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2021년 8월 석 사 학 위 논 문

Activation of Autophagy by

Mangiferin Protects Auditory Hair Cells from Oxidative stress Induced

Ototoxicity

조선대학교 대학원

글로벌바이오융합학과

임 경 민

[UCI]I804:24011-200000490139

[UCI]I804:24011-200000490139

(3)

Activation of Autophagy by

Mangiferin Protects Auditory Hair Cells from Oxidative stress Induced

Ototoxicity

망기페린의 자가포식 활성화를 통해 산화 스트레스로 유도된 청각 유모세포 손상에 대한 보호 효과

2021 년 8 월 27 일

조선대학교 대학원

글로벌바이오융합학과

임 경 민

(4)

Activation of Autophagy by

Mangiferin Protects Auditory Hair Cells from Oxidative stress Induced

Ototoxicity

지도교수 조광원

이 논문을 이학석사학위 신청 논문으로 제출함

2021년 4월

조선대학교 대학원

글로벌바이오융합학과

임 경 민

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임경민의 석사학위논문을 인준함

위원장 조선대학교 교 수 전 택 중 (인)

위 원 조선대학교 교 수 이 준 식 (인)

위 원 조선대학교 교 수 조 광 원 (인)

2021년 5월

조선대학교 대학원

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i

CONTENTS

LIST OF TABLES………...……….

LIST OF FIGURES……….……….

ABBREVIATIONS……….…..………

ABSTRACT………..………

국문초록 ..………..………..…

I. INTRODUCTION………...……….…..…

II. MATERIALS AND METHODS……..……..…

II-1. Cell culture..….……..………...………...….……….…

II-2. Cell viability assay.………..……...…………...……

II-3. Intracellular ROS detection.….……….….

II-4. Autophagy activity analysis………….………...

II-5. Immunoblot analysis……..……….…..……..…..

II-6. Statistical analysis………..……….…..……..…..

III. RESULTS………...………

iii iv

V 1

3

5 9

9 9 10 10 10 11

13

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ii

III-1. Estimating the concentration of MAG and H

2

O

2

to be used on HEI-OC1 cells……..……….………

III-2. MAG protects HEI-OC1 Cells from H

2

O

2

-induced oxidative stress………...

III-3. MAG treatment reduces intracellular ROS levels in HEI-OC1 cells………

III-4. MAG activates autophagy in HEI-OC1 cells………...…

III-5. MAG enhances autophagy activation by AMPK signaling pathway in HEI-OC1 cells.………....……...

III-6. MAG restores autophagy flux in HEI-OC1 cells……….

IV. DISCUSSION………

V. REFERENCES………

VI. Acknowledgements………...………

13

16

19 22

24 27

30

34

39

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iii

LIST OF TABLES

Table 1. Primary antibodies used in immunoblot analysis…………... 12

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iv

LIST OF FIGURES

Fig 1. Hair cell damage pathway……….

Fig 2. Determination of concentration of MAG and H2O2 on HEI-OC1 cells………...

Fig 3. Cell protective effects of MAG in H2O2-treated HEI-OC1 cells.

Fig 4. MAG treatment reduces intracellular ROS levels in HEI-OC1 cells……….

Fig 5. Autophagy activity of MAG in HEI-OC1 Cells...

Fig 6. MAG enhances AMPK activation………...

Fig 7. MAG repairs autophagic flux damaged by oxidative stress…………

Fig 8. Schematic model of the pathway by which MAG protects hair cells from oxidative stress...

8

14 17

20 23 25 28

33

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v

ABBREVIATIONS

AMPK AMP-activated protein kinase DCF 2’-7’dichlorofluorescin

DCF-DA 2’-7’dichlorofluorescin diacetate DMEM Dulbecco’s modified eagle’s medium ECL Enhanced chemiluminescence

EDTA Ethylenediaminetetraacetic acid FBS Fetal Bovine Serum

GAPDH Glyceraldehyde 3-phosphate dehydrogenase H2O2 Hydrogen peroxide

LC3 Microtubule-associated protein light chain 3 MAG Mangiferin

mTOR Mammalian target of rapamycin PBS Phosphate-buggered saline

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vi

PMSF Phenylmethylsulfonyl fluoride RIPA Radioimmunoprecipitation assay ROS Reactive oxygen species

SDS Sodium Dodecyl Sulfate SOD1 Superoxide dismutase 1 SQSTM1 Sequestosome-1

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ABSTRACT

Activation of Autophagy by Mangiferin Protects Auditory Hair Cells from Oxidative stress Induced Ototoxicity

Gyeongmin Lim Advisor: Prof. Gwang-Won Cho, Ph.D.

