Effect of Oral Administration of 3,3′-Diindolylmethane on Dextran

Experimental colitis was induced in mice by administering 3% dextran sulfate sodium (DSS) via drinking water. DIM remarkably attenuated the clinical s...
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Effect of Oral Administration of 3,3′-Diindolylmethane on Dextran Sodium Sulfate-Induced Acute Colitis in Mice Eun-Joo Jeon,† Munkhtugs Davaatseren,§ Jin-Taek Hwang,†,# Jae Ho Park,†,# Haeng Jeon Hur,† Ae Sin Lee,† and Mi Jeong Sung*,†,# †

Research Division Emerging Innovative Technology, Korea Food Research Institute, Songnam, Keongki, Republic of Korea Department of Food Science and Technology, Chung-ang University, Ansung, Keongki, Republic of Korea # Food Biotechnology, University of Science and Technology, Daejeon, Republic of Korea §

ABSTRACT: In patients with inflammatory bowel disease (IBD), inflammation is induced and maintained by lymphangiogenesis and angiogenesis. 3,3′-Diindolylmethane (DIM) is a natural product formed in acidic conditions from indole-3-carbinol in cruciferous vegetables, and it is known for its chemotherapeutic activity. This study evaluated DIM’s effects on angiogenesis, lymphangiogenesis, and inflammation in a mouse colitis model. Experimental colitis was induced in mice by administering 3% dextran sulfate sodium (DSS) via drinking water. DIM remarkably attenuated the clinical signs and histological characteristics in mice with DSS-induced colitis. DIM suppressed neutrophil infiltration and pro-inflammatory cytokines. Moreover, it significantly suppressed the expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)-2, indicating that the mechanism may be related to the repression of pro-angiogenesis activity. DIM also remarkably suppressed the expression of VEGF-C, VEGF-D, VEGFR-3, and angiopoietin-2; thus, the mechanism may also be related to the suppression of lymphangiogenesis. Therefore, DIM is a possible treatment option for inflammation of the intestine and associated angiogenesis and lymphangiogenesis KEYWORDS: 3,3′-diindolylmethane, inflammatory bowel disease, dextran sodium sulfate, angiogenesis, lymphangiogenesis

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INTRODUCTION Inflammatory bowel diseases (IBDs) (e.g., Crohn’s disease (CD) and ulcerative colitis (UC)) are distinguished by chronic inflammation. Widespread tissue injury and remodeling caused by tissue edema, the loss of epithelial integrity, inflammatory cell infiltration, and increased angiogenesis are all associated with IBD. Angiogenesis is reportedly an essential contributory cause of tissue injury in patients with IBD.1 Recently, several studies have demonstrated that lymphatic vessel (LV) dysfunction leads to the resolution of intestinal inflammation.2 In addition, in clinical and experimental studies on IBD, neoangiogenesis, vascular injury, and lymphangiogenesis were increased.3,4 Accumulating evidence indicates that lymphangiogenesis and angiogenesis have key roles in the development and advancement of clinical and experimental IBD.5,6 Therefore, it has been proposed that decreasing angiogenesis and lymphangiogenesis will lead to a decrease in IBD-induced intestinal inflammation. Many previous studies have identified the main inhibitors and stimulators of angiogenesis and lymphangiogenesis, as well as components of the vascular endothelial growth factor (VEGF) family. 7,8 VEGF-A binds to VEGF receptor (VEGFR)-1 and VEGFR-2, and placental growth factor and VEGF-B bind only to VEGFR-1. VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3. VEGF-A is a main factor of pathogenic angiogenesis in IBD and chronic inflammation.9,10 VEGF-A overexpression can trigger the expression of VEGF-C and VEGF-D. The lymphatic vascular system plays a crucial role in the maintenance of tissue fluid balance, immune surveillance, and the absorption of nutrients, and it is involved © XXXX American Chemical Society

