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ORIGINAL PAPER
Green Tea Polyphenols Protects Cochlear Hair Cellsfrom Ototoxicity by Inhibiting Notch Signalling
Lin-Tao Gu1 • Jia Yang2 • Shi-Zheng Su3 • Wen-Wen Liu4 • Zhong-Gang Shi2 •
Qi-Rong Wang1
Received: 17 February 2015 / Revised: 6 April 2015 / Accepted: 15 April 2015
� Springer Science+Business Media New York 2015
Abstract Notch signalling pathway plays an essential
role in the development of cochlea, which inhibits the
proliferation of hair cells. Epigallocatechin-3-gallate
(EGCG) is the most abundant polyphenol in green tea,
which presents strong antioxidant activation and has been
applied for anti-cancer and anti-inflammatory. In this
study, we treated the cochlear explant cultures with EGCG
and found that EGCG can protect cochlear hair cells from
ototoxic drug gentamicin. We demonstrated that EGCG
could down-regulate the expression of Notch signalling
pathway target genes, such as Hes1, Hes5, Hey1 and Hey5.
However, the Notch pathway ligands such as Delta1, Jag1
and Jag2 were not affected by EGCG. To further illustrate
the mechanism of recover cochlear hair cells, we demon-
strated that EGCG inhibited the activity of c-secrectase to
suppress Notch signalling pathway and promoted the pro-
liferation and regeneration of hair cells in the damaged
cochlea. Our results suggest for the first time the role of
EGCG as an inhibitor of the Notch signalling pathway, and
support its potential value in hearing-impaired recovery in
clinical therapy.
Keywords Cochlear hair cells � Notch signalling
pathway � Polyphenol � EGCG � c-Secrectase
Abbreviations
IHCs Inner hair cells
EGCG Epigallocatechin-3-gallate
NICD Notch intracellular domain
Introduction
In all mammalians, the sensory receptors of both the au-
ditory system and the vestibular system are cochlear hair
cells. Cochlear hair cells are arranged in four rows in the
organ of corti along the entire length of the cochlear coil.
The row of cells close to the centre of cochlea are called
inner hair cells (IHCs), which provide the main neural
output of the cochlea [1, 2]. The other three rows consist of
outer hair cells and receive neural input from the brain,
which detect movement through mechanotransduction as
part of the cochlea’s mechanical pre-amplifier. Damage to
these hair cells resulted in decreased hearing sensitivity and
sensorineural hearing loss [3]. Because human hair cells
are terminally differentiated and are incapable of regen-
eration, this damage could lead to permanent hearing loss.
There are two avenues for the generation of new hair cells
[4, 5]: one is the differentiation from inner ear stem cells;
another is the trans-differentiation from supporting cells. A
series of specific genes and signalling pathways control
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11064-015-1584-3) contains supplementarymaterial, which is available to authorized users.
& Qi-Rong Wang
1 Department of Otolaryngology-Head and Neck Surgery,
Qianfo Shan Hospital Affiliated to Shandong University,
Jinan 250014, People’s Republic of China
2 Department of Otolaryngology-Head and Neck Surgery,
Zhangqiu People’s Hospital, Jinan 250200,
People’s Republic of China
3 Department of Ophthalmology, Zhangqiu People’s Hospital,
Jinan 250200, People’s Republic of China
4 Department of Otolaryngology-Head and Neck Surgery,
Provincial Hospital Affiliated to Shandong University,
Jinan 250022, People’s Republic of China
123
Neurochem Res
DOI 10.1007/s11064-015-1584-3
these two avenues. In 1999, Bermingham et al. have re-
ported that the gene Atoh1 (as also known as Math-1)
serves to initiate inductive signals that regulate the devel-
opment for hair cell in mice. In subsequent studies, the
forced expression of Atoh1 in the mammalian adult inner
ear has been reported to cause the trans-differentiation of
supporting cells into hair cells [6–9]. Chen and Segil [10]
have reported the cyclin-dependent kinase inhibitor p27
(Kip1) plays an essential role in the developmental control
of progenitors of the hair cells and supporting cells. White
et al. [11] have found the relationship between the ex-
pression of p27 (Kip1) and the proliferative capacity of
supporting cells, which suggest p27 (Kip1) may be po-
tential target for controlling the trans-differentiation of
supporting cells into hair cell. Lanford et al. [12] have
firstly demonstrated that the Notch pathway regulate the
development of progenitor cells into hair cells, which
support a role for the Notch pathway in the development of
the cochlea.
