PP1 activity in the mouse cochlea

Accepted Manuscript Pomegranate peel extract attenuates D-galactose-induced oxidative stress and hearing loss by regulating PNUTS/PP1 activity in the mouse cochlea Shuangyue Liu, Tao Xu, Xidi Wu, Yuhan Lin, Dongyan Bao, Yang Di, Tingting Ma, Yan Dang, Peili Jia, Jianqiao Xian, Aimei Wang, Yongxin Liu PII:

S0197-4580(17)30240-3

DOI:

10.1016/j.neurobiolaging.2017.07.007

Reference:

NBA 9985

To appear in:

Neurobiology of Aging

Received Date: 27 January 2017 Revised Date:

11 July 2017

Accepted Date: 15 July 2017

Please cite this article as: Liu, S., Xu, T., Wu, X., Lin, Y., Bao, D., Di, Y., Ma, T., Dang, Y., Jia, P., Xian, J., Wang, A., Liu, Y., Pomegranate peel extract attenuates D-galactose-induced oxidative stress and hearing loss by regulating PNUTS/PP1 activity in the mouse cochlea, Neurobiology of Aging (2017), doi: 10.1016/j.neurobiolaging.2017.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 1

Pomegranate peel extract attenuates D-galactose-induced oxidative

2

stress and hearing loss by regulating PNUTS/PP1 activity in the

3

mouse cochlea

RI PT

4

Shuangyue Liu a, 1, Tao Xu b, 1, Xidi Wu a, Yuhan Lin a , Dongyan Bao a , Yang Di a ,

6

Tingting Ma a , Yan Dang a , Peili Jia a , Jianqiao Xian a , Aimei Wang a, * ,

7

Yongxin Liu c, *

8

a

Department of Physiology, Jinzhou Medical University, Jinzhou 121000 P.R. China;

9

b

Life Science Institute, Jinzhou Medical University, Jinzhou 121000 P.R. China;

10

c

11

Jinzhou Medical University, Jinzhou 121000 P.R. China

12

*

13

University, No.40, Section 3, Songpo Road, Linghe District, Jinzhou, 121000,

14

P.R.China. Tel. : +86 0416 4673179 ; fax : +86 0416 4673670 ; Yongxin Liu,

15

Department of Otorhinolaryngology-Head and Neck Surgery, the First Hospital of

16

Jinzhou Medical University, No.2, Section 5, People Street, Guta District, Jinzhou,

17

121000, P.R.China.

18

E-mail: [email protected](A. Wang); [email protected](Y. Liu)

19

1

M AN U

SC

5

Department of Otorhinolaryngology-Head and Neck Surgery, the First Hospital of

AC C

EP

TE D

Corresponding author at : Aimei Wang, Department of Physiology, Jinzhou Medical

These authors contributed equally to this work.

20 21

1

ACCEPTED MANUSCRIPT Abbreviations: ABR, auditory brainstem response; ARHL, age-related hearing loss;

23

D-gal, D-galactose; ELISA, enzyme-linked immunosorbent assay; IHC, inner hair cell;

24

MDA, malondialdehyde; miR-34a, microRNA-34a; OC, organ of Corti; OHC, outer

25

hair cell; PBS, phosphate-buffered saline; PP1, protein phosphatase 1; PPE,

26

pomegranate peel extract; PNUTS, protein phosphatase 1 nuclear targeting subunit;

27

TBS, Tris(hydroxymethyl)aminomethane-buffered saline; SGN, spiral ganglion

28

neuron; Stria, striavascularis; T-SOD, total-superoxide dismutase

29 30

Abstract

M AN U

SC

RI PT

22

31

Oxidative stress is considered to be a major contributor to age-related hearing loss

32

(ARHL). Here, we investigated whether pomegranate peel extract (PPE) protected

33

against

34

D-galactose-induced accelerated aging mice. The aging mice exhibited an increase in

35

hearing threshold shifts and hair cells loss, which were improved in the PPE-treated

36

aging mice. The aging mice also exhibited an increase in 4-HNE, the expression of

37

protein phosphatase 1 nuclear targeting subunit (PNUTS), p53 and caspase-3 and a

38

decrease in protein phosphatase 1 (PP1) and MDM2 in the cochlea. PPE treatment

39

reversed the changes in above mentioned molecules. Our results suggested that PPE

40

can protect against ARHL, the underlying mechanisms may involve in the inhibition

41

of oxidative damage of cochlea, possibly by regulating PNUTS/PP1 pathway. The

42

results from the present study provide a new therapeutic strategy to use PPE for

43

prevention of ARHL.

loss

by

decreased

oxidative

stress

in

the

cochlea

of

AC C

EP

TE D

hearing

44 2

ACCEPTED MANUSCRIPT 45

Keywords: 4-hydroxynonenal; Age-related hearing loss; Cochlea; D-galactose;

46

Pomegranate peel extract; Protein phosphatase 1 nuclear targeting subunit

47

AC C

EP

TE D

M AN U

SC

RI PT

48

3

ACCEPTED MANUSCRIPT 49

1. Introduction Age-related hearing loss (ARHL) is the progressive loss of hearing associated

51

with aging and the most common sensory disorder in the elderly population. The

52

prevalence of ARHL is approximately 30% at over the age of 65 years, and its

53

prevalence is expected to increase as the elderly population grows (Homans et al.,

54

2017). The factors that contribute to ARHL is complex, including environmental,

55

medical, and genetic factors(Fischer et al., 2016; Halonen et al., 2016; Ohmen et al.,

56

2014).

SC

RI PT

50

The presence of different reactive oxygen species(ROS) of oxidative changes in

58

individual tissues of the aging cochlea. Increasing evidence has been shown that ROS

59

play a critical role in the process cochlear senescence and the pathogenesis of ARHL

60

(Fujimoto and Yamasoba, 2014; Menardo et al., 2012). 4-hydroxynonenal (4-HNE), a

61

key end-product of lipid peroxidation (Muzio et al., 2016), which increased has

62

regulated the progression of ARHL (Jiang et al., 2007; Takumida et al., 2009;

63

Kinoshita et al., 2013).

EP

TE D

M AN U

57

Protein phosphatase 1 nuclear targeting subunit (PNUTS) is one of the major

65

proteins that regulate nuclear protein phosphatase 1 (PP1) expression. It has been

66

reported to be downregulated with age (Choy et al., 2014). PNUTS is ubiquitously and

67

highly expressed in the brain, co-localized with chromatin during telophase (Kim et

68

al., 2003). The PNUTS/PP1 holoenzyme plays a key role in various nuclear processes,

69

including DNA damage and apoptosis (De Leon et al., 2010). PNUTS directly inhibits

70

the activity of p53 (a master regulator of apoptosis) in response to cellular stress (Lee

AC C

64

4

ACCEPTED MANUSCRIPT 71

et al., 2007), recent studies showed that p53 expression was increased in aged mouse

72

models of ARHL (Li et al., 2016). Pomegranate peel, a by-product of the pomegranate juice industry with potential

74

health effects, it has been used for centuries as a therapeutic agent for the treatment of

75

inflammatory diseases in traditional Chinese medicine. Pomegranate trees

76

(Punicagranatum L.) produce deciduous fruit and belong to the Lythraceae family.

