Brazilian propolis extract reduces intestinal barrier defects and inflammation in a colitic mouse model

N U TR IT ION RE S EAR CH 6 9 ( 2 01 9 ) 3 0 –4 1

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Brazilian propolis extract reduces intestinal barrier defects and inflammation in a colitic mouse model Yuki Shimizu a , Takuya Suzuki a, b,⁎ a Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan b Program of Food and AgriLife Science, Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, HigashiHiroshima 739-8528, Japan

ARTI CLE I NFO

A BS TRACT

Article history:

Brazilian propolis is rich in cinnamic acid derivatives and reportedly reduces intestinal

Received 12 April 2019

inflammation in rodents; however, the underlying mechanisms remain unclear. We

Revised 31 May 2019

hypothesized that the regulation of tight junction (TJ) barrier, Th17 cell differentiation,

Accepted 25 July 2019

and/or, macrophage activation by cinnamic acid derivatives were involved in the propolismediated anti-inflammatory effect. Mice were orally administered 2% dextran sodium

Keywords:

sulfate (DSS) in combination with either the feeding control or a diet containing 0.3%

Propolis

ethanol extract of Brazilian propolis for 9 days. DSS administration induced acute colitis in

Colitis

mice, whereas the propolis extract mitigated DSS-induced weight loss; colon shortening;

Inflammation

increased plasma levels of lipopolysaccharide-binding protein; reduced expression of TJ

Tight junction

proteins, such as zonula occludens, junctional adhesion molecule-A, occludin, and

Macrophage

claudins; and increased expression of inflammatory cytokines, such as tumor necrosis

Interleukin 17

factor (TNF) α, interleukin (IL) 6, and IL-17a. Cinnamic acid derivatives, such as artepillin C and caffeic acid phenethyl ester, present in the propolis extract suppressed the IL-17 production from cultured murine splenocytes through decreased retinoic acid-related orphan receptor gT expression. Baccharin, drupanin, and culifolin, which are also present in Brazilian propolis, reduced the TNF-α and/or IL-6 production by suppressing inflammatory signaling in murine RAW 264.7 macrophages. Taken together, the regulation of Th17 differentiation and macrophage activation by cinnamic acid derivatives, at least in part, contribute to the anti-inflammatory effect mediated by Brazilian propolis. © 2019 Elsevier Inc. All rights reserved.

Abbreviations: CAPE, caffeic acid phenethyl ester; Cxcl-2, chemokine C-X-C motif ligand-2; DSS, dextran sodium sulfate; ELISA, enzyme-linked immunosorbent assay; FD, fluorescein isothiocyanate-labeled dextran; HBSS, Hanks’ balanced salt solution; IBD, inflammatory bowel disease; IL, interleukin; JAM, junctional adhesion molecule; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; RORγT, retinoic acid-related orphan receptor γT; SEM, standard error of the mean; TER, transepithelial electrical resistance; TGF, transforming growth factor; Th cell, helper T cell; TJ, tight junction; TNF, tumor necrosis factor; ZO, zonula occludens.. ⁎ Corresponding author at: Program of Food and AgriLife Science, Graduate School of Integrated Sciences for Life, Hiroshima University, 14-4, Kagamiyama, Higashi-Hiroshima, 739-8528, Japan. Tel.: +81 82 424 7984; fax: +81 82 424 7916. E-mail address: [email protected] (T. Suzuki). https://doi.org/10.1016/j.nutres.2019.07.003 0271-5317/© 2019 Elsevier Inc. All rights reserved.

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1.