The Notch signalling pathway is a highly conserved
cell signalling system in multicellular organisms and
controls numerous cellular processes, such as cell differ-
entiation, proliferation and apoptosis. As a transmem-
brane receptor, Notch plays an important role in
mediating critically cellular functions by cell to cell
communications [13]. Activation of Notch upon DSL
(Delta/Serrate/Lag-2) ligands induces proteolytic pro-
cessing of Notch receptor, within its extracellular domain
(NECD) via a disintegrin and metalloprotease (ADAM),
which facilitates c-secrectase proteolysis to release the
Notch intracellular domain (NICD) from the plasma
membrane [14, 15]. The NICD then translocates to the
nucleus and interacts with the DNA-binding protein CSL
(CBF1, SuH, LAG-1), and participates in the transcrip-
tional regulation of target genes, such as Hes1, Hes2,
Hey1 and Hey2 [16, 17]. With the further research of the
development of cochlear hair cell, various studies have
demonstrated Notch signalling pathway plays essential
roles in the regulation of the cochlear hair cell. In 1998,
both of Adam et al. [18] and Haddon et al. [19] have
reported the lateral inhibition mediated by Notch sig-
nalling pathway in the control of hair cell differentiation,
which plays an essential role in the development of ver-
tebrate inner ear. In 1999, Lanford et al. have demon-
strated the same inhibition in the differentiation of
mammalian hair cell by Notch signalling pathway. In their
studies, Notch signalling pathway exerts lateral inhibition
in the development of cochlear mosaic, which plays an
important role in the determination of various cell fates.
According to the recent studies, inactivation of the Notch
signalling pathway can promote the proliferation of hair
cells [7, 20–22]. Therefore, the inhibitors of c-secrectasemight promote the proliferation and regeneration of hair
cells in the damaged cochlea by targeting the Notch re-
ceptors to suppress the Notch activity [22].
In recent years, tea has attracted increasing attention for
health and delaying aging because of their powerful an-
tioxidant properties. Tea polyphenols (include catechins,
theaflavins, tannins and flavonoids) extracted from tea
leaves have been extensively used for the prevention and
treatment of human cancers [23]. Among the various
polyphenols in tea, the most significant phytochemical is
Epigallocatechin gallate (EGCG). EGCG is a type of
catechin, which is the most abundant, and active compound
and presents much stronger antioxidant activation than
Vitamin C [24]. In biochemical research fields, the benefits
of EGCG have begun to emerge [25]. In 1998, Pietta et al.
[26] have found that EGCG has the ability to inhibit lipid
peroxidation, which may decrease the risk of heart disease.
In 2000, Fujiki et al. [27] have suggested EGCG can inhibit
the release of tumor necrosis factor-alpha causing the
suppression of tumor promotion. Besides cancer and heart
disease, lots of studies have reported EGCG can confer
protection against other diseases, such as osteoporosis [28],
liver and kidney damage [29], bacterial infection [30] and
viral infection [31].
In the present work, we demonstrated EGCG can pro-
mote the regeneration of cochlear hair cells damaged by
ototoxic drug gentamicin. From immunostaining assay, we
found the number of cochlear hair cells remained the same
by EGCG treatment after gentamicin insult. Mechan-
otransduction currents recordings from cochlear hair cells
indicate that EGCG not only protects the number of
cochlear hair cells but also their functions. Furthermore, we
provide evidence that the protective effect of EGCG on
cochlear hair cells was due to its inhibition of Notch
pathway. These findings offer insights into the function of
EGCG in regeneration of cochlear hair cells and support
the medical value of tea polyphenols on the hearing-im-
paired recovery.
Materials and Methods
Ethics Statement
This study was carried out 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 Qianfo Shan Hospital Affiliated
to Shandong University. The IACUC committee members
at Qianfo Shan Hospital Affiliated to Shandong University
approved this study. All surgery was performed under
sodium pentobarbital anesthesia, and all efforts were made
to minimize suffering.
Neurochem Res
123
Experimental Animals
Transgenic mice carrying GFP under the control of an
enhancer from the Atoh1 gene (Atoh1/GFP) were purchase
from Cyagen, Shanghai. The promoter of Atoh1/GFP
transgene contains about 1.4 kb sequence from the Math1
enhancer which is fused to the reporter gene BGnEGFP.
The reporter gene BGnEGFP contains the positive au-
toregulatory elements, the h-actin promoter, and a nuclear
localization signal for GFP [32, 33]. Transgenic mice were
identified by direct observation of GFP-mediated fluores-
cence. The following primers were used for PCR based
genotyping: 50 CGAAGGCTACGTCCAGGAGCGCAC
CAT 30 and 50 GCACGGGGCCGTCGCCGATGGGGG
TGTTCTGC 30.
Cochlear Tissue Isolation and In Vitro Culture
According to the procedure from published reports [33,
34], the timed mated pregnant mice at E13 to E14.5 were
sacrificed and the embryos were isolated and placed in PBS
(GIBCO). Then unfixed bulla from embryos were dissected
and incubated in calcium-magnesium-free PBS (Invitro-
gen) containing dispase (1 mg/ml; Invitrogen) and colla-
genase (1 mg/ml; Worthington) to free the cochlear duct
from the surrounding condensed mesenchyme for
10–15 min. After that, the enzyme solution was replaced
with Hank’s balanced salt solution (HBSS, Life Tech-
nologies) and the spiral ganglia, Reissner’s membrane, and
the most basal cochlear segment should be removed to
obtain a flat cochlear surface preparation. For RT-PCR
experiments, both the most cochlear base and the cochlear
apex were removed, only the cochlear mid-turn was used.
