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Lactobacillus plantarum NCU116 Attenuates Cyclophosphamide-induced Immunosuppression and Regulate Th17/Treg Cells Immune Responses in Mice Junhua Xie, Shaoping Nie, Qiang Yu, Junyi Yin, Tao Xiong, Deming Gong, and Mingyong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06177 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Journal of Agricultural and Food Chemistry

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Lactobacillus plantarum NCU116 Attenuates Cyclophosphamide-induced

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Immunosuppression and Regulate Th17/Treg Cells Immune Responses in Mice

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Junhua Xie,† Shaoping Nie,†,* Qiang Yu,† Junyi Yin,† Tao Xiong,† Deming Gong,†,‡

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and Mingyong Xie†

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State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

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School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand

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*

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Professor Shao-Ping Nie, PhD

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Tel & Fax: +86-791-88304452 (S. P. NIE)

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E-mail address: [email protected]

Corresponding author:

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Short title: The immunoregulatory activity of Lactobacillus plantarum NCU116

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ACS Paragon Plus Environment


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ABSTRACT

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The balance of T helper cells 17 (Th17)/ Regulatory T cells (Treg) plays a key role

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in maintaining a normal immune response. It is well known that cyclophosphamide

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(CTX) applied at high dose often damage the immune system by inhibition the

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immune cell proliferation. In this study, the immunomodulating effects of

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Lactobacillus plantarum NCU116 in CTX-induced immunosuppression mice were

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investigated. Results showed that the levels of cytokines interleukin (IL)-17 and IL-21

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were significantly increased after 10 days of treatment with high dose of NCU116

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group (46.92 ± 4.28 and 119.92 ± 10.89, respectively) compared with model group

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(36.20 ± 2.63, 61.00 ± 6.92, respectively), and the levels of cytokines IL-23 and

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TGF-β3 of the three NCU116 treatment groups were significantly higher than that of

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the model group (90.48 ± 6.33, 140.45 ± 14.30, respectively) (p < 0.05) and close to

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62% and 69% of the normal group’s level (140.98 ± 14.74, 266.95 ± 23.11,

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respectively) at 10 days. The bacterium was also found to increase the expression

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levels of Th17 immune response and Treg immune response specific transcription

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factors RORγt and Foxp3. In addition, the bacterium significantly increased the

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number of CD4+T cells and dendrtic cells (DCs), and upregulated mRNA expression

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of Toll-like receptors (TLRs). These findings demonstrated that NCU116 had the

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potential ability to enhance intestine mucosa immunity and regulate the Th17/Treg

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balance, which may be attributed to the TLRs pathway in DCs.

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Keywords: Lactobacillus plantarum NCU116, immunosuppression, Th17/Treg

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balance, dendritic cells, Toll-like receptor 2


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INTRODUCTION

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Cyclophosphamide (CTX) is one of alkylating cytotoxic drugs whose therapeutic

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efficacy of cancer and autoimmune diseases is due in part to their ability to affects

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immune responses1. CTX, particularly a combination of high-dose and long times,

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often leads to immunosuppression which can further lead to intestinal problems, such

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as virus infection intestinal microflora imbalance2-4. However, a healthy environment

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can be recovered with probiotics supplementation, consisting mainly of Lactobacillus

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and Bifidobacterium species, which promote the host health by excluding pathogenic

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bacterium and modulating the immune responses from the intestinal epithelial cells5.

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Immune disorder is considered to be the main pathogenesis of autoimmune diseases,

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such as inflammatory bowel disease (IBD)6, 7 and rheumatoid arthritis (RA)8. The

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balance of the two kinds of special CD4+T cell subsets, T helper cell 17 (Th17) and

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regulator T cell (Treg) is the important factor of maintaining a normal immune

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response8. Th17 cells are characterized by expression and production of RORγt and

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IL17 that mediate inflammatory response9. They play critical roles in host defence

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reactions and tissue inflammation responses10. Foxp3 was specifically expressed in

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Treg cells11. In addition, Treg cells had a special regulatory function of immune

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response by inhibiting the activities of the other CD4+T cell subsets (Th1, Th2 and

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Th17 cells) and maintain cell immune tolerance12. Studies have shown that several

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Lactobacillus strains (Lactobacillus plantarum CGMCC 125813 and Lactobacillus

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casei DN-11400114) can induce the maturation of DCs, which can significantly

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stimulate the proliferation and activation of naive T cells, regulating the

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differentiation of naive T cells to Th17 and Treg15, 16. Furthermore, clinical studies

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have found that peripheral blood CD4+T lymphocyte apoptosis rate was lower in RA

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patients, while the ratio of Th17/Treg was increased. Moreover, the results showed

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that the breaking of balance between Th17 and Treg and the changing of cytokine

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proﬁle were the main pathogenesis of many autoimmune diseases17.