Introduction

Inflammatory bowel diseases (IBDs) are chronic, progressive, immunologically mediated intestinal disorders, mainly represented by Crohn's disease and ulcerative colitis [1]. Patients with IBDs exhibit different symptoms, such as abdominal pain, diarrhea, bloody stool, and fever. As demonstrated for many cohorts, the incidence of IBDs has been traditionally high in North America and Western Europe [1,2]. However, the incidence is currently increasing in emerging populations, such as Asian countries, hence suggesting that IBD pathogenesis is closely associated with lifestyle, namely dietary habits, and genetic factors. A case–control study demonstrated a markedly lower intake of vegetables and fruits by newly diagnosed pediatric patients with Crohn's disease than by controls without IBDs [3]. This observation may indicate that plant-derived food factors, such as phenolic compounds, have the potential to reduce the risk of IBDs. Although the pathogenesis of IBD is unclear, intestinal inflammation develops through a crosstalk of different cells in the intestine, including immune cells and epithelial cells [4]. In the early phase of disease, the impaired intestinal barrier leads to increased permeability to luminal noxious molecules, such as dietary antigens and bacterial toxins, which robustly activate the innate immune cells, including macrophages [4,5]. Activated macrophages produce proinflammatory cytokines, such as tumor necrosis factor (TNF) α and interleukin (IL) 6, stimulate the adaptive immune response and influence the lineage and activity of helper T (Th) cell subsets. Abnormally activated Th cells release different inflammatory mediators that generate the vicious cycle of inflammation, leading to chronic tissue injury and epithelial damage. Accumulating evidence suggests that Th17 cells massively infiltrate the inflamed intestines of patients with IBDs, where they produce IL-17 and other cytokines [6]. In addition, inflammatory cytokines, such as TNF-α, IL-6, and IL17, are reported to impair the intestinal epithelial barrier [7-9]. Accordingly, the treatment of different cells associated with intestinal inflammation by nutrients and other components in food may be an effective approach to reduce and treat IBDs. Propolis is a resinous substance collected by the honeybee. The honeybee produces propolis by mixing saliva and beeswax with exudates derived from tree resin, sap, bud, and bark. Consequently, the chemical composition of propolis that determines its biological activity is complex and differs based on the geographical region of botanical origin. Brazilian propolis is characterized by high amounts of cinnamic acid derivatives, such as artepillin C, baccharin, p-coumaric acid, drupanin, culifolin, and caffeic acid phenethyl ester (CAPE). Previous studies demonstrated that diet supplementation with Brazilian propolis reduces the intestinal inflammation in murine model of colitis [10-13]; however, the underlying molecular mechanisms are not fully understood. Information about the effect of propolis and cinnamic acid derivatives on the epithelial barrier structure, inflammatory reaction of macrophages, and differentiation of Th17 cells is quite limited. We hypothesized that the regulation of these cellular events by cinnamic acid derivatives plays an important role in the protection of the intestinal health by Brazilian propolis.

Table 1 – Proximate analysis of the ethanol extract of propolis g/100 g powder Protein Lipid Ash Moisture

0.7 47.0 0.4 5.9

Accordingly, in the current study, we examined the protective role of Brazilian propolis in the intestinal health in dextran sodium sulfate (DSS)-induced colitic mice. To analyze the effects of cinnamic acid derivatives on the regulation of epithelial barrier, inflammatory reaction of macrophages, and differentiation of Th17 cells, we used the intestinal Caco-2 cells, RAW 264.7 macrophage cells, and murine splenocytes, respectively.

2.

Methods and materials

2.1.

Reagents

DSS (molecular weight 36 000–50 000) was purchased from MP Biomedicals Inc (Santa Ana, CA, USA). Rabbit antibodies against zonula occludens (ZO) 1, occludin, claudin-3, claudin-4, junctional adhesion molecule (JAM) A, and goat Alexa Fluor 488-conjugated anti-rabbit IgG were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit anti-ZO-2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-pNFκB p65, pJNK1/2, pERK1/2, and pp38 MAPK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG were purchased from SeraCare (Milford, MA, USA). Cell culture reagents and supplies were from Thermo Fisher Scientific. Cinnamic acid derivatives, baccharin, drupanin, and culifolin, were kindly provided by Yamada Bee Company, Inc. (Okayama, Japan). All other chemicals were obtained from Wako Pure Chemical Industries (Osaka, Japan).

2.2.

Preparation of Brazilian propolis extract

Dried powder of propolis ethanol extracts was prepared from Brazilian propolis, as described previously [14], and kindly provided by Yamada Bee Company, Inc. The nutritional and chemical components of the ethanol extract are shown in

Table 2 – Cinnamic acid derivatives in the ethanol extract of propolis g/100 g powder Artepillin C Baccharin Drupanin p-Coumaric acid Culifolin CAPE

9.5 3.5 1.4 1.2 0.23 0.013

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Table 3 – Ingredient composition of test diet Ingredient

Control diet

a

g/kg diet b

Casein α-Corn starch c Sucrose Soybean oil Choline bitartrate L-Cystine Mineral mixture d Vitamin mixture d Cellulose e

200 529.5 100 70 2.5 3 35 10 50

a

Propolis-containing diet was prepared by adding the Brazilian propolis extract (0.3%, w/w) to the control diet. b Casein (ALACID; New Zealand Daily Board). c α-Corn starch (Amylalpha CL; Chuo-Shokuryou). d Mineral and vitamin mixtures were prepared according to the AIN-93G formulation. e Powdered cellulose (Just fiber; International Fiber Corporation).

Tables 1 and 2. The Brazilian propolis extract was standardized to contain a minimum 8.0% artepillin C.

2.3.

Animals and diets

All study protocols were pre-approved by the Animal Use Committee of Hiroshima University, and all mice were maintained in accordance with the Hiroshima University guidelines for the care and use of laboratory animals (authorization no. C15–10-3). Male BALB/c mice (7-week-old, ~21 g) were obtained from Charles River Japan (Yokohama, Japan). Throughout the study, the mice were housed in cages (3 or 4 mice/cage) under the following conditions: controlled temperature of 22 ± 2 °C; relative humidity of 40–60%; and light exposure from 08:00 to 20:00 h. The mice were allowed to acclimatize to the laboratory environment with free access to an AIN-93G-formula control diet [15] as well as distilled water for 1 week prior to the start of the experiments.

