Peng et al. BMC Molecular and Cell Biology(2023) 24:27
https://doi.org/10.1186/s12860-023-00480-7

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

D-galactose-induced mitochondrial oxidative
damage and apoptosis in the cochlear stria
vascularis of mice
Zhe Peng1,2, Chunli Zhao1,2, Zijing Yang1,2, Shusheng Gong1,2* and Zhengde Du1,2*
Abstract
Background Age-related hearing loss, known as presbycusis, is the result of auditory system degeneration.
Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play
important roles in the occurrence and progression of aging. The D-galactose (D-gal)-induced aging model is wellknown and widely utilized in aging research. Our previous studies demonstrate that administration of D-gal causes
mitochondrial oxidative damage and causes subsequent dysfunction in the cochlear ribbon synapses, which in turnleads to hearing changes and early stage presbycusis. Stria vascularis (SV) cells are vital for hearing function. However,
it is unclear to what extent D-gal induces oxidative damage and apoptosis in the cochlear SV of mice. In addition, thesource of the causative ROS in the cochlear SV has not been fully investigated.
Methods In this study, we investigated ROS generation in the cochlear SV of mice treated with D-gal. Hearing
function was measured using the auditory brainstem response (ABR). Immunofluorescence was used to examine
apoptosis and oxidative damage. Transmission electron microscopy was also used to investigate the mitochondrial
ultrastructure. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end-labeling (TUNEL) assay. Mitochondrial membrane potential (MMP) and ATP were also measured.
Results We found that D-gal-treated mice exhibited a significant shift in the mean amplitude and latency of the ABR;a remarkable increase in the levels of NADPH oxidase (NOX-2), Uncoupling protein 2 (UCP2) and cleaved caspase-3(c-Cas3) was observed, as well as an increase in the number of TUNEL-positive cells were observed in the SV of
mice. Both the expression of the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) and acommonly occurring mitochondrial DNA deletion were markedly elevated in the SV of mice that had been treatedwith D-gal to induce aging. Conversely, the ATP level and MMP were significantly reduced in D-gal-induced aging
mice. We also found alterations in the mitochondrial ultrastructure in the SV of aging mice, which include swollen anddistorted mitochondrial shape, shortened and thickened microvilli, and the accumulation of lysosomes in the SV.
Conclusion Our findings suggest that the impairment of cochlear SV during presbycusis may be caused by
mitochondrial oxidative damage and subsequent apoptosis.

*Correspondence:
Shusheng Gong
gongss@ccmu.edu.cn
Zhengde Du
duzhengde@ccmu.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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in this article, unless otherwise stated in a credit line to the data.

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

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Keywords Mitochondrial oxidative damage, Apoptosis, Stria vascularis, D-galactose, Mouse

Introduction
Age-related hearing loss (ARHL), also known as presbycusis, is the progressive loss of hearing associated withaging and is the most common sensory disorder in theelderly population [1–4]. There are four types of ARHL
involving the cochlea: sensory, neural, strial/metabolic,and cochlear conductive, according to Schuknecht’s pioneering work. Strial or metabolic presbycusis is the lossof stria vascularis (SV) cells that impairs blood supply tothe inner ear, which causes hearing loss that manifests
across all frequencies [5]. The hypothesis that vascular degeneration can play a role in presbycusis is widely
held concept [1, 6, 7]; however, the underlying biologicalmechanism remains unclear. Therefore, numerous animal models have been developed to promote the studyof molecular mechanisms of ARHL. Animal models of
D-galactose (D-gal) were widely used for this purpose[8–13]. D-gal is a common reducing sugar in vivo. At
normal levels, galactose-1-phosphate uridyltransferase
and galactokinase convert it to glucose. At high concentrations, however, it can be oxidized to aldehydes andH2O2 in the presence of galactose oxidase, causing oxidative stress in vivo [14].
In most mammalian cells, reactive oxygen species,
including hydroxyl radicals H2O2, are produced mainly
by mitochondria [15, 16]. A growing body of research
indicates that reactive oxygen species (ROS) and oxidative stress play an important role in the pathogenesis ofvarious cochlear abnormalities, especially sensorineural
hearing loss, which can be induced by ototoxicity, senility, and noise exposure [17–19].
Some studies have demonstrated that D-gal-treatedanimal models exhibit increased oxidative stress levels
and an accumulation of mitochondrial DNA (mtDNA)
common deletion (CD) in both the peripheral and central auditory systems [20–22]. A previous study on
D-gal-induced aging found that only a small number ofmarginal cells in the cochlear SV are lost due to the activation of mitochondria-dependent apoptosis [11]. The
SV is a highly vascularized tissue that lines the cochlear
lateral wall. The SV regulates cochlear fluid homeostasis and produces the endo-cochlear potential for soundtransmission. Moreover, it acts as an important bloodlabyrinth barrier, tightly regulating the passage of molecules from the blood into the cochlea. Therefore, ahealthy SV is vital for hearing function [23]. However,few studies have systematically studied D-gal-induced
oxidative damage and apoptosis in the mitochondria of
the cochlear SV of mice. In addition, the source of the
ROS that causes cochlear SV impairment has not been
fully investigated.

