Animals

Six- to eight-week-old male FVB/NJ mice with normal hearing confirmed by the auditory brainstem response (ABR) were used. The mice were housed under standard laboratory conditions on a 12 h:12 h light-dark cycle at a constant temperature (22–24°C) and relative humidity of 50%–60% with free access to water and rodent chow. The protocols were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Laboratory Animal Welfare & Ethics Committee of Zhongnan Hospital of Wuhan University.

Three groups of mice (control, cisplatin, and H-151 + cisplatin) were used. As in previous studies [16], the mice in the cisplatin group were administered furosemide and cisplatin to establish an acute ototoxicity model. First, 200 mg/kg furosemide (South Land Pharmaceutical, Guangdong, China) was injected intraperitoneally. Half an hour later, 2 mg/kg cisplatin (HY-17394, MedChemExpress, New Jersey, USA) was administered subcutaneously. One hour later, 1 mL of isotonic NaCl was injected intraperitoneally. In the H-151 + cisplatin group, 10 mg/kg H-151 (HY-112693, MedChemExpress) was injected intraperitoneally one day before the first furosemide injection and 1 h before each furosemide injection to test the protective effect of H-151 in vivo. The control group was injected with an equal volume of saline. After the final injection, the mice were allowed to recover for three days before cisplatin treatment. ABR tests were applied to detect changes in hearing.

ABR Test

The ABR test is an objective measurement of the hearing threshold. Briefly, the mice were anesthetized via an intraperitoneal injection of 50 mg/kg pentobarbital sodium and kept warm at 37°C with a thermostatic heating pad during the ABR recordings. After the mice were fully anesthetized, electrodes were subcutaneously inserted behind the tested ear, behind the contralateral ear, and at the middle of the head. The hearing threshold was assessed at three frequencies (8, 16, and 32 kHz) on a TDT System III apparatus (Tucker Davies Technologies, Gainesville, FL, USA). Each frequency was measured from 90 dB and lowered by 10 dB each time until no ABR wave II response was detected to determine the threshold for each frequency.

Immunofluorescence Staining of Cochlear Surface Preparations

In accordance with the protocol detailed by Fang and colleagues [17], we proceeded as follows: temporal bones were extracted and locally infused with a solution of 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, through the round and oval windows and then incubated with this fixative at 4°C overnight. The cochlear samples were washed in PBS a minimum of three times for 5–10 min each throughout the various stages. Following 3 days of decalcification in a 4% solution of sodium ethylenediaminetetraacetic acid (in PBS, pH 7.4) at 4 °C, the cochleae were cut into apex, middle, and base segments. These segments were subsequently mounted on 10-mm circular coverslips using Cell-Tak adhesive (#354240, BD Biosciences, New Jersey, USA). Next, the samples were permeabilized in 2% Triton X-100 for 15 min and then blocked with 10% normal donkey serum for 30 min at room temperature, followed by incubation with a primary polyclonal rabbit anti-myosin 7a antibody (#25–6790, Proteus Bioscience, Waltham, USA) at 1:500 dilution overnight at 4°C. The samples were subsequently incubated with Alexa Fluor 488-conjugated secondary antibodies (1:500, A-11008, Thermo Fisher Scientific, Waltham, USA), phalloidin (1:500, 21836, Thermo Fisher Scientific), and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) overnight at 4°C in the dark.

Following three washes with PBS, 8 μL of mounting medium (S3023, DAKO, Copenhagen, Denmark) was added to the samples. These samples were then covered with an additional round coverslip and mounted on microscope slides. Finally, the edges of the coverslips were securely sealed using nail polish. Images of the immunostained samples were acquired under a Zeiss LSM 900 microscope at 40× magnification, ensuring consistent Z-stack conditions for all images.

HC Counts

Images of the entire stretch from the apex to the base of the cochlea, highlighting myosin 7a-labeled and phalloidin-stained surfaces, were captured under a Zeiss microscope with a 10× lens. The lengths of the cochlear epithelia were measured and recorded in millimeters. Given the negligible loss of inner HCs under our cisplatin treatment conditions, the focus was on counting OHCs from the apex to the base across the full extent of the cochlear spiral. OHCs were counted from the apex to the base along the entire length of the cochlear epithelium. The percentage of HC loss in each 0.5-mm length of the epithelium was plotted as a function of the cochlear length in a cytocochleogram [18].

