Epac1 Alleviates Senescence in Auditory Hair Cells via the Ferroptosis

ABSTRACT

Objectives

The aim of this study was to investigate the changes in Epac1 and ferroptosis‐related proteins in aged mice and the House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells, with the goal of elucidating their potential role in age‐related hearing loss.

Methods

The expression of Epac1 in the cochlea of C57BL/6J mice was examined by using immunofluorescence staining and Western blot analysis. To further verify the role of Epac1 in age‐related hearing loss, small interfering RNA (siRNA) was transfected into HEI‐OC1 cells, resulting in the downregulation of Epac1.

Results

The results revealed a decrease in Epac1 expression in the cochlea of aged mice. After downregulating the level of Epac1 in vitro, a greater level of ferroptosis was observed, and the aging process was accelerated.

Conclusions

Epac1 plays a crucial role in the aging process of auditory hair cells via ferroptosis.

1. Introduction

Age‐related hearing loss (ARHL) is becoming increasingly common as the population ages. Recent studies have shown that ARHL is closely linked to Alzheimer's disease, which leads to loneliness and depression among the elderly, ultimately reducing their sense of well‐being [1, 2]. However, current clinical treatment options for ARHL are still limited and cannot offer significant improvement for many patients. Therefore, there is an urgent need to identify effective genetic targets to improve ARHL and develop safer and more effective therapeutic strategies, which could help delay organ aging and alleviate the societal burden of an aging population.

Epac (Exchange Protein directly Activated by cAMP) is a cAMP‐dependent exchange protein and a key downstream effector of cAMP signaling in cells. It is an important stress response signaling molecule that regulates physiological and pathological processes in various diseases. In neurons, changes in cAMP levels are closely associated with membrane depolarization. In mammals, Epac exists in two isoforms, Epac1 and Epac2 [3]. Previous studies have shown that cAMP fluctuations occur during cochlear hair cell excitation, and research from our group has demonstrated the pivotal role of Epac1 in a rat model of noise‐induced hearing loss [4]. Deletion of Epac1 alleviates noise‐induced hearing loss by inhibiting hair cell apoptosis. Both noise‐induced and ARHL are characterized by high‐frequency sensorineural hearing loss. Furthermore, previous studies have demonstrated that activation of the Epac1 pathway can effectively reduce the increased membrane permeability of endothelial cells that occurs with aging [5]. Resveratrol competitively inhibits cAMP degradation, activating its effector Epac1, thereby increasing intracellular calcium levels and exerting anti‐aging effects [6]. Interestingly, Epac1 has also emerged as a promising candidate in the realm of neuroprotection. Studies have shown that activation of Epac1 can reduce neuronal and retinal damage caused by ischemia/reperfusion. These findings lend strong support to the idea of Epac1 as a potential target for treating neurodegenerative diseases [7]. However, the precise link between Epac1 and ARHL remains elusive, with the underlying mechanisms still awaiting further investigation.

Ferroptosis is an iron‐dependent form of cell death, characterized by the rupture of mitochondrial membranes and the disappearance of mitochondrial cristae [8]. Several studies have shown that there is a close mechanistic relationship between ferroptosis and hearing loss, with neurodegenerative changes in the auditory cortex being alleviated by the inhibition of ferroptosis [9]. For example, a decrease in the activity of the Nrf2 signaling pathway can result in mitochondrial DNA damage and increased oxidative stress, thereby precipitating neuronal degeneration [10]. In addition, ferroptosis plays a pivotal role in the progression of both sensorineural and conductive hearing loss [11, 12]. Inhibition of GPX4‐mediated iron accumulation can effectively mitigate cisplatin‐induced hearing loss in C57BL/6J mice [13]. Ferroptosis inhibitors, such as Fer‐1, have also been shown to reduce noise‐induced hearing loss by inhibiting ferroptosis and cell lipid peroxidation assay [14]. Earlier studies focusing on auditory centers confirmed that attenuating ferroptosis can partially reverse neurodegenerative changes in the auditory cortex [9]. In previously published research by the applicant, a close association between ARHL and ferroptosis was identified. The study found that the downregulation of calcium‐activated potassium channel protein α1 (KCNMA1) expression in cochlear hair cells delays cell aging by inhibiting ferroptosis, thereby alleviating ARHL [15].

