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. 2025 Aug 20;25:645. doi: 10.1186/s12877-025-06237-5

AIMP1 exerts hearing protection role in age related hearing loss mice by regulating SIRT1 expression

Haisen Peng 1,#, Jiali Liu 1,#, Yuehui Liu 1, Chunhua Li 1, Zhilin Zhang 1, Shuihua Hu 1, Wen Xie 1,✉
PMCID: PMC12366153  PMID: 40836274

Abstract

Background

Age related hearing loss (ARHL) is a sensorineural hearing disease caused by multiple factors and its pathogenesis is still unclear. This work aims to investigate the precise role of AIMP1 in ARHL.

Methods

HEI-OC1 cells were treated with 1 mM H2O2 for 2 h to induce cell damage. CCK-8, flow cytometry and TUNEL staining examined cell viability and apoptosis. DCFH-DA fluorescence probe assessed ROS. The levels of MDA, SOD and copper were detected by kits. The expression of proteins related to mitophagy, cuproptosis were examined by western blotting and immunofluorescence. Finally, D-galactose was administered to mice to establish an ARHL model for verifying the functional role of AIMP1 in ARHL.

Results

AIMP1 and SIRT1 were down-regulated in H2O2-treated HEI-OC1 cells. AIMP1 overexpression promoted cell viability, reduced ROS and MDA levels, and increased SOD levels in H2O2-treated HEI-OC1 cells. Moreover, the levels of copper and apoptosis were decreased in H2O2-treated HEI-OC1 cells in the presence of AIMP1 overexpression. AIMP1 overexpression caused a down-regulation of cuproptosis-related proteins FDX1, DLAT, DLST, and an up-regulation of mitophagy-related proteins PINK1, Parkin, Mfn2 and Drp1 in H2O2-treated HEI-OC1 cells. Knockdown PINK1 or SIRT1 reversed the influence of AIMP1 overexpression on H2O2 induced HEI-OC1 cell damage. In vivo, AIMP1 overexpression reduced damage of cochlear tissues and partially restored hearing in ARHL mice.

Conclusion

AIMP1 up-regulated SIRT1 to promote PINK1/Parkin-mediated mitophagy and inhibit cuproptosis of cochlear hair cells in ARHL mice. Thus, AIMP1 may be a potential target for ARHL treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12877-025-06237-5.

Keywords: AIMP1, SIRT1, Age related hearing loss, Cuproptosis, Mitophagy, Oxidative stress

Introduction

Age related hearing loss (ARHL), also known as presbycusis, is a symmetrical, slowly progressive sensorineural hearing loss that occurs with age in both ears [1]. The auditory characteristics are primarily affected by high-frequency hearing, often accompanied by a significant decline in speech comprehension ability [2, 3]. According to World Health Organization data, it is expected that by 2025, the global population of elderly people over 60 years old will reach 1.2 billion, of which over 500 million will suffer from ARHL [4]. Severe hearing loss affects the communication and interaction between elderly patients and society, gradually leading to psychological problems such as cognitive impairment, social loneliness, anxiety, and depression [5, 6]. The main site of damage in ARHL is the cochlea, where hearing gradually decreases from high frequency to low frequency, accompanied by degeneration and loss of spiral ganglion neurons and hair cells [7]. Among them, irreversible apoptosis of inner ear hair cells is a key factor [8]. At present, apart from hearing aids and cochlear implants, ARHL lacks prevention and treatment strategies. Therefore, it is urgent to find effective targets for the prevention and treatment of ARHL.

Oxidative stress is closely related to the pathophysiology of ARHL. The expression of oxidative stress-related proteins or factors are increases in ARHL, while the expression of antioxidant-related proteins or factors are decreases [9, 10]. Under oxidative stress stimulation, cells produce a large amount of reactive oxygen species (ROS), and activate downstream caspase-3 induced cell apoptosis. Apoptotic cells release a large amount of ROS, thereby exacerbating auditory sensory cell damage and promoting ARHL progression [11, 12]. The accumulation of ROS, along with weakened antioxidant defense and impaired oxidative repair, can trigger mitochondrial dysfunction [13]. To maintain the homeostasis of the intracellular environment, activation of mitophagy can target and clear damaged or abnormal mitochondria [14]. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that regulates cellular metabolism, stress responses, and aging by modulating key transcription factors and metabolic enzymes [15]. Previous study has indicated that SIRT1 is involved in the maintenance of mitochondrial function and oxidative stress injury of cochlear hair cells [10]. PINK1 and Parkin, a cytosolic ubiquitin ligase, play a central role in mitophagy by tagging damaged mitochondria for degradation [16]. Research has found that SIRT1 activates PINK1/Parkin-mediated mitophagy to participate in the progression of various diseases [17, 18]. PINK1/Parkin-mediated mitophagy is related to ARHL [19]. It is still unclear whether SIRT1 can affect oxidative stress and PINK1/Parkin mediated mitophagy to regulate ARHL.

