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Introduction

Age-associated hearing loss, also known as presbycusis, is characterised by an age-dependent decline of auditory function associated with loss of sensory hair cells, spiral ganglion neurons and stria vascularis cells in the cochleae of the inner ear (1,2). However, the exact pathogenesis of age-related hearing loss remains to be elucidated.

As the cochleae tissue is not acquirable from humans during life, and the genetic and environmental background of individuals with hearing loss is heterogenous, the investigation of presbycusis is relatively limited. Natural aging can be experimentally modelled by the chronic administration of D-galactose (D-gal). Animals treated in this way exhibit a reduction in the activity of antioxidant enzymes (3–5), dysfunctional mitochondria (6–8), increased apoptosis (9,10) and neurotoxicity (7,11,12). Consequently, these animals exhibit a shortened lifespan (13), poor learning and memory (14–16) and an attenuated immune response (17–19). These characteristics are considered to be associated with an increase in oxidative stress caused by a metabolic disturbance. Previous studies have established a mimetic aging model in the cochleae of rats following 8 weeks of D-gal treatment, and demonstrated that the activity levels of antioxidant enzymes decreased and those of lipid peroxidation increased in this model (20–22). Furthermore, the levels of mitochondrial DNA (mtDNA) common deletion (CD) were significantly increased in the cochleae of the D-gal-treated rats (20–24). However, the sources of reactive oxygen species (ROS) and the effects of mtDNA CD in the cochleae of rats from this model remain to be fully elucidated.

In addition to mitochondria, the NADPH oxidase (NOX) system is one of the predominant ROS-generating sites, and it is now clear that NOX is not restricted to the immune system, and that alternative isoforms may be active in several other cell types as essential components of cellular signalling, gene expression regulation and cell differentiation (25). These enzymes share the capacity to transport electrons across the plasma membrane and to generate superoxide and other downstream ROS (25). A previous study reported that the expression levels of NOX3 are higher in the cochleae than in any other tissue (26). NOX3 forms a functional complex with P22phox to produce superoxide (27). Previous studies have demonstrated that NOX3 is a relevant source of ROS generation in the cochleae, and that NOX3-dependent ROS generation may contribute to hearing loss in response to ototoxic drugs (26,28–30).

Apoptosis may be important in the age-related decline of physiological function in several organs (31), including aging in the cochleae (32,33). A previous investigation demonstrated that D-gal-induced apoptotic cells are significantly increased in the cochlear section of newborn rats (34). Previous studies have also reported that apoptotic cells immediately increase in the central auditory system of adult rats following 8 weeks of treatment with D-gal (35,36). Du et al (37) reported that apoptotic cells increase in the peripheral auditory system of D-gal-treated aging rats following 12 months of treatment. However, whether 8 weeks of treatment with D-gal immediately causes apoptosis in the cochleae of adult rats has not been investigated. In the present study, the accumulation of mtDNA CD, mitochondrial ultrastructural changes and changes in the expression levels of 8-OHdG, NOX3, P22phox and cleaved caspase 3 (C-cas3) were investigated, as well as the occurrence of apoptosis in the cochleae of rats exposed to D-gal for 8 weeks. Furthermore, the present study also investigated the possible mechanism underlying presbycusis using D-gal-induced aging rats.

