l-Arginine Protects Ovine Intestinal Epithelial Cells from

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Bioactive Constituents, Metabolites, and Functions

L-Arginine protects ovine intestinal epithelial cells from lipopolysaccharides-induced apoptosis through alleviating oxidative stress Hao Zhang, Along Peng, Yin Yu, Shuang Guo, Mengzhi Wang, and Hongrong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06739 • Publication Date (Web): 27 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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Journal of Agricultural and Food Chemistry

L-Arginine protects ovine intestinal epithelial cells from lipopolysaccharides-induced apoptosis through alleviating oxidative stress Hao Zhang,†,‡ Along Peng,†,‡ Yin Yu,†,‡ Shuang Guo,†,‡ Mengzhi Wang,†,‡ and Hongrong Wang*,†,‡ †Laboratory

of Metabolic Manipulation of Herbivorous Animal Nutrition,

College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P. R. China ‡Joint

International Research Laboratory of Agriculture and Agri-Product Safety,

the Ministry of Education of China, Yangzhou University, Yangzhou 225009, P. R. China.

*Address correspondence to HR-W (e-mail: [email protected]).

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ACS Paragon Plus Environment


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ABSTRACT: This research aims to explore the effect of l-arginine （ Arg ） upon

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lipopolysaccharides (LPS)-induced induction of the oxidative stress as well as

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subsequent apoptosis within ovine intestinal epithelial cells (IOECs). Through a 16 h

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incubation, cells were divided into four groups and the medium was replaced with

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different medium as follows: (1) Control (Con), Arg-free Dulbecco’s modified

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Eagle’s F12 Ham medium (DMEM); (2) Arg treatment, Arg-free DMEM

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supplemented with 100 µM Arg; (3) LPS treatment, Arg-free DMEM supplemented

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with 10 µg/mL LPS; (4) LPS with Arg treatment, Arg-free DMEM supplemented

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with both 10 µg/mL LPS and 100 µM Arg. After culturing for 24 hours in different

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mediums, some characteristics of cells in the four groups were measured. Addition of

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Arg increased cell viability induced with LPS compared with LPS group (p < 0.05).

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Arg significantly decreased the release of dehydrogenase (LDH) and the production

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of malonaldehyde (MDA) (p < 0.05) within IOECs challenged by the LPS. Compared

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with the LPS group, cells treated with Arg and Arg + LPS increased (p < 0.05)

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mRNA as well as protein expression of glutathione peroxidase 1 (GPx1), catalase

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(CAT), superoxide dismutase 2 (SOD2), B-cell lymphoma 2 (Bcl2), quinone

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oxidoreductase 1 (NQO1), heme oxygenase (HO-1), and nuclear factor erythroid

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2-related factor 2 (Nrf2). IOEC treatment with Arg reduced significantly (p < 0.05)

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apoptosis induced by the LPS (12.58 ± 0.79 %). The results showed that Arg

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promoted the protein expression of Nrf2, up-regulated expression of the phase II

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metabolizing enzymes (NQO1 and HO-1) as well as anti-oxidative enzymes (GPx1,

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CAT and SOD2) for alleviating oxidative injury, and protect IOECs from 2


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LPS-induced apoptosis.

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KEYWORDS:

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lipopolysaccharides

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INTRODUCTION

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Formed by one constant monolayer of differentiating and proliferating intestinal

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epithelial cells (IECs), intestinal mucosa epithelium is one basis barrier which is the

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first defense line of human body for protecting against external environment.1

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Intestinal homeostasis is kept by dynamic, and rigorously regulated epithelial cell

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migration and proliferation.2 However, oxidative stress might damage intestinal

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barrier,3 leading to one increased permeation of allergens as well as toxins.

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Considered as one imbalance state between antioxidant defense and reactive oxygen

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species (ROS) production, oxidative stress is capable of inducing apoptosis within in

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vivo and in vitro experiments.3-5 Normal cells have various mechanisms of defense,

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which include but are not limited to enzymatic antioxidant systems like glutathione

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peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and

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non-enzymatic antioxidant systems.6,7 It has been shown that lipopolysaccharides

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(LPS), which is one main integral component of gram-negative bacteria outer

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membrane, increased oxidative injury by generating lots of ROS, which could result

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in apoptosis and lipid peroxidation.8 The current research utilized LPS for establishing

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one cell injury model, in order to investigate the protective effect of l-arginine（Arg）

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upon ovine intestinal epithelial cells （IOECs）.