in many pathological processes. Lymphangiogenesis occurs when lymphatic vascular endothelial selective growth factors (i.e., VEGF-C and VEGF-D) bind to VEGFR-3.11 Lymphangiogenesis as well as increased VEGFR-3 expression are indicative of inflammatory responses, including chronic inflammatory diseases and IBD.12,13 In human and experimentally modeled IBD, angiogenesis and lymphangiogenesis occur simultaneously, resulting in neovessel growth for remodeling of the vascular structure. 3,14 However, there is a paucity of information about lymphangiogenesis in those with IBD. Therefore, we assessed the presence of lymphangiogenesis and the main drivers of angiogenesis in IBD-mediated regulators (e.g., VEGF-A and VEGF-C/-D) in mice with IBD. 3,3′-Diindolylmethane (DIM) is naturally formed in an acidic environment from indole-3-carbinol (I3C), which is found in cruciferous vegetables.15 DIM has a promising antitumor effect against various cancers.16 Some evidence has indicated that DIM induces anti-angiogenesis activity in prostatic cancer cells.17 Additionally, DIM reportedly induces anti-anti-angiogenic and inflammatory activity in endothelial cells.18 Moreover, DIM has anti-inflammatory effects in experimental colitis.19 Furthermore, in mice, it inhibits colonic inflammation as well as tumorigenesis.20 However, until recently, few studies have reported on the anti-angiogenic and/or antilymphatic effect of DIM in IBD. Accordingly, it is Received: June 10, 2016 Revised: September 26, 2016 Accepted: September 26, 2016

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DOI: 10.1021/acs.jafc.6b02604 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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molecule-1) monoclonal antibody (1:100 dilution; Chemicon, Temecula, CA, USA) and rabbit anti-mouse lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody (1:200 dilution; Angiobio, Del Mar, CA, USA) overnight at 4 °C. Fluorescein isothiocyanate-conjugated anti-hamster (1:1000 dilution) and Cy3conjugated anti-rabbit immunoglobulin G antibodies (1:1000 dilution) were used as the secondary antibody. 4′,6-Diamidino-2-phenylindole (DAPI) (1:1000 dilution; Sigma, St. Louis, MO, USA) was used for nucleic acid staining. Next, we analyzed the stained colonic tissue sections using a confocal microscope (Nikon Eclipse Ti, Thornwood, NY, USA). The density of vascularization in each colon section was calculated as the ratio of the total microvascular area as a percentage of the tissue area that was immunopositive for CD31+/LYVE-1− for blood vessels and CD31+/LYVE-1+ for lymphatic vessels at a magnification of ×200 in five regions, each amounting to a 0.21 mm2 area. Positive fluorescent signaling was defined by special excitation and emission parameters. All counts were analyzed using the ImageJ software integrated density analysis measurement tool. Analysis of Gene Expression in Colonic Samples by RT-PCR. The total RNA was isolated from the mouse colons by using a Qiagen mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s precedures. Real-time quantitative PCR was performed using the iTaq universal SYBR Green I supermix (Bio-Rad, Hercules, CA, USA) according to the supplier’s protocol. Data were normalized β-actin levels. VEGF-A sense: 5′-GCT GTA CCT CCA CCA TGC CAA C3′. VEGF-A antisense: 5′-CGC ACT CCA GGG CTT CAT CG-3′. VEGF-C sense: 5′-AGA CGG ACA CAC ATG GAG GT-3′. VEGF-C antisense: 5′-AAA GAC TCA ATG CAT GCC AC-3′. VEGF-D sense: 5′-TTG AGC GAT CAT CCC GGT C-3′. VEGF-D antisense: 5′GCG TGA GTC CAT AGG GCA A-3′. VEGFR-2 sense: 5′-TCC AGA ATC CTC TTC CAT GC-3′. VEGFR-2 antisense: 5′-AAA CCT CCT GCA AGC AAA TG-3′. VEGFR-3 sense: 5′-CTG GCA AAT GGT TAC TCC ATG A-3′. VEGFR-3 antisense: 5′-ACA ACC CGT GTG TCT TCA CTG-3′. Angiopoietin (Ang)-2 sense: 5′-GGT GAA GAG TCC AAC TAC AG-3′. Ang-2 antisense: 5′-TTG TCA TTG TCC GAA TCC TT-3′. Interleukin (IL)-1β sense: 5′-TGT AAT GAA AGA CGG CAC ACC-3′. IL-1β antisense: 5′-TCT TCT TTG GGT ATT GCT TGG-3′. IL-6 sense: 5′-TGG AGT ACC ATA GCT ACC TGG A-3′. IL-6 antisense: 5′-TGA CTC CAG CTT ATC TGT TAG GAG-3′. Interferon (IFN)-γ sense: 5′-ACT GGC AAA AGG ATG GTG A-3′. IFN-γ antisense: 5′-GCT GTT GCT GAA GAA GGT AG3′. β-Actin sense: 5′-CGT GCG TGA CAT CAA AGA GAA-3′. βactin antisense: 5′-TGG ATG CCA CAG GAT TCC AT-3′. ELISA. We homogenized the colonic tissues (10−20 mg) in cold, phosphate-buffered saline with protease inhibitor cocktails (Calbiochem, San Diego, CA, usa); we also measured the protein level. Additionally, we calculated the IL-6, IL-1β, and IFN-γ levelS by ELISA, according to the manufacturer’s procedures (R&D Systems, Minneapolis, MN, USA). The IL-6, IL-1β, and IFN-γ concentrations are expressed as picograms of cytokine per microgram of protein. MPO Activity Assay. We assessed the MPO activity as an indicator of neutrophil infiltration in the mouse colon tissues, as reported formerly with slight modifications.21 We measured the MPO activity as the level of enzyme needed to induce a change in absorbance of 1.0 unit/min/g of tissue (wet weight). Statistical Analysis. We performed the Shapiro−Wilk test to test the normality of the data. We also performed analysis of variance to compare data between two groups. Several groups were analyzed using one-way analysis of variance, followed by the post hoc t test to assess statistical significance. We considered p < 0.05 statistically significant.