The remaining cochlear tissue was cultured on SPI filter
membranes (Spi Supplies) in DMEM-F12 (Invitrogen)
with B27 supplement (Invitrogen), 2.5 ng/ml FGF2 (NIH),
5 ng/ml EGF (Sigma) and 100 U/ml Penicillin (Sigma). All
cultures were maintained in a 5 % CO2/20 % O2-hu-
midified incubator.
Drug Treatments
After 24 h cultured in the incubator, cochlear tissues (3 in a
group) were randomly divided into 3 groups of 3–5
cochlear tissues per treatment group. These 3 groups were
separately treated with PBS (as control), 40 lg/ml gen-
tamicin or 40 lg/ml gentamicin ? 40 lg/ml EGCG for
24 h.
EdU Incorporation Assay
EdU (5-ethynyl-29-deoxyuridine, Life Technologies) was
prepared in DMSO as 100 mM stock solution and used at a
final concentration of 3 mM. Click-iT Alexa Fluor 555 Kit
(Life Technologies) was used to detect incorporated EdU.
The assay was performed according to manufacturer’s
specifications.
Hair Cell Electrophysiology
The hair cell electrophysiology procedures were modified
from published reports [35, 36] as follows: after cochlear
tissues treated separately with PBS, gentamicin or gen-
tamicin ? EGCG for 24 h, utricles and cochleae were
excised, mounted on glass coverslips, and viewed on an
Axioskop FS upright microscope (Zeiss) equipped with a
633 water-immersion objective and differential interfer-
ence contrast optics. Electrophysiological recordings were
performed at room temperature in solutions containing (in
mM): 137 NaCl, 5.8 KCl, 10 HEPES, 0.7 NaH2PO4, 1.3
CaCl2, 0.9 MgCl2, and 5.6 D-glucose, vitamins (1:100) and
amino acids (1:50) as in MEM (Invitrogen), pH 7.4
(311 mOsm/kg). Recording electrodes (2–4 MX) were
pulled from R-6 glass (King Precision Glass) and filled
with (in mM): 135 KCl, 5 EGTA-KOH, 5 HEPES, 2.5
Na2ATP, 2.5 MgCl2, and 0.1 CaCl2, pH 7.4 (284 mOsm/
kg). The whole-cell, tight-seal technique was used to
record mechanotransduction currents using an Axopatch
200B (Molecular Devices) for utricles and a Multiclamp
700A amplifier (Molecular Devices) for cochleae. Cells
were held at 84 mV, a physiologically relevant holding
potential. Currents were filtered at 2–5 kHz with a low-
pass Bessel filter, digitized at at least 20 kHz with a 12-bit
acquisition board (Digidata 1322A or 1440A), and
recorded using pClamp 8.2 software (Molecular Devices).
Inner hair bundles were deflected using a stiff glass probe
mounted on a PICMA chip piezo actuator (Physik Instru-
ments) driven by a 400-mA ENV400 amplifier (Piezosys-
tem Jena) filtered with an 8-pole Bessel filter at 10–40 kHz
to eliminate residual pipette resonance.
Western Blotting Assay
For western blotting analysis, mouse fibroblasts cells were
seeded in 6-well plates and treated for 24 h with PBS,
EGCG or L685458 (Sigma). L685458 is a potent, selective,
structurally novel c-secretase inhibitor [37]. To isolate
protein, cells were washed with PBS, and harvested using
the lysis buffer (50 mM tris�Cl pH = 6.8, 2 % SDS, 6 %
glycerol, 1 % b-mercapitalethanol, 0.004 % bromophenol
blue). Protein bands in the gel were then transferred to
Nitrocellulose Blotting membranes and incubated with the
appropriate primary antibody. The antibody dilutions were
as follows: 1:1000 for Notch (ab27526) and 1:1000 for
actin (ab1801). Membranes were incubated overnight at
4 �C and washed the next day with buffer (19 PBS, 0.05 %
Neurochem Res
123
Tween 20). Goat anti-rabbit secondary antibody was used
for secondary incubation for 1 h at room temperature.
Proteins were then visualized with chemiluminescent
substrates.
RNA Isolation and Quantitative RT-PCR Statistical
Analysis
Twenty-four hours after cochlear tissues treated separately
with PBS, gentamicin or gentamicin ? EGCG, cochlear
epithelia were isolated from these treated cochlear tissues
using dispase (1 mg/ml; Invitrogen) and collagenase
(1 mg/ml; Worthington) [34]. The total RNA was extracted
from cochlear epithelia using TRIzol reagent (Invitrogen)
and the amount of RNA was quantitated by a spectropho-
tometer (Nano-Drop ND-2000). Total RNA (2 lg) was
reverse transcribed to cDNA using the reverse transcriptase
kit from Bio-Rad according to the manufacturer’s instruc-
tions. The mRNA levels of the target genes were quantified
by real time PCR using SYBR green (Life Technologies) in
an ABI Prism 7500 real-time PCR system (Applied
Biosystems). Each PCR reaction was carried out in
triplicate.
