Endoplasmic Reticulum Stress and Unfolded Protein Response

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Food and Beverage Chemistry/Biochemistry

Endoplasmic reticulum stress and UPR pathways are involved in the health-promoting effects of allicin on the jejunum Qian Jiang, Junquan Tian, Gang Liu, Yulong Yin, and Kang Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02180 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Agricultural and Food Chemistry

Endoplasmic reticulum stress and UPR pathways are involved in the health-promoting effects of allicin on the jejunum

Qian Jiang*†‡§, Junquan Tian†, Gang Liu†, Yulong Yin†, and Kang Yao†

†Laboratory

of Animal Nutritional Physiology and Metabolic Process, Institute of

Subtropical Agriculture, Chinese Academy of Sciences; ‡University

of Chinese Academy of Sciences, Beijing, 100043, P.R. China;

§Department

of Animal Science, University of Manitoba, Winnipeg, MB, Canada

R3T 2N2 * Corresponding author: Qian Jiang. E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Intestinal endoplasmic reticulum stress (ERS) triggered by adverse

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factors disturbs the normal function of the intestine. Allicin has been reported to

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promote intestinal health and development. In the present study, we established in

4

vivo (35-day-old weaned piglets, 4-week-old mice) and in vitro (IPEC-J2 cell line)

5

ERS models to explore the possible mechanisms by which allicin may benefit

6

intestinal health. This study revealed that 1) allicin supplementation improved

7

intestinal morphological indices and ameliorated mild ERS in the jejunum of the

8

weaned piglets; 2) allicin supplementation decreased cellular reactive oxygen species

9

and upregulated the XBP-1s signaling pathways in IPEC-J2 cells; 3) allicin

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supplementation reduced the prolonged ERS-mediated apoptosis of IPEC-J2 cells and

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in the jejunal tissues of the KM mice; 4) allicin supplementation enhanced the

12

intercellular junction protein levels of jejunal cells by alleviating the prolonged ERS.

13

These novel findings suggest that eating garlic could alleviate some intestinal

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malfunctions associated with ERS.

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KEYWORDS: allicin, endoplasmic reticulum stress, jejunum, IPEC-J2, XBP-1s,

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intestinal barrier, apoptosis

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INTRODUCTION

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Garlic, a commonly used ingredient in many cuisines, has been reported to

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benefit health1 and has shown antiplatelet aggregation, anti-atherosclerotic, anti-fatty

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liver, anti-oxidative, and anti-microbial effects.2 Allicin, a major component of

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garlic,3 has been revealed to promote the health and development of the intestine,4 but

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the mechanism has not been fully investigated. Previous studies have reported that

24

allicin can affect cell proliferation or apoptosis by regulating signaling pathways,

25

including those of phosphatidylinositol 3 kinase (PI3k),5-6 hypoxia-inducible factor 1

26

(HIF),7 c-Jun NH2-terminal kinase (JNK),8 signal transducer and activator of

27

transcription 3 (STAT3),9 nitric oxide synthase 3 (eNOS),10 protein kinase RNA-like

28

endoplasmic reticulum kinase (PERK),11 and caspase-3.12 Based on these studies, we

29

hypothesized that some important molecular mechanisms might be involved in the

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positive effect of allicin on intestinal health and growth.

31

Endoplasmic reticulum stress (ERS) describes a condition in which the normal

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functions of the ER (e.g., protein synthesis, folding, and calcium ion homeostasis) are

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disturbed.13 In response to ERS, a network of cellular signaling pathways involved in

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the unfolded protein response (UPR) is triggered.14 The three main UPR pathways are

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IRE-1α, PERK-CHOP, and ATF-6.15 The functional results of these pathways are 1)

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lower translation level, 2) restoration of protein folding, and 3) degradation of

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misfolded proteins.16 The PERK downstream pathway can inhibit the translation

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process in protein synthesis and limit further protein misfolding in the ER,17 and

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PERK-CHOP–mediated apoptosis can be activated when the cell is unable to survive 3



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the prolonged stress.18 Recent evidence has shown that intestinal prolonged ERS and

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its mediated signaling can be triggered by many adverse factors such as pathogen

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infection,19 redox reaction imbalance,20-21 and ingestion of oxidized fats.22 ERS and

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UPR signaling–mediated apoptosis have recently drawn considerable attention in

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cancer research.23-24 Our recent study revealed that mild ERS is triggered in the

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rapidly growing intestine of weaning piglets under normal feeding conditions, and this

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ERS and CHOP-mediated apoptosis were aggravated by E. coli infection.25 One

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previous study illustrated that ERS triggered by tunicamycin reduced the levels of

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E-cadherin and zonula occludens-1 (ZO-1) in alveolar epithelial cells.26 Based on

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these reports, we hypothesized that allicin alleviates ERS, decreases ERS-mediated

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apoptosis, and increases the level of intercellular junction proteins in ER-stressed

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intestinal cells, thereby promoting the growth and health of the intestine.

