Subscriber access provided by Kaohsiung Medical University
Food Safety and Toxicology
Resveratrol protects porcine intestinal epithelial cells from deoxynivalenol induced damage via the Nrf2 signaling pathway Jun Yang, CUI ZHU, Jin ling Ye, Yantao Lv, Li Wang, Zhuang Chen, and Zong yong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03662 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 19, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
Resveratrol protects porcine intestinal epithelial cells from deoxynivalenol induced damage via the Nrf2 signaling pathway Jun Yang†, ‡#, Cui Zhu‡#, Jinling Ye‡, Yantao Lv‡, Li Wang§, Zhuang Chen‡*, Zongyong Jiang‡, §* †College
of Animal Science, South China Agricultural University, Guangzhou 510642,
P. R. China ‡Agro-biological
Gene Research Center, Guangdong Academy of Agricultural
Sciences, Guangzhou 510640, P. R. China. §Institute
of Animal Science, Guangdong Academy of Agricultural Sciences,
Guangzhou 510640, P. R. China.
#Jun
Yang and Cui Zhu contributed equally to this work.
*Corresponding
author: Zongyong Jiang (
[email protected]) and Zhuang Chen
(
[email protected]). Tel: +86-20-87596262, Fax: +86-20-87503358.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 32
1
ABSTRACT
2
Deoxynivalenol (DON), a common mycotoxin, usually induces oxidative stress and
3
affects intestinal health of humans and animals. This study investigated the protective
4
effect of resveratrol (RES) as a natural antioxidant on alleviating the cytotoxicity
5
induced by DON using the intestinal porcine epithelial cell line (IPEC-J2). Cells were
6
incubated with RES for 24 h, then exposed to DON for another 24 h. Cell viability,
7
proliferation, apoptosis, and oxidative stress indicators were determined. In compared
8
to DON-treated cells, pre-treatment with RES (15 µM) increased the cell viability
9
(79.74% ± 2.02 vs. 90.98% ± 2.66) (P < 0.01), improved proliferation (EdU-positive
10
cells) (26.42% ± 1.12 vs. 32.05% ± 0.78) (P < 0.01), decreased accumulation of
11
intracellular reactive oxygen species (ROS) (1.68 ± 0.05 vs. 1.29 ± 0.06) (P < 0.01),
12
stabilized mitochondrial membrane potential (MMP) (8.98 ± 1.40 vs. 2.29 ± 0.76) (P
13
< 0.001), and prevented apoptosis induced by DON (13.91% ± 1.20 vs. 6.83% ± 0.52)
14
(P < 0.01). RES activated the Nrf2 signaling pathway, while transfection with Nrf2
15
siRNA abrogated the protection of RES against DON-induced cytotoxicity,
16
accumulation
17
Collectively, RES protects IPEC-J2 cells against DON-induced damage at least partly
18
via the Nrf2 signaling pathway.
19
KEYWORDS: resveratrol, deoxynivalenol, IPEC-J2 cells, oxidative damage, Nrf2
20
signaling pathway
of
intracellular
ROS,
and
mitochondria-dependent
ACS Paragon Plus Environment
apoptosis.
Page 3 of 32
Journal of Agricultural and Food Chemistry
21
INTRODUCTION
22
Deoxynivalenol (DON, vomitoxinis), a trichothecene mycotoxin, primarily occurs in
23
fungus Fusarium infected grains, and represents one of the most common mycotoxins
24
all over the world1. DON can readily enter the food chain, and affect human health
25
and animal productivity2. Exposure to DON usually causes a series of symptoms in
26
humans and animals, such as diarrhea, vomiting, reduced feed intake, lower growth
27
performance, suppression of immunological functions3, 4. Animals displayed different
28
sensitivity to DON, with swine showing the highest sensitivity among farm animals5.
29
The epithelium of small intestinal is the first defensive barrier against gut infection,
30
and is also a selective barrier which absorbs various nutrients, such as amino acids,
31
vitamins and water while prevents the passage of foreign antigens, microorganisms
32
and their toxins such as DON6. Intestinal epithelium cells, therefore, can be frequently
33
exposed to high concentrations of DON after intake of infected feed7. Exposure to
34
DON could lead to irritation or necrosis of intestinal mucosa3, increased of intestinal
35
permeability8, and induced apoptosis9, thus compromising the intestinal health of pigs.
36
Previous studies have demonstrated that oxidative damage appears to be a key causal
37
factor of the toxicity induced by DON in intestinal cells10,
38
mechanisms underlining this toxicity of DON remains largely unclear, and
39
understanding of its mechanism will help develop potential protective strategies for
40
alleviating DON-induced oxidative stress to maintain gut health and normal growth of
41
pigs.
42
The use of antioxidants has been proved to effectively attenuate the intestinal
ACS Paragon Plus Environment
11.
The molecular
Journal of Agricultural and Food Chemistry
43
oxidative damage induced by DON11,
44
resveratrol
45
capacity13,14 and many studies have shown that RES exerted protection against
46
oxidative stress and apoptosis in cells14, 15. Nuclear factor-erythroid 2-related factor-2
47
(Nrf2) plays an important regulative effect on oxidative status through ARE-mediated
48
induction of antioxidant genes13,
49
potential protection of RES from DON-induced damage in porcine intestinal
50
epithelium cells remained unknown.
51
Given the foregoing, the IPEC-J2 cell line, originated from jejunal epithelium of an
52
unsuckled neonatal piglet17, was used as an in vitro model of porcine small intestinal
53
epithelium cells. The purpose of this study was thus to investigate whether RES exerts
54
protection in IPEC-J2 cells against damage induced by DON exposure and to explore
55
the possible molecular mechanism.
56
MATERIALS AND METHODS
57
Chemicals
58
DMEM/F12 medium, fetal bovine serum (FBS), and L-glutamine were purchased
59
from Gibco (Grand Island, NY). DON (purity ≥ 98%), RES (purity ≥ 98%), dimethyl
60
sulfoxide (DMSO) and 2’,7’-dichlorofluorescein-diacetate (DCFH2-DA) were
61
obtained from Sigma-Aldrich (St. Louis, MO). The antibodies used here were
62
obtained from Abcam (Cambridge, UK).
63
Cell culture
64
The IPEC-J2 cells were kindly provided by Prof. Daiwen Chen (Sichuan Agricultural
(3,5,4’-trihydroxystilbene,
16.
12.
Page 4 of 32
A natural polyphenolic antioxidant,
RES),
possesses
efficient
antioxidant
The possible role of Nrf2 in mediating the
ACS Paragon Plus Environment
Page 5 of 32
Journal of Agricultural and Food Chemistry
65
University, Ya’an, China), and cultured in DMEM/ F12 containing 10% FBS and 2
66
mM L-glutamine, and maintained at 37°C with 5% CO2.
67
Cell viability
68
IPEC-J2 cells were cultured in 96-well plates for DON or RES treatment. DON was
69
added to the cells for 24 h at concentrations of 0, 0.25, 0.5, 1, 2 and 4 μg/mL (0, 0.84,
70
1.68, 3.36, 6.72 and 13.44 µM) dissolved in DMSO (final solvent concentration
