Ochratoxin A-Induced Apoptosis of IPEC-J2 Cells through ROS

Nov 14, 2017 - Hong Wang†‡, Ying Chen†‡, Nianhui Zhai†‡, Xingxiang Chen†‡, Fang Gan†‡, Hu Li†‡, and Kehe Huang†‡. †Colle...
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Ochratoxin A induced apoptosis of IPEC-J2 cells through ROSmediated mitochondrial permeability transition pore opening pathway Hong Wang, Ying Chen, Nianhui Zhai, Xingxiang Chen, Fang Gan, Hu Li, and Kehe Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04434 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Journal of Agricultural and Food Chemistry

Ochratoxin A induced apoptosis of IPEC-J2 cells through ROS-mediated mitochondrial permeability transition pore opening pathway Hong Wang

a, b

, Ying Chen a, b, Nianhui Zhai a, b, Xingxiang Chen

a, b

, Fang Gan a, b, Hu Li a, b,

Kehe Huang a, b * a

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu

Province, China b

Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing

Agricultural University, Nanjing 210095, Jiangsu Province, China

*Corresponding author: Prof. Kehe Huang Tel: +86-025-84395507; Fax: +86-25-84398669 E-mail address: [email protected]

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ABSTRACT

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With the purpose to explore the mechanisms associated with the intestinal toxicity of

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Ochratoxin A (OTA), an intestinal porcine epithelial cell line (IPEC-J2) was applied in this

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study as in vitro models for intestinal epithelium. The results confirmed that OTA induced

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IPEC-J2 cell toxicity by MTT assay and apoptosis by Hoechst 33258 staining and flow

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cytometer analysis. And then, we observed that OTA induced the mitochondrial reactive

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oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP)

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opening by confocal microscopy. Western blot showed that OTA induced cytochrome c (cyt-c)

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release and caspase-3 activation, which could be suppressed by inhibition of mPTP opening

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with cyclosporin A. Treatment with Mito-TEMPO, the mitochondria-targeted ROS scavenger,

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blocked OTA-induced mitochondrial ROS generation and mPTP opening and prevented cyt-c

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release, caspase-3 activation and apoptosis in IPEC-J2 cells.

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KEYWORDS: Ochratoxin A; IPEC-J2 cells; mitochondrial reactive oxygen species;

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mitochondrial permeability transition pore opening; apoptosis.

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INTRODUCTION

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Ochratoxin A (OTA), a natural occurring mycotoxin produced by fungi of Aspergillus and

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Penicillium genera, can contaminate feed and food and raw materials, such as forages,

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cereals, fruits and pork1. Because of the wide distribution of Aspergillus and Penicillium,

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animals or humans are at high risk of exposure to OTA through ingestion of contaminated

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feed and foodstuff. OTA has been shown to have a diversity of toxic effects, such as

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nephrotoxicity, immunotoxicity, hepatotoxicity, teratogenicity and carcinogenicity2, 3. OTA

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contamination of feed and foodstuff are thus considered to affect animal and human health

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and bring economic losses.

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As known, the intestinal epithelium represents the first barrier to the access of microbial

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pathogens, food contaminants and natural toxins to the whole body4. Due to its location and

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function, the intestinal epithelium is delicate and vulnerable, and this constantly renewing

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organ may be a potential target organ for food-associated mycotoxins5. OTA has been found

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to damage the intestinal epithelium in vivo studies in chicken, rat6-8. The cytotoxicity of OTA

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has also been reported in vitro studies with some transformed human intestinal epithelial cell

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line, including Caco-2 and HT-29-D4 cells9, 10. However, the research on OTA toxicity in the

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intestinal epithelium is still relatively lack and the mechanisms responsible for the intestinal

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toxicity is still poorly understood.

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It has been found that OTA could inhibit mitochondrial respiration chain and promotes

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reactive oxygen species (ROS) generation11, 12. ROS-mediated oxidative stress is generally

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considered be a potential common mediator of apoptosis13,

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. The role of ROS in

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OTA-mediated cytotoxicity has been reported in various cell types. Bhat et al. found OTA

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elevated ROS generation and then induced apoptosis in Neuro-2a cells15. Palma et al. showed

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that ochratoxin A could promotes the production of oxidative stress, which causes

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mutagenesis in mammalian cells16. Thus we suppose that the intestinal toxicity of OTA may

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be associated with its generation of ROS.