reasonable to hypothesize that the anti-angiogenic and antilymphatic activity of DIM can also significantly affect inflammation associated with numerous chronic inflammatory conditions. Accordingly, we assessed the preventive effect of DIM on mucosal inflammation and angiogenesis/lymphangiogenesis in mice with dextran sulfate sodium (DSS)-induced colitis.

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MATERIALS AND METHODS

Dextran Sodium Sulfate Model of Mouse Colitis. We purchased 30 male C57BL/6 mice (age, 8 weeks old; weight, 20−22 g) from Charles River (Seoul, Korea). We maintained the mice on a 12 h light/12 h dark cycle at a continuous temperature of 22 °C under specific pathogen-free environments. Mice consumed a standard diet, and they were permitted unrestricted access to drinking water. An acute colitis model was induced by administrating 3% DSS (w/v; MW 36,000−50,000 kDa; MP Biochemicals, Aurora, OH, USA) in fresh tap water ad libitum for 7 days. Mice were matched by body weight, and then they were randomly divided into three groups. Mice in group 1 (control (Cont), n = 10) were given drinking water without DSS. Along with DSS treatment, the mice in group 2 received DSS and a corn oil vehicle (DSS, n = 10), and those in group 3 were given DSS and a daily oral gavage of DIM (20 mg/kg, once daily) suspended in corn oil (DSS + D20, n = 10). Mice in the Cont and DSS groups received corn oil by oral gavage once per day. The dose of DIM was chosen on the basis of the previously demonstrated protective effects of DIM in an inflammatory disease model.20 We evaluated the following variables daily during DSS treatment: gross rectal blooding, body weight, survival, and stool consistency. On day 7, we anesthetized the mice using ketamine/xylazine, and we collected a blood sample by cardiac puncture. Subsequently, the entire colon was removed from all sacrificed mice, and the length of the colon was measured and recorded. Each colon sample was divided equally into three parts. Each colon sample was fixed in 10% formalin for histological analysis and immunofluorescence staining, or each colon sample was frozen at −70 °C for a myeloperoxidase (MPO) activity assay, real-time quantitative (RT)-PCR assay, and enzyme-linked immunosorbent assay (ELISA). All animal procedures described herein were performed in agreement with the guidelines for the Institutional Animal Care and Use Committee of the Korea Food Research Institute. Assessment of Disease Activity. A blinded observer evaluated the disease activity index (DAI) daily. We determined the DAI using the following variables: stool blood (0, negative; 2, positive; and 4, gross bleeding), changes in weight (0, 15%), and stool consistency (0, normal; 2, loose stools; and 4, diarrhea).21 Histopathological Colitis Score. We embedded formalin-fixed distal colon tissues in paraffin, and then we sliced them into 5 μm thick sections. Next, the colon slices of each sample were stained using hematoxylin and eosin (H&E). Light microscopy (×40 magnification) with four randomly chosen non-overlapping fields was used to analyze the stained sections, which were assigned a colitis severity score by an examiner using methods described previously.22 In short, the degree of colitis was scored on the basis of the following parameters: extent of the injury (0, none; 1, mucosa; 2, mucosa and submucosa; and 3, transmural), inflammation severity (0, none; 1, mild; 2, moderate; and 3, severe), and crypt damage (0, none; 1, basal one-third damaged; 2, basal two-thirds damaged; 3, crypt loss and the presence of surface epithelium; and 4, loss of the entire crypt and epithelium). Then the degrees for each of these aforementioned parameters were multiplied by an extent score that represented the percentage of each parameter that had a given feature as follows: 1, 0−25%; 2, 26−50%; 3, 51−75%; and 4, 76−100%. We defined the total score as the sum of the three parameters. The bottom limit total colitis score was 0; the upper limit total colitis score was 40. Immunofluorescence Analysis. We sliced the colon tissue into 10 μm sections. Tissue sections were washed with PBS and then blocked with donkey serum. Samples were double stained with hamster anti-mouse CD31 (platelet/endothelial cell adhesion