Department of Integrative Biological Sciences, Graduate School of Chosun University

Oxidative stress is a major causes of auditory hair cells degeneration. Once the auditory hair cells are degenerated, it will lead to hearing loss. Eliminating the intracellular reactive oxygen species (ROS) may preserve the auditory hair cell’s function by reducing the damages of various macromolecules like DNA, RNA, and protein. This could be achieved by the activation of autophagy mechanism. Autophagy is an essential and highly conserved pathway that plays an important role in the maintenance of cellular function and viability upon stress. Recently, many studies were conducted, with an aim reduce oxidative stress caused by ototoxicity using Autophagy. Mangiferin (MAG) is a xanthonoid isolated from mango leaves, bark, and fruit. It has shown pharmacological effects against a variety of diseases in various preclinical studies. In this study, I have investigated the effect of MAG on oxidative stress in auditory hair cells and described its molecular mechanisms.

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Through experiments, I have found that MAG protects HEI-OC1 cells against hydrogen peroxide (H2O2) induced oxidative stress. The protective effect was confirmed by immunoblot analysis for cleaved caspase-3 apoptosis-related protein. Pre-treating the cells with MAG reduced cleaved caspase-3 expression. I also performed DCFH-DA assay to confirm ROS activity. The results clearly showed the reduction of ROS in the MAG treated cells. Autophagy was visualized by CYTO-ID staining and the activity of autophagy was determined. MAG enhanced Autophagy by activating the AMP-activated protein kinase (AMPK) signaling pathway. The activity of autophagy was confirmed by immunoblot analysis for autophagy flux-related proteins like Microtubule-associated protein light chain 3 (LC3) conversion and Sequestosome-1 (SQSTM1) degeneration.

In conclusion, MAG have a potential antioxidant effect resulting in the protection of HEI-OC1 cells from oxidative stress. Moreover, I have observed that pre-treatment of MAG activates autophagy and restores autophagy flux. Therefore, I suggested that MAG could be effective against damage caused by oxidative stress.

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국문초록

망기페린의 자가포식 활성화를 통해 산화 스트레스로 유도된 청각 유모세포 손상에 대한 보호 효과

임 경 민

지도교수

: 조 광 원

글로벌바이오융합학과 조선대학교 대학원

산화 스트레스는 청각 유모세포 퇴화의 주요 원인이다. 청각 유모세포가 퇴 화되면 청력 손실로 이어진다. 세포 내 활성 산소종 (ROS)을 제거하면 DNA, RNA 및 단백질과 같은 다양한 거대 분자의 손상을 줄여 청각 유모세포의 기능을 보존할 수 있다. 이것은 자가포식 메커니즘의 활성화에 의해 달성 될 수 있다. 자가포식은 스트레스에 대한 세포 기능 및 생존력 유지에 중요한 역할을 하는 필수적이고 진화적으로 잘 보존된 경로이다. 최근 자가포식을 이용하여 이독성으로 인한 산화 스트레스를 줄이기 위한 많은 연구가 보고 되었다. 망기페린(MAG)은 망고나무의 잎, 나무껍질 및 과일에서 분리된 크 산토노이드로, 전임상 연구에서 여러 질병에 대한 약리학적 효과가 알려졌다.

이 연구에서 청각 유모세포의 산화 스트레스에 대한 MAG의 효과를 분석하 고 분자 메커니즘을 조사하였다.

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실험을 통해 MAG이 과산화수소(H2O2)로 유도된 산화 스트레스로부터 HEI-

OC1 세포를 보호한다는 것을 발견했다. 보호 효과는 세포 사멸 관련 단백질 인 절단된 카스파제-3에 대한 면역 블롯 분석을 통해 확인되었으며, MAG로 세포를 전처리하면 절단된 카스파제-3 발현이 감소했다. 그런 다음 DCFH- DA 분석법을 이용하여 ROS 활성을 확인하였다. 결과는 MAG이 처리된 세 포에서 ROS가 감소하는 것으로 나타났다. 자가포식은 Cyto-ID 염색으로 시 각화 되었으며 자가포식의 활성을 확인하였다. MAG는 AMP 활성 단백질 키 나아제 (AMPK) 신호 전달 경로를 활성화하여 자가포식을 강화하였다. 자가

포식의 활성은 미세소관 연관 단백질 경연쇄 3 (LC3) 전환 및

Sequestosome-1 (SQSTM1) 퇴화와 같은 자가포식 흐름 관련 단백질에 대 한 면역 블롯 분석으로 확인하였다.

