Oral Administration of Bovine Lactoferrin-Derived Lactoferricin (Lfcin

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Bioactive Constituents, Metabolites, and Functions

Oral administration of bovine lactoferrin derived lactoferricin (Lfcin) B could attenuate enterohemorrhagic Escherichia coli O157:H7 induced intestinal disease through improving intestinal barrier function and microbiota Zhang Haiwen, Hua Rui, Zhang Bingxi, Guan Qingfeng, Zeng Jifeng, Wang Xuemei, and Wang Beibei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00861 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Journal of Agricultural and Food Chemistry

Oral administration of bovine lactoferrin derived lactoferricin (Lfcin) B could attenuate enterohemorrhagic Escherichia coli O157:H7 induced intestinal disease through improving intestinal barrier function and microbiota Zhang Haiwen*12, Hua Rui1, Zhang Bingxi1, Guan Qingfeng12, Zeng Jifeng12, Wang Xuemei12, Wang Beibei *2 1. Key laboratory of Tropical Animal Breeding and Epidemic Disease Research of Hainan Province, Hainan University, Haikou, Hainan, 570228, People’s Republic of China 2. Key Laboratory of Tropical Biological Resources of Ministry of Education, Haikou, Hainan, 570228, People’s Republic of China

*Zhang

Haiwen., (Tel: +86-13034929537; Fax: +86-13034929537. E-mail:

[email protected])

1

ACS Paragon Plus Environment


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ABSTRACT: Lactoferricin (Lfcin) B, derived from lactoferrin in whey, has attracted

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considerable attention for its multiple biological functions. Zoonotic enterohemorrhagic

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Escherichia coli (EHEC) O157:H7 has adverse effects on intestinal epithelial barrier

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function, leading to serious intestinal disease. In this study, EHEC O157:H7-induced

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intestinal dysfunction model was developed to investigate the effects of Lfcin B on

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EHEC O157:H7-induced epithelial barrier disruption and microbiota dysbiosis. Results

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showed that the inflammatory infiltration indexes in the jejunum of Lfcin B-treated

8

animals were significantly decreased. Lfcin B administration also significantly

9

improved ZO-1 and occludin expression following O157:H7-induced injury. Finally,

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microbiota analysis of the cecal samples revealed that Lfcin B inhibited the O157:H7-

11

induced abnormal increase in Bacteroides. Therefore, Lfcin B efficiently attenuated

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O157:H7-induced epithelial barrier damage and dysregulation of inflammation status,

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while maintaining microbiota homeostasis in the intestine, indicating that it may be an

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excellent food source for prevention and therapy of EHEC O157:H7-related intestinal

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dysfunction.

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KETWORDS: Lfcin B; enterohaemorrhagic Escherichia coli O157:H7; barrier

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function; microbiota; immune regulation; intestinal dysfunction

18 19 20 21 2


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INTRODUCTION

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Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is one of the most

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prevalent bacterial causes of diarrhea of humans, especially children.1 The pathogenic

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success of EHEC O157:H7 is aided by its ability to tightly adhere to the intestinal

26

mucosa, where it forms attaching and effacing lesions.2 Attached bacteria cause damage

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to the intestinal microstructure, including tight junction proteins, increasing the

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permeability of the intestine and leading to non-bloody diarrhea, especially during the

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weaning period.3 In addition, Shiga toxin (Stx), one of the many virulence factors of

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EHEC O157:H7, can be transported in blood, resulting in systemic infection.4 Stx

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production is also responsible for hemolytic uremic syndrome (HUS), which results in

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acute kidney injury in children.5

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Bovine lactoferrin (bLF), a bioavailable protein extracted from milk or whey,

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reportedly plays a role in enhancing immunity, supporting digestive health, and

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stimulating iron absorption and homeostasis.6 Lactoferricin (Lfcin) B, a peptide

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produced by gastric pepsin digestion of bLF, shows broad antibacterial activity against

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most Gram-positive and Gram-negative bacteria.7 Lfcin B has a looped primary

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structure consisting of 25 amino acid residues (positions 17–41 of bLF).8 To date, the

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best-studied lactoferricins are those derived from bovine and human lactoferrin,

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referred to as Lfcin B and Lfcin H, respectively. The attainability and relatively low

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cost of Lfcin B make it a promising candidate for inclusion in functional foods with