2.4.

DSS-induced colitis mouse model

BALB/c mice (n = 21) were randomly divided into three groups: control, DSS, and DSS + Propolis (n = 7 per group). The DSS + Propolis group animals were fed a diet containing 0.3% (w/w) propolis extract for 9 d of the experimental period (Table 3). The control and DSS groups were fed the control diet during the experimental period. The DSS and DSS + Propolis groups were administered 2% (w/v) DSS in drinking water, whereas the control group was administered only distilled water during the experimental period. Body weights and stool scores of mice were determined every day after the start of DSS administration, as described in section 2.5. At the end of the experiment, the mice were placed under isoflurane anesthesia and blood was collected from the abdominal vein to measure lipopolysaccharide (LPS)binding protein (LBP) levels. The mice were then euthanized by exsanguination. Plasma LBP levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Biometec GmbH, Greifswald, Germany). The colon was dissected, and its length was measured. To evaluate the

colonic barrier integrity, an intestinal permeability test was performed, as described in section 2.6. Colonic tissues were also subjected to histological, immunoblotting, immunofluorescence, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses, as described in sections 2.10–2.13.

2.5.

Stool evaluation scores

To assess the severity of colitis, the stool scores were determined, as described previously [16,17]. Briefly, the stool score was defined as the sum of the diarrheal stool score (0, normal stool; 1, mildly soft stool; 2, very soft stool; and 3, watery stool) and the bloody stool score (0, normal color stool; 1, brown color stool; 2, reddish color stool; and 3, bloody stool).

2.6.

Intestinal permeability test of the mouse colon

Colonic permeability to fluorescein isothiocyanate-labeled dextran with a molecular weight of 4000 (FD-4) was evaluated in the everted mouse colon. Briefly, the colon was everted with a blunt plastic rod and tied with a surgical suture at one end. The other end was tied after injecting 0.2 mL of Hanks' balanced salt solution supplemented with 5.6 mmol/L Dglucose and 4 mmol/L glutamine (HBSS). The everted colon was incubated in HBSS supplemented with 25 μmol/L FD-4 for 30 min at 37 °C in a water bath shaken at 50 oscillation/min. After incubation, the inner solution was collected and the fluorescence of FD-4 in the inner solution was determined using a fluorescence plate reader (ARVO X4; Perkin Elmer, Waltham, MA, USA). The colonic permeability to FD-4 was calculated as pmol/cm colon/min.

2.7. Evaluation of Th17 differentiation in murine splenocyte culture The effect of cinnamic acid derivatives from the propolis on Th17 differentiation and IL-17 production was evaluated using murine splenocytes [18]. Mice were anesthetized using isoflurane and euthanized by exsanguination. The spleen was then dissected from mice and splenocyte suspensions were prepared by physical disintegration, as described previously [18]. To induce Th17 differentiation and IL-17 production, splenocytes were incubated with 2 ng/mL transforming growth factor (TGF) β and 20 ng/mL IL-6 at 37 °C for 72 h in 120 μL of RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 10 μmol/L 2mercaptoethanol, 10 mmol/L HEPES, 50 U/mL penicillin, and 50μg/mL streptomycin. Individual cinnamic acid derivatives, artepillin C (2.5–25 μmol/L), baccharin (25 μmol/L), drupanin (25 μmol/L), p-coumaric acid (25 μmol/L), culifolin (25 μmol/L), and CAPE (2.5–25 μmol/L), were added to the culture 30 min before stimulation with TGF-β and IL-6. Production of IL-17 in the cell culture supernatant after 72 h was determined by using a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA). Splenocytes cultured for 72 h were analyzed by qRTPCR, as described in sections 2.13.

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2.8. Evaluation of the inflammatory reaction in RAW 264.7 cells To examine the effect of cinnamic acid derivatives present in propolis on the inflammatory reaction in macrophages, the RAW 264.7 murine macrophage cell line was used (American Type Culture Collection, Manassas, VA, USA). RAW 264.7 cells were propagated and maintained under standard cell culture conditions in DMEM supplemented with 10% fetal bovine serum. Cells were stimulated with LPS (100 ng/mL) for 6 h, and the production of TNF-α and IL-6 in the cell culture supernatant was measured using a commercially available ELISA kit (Thermo Fischer Scientific). Individual cinnamic acid derivatives, artepillin C (50 μmol/L), baccharin (1–30 μmol/L), drupanin (1–30 μmol/L), pcoumaric acid (50 μmol/L), culifolin (1–30 μmol/L), and CAPE (50 μmol/L), were added to the culture 30 min before stimulation with LPS. Cells cultured for 30 min and 6 h were analyzed by immunoblotting, as described in section 2.11.