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It is known that excessive production of ROS could directly induce mitochondrial

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permeability transition pore (MPTP) opening17. Specially, the opening of mPTP leads to the

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increase in mitochondrial permeability and the release of cytochrome c (cyt-c) from the

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mitochondria to the cytosol18. In general, cyt-c is stably located in the mitochondrial

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intermembrane/intercristae spaces, which works as an electron donor for cyt-c oxidase.

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However, during early stage of apoptosis, it also releases from from the mitochondria to the

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cytosol, where it helps to activate the caspase-319. Caspase-3 is one of the most commonly

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shared downstream executioner in extrinsic and intrinsic apoptotic pathways20, 21. Normally,

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it is an inactive, exclusively cytosolic homodimer. While, during apoptosis, it is activated

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through cleavage into active fragment (cleaved caspase-3)22.

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The IPEC-J2 cell line, a non-transformed intestinal cell line originally derived from jejunal

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epithelia isolated from a neonatal, unsuckled piglet23, 24, was applied as an in vitro model of

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jejunal epithelial cells in this study. The aim of this study was to determine the toxicity of

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OTA on IPEC-J2 cells and provide mechanistic explanations for its toxic effects on the

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jejunal epithelial cells.

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MATERIALS AND METHODS 4 ACS Paragon Plus Environment

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Chemicals

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EGTA, Mito-TEMPO, Ochratoxin A (OTA), trypsin, cyclosporin A (CsA), CoCl2 were

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obtained from Sigma-Aldrich, USA. Annexin V-FITC apoptosis detection kit was purchased

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from Becton Dickinson Company, USA. Enhanced chemiluminescence (ECL) kit, Dimethyl

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit, Sulphoxide

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(DMSO), Bicinchoninic acid (BCA) protein assay kit and Hoechst 33258 were obtained from

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Beyotime Institute of Biotechnology (Haimen, China). Mito-tracker Red, MitoSOX Red and

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calcein AM were purchased from molecular probes (Eugene, OR, USA). USA. DMEM/F-12

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medium and fetal bovine serum were purchased from Thermo Fisher Scientific (MA, USA).

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Cell culture and OTA treatment

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The IPEC-J2 cells (kindly provided by Professor. Zhanyong Wei, Henan Agricultural

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University, Zhengzhou, China) were cultured in an incubator at 37 °C with 5% CO2 using

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DMEM/F12 medium, which contained HEPES (25 mM), Streptomycin (100 µg/ml),

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Penicillin (100 U/ml) and 10% fetal bovine serum.

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Differentiated IPEC-J2 cells are less susceptible to mycotoxins compared to proliferating

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cells25 26. In this study, we used the proliferating IPEC-J2 cells to investigate the cytotoxicity

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of OTA. More specifically, IPEC-J2 cells were seeded on 6- or 96-well plates in DMEM/F12

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supplemented with 10% FBS and then exposed to different doses of OTA when cells

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proliferated to 70 - 80% confluence. Purified OTA purchased from Sigma-Aldrich was

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diluted in DMSO to make a stock solution and further diluted in the culture media at the

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indicated concentrations. IPEC-J2 cells were seeded on 96-wells plates and then exposed to

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OTA to increasing concentrations of OTA (0-32 µM) for 6, 12 and 24 h when the confluence

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approached 70-80%. According the MTT results, IPEC-J2 cells were exposed to 2, 4 and 8

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µM OTA for 12h in the subsequent experiments.

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Cell viability assay

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IPEC-J2 cells were plated in 96-well plates and the exposed to different doses of OTA when

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the confluence approached 70-80%. After treatments, cell viability was determined by MTT

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assay. The cells were incubated with MTT solution for 4 h, and then solubilized in DMSO.

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Absorbance was read by a spectrophotometer at 570 nm.

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Analysis of apoptosis by Hoechst 33258 staining and flow cytometry

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Hoechst 33258 was applied to detect the apoptotic morphological changes of IPEC-J2 cells.

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Cells were stained with Hoechst 33258 solution for 10 min and then washed. After staining,

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apoptotic morphological changes were detected under a fluorescence microscope (Zeiss).