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l-arginine,

apoptosis,

antioxidant,

oxidative

stress,

Previous studies have demonstrated that supplementation with bioactive 3



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substance or dietary antioxidant could reduce oxidative stress effectively.9-13 Arg is a

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semiessential amino acid that plays a key role in several metabolic pathways, such as

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in the biosynthesis of nitric oxide, creatine, protein, and polyamines.14 Arg is also a

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precursor of nitric oxide, displays prooxidant and antioxidant properties, and

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contributes to the synthesis of cellular growth factors such as putresine, spermine, and

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spermidine.15 Arg stimulates NO production within physiological ranges in the small

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intestine and other tissues,16 and NO plays an important role in regulating the

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antioxidant defense system.17 Thus, supplementation with Arg helps scavenge the

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excess ROS induced by LPS, thereby improving the balance between the production

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of ROS (e.g., superoxideanion, hydrogen peroxide, and hydroxyl radical) and the

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biological defense against the toxicity of these oxidants. Within a majority of animals,

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Arg also plays a significant role of nutritional amino acid under the condition of stress

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which could be adopted for treating health and developmental problems. Arg

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functions as one signaling molecule which translates and transmits the change of

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dietary signal to gene expression through sensing mechanism via one nutrient sensing

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signaling pathway. More and more evidence suggests that Arg-NO pathway could

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regulate the metabolisms of nutrient sensing signals and energy substrates.9, 18

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Nevertheless, there is little research on if Arg could mitigate oxidative injury as

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well as decrease apoptosis within ovine intestine. We hypothesized that Arg decreases

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the oxidative injury induced by LPS, hence preventing the apoptosis of IOEC

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oxidative injury as well as reducing apoptosis within ovine intestine. For testing our

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hypothesis, LPS was utilized for establishing a cellular injury model for examining 4


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Arg protective effect upon the oxidative injury induced by LPS and IOEC apoptosis.

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MATERIALS AND METHODS

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Reagents. Antibiotics, fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s

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F12 Ham medium (DMEM) were bought in Invitrogen (Grand Island, New York,

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USA). 2.5% of trypsin solution and Arg-free custom-made DMEM (modified DMEM,

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GIBCO) were acquired in Gibco (Carlsbad, California, USA). Plastic culture plates

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were produced by Corning Incorporated (Corning, New York, USA) and epidermal

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growth factor was acquired in BD Biosciences (Bedford, Massachusetts, USA).

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Unless indicated, each of the other chemicals was bought in Sigma-Aldrich

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Corporation (Saint Louis, Missouri, USA).

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Cell Culture. The IOECs, a cell line derived from one neonatal lamb’s jejunum with

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no suckling, was cultured as described previously.19 To grow IOECs (passages 20–25),

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the cells were cultured within 75 cm2 plastic flasks with vented capos (BD Falcon)

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within DMEM which contained 5% FBS, 100 μg/mL streptomycin, 100 U/mL

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penicillin, 1% NEAA, 4 mm/L glutamine, 15 ng/mL EGF and 1× ITS and passaged

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every 3 d as previously described.20 We changed the medium every 2 d. At the

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confluence, the cells were seeded and trypsinized within 24-cell culture plates with

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about 2×104 cells per well. All cell cultures were carried out at 37 °C in a humid

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incubator that contained 5% CO2. When the confluence of cells was between 85% and

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95%, cells were passaged routinely (at 1:3 of split ratio).

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After incubating overnight (16 h), cells were starved within Arg-free

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custom-made DMEM (modified DMEM, GIBCO) for 6 h for maximizing the total 5



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amount of Arg within cells. 5% of FBS within Arg-free DMEM offered 10 𝜇M of

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Arg.

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Effects of Arg upon Cell Viability Challenged by LPS. IOECs were seeded within

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96-well plates (1×104/well) as well as cultured within complete DMEM. After

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incubating overnight (16 h), cells were starved within Arg-free DMEM for 6 h to

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maximize the amount of Arg in the cells, then IOECs were treated for 24 h

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(determined by our previous time-dependent experiment) with (1) Con, Arg-free

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DMEM; (2) Arg treatment, Arg-free DMEM supplemented with 100 μM Arg

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(determined by our previous dose-dependent experiment); (3) LPS treatment, Arg-free

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DMEM supplemented with 10 µg/mL LPS (determined by our previous

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dose-dependent experiment) (LPS from Escherichia coli serotype 055: B5, Sigma

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Chemical Inc., Saint Louis, Missouri, USA); (4) LPS with Arg treatment, Arg-free

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DMEM supplemented with both 10 µg/mL LPS and 100 μM Arg.

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CCK-8 assay was used to determine cell viability in accordance with the protocol

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of the manufacturer. In brief, IOECs were plated within 96-well culture plates at the

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density of 6,000 cells/well 24 h before the treatment. After culturing for 24 hours in

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the different mediums, all cells in the four groups were measured. All values were

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normalized to control and computed as described previously.21

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Determination of Malonaldehyde (MDA) and Dehydrogenase (LDH). After

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culturing for 24 hours in the different mediums, we gathered 1.0 mL of cell

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supernatant and stored it at -20 ℃ until it was analyzed. We rinsed the cells within

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6-well culture plates by PBS two times, and then used RIPA Lysis Buffer R2220 6


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(with 1% of PMSF) for lysing IOECs in accordance with manufacturer’s instruction.

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Bicinchoninic acid (BCA) protein assay reagent was used to determine cellular

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protein. LDH assay kit was used to determine the level of LDH from the Nanjing

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Jiangcheng Biotechnology Institute as reported previously.22 MDA assay kit was used

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to determine the level of MDA from the Nanjing Jiangcheng Biotechnology Institute.