2.9.

Evaluation of TJ barrier in the intestinal Caco-2 cells

To examine the effects of cinnamic acid derivatives present in propolis on the intestinal barrier, human intestinal Caco-2 cells were used (American Type Culture Collection). Caco-2 cells were propagated and maintained under standard cell culture conditions, as described elsewhere [19,20]. The cells were seeded onto permeable polyester membrane filter supports (Transwell, 12-mm diameter, 0.4-μm pore size; Corning Costar Co., Cambridge, MA, USA) at a density of 0.25 × 106 cells/cm2. Then, 50 μmol/L of individual cinnamic acid derivatives (artepillin C, baccharin, drupanin, p-coumaric acid, culifolin, and CAPE) were administered to the apical wells and the cells were incubated for an additional 24 h. Transepithelial electrical resistance (TER) was measured before, and at 1, 3, 6, 12, and 24 h after the administration using a MillicellERS system (Millipore, Bedford, MA, USA). All experiments were conducted on days 13 and 14 post-seeding.

2.10.

Hematoxylin and eosin (H&E) staining

The colon tissue from mice was embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan), and frozen sections (8 μm) were prepared on glass slides. Colon sections were fixed with 4% (w/v) paraformaldehyde and subjected to H&E staining, as previously reported [21]. At least 3 sections were prepared using the colon tissue from each mouse and images were acquired using a Leica DMI 6000B with a 20× objective (Wetzlar, Germany). Histological score was evaluated

using a validated scoring system in a blinded manner [22]. Three independent parameters were measured: severity of inflammation (0, none; 1, slight; 2, moderate; 3, severe), depth of injury (0, none; 1, mucosal; 2, mucosal and submucosal; 3, trans-mural), and crypt damage (0, none; 1, basal one-third portion damaged; 2, basal two-thirds portion damaged; 3, entire crypt and epithelium lost). At least 4 optical fields were examined in a section, and the average of the scores was calculated.

2.11.

Immunoblotting

Whole colon tissue (50 mg) was homogenized in 1 mL of lysis buffer containing 1% (w/v) sodium dodecyl sulfate, 1% (v/v) Triton X-100, and 1% (w/v) sodium deoxycholate in 30 mmol/L Tris with protease and phosphatase inhibitors (pH 7.4), by using a Polytron-type homogenizer (KINEMATICA AG, Lucerne, Switzerland). For the experiments, RAW 264.7 cells were lysed in the appropriate volume of the lysis buffer. Immunoblotting analysis of ZO-1, ZO-2, JAM-A, occludin, claudin-3, claudin-4, pNFκB p65, pJNK1/2, pERK1/2, and pp38 MAPK was performed, as previously described [17,23].

2.12.

Immunofluorescence analysis

The colon tissue from mice was embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan), and frozen sections (8 μm) were prepared on glass slides. ZO-1 and claudin-3 were immunostained, as previously described [17,23]. The specimens were preserved in a mounting medium, and fluorescence was visualized using an LCM700 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany).

2.13.

qRT-PCR analysis

Total RNA from whole-colon tissue and murine splenocytes was isolated using NucleoSpin RNA II (Macherey-Nagel GmbH, Düren, Germany) and RNAiso plus (Takara Bio, Kusatus, Japan), respectively, and it was reverse-transcribed using a ReverTra Ace qPCR-RT kit (Toyobo, Osaka, Japan), according to the manufacturers' instructions. The expression of genes encoding TNF-α, IL-1β, IL-6, chemokine C-X-C motif ligand (CXCL) 2, retinoic acid-related orphan receptor gT (RORγT), cJun, and IL-17a was determined by qRT-PCR, as previously described [17,23]. The qRT-PCR was performed using a StepOne Real-Time PCR system (Thermo Fisher Scientific) and Brilliant III Ultra-Fast SYBR Green qRT-PCR

Table 4 – Primer sequences for qRT-PCR Target gene

Forward

Reverse

References

Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse

5′-GATCATCCAGTCCAGCAATG- 3’ 5′-AGTGAACTGCGCTGTCAATG-3′ 5′-TTCCTCCTTGCCTCTGATGG-3′ 5′-CTGATGCTGGTGACAACCAC-3′ 5′-TGGATTCAGAGGCAGATTCA-3′ 5′-GCCCACCATATTCCAATACCT-3′ 5′-TCGTAGCAAACCACCAAGTG-3′ 5′-TCAAGAAGGTGGTGAAGCAG-3′