결과적으로, MAG는 산화 스트레스로부터 HEI-OC1 세포를 보호하는 잠재적 인 항산화 효과를 가지고 있다. 또한, MAG의 전처리가 자가포식을 활성화하 고 자가포식 흐름을 회복시키는 것을 관찰했다. 따라서 본 연구를 통해 MAG이 산화 스트레스로 인한 손상에 효과적일 것이라고 제안한다.

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I. INTRODUCTION

Hearing loss is one of the most common sensory disorders affecting about 6.8% of the world's population and is a serious problem worldwide (Wilson et al., 2017). This hearing loss is mainly due to hair cell damage or inner ear disorders (Smouha, 2013). The goal of treating inner ear disorders is to find a cure for hearing loss caused by loss of cochlear hair cells or degeneration of spiral ganglion neurons. In these regenerative medicines, mesenchymal stem cells are considered as a good treatment option due to their important properties such as multipotency, self-renewal, immunomodulatory function, paracrine action, and wound healing (Dufner-Almeida et al., 2019). Mesenchymal stem cells can be a therapeutic tool to regenerate middle and inner ear (Maharajan et al., 2020, 2021), however it is also important to reduce damages in the cochlea hair cells along with recovering the damaged cochlea hair cells.

Hair cell damage is caused by ototoxic drug, noise, age, etc., and high levels of reactive oxygen species ROS produced by oxidative stress damage is an important mechanism of cochlear hair cell damage (Fig. 1) (Chen et al., 2014; Nelson et al., 2005;

Schacht et al., 2012). ROS induces peroxidation of polyunsaturated fatty acids, DNA degradation and protein damage, which can lead to cell dysfunction or death (Davies, 1987; Imlay et al., 1988). Unfortunately, hair cell loss is permanent in adult mammals (Bermingham-McDonogh et al., 2003). Therefore, the Autophagy activity of hair cells can be a useful strategy for preventing hearing loss (Pang et al., 2019; Ye et al., 2018).

Autophagy is an essential, homeostatic process by which cells break down unnecessary or dysfunctional cellular components (Parzych et al., 2014). In particular, it plays a function, as a cell survival pathway that generates energy-utilizing amino acids by decomposing long-lived and misfolded proteins and damaged organelles, mainly under stress conditions such as starvation and hypoxia (Esclatine et al., 2009;

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Vijayakumar et al., 2019). To understand the activity of autophagy, the concept of autophagy flux was recently introduced (Loos et al., 2014). Autophagy flux represents the overall process of autophagy from the formation of autophagosomes, recruitment of cargo and to the degradation by lysosomes. Normally, autophagy begins through membrane nucleation, which results in phagophore formation. AMP-activated protein kinase (AMPK) has been reported to modulate autophagy initiation (Tong et al., 2020).

AMPK is known to be involved in the stimulation of autophagy through mammalian target of rapamycin (mTOR) inactivation (Yan et al., 2017). In the process of autophagy, pro-LC3 is converted to LC3-I, which binds to phophatidylethanolamine and form LC3- II, which ultimately stimulates autophagosome formation (Rahman et al., 2020). LC3-II is present in both the inner and outer isolation membranes and acts as a recognition site for LC3 binding chaperones such as p62/SQSTM1 that transfers cargo to the autophagosome (Komatsu et al., 2010). After that, Autophagosomes travel along the microtubules to reach the lysosome and fuse to form autolysosomes, where the contents are broken down by lysosomal acid hydrolase. Regardless, excessive or damaged autophagic flux can contribute to cell death via "type II programmed cell death"

(Yorimitsu et al., 2005). Autophagy has proven to be a protective factor for hair cell survival in various types of hearing loss (He et al., 2017). Therefore, it indicates that autophagy plays an important role in regulating long-term physiological processes.

Mangiferin (MAG) is a natural polyphenol with a C-glycosylxanthone structure that is present at considerable levels in higher plants and other parts of mango fruit such as bark, stems, leaves, bark, and kernels (Matkowski et al., 2013). There are many phenolic hydroxyl groups in MAG that are essential for removing reactive oxygen species. In recent years, it has gained special attention due to its antiviral, anticancer, antioxidant, antidiabetic, anti-aging, hepatoprotective and analgesic effects (Dar et al., 2005; Guha et

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al., 1996; Miura et al., 2001). In addition, MAG regulates AMPK activity, which is involved in improving metabolic disorders (Niu et al., 2012).

I treated H2O2by assuming that the existing ototoxicity was due to oxidative stress.