Heavy metal ions are essential micronutrients in the organism, and too little or too much of them can lead to cell death [20]. Copper is a key catalytic cofactor in various biological processes, including mitochondrial respiration, antioxidant defense, and biological compound synthesis [21]. Cuproptosis is a programmed cell death closely related to mitochondrial metabolism [22]. Dihydrolipoamide S-acetyltransferase (DLAT) and dihydrolipoamide S-succinyltransferase (DLST) are core components of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, respectively, linking glycolysis and the tricarboxylic acid cycle [23, 24]. Ferredoxin 1 (FDX1) is an iron-sulfur protein essential for mitochondrial electron transport and steroidogenesis [25]. During the process of cuproptosis, the acylation of DLAT and DLST decreases, as well as Cu-induced the oligomerization of tricarboxylic acid cyclic acylated proteins. On the other hand, FDX1 causes instability and overall reduction of Fe-S cluster proteins, which lead to protein toxicity stress and mitochondrial dysfunction [26]. Considering the close relationship between cuproptosis and mitochondrial metabolism, whether cuproptosis participate in the progression of ARHL attracted our attention.

Aminoacyl tRNA synthase interacting multifunctional protein 1 (AIMP1), also known as p43 protein, an emerging therapeutic protein functioning at the systems level [27]. AIMP1 regulates inflammation, angiogenesis, and cell survival through cytokine-like activities [27, 28]. Studies have demonstrated that AIMP1 accelerates the progression of multiple myeloma through activation of the MAPK signaling pathway [29]. Additionally, under stress condition, AIMP1 effectively induces M1 microglial activation via JNK and p38/NF-κB-dependent pathways, thereby promoting neuroinflammation [30]. Activation of SIRT1 has been shown to suppress both MAPK and NF-κB pathways in psoriasis-associated oxidative stress [31]. However, the role of AIMP1 in ARHL and its underlying regulatory mechanisms remain unclear. In this work, we attempted to investigate whether AIMP1 may affect PINK1/Parkin-mediated mitophagy and cuproptosis of cochlear hair cells through SIRT1, thereby playing a hearing protection role in ARHL.

Methods

Animals

C57BL/6 male (5–6 weeks) were obtained from SLAC Laboratory Animal Co., Ltd., (Shanghai, China) and housed under SPF conditions with constant temperature (20–24 °C) and constant humidity (40–60%). All animal experiments were conducted in compliance with the National Institutes of Health (NIH) policies in the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University.

Animal groups and treatment

Thirty mice were divided into five groups: CON, Model, Model + Sham, Model + AAV-NC, and Model + AAV-AIMP1 (n = 6 mice/group). The ARHL mouse model was established by D-galactose induction as previously described [32]. Mice were intraperitoneally injected with 150 mg/kg/d D-galactose (MedChemExpress, Monmouth Junction, NJ, USA) for 60 days. For AIMP1 overexpression, adeno-associated virus (AAV)-AIMP1 and negative control (AAV-NC) vectors were constructed using AAV9 serotype with CMV promoter, designed and packaged by VectorBuilder (www.vectorbuilder.com). Both vectors were purified to a final titer of 1 × 10¹² v.g./mL. AAV injections were performed on day 0 of D-galactose induction in all treated groups. Under isoflurane anesthesia, mice were placed on a heating pad. After shaving and disinfecting the postauricular area, the bulla was exposed by blunt dissection. The round window membrane (RWM) was identified using the facial nerve, cochlear basal turn, and stapedial artery as anatomical landmarks. Using a 50 µL glass microsyringe, 1 µL AAV was injected through the RWM at 0.2 µL/min. The injection site was sealed with muscle tissue, and the skin incision closed with Vetbond tissue adhesive (#1469SB, Phymep) [33]. Following injection of AAV, mice were intramuscularly injected with 200,000 IU/d penicillin for 3 days. Sham-operated mice underwent the same surgical procedure and injected with normal saline. Normal mice intraperitoneally injected with normal saline as control. Following model establishment, auditory brainstem response (ABR) thresholds of mice were measured electrophysiologically in all experimental groups.

After anesthesia with isoflurane inhalation, mice were subjected to gradual CO2 displacement (20–30% chamber volume per minute) until respiratory arrest was confirmed, followed by an additional 5-minute exposure to ensure irreversible death. Subsequently, the cochlear tissues were meticulously dissected using fine forceps. 6 mice per group were used for cochlear tissue collection. For histology, cochlear tissues from 3 mice per group were immediately fixed in 4% paraformaldehyde for 48 h, followed by decalcification. The cochlear tissues from the remaining 3 mice per group were snap-frozen at −80 °C for subsequent mRNA or protein analysis.

ABR electrophysiological test

ABRs were recorded 8 weeks post-AAV injection in an open-field system using TDT MF1-1250 speakers (n = 6 mice/group). Signals were amplified 50,000× and bandpass-filtered (100–3000 Hz). Mice were anesthetized by inhalation of isoflurane. Mice were placed on a heating pad to maintain the core temperature (37 °C) of mice. The needle electrodes were inserted subcutaneously at the vertex, under the pinna of the left ear and the right ear of mice. ABRs were measured at frequencies of 8, 16, 28 and 40 kHz with tone-burst stimuli reducing levels at 100 dB in a soundproof booth. Each mouse was tested for its binaural ABR, with at least one ear having an ABR threshold greater than or equal to 15 dB, indicating the successful preparation of a mouse model of hearing loss. The ABR threshold was detected using the TDT II System and BioSig software (Tucker Davis Technologies, MathWorks, Naples, FL, USA).