5′-GTGTTCTGGCTATGCAGTTCAG-3′ 5′-ACTTTTTGACCGCCCTTGAG-3′ 5′-ATGTGCTGGTGCTTCATTCA-3′ 5′-TCCACGATTTCCCAGAGAAC-3′ 5′-CAGTTTGGGACCCCTTTACA-3′ 5′-GGTTTCCTCAAAACGAAGTCC-3′ 5′-CTTTGAGATCCATGCCGTTG-3′ 5′-AAGGTGGAAGAGTGGGAGTTG-3′

This paper 16 17 16 16 18 17 16

cJun Cxcl2 Il1b Il6 Il17a RORγT Tnfa Gapdh

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Master Mix (Agilent Technologies, Santa Clara, CA, USA). The primer sequences are listed in Table 4. Data were analyzed by the comparative threshold cycle (ΔΔCT) method and were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase gene, the internal control gene.

2.14.

analyses required logarithmic transformations to achieve homogeneity. Differences at P < .05 were considered significant. The sample size was calculated using the POWER procedure for one-way ANOVA, considering P < .05 with a power of 0.80 (SAS Institute, Cary, NC, USA) and using the results from our previous studies.

Statistical analyses

All data are presented as the means and standard error of the means (SEM). Statistical analyses were performed by one-way ANOVA followed by the Tukey–Kramer post-hoc test by using the Statcel 3 program (OMS Publishing, Saitama, Japan). Bartlett's test for homogeneity of variances was performed to determine whether the variances were equal; some

Body weight change (%)

A 15

Control DSS DSS+Propolis

10 5

#

#

0

*

-5 -10

*

-15 0

1

2

3

4

5

6

7

8

9

Time after start of DSS administration (d)

B

8

* Control DSS DSS+Propolis

Stool score

6

* *

4

*#

* * 2

# * *# *

* * *

0 0

1

2

3

4

5

6

7

8

9

3.

Results

3.1. Effect of propolis extract on body weight, clinical score, colon length, and mucosal structure in DSS-administered mice DSS administration reduced the body weight of mice. The body weight changes in the DSS group on days 8 and 9 were smaller than those in the control group (Fig. 1A). Feeding propolis partially restored the DSS-induced body weight loss. The values in the DSS + Propolis group on days 8 and 9 were higher than those in the DSS group. The stool scores in the DSS and DSS + Propolis groups on and after day 4 were higher than those in the control group (Fig. 1B). However, the scores in the DSS + Propolis group on and after day 7 were lower than those in the DSS group. Colon shortening is associated with histological damage in DSS-induced colitic mice [15]. Feeding propolis partially restored the DSS-induced colon shortening (Fig. 1C). The histological evaluation of colonic tissues using H&E staining demonstrated that the DSS administration induced pathological features of colitis such as mucosal infiltration of inflammatory cells, ulcer, and crypt loss (Fig. 2). However, propolis administration reduced these alterations, and the histological score in the DSS + Propolis group was lower than that in the DSS group. These observations indicated that supplementation with the propolis extract attenuated the development of DSS-induced colitis in mice, in agreement with previous studies [10,11].

3.2. Effect of propolis extract on the colonic barrier in DSS-administered mice

Time after start of DSS administration (d)

C Colonlength (cm)

5 4

*

* #

3 2 1 0 Control

DSS

Colonic hyperpermeability is closely associated with IBD pathogenesis [17,23]. The colonic permeability to macromolecules, evaluated using everted sacs of colonic tissue, in the DSS group was approximately 6 times greater than that in the control group (Fig. 3). Further, feeding propolis partially, but effectively, reduced the DSS-induced hyperpermeability of the colon. The increased plasma levels of LBP, an LPS transporter, induced by DSS administration were also suppressed by propolis. These findings demonstrated that propolis supplementation protected the colonic barrier in the DSS-induced colitic mice.

DSS+Propolis

Fig. 1 – The effect of propolis extract on DSS-induced body weight loss and colonic damage in mice. Body weight change (A), stool score (B), and colon length (C) were evaluated in mice fed diets with and without the propolis extract, with or without DSS administration. The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post hoc test.

3.3. Effect of propolis extract on colonic TJ protein levels in DSS-administered mice The TJ structure is an essential component of the intestinal barrier [5]. Immunoblotting revealed that DSS administration impaired the colonic expression of different TJ proteins, such as ZO-1, ZO-2, occludin, JAM-A, claudin-3, and claudin-4; the levels of these TJ proteins in the DSS group were lower than

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N U TR IT ION RE S EA RCH 6 9 ( 2 01 9 ) 3 0 –4 1

A

B *

Histological score

Control

12 10 8

* #

6 4

DSS

2 0 DSS

DSS+Propolis

DSS+Propolis

Control

A

5

FD4 permeability (pmol/cm x min)

Fig. 2 – The effect of propolis extract on DSS-induced histological alterations in colonic tissues of mice. (A) Sectioned colonic tissues of mice were stained with hematoxylin and eosin. Images were acquired using a digital inverted microscope with a 20× objective. Each histological image is representative of 7 mice. (B) Histopathological evaluations were performed in a blinded manner. The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test.