In summary, we hypothesize that Epac1 channel activation can inhibit the iron death process of hair cells and achieve the effect of delaying cochlear hair cell aging. This study will reveal that Epac1 may be an effective new target for age‐related deafness and provide new strategies and means for clinical prevention and treatment of age‐related deafness.

2. Method

2.1. Animals

There were 10 C57BL/6J mice purchased from Hefei Qingyuan Biotechnology Co. Ltd. They were evenly divided into two groups: the young mice (2 months) and the old mice (12 months). These animal experiments have been approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (2024‐N(A)‐401). The experimental mice were maintained in a specific pathogen‐free environment at 20°C–22°C, with free access to food and water to ensure their health. After a 1‐week acclimation period, the ABR test was used to assess the auditory function of the mice. During the testing process, we strictly adhered to ethical standards and ensured that the suffering of the mice was minimized as much as possible. The mice were killed by inhalation of excessive isoflurane after testing.

2.2. Auditory Brainstem Response

Pentobarbital (50 mg/kg) was injected to induce general anesthesia. Tucker‐Davis Technology System hardware and software were used for ABR tests (New York, USA). Once the C57BL/6J mice were anesthetized, subdermal needle electrodes were placed subcutaneously at the vertex, beneath the left ear (reference), and on the right ear (ground). The frequencies measured during the ABR tests included 4, 8, 16, 24, and 32 kHz. An average response to 520 stimuli was obtained by reducing the stimulus sound intensity at intervals near the threshold from 90 to 10 dB SPL. After the electrophysiological response to the stimulus, the sound disappears. The lowest stimulus level that shows a response was regarded as the threshold.

2.3. Immunofluorescence Staining

The cochlea from the dead mice were separated from the temporal bone and injected with 4% paraformaldehyde overnight at 4°C. The cochlea was immersed in 10% EDTA for decalcification for 5 days. After decalcification, remove the softened ear sacs, stria vascularis, tectorial membrane, and Reissner's membrane under the stereomicroscope. We permeabilized the cochlear basilar membrane for 1 h at room temperature with 0.3% Triton X‐100. To reduce non‐specific binding, we blocked with goat serum at room temperature for 1 h. The cochlear basilar membrane is incubated overnight at 4°C with an anti‐Epac1 antibody and an anti‐myoⅦa antibody (Table 1). After washing the specimens three times with PBS, the specimens are mixed with secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Abcam, USA) at a concentration of 1:100. This step is performed at room temperature and incubated in the dark for 1 h. Then, the samples are counterstained with Dapi (Yeasen, Shanghai, China). under dark conditions at room temperature for 5 min. Finally, the samples are observed and imaged using the Leica DMI8 imaging system (Leica, Germany).

Table 1.

Information about antibodies in this study.

Antibody	Ratio	Brand	Art. no.
GAPDH	1:5000	Proteintech	10494‐1‐AP
Epac1	1:1000	Abcam	ab109415
P21	1:1000	Cell Signaling	2947S
P16	1:1000	Cell Signaling	80772S
GPX4	1:1000	Abcam	ab125066
FACL4	1:2000	Proteintech	22401‐1‐AP

2.4. Cell Culture and Drug Treatment

The House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells, which is an inner ear cell line donated by Professor Hao Xiong at Sun Yat‐sen University, were used in this experiment. HEI‐OC1 cells were cultured in DMEM/high glucose (Gibco, China) supplemented with 10% fetal bovine serum (Gibco, Australia) and 1% penicillin–streptomycin–amphotericin B Solution (Gibco, USA) in an incubator at 33°C with 10% CO₂. To investigate the optimal concentration and duration for D‐galactose‐induced cellular senescence, this study divided the cells into three groups for systematic analysis: no D‐galactose treated as control group, 15 mg/mL D‐galactose treatment for 48 h group, 20 mg/mL D‐galactose treatment for 48 h group, To investigate the key role of Epac1 gene in HEI‐OC1 cells, we designed siRNA‐Epac1 (sequence information at Table 2) to knock down the expression of Epac1 in HEI‐OC1 cells.