77

Both the rind of the fruit and the bark of the tree have been used in traditional

78

medicines for treating diarrhea, dysentery, and intestinal parasites. The major

79

phytochemicals in pomegranate peel extract (PPE) are ellagitannins, including

80

punicalagin, which is an antioxidant with potent free radical-scavenging

81

properties(Akhtar et al., 2015). Previous studies have been conducted to examine the

82

effects of PPE on various oxidative stress-induced diseases, including vascular

83

remodeling, neurodegenerative diseases, and obesity (Dos Santos et al., 2016;

84

Morzelle et al., 2016; Neyrinck et al., 2013). Although PPE can exert protective

85

effects in patients with drug-induced hearing loss (Kahya et al., 2014; Yazici et al.,

86

2012), the role of PPE in protecting hair cells during ARHL pathogenesis has not

87

been fully elucidated. In the present study, we investigated whether PPE protected

88

against hearing loss by inhibition of oxidative stress-regulated PNUTS/PP1 pathway

89

activation in the cochlea of D-galactose-induced inner ear accelerated aging mice.

AC C

EP

TE D

M AN U

SC

RI PT

73

90 91 92 5

ACCEPTED MANUSCRIPT 93 94

2. Methods

95

2.1. Animals and drugs BALB/c mice(4-week-old) were purchased from The Vital River Laboratory

97

(Beijing, China). Experiments were performed in accordance with protocols approved

98

by the Animal Care and Use Committee of Jinzhou Medical University. All mice were

99

housed in an air-conditioned animal facility at 23ºC, with 50%–60% relative humidity

100

under a 12 h light/dark cycle, and were fed with standard rodent chow and water.

101

Before the first treatment, the mice were acclimated for 4 weeks.

M AN U

SC

RI PT

96

D-gal was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). PPE

103

was purchased from Xi`an Acetar Bio-Tech Inc. (Xi`an, Shaanxi Province, China).

104

PPE is a standardized pomegranate peel extract that was provided as a dietary

105

supplement. It is produced from pomegranate fruits grown in Xi`an by Lintong Farm.

106

PPE is produced in a two-step process: (1) a methanolic extract of pomegranate peel

107

powder was prepared and (2) solid-phase extraction was performed to produce a

108

powder with a high concentration of polyphenols. The powdered extract used in this

109

study contained 98% ellagic acid on average (Supplementary table 1).

111

EP

AC C

110

TE D

102

2.2. Experimental groups

112

BALB/c mice were used to establish D-gal-induced inner ear aging model (Wu

113

et al., 2012). To determine the best time point of the hearing loss in D-gal-induced

114

accelerated aging mice, 2 months old mice were randomly divided into a normal 6

ACCEPTED MANUSCRIPT saline (NS, n=141) and a D-gal-induced aging group (n=126). The mice in all D-gal

116

groups were subcutaneously injected with D-gal (800 mg/kg/day) for 2 months, and

117

maintained on a normal mice diet for 2-5 months after the last injection. The mice in

118

NS group were received with the same volume of 0.9% saline injection on the same

119

schedule. The mice were sacrificed at the ages of 2, 4, 6 and 9 months (Fig. 1A). Our

120

results showed that the most significant hearing loss occurred at 9 months of age at 8

121

kHz,12 kHz, and 24 kHz frequencies in D-gal mice. Therefore, we used 9-month-old

122

D-gal mice as D-gal-induced inner ear aging model mice. Another set of mice were

123

used to investigate the protective effect of PPE for hearing loss(Shukla, et al., 2008),

124

2-month-old mice were randomly divided into four groups: 1) normal control group

125

(NS, n=18): the mice received with subcutaneous injection of NS; 2) D-gal-induced

126

accelerated aging group(D-gal, n=35): the mice received with subcutaneous injection

127

of D-gal (800 mg/kg/day) for 2 months, and maintained on a normal mice diet until

128

9-month-old after the last injection; 3) D-gal plus PPE treatment group (D-gal+PPE,

129

n=42):the mice received with subcutaneous injection of D-gal (800 mg/kg/day) plus

130

PPE treatment (17 mg/kg/day or 34 mg/kg/day, by gavage) for 2 months, then stopped

131

the D-gal treatment and continued PPE treatment for 2 months, thereafter, maintained

132

a normal mice diet for another 3 months after the last injection; 4) normal control

133

mice treated with PPE (PPE, n=42), the normal mice were administered PPE (34

134

mg/kg/day) for 4 months, then maintained a normal mice diet for another 3 months

135

(Fig. 3A). The mice were sacrificed at 9 months of age.

AC C

EP

TE D

M AN U

SC

RI PT

115

136 7

ACCEPTED MANUSCRIPT 137

2.3. Auditory brainstem responses (ABRs) ABRs were measured with a tone burst stimulus at 8, 12, and 24 kHz using

139

Intelligent Hearing Systems hardware and software (Smart EP & OAE, Miami, FL,

140

USA) at 2, 4, 6, and 9 months of age. Mice were anesthetized with 1% pentobarbital

141

sodium (90 mg/kg, Sigma) and placed on a warm heating platform. Needle electrodes

142

were placed subcutaneously at the vertex (non-inverting or active), ipsilateral ear

143

(reference), and contralateral ear (ground). At each frequency, the sound level was

144

decreased in 5-dB steps from the 100 to 5 dB sound-pressure level. The hearing

145

threshold was defined as the lowest level of sound that produced a detectable ABR.

146

After measuring the ABRs, the mice were sacrificed by decapitation. The cochleae

147

from the same mice were harvested for histological analyses.

M AN U

SC

RI PT

138

TE D

148 149

2.4.

150

malondialdehyde (MDA) level

of

total-superoxide

dismutase

(T-SOD)

activity

and

EP

Measurements

Cochlear T-SOD activities and MDA concentrations were measured using T-SOD

152

and MDA assay kits (Jiancheng, Nanjing, China) according to the manufacturer’s

153

instructions. Briefly, two cochleae from individual mice were pooled for each sample.

154

Cochlear tissues were homogenized in 50 mmol/L phosphate buffer (pH 7.4) using a

155

medium throughput ball mill (DHS Life Science & Technology Co., Ltd. Beijing,

156

China). The homogenate was centrifuged at 1,500 rpm for 13 min. Pyrogallol solution

157

(10 mmol/L, 2 mL) was added to various concentrations of tissue supernatants, and

AC C

151

8

ACCEPTED MANUSCRIPT 158

the rate of autoxidation was measured spectrophotometrically at 420 nm.

159 160

2.5. HNE-His adduct assays Cochlear 4-HNE levels were determined using the HNE-His Adduct ELISA Kit

162

(Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.

163

The 4-HNE activity was measured using a Varioskan Flash Spectral Scanning

164

Multimode Reader (Thermo Scientific, Waltham, MA, USA).

SC

RI PT

161

M AN U

165 166

2.6. Surface preparations and fluorescein isothiocyanate staining of cochlear

167

epithelia for hair cell counts

Briefly, the temporal bones were removed and the cochlea was immersed in 4%

169

paraformaldehyde and incubated overnight at 4°C. The fixed cochlea was then

170

decalcified in a solution of sodium ethylene diamine tetraacetic acid for 5 days and

171

maintained at 4°C. The cochlear epithelia were dissected from the cochleae in

172

ice-cold phosphate-buffered saline (PBS), and the explants were incubated overnight

173

at 4°C with a primary antibody (1: 100, rabbit polyclonal anti-myosin VIIa; Abcam,

174

Cambridge, UK) and then for 1 h with an appropriate fluorescent secondary antibody

175

(1: 100, Cell Signaling Technology [CST], Boston, MA, USA). The cochlear epithelia

176

were then mounted on slides with Fluoromount-G mounting medium. Images were

177

obtained using an Olympus DP72 camera equipped with cellSens imaging software.

178

Hair cells were counted from the captured images using an Olympus light microscope

179

(400× magnification). By raising and lowering the focal plane, it was possible to

AC C

EP

TE D

168

9

ACCEPTED MANUSCRIPT determine if the hair cell nucleus, cuticular plate, and stereocilia bundle were present.