Here, we investigated the expression of nitrous oxide(NOX2), uncoupling protein 2 (UCP2), and the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine(8-OHdG) in the SV, as well as changes in mitochondrial
membrane potential (MMP), ATP, and mtDNA CD levels
were investigated in mice with D-gal-induced aging. To
determine the activity of caspase-3, the proteolytic cleavage product of caspase-3 (an N-terminated peptide substrate) was evaluated. DNA fragmentation in the nucleus
was detected by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end-labeling (TUNEL). The
ultrastructural variation of target cells was observed bytransmission electron microscopy (TEM).
In this study, we hypothesized that nitric oxide andmitochondria-dependent ROS generation, mtDNA oxidative damage, and apoptosis in cochlear SV may be
among the primary causes of auditory system degeneration in D-gal induced aging.

Materials and methods
Animals

60 male C57BL/6J mice aged 5 weeks were obtained from
Capital Medical University’s Experimental Animal Center. The mice were raised in an environment at a constanttemperature (22–23 °C) and humidity (60 ± 5%), on a
12-h light/dark cycle, with free access to food and water.
All mice were acclimated for a week before being divided
into three groups (n = 20 per group): control, low-dose
D-gal (D-gal-L) and high-dose D-gal (D-gal-H) groups.
The mice in the control group received a subcutaneous
injection of 0.9% saline (the D-gal vehicle) once daily for
six weeks. The second and third groups of mice received
a subcutaneous injection of D-gal (Sigma-Aldrich, St.
Louis, MO, USA) once daily for 6 weeks at a dose of 500
and 1000 mg/kg body weight, respectively. In compliance with the National Institutes of Health’s Guidelines
for the Care and Use of Laboratory Animals, every effort
was made to minimize animal suffering and reduce thenumber of animals used. The Capital Medical University Committee on the Ethics of Animal Experiments
approved the study’s protocol.
Auditory function evaluation

Auditory brainstem response (ABR) was used to analyze
the hearing function in all animals. The method of ABR
testing has been described previously [24]. Briefly, it was
conducted in an anechoic room using BioSigRZ software (Tucker-Davis Technologies, Alachua, FL, USA). A
total of 18 mice (n = 6 per group) were anesthetized byintraperitoneal injection of a ketamine (100 mg/kg) andxylazine (10 mg/kg) mixture. Before testing, the externalPeng et al. BMC Molecular and Cell Biology(2023) 24:27

auditory canal and tympanic membrane (TM) were
examined with an electric otoscope. Mice with acute otitis externa (AOE) or otitis media were excluded from thestudy.
The sound delivery tube of an inserted earphone wastightly fitted into the external auditory canal. Subcutaneous needle electrodes were used to record the ABR
response. Three stainless steel recording electrodes
were subcutaneously inserted posterior to the tested
pinna (recording), vertex (reference), and contralateralpinna (ground). A TDT System 3 (Tucker-Davis Technologies) was used to measure ABR responses to a tone
burst stimulus at 8 kHz, 16 and 32 kHz, starting at a
90-dB sound pressure level (SPL) and decreasing in 5-dB
steps. The ABR threshold for each frequency was determined, which is the lowest SPL that will reliably produce
an ABR recording with one or more distinct waves thatcan be easily distinguished by visual inspection of the
waveforms. To confirm the uniformity of the waveforms,
the procedure was repeated at low SPLs close to thethreshold.
Immunohistochemistry of cochlear frozen sections