Table 2.

Information about small interfering RNA (siRNA) sequence in this study.

Categories	Sequence (5′ to 3′)
siRNA1	GGAACAGUAUCCAGAGAGATT
	UCUCUCUGGAUACUGUUCCTT
siRNA2	UCAAGGAUGUGGAAGCAAATT
	UUUGCUUCCACAUCCUUGATT
siRNA3	CCACGUUGCACGAGGGAGATT
	UCUCCCUCGUGCAACGUGGTT

2.5. siRNA Transfection in HEI‐OC1 Cells

siRNA‐Epac1 was designed to knock down the expression of Epac1 in HEI‐OC1 cells. The cells were cultured for 18–24 h in a six‐well plate to ensure that the monolayer cell density reached the optimal 60%–70% confluency at the time of transfection. Complete culture medium with serum and antibiotics was freshly added to each well 30 min before transfection. Prior to transfection, two EP tubes were prepared, each containing 100 µL of culture medium. Two microliters of siRNA were added to the first tube and 3 µL of PepMute transfection reagent (SignaGen, USA) was added to the second tube. Both solutions were incubated at room temperature for 5 min. Then, the contents of the two tubes were combined and incubated at room temperature for 15 min. The mixture was added dropwise to the six‐well plate. After 24 h, the culture medium was replaced. Transfection was terminated after 48 h. The efficiency of transfection was confirmed via Western blot analysis and Immunofluorescence staining.

2.6. shRNA Transfection in HEI‐OC1 Cells

Before transfecting shRNA into HEI‐OC‐1 cells, the cells were cultured in a six‐well plate to an appropriate growth density (typically 60%–70%). One microliter of shRNA plasmid DNA was diluted in 200 µL of Opti‐MEM reduced serum medium. Then, 5 µL of EL Transfection Reagent (TransGen Biotech, Beijing, China) was added to the diluted shRNA plasmid. The mixture was gently vortexed and incubated at room temperature for 15 min to allow complex formation. The DNA‐transfection reagent complexes were added dropwise to the cell cultures. Cells were maintained in a humidified 37°C, 5% CO₂ incubator. Transfection efficiency was evaluated 48 h post‐transfection using appropriate assessment methods.

2.7. Western Blot Analysis

Cold RIPA lysis buffer (Thermo Fisher, USA) was used with PMSF (TargetMol, USA) to lyse HEI‐OC1 cells and the cochlea of mice. The cochlea samples were homogenized for 60 s at 70 Hz using an automatic sample grinder (Sakezi, Anhui, China). After incubating on ice for 30 min, the processed samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was stored as total protein. Using the BCA protein concentration assay kit (Abbkine, Wuhan, China) to detect protein concentration. SDS‐polyacrylamide gel electrophoresis separated proteins, which were then transferred to PVDF membranes (Millipore, USA) blocked with QuickBlock Blocking Buffer (Beyotime, Shanghai, China). The blots were incubated with Epac1, p16, p21, GPX4, FACL4, and GAPDH (Table 1) primary antibodies at 4°C overnight. The next day, the sample was incubated with the secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 h, and the immune reaction bands were detected using enhanced chemiluminescence. The band intensities were quantified and normalized by using ImageJ software, with GAPDH as the loading control.