Cell Counts
Hair cells were quantified after the cochlear tissues treated
with PBS, gentamicin or gentamicin ? EGCG and identi-
fied by Atoh1/GFP expression. High-power images of
cochlear explants were assembled and analysed in Adobe
PhotoShop CS3. ImageJ software (NIH) was used to
measure the length of analysed cochlear segments and hair
cell density (cells per 100 micron) was then calculated for
each segment. The total number of inner hair cells or outer
hair cells was counted in each of the four cochlear seg-
ments of 1200–1400 mm (apical, mid-apical, mid-basal,
and basal). Values are presented as mean ± standard error
(SEM) (n = 3, cochlear explants per condition were
analyzed from two independent experiments). Two-tailed
unpaired Student’s t tests were used to determine confi-
dence interval. p B 0.05 was considered as significant
differences.
Statistical Analysis
Two-tailed Student’s t tests were used to determine confi-
dence interval. The statistical differences between the ex-
perimental and control groups were analyzed by one-way
analysis of variance (ANOVA). Values are presented as
mean ± standard error (SEM). Statistical significance is
indicated as *p B 0.05, **p B 0.01, ***p B 0.001.
*p B 0.05 was considered as significant difference,
**p B 0.01 was considered as very significant difference,
***p B 0.001 was considered as extremely significant
difference.
Results and Discussion
Chemical Structure of EGCG and DIC Image
of Stage P2 Isolated Mouse Cochlear Explant
Tea is a popular worldwide beverage with numerous po-
tential beneficial effects such as refreshing and antioxidant
properties. The main active compounds in green tea are
polyphenols. In this study, we aimed to study the role of
EGCG (Fig. 1a), which is the most abundant and powerful
antioxidant in polyphenols in green tea leaves, in regen-
eration of cochlear hair cells. According to the procedure
from published reports [33, 34], we isolated the cochlear
tissues from the embryos of timed mated pregnant Atoh1/
GFP transgenic mice at E13 to E14.5 and cultured these
tissues in vitro. To further observe the cochlear tissues
carefully, the specimens were mounted in Slow Fade and
were captured the differential interference contrast (DIC)
and fluorescent images by an inverted microscope and a
cooled CCD camera. In Fig. 1b, the structure of the
cochlear tissues was shown by the DIC image, including
apex (AP), mid apex (MA), mid base (MB) and base (BA)
parts. Then we tested the expression of Atoh1/GFP in the
isolated cochlear tissues. The Atoh1/GFP was expressed in
inner and outer hair cell nuclei clearly (Fig. 1c).
EGCG Rescues Cochlear Hair Cell Number
from Gentamicin Damage
Gentamicin is an aminoglycoside antibiotic to treat many
types of bacterial infections, which is composed of a
mixture of related gentamicin components and fractions.
However, gentamicin can cause severe hearing and kidney
problems. Therefore gentamicin is frequently used as the
agent to induce damage in studies on cochlear hair cell
protection [38, 39]. Prior to our major experiments, we
examined the dosage effects of gentamicin and EGCG in
the culture, and found their respectively optimal concen-
tration both to be 40 lg/ml (supplementary Fig. S1),
therefore these dosages have been used throughout the
study. Therefore to examine whether EGCG could protect
the hair cells from damage caused by gentamicin, we
treated cochlear explant cultures with PBS as control,
gentamicin or gentamicin ? EGCG respectively (Fig. 2).
In the PBS treated group, the highly organised structure
composed by neat and orderly cells of cochlea could be
clearly seen, with three regular rows of outer hair cells
(OHC) and one regular row of inner hair cells (IHC).
Following the treatment with gentamicin, the hair cells
Neurochem Res
123
showed serious signs of damage and the number of hair
cells were significantly decreased, causing obvious struc-
tural lesions in cochlea. In the third group treated with the
combination of gentamicin and EGCG, the structure lesion
was rescued (Fig. 2a). Quantitative analysis in Fig. 2b di-
rectly indicated an 80 % reduction of hair cells cause by
gentamicin compared to the control. EGCG exhibited ex-
cellent protection against the damage caused by gentam-
icin, the number of hair cells has remained almost the same
as the control. In order to rule out the possibility that
EGCG only abolishes the adverse effect of gentamicin
rather than promoting hair cell proliferation originated
from dividing cells, EdU incorporation assay was per-
formed, where EdU can be actively incorporated into
newly synthesized DNA of dividing cells. No EdU labeled
hair cells were observed in control or hair cell-ablated
culture treated gentamicin, whereas a number of hair cells
treated with the combination of gentamicin and EGCG are
positively stained for EdU (Fig. 2c). These results indi-
cated EGCG regenerates cochlear hair cells from damages
induced by ototoxic drug gentamicin.
