodium (90 mg/kg) and kept warm with a heating pad during ABR recording. A subdermal (active) needle electrode was inserted at the vertex, while ground and reference electrodes were inserted subdermally in the loose skin beneath the pinnae of opposite ears. The technique used to record ABRs has been previously described in detail (34). The ABR waveforms were averaged over a 10 msec time window using the Smart EP & OAE auditory evoked potential recording system software (Launch Pad 2.32 HIS program). The sound intensity was varied at 5 dB intervals near the hearing threshold. The differences in ABR thresholds shift for each frequency between the starting and the terminal points of the experimental time course were noted. The threshold was determined off-line by two independent, experimentally blinded observers based on the ABR records. In addition, the mice were euthanized, and double cochleae were removed for further analysis.

Immediately after completion of the ABR test, the animals were killed by cervical dislocation under anesthesia with an intraperitoneal injection of pentobarbital sodium (90 mg/kg). An array of criteria was used to verify the death of animals, including absence of respiration and heartbeat and loss of corneal and palpebral reflexes.

The temporal bones were removed, and the cochlea was immersed in 4% paraformaldehyde and incubated overnight at 4°C. The cochlea was decalcified in 4% EDTA for 7 days at 4°C. Subsequently, the basilar membrane was dissected under a dissecting microscope, and the stria vascularis and tectorial membrane were removed. The basilar membrane was then stained with phalloidin-tetramethylrhodamine (TRITC; Sigma-Aldrich; Merck KGaA) for 20 min at 20-24°C and protected from light. The specimens were then rinsed three times with 0.01 M phosphate-buffered saline (PBS; pH 7.4) and then mounted on slides. Images were obtained using the TCS-SP5 II laser-scanning confocal microscope (Leica Biosystems GmbH). Outer hair cells were counted from the captured images using a fluorescent BX41 microscope (Olympus Corporation) at ×200 magnification in 20 consecutive fields from the apex to the basal turn along the entire length of the cochlear epithelium. The percentages of outer hair cell loss in each 0.5-mm length of epithelium were plotted as a function of the cochlear length as a cytocochleogram (34).

After the last auditory brainstem response test, five mice from each group were decapitated under abdominal anesthesia using pentobarbital sodium (90 mg/kg), and the temporal bones were removed immediately. The round and oval windows were opened, cochleae were perfused with 40 g/l paraformaldehyde (pH 7.4), and the specimens were immersed in the 4% paraformaldehyde for 24 h at 4°C. Following decalcification in 40 g/l EDTA solution (pH 7.4) for 5 days at 4°C, the cochleae were dehydrated with ethanol, cleared with xylene and embedded in paraffin wax. Serial sections (5-µm thickness) were prepared, dewaxed, rehydrated in descending alcohol series, rinsed with distilled water and treated with high pressure (125°C; 103 kPa; 3 min) to retrieve antigens. Endogenous peroxidase activity was blocked with 3 g/l H₂O₂ for 10 min at 20-24°C, followed by a PBS wash. After incubation with normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 15 min at 20-24°C, tissue sections were stained overnight at 4°C with primary antibodies for TRPV1 (1:500; cat. no. ab6166; Abcam) and calpain 2 (1:1,000; cat. no. sc-373967; Santa Cruz Biotechnology, Inc.). After washing with PBS, sections were stained with horseradish peroxidase-conjugated-biotinylated goat anti-rabbit immunoglobulin G (IgG) secondary anti-body (1:1,000; cat. no. ab205718; Abcam) for 1 h at 20-24°C. Following washing with PBS, color was developed with 0.05% 3,3'-diaminobenzidine for 3 min at 20-24°C. Sections were counterstained with hematoxylin for 10 min at 20-24°C, dehydrated with gradient concentrations of ethanol, vitrified with xylene and mounted with neutral gum. PBS was used as a negative control throughout the study. The sections were observed by using a BX41 fluorescent microscope at ×400 magnification (Olympus Corporation). Relative optical intensity was determined using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

Total cochlea tissue was homogenized in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease inhibitor cocktail tablets and phosphatase inhibitors cocktail (Roche Diagnostics GmbH). The tissue homogenate was sonicated for 30 sec and centrifuged at 14,000 × g at 4°C for 30 min to extract the supernatant. Protein concentrations were determined using a bicinchoninic acid kit. The protein samples (50 µg/lane) were separated using SDS-PAGE (7.5% for TRPV1 and calpain 2; 10% for 4-HNE and procaspase 3 and 12.5% for cleaved caspase 3). Following electrophoresis, the proteins were transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Inc.). The membranes were then blocked with 10% bovine serum albumin for 1 h at 4°C and incubated overnight at 4°C with the following primary antibodies: Anti-TRPV1 (1:500; cat. no. ab6166; Abcam), anti-calpain 2 (1:1,000; cat. no. sc-373967; Santa Cruz Biotechnology, Inc.), anti-4-HNE (1:800; cat. no. ab48506; Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. ab13847; Abcam) and anti-caspase-3 (1:1,000; cat. no. sc-7272; Santa Cruz Biotechnology, Inc.). After washing three times with TBS-0.05% Tween-20, membranes were incubated with IRDye 800CW goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. 925-32211; LI-COR Biosciences.) for 1 h at 4°C. Following extensive washing, the immunoreactive bands were visualized by ECL. The Odyssey CLx Infrared imaging system (LI-COR Biosciences) was used to analyze the optical density value of immunoreactive bands. β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) was used as a loading control.