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In this study, we established in vivo (35-day-old weaned piglets, 4-week-old

53

mice) and in vitro (IPEC-J2 cell line) ERS models to explore the possible mechanisms

54

by which allicin may promote intestinal health. The levels of ERS signaling proteins

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(GRP78, p-eIF-2α, IRE-1α, XBP-1s, ATF6, and CHOP) and intercellular junction

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proteins (E-cadherin and ZO-1) in jejunal tissues/cells were analyzed. The intestinal

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porcine epithelial cell line IPEC-J2 derived from porcine jejunum was used as the in

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vitro model, tunicamycin (TM) was used as an ERS inducer, and STF-08301027 was

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used as an XBP-1s inhibitor to verify the precise mechanism of allicin involvement in

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ERS and its mediated pathways. To our knowledge, this is the first study to correlate

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the UPR pathways with the positive effects of allicin on intestinal health, and our 4


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findings could be applied to exploit allicin in alleviating the intestinal malfunction

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associated with ERS.

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MATERIALS AND METHODS

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Animal models and treatments. This animal experiments in the study were

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conducted with the approval of the Animal Welfare Committee of the Institute of

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Subtropical Agriculture (Permit No. ISA000263 for pig experiment, and No.

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ISA000267 for the mice experiment), Chinese Academy of Sciences. The intestinal

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samples of 35- and 120-day-old pigs were obtained from our recent animal studies. In

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the experiment on weaned piglets, 16 piglets with 35-day-old were dietary

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supplemented with or without 0.5% allicin during the 21days experimental period (8

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piglets in each group). The Chinese Kun Ming (KM) mice used in this study were

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bought from the experimental animal co. LTD of Hunan Slyke. KM mice (4wks, 16

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mice, 8 mice in each group) were daily supplemented with tunicamycin (2.0 g L-1) in

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the drinking water to induce the prolonged ERS in the intestine. The mice in the

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experimental group were supplemented with allicin (allicin was dissolved in alcohol

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and diluted in the saline, 2.0 g/kg body weight, once a day in the morning) by

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intragastric infusion using the lavage needle, the 8 mice in the control group were

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intragastric supplemented with equal volume of saline accordingly. After these

81

treatments, the animals were slaughtered to collect the samples of jejunum for further

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analysis. The entire intestines and viscera for each animal were rapidly removed.

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After washing the enteric cavity with phosphate buffer solution (PBS), the jejunum 5



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samples were immediately frozen in liquid nitrogen and stored at −80 °C for

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subsequent analysis of protein expression by western blotting. The tissue samples

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were fixed with 4.0% paraformaldehyde for 24 hours and then embedded in paraffin

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for the subsequent analysis. Each sample was sliced into 5.0 μm sections and all of the

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experiments were performed in triplicate.

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Chemicals. The allicin (CAS 539-86-6) was obtained from Santa Cruz

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Biotechnology (Dallas, TX, USA). Dulbecco’s modified Eagle medium (DMEM),

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fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) required for the

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cell cultures were obtained from GIBCO (Carlsbad, CA, USA). The cell culture plates

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were manufactured by Corning Inc. (Corning, NY, USA). The CCK-8 and DAPI

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working solution were purchased from Beyotime Biotechnology (Shanghai, CHN).

95

Tunicamycin (TM), STF-083010 and dimethylsulfoxide (DMSO) were purchased

96

from Sigma (Saint Louis, MO, USA). The antibodies against XBP-1s, p-eIF-2α,

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eIF-2α, GAPDH and zonula occludens-1 were obtained from Santa Cruz

98

Biotechnology, and the antibodies against ATF6, IRE-1α, GRP78, CHOP, ATF4,

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caspase-12, E-cadherin, and beta-actin were obtained from Abcam (Cambridge, MA,

100 101

USA). Cell culture and treatments. The intestinal cell line IPEC-J2 (C0668, passage 4)

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was bought from Shanghai Guan Zhi bio-engineering co. LTD. Cells were cultured in

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the plastic culture flasks (25 cm2). The growth culture medium consists of DMEM-H

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containing 10% FBS, 5 mM L-glutamine, 100 U mL-1 penicillin, and 100 μg mL-1

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streptomycin. At reached to 80% confluence, cells were trypsinized and seeded in 6


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6-well (1 × 104 cells per well) /96 wells culture plates (1 × 102 cells per well) and kept

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in a 5.0% CO2 humidified incubator at 37 °C. After overnight incubation, the culture

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medium was replaced by basal medium (blank control) or basal medium + 0.5 μg

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mL-1 /1.0 μg mL-1 tunicamycin (ERS group) to induce ERS. To induce the in vitro

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mild ERS model, IPEC-J2 cells were pretreated with basal medium containing 0.5 μg

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mL-1 tunicamycin for 6 hours. To induce the in vitro prolonged ERS model, IPEC-J2

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cells were cultured with basal medium containing 1.0 μg mL-1 tunicamycin for 24

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hours. In the oxidative stress model of IPEC-J2 cells, the cells were pretreated with

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0.5 mM H2O2 for 6 hours. In the allicin supplemented groups, 2.0 μg mL-1 allicin was

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supplemented to the culture medium after the mild ERS induction, after oxidative

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stress induction, or at the 12h time point of prolonged ERS induction.