Mice (18 per group, n = 6) were anesthetized with ketamine and xylazine before being euthanized via cervicaldislocation. Cochleae were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, then washed three times
with PBS before being decalcified in 10% EDTA for 48 h
at 4 °C. Samples were dehydrated in a sucrose gradient of
20 and 30% for 1 h each. Samples were embedded in optimal cutting temperature compound before being sliced
at 20 °C with a thickness of 10 μm using a Leica Cryastat (Wetzlar, Germany) and mounted on Superfrost Plus
microscopic slides (KeyGEN Biotech, Nanjing, China).
One side of the cochleae were used for immunohistochemistry, and the other side for TUNEL staining. The
expression of NOX2, UCP2, 8-OHdG, cleaved caspase 3(c-Cas3) were evaluated using immunohistochemistry.
Cochlear sections were washed and then incubated with
0.3% Triton X-100 solution (Sigma-Aldrich) for 30 min atroom temperature. They were washed again and blockedwith 10% goat serum for 1 h at room temperature andincubated overnight at 4 °C with primary antibodies:monoclonal mouse anti-8-OHdG (diluted 1:200; Abcam,
USA, Cat.No#ab62623), anti-NOX2 (diluted 1:200; Invitrogen, Waltham, MA, USA, Cat.No#PA5-76034,), antiUCP2 (diluted 1:200; Invitrogen,Cat.No#PA5-103176)
and anti-C-cas3 (diluted 1:200; CST, Danvers, MA,
USA, Cat.No#Asp175). The slides were then washedand incubated in the dark for 2 h with Alexa Fluor 568-,
Alexa Fluor 488-, or Alexa Fluor 594-conjugated secondary antibodies at a dilution of 1:300. The sections were
washed and mounted using mounting media containing 4′,6-diamidino-2-phenylindole (DAPI; ZSGB-BIO,

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Beijing, China). Following a wash with PBS, the sections
were examined using a laser scanning confocal microscope (Leica TCS SP8). The expression levels of 8-OHdG,
NOX2, UCP2 and c-Cas3 were analyzed using the ImagePro Plus 6.0 software (Media Cybernetics, Rockville, MD,
USA). Sections were treated the same way in the negative
control, but the primary antibody incubation step was
skipped.
DNA isolation and mitochondrial DNA (mtDNA) 3860-bp
deletion assay

The method of DNA isolation and mtDNA deletionhas been described previously [25, 26]. The accumulation of mtDNA was evaluated in 12 mice (n = 4 pergroup) using real-time PCR assay. DNA extraction byusing DNA isolation kit (Tiangen Biotech Co., Beijing,
China). GeneQuant pro DNA/RNA Calculator (Amersham Pharmacia Biotech, Staffanstorp, Sweden) wasused to measure DNA concentration. 12 S rRNA gene
was used as control (GenBank: NC_006914). The primers
used to amplify the mtDNA 3860-bp deletion and 12 S
rRNA were previously described [25]. PCR amplification was performed on a StepOne Real-Time PCR System(Applied Biosystems, Waltham, MA, USA). The cycling
conditions were as follows: 95 °C for 10 min, 40 cycles at
95 °C for 15 s and 60 °C for 1 min. The cycle number atwhich a significant increase in normalized fluorescence
was first detected was designated as the threshold cycle
number (Ct). The ratio of the mtDNA 3860-bp deletion
to total mtDNA was calculated as ΔCt = (Ct3860-bp
deletion − Ct12S rRNA), and the relative expression was
calculated as 2 − ΔΔCt. The PCR products of the mtDNA
3860-bp deletion were cloned and verified with ABI
Prism 377XL sequencer (Applied Biosystems).
Transmission electron microscopy

The ultrastructure of the mitochondria in the cochlea SV
was observed using TEM. Twelve mice (n = 4 per group)
were anesthetized with ketamine and xylazine before
being euthanized via cervical dislocation. Then cochleae
from each mouse were removed, treated with 2.5% glutaraldehyde and fixed overnight at 4 °C. The following
day, the cochleae were washed with PBS and placed in
10% EDTA for decalcification for 72 h. The SV was carefully dissected and harvested from the lateral wall of thecochlea. After fixation in 1% osmium tetroxide (Maijin
Biotechnology, Nanjing, China) for 2 h, the SV was dehydrated using graded ethanol or acetone, followed by a 2-h
immersion in acetone/Epon 812 mixture (1:1), and a 10-h
immersion in Epon 812 at 80 °C. Ultrathin Sect. (50 nm)
were taken serially on copper grids and were stained withuranyl acetate and lead citrate., A FEI TecnaiG212transmission electron microscope (Philips, Amsterdam,

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

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Netherlands) was used to examine the ultrastructure of
the stained sections.

analyses (SPSS, Inc., Chicago, IL, USA). A statisticallysignificant difference was defined as P < 0.05.