Based on the results of various studies, I have hypothesized that MAG would restore autophagy flux in HEI-OC1 cells damaged by H2O2. I also hypothesized that Autophagy would be an important regulator of ototoxicity that induces oxidative stress. The purpose of this study is to develop a substance that can prevent hearing loss due to oxidative stress by restoring autophagy flux through in vitro studies.

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Fig. 1. Hair cell damage pathway.

Factors such as noise exposure, aging, and ototoxic drugs cause an imbalance in the cochlea's antioxidant defense system. ROS produced by imbalance causes hair cell degeneration and as a result leads to hearing loss.

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II. MATERIALS AND METHODS

II-1. Cell culture

HEI-OC1 cells, an auditory cell line derived from the auditory organ of a transgenic mouse, which possess hair cell-like properties, endogenously express prestin, the paradigmatic motor protein of outer hair cells. Cells were cultured in high-glucose DMEM (Gibco BRL, NY, USA) supplemented with 10% FBS (Thermo Fisher Scientific, ON, Canada) at 33°C and 5% CO2in a humidified atmosphere without antibiotics. SH- SY5Y cells were cultured in DMEM/F12 growth media supplemented with 10% FBS and 1% Penicillin-Streptomycin (Invitrogen, Massachusetts, USA) at 37°C and 5% CO2in a humidified atmosphere.

II-2. Cell viability assay

Cell viability was assessed by MTT assay (Sigma, MO, USA). The HEI-OC1 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. The next day, the cells were incubated with 0–10 μg/ml of MAG (Sigma-Aldrich, MO, USA) for 12 h and then treated with hydrogen peroxide (H2O2; 0–3 mM) for 1 h, and then analyzed by the MTT assay. The SH-SY5Y cells were seeded in 96-well plates at a density of 5 × 104 cells/well.

For neuronal differentiation, the growth medium was replaced with differentiation medium, which contained 0.1% FBS, 1% Penicillin-Streptomycin, and 1 μM retinoic acid (Sigma, MO, USA), and the cells were then further incubated for 2 days. After neuronal differentiation, SH-SY5Y cells were incubated with 0-10 μg/ml of MAG for 12 h and then cell viability was analyzed by the MTT assay.

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10

II-3. Intracellular ROS detection

Intracellular ROS levels were evaluated using the cell-permeant substrate 2′,7′- dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich, MO, USA), which converts to the detectable fluorescent product 2′,7′-dichlorodihydrofluorescein (DCF) in cells. Cells were seeded in both 24-well plates (4 × 104cells/well) and 96-well plates (5 × 103cells/well) and incubated at 33°C for 24 h. Cells (80% confluence) were then treated with MAG for 12 h at 33°C and incubated with 20 μM DCFH-DA at 33°C for 30min. After washing with PBS, the cells were incubated with 1mM H2O2for 1 h at 33°C.

Levels of intracellular ROS were observed under a Nikon Eclipse Ti2 fluorescence microscope (Nikon, Tokyo, Japan) and captured by a DS-Ri2 digital camera (Nikon, Japan).

II-4. Autophagy activity analysis

Autophagosomes were measured by CYTO-ID® autophagy detection kit (Enzo Life Sciences, NY, USA). HEI-OC1 cells were seeded in 24-well plate (4 × 104cells/well) for 24 h and then 2 μg/ml MAG 2 or 500 nM Rapamycin was treated with 30 μM Chloroquine for 12 h. After treatment, the medium was removed and the Cyto-ID assay was proceeded according to the manufacturer’s instruction. Cells were visualized by fluorescence microscopy with a Nikon Eclipse Ti2 fluorescence microscope (Nikon, Tokyo, Japan) and captured by a DS-Ri2 digital camera (Nikon, Japan).

II-5. Immunoblot analysis

Total proteins were extracted from HEI-OC1 cells with 50 μl of RIPA buffer (Santa Cruz Biotechnology, TX, USA) containing PMSF (Enzo Life Sciences, NY, USA), inhibitor cocktail (Thermo Fisher Scientific, MA, U.S.A) for 30 min on ice and then

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11

centrifuged at 16,000 rpm for 20 min. Protein concentration were measured by BSA assay kit (Thermo Fisher Scientific, MA, U.S.A). Protein samples were then analyzed by Immunoblot analysis with specific antibodies for AMPK, p-AMPK (Cell Signaling Technology, MA, USA), SOD1, LC3, SQSTM1, GAPDH (Santa Cruz Biotechnology, CA, USA) and cleaved caspase-3 (Merck Millipore, CA, USA) for 16 h at 4°C. The appropriated horse radish peroxidase-conjugated anti-goat, anti-mouse and anti-rabbit were used as secondary antibodies (Santa Cruz Biotechnology, CA, USA). Band detection were used for enhanced chemiluminescence (ECL) (GE Healthcare, Buckinghamshire, UK) detection system and exposed to X-ray films.