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Cell viability determination. Cells were cultured in the 96-well plates. After the

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specific treatments as described in the design section, cell counting kit-8 was used to

119

determine the cell viability, according to the protocol. The experimental procedures

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were described in our previous study.25

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Cell apoptosis determination. TUNEL staining is performed to determine cell

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apoptosis of jejunal tissues and IPEC-J2 cells. The determination was conducted

123

according to the protocols. In brief, cells or tissue slices were permeabilized in 0.01%

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Triton X-100, then washed them with PBS and stained with 1:10 TUNEL working

125

solution for 1 hour at 37 °C. After washing the samples with PBS for 3 times, capture

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the images with a fluorescence microscope. Nuclei were stained by DAPI and shown

127

in blue color, and TUNEL-positive nuclei were stained by TUNEL kit and shown in 7



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green color. The relative abundance of TUNEL-positive fluorescent intensity (shown

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as green intensity / blue intensity) was used as the indicator of cell apoptosis. Five or

130

more microscopic fields were randomly selected for the image capture at 400×

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magnification.

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Cell proliferation analysis. After the treatments, cell proliferation was

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determined by Cell-lightTM EdU Kit (Rui Bo Biotechnology, CHN), the procedures

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are detailly described in our previous study.28 After the incubation process of EdU

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staining according to the product manual, a fluorescence microscope was used to

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capture the image of EdU-positive cells stained with Apollo® 567 Hoechst 33342

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(red fluorescent). Five or more different microscopic fields were randomly selected

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for the capture at 400× magnification. Nuclei are stained by DAPI and shown in blue

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color. The relative abundance of EdU-positive fluorescent intensity (shown as red

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intensity/blue intensity) was used as the indicator of cell proliferation.

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Measurement of intracellular ROS concentration and anti-oxidative

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parameters. Cellular Reactive Oxygen Species Detection Assay Kit (ab186027,

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Abcam) was used for determining the intracellular ROS concentration of IPEC-J2

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cells. After the specific treatments in 96-well cell culture plate, cells were incubated

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with FBS-free media containing 20 µM DCFH-DA for 20 minutes in the dark cell

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incubator. Then washed cells 3 times in an FBS-free medium, the fluorescence

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intensity (excitation wavelength: 520nm, emission wavelength: 605nm) of each well

148

was determined by a fluorescence microplate reader FlexStation (Molecular Devices).

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The ROS concentration of intracellular ROS was evaluated by this value of relative 8


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fluorescence units (RFU). The levels of malondialdehyde (MDA) and sodium oxide

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dismutase (SOD) in the cells were measured by ELISA kits (Nanjing Jiancheng, CHN)

152

according to the manufacturer’s protocols.

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Western blotting analysis. After the treatments, the cells were washed three

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times in PBS. The samples were lysed in an ice-cold buffer with a complete protease

155

inhibitor cocktail, and immunoblotting assays were performed as previously described

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29.

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MA, USA) under the recommended conditions. The protein band densities were

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normalized to the specific loading control protein band (beta-actin, eIF-2α) and

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quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).

The blots were examined using the ECL Plus detection system (Thermo, Waltham,

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Statistical analysis. All data are representative of at least three independent

161

experiments. Data are expressed as means ± SD. Statistical analysis was performed

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by one-way ANOVA using the GraphPad software. P-values of less than 0.05 were

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considered statistically significant.

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RESULTS

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ERS occurs in the jejunum of weaned piglets but not fattening piglets. To

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investigate the roles of ERS and UPR signaling in the development of porcine

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jejunum, we compared the levels of ERS markers in weaned piglets and fattening pigs.

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Results of immunofluorescence (Figure 1A, B) and western blot (Figure 1C, D)

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showed that ERS makers (GRP78 and CHOP) were significantly higher expressed in

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the jejunum of weaned piglets when compared with these of the fattening pigs. 9



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Allicin increased the intestinal morphological indices and downregulated

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PERK pathway in the jejunum of weaned piglets. As shown in figure 2A and B,

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0.5% allicin dietary supplementation for 21 days increased the ratio of villous height

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to crypt depth of jejunum when compare with the control group. The levels of

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phosphorylation eIF-2α and ATF4 in jejunum were significantly decreased by the

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0.5% allicin dietary supplementation (figure 2C, D). The levels of GRP78 and

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apoptotic markers (CHOP, and caspase-12) were not affected by the allicin diet

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(figure2E, F).

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IRE-1/XBP-1s signaling was activated by allicin in the mild ER-stressed

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jejunum and IPEC-J2 cells. We furtherly analyzed the protein expression levels of

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IRE-1α and XBP-1s in the jejunal tissue of weaned piglets fed with basic diet or

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0.25% allicin diet. As shown in figure 3, the relative abundances of IRE-1α and

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XBP-1s in the jejunal tissue (Figure 3A, B) were significantly increased by allicin.

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This result matches the in vitro result which showing that allicin increased

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expressions of XBP-1s, IRE-1α and decreased expressions of GRP78, ATF-4 and

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p-eIF-2α in mild ER-stressed intestinal cells (Figure 3C-E). The solo allicin treatment

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did not affect the protein expressions of XBP-1s or GRP78 (Figure 3C, D). The allicin

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did not affect the expressions of CHOP, eIF-2α or caspase-12 in the TM-pretreated

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and the non-pretreated IPEC-J2 cells.