2.8. Assessment and Staining of Senescence‐Associated (SA)‐β‐gal

The activity of SA‐β‐gal was evaluated by using a senescence β‐galactosidase staining kit (Beyotime, Shanghai, China), following the instructions provided by the manufacturer. The HEI‐OC1 cells were seeded in six‐well plates. The HEI‐OC1 cells were washed three times and fixed with 4% PFA for 15 min at room temperature. In the following step, wash the cells with PBS and then stain them with a freshly prepared β‐gal detection solution at 37°C overnight. Images were captured under the microscope in the next day.

2.9. Lipid Peroxidation Level Assay

Appropriate numbers of cells were centrifuged at 600 g for 5 min at room temperature, and the supernatant was discarded. The cells were resuspended in an appropriate volume of BODIPY 581/591 C11 staining (Beyotime, Shanghai, China) working solution to achieve a cell density of 1–10 million cells/mL. The cells were incubated at 37°C for 20 min. After incubation, the cells were centrifuged at 600 g for 3–4 min at 4°C, and the supernatant was removed. The cells were washed twice with PBS. Then, the cells were resuspended in 1 mL PBS and centrifuged again at 600 g for 3 × 4 min at 4°C to collect the pellet, discarding the supernatant. This washing step was repeated once more with 1 mL PBS, followed by another centrifugation at 600 g for 3–4 min at 4°C. After the final wash, the cells were resuspended in PBS and observed under a fluorescence microscope.

2.10. ROS Assay

The ROS levels were measured using the CellROX‐orange probe (Thermo Fisher, USA). Briefly, cells were seeded in six‐well plates and treated with various conditions. After treatment, the cells were incubated with 5 μM CellROX orange in the culture medium at 37°C for 30 min. After incubation, the cells were washed three times with PBS, and DAPI (Yeasen, Shanghai, China) was added to each well for 5 min, followed by washing with PBS. The immunofluorescence signals were detected using a fluorescence microscope.

2.11. Statistical Analysis

There were at least three replications of each experiment. All data are presented as mean ± SD. The statistical software GraphPad Prism 6 and Microsoft Excel were used to analyze the significance of the data. Two‐tailed, unpaired t‐tests were used to determine statistical significance between two groups. One‐way ANOVA was used to analyze differences between the groups. p < 0.05 was considered statistically significant.

3. Result

3.1. Epac1 Is Expressed in the Inner Ear Hair Cells, and Its Expression Decreases With Age in the Cochlea, Accompanied by an Increase in Ferroptosis Levels

In this study, we used C57BL/6J male mice and divided them into two groups based on age: the young group (2‐week‐old) and the old group (12‐week‐old). The average ABR threshold of the young group was significantly lower than that of the old group (Figure 1A,B). Next, we analyzed the differences in the expression of Epac1 between the two age groups of mice. Immunofluorescence staining of the cochlear basilar membrane revealed that the arrangement of cochlear hair cells in 12‐month‐old mice was more disordered. The old mice were noticeably missing more hair cells than the young mice (Figure 1C). Furthermore, we found that most of the missing hair cells in the old mice were outer hair cells, while the younger group presented no such defects (Figure 1D).

Figure 1.

Comparison of auditory brainstem response (ABR) thresholds and Epac1 fluorescence intensity in cochlear hair cells of C57BL/6J mice. (A) ABR thresholds in C57BL/6J young and old mice at click and 4, 8, 16, 24, 32 kHz (**p < 0.01). (B) Representative click ABR traces between 20 and 90 dB in young mice and old mice. (C) The immunofluorescence staining of Epac1 (red) and myoⅦa (green) in the cochlear basilar membrane and DAPI (blue) was used to stain the nuclei, OHC is out hair cell, and IHC is inner hair cell. (D) Counts of OHC. in young and old mice, **p < 0.01. (E) Counts of IHCs in young and old mice, **p < 0.01.