TUNEL assay

Results

A TUNEL staining kit (Roche Molecular Biochemicals,
Mannheim, Germany, Cat No#11,684,817,910) was used
to identify apoptotic cells in situ. The labeling reaction,
which contained terminal deoxynucleotidyl transferase,
was carried out at 37 °C for 60 min in a humidity chamber after treatment with 10% goat serum in 0.1% Triton
X-100 for 3 min. DAPI staining solution was used tocounterstain the nuclei for 5 min at room temperature.
The sections were cleaned with PBS and then examinedwith a laser scanning confocal microscope (Leica TCS
SP8).

Auditory impairment induced by D-gal

ATP and MMP measurement

Immunohistochemistry staining of cochlear SV showedthat expression levels of NOX2, UCP2 and 8-OHdG proteins were significantly higher in the D-gal treatmentgroups than in the control group (Figs. 2 and 3). In addition, 8-OHdG was predominantly distributed in the cytoplasm of SV cells, revealed as red dotted fluorescence in
Fig. 3A, and was more prevalent in the D-gal-H group.

The luciferin-luciferase system was employed to quantify the ATP levels in these specimens according to the
manufacturer’s instructions (Beyotime, Jiangsu, China,
Cat.No#S0027). The relative ATP content of each specimen was determined by measuring the bioluminescentintensity with a microplate reader. A mitochondria isolation kit (Beyotime, Cat.No#C2006) was used to isolate
mitochondria from each specimen in order to measure
the MMP. Then, in accordance with the manufacturer’s
instructions, the JC-1 probe was used to evaluate changes
in MMP.
Statistical analysis

Data are presented as mean ± standard deviation. Oneway analysis of variance was used to determine statistical significance, and a post hoc least significant difference
test was used to assess statistical differences between
groups. SPSS 13.0 software was used to conduct the

The ABR test was used to assess the auditory function
in control and D-gal-treated mice. Five vertex-positive
peaks make up the mouse ABR waves. We discoveredthat the D-gal-induced ABR waves I–IV had a poorer
amplitude and a longer latency than in the control mice
(Fig. 1). Thus D-gal-induced aging does not lead to ABRthreshold changes, but alters the amplitude and latency,
which was particularly evident in the D-gal-H group.
D-gal induces an increase in protein expression of NOX2,
UCP2, and 8-OHdG in SV

D-gal induces an increase in mtDNA CD level in SV

The mtDNA CD level in the cochlear SV was measured
by qPCR. A dual-labeled fluorescent DNA probe wasdesigned to specifically identify the fusion sequence,
which was only present in mutant mtDNA that harboredCDs. As shown in Fig. 3C, the accumulation of mtDNA
CDs was significantly higher in the D-gal-treated groups
than in the control group (P < 0.01).

Fig. 1  A schematic representation of the scala media and auditory function parameters of the mice in the control, D-gal-L, and D-gal-H groups. (A)Scheme showing that the Strial Marginal Cells (SMCs) in the lateral wall of the cochlea are critical for maintaining the K+ cycle and cochlear homeostasis.
(B) Representative wave shapes of the ABR for the control, D-gal-L, and D-gal-H groups. The mean amplitudes of the ABR waves I–V were lower in the
D-gal-L and D-gal-H groups than in the control group. The mean latency values of the ABR waves I–V were higher in the D-gal-L and D-gal-H groups thanin the control group

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

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Fig. 2 The NOX2 and UCP2 protein expression levels in the SV of the mice of different groups. (A) Representative images showing the expression andlocalization of NOX2 and UCP2 in the SV of mice in the control, D-gal-L, and D-gal-H groups. Scale bars = 25 μm. (Green: NOX2; red: UCP2; blue: DAPI). (B)NOX2 protein levels in the D-gal-L group were significantly higher than in the control group but lower compared to that of the D-gal-H group. (C) UCP2
protein levels in the SV of the mice of the D-gal-L group were significantly higher than those of the control group but lower compared to that of the Dgal-H group. Data are expressed as the mean ± SD of 6 mice per group. **, P < 0.01 vs. the control group; ^^, P < 0.01 vs. the D-gal-L group