Materials and methods

Animals and treatments

A total of 60 1 month old male Sprague-Dawley rats were obtained from the Experimental Animal Centre of the Guangxi Medical University (Guangxi, China). The rats were individually housed in a temperature-controlled (20–22°C) room with a 12 h light/dark cycle, and were provided with free access to food and drinking water. The body weights of the experimental animals were monitored during the experiment as a general measure of health. The injection of D-gal (Sigma-Aldrich, St. Louis, MO, USA) to induce aging was administered, according to an established method (37). Following acclimation for 2 weeks, the rats were randomly divided into three groups: (1) D-gal(H) group, injected subcutaneously with 500 mg/kg D-gal once a day for 8 weeks; (2) D-gal(L) group, injected subcutaneously with 150 mg/kg D-gal once a day for 8 weeks; (3) control group, which were administered with an equal volume of vehicle (0.9% saline) for 8 weeks. Following the experimentation period, the rats were anaesthetised with intraperitoneally injected ketamine (30 mg/kg; Maijin Biotechnology, Hubei, China) and intramuscular injected chloropromazine (15 mg/kg; Maijin Biotechnology), and blood samples (6 ml/rat) were obtained from the heart. Serum was obtained by centrifugation at 800 × g for 15 min at 4°C, and stored at −80°C until the assessments of H2O2, total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) levels were performed. The cochleae were dissected and used for the extraction of total RNA, genomic DNA and protein. Alternatively, the cochleae were perfused with 2.5% glutaraldehyde (Maijin Biotechnology) for morphological investigation using transmission electron microscopy (TEM), or with 4% paraformaldehyde (Maijin Biotechnology) for immunohistochemical analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling (TUNEL) staining. All experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Guangxi Medical University.

Serum H2O2, T-SOD activity and MDA assays

Using the serum from 30 rats (n=10 per group), the levels of H2O2, T-SOD activity and MDA were quantified using H2O2 Assay, T-SOD Assay and MDA Assay kits, respectively (Nanjing Jiancheng Chemical Industrial Co., Ltd, Nanjing, China), according to the manufacturer's instructions.

DNA isolation and determination of mtDNA CD

Following the final injection, 18 rats (n=6 per group) were euthanised under deep anaesthesia with chlorpromazine (15 mg/kg; Maijin Biotechnology) and ketamine hydrochloride (30 mg/kg; Maijin Biotechnology), and the cochlea from both sides of each rat were rapidly removed. The soft tissue samples were then harvested from the cochleae using an anatomical microscope (Nikon Corporation, Tokyo, Japan). Samples were stored at −80°C until experimentation. The cochlea from one side was used for mtDNA analysis and that from the other side was used for RNA extraction. Total DNA was extracted using a Genomic DNA Purification kit (Tiangen Biotech Co., Ltd, Beijing, China), according to the manufacturer's instructions. The DNA concentration of each specimen was measured using a GeneQuant pro DNA/RNA Calculator (BioChrom, Cambridge, UK). The quantity of the mtDNA CD was determined using a TaqMan polymerase chain reaction (PCR) assay kit (Takara Biotechnology Co., Ltd., Dalian, China). Due to the fact that the D-Loop region is rarely deleted, it can represent the conserved segment. Primers and probes for the mtDNA D-loop and the mtDNA CD have previously been described (38), and were as follows: Forward, 5′-GGTTCTTACTTCAGGGCCATCA-3′; reverse, 5′-GATTAGACCCGTTACCATCGAGAT-3′ for the mtDNA D-loop primers and 5′-FAM-TTGGTTCATCGTCCATACG TTCCCCTTA-TAMRA-3′ for the probe; and forward, 5′-AAGGACGAACCTGAGCCCTAATA-3′; reverse, 5′-CGAAGTAGATGATCCGTATGCTGTA-3′ for the mtDNA CD primers and 5′-FAM-TCACTTTAATCGCCAC ATCCATAACTGCTGT-TAMRA-3′ for the probe. The PCR amplification was performed on a StepOnePlus™ Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) in a 20 µl reaction volume consisting of 10 µl 2X TaqMan PCR mix (Takara Biotechnology Co., Ltd.), 0.4 µl 50X ROX reference dye, 0.4 µl of each forward and reverse primer (10 µM), 0.2 µl of each probe (10 µM), 4 µl of the sample DNA (10 ng/µl) and 4.6 µl distilled water. The cycling conditions comprised an initial phase at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec. The cycle number at which a significant increase in the normalised fluorescence was first detected was designated as the threshold cycle (Ct). The ratio of mtDNA CD to mtDNA was calculated using the following equation: ΔCt = CtmtDNA deletion − CtmtDNA D-loop. The relative expression (RE) was calculated to indicate the factorial difference in the deletions between the experimental groups and the control group. The RE was calculated using the 2−ΔΔCt method, where ΔΔCt = ΔCtmtDNA deletion in experimental group − ΔCtmtDNAdeletion in control group.

