3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in

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Bioactive Constituents, Metabolites, and Functions

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in Cultured Human Intestinal Cells and the Model Animal Caenorhabditis elegans Joo Yeon Kim, Tram Anh Ngoc Le, So Young Lee, Dae-Geun Song, SungChul Hong, Kwang Hyun Cha, Jae Wook Lee, Cheol-Ho Pan, and Kyungsu Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03039 • Publication Date (Web): 28 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

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Journal of Agricultural and Food Chemistry

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3,3'-Diindolylmethane Improves Intestinal Permeability

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Dysfunction in Cultured Human Intestinal Cells and the

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Model Animal Caenorhabditis elegans

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Joo Yeon Kim,† Tram Anh Ngoc Le,† So Young Lee,† Dae-Geun Song,† Sung-Chul

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Hong,† Kwang Hyun Cha,† Jae Wook Lee,‡,§ Cheol-Ho Pan,†, § and Kyungsu Kang*,†,§

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†Natural

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Technology, Gangneung, Gangwon-do 25451, Republic of Korea.

Product Informatics Research Center, Korea Institute of Science and

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‡Natural

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Gangneung, Gangwon-do 25451, Republic of Korea.

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§Division

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Science and Technology (UST), Republic of Korea.

Products Research Center, Korea Institute of Science and Technology,

of Bio-Medical Science & Technology, KIST School, Korea University of

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■ AUTHOR INFORMATION

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*Corresponding Author

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K.K., Tel.: +82-33-650-3657, Fax: +82-33-650-3679, E-mail: [email protected]

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Funding

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This study was supported by KIST intramural research grants (2E29563, 2Z05620)

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and the Bio & Medical Technology Development Program of the National Research

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Foundation funded by the Ministry of Science & ICT (NRF-2016 M3A9D3915857).

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Notes

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The authors have no competing financial interests to declare.

ACS Paragon Plus Environment


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ABSTRACT: 3,3'-Diindolylmethane (DIM), a digestive metabolite originating from

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cruciferous vegetables, has dietary potential for the treatment of various human

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intestinal diseases. Although intestinal permeability dysfunction is closely related to

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the initiation and progression of human intestinal inflammatory diseases (IBDs), the

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effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM

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on the intestinal permeability of human intestinal cell monolayers and the animal

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model Caenorhabditis elegans, which were treated with IL-1β and Pseudomonas

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aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the

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intestinal permeability of differentiated Caco-2 cells by enhancing the expression of

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tight junction proteins (including occludin and ZO-1). Compared to the IL-1β single

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treatment (551.0 ± 49.0 Ω·cm2), DIM (10 μM) significantly increased the

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transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4

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Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and

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extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-

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treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ±

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1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment

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of leaky gut syndrome by improving intestinal permeability.

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KEYWORDS: Caenorhabditis elegans, 3,3'-diindolylmethane, inflammatory bowel

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disease, interleukin-1β, intestinal permeability

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■ INTRODUCTION

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Inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis,

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are chronic inflammatory gastrointestinal diseases, and the incidence of IBD is

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increasing worldwide. Environmental and genetic factors, including smoking, stress,

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medication, and diet, affect the incidence and severity of these diseases. Unhealthy

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western diets, which include fast food, high-sugar and low-fiber foods, red processed

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meats, and few vegetables, can induce intestinal microbial dysbiosis, intestinal

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permeability dysfunction, chronic intestinal inflammation, and ultimately IBDs.1,

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Thus, dietary supplementation of healthy phytochemicals is considered a favorable

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alternative to conventional steroid-sparing and immunomodulatory drugs.3

2

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The well-known metabolite 3,3-diindolylmethane (DIM) is generated during the

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digestion of indole-3-carbinol, which is abundant in cruciferous vegetables including

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broccoli, cabbage, and kale.4,

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inflammatory compound6 that exerts preventive and therapeutic effects against

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various human colorectal cancers as well as experimental colitis in mice.7-10 The

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detailed molecular and immunological mechanism underlying the preventive effects

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of DIM against colorectal cancer and IBDs has been elucidated using a cultured colon

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cancer cell model8, 11 and an in vivo mouse model,9, 10 respectively. However, even

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though intestinal permeability dysfunction is directly related to the pathogenesis of

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IBDs, the effect of DIM on intestinal permeability is unclear.