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The quantitative analysis of apoptosis was determined by flow cytometry analysis. After

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being washed with cold-PBS, IPEC-J2 cells were incubated with FITC-annexin V and PI

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following the manufacturer’s instructions. The percent of apoptosis were analyzed using

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FACSCalibur flow cytometry (BD Biosciences).

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Measurement of ROS production

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H2DCF-DA probe was applied to detect the intracellular ROS levels. Cells were incubated

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with H2DCF-DA (10 µM) for 30min at 37 °C in dark. H2DCFDA could be oxidized into

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green fluorescent DCF by ROS generation27. To confirm the primary site of ROS production,

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cells were also staining with 50 nM Mito-tracker Red. The mitochondrial ROS levels were

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detected using MitoSOX red (5 µM). Cells were staining with MitoSOX red at 37°C for 20

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min. The fluorescence signals of DCF, Mito-tracker Red and MitoSOX Red were detected by

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confocal microscope (Zeiss).

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Assessment of mPTP opening

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The mPTP opening was detected by the calcein-AM/cobalt assay28. After treatment, cells

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were incubated with calcein-AM (1 µM) for at 37 °C 30 min, and then exposed to CoCl2 (1

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mM) for another 10 min. The calcein loaded into mitochondria was preserved and the

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cytosolic calcein was quenched by CoCl2. The fluorescence signal was visualized confocal

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microscope (Zeiss).

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Western blotting analysis

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After treatment, cells were harvested and then lysed in cold-RIPA buffer (Beyotime, China)

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to obtain the total cell lysates. Meanwhile, the cytosolic protein fraction for detecting cyt-c

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was separated by a cytoplasmic protein extraction kit following the manufacturer’s

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instruction (Beyotime, China). 40 µg of protein samples were prepared for electrophoresis on

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SDS-PAGE, and then transferred to PVDF membranes (Millipore, Molsheim, France). The

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membranes were incubated with primary antibodies: rabbit anti-cyt-c (#11940, 1:1000, CST),

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rabbit anti-cleaved caspase-3(#9664,1:1000, CST) and rabbit anti-β-Actin (#4970, 1:1000,

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CST) overnight at 4 °C and then, the membranes were incubated with HRP-labeled Goat

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Anti-Rabbit IgG (A0208, Beyotime, China) for 1 h at room temperature. Finally,

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immunoreactivity was detected by ECL chemoluminescence kit. The blots were visualized

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by a Luminescent Image Analyzer (FUJIFILM LAS-4000). The density of each band was

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quantified using Quantity One software (Biorad, France) and normalized to its respective

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loading control (β-actin). The final data were expressed as the ratio of the intensity of the

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protein in treated cells to that of the corresponding protein in control cells. Each test was

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performed in four experiments with different batches of cells.

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Data presentation

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Experiments were performed at least three times with similar results. Data are presented as

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the mean ± SEM of the indicated number of replicates. Statistical comparisons were made

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using one-way analysis of variance (ANOVA) (Scheffe’s F test) after ascertaining the

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homogeneity of variance between the treatments, and P < 0.05 was regarded as significant.

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RESULTS

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Cytotoxic effects of OTA on IPEC-J2 cells viability

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To investigate OTA-induced cytotoxicity, IPEC-J2 cells were exposed to increasing

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concentrations of OTA (0-32 µM) for 6, 12 and 12 h. MTT assay was applied to measure the

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viability of cells. As shown in Figure 1, higher concentrations of OTA and longer exposure

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times exhibited more serious cytotoxicity in IPEC-J2 cells. It was observed that OTA

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significantly effected the viability of IPEC-J2 cells at 16 and 32 µM for 6 h (Figure 1a). For

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the data regarding 12 h treatment, OTA induced a significant decrease in viability of IPEC-J2

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cells at concentrations between 2 and 32 µM (Figure 1b). The IC50 value (evaluated after 12h)

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of OTA against IPEC-J2 cells was 19.3 µM. 24 h treatment with OTA resulted more apparent

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reduction in viability of cells (Figure 1c). However, we observed that most cells were

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detached after 24 h treatment with 4 µM OTA and above, which made it unfavorable for the

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following testes. In the subsequent experiments, IPEC-J2 cells were exposed to 2, 4 and 8

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µM OTA for 12 h.