TUNEL staining

Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China) was used to detect hair cell apoptosis of cochlear tissues. Paraffin sections of cochlear tissues were subjected to dewaxing and hydration. The sections were treated with 0.1% Triton, and stained with TUNEL detection reagent containing TdT enzyme and Biotin-dUTP or anti-Myosin 7a (#bs-7761R, Bioss, Beijing, China) at 4 °C in darkness overnight. Sections were stained with Streptavidin-HRP at room temperature for 30 min, and then incubated with 4′6-Diamadino-2-phenylindole (DAPI) at room temperature for 5 min. Cell apoptosis in cochlear tissues was observed under a fluorescence microscope.

Hematoxylin eosin (HE) staining

The cochlear tissue fixed with 4% paraformaldehyde was decalcified, followed by paraffin embedding. Then, the intact tissue was cut into wax slices with a thickness of 4 μm. The wax slices after drying were dewaxed. After rinsing with distilled water, sections were successively stained with hematoxylin (Beyotime, Shanghai, China) for 5 min and eosin (Beyotime) for 5 s. After being washed with distilled water again, the slices were dehydrated and sealed. Subsequently, the staining of the tissue was observed with a microscope.

Cell culture

House Ear Institute-Organ of Corti 1 (HEI-OC1) cells [34] were purchased from BFB Life Science (#BFN60808695, Shanghai, China) and cultured in DMEM medium (Gibco, Waltham, USA) in an incubator at 33 °C with 10% CO2. The medium contained 10% FBS and 1% penicillin & streptomycinin solution. HEI-OC1 cells were stimulated with 1 mM H2O2 for 2 h.

Cell transfection

The pcDNA3.1 vector carrying AIMP1 was constructed for AIMP1 overexpression. Small interference RNA (siRNA) specially targeting AIMP1 (si-AIMP1), PINK1 (si-PINK1) or SIRT1 (si-SIRT1) were constructed. The empty vector (Vector) and scrambled siRNA (si-NC) served as control. These vectors were obtained from Genepharma. HEI-OC1 cells were seeded into 6-well plate, and then transfected with 4 µg vector and 10 µL lipofectamine 2000 reagent (Thermo Fisher Scientific, San Jose, CA, USA).

Quantitative real-time PCR (qRT-PCR)

HEI-OC1 cells and cochlear tissues were treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction. Total RNA was reverse transcripted to cDNA utilizing PrimeScript™ RT reagent Kit (Takara, Beijing, China). The relative expression of mRNA was detected by performing PCR reaction applying TB Green® Premix Ex Taq™ II (Takara). Primer sequences (5’−3’) used in qRT-PCR were listed as follows: AIMP1: Forward-TTTCTCTGCCGATTCTGGGGA and Reverse-CCT GCT GCT TGA GAT ATT CGA T; and SIRT1: Forward-TGA TTG GCA CCG ATC CTC G and Reverse-CCA CAG CGT CAT ATC ATC CAG; and GAPDH Forward-GGA AGC TTG TCA TCA ATG GAA ATC and Reverse-TGA TGA CCC TTT TGG CTC CC.

Western blotting (WB)

Total proteins were extracted from HEI-OC1 cells and cochlear tissues utilizing RIPA lysis reagent (Biosharp, Hefei, China), and its concentration was detected by BCA protein assay kit (Biosharp). Protein samples were separated by 10% SDS-PAGE gel electrophoresis, and transferred on PVDF membrane (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibody at 4℃ overnight, including anti-AIMP1 (#ab188320; Abcam, Cambridge, MA, USA), anti-SIRT1 (#ab110304, RRID: AB_10864359; Abcam), anti-PINK1 (#BD-PN2037; Biodragon, Suzhou, China), anti-Parkin (#BD-PT3593; Abcam), anti-Mfn2 (#ab56889, RRID: AB_2142629; Abcam), anti-Drp1 (#BD-PT1414; Biodragon), anti-LC3 (#ab192890, RRID: AB_2827794; Abcam), anti-ATG4D (#ab237751; Abcam), anti-Beclin1 (#ab207612, RRID: AB_2692326; Abcam), anti-FDX1 (#ab108257, RRID: AB_10862209; Abcam), anti-DLAT (#A04469-2, RRID: AB_3081676; Boster, Wuhan, China), anti-DLST (#ab177934, RRID: AB_3662088; Abcam), anti-GAPDH (#ab181602, RRID: AB_2630358; Abcam). The membranes were stained with secondary antibody IgG-HRP (#ab0101/#ab0102; Abcam) at 37℃ for 1 h. The WB bands were analyzed by Image J software.

CCK-8 assay

HEI-OC1 cells were seeded into 96-well plate and incubated at 37℃ for 12 h. CCK-8 reagent was assessed into each well and incubated at 37℃ for 1 h. The absorbance value of each well was detected by Multiskan FC microplate reader (Thermo Fisher Scientific).

Flow cytometry

Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) was applied to detect apoptosis of HEI-OC1 cells. HEI-OC1 cells were collected and washed with PBS. Cells (1 × 105) were resuspended in Annexin V-FITC binding buffer, and then stained with 5µL Annexin V-FITC and 10 µL PI at room temperature in darkness for 20 min. Apoptotic cells were assessed on a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).