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RESULTS DIM Attenuates the Severity of DSS-Induced Acute Colitis. In the investigation of whether DIM exerted an inhibitory effect in the DSS-induced colitis mouse model, DSS treatment signified the well-known clinical symptoms of colitis (e.g., weight loss, diarrhea, and rectal bleeding). Compared to the control mice, body weight was significantly decreased in the DSS-treated mice. Compared to DSS only, DIM administration B

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Figure 1. 3,3′-Diindolylmethane (DIM) attenuates clinical symptoms in a mouse colitis model. (A) Changes in the body weights and (B) clinical scores of DIM-treated mice (receiving 3% dextran sulfate sodium (DSS)) and the control mice were monitored daily. The body weight values are expressed as a percentage of the starting body weight. (C) Colons were excised from the mice, and their lengths were measured 7 days after initiation of DSS administration. (D) Macroscopic features of the colons. Cont, control group (without DSS); DSS, 3% DSS administration group; DSS+D20, 3% DSS with DIM at 20 mg/kg per day group. The data shown are an aggregate of three independent experiments and are expressed as a mean ± standard deviation (n = 10 per group). (∗) p < 0.05, DSS versus cont; (#) p < 0.05, DSS+D20 versus DSS.

Figure 2. 3,3′-Diindolylmethane (DIM) reduces microscopic colon damage and inflammatory cell infiltration during colitis in mice. (A) Colons were excised 7 days after initiation of the administration of dextran sulfate sodium (DSS), and they were sectioned and stained with hematoxylin and eosin. Cont, control group (without DSS); DSS, 3% DSS administration group; DSS+D20, 3% DSS with DIM at 20 mg/kg per day group. Original magnification, ×10. White, red, and yellow arrows indicate the crypt, cell infiltration, and epithelium, respectively. (B) Histopathological scores of the analyzed slides (n = 10 per group). (C) Measurement of myeloperoxidase activity. The bars represent the mean ± standard deviation from four randomly nonoverlapping fields per mouse (n = 10 per group). (∗) p < 0.05, DSS versus Cont; (#) p < 0.05, DSS+D20 versus DSS. Scale bar = 100 μm (A, bottom right).

DIM Reduces Microscopic Colon Damage and Inflammatory Cell Infiltration during DSS-Induced Colitis in Mice. DSS treatment induced inflammatory changes typical of colitis, such as ulceration, crypt dilation, goblet cell depletion, and infiltration of inflammatory cells, in the colonic architecture. Mice that received DIM treatment concurrent with DSS had obviously decreased cell infiltration and reduced mucosal injury and edema compared to DSS-induced mice

concurrent with DSS resulted in less weight loss (Figure 1A). The DAI was also significantly decreased with DIM compared to DSS (Figure 1B). Colonic shortening was a sign of DSSinduced colitis, as represented by the shorter colon length in the DSS-treated mice compared to the control mice. Compared to DSS, DIM treatment was associated with significantly decreased colonic shortening (Figure 1C,D). C