Rescue of the Gentamicin Damaged Hair Cell
Mechanotransduction by EGCG
According to the fluorescent images and cell counting ex-
periments, EGCG exhibit powerful ability to protect
cochlear hair cells from the damage caused by gentamicin,
evident by the number of hair cells as well as cochlea
structure. We further examined whether the cochlear hair
cells rescued by EGCG retain their mechanosensory
functions. We recorded mechanotransduction currents from
the cochlear hair cells in the same three experimental
groups (Fig. 3). To avoid using hair bundles damaged
during dissection, we selected the hair cells from the mid
apex cochlea for analysis. The three groups of hair cells
were bathed in 1.3 mM Ca2? with no other permeant ca-
tions. As expected, the hair cells treated with gentamicin
had significantly attenuated transduction currents, reduced
to only one third of the control group. However, the hair
cells treated with gentamicin ? EGCG had normal trans-
duction currents as control. These data provided strong
evidence that, apart from rescuing cochlea structural
Fig. 1 Epigallocatechin gallate and mouse cochlear explant.
a Chemical structure of epigallocatechin gallate (EGCG). b DIC
image of stage P2 isolated mouse cochlear explant. White lines
subdivide auditory sensory epithelium into apex (AP), mid apex
(MA), mid base (MB) and base (BA). c Atoh1/GFP is expressed in
inner and outer hair cell nuclei of stage P2 isolated mouse cochlear
explant. Scale bar 100 lm
Fig. 2 EGCG protects cochlear hair cells from ototoxic drug
gentamicin. a Hair cell from mid apex segment of cochlear (Fig. 1c,
white box) showing OHC, outer hair cells (OHC) and inner hair cells
(IHC) labeled with Atoh1/GFP (green) were treated for 24 h with
control (pbs), gentamicin (40 lg/ml in PBS) and gentam-
icin ? EGCG (both at 40 lg/ml in PBS). b Quantification of hair
cells treated with control, gentamicin and gentamicin ? EGCG
respectively as in a. c Hair cell from mid apex segment of cochlear
treated as in a, labeled with Atoh1/GFP (green) and EdU (red). Data
were presented by the mean ± SEM. *p\ 0.05 versus control. n.s.
not significant versus control. Scale bar 25 lm (Color figure online)
Neurochem Res
123
lesions damaged by gentamicin, EGCG also recovers the
mechanosensory function of the hair cells.
EGCG Efficiently Inhibits the Activity of Notch
Signalling Pathway
The differentiation and proliferation of hair cells have been
shown to be controlled by Notch signalling pathway. The
inactivation of the Notch signalling pathway can promote
the proliferation of hair cells. To further study the
mechanisms of EGCG protection of hair cells, we focused
on four Notch pathway target genes (Hes1, Hes5, Hey1 and
Hey5), which are also negative regulators of neurogenesis
[40–42]. In Fig. 4, we analysed the expression of Hes1,
Hes5, Hey1 and Hey5 in these 3 groups (the hair cells are
separately treated with PBS, gentamicin or gentam-
icin ? EGCG) by RT-PCR assay. EGCG treatment alone
didn’t have any effect on the expression levels of these
Notch effector genes (data not shown). As shown in Fig. 4,
compared to the control, we found these four genes had no
obvious changes at mRNA level when the hair cells were
treated with gentamicin. Whereas in the gentam-
icin ? EGCG group of hair cells, the mRNA levels of all
these four genes have been significantly down-regulated.
Therefore, EGCG can negatively regulate the expression of
Hes1, Hes5, Hey1 and Hey5. These results suggest that
EGCG may inhibit the expression of the target genes of
Notch signalling to cause the inactivation of Notch
signalling.
To further study how EGCG down-regulated Notch
signalling pathway, we analysed the expression of the
Notch pathway ligands Delta1, Jag1 and Jag2 in the 3
groups (the hair cells are separately treated with PBS,
gentamicin or gentamicin ? EGCG) by RT-PCR. In
Fig. 5a, compared to the control, the mRNA level of
Delta1, Jag1 and Jag2 had been down-regulated by Gen-
tamicin. However, we found that this down-regulation was
due to the reduced number of hair cells rather than low
expression of genes in intracellular level, since the protein
levels of Delta1 in individual hair cells were not affected
(data not shown). It was worth noting that, in the gen-
tamicin ? EGCG treated group, there was no obvious
change of the mRNA levels of these 3 genes compared to
the control. According to the results of Figs. 4 and 5a, we
hypothesized that the inhibitory effect of EGCG on Notch
signalling was not conferred by interfering with either its
upstream ligands or downstream effectors, but rather on the
Notch receptor itself. One of the top candidates of EGCG
target we examined was c-secrectase. To test our hy-
pothesis, we employed L685458, known inhibitor of c-secrectase [37], and compared its effect with EGCG in
mouse fibroblasts. The cells were separately treated with
PBS (as control), EGCG or L685458 for 24 h, and the
release of NICD was analysed by Western Blot assay. In
Fig. 5b, there was clear NICD bands (indicated by the ar-
row) in the control group, whereas in the EGCG or
L685458 treated groups there were no NICD bands. This
Fig. 3 Mechanotransduction currents recorded from cochlear hair
cells. a Representative families of currents recorded at -84 mV from
hair cells after control, gentamicin, gentamicin ? EGCG treatments
as indicated above. Cells were from the mid apex cochlears bathed in
1.3 mM Ca2?. The scale bar applies to all current families. b Stimulus
protocol as used in a
Fig. 4 EGCG treatment down-regulated Notch signalling pathway.