Detection of ROS, SOD and MDA

ROS Assay Kit (Beyotime), Superoxide dismutase (SOD) Activity Assay Kit (Solarbio, Beijing, China), Malondialdehyde (MDA) Content Assay Kit (Solarbio) and Cell MDA assay kit (Colorimetric method) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to detect the levels of ROS, SOD and MDA in HEI-OC1 cells and cochlear tissues following the manufacturer’s instructions.

Immunofluorescence analysis

HEI-OC1 cells were fixed with 4% paraformaldehyde, and then treated with 0.5% Triton X-100 for 20 min. After that, cells were blocked with 3% bovine serum albumin (BSA) for 30 min. Cells were incubated with anti-PINK1 (#712654, RRID: AB_2848250; Thermo Fisher Scientific) or anti-Parkin (#66674-1-Ig, RRID: AB_2882028; Proteintech) at 4 °C overnight, and then stained with goat anti-rabbit Cy3-IgG (#A0516, RRID: AB_2893015; Beyotime), goat anti-mouse FITC-IgG (#A0568, RRID: AB_2893016; Beyotime) 37 °C for 1 h. DAPI was used to stain cell nuclei. The fluorescence intensity of PINK1 and Parkin in hippocampus was observed under fluorescence microscope (Olympus, Tokyo, Japan).

Statistical analysis

Each assay was performed for 3 times. The experimental assessments were conducted by investigators blinded to group allocation. Animals with unsuccessful model establishment were excluded from final analysis. No data were excluded in this study. Data were analyzed by SPSS 22.0 statistical software (IBM, Armonk, NY, USA) and expressed as mean ± standard deviation. Normality was tested using the Shapiro-Wilk test, and homogeneity of variance was assessed by Levene’s test. Data with normal distribution and equal variance were analyzed by t-test (two groups) or one-way ANOVA with Tukey’s test (multiple groups). For two-group comparisons, non-normal data used Mann-Whitney U test and normal but unequal variance data used Welch’s t-test. Multi-group non-normally distributed data were analyzed using the Kruskal-Wallis test, while normally distributed but heteroscedastic data were analyzed using the Brown-Forsythe test. P < 0.05 was considered as a significant difference.

Results

AIMP1 affected cell viability and oxidative stress of H2O2-treated HEI-OC1 cells

To determine the role of AIMP1 and SIRT1 in ARHL, HEI-OC1 cells were treated with H2O2 to mimic ARHL conditions, and detected the expression of AIMP1 and SIRT1 in HEI-OC1 cells. Results of qRT-PCR and WB revealed that AIMP1 and SIRT1 were down-regulated in H2O2-treated HEI-OC1 cells (Fig. 1A, B). Then, we overexpressed or silenced AIMP1 in HEI-OC1 cells to investigate its influence on oxidative stress. AIMP1 overexpression caused an up-regulation of AIMP1 and SIRT1, whereas AIMP1 silencing led to a down-regulation of AIMP1 and SIRT1 in H2O2-treated HEI-OC1 cells (Fig. 2A-C). To evaluate AIMP1’s role in cell viability, CCK-8 assays were performed in HEI-OC1 cells. As shown in Fig. 2D, H2O2 treatment reduced cell viability by 51%. Notably, AIMP1 overexpression restored viability to 82% cell survival, while AIMP1 deficiency exacerbated the H2O2-induced damage, further reducing viability to 31% cell survival. Additionally, oxidative stress-related indexs ROS, SOD and MDA were examined. The levels of ROS and MDA were increased, the levels of SOD were decreased in HEI-OC1 cells following H2O2 treatment. The levels of ROS and MDA in H2O2-treated HEI-OC1 cells were reduced by AIMP1 overexpression, while promoted by AIMP1 knockdown. The levels of SOD were enhanced by AIMP1 overexpression, whereas reduced by AIMP1 deficiency (Fig. 2E-G). All these data indicated that AIMP1 affected cell viability and oxidative stress of H2O2-treated HEI-OC1 cells.

Fig. 1.

Fig. 1

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AIMP1 and SIRT1 were down-regulated in H2 O2 -treated HEI-OC1 cells. HEI-OC1 cells were treated with 1 mM H2O2 for 2 h. Normal HEI-OC1 cells served as control. A, B qRT-PCR and WB examined the expression of AIMP1 and SIRT1 in HEI-OC1 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group

Fig. 2.

Fig. 2

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AIMP1 affected cell viability and oxidative stress of H2 O2 -treated HEI-OC1 cells. HEI-OC1 cells were transfected with AIMP1-OE, Vector, si-NC, si-AIMP1, and then treated with 1 mM H2O2 for 2 h. A-C qRT-PCR and WB examined the expression of AIMP1 and SIRT1 in HEI-OC1 cells. D CCK-8 assay detected cell viability of HEI-OC1 cells. E DCFH-DA fluorescence probe assessed ROS in HEI-OC1 cells. F, G The levels of SOD and MDA in HEI-OC1 cells were detected. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group/H2O2 + Vector group/H2O2 + si-NC group