DOI: 10.1021/acs.jafc.6b02604 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. 3,3′-Diindolylmethane (DIM) decreases angiogenesis and lymphangiogenesis in the colitis model. Colon sections were stained using the CD31 antibody (A), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (B), and CD31/LYVE-1/DAPI antibody (×200 magnification) (C). CD31 and LYVE-1 were combined for blood and lymphatic vessels (D). CD31+/LYVE-1− (red arrow) indicates the blood vessels, and CD31+/LYVE-1+ (yellow arrow) indicates the lymphatic vessels. Cont, control group (without dextran sulfate sodium (DSS)); DSS, 3% DSS administration group; DSS+D20; 3% DSS with DIM at 20 mg/kg per day group. (E) Analysis of the area densities of CD31+/LYVE-1− blood vessels (black bar) and CD31+/LYVE-1+ lymphatic vessels (gray bar). The bars represent the mean ± standard deviation from five randomly nonoverlapping fields per mouse (n = 10 per group). (∗) p < 0.05, DSS versus Cont; (#) p < 0.05, DSS+D20 versus DSS. Scale bar = 100 μm (A, bottom right).

performed real-time PCR analysis to evaluate the levels of VEGF-A and VEGF-R2 in colonic tissues. Compared to the control mice, DSS-treated mice had dramatically increased VEGF-A and VEGF-R2 levels, whereas concurrent DIM-treated mice had significantly decreased VEGF-A and VEGF-R2 mRNA levels (Figure 4). DIM Decreases VEGF-C, VEGF-D, VEGFR3, and Ang-2 Expression during Colitis. VEGF-C and VEGF-D are recognized lymphangiogenic factors that can induce lymphangiogenesis by binding to VEGFR-3.27 We evaluated the effect of DIM on lymphangiogenesis during DSS-induced colitis.

(Figure 2A). Compared to control mice, DSS-treated mice had an obviously increased histological score for inflammation relative to the control mice. However, DIM-treated mice had obviously inhibited inflammation (Figure 2B). Tissue injury and inflammatory signals in experimental colitis are mostly mediated neutrophils.23,24 Accordingly, the effects of DIM on inflammatory cell infiltration in DSS-induced colitis were determined by analyzing the MPO activity. In general, the MPO levels were associated with the level of neutrophil infiltration. In our study, the MPO activity level in the control group was 6.3 ± 2.2, whereas that in the DSS group was markedly increased to 34.8 ± 4.6 (p < 0.05). Additionally, treatment with DIM significantly reduced the MPO activity levels to 18.8 ± 2.5 (p < 0.05) (Figure 2C). DIM Decreases Angiogenesis and Lymphangiogenesis during Colitis. Lymphangiogenesis and angiogenesis have important functions in colon inflammation.3,6 To investigate the effects of DIM on angiogenesis and lymphangiogenesis, we used CD31 staining as a panendothelial marker25,26 and LYVE1 as a specific lymphatic vessel endothelial cell marker,6 and then we analyzed the density of CD31+/LYVE-1 blood vessels and CD31+/LYVE-1+ lymphatic vessels in the colon sections (Figure 3). Compared to the control group (6.3 ± 1.1), the DSS group (17.5 ± 1.4) had a demonstrably increased CD31+/ LYVE-1 blood vessel density (p < 0.05), whereas the DIM group (9 ± 1.9) had a significantly decreased mucosal vessel density (p < 0.05). Compared to the control group (8.1 ± 1.2), the DSS group (23 ± 2.1) had a significantly increased number of mucosal vessels (p < 0.05), whereas the DIM group (11.1 ± 1.7) had a significantly decreased number of mucosal vessels (p < 0.05). DIM Decreases VEGF-A and VEGF-R2 Expression during Colitis. VEGF-A is a well-known important factor of pathogenic angiogenesis.4 Therefore, we investigated the effect of DIM on angiogenesis during DSS-induced colitis. We

Figure 4. 3,3′-Diindolylmethane (DIM) decreases the expression of VEGF-A and VEGFR-2 in colitis model. (A) Vascular endothelial growth factor (VEGF)-A and (B) VEGF receptor (R)-2 mRNA expression were determined by real-time PCR in colon samples from the control group (Cont; without DSS), 3% DSS administration group (DSS), and 3% DSS with DIM at 20 mg/kg/day group (DSS+D20). β-Actin was used as an internal control. The ratio relative to the control is arbitrarily presented as 1. The bars represent the mean ± standard deviation from three experiments (n = 10 per group). (∗) p < 0.05, DSS versus Cont; (#) p < 0.05, DSS+D20 versus DSS. D