181

The lengths of the cochlear epithelia were measured and documented in millimeters.

182

A hair cell was considered present if both the stereocilia bundle and nucleus were

183

clearly visible, and not present if either was absent. Both outer hair cells (OHCs) and

184

inner hair cells (IHCs) were counted from the apex to the base along the entire length

185

of the cochlear epithelium. The percentage of hair cell loss in each 0.5-mm length of

186

the epithelium was plotted as a function of the cochlear length in a cytocochleogram

187

(Ding et al., 2016; Hill et al., 2016).

189

2.7. Immunofluorescence

M AN U

188

SC

RI PT

180

The expression of 4-HNE and PNUTS was examined by immunofluorescence. The

191

cochleae were post-fixed with 4% paraformaldehyde overnight at 4°C. After rinsing

192

with distilled water, all cochleae were dehydrated in a graded alcohol series and

193

immersed in paraffin after clearing in xylene. Following deparaffinization,

194

rehydration, and antigen retrieval, we used goat serum albumin to block nonspecific

195

binding. The sections or the cochlear epithelia were then incubated for 12 h at 4°C

196

with an anti-PNUTS IgG (1: 200, CST), anti-4-HNE (1: 100, Abcam), or anti-cleaved

197

caspase-3 (1: 400, CST) antibody. The sections or cochlear epithelia were then

198

incubated with a secondary antibody for 1 h after repeated washing with PBS. The

199

sections or cochlear epithelia were mounted on slides with DAPI-Fluoromount-G

200

mounting medium. Sections or cochlear epithelia processed without a primary

201

antibody were used as a negative control. Relative fluorescence intensity was

AC C

EP

TE D

190

10

ACCEPTED MANUSCRIPT 202

determined with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville,

203

MD, USA).

204

2.8. Extraction of total cochlear RNA for quantitative real-time PCR

RI PT

205

The cochleae were rapidly removed and isolated in RNAlater (Invitrogen, Carlsbad,

207

CA, USA). Two cochleae from a single mouse were pooled and placed in a 2-ml tube

208

with 100 µl of RNAlater and immediately crushed using forceps. One milliliter of

209

TRIzol (Invitrogen) was added to each sample, followed by homogenization with a

210

TL2010 Tissue Grinder (HEROS Technology, Inc., Beijing, China). RNA

211

concentrations were determined using a NanoDrop 2000 spectrophotometer.

212

First-strand cDNA was prepared by reverse transcribing total RNA using the

213

Superscript III First-Strand Synthesis System (Invitrogen). Primers with the following

214

sequences

215

5′-AAAACGAGCACAGAACCA-3′

and

reverse:

216

5′-GGCTCCAAAGAGGCTGTAT-3′;

PP1,

forward:

217

5′-CCTCATGGGTCATTACAG-3′

and

reverse:

218

5′-CTCAATACCCTGGATTTCTCT-3′;

β-actin,

forward:

219

5′-CATCCGTAAAGACCTCTATGCCAAC-3′

220

5′-ATGGAGCCACCGATCCACA-3′. Relative expression levels were analyzed using

221

the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA,

222

USA).

used

for

amplification:

AC C

EP

were

TE D

M AN U

SC

206

223 11

PNUTS,

and

forward:

reverse:

ACCEPTED MANUSCRIPT 224

2.9. Western blot analysis Four cochleae from 2 mice were pooled for a single sample. After the

226

homogenaztion, the supernatant (30 µg protein) were separated by sodium dodecyl

227

sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were

228

transferred onto a nitrocellulose membrane (Merck Millipore, Darmstadt, Germany)

229

and

230

aminomethane-buffered saline (TBS) plus 0.1% Tween 20 (TBS-T). The membranes

231

were incubated with anti-4-HNE (1: 500; Abcam), anti-PNUTS (1: 400; CST),

232

anti-PP1 (1: 500, CST), anti-MDM2 (1: 500; CST), anti-p53 (1: 500; CST), or

233

anti-GAPDH (1: 3000; Millipore) antibodies at 4°C overnight, and then washed 3

234

times (10 min each) with TBS-T buffer. The membranes were then incubated with an

235

appropriate IRDye® near-infrared fluorescent secondary antibodies (LI-COR

236

Biosciences, Lincoln, NE, USA) at a 1: 25,000 dilution for 1 h. The protein levels

237

were analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences,

238

Lincoln, NE, USA).

240 241

2%

bovine

serum

albumin

in

Tris(hydroxymethyl)

EP

TE D

M AN U

SC

with

AC C

239

blocked

RI PT

225

2.10. Statistical analysis Data were analyzed using GraphPad software 5.0 and Microsoft Excel in

242

Windows. Data from different groups were analyzed based on the variability of

243

measurements and the magnitude of differences between them. The following

244

statistical analyses were used: one-way analysis of variance (ANOVA) with Tukey’s

245

multiple-comparisons test was used for the protein expression of 4-HNE and PNUTS, 12

ACCEPTED MANUSCRIPT 246

Bonferroni’s post-hoc test for the gene expression, an unpaired t test for ABR

247

thresholds, One-sample t tests for hair cell counts. p< 0.05 were considered

248

statistically significant. All experiments were independently repeated at least 3 times.

250

3. Results

251

3.1. D-gal induced senescence in the cochlea

RI PT

249

D-gal-induced inner ear aging model has established by injecting overdoses of

253

D-gal to induce the aging in the central and peripheral auditory system of mice and

254

rats. To determine the optimal time point of the hearing loss in D-gal-induced inner

255

ear aging mice model, ABR thresholds were measured at 2, 4, 6 and 9 months of age

256

at 8, 12 and 24kHz. The experimental protocol for establishing the aging model is

257

shown in Fig. 1A. As shown in fig. 1B, treatment with D-gal for 2 months (at

258

4-month-old mice) did not alter the ARB threshold at low or high frequencies, as

259

compared with normal control group. However, after withdrawal of D-gal, ABR

260

threshold was gradually increased with aging in D-gal-treated mice, with a peak at

261

9-month-old mice. Hearing loss (increased ABR) appeared more early in high

262

frequency. The NS mice at 9-month-old had no significant loss in hair cells from the

263

basal region of the cochlea. However, 9-month-old mice with D-gal treatment had a

264

nearly 80% loss of OHC (76.7 ± 9.9%) and an 80% loss of IHC (75.3 ± 8.3%) lesions in

265

the basal region (n= 5, Fig. 1C and D). Those results indicate that D-gal-induced mice

266

developed significant hearing loss. The results from the present study are consistent

267

with other previous studies (Du et al., 2012; Wu et al., 2012; Sun et al., 2015). SOD

AC C

EP

TE D

M AN U

SC

252

13

ACCEPTED MANUSCRIPT (superoxide dismutase) is an important antioxidant enzyme, as shown in figure 1E,

269

total-SOD (T-SOD) activity were gradually reduced in NS and D-gal-treated mice

270

with aging, with a 58% of maximal reduction in 9-month-old D-gal mice compared to

271

the match age NS mice. MDA is a marker of oxidative lipid peroxidation, which was

272

increased in the cochlea of NS with age, and MDA was dramatically increased in

273

D-gal-treated mice (n=6, Fig. 1E and F), with the MDA levels increased by 2.49-fold

274

(p< 0.01), 3.21-fold (p< 0.01), and 1.98-fold (p< 0.01) at 4, 6, and 9 months,

275

respectively, compared to those of the age-matched NS group.

