All animal procedures were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (39). The experimental procedures were performed with the approval of the Committee on the Ethics of Animal Care and Use of Huazhong University of Science and Technology (Wuhan, China; permit no. IACUC S512).

A total of 180 male Sprague Dawley (SD) rats (70.10±11.46 g, 3-weeks old) were obtained from the Experimental Animal Centre of Tongji Medical College, Huazhong University of Science and Technology. The rats were individually maintained in a temperature-controlled (23±2°C with 50–60% relative humidity) room with a 12-h light/dark cycle and free access to water and food. Prior to treatment, the rats were allowed to acclimate for 1 week and were then randomly divided into a control group (n=90) and a mimetic aging group (n=90). The rats in the mimetic aging group were treated with D-gal (500 mg/kg/day; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) via subcutaneous injection for 8 weeks. The rats in the control group were injected with normal saline (NS; 500 mg/kg/day) on the same schedule. After the 8-week injection protocol, the rats in the two groups were further divided into a 3-month-old subgroup (just after the last injection), a 9-month-old subgroup (6 months after the last injection), and a 15-month-old subgroup (12 months after the last injection), in which the rats were sacrificed at 3, 9 and 15 months, respectively.

Total DNA of the auditory cortex was extracted using a Genomic DNA Purification kit (Tiangen Biotech Co., Ltd., Beijing, China), and TaqMan (Tiangen Biotech Co., Ltd.) quantitative polymerase chain reaction (qPCR) analysis was used to determine the proportion of the mtDNA common deletion (CD). The D-Loop region copy was used as the conservative segment. The primers and probes for the mtDNA CD and mtDNA D-loop are listed in Table I, as previously described (40). PCR amplification was performed using a LC-480 real-time PCR system (Roche Diagnostics, Basel, Switzerland) in a 20-μl reaction volume. The cycling conditions included 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. ΔCq (Cq_deletion−Cq_D-loop) was used to reflect the abundance of the mtDNA 4,834-bp deletion. The relative expression indicating the factorial difference in the deletions between the NS group and the D-gal-treated group was calculated using the 2^−ΔΔCq method (41), where ΔΔCq=ΔCq_{mtDNA deletion in D-gal-treated group} − ΔCq_{mtDNA deletion in NS group}.

Following deep anesthesia, a total of 36 rats (n=6/subgroup) were perfused transcardially with 2.5% glutaraldehyde subsequent to brief perfusion with 0.9% sodium chloride. Following perfusion, the brain was dissected from the skull, and the auditory cortex was separated from the brain. Subsequently, the auditory cortex was fixed with the 2.5% glutaraldehyde at 4°C for 12 h, and further fixation was performed using 1% osmium tetroxide at 4°C. After 2 h, the auditory cortex was dehydrated using a series of ascending graded ethanol (50, 70, 80, 85, 90, 95 and 100%). Next, the auditory cortex was treated with propylene oxide for ~0.5 h at room temperature and then embedded in graded araldite (A3183; Sigma-Aldrich; Merck KGaA; 1/3 and 1/2, pure araldite) for 12 h per step at 37°C. Following this, the embedded samples were placed in a 60°C incubator for 48 h. Serial ultrathin sections (60–100 nm) were collected on copper grids and stained at room temperature with uranyl acetate for 30 min and then lead citrate for 10 min. A Tecnai G²20 TWIN (FEI; Thermo Fisher Scientific, Inc., Waltham, MA, USA) transmission electron microscope was used to observe the ultrastructure of the sections at ×1,700 and ×5,000 magnification.