The organ culture procedures were modified from a previously published report (35). In brief, Cochlear explants were isolated from newborn 3-day-old BALB/c mice. BALB/c pups were euthanized following antisepsis using 75% ethanol. The two otic capsules were extracted from surrounding tissue and immersed ice-cold Hank's balanced salt solution (HBSS; cat. nos. CC014 and CC016; M&C Gene Biotechnology). The lateral wall tissues (stria vascularis and spiral ligament) and the auditory nerve bundle were micro-dissected for sterile dissection under a binocular microscope (Olympus SZX7; Olympus Corporation). The explants were carefully placed onto a prepared culture dish containing a 15-µl polymerized drop of rat tail collagen in 1 ml culture medium consisting of DMEM/F12 (Invitrogen; Thermo Fisher Scientific, Inc.), 1% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.), and 10 U/ml penicillin G. Following 4 h of incubation (37°C, 5% CO₂), an additional 1 ml medium was added to submerge the explants.

Cell-permeable Ca²⁺ indicator Fluo-4 AM (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) were used to monitor levels of intracellular Ca²⁺, based on the literature (36). Following immersion in HBSS without Ca²⁺ and Mg²⁺ for 20 min at 20-24°C, the organ explants were immersed in dye (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) for 25 min following Fluo4-AM loading at 24°C. Subsequently, the explants were fixed in 4% paraformaldehyde for 30 min at 20-24°C. Phalloidin (1:200) was applied for 1 h at 20-24°C and protected from light. For measurement of Fluo-4 Ca²⁺ signals, organs were imaged under a BX41 microscope at ×400 magnification (Olympus Corporation) with epifluorescence, and the images were obtained using a TCS-SP5II laser-scanning confocal micro-scope (×400 magnification; Leica Biosystems GmbH) in 20 consecutive fields.

Preparation of cochlear paraffin sections (5-µm thickness), deparaffinization, rehydration, antigen retrieval and blocking nonspecific binding were performed as described in a previous study (34). Tissue sections were incubated with rabbit anti-4-HNE antibody (1:200; cat. no. ab46545; Abcam), followed by Cy3-labeled goat anti-rabbit IgG secondary antibody (1:500; cat. no. A0516; Beyotime Institute of Biotechnology) incubation for 1 h at 24°C. Immunofluorescence images were obtained via TCS-SP5II laser-scanning confocal microscopy (×400 magnification; Leica Biosystems GmbH). Relative optical intensity was determined using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The human ovarian cancer cell line SKOV3 (Peking Union Medical College, Beijing) were seeded at a density of 3×10³ cells/well in 96-well plates and treated with different concentrations (1, 2, 3, 4, 8, 10 and 16 µg/ml) of CDDP. Each concentration treatment was repeated in six separate wells. Cells treated with RPMI 1640 media (100 ul; Sigma-Aldrich; Merck KGaA) alone acted as a control. The cells were incubated at 37°C with 5% CO₂ for 24 h, and MTT reagent (20 µl; 5 mg/ml in PBS; Sigma-Aldrich; Merck KGaA) was added to each well and incubated at 37°C with 5% CO₂ for 4 h. The absorbance in each well was measured at a wavelength of 570 nm using an enzyme-linked immunoassay microplate reader. According to the tumor cell growth inhibition rate, 4 µg/ml of CDDP was determined as optimal inhibitory concentration. Subsequently, SKOV3 cells were seeded at a density of 3×10³ cells/well in a 96-well plate and treated with CDDP (4 µg/ml). Different concentrations (20, 40, 80 and 160 mol/l) of UA were added later. Each concentration of UA treatment was repeated in six separate wells. The cells were incubated at 37°C with 5% CO₂ for 24 h, and MTT reagent (20 µl; 5 mg/ml in PBS; Sigma-Aldrich; Merck KGaA) was added to each well and incubated at 37°C with 5% CO₂ for 4 h. The absorbance in each well was measured at a wavelength of 570 nm using an enzyme-linked immunoassay microplate reader (BioTek Instruments, Inc.).

Data are presented as the mean ± SD and were analyzed using SPSS 17.0 software (SPSS, Inc.). Intergroup differences in mean values were compared by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether UA could prevent CDDP-induced hearing loss, the ABR test was performed on mice. In the control group, ABR thresholds at 8, 12, and 24 kHz were observed to be stable (0.48±1.43, 0.50±1.37 and 0.67±1.44 dB, respectively). However, ABR threshold shifts in the CDDP group were significantly increased at 8, 12 and 24 kHz (25.78±2.06, 27.59±2.86 and 28.51±2.32 dB, respectively), compared with the control group (P<0.01). In the UA + CDDP group, ABR threshold shifts significantly reduced at 8, 12 and 24 kHz (P<0.01; 3.93±1.19, 4.63±1.16 and 5.38±1.36 dB, respectively), compared with the CDDP group. In the UA group, UA treatment alone did not affect the ABR threshold shifts compared with the control group. These results suggested that UA can mitigate CDDP-induced hearing loss in mice (Fig. 1B).