DOI: 10.1021/acs.jafc.6b02604 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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effectively mimics human IBDs such as mucosal damage, superficial ulceration, the production of pro-inflammatory and other inflammatory mediators, and leukocyte infiltration.30 Importantly, DSS-induced colitis demonstrates the involvement of various genes that have a role in lymphangiogenesis and angiogenesis.31 Blood microvascular and lymphatic networks have essential functions in regulating interstitial fluid balance and trafficking leukocytes in inflammatory conditions.32 Studies have shown that angiogenesis and lymphangiogenesis are fundamental components of chronic inflammation such as IBD.6,33 Widespread angiogenesis and microvasculature remodeling are inherent to tissue remodeling that arises in inflamed intestines in human IBD.34 Additionally, lymphangiogenesis has been reported to lag angiogenesis during inflammation.35,36 IBD represents the elimination of accumulating interstitial fluid, inflammatory cells, and mediators by lymphatic expansion. A recent study indicated that patients with IBD have significantly increased lymphatic vessels.37 Alexander et al. reported that blood vessel density and lymphatic density increase during the development of experimental colitis in mice.31 In addition, VEGF164b therapy inhibits TNBS-induced inflammation, angiogenesis, and lymphangiogenesis.38 Furthermore, mice that were deficient in Ang-2 had blocked blood and lymphatic vascular remodeling.39 In this study, we stained the CD31 and LYVE-1 antibody and quantified changes in the blood vessel by CD31+LYVE-1− and the lymphatic vessels by CD31+LYVE-1. We found that the number of blood vessels in DSS mice increased by 2.3-fold compared to the control mice, whereas mice treated with DIM demonstrated a significant inhibition of blood vessel density. The number of lymphatic vessels in DSS mice also increased by 2.4-fold compared to the control mice, whereas mice treated with DIM demonstrated a significant inhibition of lymphatic vessel density (Figure 3). Our data imply that DIM has an inhibitory effect on DSS-induced angiogenesis and lymphangiogenesis. In addition, some recent studies showed that the relative proportion of angiogenesis and lymphangiogenesis might indicate the degree of severity of inflammation. In cases of acute colitis, the expansion of lymphatics is proportionately greater than that of blood during angiogenesis. Accordingly, this significant increase in lymphatics may be due to compensation for a leaky blood vessel.39 In our study, DSS-treated mice had significantly increased blood and lymphatics compared to the control mice, and similar to other studies, our study also demonstrated a greater lymphatic vessel density compared to the blood density in DSStreated mice. Yet, DIM-treated mice had a decreased blood and lymphatic vessel density after DSS treatment. Therefore, DIM also causes the opposite of lymphatic compensation for leaky blood vessels in DSS-induced colitis. Recent findings suggest that infiltration of neutrophils in inflammatory conditions may be promoted by angiogenesis and that these events have a causal relationship.40 Additionally, inflammatory-induced lymphangiogenesis contributes to neutrophil infiltration.41 In the present study, DIM with DSS reduced neutrophil infiltration significantly compared to DSS only (Figure 2C). The decreased neutrophil content by DIM may reflect the reduction in DSS-induced inflammatory angiogenesis, tissue inflammation, and lymphangiogenesis. Hence, these data suggest that DIM may significantly regulate the pro-inflammatory activity in cases of DSS-induced colitis. We also found that DSS-treated mice showed weight loss, a shortened colon, an increased DAI score, and histopathological

Therefore, we used real-time PCR analysis to evaluate the VEGF-C, VEGF-D, VEGFR-3, and Ang-2 levels in the colonic tissues. As we anticipated, compared to control mice, DSStreated mice had increased VEGF-C, VEGF-D, VEGFR-3, and Ang-2 levels, whereas DIM-treated mice had significantly decreased VEGF-C, VEGF-D, VEGFR-3, and Ang-2 mRNA levels (Figure 5).

