Methods

Animals

C57BL/6 J mice (male, 7 weeks old) were purchased from SPF Biotech (Beijing, China). Gadd45a conditional knockout (cKO) mice were obtained from Cyagen Biosciences, Inc. (Suzhou, China). GFICre mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All the mice were housed in a standard environment with a temperature of 22 °C under a 12/12 h light-dark cycle and a constant humidity of 50%–60%. The mice had free access to food and water. The cisplatin used for animal treatment was pharmaceutical-grade cisplatin for injection, obtained from Qilu Pharmaceutical (Hainan, China) Co., Ltd. (specification: 20 mg). The methods and primers used for mouse genotyping used in this experiment are shown in ¹: ¹. All animal procedures were approved by the Laboratory Animal Welfare & Ethics Committee of Zhongnan Hospital of Wuhan University (WP20240254) and were performed in accordance with the relevant institutional guidelines.

Gene knockout cell line construction and cell culture

The construction of gene knockout cell lines was performed as previously described in our earlier studies [31], [32], [33]. The immortalized OC1 cell line, which expresses multiple hair cell markers originating from the cochlear sensory epithelium, was utilized in our experiments. This cell line has been used in several previous studies [34], [35], [36], [37], [38], [39]. The OC1 cell line was cultured in high-glucose DMEM (Gibco, Life Technologies, Australia Pty Ltd., Australia) supplemented with 10% fetal bovine serum (F8318, Sigma, Darmstadt, Germany) in a 5% CO₂ incubator at 37 °C. The sequences of the siRNAs and primers used in this experiment are shown in ¹: ¹.

Auditory brainstem response test

The auditory brainstem response (ABR) test serves as an objective method for measuring hearing thresholds. Specifically, normal hearing ability was ensured before each treatment, as previously described [40], [41]. We first measured the body weights of the mice, after which ketamine (100 mg/kg) and xylazine (25 mg/kg) were injected intraperitoneally to anesthetize the mice. Following satisfactory anesthesia induction (as indicated by hand-pinching mouse toes without painful reflex movements), recording electrodes were inserted subcutaneously. The hearing thresholds of the mice were then tested at 8, 16, and 32 kHz frequencies and clicks using a Tucker-Davis Technologies (TDT) RZ6 system (Alachua, FL, USA).

RNA-sequencing, cell culture supernatant proteome-sequencing, and bioinformatic analyses

OC1 and Gadd45a-/- cells were seeded separately in 10 cm plates and harvested upon reaching 90%–95% confluence. The cells were subjected to transcriptome sequencing, and the cell culture supernatant was used for proteome sequencing at Novogene Biotechnology Co. Ltd. (Beijing, China). Differentially expressed genes were identified based on the sequencing results. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were subsequently performed for the differentially expressed genes to identify significantly enriched biological processes and signaling pathways. Terms and pathways with an adjusted P-value<0.05 were considered statistically significant. The JASPAR database (https://jaspar.elixir.no) was searched to assess whether GADD45A has a reported transcription factor-binding profile and to predict potential shared transcription factors regulating CXCL1, CXCL3, and CXCL10.

Immunofluorescence staining

The mice were sacrificed following ABR testing. The cochlea was then removed, and a hole was made in the apex of the cochlea. Paraformaldehyde (PFA) (4%) was slowly injected into the cochlea through the oval window using a 1 ml syringe. The outflow of PFA from the apex indicated that the entire cochlea was filled. After 1.5 h of fixation in 4% PFA on a shaker at room temperature, the cochleae were decalcified in 10% EDTA solution at 4 °C on a shaker. The EDTA solution was refreshed daily for 4 consecutive d. Following decalcification, the auditory epithelium was divided into two sections (apex-middle and base-hook) using ophthalmic scissors (Jinzhong Surgical Instrument, China). The cochlear basilar membranes were carefully isolated with dissecting forceps (Dumont, Switzerland) and pathology scalpels (Feather, Japan). The isolated cochlear basilar membranes were then allowed to adhere to 9-mm diameter coverslips with Cell-Tak (354240; Corning, USA), perforated with 2% Triton X-100 for 15 min at room temperature, and then blocked with 10% donkey serum at room temperature for 1 h. The membranes were subsequently washed three times with phosphate-buffered saline (PBS) for 5 min each and then incubated overnight with primary antibodies. The information on the antibodies used in this study is provided in ¹: ¹.