Cell Culture

HEI-OC1 is a mouse auditory cell line available for research purposes [19]. HEI-OC1 cells were cultured in a 33°C incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (F8318, Sigma, Darmstadt, Germany) without antibiotics. Cultured cells were subcultured with 0.25% trypsin/EDTA (Thermo Fisher Scientific) after reaching 80% confluence.

Stable Cell Line Generation

Stable STING-EGFP-expressing HEI-OC1 cells were generated by transduction with the pcSLenti-CMV-Tmem173-linker-EGFP-3×FLAG-PGK-puro-WPRE3 lentivirus (OBiO Technology, Shanghai, China). Briefly, 2 × 105 cells were incubated in a virus-containing culture medium (MOI = 40:1) and polybrene (10 µg/mL) for 24 h. Then, the virus-containing medium was removed. After an additional 48 h of incubation, HEI-OC1 cells stably expressing STING through lentivirus transduction were selected with 10 μg/mL puromycin (Yeasen, Shanghai, China). After this, stable STING-EGFP-expressing HEI-OC1 cells were washed twice in PBS, incubated with 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.5% Triton X-100 for 10 min, blocked for 1 h with 10% donkey serum, and then incubated with an anti-GM130 antibody (1:500, 610822, BD Biosciences) overnight at 4°C in the dark. The next day, the cells were washed three times in PBS and incubated with an Alexa Fluor 594-conjugated secondary antibody (1:500, A-11012, Thermo Fisher Scientific) and DAPI overnight at 4°C in the dark. The coverslips were mounted on slides with a drop of fluorescence mounting medium (Dako, S3023) and sealed with clear nail polish. Labeling patterns were observed under a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany).

Transfection

siRNA-STING was designed and synthesized by OBiO Technology (Shanghai, China) to inhibit STING expression. HEI-OC1 cells were cultured on plates for 24 h and transfected with the STING or control siRNA using the Lipofectamine RNAiMAX reagent (13778150, Thermo Fisher Scientific). The transfected cells were exposed to cisplatin or a control medium, incubated for 24 h, and harvested for analysis. The following siRNAs were used to knock down STING expression: sense 5′-GGAUCCGAAUGUUCAAUCAGCTT-3′ and antisense 5′-GCUGAUUGAACAUUCGGAUCCTT-3′.

RNA-Seq

HEI-OC1 cells maintained for 24 h in a culture medium served as the control group and those cultured for 24 h in 30 μmol/L cisplatin served as the treatment group; the cells were subjected to transcriptome sequencing at Wefindbio Biotechnology Co., Ltd. (Wuhan, China) (three independent replicates per group).

Quantitative Real-time PCR

Total RNA was extracted from cells using TRIzol reagent (R411-01, Vazyme, Nanjing, China) according to the manufacturer’s protocol, and complementary DNA (cDNA) was synthesized from the purified RNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323, Vazyme). Quantitative RT‒PCR was applied using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (11184, Yeasen, Shanghai, China) on a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). ACTIN was used as a housekeeping gene for normalization, and the 2−ΔΔCt method was used to assess relative gene expression. The sequences of the primers used are listed in Table 1.

Table 1 Primers for real-time PCR.Western Blot

The samples were lysed in RIPA buffer (P0013B, Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride, phosphatase inhibitor (4906837001, Roche, Basel, Switzerland), phosphatase inhibitor cocktail 2 (P5726, Roche), and phosphatase inhibitor cocktail 3 (P0044, Roche). A BCA protein quantification kit (P0010, Beyotime) was used to measure the total protein concentration. Protein samples (15 µg per lane) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were anti-STING (1:1000; ab288157, Abcam, Cambridge, UK); anti-cGAS (1:1000; #31659, Cell Signaling Technology, Danvers, MA, USA); anti-TBK1 (1:1000; #38066, Cell Signaling Technology); anti-phospho-TBK1 (Ser172) (1:1000; #5483, Cell Signaling Technology); anti-phospho-NF-κB p65 (Ser536) (1:1000, #3033, Cell Signaling Technology); anti-NF-κB p65 (1:1000, #4764, Cell Signaling Technology); and anti-β-actin (1:1000, 20536-1-AP, ProteinTech, Wuhan, China). The membranes were then incubated with anti-rabbit secondary antibodies (ANT020, Antgene, Wuhan, China) for 1 h at room temperature. Finally, a chemiluminescence kit (E423, Vazyme) was used to detect the signals.