We further verified that Epac1 was expressed primarily in the organ of Corti by immunofluorescence staining of cochlear sections. Additionally, a significant decrease in the fluorescence intensity of Epac1 was observed in aged mice (Figure 2A). Subsequently, western blot analysis demonstrated that Epac1 expression in the cochleae of aged mice was lower than that in the cochlea of young mice. Furthermore, we assessed the levels of the ferroptosis‐related protein GPX4, and the results revealed a reduction in GPX4 expression with aging, suggesting an increase in ferroptosis levels (Figure 2B,C).

Figure 2.

Age‐related Epac1 and GPX4 expression in cochlea. (A) Immunofluorescence of Epac1 (red) in the organ of corti (×400). Nuclei were visualized by DAPI (blue); hair cells were labeled by MyoⅦA (green). (B) Western blot analysis of Epac1 expression in the cochlea at young and old mice. (C) Relative expression level of Epac1 in cochlea of young and old mice. (D) Relative expression level of GPX4 in cochlea of young and old mice.**p < 0.01.

3.2. Knockdown of Epac1 Promotes the Ferroptosis Levels and Accelerates the Aging Process in HEI‐OC1 Cells

To investigate the role of Epac1 in ARHL, D‐galactose was used to induce senescence in the HEI‐OC1 hair cell line. Figure 3 shows the changes in the level of Epac1 in the HEI‐OC1cells after siRNA and D‐gal treatment. Moreover, cells were transfected with siRNA to downregulate Epac1. We examined the expression of Epac1 in HEI‐OC1 hair cells by western blotting. The result showed that Epac1 expression levels were lower in HEI‐OC1 cells treated with D‐galactose that in control cells (Figure 3A,C). The results also revealed that the transfection of HEI‐OC1 cells with siRNA significantly reduced the protein expression of Epac1. SiRNA3 was selected for the following experiments.

Figure 3.

Changes in the level of Epac1 in the House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells after small interfering RNA (siRNA) and d‐gal treatment. (A) Western blot analysis of Epac1 in HEI‐OC1 cells after d‐gal and siRNA treatment. (B) Immunofluorescence staining images of lipid peroxidation in HEI‐OC1 cells. Oxidized state cells were labeled in green and reduced state cells were marked in orange. (C) Relative expression level of Epac1 in HEI‐OC1 cell, **p < 0.01. (D) Quantification of the immunofluorescence staining images of lipid peroxidation in (B), **p < 0.01. (E) SA‐β‐gal staining of HEI‐OC1 cells in different groups. (F) Quantification of the SA‐β‐gal staining, **p < 0.01.

Ferroptosis is a form of regulated cell death induced by iron ion‐dependent accumulation of reactive oxygen species (ROS) and peroxidized lipids. A lipid peroxidation assay can be used to determine whether excessive ROS are oxidizing lipids. Compared with those of the control group, cells transfected with siRNA exhibited a significant reduction in red fluorescence intensity, indicating that the siRNA‐Epac1 treatment reduced ferroptosis levels (Figure 3B,D). Moreover, SA‐β‐gal staining was performed to confirm whether senescence was caused by siRNA‐Epac1 in HEI‐OC1 cells. SA‐β‐gal staining revealed that, compared with control cells, siRNA‐treated cells presented increased cell size, with blue‐stained cells expressing β‐galactosidase (Figure 3E,F).

To further verify the key role of Epac1 in ferroptosis and the aging process, we detected the expression of P16, P21, GPX4, and FACL4 in HEI‐OC1 cells via western blotting. The results of western blotting revealed that the expression of GPX4 in HEI‐OC1 cells was significantly lower in siRNA‐treated cells than that in the control group. In contrast, the expression of FACL4, P16, and P21 clearly increased (Figure 4A,B). These results demonstrated that downregulation of Epac1 promoted the ferroptosis levels and accelerated the aging process in HEI‐OC1 cells.

Figure 4.