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We performed each experiment for six times.

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Flow Cytometry. After a 24 h of incubation, flow cytometry was used to determine

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apoptotic rate utilizing Annexin V-FITC apoptosis assay kits (Nanjing KeyGen

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Biotech Company, Nanjing, China) in accordance with the instruction. We defined the

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apoptotic cells as cells located at right two quadrants of all plots and flow cytometry

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(FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA)

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was used to determine the percentage. CELL Quest software was used to analyze the

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data, and we performed each experiment for six times.

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Determination of CAT, total antioxidant capacity (T-AOC), SOD, GSH-Px

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Levels. After a 24 h of incubation, ultrasound treatment was used to break up cells,

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then they were subjected to the centrifugation to obtain the supernatant. We gathered

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1.0 mL of cell supernatant and stored it at -20 ℃ until it was analyzed. We gently

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rinsed the cells within 6-well culture plates using PBS two times, then the RIPA Lysis

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Buffer R2220 (with 1% of PMSF) was utilized for lysing IOECs in accordance with

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manufacturer’s instruction. Bicinchoninic acid (BCA) protein assay reagent was used

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to determine cellular protein at 562 nm in accordance with manufacturer’s instruction.

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We stored the protein sample extracted at -20℃ until it was analyzed. SOD, GSH-Px, 7



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CAT and T-AOC assay kits from Nanjing Jiangcheng Biotechnology Institute were

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used to determine the level of SOD, GSH-Px, CAT and T-AOC within cells and the

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supernatants of cells respectively, as reported previously.23,24 We performed each

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experiment for six times.

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Real-Time PCR (RT-PCR) Analysis on Gene Expression. We isolated total RNA

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from treated cells by Trizol reagent (Sigma, Saint Louis, Missouri, USA) in

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accordance with manufacturer’s protocol. We conducted real-time PCR as previously

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described.23,25 We utilized 𝛽-actin as invariant control. We calculated the fold changes

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of the level of mRNA of targeted genes associated with 𝛽-actin as Schmittgen et al

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recommended.26 Experiments were performed 6 times. Table 1 shows the genes’

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primers (Sangon Biotech, Shanghai, China).

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Western Blotting. After LPS/Arg treatment for 24 h, we gently rinsed the cells

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within 6-well culture plates for two times, then we lysed the IOECs by RIPA Lysis

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Buffer R2220 (with 1% of PMSF) in accordance with manufacturer’s instruction.

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BCA protein assay reagent was used to determine the protein concentration of cells at

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562 nm in accordance with manufacturer’s instruction. Equivalent number of protein

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samples of the cell lysate was loaded for SDS-PAGE, then these samples were

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transferred to the membrane of PVDF which was blocked with the PBST buffer that

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contained 5% of skim milk at indoor temperature for 1 h, followed by overnight

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hybridization at 4 ℃

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anti-GPx1, anti-SOD2, anti-Nrf2, anti-HO-1, anti-NQO1, anti-P53, anti-Fas, anti-Bcl2,

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anti-Bax, anti-β-actin and anti-Caspase 3. After incubating for 1 h with the secondary

along with the indicated primary antibodies: anti-CAT,

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antibody (HRP goat anti-rabbit IgG), we detected signals by enhanced

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chemiluminescence kits (ECL-Plus, Thermo, Waltham, Massachusetts, USA), then

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we scanned them for fluorescence detection by BioRad gel detection system.

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densitometric value was normalized to β-actin as well as expressed as one relative

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level to the control value. We conducted each experiment for six times. 24

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Statistical Analysis. Results, expressed as means with pooled standard error of the

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means (SEMs), were analyzed using 1-factor ANOVA. We performed Duncan’s

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multiple range test for measuring the difference between different treatment methods.

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Statistical analysis was carried out through the SAS software of 9.2 version.

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Probability value ≤ 0.05 was used to represent significance.

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RESULTS

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Cell Growth. In comparison with the Con, addition of LPS dramatically decreased

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the cell viability (p < 0.05) (Table 2). Addition of arginine increased cell viability

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induced with LPS in comparison to LPS group (p < 0.05). Relative to Con group, cell

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viability was greater (p < 0.05) within Arg group.

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LDH and MDA. The amount of LDH that was released in cells and medium was

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larger (p < 0.05) within IOECs with LPS treatment (Table 3), and MDA level within

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cells and medium also was higher (p < 0.05) within IOECs with LPS treatment. Arg

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significantly reduced the release of LDG and production of MDA (p < 0.05) within

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IOECs challenged by the LPS.

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Antioxidation Capacity. In comparison with the Con, addition of LPS significantly

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reduced CAT, T-AOC, SOD and GSH-PX within supernatants and cells (p < 0.05) 9


Each


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(Table 4). While the cells with Arg and LPS treatment markedly raised the level of

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CAT, T-AOC, SOD and GSH-PX in comparison with those with LPS treatment (p

l-Arginine Protects Ovine Intestinal Epithelial Cells from

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