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Previous studies have reported that some bacterium were able to induce the

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production of anti-inflammatory cytokines by regulating Th17/Treg balance to a

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Treg-dominant state in mice18. Lactobacillus plantarum NCU116 (NCU116), a newly

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identified bacterium, was isolated from pickled vegetables in our laboratory

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was reported to exhibit several bioactivities in vitro20, 21 and in vivo22-25. However,

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little is known about the effects of NCU116 on the regulation of the Th17/Treg

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balance in immunosuppressive mice. As such, we aimed to investigate the effects of

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NCU116 on CD4+T cell differentiation in CTX-induced immunosuppressive mice.

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and

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MATERIALS AND METHODS

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Chemicals. Man-Rogosa-Sharpe (MRS) was purchased from Land Bridge

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Technology (Beijing, China). CTX (No.07112221) was purchased from Jiangsu

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Hengrui Medicine Co., Ltd (Jiangsu, China). Nucleoprotein extracted kit was

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purchased from KeyGEN BioTECH (Jiangsu, China). SDS-PAGE Gel preparation

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kit, Bicinchonininc acid (BCA) assay kit and Enhanced Chemiluminescence 4


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Detection kit were purchased from Beyotime Institute of Biotechnology (Shanghai,

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China). Enzyme-Linked Immunosorbent Assay (ELISA)-based cytokine kits,

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anti-RORγt and anti-Foxp3 antibodies were purchased from Boster Bioengineering

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(Wuhan, China). Anti-β-actin, HRP-conjugated goat anti-rabbit secondary antibodies

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and DAB staining kit were purchased from ZSGB Biotechnology (Beijing, China).

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Rat anti-mouse CD4 CD8α, dendritic cells (DCs) marker DCIR2 (33D1) and

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HRP-conjugated mouse anti-rat IgG secondary antibodies were purchased from

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eBioscience (San Diego, CA, USA). RevertAid First Strand cDNA Synthesis Kit was

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purchased from Thermo Fisher Scientific (Vilnius, Lithuania). GoTaq® qPCR

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Master Mix was purchased from Promega Biotechnology (Madison, USA). All other

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reagents used were of analytical grade and purchased from Shanghai Chemicals and

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Reagents Co. (Shanghai, China).

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Preparation of Bacterial Strain. Freeze-dried NCU116 powder (Provided by

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State Key Laboratory of Food Science and Technology of Nanchang University) and

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Bifidobacterium BB12 (BB12) (Purchased from Chr. Hansen, Denmark) were

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suspended in sterile saline (sodium chloride). In the pre-experiment, the approximate

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concentrations of viable bacteria were assessed by plate count method. According to

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the measured content of viable cells, the bacterial strain was diluted in sterile saline

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to produce suspensions of designated doses for oral administration.

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Animals and Experimental Design. Female Specific Pathogen-Free (SPF)

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BALB/c mice weighing 20.0 ± 2.0 g (6-8 w) were purchased from Hunan Slac 5



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Jingda Laboratory Animal Co. Ltd. [Changsha, China, Certificate number: SCXK

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(Xiang) 2012-0003]. All mice used in the present study were cared for in accordance

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with the Guidelines for the Care and Use of Laboratory Animals published by the

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U.S. National Institutes of Health (NIH Publication 85-23, 1996), and all

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experimental procedures were approved by the Animal Care Review Committee,

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Nanchang University.

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The mice were housed in a room with controlled temperature of 23 ± 1 °C, relative

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humidity of 50 ± 5%, and a 12 h light/12 h dark cycle. All animals were randomly

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divided into six groups (n = 10): normal control group (NIM), model control group

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(IM), IM treated with 1010 CFU/mL NCU116 (NCU-H) group, IM treated with 109

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CFU/mL NCU116 (NCU-M) group, IM treated with 108 CFU/mL NCU116 (NCU-L)

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group and IM treated with 109 CFU/mL BB12 as positive control group (BB12). The

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immunosuppression mice was induced by intraperitoneal injection of CTX (80 mg/kg)

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once a day for three days, while the NIM group was subjected to intraperitoneal

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injection of saline as a control. All mice had free access to tap water and food (ad

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libitum). The NIM and IM groups were given oral administration of saline once a day,

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whereas the other four groups were orally given the probiotics at 10 mL/kg body

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weight once daily over a 20 days period. On Day 10 and 20, the mice were sacrificed

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and the small intestines were isolated for further analysis.

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Measurement of Cytokines in Small Intestine. The supernatant of small

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intestinal tissue homogenate was harvested (3000 rpm/min, 15 min), and the levels 6


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of IL-17, IL-21, IL-22 and TGF-β3 were measured using ELISA-based cytokine kits,

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in accordance with the manufacturer’s instructions. Data are expressed as the mean

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cytokine level minus background (pg/mL) of the homogenate supernatant using

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standard cytokines provided in the kits.

