Accepted Manuscript

Panax ginseng (Korea Red Ginseng) repairs diabetic sensorineural damage through
promotion of the NGF pathway in diabetic zebrafish

Youn Hee Nam, Hyo Won Moon, Yeong Ro Lee, Eun Young Kim, Isabel Rodriguez,
Seo Yule Jeong, Rodrigo Castañeda, Ji-Ho Park, Se-Young Choung, Bin Na Hong,
Tong Ho Kang
PII: S1226-8453(17)30277-4
DOI: 10.1016/j.jgr.2018.02.006
Reference: JGR 345

To appear in: Journal of Ginseng Research

Received Date: 5 September 2017

Revised Date: 31 January 2018
Accepted Date: 12 February 2018

Please cite this article as: Nam YH, Moon HW, Lee YR, Kim EY, Rodriguez I, Jeong SY, Castañeda
R, Park J-H, Choung S-Y, Hong BN, Kang TH, Panax ginseng (Korea Red Ginseng) repairs diabetic
sensorineural damage through promotion of the NGF pathway in diabetic zebrafish, Journal of Ginseng
Research (2018), doi: 10.1016/j.jgr.2018.02.006.

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 ACCEPTED MANUSCRIPT

1 Panax ginseng (Korea Red Ginseng) repairs diabetic sensorineural damage

2 through promotion of the NGF pathway in diabetic zebrafish

4 Youn Hee Nam1,☆, Hyo Won Moon1,☆, Yeong Ro Lee1, Eun Young Kim1, Isabel Rodriguez1,

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5 Seo Yule Jeong1, Rodrigo Castañeda1, Ji-Ho Park2, Se-Young Choung3, Bin Na Hong1 , Tong

6 Ho Kang1,*

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7

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8 Department of Oriental Medicine Biotechnology, College of Life Sciences and Graduate

9 School of Biotechnology, Kyung Hee University, Global Campus, Gyeonggi 446-701,

10 Republic of Korea
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11 Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-

12 701, Republic of Korea

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13 Department of Preventive Pharmacy and Toxicology, College of Pharmacy, Kyung Hee
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14 University, Seoul 130-701, Republic of Korea

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15
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16 Corresponding author: Graduate School of Biotechnology, Department of Oriental Medicine
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17 Biotechnology, Kyung Hee University, Gyeonggi 446-701, Republic of Korea. Tel.: +82-31-

18 201-3862; fax: +82-303-0300-0030

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19 E-mail address: [email protected]

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20

☆
21 The first two authors contributed equally to this work.

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25 ABSTRACT

26 Backgroud: Diabetic sensorineural damage is a complication of the sensory neural system,

27 resulting from long-term hyperglycemia. Red ginseng (RG) has shown efficacy for treatment

28 of various diseases, including diabetes mellitus; however, there is little research about its

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29 benefit for treating sensorineural damage. Therefore, we aim to evaluate RG efficacy in

30 alloxan-induced diabetic neuromast (AIDN) zebrafish.

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31 Methods: In this study, we developed and validated an AIDN zebrafish model. To assess RG

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32 effectiveness, we observed morphological changes in live neuromast zebrafish. Also,

33 zebrafish has been observed to have an ultrastructure of hair-cell cilia under SEM. Thus, we

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34 recorded these physiological traits to assess hair cell function. Finally, we confirmed that RG
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35 promoted neuromast recovery via NGF signaling pathway markers.

36 Results: First, we established an AIDN zebrafish model. Using this model, we showed via
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37 live neuromast imaging that RG fostered recovery of sensorineural damage. Damaged hair

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cell cilia were recovered in AIDN zebrafish. Furthermore, RG rescued damaged hair cell

39 function through cell membrane ion balance.

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40 Conclusion: Our data suggest that RG potentially facilitates recovery in AIDN zebrafish, and
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41 its mechanism seems to be promotion of the Nerve growth factor (NGF) pathway through

42 increased expression of trkA, TRPV1 and p-MAPK.

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45 Keywords: Red ginseng; Zebrafish; Diabetic sensorineural damage; Alloxan; Neuromast

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50 1. Introduction

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52 Panax ginseng has been use for thousands of years in Asian countries for diabetic therapy.

53 Red ginseng (RG) is prepared by steaming the roots of Panax ginseng. RG and its

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54 ginsenosides have broad pharmacological value, including anti-cancer [1], anti-inflammatory

55 [2], and anti-diabetes properties [3], as well as the ability to improve chronic liver disease [4].

