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Eupatilin induces Sestrin2-dependent autophagy to prevent oxidative stress

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Abstract

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) has many pharmacological activities including anti-inflammation, anti-oxidant and anti-cancer effects. Autophagy is the basic cellular machinery involving the digestion of damaged cellular components. In the present study, we investigated the protection effects of eupatilin against arachidonic acid (AA) and iron-induced oxidative stress in HepG2 cells and tried to elucidate the molecular mechanisms responsible. Eupatilin increased cell viability against AA + iron in a concentration-dependent manner and prevented mitochondrial dysfunction and reactive oxygen species (ROS) production. In addition, AA + iron increased the levels of pro-apoptotic proteins and these changes were prevented by eupatilin. Eupatilin also induced autophagy, as evidenced by the accumulation of microtubule-associated protein 1 light chain3-II and the detection of autophagic vacuoles. Furthermore, the protective effects of eupatilin on mitochondrial dysfunction and ROS production were significantly abolished by autophagy inhibitors. Eupatilin also increased the mRNA level of sestrin-2 and its promoter-driven reporter gene activity, which resulted in the up-regulation of sestrin-2 protein. Finally, gene silencing using sestrin-2 siRNA and the ectopic expression of recombinant adenoviral sestrin-2 indicated that sestrin-2 induction by eupatilin was required for autophagy-mediated cytoprotection against AA + iron. Our results suggest that eupatilin activates sestrin-2-dependent autophagy, thereby preventing oxidative stress induced by AA + iron.

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Abbreviations

3-MA:

3-Methyladenine

AA:

Arachidonic acid

AMPK:

AMP-activated protein kinase

AO:

Acridine orange

Baf. A1:

Bafilomycin A1

DCF:

Dichlorfluorescein

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GFP:

Green fluorescence protein

LC3:

Microtubule-associated protein 1 light chain 3

MMP:

Mitochondrial membrane potential

mTOR:

Mammalian target of rapamycin

mTORC1:

Mammalian target of rapamycin complex 1

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

Nrf2:

NF-E2-related factor 2

p70S6K1:

p70 ribosomal S6 protein kinase 1

PARP:

Poly(ADP-ribose)polymerase

PBS:

Phosphate buffered saline

PI:

Propidium iodide

Rh123:

Rhodamine123

ROS:

Reactive oxygen species

Sesn:

Sestrin

si-Control:

siRNA control

ULK1:

Unc-51-like protein kinase1

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Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government [MSIP] (No. 2012R1A5A2A42671316). K. H. Jegal would like to thank the degree program of Master at Daegu Haany University for completing the thesis through this work. The authors express thanks to Mr. Seung Wook Yi (Shin Woo Tech, Daegu, Korea) for the technical assistance of fluorescence microscope analysis.

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Authors and Affiliations

  1. Department of Herbal Formulation, MRC-GHF, College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do, 38610, Republic of Korea

    Kyung Hwan Jegal, Hae Li Ko, Sang Mi Park, Sung Hui Byun, Il Je Cho & Sang Chan Kim

  2. College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, 08826, Republic of Korea

    Keon Wook Kang

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  1. Kyung Hwan Jegal
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Jegal, K.H., Ko, H.L., Park, S.M. et al. Eupatilin induces Sestrin2-dependent autophagy to prevent oxidative stress. Apoptosis 21, 642–656 (2016). https://doi.org/10.1007/s10495-016-1233-6

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