4

*

3

#

2 1 0 Control

Plasma LBP (μg/ml)

B

DSS

DSS+Propolis

100

3.4. Effect of propolis extract on colonic cytokine expression in DSS-administered mice

*

80 60 40

those in the control group (Fig. 4A). Propolis administration mitigated the DSS-induced decrease of these TJ protein levels. Immunofluorescence microscopy indicated that ZO-1 was located at the apical region of the lateral membrane of colonic epithelial cells, with a continuous belt-like structure in the control mice (Fig. 4B). Claudin-3 was located at the basal membrane as well as the lateral membrane of epithelial cells in the colon of the control mice. DSS administration decreased the fluorescence intensity of ZO-1 and claudin-3, and it induced discontinuous protein patterns. The expression and localization of ZO-1 and claudin-3 in the DSS + Propolis group were relatively intact.

#

20

DSS administration enhanced the gene expression of proinflammatory cytokines TNF-α, IL-1β, IL-6, CXCL-2, and IL-17a in the colon, indicating colonic inflammation (Fig. 5). Propolis administration suppressed the expression of these proinflammatory cytokines, but the suppression of IL-6 and CXCL-2 expression was not statistically significant.

0 Control

DSS

DSS+Propolis

Fig. 3 – The effect of propolis extract on DSS-induced increase in the colonic permeability and plasma LBP levels in mice. Colonic permeability to fluorescein isothiocyanate-labeled dextran (FD4, A) and plasma LBP levels (B) were determined in mice fed diets with and without the propolis extract, with or without DSS administration. The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test.

3.5. Effect of propolis components on Th17 differentiation in murine splenocytes Stimulation with TGF-β and IL-6 increased IL-17 protein production in murine splenocytes, indicating Th17 differentiation (Fig. 6A). The IL-17 production induced by TGF-β and IL-6 was suppressed by all the cinnamic acid derivatives tested; however, the suppressive potency varied. The suppression by artepillin C and CAPE was much more potent than that of other compounds tested and was dose-

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N U TR IT ION RE S EAR CH 6 9 ( 2 01 9 ) 3 0 –4 1

Control

A

DSS

DSS+Propolis

β-actin

Protein density (AU)

1.2 1

#

#

0.8

#

*#

*

*#

0.6

*# *

0.4

*

*

* *

0.2 0 ZO-1

B

ZO-2

DSS

JAM-A

Claudin-3

Claudin-4

DSS+Propolis

Claudin-3

ZO-1

Control

Occludin

Fig. 4 – The effect of propolis extract on TJ protein levels in the colon of DSS-administered mice. Mice were fed diets with and without the propolis extract, with or without DSS administration. Protein levels of ZO-1, ZO-2, occludin, JAM-A, claudin-3, and claudin-4 in the colon were determined by immunoblotting (A). Each immunoblot image is representative of 7 mice. The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test. (B) Immunolocalization of ZO-1 and claudin-3 in the colon was analyzed by immunofluorescent microscopy. Representative images of 7 mice for each treatment are shown. Tukey–Kramer post-hoc test.

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N U TR IT ION RE S EA RCH 6 9 ( 2 01 9 ) 3 0 –4 1

A

C 20

*

8 6 4

#

2 0 DSS

10

# 5

DSS+ Propolis

Control

E

1000

IL-17a mRNA (AU)

CXCL-2 mRNA (AU)

D

15

750 500 250

0 Control

*

750 500 250 0

*

1000

*

IL-6 mRNA (AU)

IL-1β mRNA (AU)

TNF-α mRNA (AU)

B 10

DSS

0

DSS+ Propolis

Control

DSS

DSS+ Propolis

15

* 10

# 5

0 Control

DSS

DSS+ Propolis

Control

DSS

DSS+ Propolis

Fig. 5 – The effect of propolis extract on inflammatory cytokine expression in the colon of DSS-administered mice. The expression of Tnfa (A), Il1b (B), Il6 (C), Cxcl2 (D), and Il17a (E) genes in the colon of mice fed diets with and without the propolis extract, with or without DSS administration, was determined by qRT-PCR analysis. The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test.