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56 However, little is known about the potential efficacy of RG for the diabetic sensorineural

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57 system.

58 Neuromasts located at the surface from the head to the trunk of fish and aquatic amphibians

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59 are considered sensory organs [5]. Due to their physical location, neuromasts are easily
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60 visualized [6]. Neuromasts are able to detect and localize water movement around the body

61 and are implicated in several behaviors [7]. Each neuromast is composed of a central core of
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62 hair cells surrounded by progenitor cells and mantle cells, which are supporting cells.

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Neuromasts are innervated by the peripheral projections of afferent neurons located in the

64 posterior lateral line ganglion [8, 9, 10, 11]. In our model, the zebrafish peripheral nerve was
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65 damaged by diabetic sensorineural damage. Hyperglycemia induces oxidative stress, which is

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66 the underlying cause of development and progression of diabetic sensorineural damage, an

67 important complication of diabetes [12, 13, 14]. Nerve growth factor (NGF) is among the
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68 most important molecular targets of diabetic peripheral neurophaty, reduced levels or activity
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69 of NGF play a key role in diabetic neuropathy pathogenesis. [32, 33]. There are 2 NGF

70 receptors, p75 neurotrophin receptor (p75NTR) with low affinity and tropomyosin receptor

71 kinase (Trk) with high affinity for NGF, which is expressed in the peripheral nervous system

72 [34]. Moreover, NGF is associated with modulation of TRPV1 expression [27, 35], which is

73 well known to cause a sensation of scalding heat and pain during peripheral neuropathy [31].

74 Therefore, we aimed to evaluate RG efficacy for alloxan-induced diabetic sensorineural

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75 damage zebrafish and to investigate the underlying mechanisms. Thus, we investigated the

76 role of RG in trkA and TRPV1 expression and in the downstream of the NGF pathway

77 through MAPK level.

78 Consequently, we established an AIDN zebrafish model and then evaluated recovery due to

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79 RG through observation of live neuromasts. The ultrastructure of hair cell cilia in RG-treated

80 zebrafish was observed under SEM, and we performed physiological recordings to assess hair

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81 cell function. Finally, we confirmed RG-promoted neuromast recovery via NGF signaling

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82 pathway markers.

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83 2. Materials and Methods

84 2.1. Reagents and equipment

85 Alloxan monohydrate, sea salt, tricaine methane sulfonate, and tert-butanol ware purchased

86 from Sigma Chemical Co. (St. Louis, MO, USA). 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)

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87 amino)-2-deoxyglucose (2-NBDG) and YO-PRO were purchased from Invitrogen (Life

88 Technologies, Grand Island, NY, USA). An Olympus 1X70 microscope was used for

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89 fluorescence microscopy (Olympus, Japan). Focus Lite (Focus Co, Daejeon, Korea) and

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90 Image J software (National Institutes of Health, Bethesda, MD, USA) were used for image

91 analyses. Red ginseng extract was obtained from the Korea Ginseng Corporation (Taejon,

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92 Republic of Korea). The Korean red ginseng extract used in this study (crude saponin 70mg/g,
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93 solid component 60%, or more) contained Rb1 (0.46%), Rb2 (0.23%), Rc (0.28%), Rd

94 (0.09%), Re (0.12%), Rf (0.10%), Rg1 (0.07%), Rg2 (0.14%), Rg3 (0.12%), Rh1 (0.10%),
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95 and other minor ginsenosides.

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96
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97 2.2. Zebrafish maintenance

98 Adult zebrafish (wild-type) were maintained in an aquarium with a continuous re-circulating

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99 system. Fish were fed commercially available fish food and newly hatched brine shrimp

100 twice a day. Three sexually mature female and three sexually mature male zebrafish were
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101 allowed to breed in a breeding cage at night. The next morning, the embryos were placed at
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102 the bottom of the breeding cage and collected two hours post-fertilization (hpf). The embryos

103 were separated and raised in 0.03% sea salt solution in a petri dish. Zebrafish embryos were

104 maintained in a 14-hour light/10-hour dark photoperiod at 28 ˚C. Fish were cared for in

105 accordance with standard zebrafish protocols approved by the Animal Care and Use

106 Committee of Kyung Hee University (KHUASP(SE)-15-10).

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107

108 2.3. Validation of alloxan to confirm the extent of neuromast damage

109 Five days post-fertilization (dpf), wild-type zebrafish larvae were placed into 96-well plates.

110 The zebrafish larvae were treated with various concentrations of alloxan (100, 300, 500, and

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111 700 µM) for two days and for four days respectively. Afterward, each zebrafish was rinsed