Relative mRNA levels of Notch signalling pathway effector genes
(Hes1, Hes5, Hey1, Hey5) were analyzed in control, gentamicin and
gentamicin ? EGCG treated cochlear after 24 h in culture using RT-
PCR. Data were presented by the mean ± SEM. n.s. not significant
versus control. *p\ 0.05 versus gentamicin alone
Neurochem Res
123
finding suggests that EGCG acts as an inhibitor of c-se-crectase, same as L685458, to suppress the proteolysis of
Notch receptor, and in turn down-regulates Notch sig-
nalling pathway.
Mammalian cochlea is composed of highly differentiated
and stabilized cells. Under normal circumstances, these cells
are incapable of regeneration when damaged by internal or
external factors. Therefore, this damage could lead to per-
manent hearing loss. To date, ototoxicity has been reported
of some anti-cancer drugs (CDDP etc.) and anti-bacterial
drugs (gentamicin etc.). However, the protection and curing
of hearing loss are still challenging. Notch signalling path-
way is a highly conserved cell signalling system and plays
an essential role in controlling the development of cochlea.
It has been shown that inactivation of the Notch signalling
pathway can promote the proliferation of hair cells [7, 20–
22]. In the recent studies, more and more scientists devote to
explore the targets in Notch signalling pathway to recover
the regeneration of damaged hair cells.
In this study, we found that EGCG has the ability to
protect cochlear hair cells from ototoxic drug gentamicin.
Both number (Fig. 2) and function (Fig. 3) of cochlear hair
cells can be recovered by EGCG and the Notch signalling
target genes has been suppressed under the treatment of
EGCG (Fig. 4). However, the Notch pathway ligands
Delta1, Jag1 and Jag2 have no obvious change at mRNA
level with the treatment by EGCG and gentamicin
(Fig. 5a). We speculated that the inactivation of Notch
signalling pathway by EGCG was caused by the Notch
receptor itself, rather than interfering with either Notch
upstream ligands or downstream effectors. To further il-
lustrate this essential mechanism, we examined the top
candidate of EGCG target c-secrectase. Our studies re-
vealed that the suppression of Notch signalling pathway by
EGCG depends on the inactivation of c-secrectase (Fig. 5).EGCG is the most abundant, and active compound in tea
polyphenols, which presents strong antioxidant activation
and has been reported to exhibit anti-cancer [43] and anti-
inflammatory [44] activities in biochemical research. Ac-
cording to abundant studies, EGCG could play different
roles in various pathways and targets to participate the
therapies of diseases. In 2008, Shimizu et al. [45] have
reported EGCG decreased the mRNA and protein levels of
IGF-1 and IGF-2, which might be an inhibitor of critical
RTKs involved in hepatocellular carcinoma proliferation.
In our research, compared with L685458, EGCG may be-
come a potential inhibitor of c-secrectase to suppress the
Notch signalling pathway and be useful for hearing-im-
paired recovery in clinical therapy. Further studies can use
the drug-tagging system with EGCG into the needed por-
tion of the inner ear [46], thereby making EGCG to repair
the hair cell efficiently and selectively.
Conclusions
In summary, we demonstrated EGCG could protect
cochlear hair cells against the damage from gentamicin in
the cochlear explant cultures. The mechanism is based on
the inactivation of c-secrectase by EGCG and eventually
causes suppression of Notch signalling pathway. In the
near future, we envision that the drug-tagging system with
EGCG into the targeted portion of the inner ear could be
useful for clinical research and bring emerging prospects
for hearing-impaired recovery.
Acknowledgments This study was supported by the Natural Sci-
ence Foundation of Shandong province (No. ZR2011HM047) to
Fig. 5 EGCG inhibited c-secrectase activity. a Relative mRNA
levels of Notch ligand genes (Delta1, Jag1, Jag2) were analyzed in
control, gentamicin and gentamicin ? EGCG treated cochlear after
24 h in culture using RT-PCR. Data were presented by the
mean ± SEM. *p\ 0.05 versus control. #p\ 0.05 versus gentamicin
and not significant versus control. b Mouse fibroblasts were treated
with control, EGCG or L685458 for 24 h and subjected to western
blot analysis using antibodies against Notch1. Arrow head indicated
released NICD. Anti-actin was used as loading control
Neurochem Res
123
Qirong Wang and the Natural Science Foundation of Shandong pro-
vince (No. ZR2014HQ076) to Wenwen Liu.
Conflict of interest The authors declare that they have no com-
peting interests.