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specific usages. The peptide has a typical amphipathic structure with symmetric

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clustering of hydrophobic and positively-charged residues, which are directly 3



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correlated with broad antimicrobial activity.9 In addition, it has been reported that Lfcin

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B can bind to released lipopolysaccharide (LPS) from the outer membrane of Gram-

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negative bacteria10 and to teichoic acid originating from Gram-positive bacteria.11

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Antibiotics are generally required to treat EHEC-associated intestinal disease.12

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However, the increasing issue of antibiotic resistance is a global threat to the public

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health. In addition, lysis of EHEC O157:H7 cells following antibiotic treatment can

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result in the release of virulence factors such as endotoxin, Stx, and cytosine-phosphate-

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guanine (CpG)-containing oligonucleotides, all of which trigger the immune system,

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resulting in an exacerbated inflammatory response.13 However, Lfcin B has an affinity

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for released endotoxin10 and CpG-containing oligonucleotides,14 and can inhibit the

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inflammatory response by directly suppressing the release of interleukin 6 (IL-6) from

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mononuclear cells.15 Further, Lfcin B may also inhibit the inflammatory response by

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suppressing the classical complement pathway.16

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To date, the effects of bLF derived Lfcin B on EHEC O157:H7-induced intestinal

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disease and microbiota disorder are unknown, and the role of Lfcin B in the recovery

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of disrupted intestinal barrier function and intestinal microbiota imbalance has not been

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reported. Thus, the current study explored the effects of Lfcin B on epithelial barrier

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function, as well as on the inflammatory response and microbiota composition, using

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an EHEC O157:H7 mouse infection model. Our results will aid in the development of

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novel Lfcin B-based functional ingredients for the prevention of EHEC O157:H7-

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associated intestinal disease.

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MATERIALS AND METHODS 4


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Reagents. The antimicrobial peptide Lfcin B was synthesized and purified by GL

67

Biochem (Shanghai, China), and the purity was ≥ 95% as verified by reversed phase

68

liquid chromatography-mass spectrometry (RP-HPLC-MS). The powder was dissolved

69

in sterile saline and stored under -80℃ before used. TRIzol reagent (Invitrogen,

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Carlsbad, USA), Protein extraction kit, butyleyanoacrylate (BCA) kit (Keygen, Nanjing,

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China) and SYBR Green master mix (Roche, Basel, Switzerland) were used. Rabbit

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polyclonal Abs for zonula occludens-1 (ZO-1), Occludin, CD177 and F4/80, secondary

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Abs used for immunohistochemistry (goat anti-rabbit IgG conjugated with HRP), and

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for immunofluorescence (goat anti-rabbit IgG labeling with fluorescein isothiocyanate

75

(FITC) and tetramethyl rhodamine isothiocynate (TRITC) respectively) were

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purchased from Abcam (Cambridge, England). The TUNEL kit was purchased from

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Roche (Basel, Switzerland), the EHEC O157:H7 ATCC43889 was purchased from

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China General Microbiological Culture Collection Center (Beijing, China).

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Purity analysis of synthesized Lfcin B. As the Lfcin B was synthesized through

80

the solid-phase method, and the intramolecular disulphide bridge was formed by aerial

81

oxidation of cysteinyl peptides. Further the peptides were purified through HPLC on

82

the reversed-phase Delta-Pak C18 column (3.9 mm by 300 mm, 15 μm, 300 Å; Waters,

83

Milford, MA, USA), using a linear elution gradient of 0% to 50% acetonitrile with 0.1%

84

(v/v) trifluoroacetic acid at the rate of 1 mL/min for 40 min. The molecular weight of

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Lfcin B was determined with electrospray ionization mass spectrometry (ESI–MS)

86

(Michrom Bioresources, Auburn, CA, USA). In brief, full-scan MS was followed by

87

zoom scan, and then by an MS2 analysis of the largest peak in the full scan. Dynamic 5



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exclusion was used to include high-resolution data and the MS2 information for the

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secondary peaks within the scan.

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Tertiary and helical wheel structure prediction of Lfcin B. The tertiary

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structure of Lfcin B (FKCRRWQWRMKKLGAPSITCVRRAF) was predicted by the

92

online prediction tool I-TASSER (Protein Structure and Function Predictions,

93

https://zhanglab.ccmb.med.umich.edu/I-TASSER/). The helical wheel structure

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prediction was carried out through the online tool: http://wwwnmr.cabm.rutger

95

s.edu/bioinformatics/Proteomic_tools/Helical_wheel/.