112 three times with 0.03% sea salt solution. The lateral line neuromasts were labeled using 0.1%

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113 YO-PRO for 30 minutes. YO-PRO is a cyanine dye that selectively binds to DNA so that

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114 each hair cell nuclei is stained. Larvae were washed using 0.03% sea salt solution and then

115 anesthetized using 0.004% tricaine solution. The number of green fluorescent-labeled

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116 neuromasts was counted under fluorescence microscopy.
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118 2.4. Alloxan toxicity test

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119 Twenty zebrafish embryos were used for the toxicity test of alloxan. Embryos were placed

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in 6-well plates and incubated at 28.5°C under a 14-hour light/10-hour dark photoperiod.

121 Four treatments were used: alloxan at 0-µM, 100-µM, 300-µM, or 500-µM concentration.
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122 The embryos were observed under microscopy two days after treatment, and heartbeat and
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123 hatching rate were recorded.

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125 2.5. Evaluation of live neuromasts by RG on AIDN zebrafish

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126 Five dpf, wild-type zebrafish larvae were exposed to 300-µM concentrations of alloxan for

127 72 hours in a 96-well plate. Following alloxan treatment, the larvae were treated with 50

128 µg/mL or 100 µg/mL of RG extract for 12 hours. Afterward, each zebrafish was rinsed in 0.03%

129 sea salt solution. Neuromast hair cells were stained with 0.1% YO-PRO for 30 minutes. The

130 total number of lateral line neuromasts was counted under fluorescence microscopy.

131 Additionally, changes in pancreatic islet β-cells after alloxan exposure were assessed using
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132 the method described above.

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134 2.6. Scanning electron microscopy (SEM) observation of hair cell cilia in AIDN zebrafish

135 SEM was used to investigate the loss or recovery of hair cell cilia. Zebrafish larvae from

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136 each group were fixed for 12 hours in 2.5% glutaraldehyde in phosphate buffered saline at

137 4°C. Larvae were washed three times for five minutes in distilled water and then were

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138 dehydrated through serial exposure to graded concentrations of ethanol solution (25%, 50%,

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139 70%, 80%, 95%, and 100%) for 10 minutes. Next, zebrafish larvae samples were dehydrated

140 through serial exposure to graded concentration of tert-butanol in ethanol (25%, 50%, 75%,

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141 and 100%) for 10 minutes. Finally, the specimens were dried using a critical point dryer and
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142 were sputter-coated twice with platinum using an ion sputter coater (ps-1200; Tescan, Czech

143 Republic). The hair cell cilia were observed using SEM (supra55; Zeiss, Germany) at 10kV.
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2.7. Evaluation of hair cell function using physiological recordings of AIDN zebrafish

146 Five dpf, larvae were exposed to alloxan and then treated with RG as described above. Each
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147 larva was placed in a silicone Sylgard-coated cell dish containing extracellular solution (134
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148 mM NaCl, 2.9 mM KCl, 1.2 mM MgCl2, 2.1 mM CaCl2, 10 mM glucose, 10 mM HEPES

149 buffer, adjusted to a pH of 7.8 with NaOH). Each larva was anesthetized in embryo media
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150 containing 0.004% tricaine for 30 seconds. The larvae were positioned laterally and mounted
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151 with vasellin. One point was just posterior to the bladder, and the other point was near the tail

152 end of the larva. In order to record neuromast activity, larvae were immobilized with the

153 0.004% tricaine. Each neuromast’s condition was checked under fluorescence microscopy,

154 and the posterior trunk L1 neuromast resting membrane potential signal was measured with a

155 microelectrode. The microelectrode was filled with 3 M KCl, and the electrode resistance was

156 set to range between 5 and 10 M Ω in extracellular solution. Recorded values were converted
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157 to digital signals with AXOCLAMP-2B.