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OTA induced apoptosis of IPEC-J2 cells

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Hoechst 33258 staining and flow cytometry were used to detect the apoptosis in IPEC-J2

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cells. As shown in Figure 2a, typical morphological nuclear changes of apoptosis were

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observed by fluorescence microscopy of Hoechst 33258 staining. In addition, the flow

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cytometry analysis exhibited that OTA induced apoptosis of IPEC-J2 cells in a

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concentration-dependent manner (Figure 2b and c).

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OTA promoted mitochondrial ROS production and MPTP opening

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Intracellular ROS probe, H2DCF-DA, was applied to asses IPEC-J2 cells ROS production.

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Gradual increase of DCF fluorescence in IPEC-J2 cells with increasing concentrations of

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OTA was observed after the treatment, suggesting excessive ROS was accumulated.

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Compared with the control, a concentration-dependent generation of DCF fluorescence

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elevated at 12 h (Figure 3a and b). Overlay images of cells labeled with H2DCF-DA and

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Mito Tracker Red suggested that mitochondria may be the major site of intracellular ROS

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generation in IPEC-J2 cells exposed to OTA (Figure 3c). Therefore, next we utilized a

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specific indicator of mitochondrial ROS, MitoSOX Red, to detect mitochondrial ROS. As

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shown in Fig 3d and e. OTA exposure results in a significant elevation in mitochondrial ROS

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generation. The above results confirmed the contribution of mitochondria to OTA-mediated

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ROS generation in IPEC-J2 cells.

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Then we measured mPTP opening by the CoCl2-calcein fluorescence-quenching assay. On

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the opening of the mPTP, entrapped calcein is released from the mitochondrial matrix and

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CoCl2 quenched the cytosolic calcein but not that of mitochondria, which do not transport

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cobalt. As shown, a concentration-dependent reduction in mitochondrial calcein fluorescence

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was detected in OTA-treated IPEC-J2 cells (Figure 3f and g), indicated the opening of mPTP

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opening.

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OTA caused cytochrome c release and activated caspase-3 in IPEC-J2 cells

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Since OTA-induced apoptosis was accompanied by mitochondrial ROS generation and

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mPTP opening, we examined whether OTA induced apoptosis via a mitochondria-mediated

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pathway. As known, the release of cyt-c from mitochondria to cytosol and subsequent

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caspase-3 activation represent a critical step in the mitochondria-mediated apoptosis pathway.

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As shown, OTA treatment noticeable elevated the protein levels of cytosolic cyt-c and

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activated caspase-3 as evident by its cleavage (Figure 4a and b).

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Involvement of mPTP opening in OTA-induced apoptosis

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CsA, a potent inhibitor of mPTP, was applied to assess the role of MPTP opening on

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OTA-induced apoptosis. As shown in Figure 5, OTA-induced mPTP opening was markedly

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blocked by CsA. When mPTP opening was inhibited, OTA-induced cyt-c release was

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significantly decreased, indicating that OTA-induced cyt-c release was dependent on mPTP

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opening. Importantly, inhibition of mPTP opening with CsA also suppressed OTA-induced

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caspase-3 activation and apoptosis. Given the above results, it can be concluded that OTA

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induced apoptosis of IPEC-J2 cells in via the mitochondrial pathway

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OTA-induced mitochondrial ROS production and mPTP opening were reversed by

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Mito-TEMPO

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To examine the role of ROS on OTA-induced mPTP opening, cells were pre-incubated with

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the

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mitochondrial ROS production was markedly suppressed by Mito-TEMPO (Figure 6a, b).

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Besides, Mito-TEMPO significantly suppressed OTA-induced reduction in fluorescence of

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calcein, confirming the involvement of ROS on OTA-induced mPTP opening (Figure 6c, d).

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Role of mitochondrial ROS in OTA-induced mitochondria-dependent apoptosis

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To ascertain the correlation between OTA-induced ROS production and apoptosis,

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mitochondria-targeted antioxidant Mito-TEMPO was applied. As shown, Mito-TEMPO

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significantly suppressed the OTA-induced cyt-c release, caspase-3 activation and

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subsequently reversed OTA-induced apoptosis in IPEC-J2 cells (Figure 7). Overall, these

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data demonstrated that the elevation in mitochondrial ROS production acts as an important

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role in OTA-induced apoptosis.