To further determine the in vivo protective effects of UA against CDDP, TRITC staining of OHCs was performed on the basilar membrane of mouse cochlea and the number of surviving OHCs was counted. In the control and UA groups, OHCs on the basilar membrane appeared normal in structure and the staining pattern was homogeneous. Moreover, the loss rate of OHCs at each turn of the basilar membrane was <10%. In the CDDP group, the stereocilia of OHCs were characterized by loss and disorder. OHCs exhibited regional variation in their susceptibility to cisplatin, with gradual severity of OHC loss from the apical to the basal turn, and loss rates of OHCs at apical, middle and basal turns reaching 16, 41 and 61%, respectively, which was significantly higher compared with the control group (P<0.01; Fig. 1C and D). However, compared with the CDDP group, animals receiving UA + CDDP demonstrated significantly fewer OHC loss, especially at basal and middle turns, with the loss rate of OHCs at apical, middle and basal turns reaching 13, 25 and 32%, respectively (P<0.01; Fig. 1C and D). These results suggested that UA effectively protects against CDDP-induced OHC structural damage. Numerous studies showed that CDDP mainly damaged OHCs, while the damage of inner hair cells was weak (8). Hence, the present study focused on the outer hair cell change in and inner hair cell counting was not investigated.

Western blot analysis showed that the CDDP group expressed significantly higher levels of cleaved caspase-3 protein (a marker for apoptosis) compared with the control group (P<0.01; Fig. 1E). By contrast, cleaved caspase-3 expression levels in the UA + CDDP group were significantly lower compared with the CDDP group (P<0.01; Fig. 1E). However, cleaved caspase-3 expression levels in the UA group were not significantly different compared with the control group (Fig. 1E). Again, these results are consistent with the notion that UA can prevent the auditory loss observed in mice treated with CDDP.

Based on immunohistochemistry results (Figs. 2A and 3A), TRPV1 and calpain 2 were uniformly distributed on the OHCs, spiral ganglion (SG), and stria vascularis (SV) of the cochlea in the control group. By contrast, TRPV1 and calpain 2 staining was unevenly distributed in the CDDP group, and the expression levels of these proteins significantly increased compared with the control group (P<0.01; Figs. 2B and 3B). In the UA + CDDP group, TRPV1 and calpain 2 staining was uniformly distributed, and expression significantly decreased compared with the CDDP group (P<0.01, respectively; Figs. 2B and 3B). UA treatment alone did not affect TRPV1 and calpain 2 expression in the mouse cochlea (Figs. 2A and B and 3A and B). These results were consistent with the results of western blot analysis (P<0.01; Figs. 2C and 3C).

In the control group, Ca²⁺ staining was uniformly distributed in OHCs of the basement membrane (Fig. 4A). In the CDDP group, Ca²⁺ staining was unevenly distributed (Fig. 4A), and its intensity significantly increased compared with the control group (P<0.01; Fig. 4B). In the UA + CDDP group, Ca²⁺ staining was more uniformly distributed (Fig. 4A), with significantly lower intensity compared with the CDDP group (P<0.01; Fig. 4B). The results suggested that CDDP could cause Ca²⁺ overload, and that UA could effectively inhibit Ca²⁺ influx in mice cochleae.

To investigate whether UA could alleviate oxidative stress, the levels of 4-HNE were analyzed. Based on immunofluorescence results, CDDP significantly enhanced production of 4-HNE in the OHCs, SG, and SV of mouse cochlea compared with the control group (P<0.01; Fig. 5A and B). However, 4-HNE production was significantly inhibited in the UA + CDDP group compared with the CDDP group (P<0.01; Fig. 5A and B). Moreover, compared with the control group, UA treatment alone did not affect 4-HNE production in the mouse cochleae (Fig. 5B). These results were confirmed by western blot analysis of 4-HNE levels (P<0.01; Fig. 5C). The results suggested that UA could prevent an increase in oxidative stress-related markers in the cochlea of mouse treated with CDDP.

Since CDDP is used as an anticancer therapeutic, it was investigated whether UA co-treatment would impair its antitumor effects. MTT assay was performed to evaluate the viability of the human ovarian cancer cell line SKVO3 in response to CDDP, UA, and UA + CDDP. While CDDP inhibited the growth of SKVO3 cells in a dose-dependent manner (Fig. 6A), growth inhibition of SKVO3 cells was significantly enhanced following UA + CDDP (4 g/ml) treatment. Moreover, UA inhibited the growth of SKOV3 cells in a dose-dependent manner (P<0.01; Fig. 6B), suggesting that UA enhanced the antitumor effect of CDDP in vitro.