158

159 2.8. Pancreatic islet recovery by RG on AIDN zebrafish

160 Five dpf, wild-type zebrafish larvae were exposed to 300 µM of alloxan for 72 hours in a

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161 96-well plate. Following alloxan treatment, the larvae were treated with 100 µg/mL of RG

162 extract for 12 hours. RG-treated zebrafish larvae were exposed to 25 µM of 2-NBDG for 12

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163 hours, and then pancreatic islets were imaged by fluorescence microscopy. Damaged β-cell

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164 regeneration due to the effect of RG was confirmed in the ins:GFP line zebrafish based on

165 green fluorescence protein tagging in the β-cells. Transgenic zebrafish lines expressing GFP

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166 specifically in β-cells were obtained from the Zebrafish Organogenesis Mutant Bank
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167 (ZOMB). Pancreatic islets and β-cells were analyzed using Focus Lite software or Image J

168 software.
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2.9. Total RNA isolation

171 Total RNA was isolated from zebrafish larvae using Trizol Reagent (Life science) following
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172 the manufacturer’s instructions. For this, 500 µL of Trizol reagent was added to whole larva
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173 samples and then the samples were homogenized. Total RNA was separated from the sample

174 using chloroform containing amylenes (Sigma-Aldrich) and centrifugation. This was
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175 followed by treatment with 99.5% isopropanol (Samchun Chemical) and and another round
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176 of centrifugation. Finally, the sample was washed with 75% EtOH and diethyl pyrocarbonate

177 (DEPC)-treated water, and the total RNA sample was dissolved in DEPC water. The

178 remaining amount of RNA was measured with a NanoDrop 2000 (Thermo Scientific).

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180 2.10. cDNA synthesis

181 Genomic DNA was removed from the total RNA sample using DNase I (Promega) at 37˚C
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182 on a heat block for 30 minutes (thermoBATH). Next, cDNA was synthesized from 4 mg of

183 total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and

184 Oligo(dT)18 primer, following the manufacturer’s instructions.

185

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186 2.11. Quantitative real-time RT-PCR

187 Changes in candidate mRNA levels in the zebrafish fries were identified by real-time RT

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188 PCR using SYBR Green Master mix (Applied Biosystems) with the primers listed in Table 1.

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189 Real-time PCR conditions consisted of one cycle of 5 minutes at 95 ˚C; followed by 45

190 cycles of 1) 95 ˚C for 15 seconds, 2) 60 ˚C for 15 seconds, and 3) 72 ˚C for 30 seconds; and,

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191 lastly, one cycle of 10 minutes at 72 ˚C. Each real-time PCR was carried out in triplicate in a
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192 total reaction mixture of 10 µL using a Rotor gene 6000 (Qiagen). The housekeeping gene β-

193 actin was concurrently amplified in each sample as a control and was used for normalization.
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194 Finally, real-time PCR results were calculated using the -2∆∆Ct method [15].

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196 2.12. Protein extraction

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197 Whole larvae were extracted via homogenization in 300 µl of cold extraction buffer
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198 (HEPES 20 mM, KCl 100 mM, glycerol 5%, EDTA 5 mM, MgCl2 1 mM, DTT 1 mM, Triton

199 X-100 0.1%) with protease inhibitors (Roche). Next, the samples were centrifuged at 14,000
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200 rpm at 4˚C for 15 minutes. Protein levels were measured using the NanoDrop 2000 (Thermo
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201 Scientific).

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203 2.13. Western blot

204 Total protein was loaded into the SDS-PAGE and subsequently transferred to a

205 nitrocellulose membrane (Millipore). The membrane was blocked with 5% skim milk (Bio

206 Basic Canada Ins.) in TBS-T (137 mM of sodium chloride, 20 mM Tris, 0.1% Tween® 20,
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207 supplied at pH 7.6). After the blocking step, the membrane was probed with an anti-p-MAPK

208 (Cell Signaling, 1:2000), α-tubulin (EPICMICS, 1:1000), and primary antibody. p-MAPK

209 was normalized by α-tubulin, followed by the addition of horseradish peroxidase (HRP)-

210 conjugated anti-rabbit secondary antibody (Bethyl, 1:5000). Immunoreactive proteins were

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211 visualized using a WEST-One (iNtRON) and an ImageQuant™ LAS 4000 (GE Healthcare).

212 Band intensities were determined using Quantity One software (Bio-Rad).

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213

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214 2.14. Statistical analysis

215 Statistical analysis was performed using GraphPad Prism (version 5). All data are expressed

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216 as mean ± standard error of the mean (SEM). Significance was determined using one-way
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217 ANOVA followed by Tukey’s test. The probability level for statistical significance was set at

218 p < 0.05.