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DISCUSSION

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Generally, exposure to Ochratoxin A (OTA) occurs through ingestion of contaminated

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foodstuff29. The small intestine has been found to be the major site of OTA absorption, with

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maximal absorption at the proximal jejunum30. As known, the intestinal epithelium represents

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the first barrier that protect the host against the penetration of diverse food contaminants.

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Due to its location and function, the intestinal epithelium may be a potential target for the

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toxic effect of OTA after intake of OTA contaminated food and feed. It is thus of importance

mitochondria-targeted antioxidant Mito-TEMPO.

As expected,

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OTA induced

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to study the toxic effects of OTA on the intestinal epithelium.

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It has been reported that OTA could inhibit cell growth and induced cell death in various cell

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types31-33. A porcine epithelial intestinal cell line (IPEC-J2) was used as in vitro models for

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the jejunal epithelial cells in this study. We observed that OTA could induce cytotoxicity in

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IPEC-J2 cells (Figure1). This result confirmed intestinal toxicity of OTA in vitro. As known,

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cell death can generally be produced via apoptosis and necrosis34. In this study, we focused

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on the induction of apoptosis by OTA on IPEC-J2 cells. As shown in Figure2, OTA elevated

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the percentages of apoptotic cells in a dose-dependent manner. These results suggested that

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OTA-induced apoptosis may be play an important role in OTA intestinal toxicity.

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A number of mechanisms has been put forward to account for OTA toxicity, including

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inhibition of protein synthesis, inhibition of mitochondrial respiration, generation of reactive

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oxygen species (ROS)35. Data in our study exhibited that OTA exposure could induce the

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production of ROS, as indicated by the progressive increase in H2DCF-DA fluorescence

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(Figure 3a and b). The merged images of H2DCF-DA and Mito Tracker Red suggested that

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mitochondria may be the major site of intracellular ROS generation induced by OTA (Figure

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3c). In mammalian cells, mitochondria are often assumed as the main source of ROS36, 37.

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Consistent with this idea, mitochondrial ROS generation induced by OTA was confirmed by

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MitoSOX Red, which showed a significant increase in fluorescence (Figure 3d and e).

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Interestingly, mPTP opening was observed in IPEC-J2 cells during OTA exposure, as

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evidenced by a reduction in mitochondrial calcein fluorescence (Figure 3f and g).

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OTA-induced apoptosis of IPEC-J2 cells was accompanied by increased ROS generation and

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mPTP opening, which enables us to think whether OTA induced apoptosis via

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mitochondria-mediated pathway.

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It is well known that mitochondria play an important role in the regulation of cellular death38,

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39

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opening. And through integration of diverse intracellular signals, mPTP serves as a gate that,

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once switched-on, trigger the apoptotic process40, 41. Specially, the opening of mPTP leads to

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the increase in mitochondrial permeability, resulting in the release of apoptotic factors such

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as cyt-c from the mitochondria to the cytosol42, 43. The present research found that OTA

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promoted the release of cyt-c into the cytosol in IPEC-J2 cells (Figure 4a). Once cyt-c is

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released from mitochondria to cytosol, it leads to activation of the caspase cascades, and

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finally leads to apoptosis44. Caspase 3 is one of the most common apoptosis executioners45.

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In this study, our data showed that caspase-3 was activated during OTA exposure (Figure 4b).

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Moreover, the release of cyt-c, caspase-3 activation and apoptosis induced by OTA were

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markedly inhibited by CsA, an inhibitor of mPTP (Figure 5). The above results suggested

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that OTA could induced apoptosis of IPEC-J2 cells via the mitochondrial pathway.

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To

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mitochondria-targeted antioxidant, was applied to block ROS production. As expected,

248

OTA-induced ROS production was markedly suppressed by Mito-TEMPO (Figure 6a and b).