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Previous studies have revealed that DIM is an anti-

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In the present study, we exploited a cultured human intestinal cell model and a

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Caenorhabditis elegans animal model for in vitro and in vivo experiments aiming to

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evaluate the effect of DIM on intestinal permeability and gut health. To mimic IBD

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conditions, we treated the cultured human colon cells with interleukin 1-beta (IL-1β),

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an initiating proinflammatory cytokine involved in the progression of IBDs.9, 12 After



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treatment with IL-1β and DIM, we measured the production of the proinflammatory

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cytokine IL-8, intestinal permeability, and protein expression related to tight junctions.

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We then evaluated the effect of DIM on the intestinal permeability of C. elegans fed

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Pseudomonas aeruginosa, a Gram-negative inflammatory pathogenic bacterium

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found in soil, water, and some eukaryotic organisms that increases intestinal necrosis,

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induces host innate immune responses and colonizes and accumulates in the intestine

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of nematodes.13, 14 We also evaluated the effect of DIM on the lifespan of C. elegans

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fed P. aeruginosa because this parameter could be considered a direct phenotypic

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marker for determining the overall treatment effect and the adverse effects of DIM in

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vivo.

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■ MATERIALS AND METHODS

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Chemicals. DIM (purity; ≥98%), dimethyl sulfoxide (DMSO), fluorescein

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isothiocyanate (FITC)-dextran, protease inhibitor cocktail, and Tween 20 were

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purchased from Sigma (St. Louis, MO, USA). Primary claudin-1, claudin-4,

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occluding, and ZO-1 antibodies were purchased from Thermo Fisher (Rockford, IL,

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USA). Primary GAPDH antibody was purchased from Cell Signaling Technology

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(Danvers, MA, USA). Primary β-actin and horseradish peroxidase-conjugated anti-

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mouse and anti-rabbit secondary antibodies were purchased from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). DIM was dissolved in DMSO for the cellular

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and animal treatments.

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Cell Culture. The human colonic cancer cell lines HT-29 and Caco-2 were

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obtained from the American Type Culture Collection (Rockville, MD, USA) and

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cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%


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(v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL

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streptomycin. These cells were maintained in a humidified atmosphere consisting of

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95% air and 5% CO2 at 37 ℃.

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Measurement of IL-8 Production and Viability of HT-29 Cells. Human

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intestinal HT-29 cells (1 × 104 cells/well) were seeded in 96-well plates in 100 μL of

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medium and incubated for 24 h at 37 ℃. After treatment with DIM for 24 h, IL-1β (1

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ng/mL) was added, and the mixture was incubated for 5 h. The IL-8 levels in the

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culture medium were determined using the Human IL-8 ELISA kit (Bio-Legend, San

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Diego, CA, USA). The viability of HT-29 cells was continuously determined using

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the EZ-Cytox cell viability assay kit (Dogenbio, Seoul, Korea) as described

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previously.15, 16

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Measurement

of

Transepithelial

Electrical

Resistance

(TEER)

in

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Differentiated Caco-2 Cell Monolayers. To investigate the effect of DIM on

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intestinal permeability in vitro, we measured the TEER in differentiated Caco-2 cell

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monolayers using a previously described method17 with some modifications. Caco-2

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cells (3 × 105 cells per insert) were seeded in a 12-well transwell plate (Corning, 12-

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mm diameter, 0.4 µm pore size; Kennebunk, ME, USA) and incubated for 14–17 days.

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The TEER values were measured using a Millicell Electrical Resistance System (ERS)

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meter (Millipore Corporation, Bedford, MA, USA). They are calculated as TEER =

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(Rm - Ri) × A, where Rm is the transmembrane resistance of the treated group, Ri is the

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intrinsic resistance of a cell-free transwell, and A is the surface area (cm2) of the

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membrane.18 The TEER values of the Caco-2 cells after their differentiation ranged

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from 400 to 600 Ω·cm2. Then, Caco-2 cells were treated with IL-1β, DIM, or butyrate

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for 72 h to test the effect of DIM on the intestinal permeability exacerbated by

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proinflammatory cytokines. The protective effect of DIM on detergent-induced



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intestinal permeability dysfunction was measured as follows: differentiated Caco-2

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cell monolayers were pretreated with DIM or butyrate for 72 h, Tween 20 (0.5%) was

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then added, the mixture was incubated for 1 h to induce intestinal cell monolayer

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injury, and the resulting TEER value was measured.

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Measurement of FITC-Dextran Permeability in Caco-2 Cell Monolayers.