Apoptosis was detected using TUNEL staining. Following deep anesthesia, a total of 36 rats (n=6/subgroup) were transcardially perfused with 0.9% sodium chloride and 4% paraformaldehyde solution. Following the perfusion, separated brain sections were fixed with 4% paraformaldehyde solution overnight at 4°C. Following rinsing with distilled water, brain sections were dehydrated in a graded alcohol series (70, 80, 90, 95, 100, 100 and 100%), and immersed in paraffin after clearing in xylene. Serial 5-mm coronal sections containing the auditory cortex were deparaffinized in xylene and rehydrated through graded concentrations of ethanol (100, 100, 95, 90, 80 and 70%). Following a 0.5-h incubation with proteinase K (20 μg/ml; Roche Diagnostics) at 37°C, the sections were stained using an In Situ Cell Death Detection kit (Roche Diagnostics), according to the ratio 1:9 of solution A and B, and incubated at 37°C for 1 h. A DAPI staining solution (1 μg/ml; Beyotime Institute of Biotechnology, Haimen, China) was employed to counterstain the nuclei at room temperature for 10 min. The labelled cells were observed using a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan), and six fields of view for each section were observed.

The auditory cortex tissues from 6 rats in each subgroup were separated and prepared for RNA extraction using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). Following this, an aliquot of the extracted RNA was immediately reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed with a LightCycler 480 RT-PCR system (Roche Diagnostics) using SYBR-Green II PCR Master Mix (Takara Bio, Inc.). The primers were designed by Takara Bio, Inc., and are listed in Table II. β-actin was included as an endogenous control. The amplification conditions were as follows: 95°C for 5 min; 45 cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for 20 sec; followed by 95°C for 5 sec and 65°C for 60 sec. The relative mRNA expression levels in the control and mimetic aging groups were calculated using the 2^−ΔΔCq method.

The protein expression levels were examined using western blotting. A total of 36 rats (n=6/subgroup) were sacrificed, and their auditory cortices were dissected and lysed in radioimmunoprecipitation assay lysis solution (Beyotime Institute of Biotechnology) containing phosphatase inhibitors and PMSF. A BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentrations. The proteins were loaded onto 6 and 12% SDS-PAGE, separated, and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in blocking solution [5% non-fat milk (BD Biosciences, Franklin Lakes, NJ, USA) in Tris-buffered saline] for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: LC3B (ab192890; 1:1,000; Abcam, Cambridge, MA, USA), Beclin 1 (BECN1; ab217179; 1:1,000; Abcam), p62 (ab155686; 1:1,000; Abcam), cleaved caspase 3 (CASP3; 9664; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), B-cell lymphoma 2 (BCL2)-associated X protein (BAX; 34260; 1:500; Signalway Antibody LLC, College Park, MD, USA), BCL-extra large (BCL-xL; 21061; 1:500; Signalway Antibody LLC), BCL2 (ab59348; 1:500; Abcam), phosphorylated (p)-AMPK (sc-33524; 1:1,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p-mTOR (SAB4504476; 1:800; Sigma-Aldrich; Merck KGaA), p-ULK1 (14202; 1:1,000; Cell Signaling Technology, Inc.), Atg13 (ab201467; 1:1,000; Abcam), Atg5 (44254; 1:2,000; Signalway Antibody LLC), Atg7 (38148; 1:500; Signalway Antibody LLC), p-4EBP1 (2855; 1:800; Cell Signaling Technology, Inc.) and β-actin (ab8227; 1:3,000; Abcam). The membranes were incubated with the corresponding secondary antibodies (ANT019; ANT020; 1:4,000; AntGene Biotech Co., Ltd., Wuhan, China) for 60 min at room temperature. Enhanced chemiluminescent Plus (Beyotime Institute of Biotechnology) was used to visualize the membranes. The protein levels were normalized to the level of β-actin in the corresponding lanes and ImageJ 10.0 software (National Institutes of Health, Bethesda, MD, USA) was used for densitometry.