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Allicin supplementation reduces the cellular ROS concentration in the mild

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ER-stressed IPEC-J2. To determine if the antioxidant program participates in the

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ATF4 downregulation induced by allicin, after treating the mild ER-stressed IPECs 10


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with 0 or 2.0 μg mL-1 allicin for 12 hours, we determined the levels of cellular ROS,

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malondialdehyde (MDA) and sodium oxide dismutase (SOD). As shown in figure 4,

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the allicin supplemented to culture medium significantly decreased the level of ROS

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in the ER-stressed cells (Figure 4A). However, the levels of MDA and SOD in the

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ER-stressed cells were not affected by the allicin supplementation (Figure 4B-C). We

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pretreated the IPEC-J2 cells with 0.5 mM H2O2 for 6 hours, then cultured the cells in

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the normal culture medium with or without allicin for 12 hours. The cellular ROS,

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malondialdehyde (MDA) and sodium oxide dismutase (SOD) was determined.

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Results showed that allicin supplementation significantly decreased the levels of ROS

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and MDA, but increased the SOD level in the oxidative-stressed cells (Figure 4 D-F).

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IRE-1α Blockage reversed the positive effects of allicin on the mild

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ER-stressed IPEC- J2 cells. To investigate whether the IRE-1α/XBP-1s signaling is

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the fateful component in the allicin intervened ER-stressed IPEC-J2 cells. The effects

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of allicin on the ER-stressed IPEC-J2 cells were evaluated when treated the cells with

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STF-083010 (a specific IRE-1α inhibitor). The western blot analysis confirmed the

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inhibitory effect of STF-083010 on splicing XBP-1 in the ER-stressed IPEC-J2 cells

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(Figure 5A). Meanwhile, the effects of allicin on reducing the levels of p-eIF-2α

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(Figure 3E), ATF4 (Figure 3E), GRP78 (Figure 3D) and ROS (Figure 5B) in the mild

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ER-stressed IPEC-J2 cells were diminished (Figure 5C-G) by this IRE-1α pathway

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blocking.

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Allicin did not affect the proliferation of jejunal cells in the piglets or

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IPEC-J2 cells. We furtherly investigate whether any proliferation effect of allicin has 11



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on the intestinal cells. After the treatments, we decided the proliferation rate of the

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porcine jejunum and tunicamycin pre-treated IPEC-J2 cells by EdU staining, as

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shown, no proliferation effect of allicin on jejunal tissue (Figure 6A, B) or in vitro

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cells (Figure 6C, D) was observed.

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Allicin attenuated cell apoptosis and enhanced the levels of intercellular

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junction proteins in the prolonged ER-stressed jejunum of mice. As shown in

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figure 7A and B, allicin (2.0 g/ kg body weight) supplemented to the prolonged ERS

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model of KM mice significantly reduced the ratio of tunnel-positive cells by 8% in the

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jejunal tissue, which indicating cell apoptosis was reduced by this allicin. The protein

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levels of CHOP and caspase-12 in the prolonged ER-stressed jejunal tissue were

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significantly decreased by allicin supplementation, and the XBP-1s levels of the

227

jejunal tissue in the TM + allicin group were higher than the TM group (Figure 7C, D).

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In addition, this allicin supplementation significantly increased the relative

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abundances of E-cadherin and zonula occludens-1 (ZO-1) proteins by 229% and

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190%, respectively (Figure 7 E, F).

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Allicin attenuated cell apoptosis and maintained the intercellular junction

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proteins levels of the prolonged ER-stressed IPEC-J2 cells. As shown, within the

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treatments of tunicamycin, IPEC-J2 cells showed a higher apoptotic ratio (Figure 8A,

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C), decreased cell viability (Figure 8B) and increased expression of caspase-12,

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CHOP, and ATF-4 (Figure 8E). This 2.0 μg mL-1 allicin supplementation attenuated

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tunicamycin-induced apoptosis, downregulated the protein levels of caspase-12,

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CHOP, and ATF4, but significantly increased the XBP-1s level of IPEC-J2 cells 12


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(figure 8D, E). STF-083010 (5.0 μg mL-1) increased the apoptosis of allicin-treated

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ER-stressed intestinal cells (Figure 8A, C), and reversed the positive effects of allicin

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in attenuating the PERK pathway and ATF4-CHOP-caspase12 mediated cell

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apoptosis (figure 8D, E). Moreover, the 1.0 μg mL-1 tunicamycin supplementation

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significantly reduced the relative abundances of E-cadherin and zonula occludens-1

243

(ZO-1) proteins by 76.0% and 78.4%, respectively, and this allicin additionally

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supplementation reversed this downregulation of E-cadherin and ZO-1 proteins

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induced by tunicamycin (Figure 8 G, H).