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After the TEER measurement, FITC-dextran (average molecular weight = 4 kDa,

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Sigma) was added to the apical chamber, and the mixture was incubated for 2 h. The

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culture media from the bottom chambers were then collected, and the fluorescence

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(excitation wavelength at 480 nm and emission wavelength at 530 nm) was measured

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using a multifunctional microplate reader.19

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Western Blot Analysis. Caco-2 cells (4 × 105 cells/mL) were seeded on 60 mm

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dishes and incubated for 14 days to allow differentiation, and during this incubation

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period, the media were changed every 2 days. The differentiated Caco-2 cells were

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treated with DIM (10 µM) or IL-1β (50 ng/mL) for 48 h and washed twice with ice-

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cold DPBS, and the whole cells were harvested. Subsequently, 100 µL of RIPA buffer

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(supplemented with 100 mM phenylmethylsulfonyl fluoride and 1 µg/mL protease

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inhibitor cocktail) was added, and the cells were transferred to a 1.5 mL tube,

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vortexed four times and incubated for 40 min on ice. The cell lysates were then

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centrifuged at 14,000×g and 4 °C for 30 min. Then, the protein concentration of the

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supernatant was determined by the Bradford protein assay. The cell lysate was mixed

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with 4× Laemmli sample buffer (Bio-Rad, Richmond, CA, USA) and heated at 100 ℃

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for 5 min. Then, the protein samples (10–20 µg) were separated by SDS-PAGE (8 or

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15% Tris-HCl gels) at 90 V for 20 min and at 125 V for 1 h. The proteins were then

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transferred at 100 V for 100 min from the gel to a polyvinylidene fluoride membrane,

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and the membrane was incubated in 3% bovine serum albumin blocking solution. All


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other procedures were performed as described previously.20

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C. elegans Maintenance and Bacterial Strains. The model animal C. elegans

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was used to evaluate the efficacy of DIM in vivo. The nematode strains used to

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investigate intestinal permeability and lifespan were the C. elegans wild-type strain

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N2 and the mutant strain SS104.21 The worms were maintained on nematode growth

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media (NGM) plates at 20 ℃ using Escherichia coli OP50 as the food source

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according to previous methods.22, 23 P. aeruginosa PAO1, rather than E. coli OP50,

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was fed to the worms to induce intestinal inflammation. The C. elegans strains and E.

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coli OP50 were obtained from the Caenorhabditis Genetics Center (MN, USA). P.

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aeruginosa PAO1 was provided by the Korean Collection for Type Culture (Jeongeup,

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Korea).

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Measurement of FITC-Dextran Permeability in Nematodes. To investigate

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the effect of DIM on intestinal permeability in vivo, we measured the FITC-dextran

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permeability of N2 worms as previously described24 with some modifications.

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Specifically, we used four different treatment protocols to evaluate the effects of

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DIM: cotreatment with P. aeruginosa PAO1 and DIM for 72 h, cotreatment with the

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P. aeruginosa culture supernatant and DIM for 72 h, pretreatment with DIM for 48 h

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followed by treatment with P. aeruginosa for 6 h (preventive effect), and

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pretreatment with P. aeruginosa for 6 h followed by treatment with DIM for 48 h

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(therapeutic effect). First, we assessed the effect of the cotreatment with P.

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aeruginosa and DIM for 72 h on intestinal permeability by treating age-

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synchronized L4 worms with E. coli OP50, P. aeruginosa PAO1, and DIM (100 µM)

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for 72 h. We next assessed the effect of the cotreatment with P. aeruginosa culture

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supernatant and DIM for 72 h to confirm that the effect of DIM did not originate

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from the direct antimicrobial activity of DIM against P. aeruginosa. To assess this



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effect, P. aeruginosa PAO1 was cultured in LB media for 14-15 h, and the culture

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supernatant was harvested by centrifugation at 3220×g for 30 min at 4 ℃ and stored

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at 4 ℃ until use. L4 worms were fed E. coli containing the P. aeruginosa culture

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supernatant and DIM (100 µM) for 72 h. In addition, as described above, we also

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evaluated the preventive and therapeutic effects of DIM on the intestinal

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permeability of C. elegans. To assess the preventive effect of DIM, L4 worms were

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fed E. coli containing DIM (0 or 100 µM) for 48 h and then E. coli or P. aeruginosa

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for 6 h. To test the therapeutic effect of DIM, L4 worms were first fed E. coli or P.