II-6. Statistical analysis

All data are presented as mean ± standard deviation (SD) of the indicated number of experiments and significance was estimated using a Student’s t-test. Statistical comparisons between groups were analyzed using an independent t-test. p-value of < 0.05 was considered statistically significant.

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12

Table 1. Primary antibodies used in immunoblot analysis

1stantibody 2ndantibody Titer Company (Cat.NO.)

Cleaved caspase-3 Rabbit 1:200 Merck (AB3623)

SOD1 Goat 1:500 Santa Cruz (sc-8637)

AMPK Rabbit 1:500 Cell Signaling (5832)

p-AMPK Rabbit 1:500 Cell Signaing (2535)

LC3 Mouse 1:500 Santa Cruz (sc-398822)

SQSTM1 Mouse 1:500 Santa Cruz (sc-48402)

GAPDH Goat 1:1000 Santa Cruz (sc-48167)

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13

III. RESULTS

III-1. Estimating the concentration of MAG and H

2

O

2

to be used on HEI-OC1 cells.

To determine the effective concentration of H2O2in HEI-OC1 cells, H2O2was treated for 1 h, and cell viability was measured by MTT analysis (Fig. 2A). 1mM concentration of H2O2has been considered as appropriate for the study. Since hair cell is connected to spiral ganglion, cytotoxicity experiments were conducted on neuronally differentiated SH-SY5Y and HEI-OC1 cells. The cytotoxicity of MAG was evaluated by treating MAG in a dose dependent manner. The cell viability was examined by MTT analysis. No toxicity was detected at all concentrations of MAG (0-10 μg/ml) (Fig. 2B).

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14

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15

Fig. 2. Determination of concentration of MAG and H2O2on HEI-OC1 cells.

HEI-OC1 cells were treated with 0-3 mM H2O2for 1 h and cell viability was measured by MTT assay (A; t-test, * p < 0.05, mean ± SD, n = 4). HEI-OC1 cells and neuronally differentiated SH-SY5Y cells were treated with 0-10 μg/ml MAG for 12 h and evaluated by an MTT assay (B; mean ± SD, n = 3).

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16

III-2. MAG protects HEI-OC1 cells from H

2

O

2

-induced oxidative stress.

To evaluate the antioxidant effect of MAG, cells were incubated with MAG (0-10 μg / ml) for 12 h and then exposed to 1 mM H2O2 for 1 h. Viability was significantly increased in HEI-OC1 treated with 2 μg/ml MAG compared to untreated HEI-OC1 (Fig.

3A). To confirm the effect at the molecular level, apoptosis-related proteins were measured by immunoblot analysis. The expression of Cleaved caspase-3 was decreased in HEI-OC1 treated with 2 μg/ml MAG compared to untreated HEI-OC1 (Fig. 3B and 3C), suggests that cell damage due to oxidative stress was reduced by treating MAG.

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17 Fig. 3.(continued)

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Fig. 3. Cell protective effects of MAG in H2O2-treated HEI-OC1 cells.

HEI-OC1 cells were treated with 1 mM H2O2 following pre-incubation with 0-10 μg/ml MAG for 12 h and cell viability was measured by MTT assay (A ; t-test, * p<0.05, mean ± SD, n=3). 2 μg/ml MAG treated HEI-OC1 cells were incubated with 1 mM H2O2 for 1 h. Total protein was examined by immunoblot analysis with antibodies against the apoptotic protein cleaved caspase-3 and GAPDH (B). The protein expressions were quantified using the Image J software. GAPDH was used as the internal standard (C; t- test, * p<0.05, mean ± SD, n=3).

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III-3. MAG treatment reduces intracellular ROS levels in HEI-OC1 cells.

Excessive levels of ROS cause oxidative stress and cellular damage. In the H2O2

treated HEI-OC1, intracellular ROS level was investigated because MAG has a protective effect. To measure the cellular ROS level, 1 mM H2O2was treated for 1 h. ROS levels were significantly decreased in HEI-OC1 pre-treated with 2 μg/ml MAG (Fig. 4A and 4B). Antioxidant enzyme SOD1 were measured by immunoblot analysis. The expression of SOD1 was increased in HEI-OC1 treated with 2 μg/ml MAG compared to untreated HEI-OC1 (Fig. 4C). This result suggests that MAG protects hair cells by removing ROS under oxidative stress.