AIMP1 regulated mitophagy and cuproptosis of H2O2-treated HEI-OC1 cells

Whether AIMP1 can affect cuproptosis of H2O2-treated HEI-OC1 cells was explored. Flow cytometry results indicated that H2O2 treatment significantly increased apoptosis rate in HEI-OC1 cells to 33%. Importantly, AIMP1 overexpression reduced H2O2-induced apoptosis rate to 10%, while AIMP1 deficiency exacerbated the apoptotic effect, resulting in 43% apoptosis rate (Fig. 3A). Furthermore, the levels of copper were elevated in H2O2-treated HEI-OC1 cells. The levels of copper were decreased in H2O2-treated HEI-OC1 cells following transfection of AIMP1-OE. AIMP1 silencing enhanced the levels of copper in H2O2-treated HEI-OC1 cells (Fig. 3B). Mitofusin-2 (Mfn2) and dynamin-related protein 1 (DRP1) are critical regulators of mitochondrial dynamics, where Mfn2 promotes fusion while DRP1 mediates fission [35]. WB assays revealed that protein levels of PINK1, Parkin, Mfn2 and Drp1 were down-regulated in H2O2-treated HEI-OC1 cells. Up-regulation of PINK1, Parkin, Mfn2 and Drp1 was observed in H2O2-treated HEI-OC1 cells overexpressing AIMP1. AIMP1 knockdown down-regulated the expression of PINK1, Parkin, Mfn2 and Drp1 in H2O2-treated HEI-OC1 cells (Fig. 3C). Results of IF revealed that the expression of PINK1 and Parkin was decreased in H2O2-treated HEI-OC1 cells. AIMP1 overexpression enhanced the expression of PINK1 and Parkin in H2O2-treated HEI-OC1 cells. The expression of PINK1 and Parkin in H2O2-treated HEI-OC1 cells was reduced by AIMP1 knockdown (Fig. 3D, Figure S1A). LC3II/LC3I ratio reflects autophagosome formation, ATG4D is a cysteine protease processing LC3, and Beclin 1 initiates autophagosome assembly [36, 37]. The levels of LC3II/LC3I ratio, ATG4D, Beclin 1 were down-regulated and p62 level was upregulated in H2O2-treated HEI-OC1 cells, indicating a reduction of autophagy, which can lead to the accumulation of damaged organelles and cell death. AIMP1 overexpression significantly up-regulated LC3II/LC3I ratio, ATG4D, and Beclin 1 while downregulating p62 levels, whereas AIMP1 deficiency exhibited the opposite effect (Fig. 3E). Cuproptosis-related proteins FDX1, DLAT, DLST were up-regulated in H2O2-treated HEI-OC1 cells. The expression of these proteins was inhibited by AIMP1-OE, and elevated by si-AIMP1 (Fig. 3F). Thus, AIMP1 enhanced mitophagy and down-regulated cuproptosis of H2O2-treated HEI-OC1 cells.

Fig. 3.

Fig. 3

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AIMP1 regulated mitophagy and cuproptosis of H2 O2 -treated HEI-OC1 cells. HEI-OC1 cells were transfected with AIMP1-OE, Vector, si-NC, si-AIMP1, and then treated with 1 mM H2O2 for 2 h. A Flow cytometry examined apoptosis of HEI-OC1 cells. B The levels of copper in HEI-OC1 cells were assessed. C WB detected the expression of mitophagy-related proteins PINK1, Parkin, Mfn2 and Drp1 in HEI-OC1 cells. D IF staining examined the location of PINK1 and Parkin in HEI-OC1 cells. E, F WB detected the expression of autophagy-related proteins p62, LC3I, LC3II, ATG4D, Beclin 1 and cuproptosis-related proteins FDX1, DLAT, DLST in HEI-OC1 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group/H2O2 + Vector group/H2O2 + si-NC group

AIMP1 mitigated H2O2-treated HEI-OC1 cell oxidative damage via PINK1/Parkin-mediated mitophagy

To determine whether AIMP1 can affect H2O2-treated HEI-OC1 cell damage by regulating PINK1/Parkin-mediated mitophagy, HEI-OC1 cells were transfected with AIMP1-OE and si-PINK1. The expression of AIMP1 and SIRT1 was elevated in H2O2-treated HEI-OC1 cells in the presence of AIMP1-OE. PINK1 knockdown had no influence on the expression of AIMP1 and SIRT1 (Fig. 4A, B). CCK-8 assay results showed that PINK1 silencing reversed AIMP1-OE-mediated promotion of cell viability of H2O2-treated HEI-OC1 cells (Fig. 4C). The levels of ROS and MDA were decreased, the levels of SOD were elevated in H2O2-treated HEI-OC1 cells following transfection of AIMP1-OE, which was abolished by si-PINK1 (Fig. 4D-F). AIMP1 overexpression inhibited apoptosis of H2O2-treated HEI-OC1 cells. PINK1 deficiency enhanced apoptosis of H2O2-treated HEI-OC1 cells (Fig. 4G). Furthermore, the levels of copper were decreased in H2O2-treated HEI-OC1 cells overexpressing AIMP1. The levels of copper were elevated in H2O2-treated HEI-OC1 cells in the presence of AIMP1-OE + si-PINK1 (Fig. 4H). AIMP1 overexpression caused an up-regulation of PINK1, Parkin, Mfn2 and Drp1 in H2O2-treated HEI-OC1 cells, which was inhibited by PINK1 knockdown, as determined by WB and IF assays (Fig. 4I, J, M, Figure S1B). Under H2O2 treatment, AIMP1-overexpressing HEI-OC1 cells showed increased LC3II/LC3I ratio, ATG4D, and Beclin 1 with concomitant p62 reduction, whereas PINK1 silencing attenuated these autophagy markers and elevated p62 levels (Fig. 4K). Additionally, cuproptosis-related proteins FDX1, DLAT, DLST were down-regulated in H2O2-treated HEI-OC1 cells overexpressing AIMP1, which was elevated by PINK1 knockdown (Fig. 4L). Therefore, AIMP1 regulated H2O2-treated HEI-OC1 cell damage by regulating PINK1/Parkin-mediated mitophagy.