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DISCUSSION

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The ER is a central organelle associated with lipid synthesis, protein folding, and

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protein maturation. Elevated protein synthesis in the intestine may induce the

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accumulation of unfolded proteins in the ER, thus triggering mild ERS. It has been

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reported that ERS and its mediated signaling can be fully activated in the porcine

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small intestine.25,30 Rapid cell proliferation has been reported to induce spontaneous

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ERS in various tumor cells.31-32 The weaning period of piglets is a critical period for

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intestinal development during which intestinal cells rapidly proliferate, and a large

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number of histones are synthesized. We hypothesized that elevated histone synthesis

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in the intestinal cells during weaning might lead to spontaneous mild ERS. To verify

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this hypothesis, we determined the ERS markers in the jejunum of weaned piglets and

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fattening pigs to investigate differences of UPR signaling activation in the jejunum

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between these two periods. Our data reveal that ERS was triggered in the jejunum of 13



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the weaned piglets but not in the jejunum of fattening piglets, consistent with our

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expectation. This prompted us to investigate the regulatory mechanism of ERS in the

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fast-developing jejunum.

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TM triggers ER stress by inhibiting N-linked glycosylation of newly synthesized

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proteins in the ER. Glycosylation is essential in the control of both the processing and

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quality of protein folding. If hydrophobic parts of the amino acid chain cannot be

266

buried in the interior of the protein during folding, the unfolded protein response and

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related pathways will be triggered. Various studies33-35 have reported that TM can

268

induce ERS in various organs in various animal species. In this study, we added 2.0 g

269

L-1 TM to the drinking water to induce prolonged ERS in the intestines of mice

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according to a previously described method.34 We previously established a prolonged

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ERS model in IPEC-J2 cells with 1.0 μg mL-1 TM treatment.36 In this study, we

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established a mild ERS cell model by pretreating IPEC-J2 cells with 0.5 μg mL-1 TM

273

for 6 h. The protein expression of ERS markers in the mild ERS cell model is similar

274

to that in the jejunum of weaned piglets. Therefore, we propose that this mild ERS

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model of porcine intestinal cells can be applied to investigate the molecular

276

mechanism of mild ERS and ERS-mediated biological processes in the jejunum.

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Our previous study showed that allicin improved the daily weight gain of weaned

278

piglets. We surmised that allicin may improve the nutrient assimilation by promoting

279

the development of the small intestine in the weaned piglets. To further investigate the

280

role of allicin in the development of the porcine small intestine, we determined

281

intestinal morphological indices and ERS signaling in the jejunum of weaned piglets. 14


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Our data revealed that allicin increased the intestinal morphological indices and

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downregulated phosphorylation of eIF-2α and ATF4 in the jejunum of weaned piglets,

284

suggesting that allicin may improve the ability of ER to process unfolded proteins and

285

alleviate mild ERS in the intestine of weaned piglets via the UPR pathways.

286

It is well known that activation of IRE-1/XBP-1s signaling is the major adaptive

287

response to restore ER homeostasis in ER-stressed cells. XBP1s can directly activate

288

ERS target genes to facilitate the refolding and degradation of misfolded proteins and

289

ER chaperones.37 To determine whether the IRE-1α signaling pathway plays a role in

290

the downregulation of phosphorylation of eIF-2α and ATF4 in the allicin-treated

291

intestine, we further analyzed the expression levels of IRE-1α and XBP-1s in the

292

jejunal tissue of weaned piglets fed with an allicin diet and in allicin-treated

293

ER-stressed IPEC-J2 cells. These results revealed that IRE-1/XBP-1s signaling was

294

activated by allicin supplementation. Of interest, the expression of CHOP and

295

caspase-12 was relatively low in the jejunum of the weaned piglets, indicating that

296

ERS-mediated apoptosis was slightly induced, but not fully triggered within the

297

normal feeding condition.

298

ROS are important mediators of apoptosis,38 and recent findings39 have linked

299

ERS to the generation and accumulation of intracellular ROS, a state commonly

300

referred to as oxidative stress. An increase in the protein-folding load in the ER (i.e.,

301

ERS) can lead to the accumulation of ROS, thus initiating an apoptosis program. In

302

turn, the accumulation of ROS induced by ERS can activate PERK-mediated ATF4

303

encoding.40 To determine whether the antioxidant program participates in the ATF4 15



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downregulation induced by allicin, we established a mild ERS cell model and

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oxidative stress cell model using IPEC-J2 cells, treated them with 2.0 μg mL-1 allicin

306

for 12 h, and determined the levels of cellular ROS, malondialdehyde (MDA), and

307

sodium oxide dismutase (SOD). We found that allicin supplementation significantly

308

decreased the level of ROS in the ER-stressed cells but did not affect the levels of

309

MDA and SOD in ER-stressed cells. However, allicin supplementation significantly

310

decreased the levels of ROS and MDA and increased the level of SOD in the

311

oxidative-stressed cells, consistent with the findings of one previous study on the

312

anti-oxidant effect of allicin.41 This difference in activation of oxidant detoxifying

313

enzymes between the ERS model and the oxidative stress model indicates that the

314

mechanisms in which allicin participates may differ. These results suggest that allicin

315

supplementation decreased cellular ROS accumulation via ERS signaling instead of

316

by activating the oxidant detoxifying enzymes in the ER-stressed IPEC-J2 cells.