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Western blotting Analysis. Protein was extracted from small intestinal tissue

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samples following the method by the manufacturer’s instructions, and the protein

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contents of the supernatant was determined using the BCA assay, and then lysates

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(20 µg of total protein) were separated on 10% SDS-PAGE gels and transferred to

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nitrocellulose membranes. Blots were blocked for 2 h at room temperature in 5%

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bovine serum albumin (BSA) prepared in Tris-buffered saline containing 0.1%

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Tween 20 (TBST), and then incubated with the following primary rabbit polyclonal

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antibodies overnight at 4˚C: anti-Foxp3 anti-RORγt, and anti-β-actin. Following

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three 15 min washings with TBST, the membranes were incubated with secondary

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HRP-conjugated goat anti-rabbit IgG for 1 h at room temperature. After a further

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three 15 min washings, immunoreactive bands were visualized using the Enhanced

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Chemiluminescence Detection kit by following the manufacturer’s instructions.

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β-actin was used as a control. Densitometry was performed using the software

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Quantity One 4.0.

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Immunohistochemical Analysis of Immune Cells. The number of CD4+, CD8+ T

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lymphocytes and DCs were determined in the small intestine by indirect

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immunohistochemical assay following the method by Liu et al26. The rat anti-mouse 7



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CD4, CD8α polyclonal antibodies, 33D1 monoclonal antibody and mouse anti-rat

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IgG HRP antibody were used. The results are expressed as the number of positive

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cells per 10 fields of vision (1000×).

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Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

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Analysis. Total RNA was extracted from the small intestinal tissue, and cDNA was

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obtained by reverse transcription using the RevertAid First Strand cDNA Synthesis

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Kit according to the instructions of the manufacturer. The PCR reactions were

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performed by a 7900HT fast real-time PCR system (ABI, CA, USA) using GoTaq®

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qPCR Master Mix. Data analysis was carried out using the 2−∆∆CT method. The

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housekeeping gene, β-actin, was used for normalization. The sequences of the

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primers (Gencript China, Ltd., Nanjing, China) used for RT-qPCR were as follows:

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β-actin (5′ F: TGGAAATCCTGTGGCATCCAGTAAAC -3′, 5′ R: TAAAACGCAG

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CTCAGTAACAGTCCG -3′); TLR-2 (5′ F: GTCAGCTCACCGATGAAGAA -3′, 5′

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R: GAGCCCATTGAGGGTACAGT -3′); TLR-4 (5′ F: TTCAGAACTTCAGTGGC

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TGGATT -3′, 5′ R: CCATGCCTTGTCTTCAATTGTTT -3′); TLR-6 (5′ F: GAGCC

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TGAGGCATCTAGACC -3′, 5′ R: AGATGCAAGTGAGCAACTGG -3′) ; TLR-9 (5′

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F: GCATGGTGGTGCCTA TACTG -3′, 5′ R: AACACCACGAAGGCATCATA -3′).

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Statistical Analysis. The values were expressed as mean ± standard deviation

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(SD), and the data were analyzed using Two-way analysis of variance (Two-way

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ANOVA) (treatment and time) with Tukey’s test to compare the differences among

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various groups. A p < 0.05 was considered to be statistically significant. All analyses 8


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were done using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).

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RESULTS

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Cytokine Secreted by Small Intestine. As shown in Figure 1, the intervention

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with NCU116 led to significant changes of cytokine profile that the cytokines of

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IL-17, IL-21, IL-23 and TGF-β3 in the small intestine, which relate to Th17 and Treg

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cells immune response, respectively. Significant increases in the levels of IL-21

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(Figure 1B) and TGF-β3 (Figure 1D) were found in CTX-treated mice receiving

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NCU116 and BB12, and the contents of IL-17 (Figure 1A) and IL-23 (Figure 1C)

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were significantly increased in the BB12 and NCU-H groups compared with the IM

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group at Day 10 (P < 0.05). No difference was observed for IL-23 and TGF-β3

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secretion in all bacterium treatment groups at Day 20 compared with Day 10, and the

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contents of IL-17 (BB12 and NCU-H) and IL-21 (BB12) tended to decline at Day 20

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compared with Day 10 (Figure 1).

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Protein Expression Level in the Small Intestine. As shown in Figure 2, the

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SDS-PAGE gel electrophoresis images were showed in Figure 2A and 2B.

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Compared to IM group, the NCU-H, NCU-M, NCU-L and BB12 groups had obvious

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increases in protein levels of the two T-cell-specific transcription factors RORγt and

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Foxp3 at Day 10 (Figure 2C and D), and Foxp3 expression levels in the NCU-M

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and NCU-L groups at Day 20 were significantly higher than those at Day 10 (Figure

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2D) (P < 0.05). In contrast, the obvious change in the expression of RORγt was only 9



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observed in the NCU-H group at Day 20 compared with Day 10 (Figure 2C) (P

Lactobacillus plantarum NCU116 Attenuates ... - ACS Publications

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