HEI-OC1 cells (M8-0401, OriCell, Guangzhou, China), a mouse auditory cell line, were utilized in this study as an in vitro system to assess the functional response of Bhlhe40 in oxidative injury to cochlear hair cells. Following a publicly available protocol in Journal of Visualized Experiments (JOVE; http://www.jove.com/) [9,22], HEI-OC1 cells were cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM; G4510, servicebio, Wuhan, China), supplemented with 10% fetal bovine serum (FBS, 11011-8611, Tianhang, Huzhou, China) in the absence of antibiotics, and maintained at 33°C with 10% CO₂.

To construct the indicated recombinant adenovirus, the protein-coding DNA sequence (CDS) of Bhlhe40 (NM_011498) was cloned into the shuttle plasmid pDC315 (VT1500, YouBio, Hunan, China) by Auhui General Biology (Chuzhou, China). The recombinant plasmid pDC315, containing Bhlhe40-CDS, was generated by digesting the pDC315 plasmid with Nhel and Sa1I enzymes. HEK-293A cells were then co-transfected with the Bhlhe40-pDC315 plasmid and the adenovirus helper plasmid as per the manufacturer’s instructions to produce purified Bhlhe40-adenovirus (Ad-Bhlhe40) particles. Subsequently, HEI-OC1 cells were infected with Ad-Bhlhe40 particles to achieve Bhlhe40 overexpression.

HEI-OC1 cells were plated at a density of 4 × 10³/mL in a 96-well plate (5 × 10³ cells/well) and exposed to 2 mM H₂O₂ for 4, 8 or 12 h in a 33°C incubator with 10% CO₂. Following this exposure, the cells were subjected to cell counting kit-8 solution (CCK-8; KGA317, KeyGen Biotech, Nanjing, China) for 2 h at a volume of 10 μL per well. Cell viability was then assessed by measuring the absorbance at 450 nm using a BioTek® 800™ TS Absorbance Reader (BioTek, USA).

TRIpure reagent (RP1001, BioTeke, Beijing, China) was utilized for total RNA extraction as per the manufacturer’s instructions. The cDNA synthesis was carried out with M-MLV reverse transcriptase (D7160L, Beyotime, Shanghai, China) following the manufacturer’s guidelines. Real-time fluorescence quantitative PCR was conducted on ExicyclerTM 96 (Bioneer, Korea) using SYBR Green (SY1020, Solarbio, Beijing, China) and 2 × Taq PCR MasterMix (PC1150, Solarbio, Beijing, China). β-actin served as a housekeeping gene. The relative expression of Bhlhe40, Hdac1, Hdac2 and Hdac3 were calculated based on 2^−△△CT method. The PCR primers are provided as follows:

Mus Bhlhe40 forward primer (FP), AACGGAGCGAAGACAGC; reverse primer (RP), CCAAGTGACCCAAAGTAGTAAG.

Mus Hdac1 FP, GGGCACCAAGAGGAAAG; RP, GCGAATAGAACGCAGGA.

Mus Hdac2 FP, TTTTGGACCAGACTTCA; RP, CTACGACCTCCTTCACC.

Mus Hdac3 FP, ATGTATGAAGTTGGAGCAG; RP, TTTCGGACAGTGTAGCC.

Cells were lysed using RIPA lysis buffer (PR20001, Proteintech Group, Inc., Wuhan China) mixed with 1% protease inhibitor (PR20032, Proteintech Group, Inc., Wuhan, China). The lysate was then centrifuged at 10000 g for 3 min at 4°C to collect resulting supernatant. The concentration of protein was estimated using BCA Protein Concentration Assay Kit (PK10026, Proteintech Group, Inc., Wuhan, China). Samples containing 15–30 μg of proteins were loaded into each well and underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were subsequently transferred to a polyvinylidene fluoride membrane (LC2005, Thermo Scientific, Pittsburgh, PA, USA) and blocked with 5% skimmed milk in Tris-buffer-saline with Tween-20 buffer (TBST; PR20011, Proteintech Group, Inc., Wuhan, China). Protein expression levels were assessed following incubation with primary and secondary antibodies. Specifically, the membranes were probed overnight at 4°C with primary antibodies against BHLHE40 (1: 1000; 17895-1-AP, Proteintech Group, Inc., Wuhan, China), P53 (1: 1000; A0263, ABclonal Technology, Shanghai, China), P21 (1: 1000; A1483, ABclonal Technology, Shanghai, China), P16 (1: 1000; 10883-1-AP, Proteintech Group, Inc., Wuhan, China), Hdac2 (1: 1000; A2084, ABclonal Technology, Shanghai, China) or β-actin (1: 20000; 66009-1-Ig, Proteintech Group, Inc., Wuhan, China). After washing with Western Blot Liquid Detergent (PR20012, Proteintech Group, Inc., Wuhan, China), the membranes were then incubated with the appropriate secondary antibodies HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1: 10000; H + L, SA00001-2, Proteintech Group, Inc., Wuhan, China) or HRP-conjugated Affinipure Goat Anti-Mouse IgG (1: 10000; H + L, SA00001-1, Proteintech Group, Inc., Wuhan, China) for 40 min at 37°C. Following thorough rinsing of the membranes, the immune-reactive bands were visualized utilizing the Hypersensitive ECL Chemiluminescence Detection Kit (PK10003, Proteintech Group, Inc., Rosemont, IL, USA). The density value of bands were analyzed with the Gel-Pro-Analyzer software (Version 4.0, Media Cybernetics, Maryland, USA).