Fig. 4.

Fig. 4

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AIMP1 affected H2 O2 -treated HEI-OC1 cell damage by regulating PINK1/Parkin-mediated mitophagy. HEI-OC1 cells were transfected with AIMP1-OE/si-PINK1, and then treated with 1 mM H2O2 for 2 h. (A, B) qRT-PCR and WB examined the expression of AIMP1 and SIRT1 in HEI-OC1 cells. C CCK-8 assay detected cell viability of HEI-OC1 cells. D DCFH-DA fluorescence probe assessed ROS in HEI-OC1 cells. E, F The levels of SOD and MDA in HEI-OC1 cells were detected. G Flow cytometry examined apoptosis of HEI-OC1 cells. H The levels of copper in HEI-OC1 cells were assessed. I-L WB detected the expression of mitophagy-related proteins PINK1, Parkin, Mfn2, Drp1, autophagy-related proteins p62, LC3I, LC3II, ATG4D, Beclin 1 and cuproptosis-related proteins FDX1, DLAT, DLST in HEI-OC1 cells. M IF staining examined the location of PINK1 and Parkin in HEI-OC1 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. H2O2 group/H2O2 + AIMP1-OE group

AIMP1 protected HEI-OC1 cells against oxidative damage by regulating SIRT1 expression

SIRT1 silencing had no influence on AIMP1 expression, and inhibited the increased expression of SIRT1 observed in H2O2-treated HEI-OC1 cells (Fig. 5A, B). Results obtained from CCK-8 assay showed that SIRT1 silencing reversed AIMP1-OE-mediated promotion of cell viability of H2O2-treated HEI-OC1 cells (Fig. 5C). The levels of ROS and MDA were decreased, the levels of SOD were elevated in H2O2-treated HEI-OC1 cells in the presence of AIMP1-OE, which was partially reversed by SIRT1 silencing (Fig. 5D-F). Moreover, AIMP1 overexpression inhibited apoptosis and the levels of copper of H2O2-treated HEI-OC1 cells. SIRT1 silencing enhanced apoptosis and the levels of copper of H2O2-treated HEI-OC1 cells (Fig. 5G-H). AIMP1-OE-mediated up-regulation of PINK1, Parkin, Mfn2 and Drp1 in H2O2-treated HEI-OC1 cells was inhibited by SIRT1 knockdown (Fig. 5I-J, M, Figure S1C). AIMP1 overexpression caused an up-regulation of LC3II/LC3I ratio, ATG4D, Beclin 1 and a down-regulation of p62, FDX1, DLAT, DLST in H2O2-treated HEI-OC1 cells. The influence of AIMP1-OE on these proteins related to autophagy and cuproptosis was reversed by SIRT1 knockdown (Fig. 5K, L). These findings suggested that AIMP1 affected H2O2-treated HEI-OC1 cell mitophagy and cuproptosis by regulating SIRT1 expression.

Fig. 5.

Fig. 5

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AIMP1 affected H2O2 -treated HEI-OC1 cell damage by regulating SIRT1 expression. HEI-OC1 cells were transfected with AIMP1-OE/si-SIRT1, and then treated with 1 mM H2O2 for 2 h. A, B qRT-PCR and WB examined the expression of AIMP1 and SIRT1 in HEI-OC1 cells. C CCK-8 assay detected cell viability of HEI-OC1 cells. D DCFH-DA fluorescence probe assessed ROS in HEI-OC1 cells. E, F The levels of SOD and MDA in HEI-OC1 cells were detected. G Flow cytometry examined apoptosis of HEI-OC1 cells. H The levels of copper in HEI-OC1 cells were assessed. I-L WB detected the expression of mitophagy-related proteins PINK1, Parkin, Mfn2, Drp1, autophagy-related proteins p62, LC3I, LC3II, ATG4D, Beclin 1 and cuproptosis-related proteins FDX1, DLAT, DLST in HEI-OC1 cells. M IF staining examined the location of PINK1 and Parkin in HEI-OC1 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. H2O2 group/H2O2 + AIMP1-OE group