M AN U

SC

RI PT

268

276 277

3.2. 4-HNE expression in hair cells of aging mice

4-HNE is an endogenous product of lipid peroxidation. To evaluate the

279

expression of 4HNE in the hair cells, 4-HNE was immunolabeled in cochlear paraffin

280

sections. The expression of 4-HNE was minimal in the hair cells of 2-month-old NS

281

mice, was significantly increased in 9-month-old mice (data not shown). D-gal

282

treatment further increased the expression of 4HNE at 4, 6 and 9 month-old mice (n=

283

4, Fig. 2A and B).

284

9-month-old NS mice, and 4-HNE activity was further increased in D-gal treated mice

285

at 4, 6 and 9 month-old mice (n= 6, Fig. 2C). The results were further confirmed by

286

Western blot (n= 3, Fig. 2D).

EP

TE D

278

AC C

The ELISA showed that 4-HNE activity was increased in

287 288 289

3.3. PPE prevented hearing and hair cell loss in aging mice at 9-month-old As shown in Fig. 3A, the experimental protocol for investigate the protective 14

ACCEPTED MANUSCRIPT effect of PPE for hearing loss in D-gal-induced aging mice (the detailed process had

291

been described in the Method 2.2). To determine the best protection dose of PPE for

292

auditory function in D-gal-induced inner ear aging model mice, we used two doses of

293

PPE (17 and 34 mg/kg/day) in adult BALB/c mice. LD50 of oral PPE is more than 5

294

g/kg body weight in rats and mice, with no observed adverse effects following

295

administration of 600 mg/kg/day (Akhtar et al., 2015), which greatly exceeded the

296

dose used in our experiments (34 mg/kg/day). Our preliminary results showed that NS

297

mice received PPE at low or high doses did not show any signs of illness including

298

body weight loss and increased hearing threshold, and changes in fur color (data not

299

shown). PPE at two different doses significantly reduced the auditory threshold shifts

300

at all frequencies in D-gal-treated mice, reduction in the auditory threshold shifts was

301

more prominent in D-gal mice treated with high dose of PPE (Fig. S1), indicating that

302

PPE effectively prevents D-gal-induced impairment of auditory function (Fig. 3B).

303

Because high dose of PPE (34 mg/kg/day) elicited greater protection at all frequencies

304

(Fig. S1), high dose of PPE was used for all our experiments.

EP

TE D

M AN U

SC

RI PT

290

As shown in Fig. 3C and D, D-gal decreased T-SOD activities and increased

306

MDA expression. PPE prevented the decrease in T-SOD and increase in MDA

307

content in the cochlea of D-gal-induced aging mice (p< 0.01). The expression of

308

caspase-3 was observed in the basal turn of the cochlea in 9-month-old D-gal mice,

309

which was ameliorated by PPE treatment (n=5, p< 0.01, Fig. 3E). The

310

immunofluorescence showed that PPE markedly decreased 4-HNE expression in 3

311

key regions of the D-gal mice cochlea, namely the organ of Corti (OC), SGNs, and

AC C

305

15

ACCEPTED MANUSCRIPT Stria (n= 6, p< 0.01, Fig. 4A and B). In addition, western blot and ELISA analyses

313

further demonstrated that PPE decreased in 4-HNE levels and activity in the cochlear

314

of D-gal mice (p< 0.01, Fig. 4C–E).

315

3.4. PNUTS and PP1 activation in cochlear tissues in D-gal-induced aging mice

RI PT

312

To further probe the effects of PNUTS/PP1 pathway on aging, we evaluated the

317

level and location of PNUTS at several points in the D-gal induced aging mice. PNUTS,

318

one of the two most abundant nuclear specific PP1 binding proteins. The nuclear

319

function of PNUTS: PP1 holoenzyme are promotion of chromatin decondensation and

320

regulation of RNA splicing (Kim, et al., 2003; Ciurciu, et al., 2013; Choy, et al., 2014).

321

PNUTS localized predominantly to the OHCs and IHCs, SGNs, and endothelial cells of

322

the Stria vessels (n= 6, Fig. 5A). PNUTS expression significantly decreased at 4, 6 and

323

9 months in the OC of the D-gal-induced aging mice, including in the above cells of the

324

cochlea (Fig. 5B); and this decrease may be related to changes in the microvasculature

325

(Ruan et al., 2014). To further quantify changes in PNUTS and PP1 expression in the

326

cochlea, real-time PCR and western blot was used to in the D-gal mice. As shown in Fig.

327

5C, PNUTS gene expression significantly decreased at 4, 6 and 9 months in the

328

cochleae of the D-gal mice, while PP1 gene expression dramatically increased. In

329

addition, PNUTS protein expression was distinct decreased with age in NS and D-gal

330

mice, was significantly decreased in the 9-month-old mice, compared to the

331

2-month-old mice, while PP1 expression was an opposite dramatically increased (Fig.

332

5D).

333

3.5. PPE attenuated PNUTS/PP1 pathway activation in cochlear tissues in aging

AC C

EP

TE D

M AN U

SC

316

16

ACCEPTED MANUSCRIPT 334

mice at 9-month-old To further investigate the mechanism by which PPE attenuate aging-induced hair

336

cell loss, we investigated the effects of PPE on PNUTS/PP1 pathway in D-gal-treated

337

9-month-old mice. PPE significantly increased PNUTS expression in the hair cells of

338

the OC (p< 0.01), the SGNs (p< 0.01), and the Stria (p< 0.05) of D-gal mice (n= 6,

339

Fig. 6A and B), as demonstrated by immunofluorescence, and was further confirmed

340

by Western blot (Fig. 6C). PPE dramatically inhibited the expression of PP1 in D-gal

341

mice (p< 0.01, Fig. 6C and D). P53 is a key factor in maintaining genomic integrity

342

upon DNA damage and oncogene activation MDM2 promotes p53 degradation and

343

suppresses p53 transcriptional activity (Marine, et al., 2006), and P53 is a direct target

344

of PNUTS (Lee, et al., 2007). The expression of p53 was increased and MDM2

345

decreased in the cochlea of D-gal mice, as compared with the corresponding NS mice.

346

PPE significantly reduced p53 expression and enhanced MDM2 expression in the

347

cochlea of D-gal mice (p< 0.01, Fig. 6C and E).

348

350

4. Discussion

AC C

349

EP

TE D

M AN U

SC

RI PT

335

D-gal can be converted into galactitol, because galactitol cannot be metabolized

351

iv vivo, it can be accumulated in the cells, which stimulate ROS production

352

(Cuatrecasas and Segal, 1966). Chronic systemic exposure of rodents to D-gal induces

353

accelerated aging, such as advanced glycation end-product formation (Tian et al.,

354

2005), neurodegeneration (Kou et al., 2016), increased oxidative stress (Wu et al.,

355

2017), declined immunity (Mu et al., 2017), and skeletal muscle atrophy (Fan et al., 17

ACCEPTED MANUSCRIPT 2017). Thus, Oversupply of D-gal-treated by causes an acceleration of senescence and

357

has been used as an aging model. The hearing impairment, the change of oxidative

358

biomarkers, and the elevated accumulation of mitochondrial DNA common deletion

359

in the D-gal-induced inner ear aging model animals are similar with those in naturally

360

aged animals (Du et al., 2012; Zhong et al., 2012; Wu et al., 2012; Zeng et al., 2014;

361

Sun et al., 2015). With the aim of investigating the mechanisms of PPE ameliorating

362

hearing function in ARHL, we investigated using the D-gal-induced aging model. In

363

contrast, PPE reduced oxidative damage in the cochleae of D-gal-induced aging mice,

364

possibly by regulating the PNUTS/PP1 pathway and then ameliorating auditory

365

function in ARHL.