Changes in the level of ferroptosis and senescence in the House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells after small interfering RNA (siRNA) treatment. (A) Western blot analysis of Epac1, FACL4, GPX4, P21, and P16 expression after cells were transfected with siRNA. (B) Quantification of the Epac1, FACL4, GPX4, P21, and P16 in HEI‐OC1 cells, **p < 0.01.

3.3. Regulation of Epac1 Influences the Level of Ferroptosis and Aging Process in HEI‐OC1 Cells

To further explore the role of Epac1 in the ferroptosis and the aging process, shRNA‐Epac1 was applied to specifically increase the expression of Epac1 in HEI‐OC1 cells.

We determined the expression of Epac1 after different treatments by Western blotting and immunofluorescence. The experimental results are shown in Figure 5. After transfection with siRNA‐Epac1, the fluorescence intensity of Epac1 in HEI‐OC1 cells was significantly reduced. In contrast, transfection with shRNA‐Epac1 resulted in a noticeable increase in Epac1 fluorescence intensity compared with that in the control group. Furthermore, the fluorescence intensity of Epac1 was not significantly different in cells treated with both siRNA and shRNA compared with untreated cells (Figure 5A,D).

Figure 5.

Epac1 affected the level of ferroptosis and the aging process in the House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells after small interfering RNA (siRNA) and short hairpin RNA (shRNA) treatment. (A) Immunofluorescence of Epac1 (red) in HEI‐OC1 cells after different treatments. DAPI (blue) was used to stain the nuclei. (B) Immunofluorescence of CellRox (orange) in HEI‐OC1 cells after different treatments. DAPI (blue) was used to stain the nuclei. (C) Western blot analysis of Epac1, FACL4, P21, GPX4, and P16 expression in HEI‐OC1 cells after the cells were transfected with siRNA. (D) Quantitative analysis of relative fluorescence intensity in (A), **p < 0.01. (E) Quantification of the immunofluorescence of CellRox (orange) in (B), **p < 0.01. (F) Relative expression level of Epac1, FACL4, P21, GPX4, and P16 in HEI‐OC1 cells.

An increase in ROS levels is considered as an important indicator of ferroptosis. To investigate the effect of modulating Epac1 expression on cellular oxidative stress levels. CellROX staining was used to measure the level of intracellular ROS. Compared with that in the control group, the CellROX signal was significantly increased in the siRNA‐Epac1 group. In the shRNA‐Epac1 group, minimal CellROX signal was detected. The intensity of the CellROX signal in cells transfected with both siRNA and shRNA was between that in cells transfected with siRNA or shRNA alone (Figure 5B,E).

In addition, we further analyzed the expression levels of aging and ferroptosis‐related proteins. Cells treated with siRNA‐Epac1 exhibited a significant decrease in GPX4 expression, accompanied by reduced expression of FACL4 and P16, as revealed by western blotting (Figure 5C,F). However, the expression of these proteins in HEI‐OC1 cells after shRNA treatment showed the opposite trend. Furthermore, no significant differences in protein expression levels were detected between the siRNA and shRNA‐treated group, compared with the control group.

4. Discussion

In this study, we first used ABR testing to confirm that old mice experience significant hearing loss compared with young mice. This part of the study aimed to validate the precision of our naturally aged mouse model, thereby establishing a robust basis for future experiments. Next, we measured the Epac1 expression in the peripheral auditory system to confirm its critical role in the hearing system. Through the application of immunofluorescence staining and western blotting, we conclusively showed that Epac1 expression decreased with age in mice. Previous studies have shown that Epac1 plays a critical role in maintaining mitochondrial integrity, and that inhibiting Epac1 can prevent ferroptosis‐induced cell death [16]. Previous research from the same group has shown that the expression level of Epac1 is increased in a rat model of noise‐induced hearing loss [17]. We believe that ARHL and noise‐induced hearing loss differ in their underlying pathological mechanisms. ARHL is typically associated with cellular dysfunction and changes in the inner ear environment due to aging [18, 19]. Epac1 plays a role in cellular stress and repair [20, 21], and its reduced expression may indicate a decline in the regenerative capacity of inner ear cells. In contrast, noise‐induced hearing loss is caused by acute noise exposure, often accompanied by oxidative stress and inflammation [22, 23, 24, 25]. In rat models of noise‐induced hearing loss, increased expression of Epac1 may indicate an immediate cellular stress response, with its upregulation potentially facilitating the initiation of repair or protective mechanisms. These findings indicate that Epac1 plays a pivotal role as a regulatory molecule in the progression of hearing loss. In addition, these findings provide a crucial theoretical foundation for future research on the molecular mechanisms of hearing loss, such as the role of Epac1 signaling, and for the development of therapeutic strategies, including medical treatments and surgeries.