Figure 5. 3,3′-Diindolylmethane decreases the expression of VEGF-C, VEGF-D, VEGFR-3, and Ang-2 in the colitis model. (A) VEGF-A, (B) VEGF-D, (C) VEGFR3, and (D) angiopoietin (Ang)-22 mRNA expression are determined using real-time PCR in colon samples from the control group (Cont, without DSS), 3% DSS administration group (DSS), and 3% DSS with DIM at 20 mg/kg per day group (DSS +D20). β-Actin is used as an internal control. The ratio relative to the control is arbitrarily presented as 1. The bars represent the mean ± standard deviation from three experiments (n = 10 per group). (∗) p < 0.05, DSS versus Cont; (#) p < 0.05, DSS+D20 versus DSS.

DIM Decreases Colonic Protein Levels and mRNA Expression of Inflammatory Cytokines during Colitis. Reducing pro-inflammatory cytokines (e.g., IL-1β, IL-6, and IFN-γ) can clearly inhibit the severity of colitis and neutrophil/ macrophage migration.28 In the current study, we investigated the effect of DIM on pro-inflammatory cytokines in the colonic tissues by using real-time PCR and ELISA assays. The levels of IL-6, IL-1β, and IFN-γ in DSS-treated mice were markedly increased compared to the control mice. However, DIM treatment markedly inhibited the levels of IL-6, IL-1β, and IFNγ (Figure 6A−C). In addition, DSS treatment significantly increased the colonic expressions of IL-6, IL-1β, and IFN-γ mRNA. Interestingly, DIM treatment significantly decreased the expressions of IL-6, IL-1β, and IFN-γ mRNA (Figure 6B,D,E). These results indicate that the anti-inflammatory effect of DIM is likely related to its ability to release and express cytokines.

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DISCUSSION IBD is an immune-mediated, chronic, relapsing intestinal inflammatory condition; although the process underlying the development of ulcerative colitis is quite complex, DSS-induced experimental colitis is a recognized animal model.29 It E

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Figure 6. 3,3′-Diindolylmethane decreases colonic protein levels and mRNA expression of inflammatory cytokines during colitis. (A) Interleukin (IL)-1β, (B) IL-6, and (C) interferon gamma (IFN-γ) protein levels are determined by enzyme-linked immunosorbent assay in colon samples from the control group. (D) IL-1β, (E) IL-6, and (F) IFN-γ mRNA expressions are determined using real-time polymerase chain reaction in colon samples from the control group (Cont; without DSS), 3% DSS administration group (DSS), and 3% DSS with DIM at 20 mg/kg per day group (DSS+D20). β-Actin is used as an internal control. The ratio relative to the control is arbitrarily presented as 1. The bars represent the mean ± standard deviation from three experiments (n = 10 per group). (∗) p < 0.05, DSS versus Cont; (#) p < 0.05, DSS+D20 versus DSS. mRNA, messenger ribonucleic acid.

VEGFR-3 mRNA expression that was increased by DSS only (Figure 5). Therefore, we assume that DIM can reduce angiogenesis and lymphangiogenesis through inhibition of the NF-κB pathway. However, the present study did not assess the effect of DIM on the NF-κB signaling pathway. Further research should be performed to determine whether DIM has a potential antiangiogenic and antilymphatic effect on colonicinduced inflammation through the NF-κB signaling pathway. Ang-2 binds the endothelial-specific receptor tyrosine kinase-2 (Tie-2), a competitive inhibitor of Ang-1/Tie-2 signaling in angiogenesis, and modulates lymphangiogenesis.46 Additionally, Ganta et al. reported that the Ang-2 deficiency significantly reduced DSS-induced angiogenesis and lymphangiogenesis.39 In the present study, DIM with DSS significantly reduced DSS increased Ang-2 mRNA expression (Figure 5). Collectively, these data suggest that DIM inhibits DSS-induced VEGF-A/ VEGFR-2, VEGF-C, and VEGF-D/VEGFR-3 as well as Ang-2 expression, indicating that the mechanism may involve inflammatory-mediated angiogenesis and lymphangiogenesis. VEGF-A is a key element of lymphangiogenesis and angiogenesis in cases of IBD.42 Additionally, VEGF-A mediates the recruitment of macrophages that secrete VEGF-C and VEGF-D during cornea neovascularization. Thus, VEGFR2 in blood vessels binds to VEGF-A secreted by macrophage, and VEGFR3 in lymphatic vessels binds VEGF-C and VEGF-D secreted by activated macrophage. This leads to both indirectly inducing inflammatory angiogenesis and lymphangiogenesis.47 The inflammatory process is characterized by obvious features of intestinal mucosa, which includes chronic inflammatory cell infiltration of neutrophils and macrophage; this effect is mediated by the production of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and IFN-γ).48,49 Selectively blocking IL-6 and IL-1β dramatically reduces the severity of colitis and neutrophil/macrophage infiltration.50 The results showed that