M AN U

SC

RI PT

356

Oxidative stress is one of the most prominent factors that influence ARHL onset

367

and progression (Stebbings et al., 2016). Our results showed that, consistent with the

368

ABR measurements, D-gal-treated mice displayed hearing loss peak, which was

369

especially serious at high frequencies, and similar to that observed with a large number

370

of hair cell loss, especially in the cochlear basal turn. The changes of these two

371

age-related biochemical markers (MDA and SOD）in the cochlea of D-gal mice,

372

indicative of increased oxidative stress during aging. These results indicated that we

373

established an inner ear aging mice model (Wu et al., 2012), and determined the best

374

time point of the hearing loss in the aging model. The lipid peroxidation product,

375

4-HNE, is recognized as a biomarker of oxidative stress (Pillon et al., 2012). Elevated

376

levels of 4-HNE were consistent with those that occur during the natural aging process

377

in animals and humans (Nam et al., 2014). The results demonstrated that D-gal

AC C

EP

TE D

366

18

ACCEPTED MANUSCRIPT 378

administration drastically increased 4-HNE expression and activity in the cochlea.

379

These results were consistent with the courses of ROS and oxidative changes in aging

380

cochlea of 18 months old mice (Jiang et al., 2007). PPE is an antioxidant and a potent free-radical scavenger. The antioxidant activity

382

of PPE is beneficial for the whole organism. It reports that treatment with pomegranate

383

peel could prevent aluminum-induced oxidative stress and histopathological alterations

384

in brain of female rats (Abdel Moneim. 2012). PPE completely inhibites chronic mild

385

stress-induced increase in MDA in the blood and brain (Naveen et al., 2013). PPE is

386

also shown neuroprotective effects, and prevention of neurodegenerative process in a

387

mouse model of neurodegeneration (Morzelle MC et al., 2016). PPE exhibits a

388

hepatoprotective effect through possessing potent antioxidant compounds on murine

389

malaria-induced hepatic injury (Hafiz et al., 2016). In addition, urolithin A (an

390

end-product of ellagic acid metabolism, and ellagic acid is known a main ingredient in

391

PPE) enhanced muscle strength and robustly augmented running endurance in aged

392

mice (Ryu et al., 2016). Our results also showed that PPE remarkably exerted a

393

protective effect in the cochlea of D-gal-treated aging mice. Our results demonstrated

394

that PPE inhibited D-gal-induced inner ear aging, led to hair cell survive, and result in

395

reducing the occurrence of ARHL. Corresponding to our previous study in

396

amikacin-induced ototoxicity mice (Liu et al., 2017), PPE prevented the hair cells loss

397

and hearing impairment caused by aging or drugs.

AC C

EP

TE D

M AN U

SC

RI PT

381

398

In the present study, we demonstrated that a decreasing PNUTS expression in the

399

cochlea of D-gal-induced aging mice was associated with damage in hair cells and 19

ACCEPTED MANUSCRIPT auditory function. We found that PPE enhanced PNUTS expression, decreased PP1 and

401

p53 expression, and reduced damage in the cochlear hair cells of D-gal mice. PNUTS

402

gene and protein expression were downregulated in the heart cells of aging mice ( Boon

403

et al., 2013). In contrast, PP1 activity increased in D-gal-induced aging mice. PNUTS

404

actively regulates PP1 substrate specificity by blocking a subset of substrates from

405

binding PP1 (Kim et al., 2003). Importantly, PNUTS also directly regulates the activity

406

of p53, a key regulator of apoptosis. Specifically, PNUTS inhibited the PP1-mediated

407

dephosphorylation of Ser15 of p53 and Ser395 of MDM2, which resulted in increased

408

stability and transcriptional activity of p53 and enhanced degradation of MDM2(Choy

409

et al., 2014). In this study, we demonstrated that the level of p53 in the cochlea of

410

D-gal-induced aging mice significantly increased, compared to NS mice, while the

411

MDM2 level decreased. Previous findings showed that p53 was activated through a

412

p53-dependent pathway after exposure to oxidative stress in aging mice (Clements et

413

al., 2013), and p53 upregulation stimulated the death of developing epithelial

414

supporting cells of the auditory organ, leading to auditory disturbances (Xiong et al.,

415

2015; Laos et al., 2017). Further investigations are required to determine whether PPE

416

regulates PNUTS/PP1 expression in the same manner as intrinsic aging. Taken

417

together, these data indicate that PPE may potentially be used as an alternative

418

treatment for lowering ARHL.

AC C

EP

TE D

M AN U

SC

RI PT

400

419

In conclusion, PPE alleviate D-gal-induced oxidative stress and hearing loss by

420

regulating PNUTS/PP1 pathway activity in the mouse cochlea. PPE exerts

421

pronounced therapeutic effects, thus, can be used to prevent complications associated 20

ACCEPTED MANUSCRIPT 422

with ARHL. We believe that the present study provided a new therapeutic strategy to

423

use PPE for prevention of ARHL.

424

426

Conflicts of interest

RI PT

425

None

427

Acknowledgements

SC

428

We would like to thank Prof. Jochen Schacht and Su-Hua Sha for providing the

430

cytocochleogram software. We would like to thank Prof. Ming-Sheng Zhou for

431

providing the revised suggestions. This work was supported by the National Natural

432

ScienceFoundation of China (Grant Number 81674036), LiaoningEducation Program

433

(Grant Number L2015316), and Liaoning Science and Technology Program (Grant

434

Number 2014022029,2015020332).

437 438 439

TE D

EP

436

AC C

435

M AN U

429

440 441 442 21

ACCEPTED MANUSCRIPT 443 444

Reference

446

Abdel Moneim AE. 2012. Evaluating the potential role of pomegranate peel in

447

aluminum-induced oxidative stress and histopathological alterations in brain of

448

female

449

10.1007/s12011-012-9498-2.

Trace

Elem

Res

150(1-3):

328-36.

doi:

SC

Biol

Akhtar S, Ismail T, Fraternale D, Sestili P. 2015. Pomegranate peel and peel extracts:

M AN U

450

rats.

RI PT

445

451

chemistry

452

10.1016/j.foodchem.2014.11.035

and

food

features.

Food

Chem

1;174:417-25.

doi:

Boon, R.A., Iekushi, K., Lechner, S., Seeger, T., Fischer, A., Heydt, S., Kaluza, D.,

454

Treguer, K., Carmona, G., Bonauer, A., Horrevoets, A.J., Didier, N., Girmatsion,

455

Z., Biliczki, P., Ehrlich, J.R., Katus, H.A., Muller, O.J., Potente, M., Zeiher,

456

A.M., Hermeking, H., Dimmeler, S. 2013. MicroRNA-34a regulates cardiac

457

ageing and function. Nature 495(7439), 107-10. doi:10.1038/nature11919.

458

Choy M.S., Hieke M., Kumar G.S., Lewis G.R., Gonzalez-DeWhitt K.R., Kessler R.P.,

460 461 462

EP

AC C

459

TE D

453

Stein B.J., Hessenberger M., Nairn A.C., Peti W., Page R. 2014. Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code. Proc Natl Acad Sci U S A. 111(11), 4097-102. doi:10.1073/pnas.1317395111.

463

Ciurciu A, Duncalf L, Jonchere V, Lansdale N, Vasieva O, Glenday P, Rudenko A,

464

Vissi E, Cobbe N, Alphey L, Bennett D. 2013. PNUTS/PP1 regulates 22

ACCEPTED MANUSCRIPT 465

RNAPII-mediated gene expression and is necessary for developmental growth.

466

PLoS Genet. 9(10): e1003885. doi: 10.1371/journal.pgen.1003885. Cuatrecasas P, Segal S. 1966. Galactose conversion to D-xylulose: an alternate route

468

of

469

10.1126/science.153.3735.549.

galactose

metabolism.