Ferroptosis is a form of cell death closely associated with lipid peroxidation and oxidative stress, characterized by the abnormal accumulation of iron ions in cells, leading to the production of lipid peroxides and ultimately triggering cell death [26]. The results in Figure 3C indicate that downregulation of Epac1 significantly increases lipid peroxidation levels. To explore the impact of Epac1 on ferroptosis and aging, we induced aging using v‐galactose and knocked down Epac1 with siRNA to establish an aging model in HEI‐OC1 cells. The experimental results show that Epac1 expression decreases with cellular aging, and the downregulation of Epac1 induces ferroptosis and accelerates cell aging, suggesting that Epac1 plays an important role in this process. β‐Galactosidase staining revealed an increased number of aging cells in the siRNA‐treated group, further confirming the elevation of aging levels.

Furthermore, our study identified key ferroptosis biomarkers, including GPX4 and FACL4. Glutathione peroxidase 4 (GPX4) is a pivotal enzyme that regulates the process of ferroptosis, and its function is closely related to the elimination of lipid peroxides. A decrease in GPX4 activity or its inhibition can trigger ferroptosis [26, 27, 28]. Based on the western blotting data, we observed a significant reduction in GPX4 expression in the siRNA‐Epac1 group. The decrease in GPX4, which prevents lipid peroxidation, indicates an increase in ferroptosis. Concurrently, we noted a marked increase in FACL4 expression, which may be associated with the regulation of lipid metabolism during ferroptosis. FACL4 is a crucial enzyme that significantly impacts ferroptosis. By modulating lipid metabolism, it can exert a profound influence on cellular sensitivity to ferroptosis [29]. Based on these data, we found that ferroptosis levels increase with age in vitro. Moreover, we detected senescence biomarkers including P16 and P21. P16 and P21 are commonly used biomarkers of cellular senescence. In senescent cells, the expression levels of P16 and P21 are markedly elevated [30, 31]. The western blotting results indicated that the levels of P16 and P21 notably increased following siRNA downregulation of Epac1. This finding implies that variations in senescence correspond with changes in ferroptosis levels. Previous studies have indicated that downregulating KCNMA1 accelerates the aging of auditory hair cells in mice, with ferroptosis playing a crucial role in this process. These results are consistent with our findings [32]. It is widely recognized that Epac1 is primarily involved in the cAMP signaling pathway, and its relationship with GPX4 seems not to be direct [33]. We speculate that Epac1 may indirectly influence GPX4 expression by regulating downstream signaling molecules. Studies show that Epac1 regulates CREB activity, which in turn activates the GPX4 transcription and inhibits ferroptosis [34]. In addition, Epac1 regulate CREB through the Rap1‐ERK1/2 pathway [35]. Based on this background, we propose that Epac1 may significantly impact the antioxidant stress response and defense against ferroptosis by indirectly enhancing GPX4 expression via the CREB pathway.