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20 Fig. 4. (continued)

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Fig. 4. MAG treatment reduces intracellular ROS levels in HEI-OC1 cells.

Intracellular ROS were stained with DCFH-DA and labeled with green fluorescent. The ROS levels were observed with a fluorescence microscope. The ROS level was increased in H2O2-treated HEI-OC1 cells, and significantly decreased in HEI-OC1 cells pre-treated with MAG (A). Fluorescence levels were quantified with a fluorescence ELISA plate reader (B; t-test, * p<0.05, mean ± SD, n=3). The expression of antioxidant enzyme SOD1 was examined by immunoblot analysis. GAPDH was used as the internal standard (C).

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III-4. MAG activates autophagy in HEI-OC1 Cells.

To determine whether MAG could influence autophagy, I checked autophagy activity.

Cyto-ID assay was used to measure the autophagy activity of MAG. Upon observation 2 μg/ml MAG and positive control 500 nM rapamycin treated cells showed the activity of autophagy (Fig. 5A ~ 5C). When compared with the positive control, the autophagy activity of MAG was confirmed. This results suggest that MAG can eliminate intracellular ROS by activating autophagy.

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Fig. 5. Autophagy activity of MAG in HEI-OC1 cells.

Autophagosome were stained with CYTO-ID and the nucleus were stained with hoechst 33342 (blue), and visualized under fluorescence microscopy. Control was treated with only 30 μM Chloroquine in HEI-OC1 cells (A). 2 μg/ml MAG or 500 nM Rapamycin were treated along with 30 μM Chloroquine for 12 h in HEI-OC1 cells (B and C).

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III-5. MAG enhances autophagy activation by AMPK signaling pathway in HEI-OC1 cells.

As I observed the Autophagy activity of MAG, I also wanted to confirm the level of p-AMPK through the immunoblot analysis. AMPK works as an important regulator of autophagy. In the H2O2treated group, the level of AMPK was significantly increased than the control group (Fig. 6A and 6B). However, when compared with the MAG treated cells, p-AMPK expression was significantly reduced in the H2O2treated cells. This result shows that the activity of autophagy became more active upon the treatment of MAG.

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26 Fig. 6. MAG enhances AMPK activation.

The proteins were targeted with relative antibodies specific for p-AMPK and AMPK.

GAPDH was used as loading control. The expression of p-AMPK was examined by immunoblot analysis. p-AMPK expression levels were quantified by using ImageJ software (A and B; t-test, * p<0.05, mean ± SD, n=3). The expression of p-AMPK was normalized with AMPK.

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III-6. MAG restores autophagy flux in HEI-OC1 cells.

I conducted the later experiments in terms of autophagy flux. Formation of LC3-II and the degradation of SQSTM1 were checked by immunoblot Analysis (Fig. 7A to 7C).

Expression of LC3-II was increased in the group treated with H2O2 and MAG but SQSTM1 level was increased only in the group treated with H2O2. This result shows the failure of autophagy flux in H2O2group. In contrast, SQSTM1 was decreased in the group treated with MAG proving the degradation has happened and the entire autophagy flux mechanism was completed. The result suggests that MAG activates autophagy through AMPK pathway and also aid in the completion of autophagy flux. Thus, MAG protects the HEI-OC1 cells from oxidative stress.

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Fig. 7. MAG repairs autophagic flux damaged by oxidative stress.

The expression of autophagy related proteins LC3 and SQSTM1 was examined by immunoblot analysis. Autophagy related protein expression levels were quantified using ImageJ software. In the group pre-treated with MAG, SQSTM1 was observed to be decomposed, indicating the recovery of Autophagy flux. (A~C; t-test, * p<0.05, mean ± SD, n=3). The expression level of proteins were normalized with GAPDH.

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IV. DISCUSSION

Recent research on the role of autophagy mechanisms in auditory cells has focused primarily on using the antioxidant capability of autophagy to protect hair cells and hearing (Fang et al., 2014; He et al., 2017; Tsuchihashi et al., 2015; Yuan et al., 2015). Oxidative stress can also be observed in hearing loss caused by many endogenous factors such as aging (Fujimoto et al., 2014), ischemia (Yang et al., 2017), exogenous factors such as ototoxic drugs (Jiang et al., 2016) and noise exposure (Umugire et al., 2019). Oxidative stress produces intracellular ROS and it leads to hair cell degeneration and hearing loss (Fig. 1). Antioxidants have been applied for the Sensorineural hearing loss treatment and have been proven to have therapeutic potential (Kil et al., 2017; Lu et al., 2011; Yuan et al., 2015). Based on these evidences, the experiment was conducted under the assumption that the main factor which damages hair cells is excessive ROS formation by oxidative stress. Here, I have shown that MAG, a natural compound extracted from Mangifera indica, can act as a drug for hearing loss.