TEM

The ultrastructure of the mitochondria in the spiral ganglion cell (SGC) of the cochleae was observed using TEM. A total of 12 rats (n=4 per group) were sacrificed, and both cochleae from each rat were removed, treated with 2.5% glutaraldehyde and fixed overnight at 4°C. The following day, the cochleae were washed with 0.1 M phosphate-buffered saline (PBS) and placed in 10% ethylenediaminetetraacetic acid solution (EDTA; Maijin Biotechnology) for decalcification for 3 days. The spiral ganglion (SG) was carefully dissected and harvested from the cochleae using an anatomical microscope. Following post-fixation in 1% osmium tetroxide (Maijin Biotechnology) for 2 h at room temperature, the SG was dehydrated using graded ethanol or acetone (50, 70, 80, 90 and 100%), immersed in an acetone/Epon 812 mixture (1:1) for 2 h, followed by immersion in Epon 812 for 2 h and final embedding in Epon 812 for 10 h at 80°C. Serial ultrathin sections (50 nm) were collected on copper grids and stained with uranyl acetate and lead citrate. The ultrastructure of the stained sections were examined using a FEI TecnaiG212 TEM (Philips, Amsterdam, Netherlands).

RNA preparation and reverse transcription-quantitative (RT-q) PCR

The mRNA expression levels of NOX3 and P22phox were determined using RT-qPCR. Total RNA was extracted using TRIzol® reagent (Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. cDNA was reverse transcribed using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.). The RNA and cDNA of each sample were analysed using a GeneQuant pro DNA/RNA calculator to assess the concentrations and purity. The cDNA samples (n=6/group) were stored at −20°C until further use. RT-qPCR was performed using real-time SYBR Green PCR technology with a StepOnePlus™ Real-Time PCR system (Applied Biosystems Life Technologies). The primer pairs for NOX3, P22phox and the internal standard (β-actin) were as follows: NOX3, forward 5′-TCGACGAATGGCAGGAAGC-3′ and reverse 5′-ATGGATGGGCACTGGATAAAG-3′; P22phox, forward 5′-ACCGTCTGCTTGGCCATTG-3′ and reverse 5′-TCAATGGGAGTCCACTGCTCAC-3′; and β-actin, forward 5′-CCTGGAGAAGAGCTATGAGC-3′; and reverse 5′-ACAGGATTCCATACCCAGG-3′. The amplification thermocycling conditions were as follows: 30 sec at 95°C, 40 cycles of 5 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. An internal standard was used to normalise the relative gene expression levels. Subsequent melting curve analysis was performed for each gene, and the specificity and integrity of the PCR products were confirmed by the presence of a single peak. The relative expression levels were calculated from the variations in Ct values between the target mRNA and the internal standard (β-actin). Changes in the relative mRNA expression levels between the experimental and control groups were analysed using the 2−ΔΔCt method, as previously reported (39).

Immunohistochemical analysis

The expression of 8-hydroxy-2-deoxyguanosine (8-OHdG) expression was analysed using immunohistochemistry. A total of 12 rats (n=4 per group) were sacrificed, and the cochleae from each rat removed and fixed with 4% buffered-paraformaldehyde overnight, followed by decalcification with 10% EDTA in PBS for 2 weeks, dehydration and embedding in paraffin wax. The cochlea from one side was used for immunohistochemical analysis, and the cochlea from the other side was used for the TUNEL assay. A 5 µm section was deparaffinised in xylene and rehydrated through graded concentrations of ethanol. The samples were incubated with mouse monoclonal anti-8-OHdG antibody (1:4,000; Abcam, Cambridge, MA, USA) overnight at 4°C. The samples were then incubated with CY3-labelled goat anti-mouse secondary antibody (1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 30 min at room temperature. The nuclei were counterstained with DAPI staining solution (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at room temperature. For immunofluorescence imaging, the slides were visualised using a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan) and analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Western blot analysis