AIMP1 overexpression promoted hearing function in ARHL mice

Finally, an ARHL mouse model was constructed to investigate the influence of AIMP1 on hearing function. As shown in Fig. 6A, the ABR threshold was elevated in ARHL mice. AIMP1 overexpression reduced the ABR threshold in ARHL mice. We also observed significant hair cell loss and structural damage in the ARHL mice compared to control group. AIMP1 overexpression significantly attenuated ARHL-induced hair cell loss and cochlear structural damage (Fig. 6B). Results of qRT-PCR and WB showed that the expression of AIMP1 and SIRT1 was decreased in cochlear tissues of ARHL mice. AIMP1 overexpression enhanced the expression of AIMP1 and SIRT1 in ARHL mice (Fig. 6C, D). ARHL mice exhibited significantly elevated ROS and MDA levels alongside reduced SOD level. AAV-mediated AIMP1 overexpression effectively reversed these oxidative stress markers, increasing SOD level while reducing both ROS and MDA levels (Fig. 6E-G). Furthermore, cochlear cell apoptosis and copper levels were elevated in ARHL mice, but these effects were suppressed by AIMP1 overexpression (Fig. 6H, I). In ARHL mice, we observed downregulation of mitophagy-related proteins (PINK1, Parkin, Mfn2, Drp1) and autophagy markers (LC3II/LC3I ratio, ATG4D, Beclin 1), accompanied by p62 accumulation, while cuproptosis-related proteins (FDX1, DLAT, DLST) were significantly upregulated. Remarkably, AIMP1 overexpression reversed these alterations, enhancing the levels of PINK1, Parkin, Mfn2, Drp1, LC3II/LC3I, ATG4D, and Beclin 1 while reducing the levels of p62, FDX1, DLAT, and DLST) (Fig. 6J-N). These findings indicated that AIMP1 overexpression enhanced mitophagy, suppressed cuproptosis, and promoted hearing function in ARHL mice.

Fig. 6.

Fig. 6

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AIMP1 overexpression promoted hearing function in ARHL mice. Mice were administrated with D-galactose to construct ARHL model. ARHL model were injected with AAV-AIMP1 or AAV-NC. A Auditory brainstem response (ABR) electrophysiological test examined the ABR threshold. B H&E staining was performed to evaluate cochlear histopathology. C, D qRT-PCR and WB examined the expression of AIMP1 and SIRT1 in cochlear tissues. E DCFH-DA fluorescence probe assessed ROS in cochlear tissues. F, G The levels of SOD and MDA in cochlear tissues were detected. H TUNEL staining assessed apoptosis of cochlear tissues. I The levels of copper in cochlear tissues were assessed. J-N WB detected the expression of mitophagy-related proteins PINK1, Parkin, Mfn2, Drp1, autophagy-related proteins LC3I, LC3II, ATG4D, Beclin 1 and cuproptosis-related proteins FDX1, DLAT, DLST in cochlear tissues. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CON group/Model + AAV-NC group

Discussion

ARHL is a common disorder of the auditory system function that occurs during the aging process in mammals. In this work, we confirmed that abnormally low expression of AIMP1 and SIRT1 was closely associated with the progression of ARHL. AIMP1 overexpression promoted cell viability and mitophagy, and reduced cuproptosis and oxidative stress of H2O2-treated HEI-OC1 cells. The influence conferred by AIMP1 overexpression on H2O2-treated HEI-OC1 cells was reversed by knockdown PINK1 or SIRT1. In vivo, AIMP1 overexpression reduced cuproptosis and oxidative stress, enhanced mitophagy of cochlear tissues, and exerted a role of hearing protection in ARHL mice. Thus, AIMP1 may be a potential target for ARHL.

AIMP1 was first discovered to be located at the center of a large aminoacyl tRNA synthetase complex (MSC) and involved in MSC synthesis. In addition to participating in protein synthesis, AIMP1 also has various biological activities, such as participating in intracellular and extracellular immune cell regulation, angiogenesis, wound healing, and blood glucose homeostasis [38]. Previous studies have demonstrated that AIMP1 inhibition significantly ameliorates nephritis in lupus-prone mice and preserves cognitive function in Alzheimer’s disease models [39, 40]. Furthermore, AIMP1 has been shown to modulate tumor progression through immune cell regulation [41, 42]. In the present work, we provided the first evidence for the hearing-protective role of AIMP1 in ARHL and elucidated its underlying mechanism. AIMP1 overexpression reversed H2O2 treatment induced damage of HEI-OC1 cells. AIMP1 silencing led to an opposite result. In vivo, AIMP1 overexpression reduced cuproptosis and oxidative stress, enhanced mitophagy of cochlear tissues, and protected the hearing function in ARHL mice, which may attribute to regulate SIRT1.

A large number of studies have shown that the occurrence of hearing loss is mainly related to oxidative stress damage [43, 44]. The aggregated ROS will attack the mitochondrial respiratory chain, mitochondrial DNA, and mitochondrial membrane potential. and the damaged mitochondria exacerbates the release of ROS, thereby exacerbating the occurrence of oxidative stress [45, 46]. Mitophagy is an important mechanism for maintaining mitochondrial quality and functional stability. It selectively clears damaged mitochondria, reduces ROS deposition, and thus reduces oxidative stress damage [47]. Mitophagy can be regulated by PINK1/Parkin signaling pathway, thereby maintaining normal cellular physiological function [48]. In this work, the levels of ROS and MDA were increased, the levels of SOD were decreased in H2O2-treated HEI-OC1 cells, indicating that H2O2 induced oxidative stress damage of HEI-OC1 cells. Furthermore, the expression of mitophagy-related proteins PINK1, Parkin, Mfn2 and Drp1 were decreased in H2O2-treated HEI-OC1 cells. It indicated that H2O2 treatment caused oxidative stress damage to HEI-OC1 cells, and then attacked mitochondrial function. AIMP1 overexpression reduced oxidative stress and enhanced mitophagy of H2O2-treated HEI-OC1 cells. PINK1 knockdown reversed the influence of AIMP1 overexpression on H2O2-treated HEI-OC1 cells. AIMP1 overexpression may enhance the expression of PINK1 and Parkin, and then activate PINK1/Parkin-mediated mitophagy in H2O2-treated HEI-OC1 cells. Thus, increased mitophagy may contribute to maintain the structure and function of mitochondria, clears damaged mitochondria, and reduces ROS deposition in cochlear hair cells, thereby reduced cochlear hair cell damage.