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221 3. Results
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222 3.1. Optimization of alloxan for neuromast damage in zebrafish

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223 This study was designed to produce alloxan-induced neuromast damage in zebrafish. We

224 confirmed the extent of neuromast damage cause by alloxan. As shown in Fig. 1 A, alloxan
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225 concentration was validated in five-dpf zebrafish larvae. The trunk neuromasts (Fig. 1 B)
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226 were analyzed for damage by alloxan after two days and four days of exposure to various

227 doses (100 µM, 300 µM, and 500 µM). Two days and four days after alloxan treatment, the

228 trunk neuromasts decreased in a dose- and time-dependent manner (Figs. 1 C, D). Normal

229 zebrafish were observed to have 19.3 ± 1.0 neuromasts, while after two days, the 100-µM,

230 300-µM, and 500-µM alloxan-treated zebrafish had significantly fewer neuromasts: 14.7 ±

231 1.5 (p=0.0044), 13.5 ± 1.3 (p=0.0004), and 8.3 ± 1.5 (p<0.001), respectively. After four days,
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232 normal zebrafish were observed to have an average of 17.0 ± 1.0 neuromasts. The 100-µM,

233 300-µM, and 500-µM alloxan-treated zebrafish had significantly fewer neuromasts: 11.3 ±

234 2.5 (p=0.0223), 9.3 ± 2.5 (p=0.0080), and 0.5 ± 0.6 (p<0.001), respectively.

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236

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237 3.2. Evaluation of otic 1 hair cell (O1), lateral neuromast 1 (L1), lateral neuromast 4 (L4),

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238 and terminal neuromast (T) parts on alloxan-induced diabetic neuromast (AIDN) zebrafish

239 To study O1, L1, L4, and T damage in neuromasts after alloxan-induced damage in

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240 zebrafish, we observed each part after two and found days using 0.1% YO-PRO. As shown

241 (Fig. 2 A), several alloxan concentrations were applied to five-dpf zebrafish larvae. The otic 1

242
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hair cell in cranial neuromasts, lateral neuromasts 1 and 4, and terminal neuromasts in
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243 zebrafish (Fig. 2 B) were analyzed for damage after two and four days of exposure to several
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244 alloxan doses (100 µM, 300 µM, 500 µM, and 700 µM). After two and four days alloxan

245 treatment, O1, L1, L4, and T number were decreased in a dose- and time-dependent manner.
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246 The greatest damage was observed on the terminal part (Figs. 2 C-E).
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247

248 3.3. Alloxan toxicity test

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249 To evaluate alloxan toxicity, we measured heartbeat and hatching rate. Alloxan-treated
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250 zebrafish had significantly decreased heartbeat compared with normal zebrafish (Fig. 3 A).
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251 Furthermore, alloxan-treated zebrafish exhibited a dose-dependent decrease in hatching rate

252 (Fig. 3 B).

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254 3.4. Recovery of live neuromasts by RG in AIDN zebrafish

255 To evaluate the efficacy of RG, we investigated trunk neuromasts after alloxan with RG at

256 50 µg/mL, and 100 µg/mL doses. The normal group had 20.0 ± 0.6 neuromasts. The alloxan-
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257 treated group had significantly fewer neuromasts by 12.9 ± 1.8 (p<0.001). The RG-treated

258 group showed increased neuromast numbers compared with the alloxan-treated group.

259 Specially, the 50 µg/mL and 100 µg/mL RG-treated groups showed significantly higher

260 numbers by 16.0 ± 2.8 (p=0.0284) and 17.4 ± 1.0 (p<0.001), respectively (Fig. 4).

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261

262 3.5. Effect of RG on hair cell cilia

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263 To investigate the loss or recovery of cilia, we assessed the morphological differences of

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264 cilia among groups under SEM (Fig. 5). Each neuromast was composed of sensory hair cells

265 with a covered cupula. Cilia bundles were observed in the normal group, whereas cilia

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266 bundles of hair cells were destroyed in specimens treated with 300 µM alloxan. However,
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267 cilia bundles from hair cells in larvae were recovered with the RG treatment (100 µg/mL).

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269 3.6. Effect of RG on resting membrane potential of neuromast hair cells

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The resting membrane potential of L1 hair cells was measured to investigate electrical

271 activity using the physiological recording method. Resting membrane potential was defined
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272 as the membrane voltage measured by a current clamp with no applied current. The hair cells
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273 were accessible via microelectrode, and the resting membrane value was recorded easily (Fig.