To calculate the number of cochlear hair cells lost, the samples were incubated with an anti-myosin VIIa antibody (#25-6790; 1:1000, Proteus Biosciences, USA) overnight at 4 °C. After three washes with PBS, the samples were stained with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (A-11008; 1:500, Invitrogen, Thermo Fisher Scientific, USA) and phalloidin conjugated to Alexa Fluor 594 (A-12381; 1:500, Invitrogen, Thermo Fisher Scientific, USA) overnight at 4 °C. Following three additional washes with PBS, the nuclei were stained with DAPI (10236276001; Roche, Switzerland) diluted 1:1000 for 30 min at room temperature. Phalloidin-labeled hair cell stereocilia and myosin VIIa-labeled cell cytoplasm were observed under a fluorescence confocal microscope using appropriate filters.

To examine the synapses of inner hair cells (IHCs), the cochlear basilar membranes were first isolated and then treated with 3% Triton X-100 for 30 min at room temperature to permeabilize the tissues. The membranes were subsequently blocked with 10% donkey serum at room temperature for 1 h. After blocking, the samples were washed three times with PBS for 5 min each. The samples were then incubated overnight at 37 °C with primary antibodies, including an anti-C-terminal binding protein 2 (CtBP2) IgG1 antibody (#612044; 1:200, BD Biosciences, USA) and an anti-AMPA-type glutamate receptor subunit 2 (GluR2) IgG2a antibody (#MAB397; 1:1000, Millipore, USA). The samples were subsequently washed three times with PBS for 5 min each. Next, the samples were incubated at 37 °C for 1 h with the following secondary antibodies: Alexa Fluor 594-conjugated goat anti-mouse IgG1 (A-21125; 1:1000, Invitrogen, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG2a (A-21131; 1:1000, Invitrogen, USA). This secondary antibody incubation was repeated for 1 h at 37 °C. After synaptic staining, the hair cells were stained with an anti-myosin VIIa antibody (#25-6790; 1:1000, Proteus Biosciences, USA) overnight at 4 °C. Finally, the cochlear basilar membranes were washed three times with PBS for 5 min each and incubated overnight at 4 °C with a secondary antibody: Alexa Fluor 647-conjugated goat anti-rabbit IgG (A-21235; 1:1000, Invitrogen, USA). Finally, all cochlear basilar membranes were washed three times with PBS for 5 min each, and 15 μl Dako mounting medium (Dako, Denmark) was added to each coverslip, which was then covered with another coverslip. The edges were sealed with nail polish. The cochlear basilar membrane was photographed using a confocal microscope (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany).

Neonatal cochlear membrane culture

Cochlear organotypic cultures were prepared from postnatal day 3 (P3) SD rat pups as described in our previous publications [42], [43], [44]. Following incubation of the cochlear membranes with cisplatin for 24 and 48 h, three cochlear membranes were harvested using dissecting forceps and placed into a 1.5 ml Eppendorf tube as one sample. Subsequently, 200 μl of RIPA lysis buffer (P0013B; Beyotime Biotech Inc., China) supplemented with protease inhibitor cocktail tablets (04693159001; Roche, Switzerland) was added to the tube. The samples were then homogenized using a freezing-type grinding instrument and subjected to Western blotting analysis.

Injection of siRNA into the inner ear through posterior semicircular canal

To evaluate the stability and retention time of siRNA in the inner ear, 5’-fluorescein amidite-labeled siRNA (5’-FAM-siRNA), with an excitation wavelength of 494 nm and an emission wavelength of 520 nm, was mixed with Invivofectamine 3.0 Reagent (Invitrogen, IVF3001, Thermo Fisher Scientific, USA) according to the complexation protocol. The siRNA was then injected into the inner ear via the posterior semicircular canal (PSC). An in vivo imaging system (IVIS SPECTRUM, PerkinElmer, USA) was used to evaluate the retention effect. Similarly, Gadd45a siRNA (catalog No. s64885; Thermo Fisher Scientific, USA) was injected into the left ear through the PSC for subsequent experiments.

Other experimental details are provided in the ¹: ¹.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 9.0; La Jolla, CA, USA). All the data are presented as the mean±standard deviation (SD). A paired t-test was used to compare the data between two groups. One-way analysis of variance (ANOVA) was used to assess differences among multiple groups. Tukey’s multiple comparisons test was used to assess differences between groups. P-values of <0.05 were considered statistically significant.

Footnotes

1. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.mmr.2026.100063. ↩ ↩² ↩³ ↩⁴ ↩⁵ ↩⁶ ↩⁷ ↩⁸