Cuproptosis is a copper-dependent programmed death mode [21]. We found that cuproptosis-related proteins FDX1, DLAT, DLST were up-regulated in H2O2-treated HEI-OC1 cells, which was inhibited by AIMP1 overexpression. A previous research has shown that copper oxidize ascorbic acid react with H2O2 to produce more destructive ROS when transported into cells through a copper carrier Elesclomol [49, 50]. Excess copper may have more functions, including blocking G1 phase cells, damaging DNA, and clarifying mitochondrial membrane potential, thereby inducing cell death [51]. We speculated that H2O2 treatment may enhanced the levels of copper in HEI-OC1 cells. Excess copper enhanced ROS levels to induce oxidative stress damage, and caused mitochondria dysfunction in cochlear hair cells. AIMP1 overexpression reduced cuproptosis to protect function of cochlear hair cells.

SIRT1 widely distributed in various organs and tissues throughout the organism, involved in anti-aging, anti-inflammatory, antioxidant, and cell cycle regulation in the brain, liver, and other important organs [52, 53]. A previous study has shown that SIRT1 also exists in the cochlea [10]. In the mouse cochlea, SIRT1 is expressed in the edge cells and intermediate cells of the stria vascularis, spiral ligaments, spiral ganglion cells, and inner and outer hair cells [54]. The expression of SIRT1 is significantly lower in elderly mice than in young mice [55]. These results confirm a close correlation between SIRT1 and ARHL [56]. The present work further confirmed the role of SIRT1 in ARHL. AIMP1 and SIRT1 were down-regulated in H2O2-treated HEI-OC1 cells. SIRT1 silencing reversed the positive impact of AIMP1 overexpression on oxidative stress, mitophagy and cuproptosis of H2O2-treated HEI-OC1 cell. Thus, AIMP1 overexpression protected hearing function in ARHL mice by regulating SIRT1.

This work demonstrated that AIMP1 influences mitochondrial autophagy and cuproptosis of cochlear hair cells by regulating SIRT1 expression in ARHL mice, providing a potential target for ARHL treatment. However, there are still some limitations. AIMP1 overexpression only resulted in partial improvement of ABR in ARHL mice. In the D-galactose-induced ARHL model, oxidative stress and apoptosis serve as primary pathological mechanisms [57–59]. Although up-regulation of SIRT1 was confirmed to alleviate D-galactose-induced oxidative stress and apoptosis through modulation of NF-κB and mTOR signaling pathways [60, 61]. SIRT1 activation only partially inhibits apoptosis without fully restoring cellular function in hyperglycemia-induced apoptosis models [62]. Additionally, different doses of D-galactose treatment have varying effects on hair cells [63]. Therefore, the therapeutic effect of AIMP1-mediated SIRT1 up-regulation may be influenced by experimental conditions as well as other pathways beyond SIRT1. Future studies could explore combination therapies targeting multiple pathways to achieve more robust hearing recovery in ARHL mice.

Supplementary Information

Supplementary Material 1. (3.2MB, pdf)
Supplementary Material 2. (116.5KB, pdf)

Abbreviations

AAV

Adeno-associated virus

ABR

Auditory brainstem response

AIMP1

Aminoacyl tRNA synthase interacting multifunctional protein 1

DAPI

4′6-Diamadino-2-phenylindole

ELISA

Enzyme-linked immuno sorbent assay

MDA

Malondialdehyde

qRT-PCR

quantitative real-time PCR

ROS

Reactive Oxygen Species

siRNA

Small interference RNA

SIRT1

Sirtuin 1

SOD

Superoxide dismutase

WB

Western blotting

Authors' contributions

Conceptualization: H. P., J. L.; Methodology: H. P., J. L.; Investigation: H. P., Y. L.; Data Curation: H. P., J. L., Y. L.; Formal analysis: H. P., J. L., Y. L., C. L., Z. Z.; Software: C. L., Z. Z.; Validation: S. H.; Resources: W. X.; Supervision: W. X.; Project administration: W. X.; Funding acquisition: W. X.; Writing - original draft preparation: H. P., J. L., Y. L.; (V) Writing - review and editing: H. P., J. L., Y. L., C. L., Z. Z., W. X.

Funding

This work was supported by the Natural Science Foundation of Jiangxi Province (Grant No. 20224BAB206051).

Data availability

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in compliance with the National Institutes of Health (NIH) policies in the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Haisen Peng and Jiali Liu contributed equally to this work and are co-first authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (3.2MB, pdf)
Supplementary Material 2. (116.5KB, pdf)

Data Availability Statement

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

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