Science

153(3735),

549-51.

doi:

RI PT

467

Clements ME, Chaber CJ, Ledbetter SR, Zuk A. 2013. Increased cellular senescence

471

and vascular rarefaction exacerbate the progression of kidney fibrosis in aged

472

mice following transient ischemic injury. PLoS One 8(8), e70464. doi:

473

10.1371/journal.pone.0070464.

M AN U

SC

470

De Leon G., Cavino M., D'Angelo M., Krucher N.A. 2010. PNUTS knockdown

475

potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells.

476

International journal of oncology 36(5), 1269-75.

TE D

474

Ding D, Jiang H, Chen GD, Longo-Guess C, Muthaiah VP, Tian C, Sheppard A, Salvi

478

R, Johnson KR. 2016. N-acetyl-cysteine prevents age-related hearing loss and

479

the progressive loss of inner hair cells in γ-glutamyl transferase 1 deficient mice.

480

Aging (Albany NY) 8(4), 730-50. doi: 10.18632/aging.100927.

AC C

EP

477

481

Dos Santos R.L., Dellacqua L.O., Delgado N.T., Rouver W.N., Podratz P.L., Lima

482

L.C., Piccin M.P., Meyrelles S.S., Mauad H., Graceli J.B., Moyses M.R. 2016.

483

Pomegranate peel extract attenuates oxidative stress by decreasing coronary

484

angiotensin-converting enzyme (ACE) activity in hypertensive female rats.

485

Journal of toxicology and environmental health Part A 79(21), 998-1007.

486

doi:10.1080/15287394.2016.1213690. 23

ACCEPTED MANUSCRIPT Du Z, Yang Y, Hu Y, Sun Y, Zhang S, Peng W, Zhong Y, Huang X, Kong W. 2012. A

488

long-term high-fat diet increases oxidative stress, mitochondrial damage and

489

apoptosis in the inner ear of D-galactose-induced aging rats. Hear Res 287(1-2),

490

15-24. doi: 10.1016/j.heares.2012.04.012.

RI PT

487

Fan J, Yang X, Li J, Shu Z, Dai J, Liu X, Li B, Jia S, Kou X, Yang Y, Chen N. 2017.

492

Spermidine coupled with exercise rescues skeletal muscle atrophy from

493

D-gal-induced aging rats through enhanced autophagy and reduced apoptosis

494

via AMPK-FOXO3a signal pathway. Oncotarget. 8(11):17475-17490. doi:

495

10.18632/oncotarget.15728.

497

M AN U

496

SC

491

Fischer N., Weber B., Riechelmann H. 2016. Presbycusis - Age Related Hearing Loss. Laryngo- rhino- otologie 95(7), 497-510. doi:10.1055/s-0042-106918. Fujimoto C., Yamasoba, T. 2014. Oxidative stresses and mitochondrial dysfunction in

499

age-related hearing loss. Oxidative medicine and cellular longevity 2014,

500

582849. doi:10.1155/2014/582849.

TE D

498

Hafiz TA, Mubaraki MA, Al-Quraishy S, Dkhil MA. 2016. The potential role of

502

Punica granatum treatment on murine malaria-induced hepatic injury and

504

AC C

503

EP

501

oxidative

stress.

Parasitol

Res.

115(4):1427-33.

doi:

10.1007/s00436-015-4876-2.

505

Halonen J., Hinton A.S., Frisina R.D., Ding B., Zhu X., Walton J.P. 2016. Long-term

506

treatment with aldosterone slows the progression of age-related hearing loss.

507

Hearing research 336, 63-71. doi:10.1016/j.heares.2016.05.001.

508

Hill K, Yuan H, Wang X, Sha SH. 2016. Noise-Induced Loss of Hair Cells and 24

ACCEPTED MANUSCRIPT 509

Cochlear Synaptopathy Are Mediated by the Activation of AMPK. J Neurosci

510

36(28), 7497-510. doi: 10.1523/JNEUROSCI.0782-16.2016. Homans NC, Metselaar RM, Dingemanse JG, van der Schroeff MP, Brocaar MP,

512

Wieringa MH, Baatenburg de Jong RJ, Hofman A, Goedegebure A. 2017.

513

Prevalence of age-related hearing loss, including sex differences, in older adults

514

in a large cohort study. Laryngoscope 127(3):725-730. doi: 10.1002/lary.26150.

RI PT

511

Jiang H, Talaska AE, Schacht J, Sha SH. 2007. Oxidative imbalance in the aging inner

516

ear. Neurobiol Aging28(10):1605-12.doi:10.1016/j.neurobiolaging.2006.06.025.

517

Kahya V., Ozucer B., Dogan R., Meric A., Yuksel M., Gedikli O., Ozturan O. 2014.

518

Pomegranate extract: a potential protector against aminoglycoside ototoxicity.

519

The

520

doi:10.1017/S0022215113003460.

of

laryngology

and

otology

128(1),

43-8.

TE D

Journal

M AN U

SC

515

Kim Y.M., Watanabe T., Allen P.B., Kim Y.M., Lee S.J., Greengard P., Nairn A.C.,

522

Kwon Y.G. 2003. PNUTS, a protein phosphatase 1 (PP1) nuclear targeting

523

subunit. Characterization of its PP1- and RNA-binding domains and regulation

524

by phosphorylation. The Journal of biological chemistry 278(16), 13819-28.

AC C

525

EP

521

doi:10.1074/jbc.M209621200.

526

Kinoshita M, Sakamoto T, Kashio A, Shimizu T, Yamasoba T. 2013. Age-related

527

hearing loss in Mn-SOD heterozygous knockout mice. Oxid Med Cell Longev

528

2013:325702. doi: 10.1155/2013/325702.

529

Kou X, Liu X, Chen X, Li J, Yang X, Fan J, Yang Y, Chen N. 2016. Ampelopsin

530

attenuates brain aging of D-gal-induced rats through miR-34a-mediated 25

ACCEPTED MANUSCRIPT 531

SIRT1/mTOR

532

10.18632/oncotarget.12811.

signal

pathway.

Oncotarget.

7(46):74484-74495.

doi:

Laos M, Sulg M, Herranen A, Anttonen T, Pirvola U. 2017. Indispensable role of

534

Mdm2/p53 interaction during the embryonic and postnatal inner ear

535

development. Sci Rep

RI PT

533

9;7, 42216. doi: 10.1038/srep42216.

Lee S.J., Lim C.J., Min J.K., Lee J.K., Kim Y.M., Lee J.Y., Won M.H., Kwon Y.G.

537

2007. Protein phosphatase 1 nuclear targeting subunit is a hypoxia inducible

538

gene: its role in post-translational modification of p53 and MDM2. Cell death

539

and differentiation 14(6), 1106-16. doi:10.1038/sj.cdd.4402111.

M AN U

SC

536

Li J., Cai D., Yao X., Zhang Y., Chen L., Jing P., Wang L., Wang Y. 2016. Protective

541

Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through

542

Attenuating Oxidative Stress and the Wnt/beta-Catenin Signaling Pathway in a

543

Mouse Model of d-Galactose-induced Aging. International journal of molecular

544

sciences 17(6). doi:10.3390/ijms17060849.

TE D

540

Liu S, Zhang X, Sun M, Xu T, Wang A. 2017. FoxO3a plays a key role in the

546

protective effects of pomegranate peel extract against amikacin-induced

AC C

547

EP

545

ototoxicity. Int J Mol Med 40(1):175-181. doi: 10.3892/ijmm.2017.3003.