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Mouse model of EHEC O157:H7 induced disease. Forty-eight C57/BL6 male

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mice with the averaged weight of 18 g (6-8 weeks) were obtained from the Laboratory

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Animal Center of the Chinese Academy of Sciences (Shanghai, China) and were

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individually housed and maintained on a 12:12 h light-dark cycle under specific

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pathogen free conditions, with free access to feed and water throughout the

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experimental period. After one week adaptive period, the mice were randomly divided

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into four groups, each group had twelve mice. The experimental period lasted for ten

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days, at the first step, the Lfcin B and Lfcin B+O157 groups were intragastric

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administrated with 0.5 mg/kg body weight Lfcin B dissolved in 0.25 mL PBS at day 1,

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5, 9, while the CK and O157 groups were administered equal volume of PBS. Since 1

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hour after the Lfcin B administration, the O157 and Lfcin B+O157 groups were orally

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administered 0.2 mL PBS containing 1×106 colony-forming unit (CFU) of EHEC

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O157:H7, while the CK and Lfcin B groups were administered equal volume of PBS.

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At day 10, all the mice were euthanized, the serum was separated and tissues were 6


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collected for further analysis. The Animal Care and Use Committee of Hainan

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University approved all experiments, and the experimental process was conducted

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strictly in accordance with the Guidelines for the Care and Use of Animals for Research

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and Teaching at Hainan University.

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Body weight, survival rate and index of immune organs. The average body

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weight of mice in each group at even day and finally survival rate at day 10 were

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calculated. Mice were sacrificed and the spleen and thymus were isolated (n=6). The

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blood on organ surfaces was drained with filter paper before weighing, and the immune

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organs index (IOX) was calculated using the following formula:

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IOX= weight of immune organs (mg)/body weight (g) Bacterial shedding and translocation analysis. On day 1, 3, 5, 7 and 9, the

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feces of each group were randomly collected, and the colonies of E.coli were

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determined by plating series dilutions on Eosin Methylene Blue (EMB) agar plates

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(Hopebio, Beijing, China). On day 10, the spleen and liver tissues were removed

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aseptically from six mice of each group and homogenized in 4 ℃ cold PBS, the

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numbers of CFU were determined by plating series dilutions on EMB agar plates.

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Trans epithelial electric resistance measurement in jejunum. The multichannel

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voltage-current clamp (Physiologic Instruments, santiago maior, USA) was used to

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determine the trans epithelial electrical potential of fresh jejunum tissues, following the

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protocols described previously.17, 18 Fresh jejunum tissues were excised from mice and

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immediately immersed in oxygenated Krebs’s buffer, then mounted onto Ussing

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chambers (World Precision Instruments, Narco Scientific, Mississauga, Ontario, 7



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Canada). The Ussing chambers were equipped with two pairs of Ag/AgCl electrodes

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connected to the chambers via 3 M KCl/3.5% agar bridges, to measure the potential

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difference (PD) and passing current (I). For each test, the PD value was clamped to 20

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mV, and the necessary current was recorded, the electrical resistance was determined

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according to Ohm’s law: [R= (20 mV - PD)/I]. To ensure comparability of trans

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epithelial electric resistance (TEER) measurements of each group, the differences

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between the effective exposed area of the epithelium and the apparent exposed tissue

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area were normalized by presenting all measurements as a percentage of the TEER

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value at the end of the equilibration period for each tissue insert.

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The determination of FITC-dextran in serum. To evaluate the permeability of

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the intestinal lining, 250 μL of FITC-dextran (Sigma-Aldrich, St Louis, USA) was

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intragastrically administrated to six mice of each group 4 h before sampling at day 10.

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The serum was collected and added into a 96-well plate avoiding light, fluorescence

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intensity was detected with a microplate reader (Spectrumax M5 molecular devices,

146

Sunnyvale, USA), with the excitation and emission wavelength of 488 nm and 525 nm,

147

respectively.