In this study, in order to determine the antioxidant and autophagy activation properties of MAG, HEI-OC1 cells were treated with MAG and protective effect was evaluated against oxidative stress via H2O2. MAG did not show cytotoxicity in HEI-OC1 cells and neuronally differentiated SH-SY5Y cells (Fig. 2). Therefore, it provides a reason for use in animal testing. MAG treatment enhanced protection against oxidative stress in HEI- OC1 cells. Apoptosis-related protein cleaved-caspase3 expression was increased during H2O2treatment and this was decreased when HEI-OC1 cells were pre-treated with MAG (Fig. 3). As a result, MAG shows better protection against apoptosis.

Cells exposed to excessive oxidative stress accumulate intracellular ROS due to an imbalance in the antioxidant system. I confirmed that HEI-OC1 pre-treated with MAG protects against oxidative stress, therefore I measured ROS levels. As expected, I

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observed a significant decrease in ROS levels in HEI-OC1 pre-treated with MAG. AS a result of checking whether the reduced ROS level is due to production of antioxidant enzyme SOD1. It was confirmed that the expression of SOD1 was increased (Fig. 4).

Autophagy is an endogenous process that removes damaged organelles and maintains essential cellular homeostasis (Galluzzi et al., 2019). This autophagy mechanism not only removes damaged and oxidized proteins, also removes damaged mitochondria. Thus, autophagy can protect cells by breaking down proteins and ROS that have already been oxidized. Therefore, autophagy is an ideal antioxidant tool that directly eliminates damaged mitochondria which continuously release ROS (Giordano et al., 2014). Also, the activity of autophagy is important because if auditory cells are damaged, they do not regenerated in adult mammals. To prove that this significant reduction in oxidative stress and ROS is related to autophagy, the autophagy activity of MAG was measured. It was confirmed that MAG pretreatment increased Autophagy when compared to the positive control rapamycin (Fig. 5).

The AMPK signaling pathway is an important autophagy regulator, and AMPK triggers autophagy by inhibiting mTOR and directly promoting p-ULK1. I investigated the level of p-AMPK under oxidative stress and observed that p-AMPK significantly increased in MAG pre-treated HEI-OC1 (Fig. 6). In order to prove that the protective effect is due to the autophagy activity, the damage to the autophagy flux have been checked by treating H2O2 in HEI-OC1 pre-treated with MAG. It is known that H2O2

inhibits autophagy flux by impairing the fusion between autophagosomes and lysosomes (Abdullah et al., 2020; Jiang et al., 2013). I have performed immunoblot analysis to detect autophagy flux via the combined analysis of LC3-II and SQSTM1 levels. In only H2O2

treated group, autophagy flux was damaged and this was confirmed by the increase in the expression of autophagy related proteins SQSTM1 and LC3-II. However in the MAG pre-treated group, the autophagy flux was not damaged, as the expression of SQSTM1

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was decreased and the LC3-II expression was increased (Fig. 7). This suggests that MAG can repair organs damaged by intracellular ROS by preventing autophagy flux which was damaged under oxidative stress (Fig. 8). In conclusion, this study shows that MAG activates antioxidant effects and autophagy during oxidative stress. I suggest that MAG may provide a better therapeutic effect on hearing recovery.

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Fig. 8. Schematic model of the pathway by which MAG protects hair cells from oxidative stress.

MAG enters the cell and activates AMPK to activate autophagy. When autophagy is activated, it forms autophagosome and later derives into autolysosome after fusing with lysosome, this is called autophagic flux, and it can suppress apoptosis by reducing intracellular ROS. However, when subjected to oxidative stress, autophagic flux is damaged and cannot function properly, but MAG can restore damaged autophagic flux to suppress apoptosis.

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VI. Acknowledgements

2018년 3월에 실험실에 들어와서 어느덧 3년이라는 시간이 흘렀습니다.

처음 실험실에 들어왔을 때는 모든 것이 새롭고 어려웠지만, 열심히 배우며 석사 졸업이라는 문까지 온 것 같습니다. 석사 졸업 이후에 박사 과정을 시 작하겠지만, 지금까지 고마운 분들께 짧게나마 글로 마음을 전하고자 합니다.