Flow Cytometry for Apoptosis Detection

HEI-OC1 cells were stained using a FITC Annexin V Apoptosis Detection Kit (556547, BD Biosciences). Annexin V was used to analyze apoptosis, and PI was used to differentiate between live and dead cells. The samples were trypsinized and collected via centrifugation at 1,000 rpm for 5 min, washed with PBS, and resuspended in binding buffer. Then, Annexin V and PI were added, and the mixture was gently vortexed and incubated for 15 min in the dark at room temperature. Flow CytExpert (CytoFLEX, Beckman, Brea, CA, USA) was promptly used to analyze the samples.

Flow Cytometry for Mitochondrial Membrane Potential Measurement

Image-iT™ TMRM Reagent (I34361, Thermo Fisher Scientific) was used as an indicator of the mitochondrial membrane potential. After the HEI-OC1 cells were collected, they were washed with PBS, incubated for 30 min at 37°C in the dark, and analyzed via CytExpert (CytoFLEX, Beckman).

Mitochondrial ROS Assay

MitoSOX Red (M36008, Thermo Fisher Scientific) was used to analyze mitochondrial ROS production. First, we measured mitochondrial ROS levels using flow cytometry. After trypsinization, the HEI-OC1 cells were collected via centrifugation and washed with PBS. The cell pellets were then resuspended in a solution containing MitoSOX Red for 15 min at 37°C in the dark and analyzed via CytExpert (CytoFLEX, Beckman). In addition, we assessed mitochondrial ROS levels via fluorescence staining. After treatment and washing with PBS, the cells were stained with MitoSOX Red in prewarmed serum-free DMEM in the dark for 30 min in an incubator. Images were acquired under a Zeiss LSM 900 confocal microscope.

Transmission Electron Microscopy

HEI-OC1 cells were fixed in 2.5% glutaraldehyde (Sigma‒Aldrich) for 24 h at 4°C in the dark and 1% osmic acid (Sigma‒Aldrich) for 1–2 h, dehydrated with acetone (Sinopharm Chemical Reagent), and embedded in Araldite CY212 (TAAB). Ultrathin sections were stained with uranyl acetate in alcohol (Polysciences) and alkaline lead citrate (Sigma‒Aldrich). The sections were gently washed with distilled water and examined under a Hitachi HT 7800 transmission electron microscope (Hitachi Ltd, Tokyo, Japan) by Servicebio Technology (Wuhan, China).

Immunofluorescence Staining for Mitochondrial DNA Release

The method used to detect mitochondrial DNA release was adapted from previously published methods [20, 21]. HEI-OC1 cells were incubated with the indicated treatments, washed twice with PBS, incubated with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.5% Triton X-100 for 10 min, blocked for 1 h with 10% donkey serum, and then incubated with an anti-dsDNA antibody (1:200, 690014, Progen, Heidelberg, Germany) and an anti-TOM20 antibody (1:500, 11802-1-AP, ProteinTech) overnight at 4°C. The following day, the cells were washed three times with PBS and incubated with an Alexa Fluor 488-conjugated secondary antibody (1:500, Thermo Fisher, A-11001), Alexa Fluor 594-conjugated secondary antibody (1:500, Thermo Fisher, A-11012), and DAPI overnight at 4°C in the dark. The coverslips were mounted on slides with a drop of fluorescence mounting medium (S3023, Dako) and sealed with clear nail polish. Labeling patterns were examined under a Zeiss LSM880 with an Airyscan confocal microscope. The number of DNA foci outside the nucleus and the mitochondrial perimeter were measured manually to quantify the number of cytosolic DNA foci.

TUNEL Assay

The TUNEL assay was applied according to the protocols of the In Situ Cell Death Detection Kit TMR Red (1215679291, Roche). After staining with DAPI and the required antibodies, a confocal laser scanning microscope (Leica TCS SP8) was used for visualization.

Statistical Analysis

All of the data are presented as the mean ± SD. All of the experiments were repeated at least three times. Statistics were calculated using Microsoft Excel and GraphPad Prism 9. Statistical significance was determined using a two-tailed unpaired t test when two groups were compared and using one-way ANOVA and Dunnett's multiple comparisons test when more than two groups were compared. P values <0.05 were considered to indicate statistical significance.