D-gal induces a decrease in ATP level and MMP in cochlea

Alterations in the mitochondrial ultrastructure in the SV

To further assess mitochondrial function, we measured ATP level and MMP using a colorimetric test kit.
ATP levels were 14.17 ± 0.72, 11.33 ± 1.1 and 9.15 ± 0.8
9.15 ± 0.8 nmol/mg in control, D-gal-L and D-gal-Hgroups, respectively. ATP levels in the D-gal-L and
D-gal-H groups were significantly lower than in control
group (Fig. 4B). MMP production levels were 8.69 ± 0.31,
6.89 ± 0.5, and 5.87 ± 0.5 nmol/mg in control, D-gal-L, andD-gal-H groups, respectively. The MMP in the D-gal-L
and D-gal-H groups were significantly lower than in control group (Fig. 4C).

To investigate changes in the ultrastructure of mitochondria in SV, we compared the mitochondrial morphology
in the three groups (Fig. 4A). The TEM results revealed
that the SV of the mice in the control group was richin mitochondria, which had a normal shape and size; awith normal microvilli. In contrast, numerous mitochondria in the SV of mice in the D-gal-L and D-gal-Hgroups were swollen and had a reduced electron density
in their matrix. In the D-gal-L group, the microvilli werereduced or shortened, and most microvilli were missingin the D-gal-H group. We also observed cytolysosomes

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

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Fig. 3 The 8-OHdG expression in the SV and quantification of the mtDNA CDs. (A) Representative images showing the expression and localization of
8-OHdG in the SV of mice in the control, D-gal-L, and D-gal-H groups. Scale bars = 25 μm. (Red: 8-OhdG; blue: DAPI). (B) The protein expression of 8-OHdG
in the SV of the D-gal-L group was significantly higher than that of the control group mice but lower compared to that of the D-gal-H group. (C) The
mtDNA CD level in the cochleae of mice in the D-gal-L group was significantly higher than that in the mice of the control group but lower compared tothat of the D-gal-H group. Data are expressed as the mean ± SD of 6 mice per group. **, P < 0.01 vs. the control group; ^, P < 0.05 vs. the D-gal-L group;^^, P < 0.01 vs. the D-gal-L groupcontaining mitochondrial remnants in the D-gal-Hgroup.
D-gal induces an increase in the c-Cas3 protein expression
in the SV

The protein expression level of c-Cas3 in the cochlear SV
was investigated by immunohistochemistry. As shown in
Fig. 5A and B, its expression in the D-gal treated groups
was significantly higher than in the control group.

D-gal induces an increase in TUNEL-positive cells in the SV

Apoptotic activity was observed in the SV compartmentsof D-gal treated mice (Fig. 5A). The number of TUNELpositive cells in the cochlear SV was significantly higher
in the D-gal-treated groups than in the control group(Fig. 5C, P < 0.01).

Discussion
Our main findings revealed that D-gal induced oxidative damage and apoptosis of SV mitochondria in mousecochlea. According to the mitochondrial theory of aging,mitochondrial DNA is highly susceptible to oxidative

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

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Fig. 4 Alterations in the mitochondrial ultrastructure of the SV and quantification of the ATP level and MMP in the cochlea. (A) The mitochondria in thecontrol group were normal (arrows), and those in the D-gal-L and D-gal-H groups had a swollen shape and reduced electron density in their matrix (arrowheads). Severely degenerated mitochondria are indicated with double arrows. In the control group, the microvilli in mitochondria of marginal cells
were normal (circle); in the D-gal-L group, the microvilli were reduced or shortened and in the D-gal-H group, most microvilli were missing. Moreover,cytolysosomes containing mitochondrial remnants were found (asterisk) in the D-gal-H group. N, nucleus; n = 4 mice per group. Scale bar = 0.5 μm. (B)The ATP level in the cochleae of the mice in the D-gal-L group was significantly lower than that of the control group but significantly higher than that of
the D-gal-L group. (C) The MMP in the cochleae of the mice in the D-gal-L group was significantly lower than that of the control group but significantly
higher than that of the D-gal-L group. Data are expressed as the mean ± SD of 6 mice per group. **, P < 0.01 vs. the control group; ^, P < 0.05 vs. the D-gal-Lgroup; ^^, P < 0.05 vs. the D-gal-L group