References
1. Romand R (1997) Proliferation of auditory hair cells in organ-
otypic cultures of the cochlea. Eur J Cell Biol 74:138
2. Borg E, Viberg A (2002) Extra inner hair cells in the developing
rabbit cochlea. Hear Res 172:10–13
3. Bodmer D (2008) Protection, regeneration and replacement of
hair cells in the cochlea: implications for the future treatment of
sensorineural hearing loss. Swiss Med Wkly 138:708–712
4. Geleoc GS, Holt JR (2014) Sound strategies for hearing
restoration. Science 344:1241062
5. Liu QW, Chen P, Wang JF (2014) Molecular mechanisms and
potentials for differentiating inner ear stem cells into sensory hair
cells. Dev Biol 390:93–101
6. Cai TT, Seymour ML, Zhang HY, Pereira FA, Groves AK (2013)
Conditional deletion of Atoh1 reveals distinct critical periods for
survival and function of hair cells in the organ of Corti. J Neu-
rosci 33:10110–10122
7. Groves AK (2010) The challenge of hair cell regeneration. Exp
Biol Med 235:434–446
8. Chonko KT, Jahan I, Stone J, Wright MC, Fujiyama T, Hoshino
M, Fritzsch B, Maricich SM (2013) Atoh1 directs hair cell dif-
ferentiation and survival in the late embryonic mouse inner ear.
Dev Biol 381:401–410
9. Yang SM, Chen W, Guo WW, Jia SP, Sun JH, Liu HZ, Young
WY, He DZZ (2012) Regeneration of stereocilia of hair cells by
forced Atoh1 expression in the adult mammalian cochlea. Plos
One 7:e46355
10. Chen P, Segil N (1999) p27(Kip1) links cell proliferation to
morphogenesis in the developing organ of Corti. Development
126:1581–1590
11. White PM, Doetzlhofer A, Lee YS, Groves AK, Segil N (2006)
Mammalian cochlear supporting cells can divide and trans-dif-
ferentiate into hair cells. Nature 441:984–987
12. Lanford PJ, Lan Y, Jiang RL, Lindsell C, Weinmaster G, Gridley
T, Kelley MW (1999) Notch signalling pathway mediates hair
cell development in mammalian cochlea. Nat Genet 21:289–292
13. Baron M (2003) An overview of the Notch signalling pathway.
Semin Cell Dev Biol 14:113–119
14. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR,
Cumano A, Roux P, Black RA, Israel A (2000) A novel prote-
olytic cleavage involved in Notch signaling: the role of the dis-
integrin-metalloprotease TACE. Mol Cell 5:207–216
15. Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tian XL,
Pan DJ, Ray WJ, Kopan R (2000) A ligand-induced extracellular
cleavage regulates gamma-secretase-like proteolytic activation of
Notch1. Mol Cell 5:197–206
16. D’Souza B, Miyamoto A, Weinmaster G (2008) The many facets
of Notch ligands. Oncogene 27:5148–5167
17. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M
(2004) The Notch target genes Hey1 and Hey2 are required for
embryonic vascular development. Gene Dev 18:901–911
18. Adam J, Myat A, Le Roux I, Eddison M, Henrique D, Ish-
Horowicz D, Lewis J (1998) Cell fate choices and the expression
of Notch, Delta and Serrate homologues in the chick inner ear:
parallels with Drosophila sense-organ development. Develop-
ment 125:4645–4654
19. Haddon C, Jiang YJ, Smithers L, Lewis J (1998) Delta–Notch
signalling and the patterning of sensory cell differentiation in the
zebrafish ear: evidence from the mind bomb mutant. Develop-
ment 125:4637–4644
20. Li W, Wu J, Yang J, Sun S, Chai R, Chen Z, Li H (2014) Notch
inhibition induces mitotically generated hair cells in mammalian
cochleae via activating the Wnt pathway. In: Proceedings of the
National Academy of Sciences of the United States of America
21. Jeon SJ, Fujioka M, Kim SC, Edge ASB (2011) Notch signaling
alters sensory or neuronal cell fate specification of inner ear stem
cells. J Neurosci 31:8351–8358
22. Mizutari K, Fujioka M, Hosoya M, Bramhall N, Okano HJ,
Okano H, Edge AS (2013) Notch inhibition induces cochlear hair
cell regeneration and recovery of hearing after acoustic trauma.
Neuron 77:58–69
23. Kanwar J, Taskeen M, Mohammad I, Huo C, Chan TH, Dou QP
(2012) Recent advances on tea polyphenols. Front Biosci (Elite
Ed) 4:111–131
24. Shah A, Kurlandsky S (2007) Green tea and bilberry extracts but
not vitamin C protect retinal epithelial cells from oxidative
damage. FASEB J 21:A172
25. Mukhtar H, Ahmad N (2000) Tea polyphenols: prevention of
cancer and optimizing health. Am J Clin Nutr 71:1698S–1702S
(discussion 1703S–1694S)
26. Pietta P, Simonetti P, Gardana C, Brusamolino A, Morazzoni P,
Bombardelli E (1998) Relationship between rate and extent of
catechin absorption and plasma antioxidant status. Biochem Mol
Biol Int 46:895–903
27. Fujiki H, Suganuma M, Okabe S, Sueoka E, Suga K, Imai K,
Nakachi K (2000) A new concept of tumor promotion by tumor
necrosis factor-alpha, and cancer preventive agents (-)-epigal-
locatechin gallate and green tea: a review. Cancer Detect Prev
24:91–99
28. Jin P, Wu HY, Xu GJ, Zheng L, Zhao JM (2014) Epigallocatechin-
3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic
differentiation of mesenchymal stem cells from human bone
marrow: an in vitro study. Cell Tissue Res 356:381–390
29. Niu YC, Na LX, Feng RN, Gong LY, Zhao Y, Li Q, Li Y, Sun
CH (2013) The phytochemical, EGCG, extends lifespan by re-
ducing liver and kidney function damage and improving age-
associated inflammation and oxidative stress in healthy rats.