317

The PERK pathway can activate an antioxidant program by preferentially

318

translating mRNA and encoding the bZIP-containing transcription factor ATF4.42

319

Based on the finding that allicin decreases cellular ROS in ER-stressed cells, we

320

surmised that the p-eIF-2α downregulation by allicin might contribute to the

321

decreased level of ROS and the relaxation of this antioxidant program. To address this

322

point, we added H2O2 to the cell culture medium to supply excess cellular ROS during

323

the 12h treatments. We found that ATF4 was upregulated by the sole treatment with

324

H2O2 and was still downregulated by allicin. The p-eIF-2α was also downregulated by

325

allicin supplementation in IPEC-J2 cells that experienced both ERS and oxidative 16


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326

stress (data not shown). This result implies the participation of some crosstalk, but not

327

ROS-mediated ATF4 preferential encoding, in p-eIF-2α downregulation in the

328

allicin-treated ER-stressed cells. It points to a reduction in the cellular ROS in the

329

ER-stressed IPEC-J2 cells by allicin as a result of ERS alleviation, rather than as a

330

cause of p-eIF-2α downregulation.

331

STF-083010 has been reported27 to inhibit IRE1 activity without affecting kinase

332

activity under conditions of ERS. Here, STF-083010 was used as the specific IRE-1α

333

inhibitor to block the IRE-1α pathway to confirm the hypothesis that IRE-1α/XBP-1s

334

signaling is an essential component in allicin-treated ER-stressed jejunal cells. In this

335

study, we confirmed the inhibiting effect of STF-083010 on splicing of XBP-1 in

336

IPEC-J2 cells. Meanwhile, the effects of allicin in decreasing the levels of p-eIF-2α,

337

ATF4, GRP78, and ROS in the ER-stressed IPECs were diminished by STF-083010.

338

We conclude that allicin helps the jejunum alleviate mild ERS via activation of

339

IRE-1α/XBP-1s signaling.

340

One recent study43 reported that garlic extract inhibited the growth of various

341

cancer cells and upregulated GRP78 expression and PERK signaling, which seems to

342

contrast with the conclusion that allicin alleviates ERS in the jejunal cells. This

343

difference might be due to the various regulatory mechanisms of allicin in normal

344

cells and cancer cells. Similar cases were observed for allicin’s mechanism in the

345

regulation of oxidative stress. For example, previous reports have shown that allicin

346

sensitizes cancer cells to treatment by increasing the level of intracellular reactive

347

oxygen species (ROS),44 whereas allicin has been widely reported to reduce 17



Page 18 of 44

348

intracellular ROS levels and attenuate oxidative stress in normal cells.45 Another

349

possible reason for the difference is the different components and levels of allicin

350

were used between this study and the cancer study. A high concentration of allicin has

351

been shown to inhibit the proliferation of some species of cancer cells in vitro in a

352

dose-dependent manner,44,46 and proper allicin supplementation in animal feed

353

improved intestinal health.47 Consistent with these previous findings, the results of the

354

current study show that allicin improves intestinal morphological indices in porcine

355

jejunum. Recent studies48-49 reported that ERS and UPR-mediated AKT-cyclin D1

356

signaling can affect cell proliferation. One study revealed that relief of ERS promotes

357

the proliferation of pancreatic β cells via the AKT-Cyclin D1 axis.50 We speculated

358

that relief of spontaneous ERS in the intestine may improve cell proliferation, thereby

359

promoting intestinal development. This prompted us to investigate whether allicin has

360

any proliferative effect on the porcine jejunum or ERS-stressed IPEC-J2 cells.

361

However, we did not find any positive effect of allicin on the proliferation of the

362

jejunum or IPEC-J2 cells. Hence, we propose that allicin improves the intestinal

363

morphological indices of porcine jejunum possibly by attenuating ERS incidents in

364

the intestine, and the reduction of the negative influence of ERS contributes to

365

improving the intestinal morphological indices of porcine jejunum during

366

development. Another possibility is that allicin may reduce ERS-mediated apoptosis

367

when more extreme ERS been triggered in the intestine by unknown risk factors. To

368

clarify this possibility, we used models of prolonged ERS to explore the effects of

369

allicin on ERS-mediated apoptosis and associated mechanisms. In these prolonged 18


Page 19 of 44


370

ERS models, we detected that allicin reduced apoptosis not only in the TM-treated

371

jejunum in mice but also in the TM-treated IPEC-J2 cells. Caspase-12 is considered to

372

be necessary for apoptosis induced by a variety of ER-directed pro-apoptosis

373

signals,51 and CHOP signaling has been identified as a pro-apoptotic mechanism in

374

the downstream ERS and UPR pathways, as confirmed by the finding that cells

375

deficient in CHOP can survive prolonged ERS and its mediated apoptosis.52 In this

376

study, the expressions of caspase-12 and CHOP were activated in the jejunum of

377

TM-supplemented mice and in TM-treated IPEC-J2 cells. We also found that allicin

378

decreased the level of ATF4 and increased the level of XBP-1s in the prolonged ERS

379

models, and blockage of IRE-1α reversed the positive effects of allicin in attenuating

380

activation of the PERK pathway and apoptosis mediated by ATF4-CHOP-caspase12.