548

Marine JC, Francoz S, Maetens M, Wahl G, Toledo F, Lozano G. 2006. Keeping p53

549

in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death

550

Differ 13(6), 927-34. doi: 10.1038/sj.cdd.4401912.

551

Menardo J., Tang Y., Ladrech S., Lenoir M., Casas F., Michel C., Bourien J., Ruel J.,

552

Rebillard G., Maurice T., Puel J.L., Wang J. 2012. Oxidative stress, 26

ACCEPTED MANUSCRIPT 553

inflammation, and autophagic stress as the key mechanisms of premature

554

age-related hearing loss in SAMP8 mouse Cochlea. Antioxidants & redox

555

signaling 16(3), 263-74. doi:10.1089/ars.2011.4037. Morzelle M.C., Salgado J.M., Telles M., Mourelle D., Bachiega P., Buck H.S., Viel

557

T.A. 2016. Neuroprotective Effects of Pomegranate Peel Extract after Chronic

558

Infusion with Amyloid-beta Peptide in Mice. PloS one 11(11), e0166123.

559

doi:10.1371/journal.pone.0166123.

SC

RI PT

556

Mu X, Zhang Y, Li J, Xia J, Chen X, Jing P, Song X, Wang L, Wang Y. 2017.

561

Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells

562

Senescence in D-Galactose-Induced Aging Mouse Model. Stem Cells Int.

563

2017:3508907. doi: 10.1155/2017/3508907.

M AN U

560

Muzio G., Ricci M., Traverso N., Monacelli F., Oraldi M., Maggiora M., Canuto R.A.

565

2016. 4-Hydroxyhexenal and 4-hydroxynonenal are mediators of the

566

anti-cachectic effect of n-3 and n-6 polyunsaturated fatty acids on human lung

567

cancer cells. Free radical biology & medicine 99, 63-70. doi:10.1016/j.

568

freeradbiomed. 2016.07.031.

AC C

EP

TE D

564

569

Nam SM, Chung TH, Kim JW, Jung HY, Yim HS, Kim DW, Yoo DY, Nam H, Choi

570

JH, Hwang IK, Suh JG, Yoon YS. 2014. Comparison of N-methyl-D-aspartate

571

receptor subunit 1 and 4-hydroxynonenal in the hippocampus of natural and

572

chemical-induced aging accelerated mice. Neurochem Res 39(9), 1702-8. doi:

573

10.1007/s11064-014-1362-7.

574

Neyrinck AM., Van Hee V.F., Bindels LB., De Backer F., Cani PD., Delzenne NM. 27

ACCEPTED MANUSCRIPT 575

2013.

576

inflammation and hypercholesterolaemia in high-fat diet-induced obese mice:

577

potential implication of the gut microbiota. The British journal of nutrition

578

109(5), 802-9. doi:10.1017/S0007114512002206.

extract

of

pomegranate

peel

alleviates

tissue

RI PT

Polyphenol-rich

Ohmen J., Kang E.Y., Li X., Joo J.W., Hormozdiari F., Zheng Q.Y., Davis R.C., Lusis

580

A.J., Eskin E., Friedman R.A. 2014. Genome-wide association study for

581

age-related hearing loss (AHL) in the mouse: a meta-analysis. Journal of the

582

Association for Research in Otolaryngology : JARO 15(3), 335-52.

583

doi:10.1007/s10162-014-0443-2.

M AN U

SC

579

Pillon N.J., Croze M.L., Vella R.E., Soulere L., Lagarde M., Soulage C.O. 2012. The

585

lipid peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin

586

resistance in skeletal muscle through both carbonyl and oxidative stress.

587

Endocrinology 153(5), 2099-111. doi:10.1210/en.2011-1957.

TE D

584

Ruan Q, Hu X, Ao H, Ma H, Gao Z, Liu F, Kong D, Bao Z, Yu Z. 2014. The

589

neurovascular protective effects of huperzine A on D-galactose-induced

590

inflammatory damage in the rat hippocampus. Gerontology 60(5), 424-39. doi:

AC C

591

EP

588

10.1159/000358235.

592

Ryu D, Mouchiroud L, Andreux PA, Katsyuba E, Moullan N, Nicolet-Dit-Félix AA,

593

Williams EG, Jha P, Lo Sasso G, Huzard D, Aebischer P, Sandi C, Rinsch C,

594

Auwerx J. 2016. Urolithin A induces mitophagy and prolongs lifespan in C.

595

elegans and increases muscle function in rodents. Nat Med 22(8), 879-88. doi:

596

10.1038/nm.4132. 28

ACCEPTED MANUSCRIPT Stebbings KA., Choi HW., Ravindra A., Llano DA. 2016. The impact of aging,

598

hearing loss, and body weight on mouse hippocampal redox state, measured in

599

brain slices using fluorescence imaging. Neurobiology of aging 42, 101-9.

600

doi:10.1016/j.neurobiolaging.2016.03.006.

RI PT

597

Sun H.Y., Hu Y.J., Zhao X.Y., Zhong Y., Zeng L.L., Chen X.B., Yuan J., Wu J., Sun Y.,

602

Kong W., Kong W.J. 2015. Age-related changes in mitochondrial antioxidant

603

enzyme Trx2 and TXNIP-Trx2-ASK1 signal pathways in the auditory cortex of

604

a mimetic aging rat model: changes to Trx2 in the auditory cortex. The FEBS

605

journal 282(14), 2758-74. doi:10.1111/febs.13324.

M AN U

SC

601

606

Takumida M, Ishibashi T, Hamamoto T, Hirakawa K, Anniko M. 2009.

607

Age-dependent changes in the expression of klotho protein, TRPV5 and TRPV6

608

in

609

10.3109/00016480902725254.

inner

ear.Acta

Otolaryngol

129(12):1340-50.

doi:

TE D

mouse

Tian J, Ishibashi K, Ishibashi K, Reiser K, Grebe R, Biswal S, Gehlbach P, Handa JT.

611

2005. Advanced glycation endproduct-induced aging of the retinal pigment

612

epithelium and choroid: a comprehensive transcriptional response. Proc Natl

AC C

613

EP

610

Acad Sci U S A. 102(33):11846-51.

614

Wu L, Sun Y, Hu YJ, Yang Y, Yao LL, Zhou XX, Wang H, Zhang R, Huang X, Kong

615

WJ. 2012. Increased p66Shc in the inner ear of D-galactose-induced aging mice

616

with accumulation of mitochondrial DNA 3873-bp deletion: p66Shc and

617

mtDNA damage in the inner ear during aging. PLoS One 7(11), e50483. doi:

618

10.1371/journal.pone.0050483. 29

ACCEPTED MANUSCRIPT 619

Wu W, Hou CL, Mu XP, Sun C, Zhu YC, Wang MJ, Lv QZ. 2017. H2S Donor NaHS

620

Changes the Production of Endogenous H2S and NO in D-Galactose-Induced

621

Accelerated

622

10.1155/2017/5707830.

Oxid Med Cell

Longev. 2017:

5707830.

doi:

RI PT

Ageing.

Xiong, H., Pang, J., Yang, H., Dai, M., Liu, Y., Ou, Y., Huang, Q., Chen, S., Zhang, Z.,

624

Xu, Y., Lai, L., Zheng, Y. 2015. Activation of miR-34a/SIRT1/p53 signaling

625

contributes to cochlear hair cell apoptosis: implications for age-related hearing

626

loss.

627

doi:10.1016/j.neurobiolaging.2014.12.034.

of

aging

36(4),

1692-701.

M AN U

Neurobiology

SC

623

Yazici ZM., Meric A., Midi A., Arinc YV., Kahya V., Hafiz G. 2012. Reduction of

629

cisplatin ototoxicity in rats by oral administration of pomegranate extract.