* # *# *# *

*#

#

#

10

*

8

*#

6

*#

4 2

#

0 ArtepillinC 0 (µM)

0

2.5

5

#

10

25

IL-17 production (pg/ml)

C

B 7 6 5 4 3 2 1 0

IL-17 production (ng/mL)

IL-17 production (ng/mL)

A

6

*

5 4 3

*#

2

*#

1

0 CAPE 0 (µM)

0

TGF-β+IL-6

2.5

5

#

#

10

25

TGF-β+IL-6

TGF-β+IL-6

20

0 µM 0 µM

15

10 µM 10 5

E

ArtepillinC

*

TGF-β+IL-6

25 µM

*

* # #

#

# #

0 IL-17A

RORγT

cJun

mRNA expression (AU)

mRNA expression (AU)

D

35 30

CAPE

*

0 µM 0 µM 10 µM 25 µM

25 20 15 10

*#

5

TGF-β+IL-6

* #

#

*

0 IL-17a

RORγT

cJun

Fig. 6 – The effect of propolis components on Th17 response in murine splenocytes. Murine splenocytes were stimulated with TGF-β and IL-6 in the absence or presence of artepillin C (A, B, D); baccharin, drupanin, p-coumaric acid, and culifolin (A); and CAPE (A, C, E). The IL-17 production in cell culture supernatants was determined by ELISA (A–C). The expression of Th17related genes was determined by qRT-PCR (D, E). The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test.

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30

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Fig. 7 – The effect of propolis components on the inflammatory response in murine macrophages. Murine macrophage cell line, RAW 264.7, was stimulated with LPS in the absence or presence of artepillin C (A, B), baccharin (A, B, C, D), drupanin (A, B, E, F), p-coumaric acid (A, B), culifolin (A, B, G, H), and CAPE (A, B). TNF-α and IL-6 production was determined by ELISA (A–C, E, G). Gene expression of the inflammatory molecules was determined by qRT-PCR (D, F, H). The values are means ± SEM (n = 7). *P < .05 vs the control group. #P < .05 vs DSS group. Tukey–Kramer post-hoc test.

dependent (Fig. 6B and C). The qRT-PCR analysis revealed that the expression of Il17a, Rorgt, and cjun genes was higher in the cells stimulated with TGF-β and IL-6 than in the control cells (Fig. 6D and E). Artepillin C and CAPE reduced these Th17-associated mRNA levels in a dose-dependent

manner, although the suppression of Rorgt expression by 10 μmol/L artepillin C and CAPE, and the suppression of cjun by CAPE were not statistically significant.

N U TR IT ION RE S EA RCH 6 9 ( 2 01 9 ) 3 0 –4 1

3.6. Effect of propolis extract on the inflammatory reaction of RAW 264.7 cells LPS stimulation increased the production of TNF-α and IL-6 by RAW 264.7 cells (Fig. 7A and B). Among the cinnamic acid derivatives used, baccharin, drupanin, and CAPE suppressed both the LPS-induced TNF-α and IL-6 production. Culifolin suppressed the TNF-α production, but not IL-6 production. The suppressive effects of baccharin and drupanin on the LPS-induced IL-6 production and of culifolin on the LPSinduced TNF-α production were dose-dependent (Fig. 7C, E, and G). LPS stimulation induced the phosphorylation of NFκB p65, JNK1/2, ERK1/2, and p38MAPK (Fig. 7D, F, and H). Baccharin mitigated the LPS-induced phosphorylation of NFκB p65 and ERK1/2, and drupanin mitigated the phosphorylation of pNFκB p65, ERK, and p38. Culifolin mitigated the phosphorylation of the four signaling molecules examined.

3.7. Effect of propolis components on the intestinal barrier of Caco-2 cells The TER values across the monolayers formed by Caco-2 cells were between 101% and 107% of the initial value in comparison with the control treatments. Incubation with none of the cinnamic acid derivatives tested affected the measured TER values (data not shown).

4.

Discussion

In agreement with previous studies [10-13], this current study demonstrated that the ethanol extract of Brazilian propolis attenuated the development of DSS-induced colitis in mice. Interestingly, propolis supplementation reduced the impairment of epithelial TJ barrier in the colon. In addition, we showed that some cinnamic acid derivatives present in Brazilian propolis, such as artepillin C, baccharin, drupanin, culifolin, and CAPE, have the potential to suppress the inflammatory reaction in immune cells. These observations suggest that the different components of propolis cooperatively produce the protective effect of propolis on the colon by targeting specific cells. Accumulating evidence from basic and clinical studies indicates that Th17-type cytokines, such as IL-17, seem to play roles in the destruction of tissues and maintenance of inflammation in IBDs [4,6]. Indeed, IL-17 expression in the blood and tissues of patients with IBDs is up-regulated [24]. Our results showed that propolis extract supplementation reduced the IL-17a expression in colitic mice. Furthermore, all the tested cinnamic acid derivatives present in the propolis suppressed the IL-17 production to different degrees in murine splenocytes. In agreement with these findings, IL-17 suppression by propolis was also observed in a murine model of arthritis, which is another Th17-related disease [25]. Taken together, the suppression of Th17 differentiation is possibly one of the mechanisms underlying the propolis-mediated reduction of the severity of colitis. Since artepillin C and CAPE exerted the most potent effects among the cinnamic acid derivatives tested, we focused on the molecular mechanisms underlying the suppression of