The protein expression levels of NOX3, P22phox and C-cas3 were determined using western blot analysis. A total of 18 rats (n=6 per group) were sacrificed, and soft tissue samples (~500 µg) of the cochleae from each rat were dissected. Total protein was extracted using Radioimmunoprecipitation Assay Lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Protein concentrations were determined using an Enhanced Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). A total of 30 µg of each protein lysate was separated by 12% SDS-PAGE (Maijin Biotechnology) and transferred onto polyvinylidene difluoride membranes (Maijin Biotechnology). The membranes were incubated for 1 h in a blocking solution, Tris-buffered saline (TBS; Maijin Biotechnology) containing 5% skimmed milk, and then washed briefly in TBS. The membranes were subsequently incubated overnight at 4°C with the appropriate dilution of rabbit polyclonal anti-NOX3 (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-p22phox (1:100; Wuhan Boster Biological Technology, Ltd.) and rabbit monoclonal anti-C-cas3 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies. Following membrane washing with TBS, to remove excess primary antibody, the membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc.). The membranes were visualised using BeyoECL Plus (Beyotime Institute of Biotechnology). Quantification of the detected bands was performed using Image-Pro Plus 6.0 software. β-actin served as an internal control.

TUNEL assay

Apoptotic cells were detected in situ using a TUNEL POD assay kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, the tissue sections were deparaffinized through a concentration gradient of xylene and rehydrated with distilled water. Following treatment with proteinase K (20 µg/ml; Beyotime Institute of Biotechnology) in 10 mM Tris-HCl (pH 7.6) for 10 min at 37°C, the sections were washed in PBS, and the labelling reaction was performed using labelling solution containing terminal deoxynucleotidyl transferase, its buffer, and fluorescein dUTP at 37°C for 60 min in a humidity chamber. The nuclei were counterstained using DAPI staining solution for 5 min at room temperature. Following washing with PBS, the sections were examined using a laser scanning confocal microscope (C1si; Nikon Corporation, Tokyo, Japan).

Statistical analysis

The data are presented as the mean ± standard deviation. Statistical significance was determined using a one-way analysis of variance, and a least significant difference post-hoc test was used to evaluate the statistical differences between groups. Analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Oxidative stress is induced by D-gal

The serum levels of H2O2, T-SOD and MDA from the rats are summarised in Table 1. Following 8 weeks D-gal exposure, the serum levels of H2O2 and MDA were significantly higher, and the serum activity levels of T-SOD were markedly lower, compared with the control group.

Table I Serum levels of H2O2, T-SOD activity and MDA following treatment with D-gal.

Table I

Serum levels of H2O2, T-SOD activity and MDA following treatment with D-gal.

Compound	Control	D-gal (L)	D-gal (H)
H2O2 (µmol/ml)	14.65±1.78	19.37±1.82a	27.88±3.31a
T-SOD (U/ml)	146.99±7.10	118.11±3.95a	97.59±3.81a
MDA (nmol/ml)	2.44±0.56	5.04±0.93a	7.65±1.09a

{ label (or @symbol) needed for fn[@id='tfn1-mmr-12-06-7883'] } Data are expressed as means ± standard deviation (10 rats per group).

a P<0.01, vs. control group. D-gal, D-galactose; T-SOD, total superoxide dismutase; MDA, malondialdehyde; H, 500 mg/kg; L, 150 mg/kg.

Age-associated accumulation of mtDNA CD is induced by D-gal

To evaluate the level of mtDNA damage induced by D-gal in the cochleae, the levels of mtDNA CD were determined using RT-qPCR with a TaqMan probe. The dual-labelled fluorescent DNA probe used was specific for the novel fusion sequence, which was present only in mutant mtDNA, which contained the CD. As shown in Fig. 1, the levels of mtDNA CD were significantly higher in the D-gal group, compared with the control group. Compared with the control group, the accumulation of mtDNA CD in the D-gal(L) group and in the D-gal(H) group were increased by 1.45- and 2.23-fold, respectively.