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ELISA determination. The concentrations of interleukin-1β (IL-1β), interleukin-

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6 (IL-6), Tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in serum, and IL-

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1β, IL-6 and TNF-α (Boster, Wuhan, China) in jejunum were determined. The

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concentration of TNF-α (Boster), malondialdehyde (Boster), Caspase-3 (Keygen) in

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liver and kidney were determined using ELISA kit. The concentration of D-lactic acid

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(D-LA) and diamine oxidase (DAO) were determined using ELISA kit (Boster). The 8


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concentrations of enzyme myeloperoxidase (MPO), Inducible nitric oxide synthase

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(INOS) and cyclooxygenase-2 (COX-2) (Keygen) in jejunum were tested by ELISA

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kits. Total protein from colon tissues were extracted with RIPA lysis buffer (Boster),

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and the quantification of protein was assayed via the BCA kit (Keygen). All the related

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kits assays were carried out according to the manufacturer’s instructions.

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H&E staining of intestinal and organs. Intestinal tissues of the middle part of

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duodenum, jejunum, ileum and colon, and middle site of organs of liver and kidney

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were cut out to carry out hematoxylin-eosin (H&E) staining. Briefly, the above tissues

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from six individuals in each group were isolated, and immediately fixed in 4%

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paraformaldehyde solution, then embedded in paraffin, samples were sliced and stained

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with hematoxylin and eosin in turn. The morphological characteristics were observed

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with Leica NEWDM 4500BR microscope (Leica, Frankfurt, Germany) under certain

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magnifications, and the villous height and crypt depth were measured using image pro

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software (MediaCybernetics, MD, USA).

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Immunofluorescence analysis. The tissues of jejunum of each groups were

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isolated and fixed in 4% paraformaldehyde, then embedded with paraffin and sliced for

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immunofluorescence analysis. Briefly, sections of 5 mm thickness were deparaffinized

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and rehydrated and proceed antigen retrieval. The sections were then incubated in 3%

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hydrogen dioxide for 20 min without light. Then the sections were incubated with

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primary antibodies (1:200 dilution) specific for ZO-1 and Occludin (Abcam). TRITC-

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conjugated goat anti rabbit IgG for ZO-1 and FITC- conjugated goat anti rabbit IgG for

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Occludin (JIR) were added at a ratio of 1:100 and left to be incubated at room 9



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temperature for 1 h in darkness. DAPI was then used to stain the nucleus. Glycerol was

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used to mount the samples onto slides. Images were taken under Leica fluorescence

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microscope.

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Immunohistochemistry analysis. For immunohistochemical analysis of CD177

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and F4/80 of jejunum, nonspecific binding sites were blocked with PBS containing 1%

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w/v bovine serum albumin (BSA) for 30 min. Anti-CD177 and F4/80 antibodies (Santa

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Cruz, Northern California, USA) were added at a dilution of 1:100 and incubated

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overnight at 4 ℃, samples were washed four times in PBS and treated with HRP-

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conjugated rabbit anti goat IgG (JIR, Scottsdale, USA) at a ratio of 1:100, samples were

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incubated at 4 ℃ for 1 h and washed with PBS three times.

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Scanning electron microscopy. The tissue of jejunum were fixed with 2.5%

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glutaradehyde and then with 1% OSO4 for 1 h. The specimens were then dehydrated in

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a graded series of ethanol for 20 min at each step, then transferred into a mixture of

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alcohol and isoamyl acetate (V:V=1:1) for 30 min and isoamyl acetate alone for 1 h.

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Finally, the specimens were then dehydrated in a Hitachi Model HCP-2 critical point

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dryer with liquid CO2, the dehydrated specimens were coated with gold-palladium and

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visualized using a Philips Model SU8010 FASEM (HITACHI, Hitachi, Japan).

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Diaminobenzidine (DAB, DAKO, Glostrup, Denmark) was added then hematoxylin

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was used to counterstain the slices. The samples were dewatered with gradient

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alcohol, and xylene was used to increase the transparency of slides, a neutral balsam

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was applied for mounting. For evaluating the apoptosis level of jejunum tissues,

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paraffin sections were dewaxed with water and antigen retrieval was executed. TdT 10


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and dUTP (Roche, Basel, Switzerland) were mixed at a ratio of 1:9 and incubated at

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37 ℃ for 60 min, the endogenous peroxidase was blocked, and the slides were

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allowed to dry. The tissue was then covered with converter-peroxidase (converter-

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POD) (Roche) and incubated at 37 ℃ for 30 min, and washed with PBS three times.