39

Th17 differentiation by these two components. Artepillin C reduced the expression of RORγT and cJun, while CAPE reduced only the expression of RORγT. The differentiation of Th17 cells from naïve T cells requires TGF-β and IL-6. These two cytokines induce RORγT, which is a master regulator of Th17 differentiation, through cJun activation [26]. Artepillin C and CAPE may reduce the activity of JNK and cJun, respectively. Further studies should be performed to reveal the molecular mechanisms underlying the suppressive effects of artepillin C and CAPE on Th17 differentiation. Four cinnamic acid derivatives in Brazilian propolis, baccharin, drupanin, culifolin, and CAPE, apparently suppress the LPS-mediated inflammatory reaction of macrophages. LPS induces the activation of different cellular signaling, resulting in the production of inflammatory cytokines, such as TNF-α and IL-6 [27]. We examined the cellular signaling in macrophages treated with baccharin, drupanin, and culifolin, but not CAPE, because it has already been demonstrated that CAPE reduces the inflammatory reaction in macrophages [28]. While culifolin reduced the phosphorylation of all signaling molecules tested, baccharin reduced the phosphorylation of only NFκB and ERK1/2, and drupanin did not affect the phosphorylation of JNK1/2. Recognition of LPS by Toll-like receptor 4 recruits the adaptor proteins, such as myeloid differentiation factor 88, which activate the downstream signaling molecules. Baccharin and drupanin may target IκBkinase, MEK1/2, and MKK, which are the upstream molecules of NFκB, ERK1/2, and p38, respectively. On the contrary, culifolin may influence the interaction of TLR4 with adaptor proteins. We observed that none of the cinnamic acid derivatives tested influenced the intestinal barrier of Caco-2 cells, although propolis supplementation restored the colonic TJ barrier in colitic mice. This discrepancy may be explained by the conditions in the experiment involving Caco-2 cells. We examined the promotive effects of cinnamic acid derivatives on the TJ barrier in unstimulated Caco-2 cells; however, it has been reported that inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, as well as oxidative stress are associated with the barrier defect in IBDs [5,7,8,29,30]. The protective effect of cinnamic acid derivatives on the intestinal barrier against these noxious stimulants should be examined in further studies. Alternatively, the other components of Brazilian propolis may contribute to the regulation of the intestinal TJ barrier. Meanwhile, Wang et al. [27] have reported that Chinese propolis extract improves the intestinal barrier function. This discrepancy is possibly associated with the difference in the components of Brazilian and Chinese propolis preparations. Chinese propolis is reportedly rich in apigenin, chrysin, dimethoxycinnamic acid, and transisoferulic acid, as well as CAPE [12]. Data on murine splenocytes and RAW 264.7 cells presented in the current study suggest that some cinnamic acid derivatives present in the Brazilian propolis extract could potentially regulate the inflammatory response in Th cells and macrophages. However, the tested cinnamic acid derivatives exerted their effects at widely different concentrations. Artepillin C and CAPE suppressed Th17 differentiation at concentrations above 2.5 μmol/L. The anti-inflammatory effect of baccharin, drupanin, and p-coumaric acid on

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macrophages was observed at 10 and 30 μmol/L. Artepillin C is one of the most abundant cinnamic acid derivatives in the Brazilian propolis extract and accounts for approximately 10% of the powder. Baccharin, drupanin, and p-coumaric acids are also relatively abundant in the propolis extract, while CAPE and culifolin account for less than 1% of the extract. Precise abundance of these cinnamic acid derivatives in the intestinal lumen of mice receiving a propolis extract-supplemented diet should be analyzed in future studies. In addition, we did not examine the effect of the propolis extract on intestinal microbiota, which are closely associated with intestinal health and diseases. Recent studies demonstrated that feeding propolis extracts influenced the diversity and composition of intestinal microbiota in rodents. Wang et al. suggested that the propolismediated reduction in the genus Bacteroides is potentially linked to reduced severity of colitis in rats [12,13]. In conclusion, supplemental feeding of a Brazilian propolis extract reduced the development of colitis in mice. The cinnamic acid derivatives present in propolis appear to impact the inflammatory reactions in different cell types, such as macrophages and Th17 cells. The presented findings, at least in part, reveal the molecular mechanisms underlying the protective effect of propolis on intestinal health.

Acknowledgment This work was partially supported by JSPS KAKENHI (grant no. 16 K07737) and Yamada Research Grant. The funders did not play any role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The authors declare that there are no conflicts of interest. We would like to thank Editage (www.editage.jp) for English language editing. Y. S. and T. S. designed the research, conducted the study, and performed statistical analyses. T.S. wrote the manuscript and has primary responsibility for the final content. All authors have read and approved the final manuscript.

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