The cleaved CASP3, LC3, BECN1, p62 and BCL2 protein levels were also examined using immunofluorescence. Following deep anesthesia, a total of 36 rats (n=6/subgroup) were transcardially perfused, and separated brain sections were fixed with 4% paraformaldehyde solution overnight at 4°C. Subsequent to rinsing with distilled water, brain sections were dehydrated in graded alcohol series (70, 80, 90, 95, 100, 100 and 100%), and immersed in paraffin after clearing in xylene. A 5-um paraffin section was deparaffinized in xylene and rehydrated through graded concentrations of ethanol (100, 100, 95, 90, 80 and 70%). Following 10-min citrate solution antigen repair in boiling water, donkey serum albumin (ANT051; AntGene Biotech Co., Ltd.) was used to block nonspecific binding for 30 min at room temperature. Subsequently, the sections were incubated overnight at 4°C with the aforementioned primary antibodies diluted to 1:200. The sections were then incubated with a secondary antibody (ANT024; 1:200; AntGene Biotech Co., Ltd.) for 2 h after repeated washes with Tris-buffered saline-Tween-20. Sections that were not incubated with a primary antibody were used as the negative controls. A laser scanning confocal microscope (Nikon Corporation) was used to observe the staining at ×200 and ×600 magnifications.

The data were presented as the mean ± standard error of the mean. The statistical analyses were performed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Statistically significant differences between the groups were determined using one-way analysis of variance followed by Tukey’s post hoc test, and P<0.05 was considered to indicate a statistically significant difference.

CDs are also called mt 4,834-bp deletions and correspond to mtDNA 4,977-bp deletions in humans (42). Previous research has indicated an association between CD levels and presbycusis (43). TaqMan qPCR analysis demonstrated that D-gal treatment induced significantly higher CD levels in the D-gal groups at all ages compared with the levels in the age-matched NS groups (Fig. 1). In addition, the CDs accumulated with age in both the natural aging group and the mimetic aging group.

Neurons in the auditory cortex in the NS group did not display obvious ultrastructural changes in the 3- and 9-month-old rats (Fig. 2A and B). However, in the 15-month-old rats in the NS group, swollen mitochondria, disrupted myelin and lipofuscin were observed (Fig. 2C). In the D-gal-treated groups, normal nuclei, mitochondria, Golgi apparatus and rough endoplasmic reticulum were observed in the 3-month-old subgroup (Fig. 2D), whereas in the 9-month-old D-gal-treated rats, irregular nuclei, condensed chromatin, accumulated lipofuscin, swollen mitochondria and disrupted myelin were identified (Fig. 2E). Additionally, these changes were more pronounced in the 15-month-old D-gal-treated rats (Fig. 2F). Furthermore, more autophagosomes and auto-lysosomes were observed in the 15-month-old NS group and the 3- and 9-month-old D-gal subgroups, while few autophagosome and auto-lysosomes were identified in the 3- and 9-month-old NS subgroups and 15-month-old D-gal subgroup, particularly in the 15-month-old D-gal subgroup.

The detected protein level of cleaved CASP3 by western blotting and immunofluorescence assays, as well as the results of TUNEL staining (Fig. 3), revealed that apoptosis levels were significantly increased in the 9- and 15-month-old D-gal groups compared with the levels in the respective NS control groups, and this level increased with age in both groups.

The mRNA levels of LC3 and BECN1 were increased at 3 and 9 months in the D-gal-treated groups; however, these levels were decreased at 15 months compared with those in the age-matched NS control groups (Fig. 4A a–c). The same trend was observed for the protein expression levels of LC3 and BECN1 (Fig. 4A d–f). The immunofluorescence images demonstrating the protein levels of LC3 and BECN1 in the auditory cortex with aging are shown in Fig. 4B. It was demonstrated that autophagy was increased from 3 months to 15 months in the NS group, while it decreased with aging in the D-gal group.

Furthermore, in addition to LC3 and BECN1, p62 protein expression levels were detected using western blotting and immunofluorescence assays. p62 expression levels were significantly decreased in the 3-month-old D-gal subgroup compared with the levels in the age-matched NS group, while these levels were significantly increased at 15 months compared with those in the NS groups (Fig. 5). Additionally, these autophagy-related proteins demonstrated approximately equivalent levels in the 9-month-old D-gal induced mimetic aging group and 15-month-old NS group.