274 6 A). As a result, in the normal group, the mean value of resting membrane potential was -46
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275 mV. The average resting membrane value of the alloxan-treated group was significantly
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276 higher, up to -8 mV. Treatment with RG significantly decreased the resting membrane

277 potential compared with the alloxan-treated group (Figs. 6 B, C)

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279 3.7. Effect of RG on pancreatic islet

280 Alloxan is a known diabetogenic chemical and is reported to decrease β-cell mass in the

281 pancreatic islet. To investigate the pancreatic islet recovery rate according to RG presence,
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282 we used wild type zebrafish stained with 2-NBDG dye and zebrafish from the Tg transgenic

283 line (ins:GPF; Transgenic zebrafish lines expressing GFP specifically in β-cells). The

284 pancreatic islet size of the alloxan-treated group was significantly decreased by 74.6%

285 compare to the normal group (p=0.0002). In contrast, the islet size in 100-µg/ml RG-treated

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286 group increased significantly by 40.4% compared with the alloxan-treated group (p=0.0380;

287 Figs. 7 A, B). Additionally, the alloxan-treated group was confirmed to damage β-cells

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288 compared with the normal group via the relative fluorescence intensity value. Damaged β-

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289 cells were repairable with the RG treatment (100 µg/mL) (Figs. 7 C, D).

290

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291 3.8. PEPCK, TRPV1, and TrkA mRNA levels in AIDN zebrafish
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292 Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes gluconeogenesis and is regulated

293 by glucagon and insulin [16]. The PEPCK expression level in the alloxan-treated group
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294 showed a tendency to increase when compare to the normal group, whereas the PEPCK

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mRNA level tend to decrease with RG exposure (Fig. 8 A). While there are multiple

296 peripheral and central neural mechanisms of NGF-TrkA-TRPV1, one signal is considered a
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297 key mechanism. Transient receptor potential channel vanilloid subfamily type 1 (TRPV1) is
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298 ubiquitous throughout the nervous system and is a known biomarker of acute and chronic

299 pain sensation and cochlear injury [17]. TRPV1 mRNA expression was significantly
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300 enhanced 2-fold compared with the normal group, whereas elevated TRPV1 mRNA level
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301 decreased significantly when exposed to RG (Fig. 8 B). Furthermore, tropomyosin receptor

302 kinase A (TrkA) is known as a nerve growth factor receptor [18], and it also increased

303 following RG treatment (Fig. 8 C).

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305 3.9. MAPK activation by RG in AIDN zebrafish

306 Mitogen-activated protein kinase (MAPK) is related to cell survival and differentiation, and
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307 is located downstream of the NGF pathway [19, 20]. By using Western blotting, we tried to

308 detect any MAPK changes, such as MAPK phosphorylation (p-MAPK), in the NGF pathway.

309 Western blot analysis showed that p-MAPK in larvae was reduced by alloxan, whereas p-

310 MAPK was significantly elevated in the RG-treated group (Fig. 9).

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313 4. Discussion

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315 Panax ginseng is a traditional oriental medicine that has been used for over 2000 years and

316 has various beneficial effects on the human body [21]. Moreover, RG has been reported to

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317 improve hearing loss due to diabetes complications [12]. Diabetes can cause development

318 and progression of sensorineural damage. Some animal models have been established to

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319 examine the therapeutic properties of RG for diabetic subjects, but no zebrafish model has yet

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320 been reported [12, 22]. To confirm diabetic sensorineural efficacy, we used zebrafish because

321 they offer the advantage of being able to observe neuromast changes in a live model.

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322 Therefore, we tried to identify the effect of RG using live zebrafish neuromasts.
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323 First, we established diabetic sensorineural damage in the zebrafish using alloxan, which is

324 a well known experimental diabetogenic agent that causes pancreatic β-cell necrosis leading
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325 to a decreased β-cell mass and consecuently an inhibition of insulin secretion. Moreover, we

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previously reported alloxan-induced diabetic zebrafish [28, 29]. To establish diabetic

327 sensorineural damage in zebrafish, we confirmed neuromast presence in zebrafish using YO-
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328 PRO, which is a fluorescent dye for staining hair cells [23]. Neuromast hair cell death can be
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329 easily evaluated by measuring fluorescence loss [24]. The neuromast is a mechanosensory

330 organ, which comprises cranial and trunk neuromasts. Cranial neuromasts are called the
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331 anterior lateral line and are present on the head. Trunk neuromasts are called the posterior
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332 lateral line and include the neuromasts on the trunk and tail [25, 26]. Specifically, we focused