381

Thus, we propose that allicin can help jejunal cells survive the prolonged ERS by

382

enhancing the splicing of XBP-1.

383

The integrity of adherent and tight junction proteins between intestinal epithelial

384

cells is considered an indicator of a healthy intestinal barrier.53 In this study,

385

significant decreases in the levels of E-cadherin and ZO-1 were observed in the

386

prolonged ER-stressed IPEC-J2 cells, indicating that the barrier function of the

387

jejunum may be susceptible to prolonged ERS. Interestingly, allicin reversed the

388

downregulation of E-cadherin and ZO-1 induced by prolonged ERS stress in the

389

IPEC-J2 cells and in the jejunum tissue of mice, possibly because the allicin alleviates

390

ERS and maintains the normal functioning of the ER, particularly the folding of

391

intercellular junction proteins, and thus contributes to the integrity of the intestinal 19



392

Page 20 of 44

barrier.

393

In conclusion, our findings reveal that allicin administration attenuates ERS in

394

the jejunum of weaned piglets, in models of prolonged ERS in the jejunum of mice,

395

and in IPEC-J2 cell models of mild ERS and prolonged ERS. Levels of ERS signaling,

396

ERS-mediated apoptosis, and intercellular junction proteins were found to correlate

397

with the positive effects of allicin on intestinal health and development. Thus, the use

398

of garlic in cooking may alleviate the intestinal malfunction associated with ERS.

399

Garlic contains an average of 1.8% allicin, and some of the alliin in garlic can be

400

converted to allicin during cooking.54 Therefore, we recommend that adults consume

401

20 grams of garlic per day to maintain intestinal health or 80 grams per day to

402

alleviate intestinal malfunction. Further clinical studies are warranted to demonstrate

403

the curative effects of allicin in intestinal diseases induced by ERS and XBP-1s

404

inhibition.

405

ABBREVIATIONS USED

406

ATF4, Activating transcription factor 4; CCK-8, Cell Counting Kit-8; CHOP, C/EBP

407

homologous protein; DAPI, 4',6-diamidino-2-phenylindole; ERS, endoplasmic

408

reticulum stress; FI, fluorescence intensity; GRP78, glucose-regulated protein 78; HIF,

409

hypoxia-inducible factor 1; IPEC-J2, Intestinal Porcine Epithelial Cell line-J2; IRE-1α,

410

Inositol-requiring

411

malondialdehyde; p-eIF-2α, phosphorylation-eukaryotic Initiation Factor 2; PERK,

412

Protein kinase RNA-like endoplasmic reticulum kinase; PI3k, phosphatidylinositol 3

413

kinase; ROS, reactive oxygen species; SOD, sodium oxide dismutase; STAT3, signal

enzyme-1α;

JNK,

c-Jun

NH2-terminal

kinase;

MDA,

20


Page 21 of 44


414

transducer and activator of transcription 3; TM, tunicamycin; TUNNEL,

415

TdT-mediated dUTP nick end labeling; UPR, unfolded protein response; XBP-1s,

416

splicing x-box binding protein 1; ZO-1, zonula occludens-1.

417

ACKNOWLEDGEMENTS

418

The authors are thankful to the China Scholarship Council (CSC) for both financial

419

support and scholarships. The authors appreciate Dr. Chengbo Yang and Dr. Francis

420

Lin at the University of Manitoba for their help on this project.

421

AUTHOR INFORMATION

422

*Corresponding Author

423

E-mail address: [email protected] (Qian Jiang).

424

Funding

425

This work was supported by the National Natural Science Foundation of China

426

(31472107); the Youth Science Fund Project of the National Natural Science

427

Foundation of China (31702126); the Chinese Academy of Sciences ‘Hundred Talent’

428

award, the National Science Foundation for Distinguished Young Scholars of Hunan

429

Province (2016JJ1015); the Postgraduate Research and Innovation Project of Hunan

430

Province (CX2017B348); the Hunan Province “Hunan Young Science and

431

Technology Innovation Talent” Project (2015RS4053); the Hunan Agricultural

432

University

433

(YB2017002), and the Open Foundation of Key Laboratory of Agro-ecological

434

Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese

435

Academy of Sciences (ISA2016101).

Provincial

Outstanding

Doctoral

Dissertation

Cultivating

Fund

21



436

Notes

437

The authors have declared no conflict of interest.

Page 22 of 44

438 439

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621

Figure captions

622

Figure 1. The expression of ERS marker proteins in weaned piglets and fattening pigs.

623

A) Representative immunofluorescence photographs of the jejunum in weaned piglets

624

and fattening pigs. B) Relative fluorescence intensities of GRP78 and CHOP in the

625

jejunal tissues. C) Representative bands of Western blot analysis for the ERS marker

626

proteins (GRP78 and CHOP) in the jejunum from the weaned piglets and fattening

627

pigs. D) Relative abundance of GRP78 and CHOP in the jejunum. Values are

628

represented by means ± SD, n = 4. Differences between the treatments were

629

considered statistically significant when P < 0.01 (**) or P < 0.05 (*).