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aeruginosa for 6 h and then E. coli containing DIM (0 or 100 µM) for 48 h. After the

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bacterial and DIM treatment, the worms were transferred to FITC-dextran (average

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molecular weight = 10 kDa, final concentration = 20 µg/mL)-containing plates and

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incubated overnight at 20 ℃. To prepare the FITC-dextran-containing plates, FITC-

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dextran was mixed with heat-inactivated E. coli OP50,25 spread on NGM agar plates

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and allowed to dry for 1 h. After FITC-dextran feeding, the excess dye from the gut

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was removed by briefly washing the worms with S-buffer and allowing them to

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crawl freely on NGM plates containing live E. coli OP50 for 1 h. The nematodes

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were then rewashed with S-buffer, transferred to a 96-well polystyrene plate (Costar,

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black with clear flat bottom; Corning, NY, USA) containing 50 μL of 4%

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formaldehyde in each well to immobilize the worms and incubated for 1-2 min.

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After the 4% formaldehyde was discarded, 100 μL of the mounting media was added

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to each well. Fluorescence microscopic images were then obtained using the

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Operetta High-Content Imaging System (PerkinElmer, Waltham, MA, USA) with

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the EGFP filter set. The image data were analyzed, and the FITC fluorescence

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intensity was determined using Harmony software (ver. 3.5).

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Assay of the Lifespan of C. elegans Fed P. aeruginosa. Age-synchronized eggs


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of C. elegans SS104 were incubated on NGM agar plates containing live E. coli OP50

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at 25 ℃. Twenty-eight hours after egg preparation, approximately 240 synchronized

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L4 worms were manually transferred to fresh NGM plates supplemented with DIM (0

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or 100 µM) and P. aeruginosa PAO1. Worms were transferred every day for the first

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ten days and every other day thereafter. The worms that showed no reaction to gentle

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stimulation were scored as dead, whereas the animals that crawled off the plates were

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censored. The surviving and dead animals were counted and transferred every 1–3

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days to replenish the food. Three independent lifespan assays were performed.

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Statistical Analysis. The data are expressed as the mean ± standard deviation

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(SD) and statistically analyzed by one-way analysis of variance (ANOVA) followed

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by Tukey’s multiple comparison test by using GraphPad Prism 7.04 (La Jolla, CA,

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USA). The differences in the distributions obtained in the lifespan assays were

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statistically analyzed with the log-rank test using JMP software (version 10, SAS

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Institute, Cary, NC, USA). A value of p < 0.05 was considered statistically significant.

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■ RESULTS

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DIM Decreased IL-8 Production in Undifferentiated HT-29 Cells Treated

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with IL-1β. First, to confirm the anti-inflammatory activity of DIM on human

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intestinal cells, we measured the IL-8 production in HT-29 cells stimulated with IL-1β

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(1 ng/mL) with ELISAs. IL-1β is an inflammatory signal that mediates cytokines

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during the initiation and progression of IBD,9, 12 and IL-8 is a cytokine that is directly

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produced in response to inflammation and pathogen infection within the inflamed

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mucosa of both cultured intestinal cells and patients with IBD.26, 27 We used curcumin

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as a positive control because of its anti-inflammatory activity and ability to inhibit



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intestinal IL-8 production triggered by bacterial invasion.28 All tested concentrations

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of DIM (5, 10, 20, and 40 μM) significantly inhibited IL-8 production compared with

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that achieved with the IL-1β single treatment, although the effects of DIM (5-20 μM)

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were not dose-dependent (Figure 1A). DIM did not affect the HT-29 cell viability

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except at the highest concentration (40 μM) (Figure 1B). Based on these data, we

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conclude that DIM has anti-inflammatory activity in human intestinal epithelial cells.

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DIM Restored the Intestinal Permeability Dysfunction in Differentiated

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Caco-2 Cell Monolayers Exposed to IL-1β or Tween 20. We subsequently

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investigated the effect of DIM on intestinal permeability in human intestinal cell

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monolayers. For this purpose, we differentiated human intestinal Caco-2 cells in a

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transwell plate for more than 2 weeks because differentiated Caco-2 cells have

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morphological characteristics, such as microvilli and biochemical properties, that are

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similar to those of the actual human intestine.29 To determine the intestinal

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permeability, we measured the TEER using an electric resistance meter or quantified

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the flux of FITC-dextran using a fluorimeter. We damaged Caco-2 intestinal cell

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monolayers using IL-1β (50 ng/mL) or Tween 20 (0.5%), and butyrate, a well-known

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short-chain fatty acid that can enhance the intestinal barrier in Caco-2 cell monolayers,

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was used as a positive control.17, 19

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After differentiation of Caco-2 cell monolayers, every transwell had a similar

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intestinal permeability with TEER values ranging from 400 to 600 Ω·cm2 (Figure 2A).

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The IL-1β treatment significantly decreased the TEER value (551.0 ± 49.0 Ω·cm2, p

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in

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