630

European archives of oto-rhino-laryngology : official journal of the European

631

Federation

632

doi:10.1007/s00405-011-1582-2.

of

TE D

628

Oto-Rhino-Laryngological

Societies

269(1),

45-52.

Zeng L, Yang Y, Hu Y, Sun Y, Du Z, Xie Z, Zhou T, Kong W. 2014. Age-related

634

decrease in the mitochondrial sirtuin deacetylase Sirt3 expression associated

636

AC C

635

EP

633

with ROS accumulation in the auditory cortex of the mimetic aging rat model. PLoS One 9(2), e88019. doi: 10.1371/journal.pone.0088019.

637

Zhong Y, Hu Y, Peng W, Sun Y, Yang Y, Zhao X, Huang X, Zhang H, Kong W. 2012.

638

Age-related decline of the cytochrome c oxidase subunit expression in the

639

auditory cortex of the mimetic aging rat model associated with the common

640

deletion. Hear Res 294(1-2), 40-8. doi: 10.1016/j.heares.2012.09.006. 30

ACCEPTED MANUSCRIPT 641 642 643

Figure captions

645

Fig. 1. D-gal-induced aging cochlear model. (A) Experimental protocol used to

646

establish the aging model. (B) Thresholds measured in the NS and aging groups at 2,

647

4, 6, and 9 months (n = 25 in each group). (C) Representative immunofluorescence

648

images of hair cells stained with an anti-myosin VIIa antibody (green) in the basal

649

turn at 2, 4, 6, and 9 months post-D-gal administration. Asterisks indicate missing hair

650

cells. Scale bar: 100 µm. (D) Loss of lesions in the OHCs and IHCs of different groups

651

were observed at 2, 4, 6, and 9 months (n = 5/group). (E) and (F) T-SOD and MDA

652

were detected in the cochlea of mice in the NS and aging groups (n = 6/group). *p<

653

0.05, **p< 0.01. Data are presented as the mean ± SD. D-gal: D-galactose; D-gal

654

group, group treated with 800 mg/kg/day D-gal; IHCs: inner hair cells; MDA:

655

malondialdehyde; NS group, group treated with normal saline; OHCs: outer hair cells;

656

T-SOD: total superoxide dismutase

SC

M AN U

TE D

EP

AC C

657

RI PT

644

658

Fig. 2. 4-HNE expression in the cochleae. (A) Representative immunofluorescence

659

images of hair cells stained with a TRITC-conjugated anti-phalloidin antibody (red)

660

and an anti-4-HNE antibody (green) in the basal turn at 2, 4, 6, and 9 months

661

post-D-gal stimulation. Cell nuclei were counterstained with DAPI (blue). Scale bar:

662

10 µm. (B) Quantification of 4-HNE immunolabeling in the different groups (n = 31

ACCEPTED MANUSCRIPT 4/group). (C) 4-HNE levels in cochlear tissue lysates, as measured by ELISA (n =

664

6/group). (D) 4-HNE expression in cochlear tissue lysates, as determined by western

665

blotting (n = 3/group). *p< 0.05, **p< 0.01. Data are presented as the mean ±

666

SD.4-HNE, 4-hydroxynonenal; DAPI, 4, 6-diamidino-2-phenylindole

RI PT

663

667

Fig. 3. PPE prevented hearing and hair cell loss in aging mice at 9 months. (A)

669

Experimental protocol for examining the effect of PPE treatment on aging mice. (B)

670

Threshold shifts of PPE-treated aging mice and NS mice (n = 25/group). (C) and (D)

671

T-SOD and MDA measured in PPE-treated aging mice and control mice (n = 6/group).

672

(E) Numbers of apoptotic hair cells at the basal turn in the different treatment groups

673

at 9 months. Cleaved caspase-3 (red), myosin VIIa (green), and DAPI (blue). Hair

674

cells undergoing apoptosis (cleaved caspase-3 and myosin VIIa double-stained cells).

675

Scale bar: 20 µm. (n = 5/group) *p< 0.05, **p< 0.01. Data are presented as the mean

676

± SD.

M AN U

TE D

EP

677

SC

668

Fig. 4. PPE inhibited 4-HNE expression in the cochlear tissues of aging mice at 9

679

months. (A) After PPE treatment, immunolabeling with an FITC-conjugated

680

anti-4-HNEantibody (green) was reduced within hair cells of the OC (scale bar: 10

681

µm), the SGNs (scale bar: 20 µm), and the Stria (scale bar: 20 µm), compared to that

682

in the untreated aging group. The cell nuclei were counterstained with DAPI (blue).

683

(B) Quantification of 4-HNE immunolabeling in the different treatment groups (n =

684

6/group). (C) 4-HNE levels in cochlear tissue lysates, as measured by ELISA (n =

AC C

678

32

ACCEPTED MANUSCRIPT 685

6/group). (D) 4-HNE expression in the cochlea tissue lysates, as determined by

686

western blotting (n = 3/group). (E) 4-HNE protein levels, quantified relative to the

687

indicated NS group. **p< 0.01. Data are presented as the mean ± SD (n = 6/group).

RI PT

688

Fig. 5. Activation of PNUTS and PP1 in the cochleae of aging mice. (A) Paraffin

690

sections of the cochleae of aging mice showed immunostaining with an Alexa Fluor

691

594-conjugated anti-PNUTS antibody (red) in the OC, including in the OHCs, IHCs,

692

(scale bar: 10 µm), SGNs (scale bar: 20 µm), and Stria cells (scale bar: 20 µm) at 2, 4,

693

6, and 9 months. Cell nuclei were stained with DAPI (blue). (B) Quantification of

694

PNUTS immunolabeling in the different groups (n = 6/group) (C) PNUTS and PP1

695

mRNA levels in cochlea tissues, as determined by real-time PCR (n = 3/group) (D)

696

PNUTS and PP1 expression in the cochlea tissue lysates, as determined by western

697

blotting. *p< 0.05, **p< 0.01. Data are presented as the mean ± SD (n = 3/group). OC:

698

organ of Corti; SGNs: spiral ganglion neurons; Stria: striavascularis

M AN U

TE D

EP

699

SC

689

Fig. 6. PPE attenuated PNUTS/PP1 activation in the cochlear tissues of aging mice at

701

9 months. (A) After PPE treatment, immunostaining with an Alexa Fluor

702

594-conjugated anti-PNUTS antibody (red) was reduced in the hair cells of the OC

703

(scale bar: 10 µm), the SGNs (scale bar: 20 µm), and the Stria (scale bar: 20 µm),

704

compared to that in the untreated aging group. (B) Quantification of immunolabeled

705

PNUTS in the different groups (n = 6/group). (C) Expression of PNUTS in cochlear

706

tissue lysates, as determined by western blotting. (D) PNUTS and PP1 protein levels,

AC C

700

33

ACCEPTED MANUSCRIPT 707

and (E) MDM2 and p53 levels were quantified relative to the indicated controls (n =

708

3/group). *p< 0.05, **p< 0.01. Data are presented as the mean ± SD.

709

Fig. S1. Threshold shifts of two doses of PPE-treated D-gal induced aging mice. (n =

711

25/group), *p< 0.05, **p< 0.01, Data are presented as the mean ± SD.

AC C

EP

TE D

M AN U

SC

RI PT

710

34

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights 4-HNE was increased in cochlear hair cells of D-gal-induced mimetic aging mice.

•

PNUTS expression decreased in D-gal-induced mimetic aging mice.

•

PPE reduce oxidative damage to hair cells by regulating PNUTS/PP1 signaling.

AC C

EP

TE D

M AN U

SC

RI PT

•