damage during aging due to excess ROS production,
decreased antioxidant levels, and a lack of protective histone proteins [27]. Du et al. found that the NOX2 system
is a prominent generator of ROS in the cochlea of D-gal
induced aging rats [11]. NOX2 is an enzyme complex that
produces ROS by transferring electrons from NADPH tooxygen [28]. Also, it has been demonstrated that NOX2
is broadly distributed in various cells and organs, including the cochlea [29, 30]. Here, we discovered that NOX2
expression is increased in the cochlear SV of mice subjected to accelerated aging induced by D-gal (Fig. 2).
Together, these data indicate that ROS generated by
NOX2 may be, at least in part, responsible for the D-galinduced mtDNA oxidative damage in the cochlear SV.
In addition to the NADPH oxidase system, mitochondria are another major source of ROS generation in vivo
[27]. Mitochondrial ROS production can result in theactivation of UCP2, which is localized to the inner mitochondrial membrane [31–33]. The level of cellular ROS
is also indirectly reflected in UCP2 expression. Previous
research had shown that rat models of D-gal-induced

aging with overexpressed UCP2 in the cochlea and central auditory system [34]. In the current investigation, we
discovered that UCP2 protein was significantly higher inthe cochlea SV of mice treated with D-gal (Fig. 2). Thisfinding suggests that mitochondrial ROS may be implicated in D-gal-induced mtDNA damage in the cochlea
SV.
There is evidence that ROS overproduction in mitochondria can result in the progressive destruction of
mtDNA [35]. One of the most common and best characterized age-associated mtDNA mutations is CD [36,37]. CD, which is used as a molecular marker to evaluate age-induced DNA damage in the inner ear [10, 21].
Here, we found that D-gal significantly elevated CD levelsof mtDNA in the cochlear SV (Fig. 3C). According to a
recent study, the degree of hearing loss in presbycusis issignificantly correlated with the CD level in the cochlea[38]. Therefore, the accumulation of the CD mutationis considered to play a crucial role in the developmentof ARHL. Moreover, the level of 8-OHdG, a biomarkerof oxidative DNA damage, is also elevated and the highPeng et al. BMC Molecular and Cell Biology(2023) 24:27

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Fig. 5 Changes in caspase-3-mediated apoptosis in the SV of mice. (A) Representative images showing the expression and localization of c-Cas3 protein
and TUNEL-positive cells in the cochlear SV of mice in the control, D-gal-L, and D-gal-H groups. Scale bars = 25 μm. The c-Cas3 protein expression in the
SV of the mice of the D-gal-L group was significantly higher than that of the control group but lower compared to that of the D-gal-H group. Comparedwith that of the D-gal-L group, a greater number of TUNEL-positive cells was found in the mice of the D-gal-H group; Almost no TUNEL-positive cells were
found in the SV of mice in the control group. (Green: c-Cas3; red: TUNEL; blue: DAPI). (B) The expression of c-Cas3 protein in the SV of the mice in the Dgal-L group was significantly higher than those of the control group but lower compared to that of the D-gal-H group. (C) The number of TUNEL-positive
cells in the SV of the mice of the D-gal-L group was significantly higher than that of the control group but lower compared to that of the D-gal-H group.
Data are expressed as the mean ± SD of 6 mice per group. **, P < 0.01 vs. the control group; ^^, P < 0.01 vs. the D-gal-L grouplevel of 8-OHdG in the cytoplasm, consistent with theview that D-gal administration leads to mtDNA oxidative damage in the cochlear SV (Fig. 3A, B). To further
evaluate the mitochondrial damage induced by oxidative
stress, we investigated the mitochondrial ultrastructuralchanges in the SV of cochleae. Swelling, deformation,vacuolization and lower electron density were foundin numerous mitochondria in the marginal cells of the
SV. In addition, the presence of cytolysosomes andthe shortening and disappearance of microvilli further
directly reflect the mitochondrial dysfunction (Fig. 4A).
Cell energy deficiency and mitochondrial malfunction
may be driven by mtDNA mutations and ultrastructural
damage. In the SV of aging mice induced by D-gal, we
demonstrated that mitochondrial ATP production had
decreased (Fig. 4B). It is well known that maintaining oxidative phosphorylation and ATP generation requires anormal MMP. The cumulative burden of ROS production
can lead to MMP breakdown, which results in energy