먼저, 실험실 생활을 하면서 연구활동을 끊임없이 지지해 주시며 부족한 저를 이끌어 주시고 지도해 주신 조광원 교수님 감사드립니다. 교수님께서 해주신 말씀과 충고들은 저의 의지를 다지게 해주는 원동력이 되었습니다.

그리고 지도교수님만큼 열정적으로 저를 가르쳐 주시고 인도해주신 장철호 교수님 감사드립니다. 또한, 저의 논문을 심사해주시고 충고와 격려를 아끼 지 않으신 전택중 교수님, 이준식 교수님 감사드립니다. 복도에서 마주칠 때 마다 해 주신 격려는 저에게 큰 도움이 되었습니다. 학부 때부터 많은 가르 침을 주신 윤성명 교수님, 조태오 교수님, 송상기 교수님, 이현화 교수님, 원 부연 교수님을 포함한 학과 교수님들께도 감사 인사 드립니다.

같이 고생한 저의 실험실 식구들에게도 감사의 말을 전하고 싶습니다. 실 험실 생활을 같이는 못하였지만 많은 조언과 격려를 해주신 신구형, 호태형 감사합니다. 한학기 먼저 졸업한 안지야 많이 알려주고 도와줘서 고마웠어.

Nagarajan, Karthi and Chitra thank you. 실험실 들어와서 열심히 하는 주호, 학부생이지만 열심히 하는 민지, 동물실험이 힘들지만 열심히 하는 수민이 와 연진이 그리고 항상 웃는 창민이도 고마웠어. 앞으로도 잘해보자. 그리고 나랑 같이 오래 했지만 다른 길을 찾아 나선 하준이, 다른 곳에서 새롭게 시 작하는 성은이도 열심히 해서 좋은 결과 꼭 얻길 바랄게.

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다른 실험실이지만 조언과 격려를 아끼지 않으신 김미은 박사님, 모르는 게 있을 때 항상 잘 알려주셔서 감사합니다. 항상 듬직한 실험실 형들인 주 원이형, 수민이형, 영빈이형, 요한이형, 현웅이형 감사합니다. 형들이 잘 챙겨 주신 덕분에 실험실 생활이 힘들지 않았습니다. 항상 밝은 모습으로 맞아주 는 진솔아 앞으로도 좋은 일만 있길 바랄게. 모르는 게 없던 준휘야 실험 팁 들이며 많은 걸 알려줘서 고마웠어. 항상 재미있게 실험실 생활을 하게 해준 동주야 덕분에 많이 웃을 수 있었다. 묵묵히 열심히 하는 원범아 박사까지 잘할 수 있을 거야. 이제 한 학기 남은 광철이, 준한이 마지막까지 열심히 해서 유종의 미를 거두기 바랄게. 같이 실험실 생활을 하고 있는 후배들 동 원이, 유석이, 대현이, 지성이, 예은이, 상철이, 영훈이 응원해줘서 고맙고 실 험실 생활 하면서 무사히 마무리 했으면 좋겠다. 꿈이 많은 찬일아 소방관 된 거 축하한다. 학부동기인 병규야 너의 조언들 덕분에 여기까지 올 수 있 었던 것 같아서 고마워. 비슷한 시기에 다른 실험실을 들어갔지만 다른 길을 택한 현우형 많이 격려도 해주고 잘 챙겨줘서 고마웠어요. 자주 실험실 찾아 와서 연구실 생활을 응원해준 후배 기연이, 잠깐이지만 같이 실험실 생활을 같이했던 차현이도 고마웠어.

마지막으로, 저를 낳아 주시고 기쁠 때나 슬플 때 항상 제 옆에서 지지해 주신 부모님께 정말 감사드립니다. 제가 여기까지 할 수 있었던 것은 저를 믿어주신 부모님 덕분 이였습니다. 우리 엄마, 아빠 항상 존경하고 사랑합니 다. 사랑하는 우리 동생 수경아 매번 응원해줘서 고마웠어. 너도 대학원 잘 마무리하길 바랄게. 그리고 사랑하는 우리 막내 동생 수연아 타지에서 대학 생활 하는게 힘들지만 스스로 잘해 나가는게 뿌듯하고 보기 좋다. 항상 저 잘되라고 말씀하신 할머니, 외할머니 감사하고 사랑합니다. 마지막으로 한분 한분 말씀을 못 드려서 죄송하지만 저를 아껴주신 친척분들께 감사합니다.

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그리고 미처 감사한 마음을 전하지 못한 분들 에게도 감사의 말씀을 드리며 항상 감사한 마음 잊지 않고 살겠습니다. 감사합니다.

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