Figure 1 Quantitative analysis of the levels of mtDNA CD in the cochleae of rats from the control and D-gal groups. The levels of mtDNA CD were significantly higher in the D-gal groups, compared with the control group. The data are expressed as the mean ± standard deviation of six rats per group. *P<0.05 and **P<0.01, vs. control group. D-gal, D-galactose; mtDNA, mitochondrial DNA; CD, common deletion.

Mitochondrial ultrastructural damage is induced by D-gal

To further investigate the mitochondrial damage induced by D-gal in the cochleae, changes to the mitochondrial ultrastructure in the SGC of the cochleae were observed using TEM. In the control group, numerous round and oval mitochondria with lamellar cristae were present, predominantly around the nucleus of the SGC (Fig. 2A). By contrast, the mitochondria in the SGC of the D-gal groups were swollen with reduced electron density in the matrix or exhibiting severe degeneration. Furthermore, the lipofuscins were also deposited in the SGC of the D-gal groups, indicating structural decay (Figs. 2B–C).

Figure 2 Changes in the mitochondrial ultrastructure of the SGC in the basal turn of the cochleae. (A) Normal mitochondria surrounding the nucleus of the SGC in the control group. (B) Mitochondria with reduced electron density (arrows) or severe degeneration (*) in the SGC of the D-gal(L) group. Lipofuscin (arrowhead) deposited in the SGC of the D-gal(L) group, indicating structural decay. (C) Lipofuscins (arrowhead) deposited in the SGC of the D-gal(H) group, indicating structural decay. (D) Mitochondria with reduced electron density (arrows) or severe degeneration (*) in the SGC of the D-gal(H) group. Scale bar=0.5 µm. D-gal, D-galactose; H, 500 mg/kg; L, 150 mg/kg; M, mitochondria; N, nucleus; SGC, spiral ganglion cell.

Oxidative mtDNA damage is induced by D-gal

To determine whether increased mtDNA CD was associated with increased oxidative stress induced by D-gal in the cochleae, the expression levels of 8-OHdG, a biomarker of DNA oxida-tive damage, were analysed using immunohistochemical analysis (Fig. 3). As shown in Fig. 3A, the expression levels of 8-OHdG were markedly increased in the cytoplasm of the cochleae cells from the D-gal-induced aging rats, compared with those of the control rats, which suggested that D-gal increased oxidative mtDNA damage in the cochleae. Compared with the control group, the immunohistochemical analysis indicated that the expression levels of 8-OHdG in the D-gal(L) and the D-gal(H) groups increased by 3.24- and 6.59-fold, respectively (Fig. 3B).

Figure 3 mtDNA oxidative damage, detected using 8-OHdG immunohistochemistry. (A) Expression and localization of 8-OHdG in the basal turn of the cochleae from the control and D-gal groups. The nuclei were labeled with DAPI. Fluorescent images of the cochleae are presented at ×200 magnification. (B) Quantification of the expression levels of 8-OHdG. The expression levels of 8-OHdG in the D-gal groups were significantly increased, compared with those in the control group. The data are expressed as the mean ± standard deviation of four rats per group. **P<0.01, vs. control group. D-gal, D-galactose; H, 500 mg/kg; L, 150 mg/kg; mtDNA, mitochondrial DNA; 8-OHdG, 8-hydroxy-2-deoxyguanosine.