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DAB was added to the slices, and distilled water was used to stop color development.

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Finally, the cell nucleus was counterstained and the slices were dehydrated and

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mounted.

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Real-time PCR for gene expression analysis. RNA samples (n=6) were extracted

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from the middle jejunum tissues, total RNA isolation and cDNA synthesis by reverse

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transcription were conducted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and

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M-MuLV reverse transcriptase kit (Fermentas, Glen BURNIE, USA) respectively. The

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mRNA levels of individual genes were measured by real-time PCR using the SYBR

210

Premix Ex Taq Kit (Takara Biotechnology, Shiga, Japan) in the ABI Step One Plus

211

Real-Time PCR system (Applied Biosystems, CA, USA), data was analyzed according

212

to the comparative threshold cycle (Ct) method and normalized to an endogenous

213

reference GAPDH. The related primers used in the experiment were listed in table 1,

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the relative expression levels of barrier function related genes (ZO-1, claudin-1,

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occludin, mucin-1, mucin-2) were analyzed on jejunum tissues.

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Intestinal microbiota analysis based on 16S rRNA sequencing. Total genomic

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DNA was extracted from sixteen cecal samples (n=4) using fecal DNA kit (Sigma-

218

Aldrich) according to the manufacturer’s instructions. V4-V5 regions of bacterial 16S

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rRNA gene (from 507 to 907) were amplified from extracted DNA using bar-coded 11



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primers 515F (5’-GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTC

221

MTTTRAGTTT-3’). The amplicons were pooled, purified and then quantified by

222

Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA).

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Subsequently, next-generation sequencing was performed by Illumina Hiseq PE250

224

(SAGENE, Guangzhou, China). The original data were filtered through removing

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reads containing more than 10% of unknown nucleotides or containing less than 80%

226

of bases with quality (Q-value)＞20. The filtered reads were then assembled into tags

227

according to overlap between paired-end with more than 10-bp overlap and less than

228

2% mismatch. The high-quality sequences were clustered into operational taxonomic

229

units (OTUs) defined at 97% similarity. Taxonomic assignments of OTUs were made

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using quantitative insights into microbial ecology (QIIME) software19 through

231

comparison with the databases of SILVA20, Greengene21 and RDP.22 The distribution

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of OTUs at different taxonomic levels were shown as box plot. Linear discriminant

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analysis (LDA) effect size (LEfSe) method was used to identify the most

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differentially abundant taxons between groups. The cladogram analysis was

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conducted based on LEfSe analysis of 16 S sequences (relative abundance≥0.1%), and

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the cladogram plot was drawn through the software of Fig Tree (v1.4.4,

237

http://tree.bio.ed.ac.uk/software /figtree/), which would help find the biomarkers of

238

corresponding groups.23

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Statistical analyses. Multiple comparison test were carried out by turkey’s HSD

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and one way analysis of variance (ANOVA) with SPSS 18.0 (SPSS, Chicago IL, USA),

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a value of P < 0.05 was considered as significant, a value of P < 0.01 was considered 12


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as highly significant, a value of P ＜ 0.001 was considered as extremely significant,

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results are expressed as mean ± standard deviation (SD).

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RESULTS AND DISCUSSION

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EHEC O157:H7 is a notorious enteric pathogen associated with outbreaks of food-

246

borne hemorrhagic colitis.24,

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O157:H7 is the causative agent of HUS in humans.26 In the study, the effects of Lfcin

248

B on EHEC O157:H7 induced epithelial barrier damage, dysregulation of inflammation

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status and imbalance of microbiota were observed. Previous studies indicated that

250

bacteroides could cleave sialic acid moieties and other sugars from mucosal

251

glycoproteins, which are then consumed by EHEC, leading to enhanced expression of

252

virulence genes and pathogenicity.27, 28 Recently, one of the most important member of

253

bacteroides-fragilis was reported to cause fatal sepsis in human.29 In the study, mice

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infected with EHEC O157:H7 caused abnormal increase of bacteroides, accompanied

255

by inflammation response in intestine and serum, it indicated that the disordered

256

intestinal microbiota caused by EHEC O157:H7 could intensify systemic inflammation,

257

we also found Lfcin B treatment effectively reduced the inflammation level, apart from

258

the anti-inflammatory effects itself, the results showed LFcin B could attenuate pro-