249

Importantly, OTA-mediated MPT opening was significantly blocked by Mito-TEMPO

250

(Figure 6c and d), which provided evidence that mitochondria generated ROS are involved in

251

mMPT induction. We next wanted to examine whether mitochondrial ROS generation is an

. Excessive production of ROS could induce mitochondrial permeability transition pore

determine

the

role

of

ROS

on

OTA-induced

apoptosis,

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upstream event of OTA-induced apoptosis of IPEC-J2 cells. Previous studies have shown

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that elevated levels of mitochondrial ROS is sufficient to trigger apoptosis38, 46. In this study,

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the release of cyt-c, caspase-3 activation and subsequent apoptosis induced by OTA were

255

markedly inhibited by Mito-TEMPO, respectively (Figure 7). These results confirmed the

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involvement of mitochondrial ROS in OTA-induced apoptosis of IPEC-J2 cells. This result

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suggests us that application of antioxidants may be a practical and effective way to protect

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the intestinal injuries induced by OTA, which will be worthy of further investigation.

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In summary, OTA induces apoptosis of IPEC-J2 cells cells via ROS-dependent mitochondrial

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apoptosis pathways by inducing mPTP opening, thereby promoting cytochrome c release and

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caspase-3 activation.

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ABBREVIATIONSA

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OTA, Ochratoxin A; ROS, reactive oxygen species; mPTP, mitochondrial permeability

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transition pore; cyt-c, cytochrome c; DMSO, Dimethyl Sulphoxide; (DMSO); BCA,

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Bicinchoninic

acid;

266

acetoxymethyl

ester;

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Medium/Nutrient Mixture F-12; FBS, fetal bovine serum.

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AUTHOR INFORMATION

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Corresponding Author

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*Tel: +86-25-84395507. Fax: +86-25-84398669. E-mail: [email protected].

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ORCID

272

Kehe Huang: 0000-0003-4132-3052

ECL,

enhanced

(Calcein-AM),

chemiluminescence; DMEM/F-12,

Calcein-AM,

Dulbecco's

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Calcein Eagle

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Funding

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This work was funded by the National Key R & D Program (2016YFD0501203), the

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National Natural Science Foundation of China (31472253) and the Priority Academic

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Program Development of Jiangsu Higher Education Institutions (Jiangsu, China).

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Notes

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The authors declare no competing financial interest.

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Figure Captions

434

Figure 1. Effect of OTA on IPEC-J2 cell viability. IPEC-J2 cells were cultured with OTA

435

for (a) 6 h, (b) 12 h and (c) 24 h at 0.5, 1, 2, 4, 8, 16 and 32 µM. MTT assay was applied to

436

determine cell viability. Data are expressed as the percentage compared to the control group

437

(set as 100%), means ± SEM of three independent experiments (n=6). *P < 0.05, **P < 0.01

438

as compared to control.

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Figure 2. OTA-induced apoptosis in IPEC-J2 cells. Cells were exposed with OTA (2, 4 and

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8 µM) for 12 h. (a) Morphology of apoptotic cell nuclei was measured by Hoechst 33258

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staining. Arrows denote condensed nuclei. Among the groups, A: control; B: 2 µM OTA; C:

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4 µM OTA; D: 8 µM OTA. (b) The representative plots of flow cytometry apoptosis. (c)

443

Quantification of apoptosis using flow cytometer. Data in (c) are presented as means ± SEM

444

of three independent experiments (n=9). *P < 0.05, **P < 0.01 as compared to control.

445

Figure 3. OTA-induced ROS production and mPTP opening in IPEC-J2 cells. IPEC-J2 cells

446

were exposed with OTA (2, 4 and 8 µM) for 12 h, and then stained with H2DCF-DA (for

447

measurement of intracellular ROS) or MitoSOX Red (for measurement of mitochondria ROS)

448

or calcein-AM (for measurement of mPTP opening). (a, b) Shown are representative

449

confocal images for DCF staining. The average fluorescence intensity was analyzed using

450

Image-Pro Plus software and expressed as the percentage compared to the control group (set

451

as 100%). (c) Cells were stained with H2DCF-DA and MitoTracker Red (red, localizes to

452

mitochondria). Overlay images (yellow) indicated mitochondria are the major source of

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intracellular ROS induced by OTA. (d, e) Shown are representative confocal images for

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MitoSOX Red staining. The average fluorescence intensity was analyzed using Image-Pro

455

Plus software and expressed as the percentage compared to the control group (set as 100%).