333 on trunk neuromast observation using 0.1% YO-PRO. We determined the optimal alloxan

334 concentration and exposure duration for causing trunk neuromast damage. Trunk neuromasts

335 were observed after exposure to 100 µM, 300µM, and 500 µM of alloxan for two and four

336 days. According to our results, alloxan caused hair cell loss in zebrafish neuromasts in a dose-

337 and time-dependent manner. Additionally, the optimal dosage and timing of alloxan exposure
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338 for damaged neuromasts in the zebrafish model were 300 µM and 72 hours, respectively. We

339 confirmed that sensitivity of hair cell loss depends on localization of the neuromast. When

340 zebrafish were exposed to alloxan at higher concentrations, the average number of otic hair

341 cells on the head, known as the anterior lateral line, slightly decreased but not significantly so.

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342 In contrast, the neuromasts of the posterior lateral line were more sensitive, significantly

343 decreasing in number.

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344 To determine whether alloxan-induced neuromast damage was reduced by RG, zebrafish

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345 larvae were treated with RG after alloxan exposure. Our data showed that RG enhanced trunk

346 neuromast recovery after alloxan-induced neuromast damage. Additionally, cilia of

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347 neuromast hair cells were severely damaged by alloxan; however, RG stimulated hair cell
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348 cilia regeneration, indicating that RG protects the mechanosensitive function of cilia.

349 We confirmed morphological changes in the hair cells of zebrafish and performed
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350 physiological recording to assess functional changes. Alloxan-treated hair cells had a more

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positive resting membrane potential than normal hair cells. Abnormal movement of ions

352 across the membrane caused by alloxan would result in a charge imbalance across the
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353 membrane. On the other hand, the resting membrane potential significantly decreased in the
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354 RG-treated group compared to the alloxan-exposed group. We predict that RG protects hair

355 cell function against induced hair cell damage by AIDN. However, the precise mechanism of
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356 RG’s effect on the resting membrane potential is still not known.
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357 In AIDN zebrafish, pancreatic islet size decreased and recovery was present after treatment

358 with RG. Furthermore, RG stimulated recovery of damaged β-cells and tend to reduce

359 PEPCK expression. Is well known that chronic hyperglycemia which occurs in diabetes,

360 causes an increase of PEPCK expression [30], therefore, any agent that diminishes its

361 expression is expected to improve sensorineural damage.

362 TRPV1 is regulated by nerve growth factor and causes a sensation of scalding heat and pain
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363 in the peripheral nervous system [31]. TRPV1 expression increased in the AIDN zebrafish

364 but decreased after RG treatment. Also, trkA mRNA level indicated that RG has a nerve-

365 protection effect. The pattern of trkA expression showed that RG has a potential neuron-

366 recovery effect and can help repair diabetic sensorineural damage. NGF stimulates MAPK

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367 activation, and our data indicated that MAPK was activated after RG treatment.

368 In conclusion, we found that RG stimulates neuromast hair cell recovery following damage

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369 caused by AIDN. We suggest that the mechanism might related to the promotion of the NGF

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370 pathway through increased expression of TRPV1, trkA, and p-MAPK. Furthermore, RG can

371 improve diabetes symptoms by protecting pancreatic islets and tend to lower PEPCK level.

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372 These results suggest that it is possible to treat both diabetes and diabetic sensorineural
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373 damage with RG.

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375

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Acknowledgements

377 This research was supported by Basic Science Research program through the National
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378 Research Foundation of Korea (NRF) funded by the ministry of Education, Science and
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379 Technology (NRF-2015R1D1A1A09060469, NRF-2016K1A1A8A01938680).

380
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381 Abbreviation list

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382 RG Red ginseng

383 AIDN Allan-induced diabetic neuromast

384 SEM Scanning electron microscopy

385 NGF Nerve growth factor

386 TrkA Topomyosin receptor kinase A

387 MAPK Mitogen-activated protein kinase

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388 dpf Days post-fertilization

389 hpf Hours post-fertilization

390 O1 Otic 1 hair cell

391 L1 Lateral neuromast 1

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392 L4 Lateral neuromast 4

393 T Terminal neuromast

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394 PEPCK Phosphoenolpyruvate carboxykinase

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395 TRPV1 Transient receptor potential channel vanilloid subfamily type 1

396

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397 References
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476 human peripheral nervous system and in painful neuropathies. Journal of the Peripheral

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478 [32] Apfel, S. C., 2002. Nerve growth factor for the treatment of diabetic neuropathy: what
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479 went wrong, what went right, and what does the future hold?. International review of
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480 neurobiology, 50, 393-413.