features. However, DIM with DSS significantly reduced the weight loss, bloody stool, DAI, and tissue injury (Figures 1 and 2). Overall, our findings indicate that DIM may ameliorate DSS-induced pathologic features of IBD. Angiogenesis and lymphangiogenesis in IBD stimulate many growth factors and their receptors. VEGF-A binds VEGFR-1 and VEGFR-2, which is a main proangiogenic factor that plays a crucial role in human and mouse colitis.41 Therefore, one of the strategies to suppress the severity of colitis is blocking the expression of VEGF-A or its receptors.6,38,42 The nuclear factor (NF)-κB signaling pathway regulates inflammation-induced angiogenesis and lymphangiogenesis.18,43 Additionally, inactivation of the NF-kB pathway affects the severity of colitis. Previously, our studies reported that poly(γ-glutamic acid) and allyl isothiocyanate attenuate DSS-induced inflammation and angiogenesis mediated by the inhibition of VEGF-A and VEGFR-2.21,22 Recently, several studies have reported that DIM suppresses VEGF expression through the NF-κB pathway in cells and inhibits DSS-induced colonic inflammatory reaction by reducing NF-κB activation.44,20 Therefore, these findings suggest that the expressions of VEGF-A and VEGFR-2 were clearly increased in DSS-induced colitis in mice, and DIM with DSS significantly reduced DSS-increased VEGF-A and VEGFR2 expression (Figure 4). Lymphangiogenesis is considered to be principally correlated with the binding of VEGF-C and VEGFD to VEGFR-3.41 Some studies have shown that lymphangiogenesis is mediated by angiogenesis in inflammatory conditions, and the overexpression of VEGF-A can promote the expressions of VEGF-C and VEGF-D, both of which are potent inducers of lymphangiogenesis and angiogenesis in IBD.38 Additionally, Flister et al. reported that inflammation drives lymphangiogenesis through the up-regulation of VEGFR-3-mediated NF-κB activation.45 In the present study, DIM with DSS significantly reduced VEGF-C, VEGF-D, and F

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Journal of Agricultural and Food Chemistry

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DIM suppressed increased neutrophil infiltration and the level of mRNA expression and protein levels of IL-1β, IL-6, and IFNγ. However, although we did not measure macrophage infiltration in this study, as in our previous study,22 we predicted that DIM also suppresses macrophage infiltration and neutrophils. Collectively, these data suggest that DIM has an indirect effect in angiogenesis and lymphangiogenesis because it inhibits VEGF-A/VEGFR2 and VEGF-C/VEGFR3 by inhibiting neutrophil and macrophage infiltration in addition to cytokines in colitis. In the current study, our findings indicated that DIM markedly attenuated the clinical signs and histological characteristics of DSS-induced colitis in mice. DIM suppressed neutrophil infiltration and pro-inflammatory cytokines by a mechanism mediated with the suppression of inflammation. Furthermore, DIM significantly suppressed the expression of VEGF-A and VEGFR-2; thus, the mechanism may be related to the suppression of proangiogenesis. Moreover, because DIM significantly reduced VEGF-C, VEGF-D, VEGFR-3, and Ang-2 expression, the mechanism may be related to the suppression of lymphangiogenesis. Overall, our data support that DIM has a preventive effect, and DIM may be a possible treatment option for intestinal inflammatory-associated lymphangiogenesis and angiogenesis.

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AUTHOR INFORMATION

Corresponding Author

*(M.J.S.) Mail: Research Division Emerging Innovative Technology, Korea Food Research Institute, 516 BaekhyunDong, Bundang-Ku, Songnam, Gyeongki 463-746, Republic of Korea. Phone: 82-31-780-9316. Fax: 82-31-709-9876. E-mail: [email protected]. Funding

This work was supported by Korea Food Research Institute of South Korea and National Research Council of Science & Technology (NST) grant by the Korea government (MSIP) (No. CAP-15-09-KIMS). Notes

The authors declare no competing financial interest.

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