456

(f, g) Shown are representative confocal images for calcein staining. The average

457

fluorescence intensity was analyzed using Image-Pro Plus software and expressed as the

458

percentage compared to the control group (set as 100%). Among the groups (a, d, f), A:

459

control; B: 2 µM OTA; C: 4 µM OTA; D: 8 µM OTA. Data in (b, e and g) represent mean ±

460

SEM of three separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control.

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Figure 4. Release of cyt-c into the cytoplasm and caspase-3 activation in OTA-treated

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IPEC-J2 cells. The protein level of cyt-c in the cytoplasm (a) and active caspase-3 (b) were

463

detected by western blot. Representative images were shown (upper panels), and quantitative

464

data (lower panels) were performed with images of four independent experiments (mean ±

465

SEM, n = 4). *P < 0.05, ** P < 0.01 as compared to control.

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Figure 5. Effect of CsA on OTA-induced apoptosis. (a, b) Cells were treated with OTA (4

467

µM) and/or CsA (5 µM) for 12 h and then incubated with calcein-am. (a) Shown are

468

representative confocal images for calcein staining. (b) The average fluorescence intensity

469

was analyzed using Image-Pro Plus software and expressed expressed as the percentage

470

compared to the control group (set as 100%). Data in (b) represent mean ± SEM of three

471

separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control. (c, d) The protein

472

level of cyt-c in the cytoplasm and active caspase-3 were detected. Representative images of

473

western blot were shown (upper panels), and quantitative data (lower panels) were performed

474

with images of four independent experiments (mean ± SEM, n = 4). The apoptosis was assed

475

using Hoechst 33258 staining (e) and flow cytometry (f). Data in (f) are presented as means ±

476

SEM of three independent experiments (n=9). *P < 0.05, **P < 0.01 as compared to control.

477

Among the groups (a, e), A: control; B: 4 µM OTA; C: 4 µM OTA and 5 µM CsA; D: 5 µM

478

CsA. D

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Figure 6. Effect of Mito-TEMPO on OTA-induced mitochondrial ROS production and

480

mPTP opening. Cells was pre-incubated with Mito-TEMPO (10 µM) for 1 h before a 12 h

481

treatment with OTA. MitoSOX Red (a) and calcein (c) flourence were detected by confocal

482

microscope. Shown are representative confocal images. The average fluorescence intensity

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was analyzed using Image-Pro Plus software and expressed expressed as the percentage

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compared to the control group (set as 100%). Data in (b and d) represent mean ± SEM of

485

three separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control. Groups (a,

486

c), A: control; B: 4 µM OTA; C: 4 µM OTA and 10 µM Mito-TEMPO; D: 10 µM

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Mito-TEMPO.

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Figure 7. Mito-TEMPO inhibited OTA-induced apoptosis. Cells was pre-incubated with

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Mito-TEMPO (10 µM) for 1 h, followed by treatment with OTA for 12 h. (a, b) Protein

490

expression levels of cyt-c in the cytoplasm, active caspase-3 and β-Actin were analyzed by

491

western blot. Representative images of western blot were shown (upper panels), and

492

quantitative data (lower panels) were performed with images of four independent

493

experiments (mean ± SEM, n = 4). The apoptosis was measured by Hoechst 33258 (c) and

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flow cytometry (d). A: control; B: 4 µM OTA; C: 4 µM OTA and 10 µM Mito-TEMPO; D:

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10 µM Mito-TEMPO. Data are presented as mean ± SEM of three separate experiments, each

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one performed in triplicate (n = 9). **P < 0.01 as compared to control group; #P < 0.05 as

497

compared with OTA alone group.

498 499 500 501 502 503

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Figure graphics: Figure 1 (a) 6h

(b) 12 h

(c) 24 h

Figure 2

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Figure 4

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(b)

(a) OTA (µM)

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OTA (µM)

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Active caspase-3

Cyt-c

β-actin

β-actin

Figure 5 (a)

(b)

(c) OTA (µM)

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4

4

0

CSA (µM)

0

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Cyt-c β-actin

(d)

(e)

OTA (µM)

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Active caspase-3

β-actin

Figure 6

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(a) OTA (µM) MitoTEMPO (µM)

0 0

4 0

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cyt-c

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OTA (µM) MitoTEMPO (µM)

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