Increased mRNA expression levels of NOX3 and P22phox are induced by D-gal

To investigate the effects of NOX3-associated oxidative stress on the mtDNA damage induced by D-gal in the cochleae, the mRNA expression levels of NOX3 and P22phox were determined using an RT-qPCR assay. As shown in Fig. 4, the mRNA expression levels of NOX3 and P22phox were significantly higher in the D-gal groups, compared with the control group. Compared with the control group, the mRNA expression levels of NOX3 in the D-gal(L) and D-gal(H) group increased by 1.57- and 2.33-fold, respectively. The mRNA expression levels of P22phox in the D-gal(L) and D-gal(H) group increased by 1.43- and 2.19-fold, respectively.

Figure 4 Quantitative analysis of the mRNA expression levels of NOX3 and P22phox in the cochleae of rats from the control and D-gal groups. The mRNA expression levels of NOX3 and P22phox were significantly increased in the D-gal groups, compared with the control group. The data are expressed as the mean ± standard deviation of six rats per group. **P<0.01, vs. control group. D-gal, D-galactose; H, 500 mg/kg; L, 150 mg/kg.

Increased protein expression levels of NOX3, P22phox and C-cas3 are induced by D-gal

To examine the protein expression levels of NOX3, P22phox and C-cas3 in the cochleae, western blot analysis was performed. As shown in Fig. 5A, the protein expression levels of NOX3, P22phox and C-cas3 were markedly increased following treatment with D-gal. Compared with the control group, the protein expression levels of NOX3, P22phox and C-cas3 in the D-gal(L) group increased by 1.92-, 2.25-and 4.18-fold, respectively. The protein expression levels of NOX3, P22phox and C-cas3 in the D-gal(H) group increased by 3.63-, 3.87- and 6.59-fold, respectively (Fig. 5B).

Figure 5 Western blot analysis and densitometric analysis of the expression levels of NOX3, P22phox and C-cas3 in the cochleae. (A) Expression levels of NOX3, P22phox and C-cas3 in the different treatment groups, determined using western blot analysis. (B) Relative protein expression levels of NOX3, P22phox and C-cas3 were significantly increased in the D-gal groups, compared with the control group. The data are expressed as the mean ± standard deviation of six rats per group. **P<0.01, vs. control group. C-cas3, cleaved caspase 3; D-gal, D-galactose; H, 500 mg/kg; L, 150 mg/kg; NOX3, NADPH oxidase 3.

Cell apoptosis is induced by D-gal

To further understand the occurrence of apoptosis induced by D-gal in the cochleae, the numbers of apoptotic cells were determined using TUNEL staining. As shown in Fig. 6, TUNEL-positive cells were located only in the cochleae of the D-gal-treated rats. A small number of TUNEL-positive cells were limited to the SV of the basal turn of the cochleae.

Figure 6 Apoptotic cells in the cochleae of the rats of the different treatment groups. No apoptotic cells were detected in the control group. Apoptotic cells (arrows) were observed in the basal turn of the cochleae of rats from the D-gal groups, determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling staining. The nuclei were labeled with DAPI. Fluorescent images of the cochleae are presented at ×200 magnification. D-gal, D-galactose; H, 500 mg/kg; L, 150 mg/kg; SV, stria vascularis; OC, organ of Corti; SG, spiral ganglion.