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482 Experimental Diabetes Research, 4(4), 271-285.

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483 [34] Hirose, M., Kuroda, Y., & Murata, E., 2016. NGF/TrkA signaling as a therapeutic target

484 for pain. Pain Practice, 16(2), 175-182.

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486 pathobiology. Molecules, 20(6), 10657-10688.

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489 Table

490 Table 1. Primer sequences for real-time RT-PCR

Gene bank

Gene Strand Sequence (5’-3’) accession

number

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TrkA Forward ACT CCA AGT TTG GCA TCC AC

(Tyrosine kinase JN_837101.1

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receptor) Reverse GGG TTC TCC ACA AAA CTG GA

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PEPCK Forward GAA CCT CCA GCA AAA CCA CA
(Phosphoenolpyruvate NM_214751.1

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carboxykinase) Reverse GTC AGC GTC ACT CCT TCA G
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Trpv1
Forward TCC AAC CCT CAA AGT CGT ATG
(transient receptor
XM_005165327.2
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potential cation channel,

Reverse TCA ATC CAA ATC GTC CCC TG
subfamily V, member 1)
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Forward CAAGCAGGAGTACGATGAGTC
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β-Actin NM_181601.4
Reverse GTC GTT TGA AGT TTC TCT GCG
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494 Figure

495

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496

497 Figure 1. (A) Scheme of experiment; (B) Lateral line for zebrafish; (C) Number of trunk neuromasts of alloxan

498 dose-dependently damaged zebrafish; (D) Fluorescent microscopic image of trunk neuromast following

499 treatment with 0.1 YO-PRO (*p < 0.05, **p < 0.01, ***p < 0.001; compared to normal fish)

500
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501
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503 Figure 2. (A) Scheme of experiment; (B) The otic 1 hair cell in cranial neuromasts, lateral neuromasts 1 and 4,

504 and terminal neuromasts in zebrafish; (C) The survival rate of each hair cell of alloxan dose-dependently

505 damaged zebrafish following two days of treatment; (D) The survival rate of each hair cell type in alloxan dose-

506 dependently damaged zebrafish following four days of treatment; (E) Fluorescent microscopic image of each

507 hair cell type following 0.1% YO-PRO treatment.

508
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509

510

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Figure 3. Toxicity of alloxan in zebrafish embryos; (A) Heartbeat per minute in zebrafish exposed to alloxan for

511 48 hours; (B) Hatching rate in zebrafish exposed to alloxan for 48 hours (*p < 0.05, ***p < 0.001; compared to

512 normal)

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514

515 Figure 4. (A) Scheme of experiment; (B) The number of trunk neuromasts in RG-treated AIDN zebrafish; (C)

516 Fluorescent microscopic image of trunk neuromasts following treatment with 0.1% YO-PRO (###p < 0.001;

517 compared to normal), (*p < 0.05, **p < 0.01; compared to alloxan)

518

519
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520

521 Figure 5. Scanning electron microscopy (SEM) images of representative neuromasts in zebrafish larva. Scale

522 bar = 3 µm

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524

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Figure 6. Measure of resting membrane potential on an L1 neuromast; (A) Lateral mounting of zebrafish shows
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526 the location of the microelectrode in the L1 neuromast; (B) Value of resting membrane potential; (C) Image of

527 an L1 neuromast (###p < 0.001; compared to normal, *p < 0.05; compared to alloxan treatment)
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529

530 Figure 7. Effect of RG on pancreatic islet; (A) Pancreatic islet size by RG; (B) Pancreatic islet image of each
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531 group; (C) Relative fluorescence intensity of pancreatic β-cells by RG; (D) Pancreatic β-cell image of each

532 group (###p < 0.001; compared to normal, *p < 0.05; compared to alloxan treatment, *p < 0.05, **p < 0.01)
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535 Figure 8. Expression of PEPCK, TRPV1, and TrkA in AIDN zebrafish (##p < 0.01; compared to normal, **p <

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536 0.01, **p < 0.001; compared to alloxan treatment)

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538

539 Figure 9. Expression of p-MAPK in AIDN zebrafish; (A) Normalization of p-MAPK level in zebrafish; (B)
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540 Western blot assay of p-MAPK and α-tubulin levels in zebrafish (###p<0.001 compared to normal, ***p<0.001

541 compared to alloxan treatment)

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