630

Figure 2. The effects of dietary allicin supplementation on the jejunal tissue of

631

weaned piglets. A) Representative HE-staining photographs of the jejunum in weaned

632

piglets. B) Relative ratios of villous height to crypt depth in the jejunal tissues. C)

633

Representative bands of Western blot analysis for the PERK pathway proteins (eIF-2α,

634

p-eIF-2α, and ATF4) in the jejunum of piglets. D) Relative abundance of eIF-2α,

635

p-eIF-2α and ATF4 in the jejunum. E) Representative bands of Western blot analysis

636

for the ERS marker proteins (GRP78 and CHOP) in the jejunum of weaned piglets. F)

637

Relative abundance of GRP78 and CHOP in the jejunum. Values are represented by

638

means ± SD, n = 4. Differences between the treatments were considered statistically

639

significant when P < 0.01 (**) or P < 0.05 (*).

640

Figure 3. The effects of allicin on ERS signaling proteins in the porcine jejunal tissue

641

and tunicamycin-pretreated intestinal porcine epithelial jejunal cell line J2 (IPEC-J2).

642

A) Representative bands of Western blot analysis for the XBP-1s, IRE-1α, and β 31



Page 32 of 44

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-actin in the jejunum tissues of the piglets supplemented with basic diet or 0.5%

644

allicin diet. B) Relative abundance of XBP-1s and IRE-1α in the jejunum. C)

645

Representative bands of Western blot analysis for the ERS signaling proteins (XBP-1s,

646

IRE-1α, GRP78, CHOP, eIF-2α, p-eIF-2α, ATF4, and Caspase-12) in the IPEC-J2

647

cells cultured with basic culture medium, 2.0 μg mL-1 allicin, tunicamycin

648

pretreatment or tunicamycin pretreatment with allicin. D) Relative protein abundance

649

of XBP-1s, IRE-1α, GRP78, CHOP in the treated IPEC-J2 cells. D) Relative protein

650

expressions of eIF-2α, p-eIF-2α, ATF4 and Caspase-12 in the treated IPEC-J2 cells.

651

Values are represented by means ± SD, n = 4. Differences between the treatments

652

were considered statistically significant when P < 0.01 (**) or P < 0.05 (*).

653

Figure 4. Effects of allicin on ROS concentration, SOD activity and MDA levels in

654

tunicamycin or hydrogen peroxide-pretreated IPEC-J2 cells. A). The relative

655

concentration of cellular ROS in the untreated cells (Control), 2.0 μg mL-1allicin

656

supplemented cells (Allicin), 0.5 μg mL-1 tunicamycin pretreated cells (TM

657

pretreated), and the 0.5 μg mL-1 tunicamycin pretreated cells with allicin

658

supplementation (TM pretreated + Allicin). B) SOD activity of the treated cells was

659

shown. C) MDA levels in control, Allicin, TM pretreated and TM pretreated + Allicin

660

cells were shown. D). The relative concentration of cellular ROS in the untreated cells

661

(Control), 2.0 μg mL-1 allicin supplemented cells (Allicin), 0.5 mM hydrogen peroxide

662

pretreated cells (H2O2 pretreated), and the 0.5 mM hydrogen peroxide pretreated cells

663

with allicin supplementation (H2O2 pretreated + Allicin). E) SOD activities in treated

664

cells were shown. F) MDA levels in the cells were shown. Relative fluorescence units 32


Page 33 of 44


665

were used as an indicator for relative concentrations of intracellular ROS. Values are

666

represented by means ± SD, n = 4. Differences between the treatments were

667

considered statistically significant when P < 0.01 (**) or P < 0.05 (*).

668

Figure 5. STF-083010 reversed the positive effects of allicin on the mild ER-stressed

669

IPEC-J2 cells. A) Representative bands of Western blot analysis for the XBP-1s levels

670

in the tunicamycin/ STF-083010 treated IPEC-J2 cells with the specific time point (0,

671

3, 6 and 12 hours). B) Relative concentration of cellular ROS in the untreated cells

672

(Control), 0.5 μg mL-1 tunicamycin pretreated cells (TM pretreated), the 0.5 μg mL-1

673

tunicamycin pretreated cells with allicin supplementation (TM pretreated + Allicin),

674

the 0.5 μg mL-1 tunicamycin-pretreated cells with allicin and STF-083010

675

supplementation (TM pretreated + Allicin + STF). C) Representative bands of

676

Western blot analysis for the p-eIF-2α, GRP78, XBP-1s, ATF-4 levels in the

677

tunicamycin pretreated IPEC-J2 cells with or without STF- 083010 supplementation.

678

E, F, G) Relative protein abundance of p-EIF-2 α , GRP78, XBP-1s, ATF-4 in the

679

specific treated IPEC-J2 cells. Values are represented by means ± SD, n = 4.

680

Differences between the treatments were considered statistically significant when P

Endoplasmic Reticulum Stress and Unfolded Protein Response

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