To further validate whether the regulation of Epac1 can influence the degree of ferroptosis and senescence, we also used shRNA to upregulate Epac1 expression in HEI‐OC1 cells. First, immunofluorescence staining was used to confirm the effectiveness of siRNA‐Epac1 and shRNA‐Epac1 in modulating Epac1 expression. Ferroptosis is driven by oxidative stress, exacerbating lipid peroxidation within the cells, ultimately resulting in membrane damage and mitochondrial dysfunction [36]. CellROX can be used to detect the accumulation of ROS, thereby assessing the extent of ferroptosis [37]. Our study also revealed that when the expression level of Epac1 was downregulated in HEI‐OC1 cells, oxidative stress levels were significantly increased. In contrast, when Epac1 levels were increased via shRNA, a marked reduction in oxidative stress levels was detected. Consistent with previous findings, the western blot results showed that when Epac1 expression was downregulated, the levels of both ferroptosis and senescence were increased compared to the control group. However, when Epac1 expression was upregulated, an expected decrease in both ferroptosis and senescence levels was observed. In contrast, the combined siRNA and shRNA treatment group presented no significant differences in ferroptosis or senescence levels compared with those of the control group.

Aging is a complex biological process characterized by the gradual loss of cellular functions and tissue degeneration [38]. The link between ferroptosis and aging has been confirmed in various cell types [36], and our results further support this relationship. We observed that when Epac1 expression was downregulated, the levels of ferroptosis and aging markers significantly increased, suggesting that Epac1 may mitigate aging by inhibiting ferroptosis. However, upregulated expression of Epac1 reduced the levels of both aging and ferroptosis markers, implying that Epac1 may indirectly delay the aging process by reducing oxidative stress and decreasing lipid peroxidation.

Owing to financial and technical limitations, in vivo validation in a mouse model was not performed in this study. Future research plans include targeting Epac1 expression through semicircular canal injection of an adenovirus to further validate these findings. Although this study suggests a potential role for Epac1 in aging and ferroptosis, the specific molecular mechanisms involved have not been thoroughly investigated. Epac1 may exert its effects through multiple signaling pathways, such as the cAMP–PKA and ERK pathways, but a detailed elucidation of these mechanisms requires further experimental validation. In this study, we focused only on ferroptosis and did not explore other mechanisms. The primary reason for this is that the focus of our research was to investigate the role of Epac1 channel activation in ferroptosis, and due to limitations in time and resources, we were unable to comprehensively cover other potential mechanisms. While we acknowledge that ARHL is a complex, multifactorial process involving the interaction of mechanisms such as apoptosis, autophagy, and inflammation, studying these mechanisms requires a broader experimental design and more extensive data support. Therefore, our study concentrated on the single mechanism of ferroptosis. Future research can expand the investigation of these mechanisms to gain a more comprehensive understanding of the onset and development of ARHL.

Our findings suggest that Epac1 plays a crucial role in aging and ferroptosis. Upregulating Epac1 expression can alleviate oxidative stress, inhibit lipid peroxidation accumulation, and slow the onset of ferroptosis, which may delay the aging process. Therefore, Epac1 may be a potential protective factor in aging and a therapeutic target for age‐related diseases. Future research should further explore the specific mechanisms by which Epac1 influences aging, oxidative stress, ferroptosis, and associated diseases, offering new insights for treating age‐related disorders.

Author Contributions

Wen‐Jun An, Xiao‐Min Tang, and Jing‐Wu Sun conceived and designed the study. Wen‐Jun An and Xiao‐Min Tang did the main experiments. Meng‐Ya Shen and Chen‐Yu Xu performed the animal experiments. Xiao‐Min Tang and Xiao‐Tao Guo analyzed and interpreted the data. Xiao‐Min Tang, Chun‐Chen Pan, and Jing‐Wu Sun provided technical support. Jing‐Wu Sun, Xiao‐Min Tang, and Jia‐Qiang Sun were responsible for reagents and materials. Wen‐Jun An drafted the article. Jing‐Wu Sun and Jia‐Qiang Sun revised the article critically. All authors had final approval of the submitted versions.

Disclosure

We verify that all data are consistently reported across text, tables, figures, and supplementary material. We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with the submission.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data will be made available on request.