The AnnexinV-FITC Kit (KGA106, Nanjing, China) from KeyGen Biotech was utilized to quantify cell apoptosis. The indicated cells were initially plated in 6-well plates at a density of 5 × 10⁵ cells per well and then exposed to 2 mM H₂O₂ for 4, 8 or 12 h. Subsequent to centrifugation at 150 g for 5 min, the supernatant was removed, and the cells were washed twice with PBS before being resuspended in 500 μL of binding buffer. Finally, the cells were incubated with Annexin V-FITC/PropidiumIodide (PI) staining reagent for 15 min at room temperature. The apoptotic cell percentage were determined using a flow cytometer (NovoCyte, Agilent, USA).

Apoptosis in HEI-OC1 cells was evaluate by analyzing DNA fragmentation using an In Situ Cell Death Detection Kit (12156792910, Roche, Basel, Switzerland) following the manufacturer’s guidelines. The cell sections were stained with DAPI solution (D106471, Aladdin, Shanghai, China) for 5 min (protected from light) to visualize the cell nucleus. Following rinsing with PBS, the images were captured using a microscope (BX53, OLYMPUS, Japan).

Apoptosis was assessed by measuring the activity of Caspase-3 in cell culture extracts with the Caspase-3 Activity Detection Kit (C1116, Beyotime, Shanghai, China) as per the manufacturer’s protocol. Based on Caspase-3 catalyze the production of pNA (p-nitroaniline, yellow) from the substrate Ac-DEVD-pNA, Caspase-3 activity was assessed by measuring the absorbance of pNA at 405 nm.

The ROS Assay Kit (KGAF019) from KeyGen Biotech in Nanjing, China utilized the red fluorescent probe dihydroethidium (DHE) to evaluate cytosolic superoxide production. Specifically, after 48 h of adenovirus infection, cells were exposed to 2 mM H₂O₂ for 8 h. Afterward, the cell culture medium was aspirated, and the cells were incubated with 10 μM of DHE probe at 37°C for 30 min in the absence of light. To ensure the elimination of any residual DHE outside the cells, the incubated cells underwent three washes with serum-free cell culture medium. Finally, the cells were observed under an Olympus DP73 microscopic photography system (OLYMPUS, Tokyo, Japan).

Cell precipitates were resuspended in PBS and lysed through sonication. The protein content determination was carried out using a BCA protein assay kit (P0011, Beyotime, Shanghai, China). SOD (A001), CAT (A007) and GPx (A005) detection were performed with assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer’s experimental procedure. The optical density (OD) readings were taken at 550 nm for SOD activity, 405 nm for CAT activity, and 412 nm for GPx activity using a UV-Vis spectrophotometer (UV752N, Shanghai Youke Instrument and Meter Co., Ltd., Shanghai, China).

JC-1 Mitochondrial Membrane Potential Assay Kit (C2006, Beyotime, Shanghai, China) was employed to determine the ∆Ψm in mitochondria. The specified cells were harvested as per the manufacturer’s experimental procedure, rinsed with PBS, and incubated with 0.5 mL of JC-1 solution for 20 min. After centrifugation at 300 g for 3 min, the cells were washed twice with JC-1 buffer (1×), and then resuspended in 500 μL of JC-1 buffer (1×) for mitochondrial membrane potential analysis using flow cytometry (NovoCyte, Agilent, USA).

Cell climbing sheets were fixed in 4% paraformaldehyde (80096618, Sinoreagen, Shanghai, China) for 15 min and permeabilized with 0.1% tritonX-100 (ST795, Beyotime, Shanghai, China) for 30 min at room temperature. Following three washes with PBS, the specimens were sealed with 1% bovine serum albumin (BSA; A602440-0050, Sangon, Shanghai, China) for 15 min at 25°C. Subsequently, they were incubated with the primary antibody anti-BHLHE40 (1: 100; 17895-1-AP, Proteintech Group, Inc., Wuhan, China) at 4°C overnight. And a secondary Cy3-Goat Anti-Rabbit IgG antibody (1: 200; SA00009-2, Proteintech Group, Inc., Wuhan, China) was then applied under darkness at a concentration of 1:200 for 60 min. DAPI was used for nuclear re-stain. Following a rinsing in PBS, fluorescent visualization of HEI-OC1 cells was performed on a BX53 microscope (Olympus, Tokyo, Japan) with the DP73 photography system.