Discussion

The results of the present study demonstrated that the levels of H2O2 and MDA increased, and the activity of T-SOD decreased in the blood of rats following 8 weeks of D-gal exposure, which indicated that an animal model of mimetic aging was successfully established by D-gal (40). The results also indicated that the accumulation of mtDNA CD was significantly increased in the cochleae following treatment with D-gal, which is concordant with the results of previous studies (20–24). Mitochondria are one of the predominant generators of ROS within the cell (41,42). The mitochondrial theory of aging states that ROS generated inside mitochondria damage key mitochondrial components, including mtDNA and respiratory chain complex proteins. This damage accumulates with time and ultimately leads to permanent age-associated mitochondrial dysfunction, which in turn contributes to the aging phenotypes (43,44). The mtDNA 4977 bp deletion in humans, also known as the CD, and the corresponding mtDNA 4834-bp deletion in rats, is the most frequent age-associated mtDNA damage, therefore, CD has been used as a biomarker for aging (38,45,46). An association between elevated mtDNA CD and presbycusis has been observed in several studies (35,47–49). Although no significant difference is observed in elevation of the auditory brainstem response (ABR) threshold between rats with mtDNA CD induced by D-gal and control rats, the hearing threshold in the rats carrying the mtDNA CD increases significantly following aminoglycoside antibiotic injection, compared with the control rats (20). These results indicate that the mtDNA CD may not directly lead to hearing loss, but rather act as a predisposing factor that enhances the sensitivity of the cochleae to aminoglycoside antibiotics (20). To further evaluate D-gal-induced mitochondrial damage in the cochleae, the present study investigated changes in the mitochondrial ultrastructure using TEM. The results indicated that numerous mitochondria were degenerated in different cells of the cochleae in rats following 8 weeks of D-gal exposure. Notably, increased accumulation of mtDNA CD and mitochondrial ultrastructural damage in the cochleae of D-gal-treated rats significantly correlated with increased expression levels of 8-OHdG, a biomarker of DNA oxidative damage (50,51). Therefore, these findings suggested that chronic D-gal treatment and the elicited oxidative stress inside mitochondria may contribute to the increased frequency of mtDNA CD and mitochondrial ultrastructural damage in the cochleae of D-gal-treated rats.

The NADPH oxidase system is another important source of ROS production (52). The expression of NOX3 is almost restricted to the cochleae (26), and NOX3-dependent super-oxide production is dependent on P22phox (27). Previously, the involvement of NOX3 in cisplatin-induced hearing loss. Knockdown of NOX3 using small interfering (si)RNA inhibited cisplatin ototoxicity, as evidenced by the protection of the outer hair cells from damage, and reduced threshold shifts in ABR in the rat (29,30). Furthermore, transtympanic administration of NOX3 siRNA reduced the expression of B cell lymphoma 2 (Bcl-2)-associated protein X (Bax), reversed the decreased expression of Bcl-2 and attenuated the apoptosis induced by cisplatin in the cochleae (29). The results of the present study demonstrated that the expression levels of NOX3 and P22phox were significantly increased in the cochleae of rats in the D-gal groups, compared with those in the control group. The overexpression of NOX3 and P22phox may partly explain the mitochondrial oxidative damage in the cochleae and the occurrence of apoptosis in the SV of the cochleae, in the rats of the D-gal groups.

Apoptosis was also induced by the accumulation of mtDNA mutations (50). In the mitochondrial signalling pathway of apoptosis, mitochondrial dysfunction can lead to permeabilization of the mitochondrial outer membrane, the release of cytochrome c into the cytosol and the activation of key effector protease, caspase-3, by proteolytic cleavage (53,54). To determine whether increased expression levels of C-cas3 is a feature in the cochleae of D-gal-induced aging rats, soft tissue samples from the cochleae of rats in the treatment groups were examined using western blot analysis. The results indicated that D-gal significantly increased the protein expression levels of C-cas3. Apoptosis is also associated with nuclear DNA fragmentation. The present study examined sections of the cochleae using a TUNEL assay, which detects apoptotic cells in situ. Although a small number of apoptotic cells were located in the SV of the basal turn of the cochleae from the D-gal-induced aging rats, the region of damage in the SV of cochleae may not be sufficient to cause hearing loss (55).

In conclusion, the findings of the present study demonstrated that a marked increase in the expression of NOX3 was involved in the accumulation of mtDNA mutations and in the activation of caspase-3-dependent apoptosis in the cochleae of D-gal-induced aging rats. NOX3 may serve as a useful therapeutic target to prevent or reduce the rate of development of presbycusis.

Acknowledgments

The present study was supported by funding from the Science and Technology Development Foundation of Shenzhen, China (grant no. JCYJ20140411092351692), the Medical Scientific Research Foundation of Guangdong Province, China (grant no. B2014370) and the Science and Technology Development Foundation of Shenzhen Nanshan District, China (grant no. 2012014).

Abbreviations:

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