By investigating the levels of CD, and cell apoptosis and neuronal degeneration at 3, 9 and 15 months in the two groups, the present results demonstrated that the aging level in the 15-month-old rats in the NS group was higher than those in 9-month-old NS subgroup. Additionally, the present results indicated that 9-month-old rats in the D-gal-treated group presented an approximately similar life status compared with that of the 15-month-old rats in NS group, which was further confirmed by immunofluorescence analyses of LC3 and BECN1. Furthermore, neuronal degeneration and cell apoptosis were more apparent in the 15-month-old D-gal subgroup than the 9-month-old D-gal subgroup. The present results revealed that the function of D-gal exposure that accelerated the aging in rats was confirmed in the present study, which is consistent with previously research (6,44). The lifespan of the majority of SD rats is 2.5–3 years, and 15 months is equivalent to the late adult period of rats and roughly equivalent to 40-year-old humans (45). In other words, rats in the 15-month-old NS subgroup and 9-month-old D-gal subgroup were classified as late adults; 15-month-old rats in the D-gal-induced mimetic aging group demonstrated more severe aging than that of other subgroups and should be classified in the old period.

Based on the present results, it was demonstrated that cell apoptosis level increased with aging in the auditory cortex of SD rats, while the level of autophagy showed an increasing trend from young to adult rats and decreased at an old age (Fig. 6).

Previous research has suggested that autophagy has an anti-apoptotic effect in injured spinal cord neurons (46). To investigate the role of autophagy in the present study, in addition to the detection of apoptosis level by TUNEL analysis and cleaved CASP3, the changes in the BCL2 family proteins (BCL2, BCL-xL and BAX) were also assessed. mRNA and protein expression levels of anti-apoptotic proteins, BCL2 and BCL-xL, as well as the ratio of BCL2/BAX and BCL-xL/BAX were significantly increased at 3 months in the mimetic aging groups but decreased at 15 months compared with those in the age-matched NS control groups. However, the mRNA and protein levels of pro-apoptotic protein, BAX, demonstrated no significant changes at 3 months in the mimetic aging groups, but increased significantly at 9 and 15 months compared with those in the age-matched NS control groups (Fig. 7A). The immunofluorescence images demonstrated that BCL2 was increased from 3 to 15 months in the NS group, while it decreased with aging in the D-gal group (Fig. 7B).

To investigate the hypotheses regarding the activation and inhibition of autophagy, the protein expression levels of the AMPK-mTOR-ULK1 signaling pathway in the auditory cortex were detected using western blotting. Representative western blots demonstrated that the expression of p-AMPK was significantly increased in a compensatory manner at 3 months, but significantly decreased at 9 and 15 months in the D-gal groups compared with its expression in the age-matched NS group. However, p-mTOR and p-ULK1 demonstrated an inverse trend with p-AMPK. Additionally, the autophagy-related protein Atg13 was significantly increased at 3 and 9 months in the D-gal group compared with the levels in the age-matched NS group (Fig. 8), which was consistent with the expression results for LC3-II and BECN1.

The 4EBP1 phosphorylation level was also investigated. As demonstrated in Fig. 9, the expression level of p-4EBP1 was significantly increased at 3 and 9 months in the D-gal groups compared with that in the age-matched NS groups. However, the autophagy-related proteins Atg5 and Atg7 were significantly increased at 3 months, while significantly decreased at 15 months in D-gal groups compared with the levels in the age-matched NS group. These results suggest that there is no obvious relevance between 4EBP1 activity and autophagy regulation. Based on these findings, it was hypothesized that p-4EBP1 was not involved in the inhibition of autophagy in the present model. Additionally, the potential relationships among AMPK, mTOR, p-ULK1 (Ser 757), autophagy and apoptosis in the present model are presented in Fig. 10.