Aging Cell 12:1041–1049
30. Gordon NC, Wareham DW (2010) Antimicrobial activity of the
green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)
against clinical isolates of Stenotrophomonas maltophilia. Int J
Antimicrob Agents 36:129–131
31. Xiao X, Yang ZQ, Shi LQ, Liu J, Chen W (2008) Antiviral effect
of epigallocatechin gallate (EGCG) on influenza A virus. China J
Chin Mater Med 33:2678–2682
32. Doetzlhofer A, White PM, Johnson JE, Segil N, Groves AK
(2004) In vitro growth and differentiation of mammalian sensory
hair cell progenitors: a requirement for EGF and periotic mes-
enchyme. Dev Biol 272:432–447
33. Chen P, Johnson JE, Zoghbi HY, Segil N (2002) The role of
Math1 in inner ear development: uncoupling the establishment of
the sensory primordium from hair cell fate determination.
Development 129:2495–2505
34. Doetzlhofer A, Basch ML, Ohyama T, Gessler M, Groves AK,
Segil N (2009) Hey2 regulation by FGF provides a Notch-inde-
pendent mechanism for maintaining pillar cell fate in the organ of
Corti. Dev cell 16:58–69
35. Kawashima Y, Geleoc GS, Kurima K, Labay V, Lelli A, Asai Y,
Makishima T, Wu DK, Della Santina CC, Holt JR, Griffith AJ
(2011) Mechanotransduction in mouse inner ear hair cells re-
quires transmembrane channel-like genes. J Clin Investig
121:4796–4809
Neurochem Res
123
36. Pan BF, Geleoc GS, Asai Y, Horwitz GC, Kurima K, Ishikawa K,
Kawashima Y, Griffith AJ, Holt JR (2013) TMC1 and TMC2 are
components of the mechanotransduction channel in hair cells of
the mammalian inner ear. Neuron 79:504–515
37. Shearman MS, Beher D, Clarke EE, Lewis HD, Harrison T, Hunt
P, Nadin A, Smith AL, Stevenson G, Castro JL (2000) L-685,458,
an aspartyl protease transition state mimic, is a potent inhibitor of
amyloid beta-protein precursor gamma-secretase activity. Bio-
chemistry 39:8698–8704
38. Hirose K, Hockenbery DM, Rubel EW (1997) Reactive oxygen
species in chick hair cells after gentamicin exposure in vitro.
Hear Res 104:1–14
39. Lombarte A, Yan HY, Popper AN, Chang JS, Platt C (1993)
Damage and regeneration of hair cell ciliary bundles in a fish ear
following treatment with gentamicin. Hear Res 64:166–174
40. Tateya T, Imayoshi I, Tateya I, Ito J, Kageyama R (2011) Co-
operative functions of Hes/Hey genes in auditory hair cell and
supporting cell development. Dev Biol 352:329–340
41. Jung JY, Avenarius MR, Adamsky S, Alpert E, Feinstein E,
Raphael Y (2013) siRNA Targeting Hes5 Augments Hair Cell
Regeneration in Aminoglycoside-damaged Mouse Utricle. Mol
Ther 21:834–841
42. Zheng JL, Shou JY, Guillemot F, Kageyama R, Gao WQ (2000)
Hes1 is a negative regulator of inner ear hair cell differentiation.
Development 127:4551–4560
43. Du GJ, Zhang ZY, Wen XD, Yu CH, Calway T, Yuan CS, Wang
CZ (2012) Epigallocatechin gallate (EGCG) is the most effective
cancer chemopreventive polyphenol in green tea. Nutrients
4:1679–1691
44. Zhong Y, Chiou YS, Pan MH, Shahidi F (2012) Anti-inflam-
matory activity of lipophilic epigallocatechin gallate (EGCG)
derivatives in LPS-stimulated murine macrophages. Food Chem
134:742–748
45. Shimizu M, Shirakami Y, Sakai H, Tatebe H, Nakagawa T, Hara
Y, Weinstein IB, Moriwaki H (2008) EGCG inhibits activation of
the insulin-like growth factor (IGF)/IGF-1 receptor axis in human
hepatocellular carcinoma cells. Cancer Lett 262:10–18
46. Kanzaki S, Fujioka M, Yasuda A, Shibata S, Nakamura M,
Okano HJ, Ogawa K, Okano H (2012) Novel in vivo imaging
analysis of an inner ear drug delivery system in mice: comparison
of inner ear drug concentrations over time after transtympanic
and systemic injections. Plos One 7:e48480
Neurochem Res
123