Peng et al. BMC Molecular and Cell Biology(2023) 24:27

deficiency and mitochondrial dysfunction. The highaccumulation of ROS leads to the destruction of MMP,
which in turn leads to energy deficiency and mitochondrial malfunction. In the present study, both the D-gal-L
and D-gal-H groups had a reduced MMP (Fig. 4C). The
concomitant decline in MMP decreases the ATP levelsin the cochlea, which is insufficient for the normal ion
transportation in the inner ear, further inducing hearing
loss [39].
Mitochondrial oxidative damage can initiate apoptosis. Apoptosis is initiated by two pathways: the intrinsic
pathway, which is initiated by a change in mitochondrial membrane permeability, and the extrinsic pathway,
which is activated by death receptors [40–42]. In theapoptosis intrinsic pathway, mitochondrial dysfunction
can result in the permeabilization of the mitochondrial
outer membrane, the release of cytochrome c into thecytosol, and activation of caspase-3 via proteolytic cleavage [43, 44]. To determine whether an increased level ofc-Cas3 is a characteristic of the cochlear SV of mice subjected to D-gal-induced aging, the expression of c-Cas3
was evaluated through immunohistochemical analysis of frozen cochlear sections. Cleaved caspase-3 waselevated in the cochlear SV in both D-gal-L and D-galH treated mice. The expression of c-Cas3 was higher inthe latter group than in the former (Fig. 5). DNA fragmentation is associated with apoptosis. We found that
TUNEL-positive cells in the cochlear SV were increased
significantly in the D-gal-treated groups. Although weobserved a higher number of apoptotic cells in the SV of
the cochleae (Fig. 5), this damage in the peripheral auditory system might be insufficient to explain the hearing
impairment [6].
Taken together, these results indicate that mitochondrial oxidative damage and the subsequent apoptosis may
be responsible for cochlear impairment. D-gal induces
mitochondrial dysfunction, which in turn, increases ROS
production. A considerable amount of ROS can trigger
progressive oxidative damage, promote the oxidation of
mtDNA and associated proteins [45], which can result in
apoptosis and impairment of the normal function of thecochleae. In recent years, some researchers have foundthat autophagy can attenuate ROS accumulation and alleviate age- and cisplatin-induced oxidative stress in theauditory systems [4]. Thus, in the process of degenerationof the auditory system, hair cells can protect themselvesfrom a certain level of damage by activating autophagy.
Our results provide evidence that supports the existenceof a NOX–ROS–DNA damage linkage and indicate that
this process leads to apoptosis of marginal cells, further
influencing cochlear microcirculation and inducing hearing loss. Also, the current study confirms that oxidative
damage to cochlear SV marginal cells is one of the main

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mechanisms in the D-gal-induced aging model, providinga theoretical basis for subsequent antioxidant therapy.
Abbreviations
ARHL	Age-related hearing loss
SV	Stria vascularis
D-gal	D-galactose
mtDNA	Mitochondrial DNA
CD	Common deletion
NOX2	Nitrous oxide
UCP2	Uncoupling protein 2ATP	Adenosine triphosphate
MMP	Mitochondrial membrane potential
8-OHdG	8-hydroxy-2-deoxyguanosine
ABR	Auditory brainstem responseTM	Tympanic membrane
AOE	Acute otitis externa
ROS	Reactive oxygen species
TUNEL	Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend-labeling
Acknowledgements
Not applicable.
Author contributions
ZP, CLZ, SSG and ZDD designed the project; ZP, CLZ and ZJY performed theexperiment; ZP and ZDD analyzed data; ZP and CLZ wrote the manuscript.
ZDD and SSG applied for technical assistance. All authors read and approved
the final manuscript.
Funding
The work was supported by the National Natural Science Foundation of China(Nos. 82171132 and 81830030), and the Beijing Municipal Administration of
Hospitals’ Youth Programme (Code: QML20200107).
Data Availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The animal experiments were approved by the Institutional Animal Care and
Use Committee of Capital Medical University (Beijing, China). The study was
performed in compliance with the guidelines of National Institutes of Health’s
Guidelines for the Care and Use of Laboratory Animals. The methods arereported in accordance with ARRIVE guidelines.
Consent for publication
Not applicable.
Author details
1
Department of Otolaryngology Head and Neck Surgery, Beijing
Friendship Hospital, Capital Medical University, No.95, Yong’an Road,
Xicheng District, Beijing 100050, China
2
Clinical Center for Hearing Loss, Capital Medical University,
Beijing 100050, China
Received: 31 January 2023 / Accepted: 2 May 2023

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