The activity levels of senescence-associated β-galactosidase (SA-β-gal), a marker of cellular senescence, were confirmed by a β-galactosidase staining kit (G1580, Solarbio, Beijing, China) according to the standard experimental procedure from manufacturer. Briefly, HEI-CO1 cells were fixed with 1 mL β-gal fixative for 15 min at 25°C, followed by incubation with 1 mL staining solution at 37°C overnight. Brightfield images were taken at 100× magnification using an Olympus IX53 microscope (OLYMPUS, Tokyo, Japan).

To investigate the potential regulatory of BHLHE40 on Hdac2 transcriptional activity, the luciferase reporter plasmid containing Hdac2 promoter region from −1000 to +100 relative to the transcription start site was constructed by inserting nucleotide fragments into the vector. For the luciferase activity assay, HEK293 cells were co-transfected with this luciferase reporter plasmid, Bhlhe40 overexpressing plasmid, and pRL-TK plasmid (KeyGen Biotech) with renilla luciferase gene. Forty-eight hours later, the luciferase activity was measured using the Luciferase Assay Kit (KGAF040, KeyGen Biotech, Nanjing, China) according to the manufacturer’s protocol.

The experiments were conducted in triplicate, with all values presented as the mean ± standard deviation (SD). Statistical analysis was performed using unpaired student’s t-test for comparisons between two groups and ordinary one-way ANOVA for comparisons among multiple groups. A p-value of less than 0.05 was considered statistically significant.

The results depicted in Figs. 1A and 1B demonstrated that HEI‑OC1 cells, when exposed to 2 mM H₂O₂ for 4, 8, and 12 h, exhibited a time-dependent pattern of cell death. Further flow cytometry analysis revealed that the exposure to 2 mM H₂O₂ led to a significant increase in apoptotic cells, with 31.91% at 8 h and 47.97% at 12 h (Figs. 1C and 1D). Additionally, a decrease in Bhlhe40 expression was observed in a time-dependent manner following H₂O₂ treatment, particularly evident after 8 h (Figs. 1E and 1F). Immunofluorescent assay results further supported this finding, showing a noticeable reduction in Bhlhe40 levels in HEI-OC1 cells exposed to 2 mM H₂O₂ for 8 h (Fig. 1G).

To investigate the relationship between H₂O₂-induced apoptosis of HEI-OC1 cells and the expression of Bhlhe40, a Bhlhe40 overexpressing HEI-OC1 cell line was generated using the pDC315 adenoviral vector system (Fig. 2A). The efficiency of Bhlhe40 overexpression was confirmed by real-time PCR and western blotting assay (Figs. 2B and 2C). Subsequent CCK‑8 assay demonstrated a significant reduction in HEI‑OC1 cell viability following H₂O₂ exposure, which was rescued by Bhlhe40 overexpression (Fig. 3A). Flow cytometry analysis indicated that Bhlhe40 overexpression also hindered H₂O₂-induced cell apoptosis (Figs. 3B and 3C), a finding further supported by followed TUNEL assays (Fig. 3D). Furthermore, activity detection revealed that H₂O₂ treatment led to an increase in Caspase-3 activity, which was counteracted by Bhlhe40 overexpression (Fig. 3E).

Subsequent analysis showed that the oxidative stress markers SOD, CAT and GPx were notably reduced in HEI‑OC1 cells after exposure to H₂O₂ (Figs. 4A–4C). Conversely, Bhlhe40 overexpression enhanced the activity of SOD, CAT and GPx (Figs. 4A–4C). Additionally, DHE probe detection indicated that Bhlhe40 overexpression alleviated the generation of ROS induced by H₂O₂ (Fig. 4D). Furthermore, JC-1 analysis revealed that H₂O₂ treatment led to a decrease in mitochondrial membrane potential, which represented the accelerated apoptosis. Nevertheless, this effect was reversed by Bhlhe40 overexpression (Fig. 4E).

The SA‑β‑gal staining showed that Bhlhe40 overexpression suppressed H₂O₂‑induced cell senescence, as evidenced by a decrease in the number of SA-β-gal-positive HEI-OC1 cells (Fig. 5A). Besides, the expression of P53, P21 and P16 was assessed in HEI‑OC1 cells. As depicted in Fig. 5B, the elevated levels of P53, P21 and P16 were observed in cells exposed to H₂O₂ but decreased after Bhlhe40 overexpression.

Histone acetylation has been previously identified as playing a role in the pathogenesis of noise-induced cochlear cell death and hearing loss [23]. In this study, we explored the effects of Bhlhe40 on the expression of histone deacetylases 1, 2, and 3 (Hdac1, Hdac2 and Hdac3). Our findings demonstrated that Bhlhe40 overexpression led to the inhibition of H₂O₂-induced Hdac2 expression, while not significantly affecting the mRNA levels of Hdac1 and Hdac3 (Fig. 6A). Utilizing the JASPAR online database (http://jaspar.genereg.net/), we identified 3 potential BHLHE40-responsive elements in the promoter region of Hdac2 (Fig. 6B). Further western blot analysis confirmed that Bhlhe40 overexpression suppressed the expression of HDAC2 protein (Fig. 6C), possibly due to the repression of Hdac2 transcription (Fig. 6D).