259

inflammatory status through inhibiting the proliferation of E.coli and recovering the

260

balance of microbiota. As the occurrence of intestinal inflammation can lead to

261

increased mucosal permeability, and damaged intestinal barrier function will in turn

262

aggravate inflammation.30 Therefore, we further investigated the effects of Lfcin B on

263

impaired intestine, our results demonstrated that Lfcin B improved the barrier function

25

In addition, as a result of Stx production, EHEC

13



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of intestine through enhancing the expression of tight junction proteins. In general, the

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results demonstrated that Lfcin B could significantly improve the damaged epithelial

266

barrier function and attenuate the pro-inflammatory status, meanwhile, it could recover

267

the balance of intestinal microbiota.

268

Lfcin B has a characteristic amphipathic structure. RP-HPLC analysis showed

269

that the purity of the Lfcin B was 97.53% (Figure 1A), which was high enough for use

270

in the animal experiments. The predicted tertiary structure of the protein showed

271

distinct β-folding, likely as a result of the disulfide linkage formed by two cysteine

272

residues (Figure 1B). The molecular weight of Lfcin B was predicted to be 3123.78 Da

273

based on ESI-MS analysis (Figure 1C). The predicted helical wheel structure of Lfcin

274

B suggested that it likely displays excellent amphipathic properties, with hydrophobic

275

and hydrophilic residues distributed symmetrically between the two sides (Figure 1D).

276

As reported previously,31 spatial structure prediction analysis of Lfcin B in the current

277

study revealed a twisted β-sheet structure with significant amphipathicity, which is a

278

common trait amongst antimicrobial peptides and is thought to support their broad-

279

spectrum antibacterial activity.32

280

Lfcin B corrects growth retardation caused by EHEC O157:H7 infection in a

281

mouse model. Clinical symptoms induced by EHEC O157:H7 in the mouse model of

282

infection included soft feces, lethargy, loss of appetite, and weakness (Data not shown).

283

As shown in Figure 2A, the average weight in of the O157-infected group was

284

obviously lower than the corresponding values for the other three groups. The O15714


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infected group showed significant weight loss (P < 0.05) compared with the CK group

286

at days 4 and 6 post-infection, with an even more significant difference in the weights

287

of the two groups observed at days 8 and 10 (P < 0.01) (Figure 2A). The survival rate

288

of each group was also calculated at the end of the experimental period. The O157-

289

infected group showed a survival rate of only 50%, compared with 80% for the Lfcin

290

B+O157 group (Figure 2B), indicating that oral administration of Lfcin B could

291

alleviate the clinical symptoms of O157 infection. Complete spleen and thymus organs

292

were collected from each animal to evaluate the immune development status of each

293

group. The organs were also weighed to calculate the corresponding organ index. The

294

spleen index of the O157-infected group was significantly lower than those of the CK

295

and Lfcin B+O157 groups (P < 0.05), while there was no significant difference between

296

the CK and Lfcin B+O157 groups (Figure 2C). The same trend was observed in the

297

results of thymus index analysis of the three groups (Figure 2D). The thymus and spleen

298

are important organs that reflect the functional status of innate immunity33. Challenge

299

with O157:H7 significantly lowered both the thymus and spleen indexes, indicating the

300

inhibition of innate immunity in the infected mice. However, Lfcin B treatment returned

301

immune organ index values to levels closer to those of the CK group, suggesting that

302

Lfcin B has a positive effect on the innate immunity of O157:H7-infected mice.

303

Lfcin B improves EHEC O157:H7 infection-induced changes in the

304

concentration of cytokines in the serum of infected mice. As shown in Figure 3, pro-

305

inflammatory cytokines IL-1β, IL-6, and TNF-α were present at significantly higher

306

concentrations in the serum of the O157-infected group compared with the Lfcin 15



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B+O157 group (P < 0.05). Further, compared with the CK group, O157-infected mice

308

showed significantly higher serum concentrations of IL-1β and IL-6 (P < 0.05) (Figure

309

3A and 3B), and even higher concentrations of TNF-α (P < 0.01). In comparison, there

310

was no significant difference in the serum concentrations of inflammatory cytokines

311

between the CK and Lfcin B+O157 groups, except in the concentration of TNF-α (P

Oral Administration of Bovine Lactoferrin-Derived Lactoferricin (Lfcin

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