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Journal of Animal Science logoLink to Journal of Animal Science
. 2020 Jul 31;98(8):skaa245. doi: 10.1093/jas/skaa245

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells

Qianru Hui 1,#, Emily Ammeter 1,#, Shangxi Liu 1, Runqiang Yang 1,2, Peng Lu 1, Ludovic Lahaye 3, Chengbo Yang 1,✉
PMCID: PMC7531220  PMID: 32735667

Abstract

Eugenol (4-allyl-2-methoxyphenol) is an essential oil component, possessing antimicrobial, anti-inflammatory, and antioxidative properties; however, the effect of eugenol on porcine gut inflammation has not yet been investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2) has been set up. Cells were pretreated with 100 μM (16.42 mg/L) eugenol for 2 h followed by 10 μg/mL LPS stimulation for 6 h. Proinflammatory cytokine secretion; reactive oxygen species; gene expression of proinflammatory cytokines, tight junction proteins, and nutrient transporters; the expression and distribution of zonula occludens-1 (ZO-1); transepithelial electrical resistance (TEER); and cell permeability were measured to investigate the effect of eugenol on inflammatory responses and gut barrier function. The results showed that eugenol pretreatment significantly suppressed the LPS-stimulated interleukin-8 level and the mRNA abundance of tumor necrosis factor-α and restored the LPS-stimulated decrease of the mRNA abundance of tight junction proteins, such as ZO-1 and occludin, and the mRNA abundance of nutrient transporters, such as B0 1 system ASC sodium-dependent neutral amino acid exchanger 2, sodium-dependent glucose transporter 1, excitatory amino acid transporter 1, and peptide transporter 1. In addition, eugenol improved the expression and even redistribution of ZO-1 and tended to increase TEER value and maintained the barrier integrity. In conclusion, a low dose of eugenol attenuated inflammatory responses and enhanced selectively permeable barrier function during LPS-induced inflammation in the IPEC-J2 cell line.

Keywords: barrier function, eugenol, inflammatory responses, porcine intestinal epithelial cells, inflammation

Introduction

Weaning is one of the most stressful events during the life of a pig because they have to encounter various stressors, including environmental, biological, social, and psychological changes. These stressors may trigger a compromised immune system and a disrupted intestinal structure and barrier function, resulting in compromised pig health, growth performance, and a series of enteric diseases with symptoms such as diarrhea and presence of gut inflammation (Campbell et al., 2013), which, if not properly treated, can lead to greater mortality and economic loss for commercial swine production (Lallès et al., 2007).

Intestinal epithelial cells (IECs) line the gastrointestinal tract (GIT) and form a physical monolayer barrier to serve as the first line of defense, which plays a key role in immunity (Kong et al., 2018). The gut barrier formed by circumferential intercellular junctions (including tight junctions, adherens junctions, and desmosomes) connecting with neighboring cells can prevent the invasion of microbial pathogens and the passage of other harmful molecules (Förster, 2008). The gut barrier also allows transcellular and paracellular movements of nutrients and molecules to be regulated in both passive and active ways (Pitman and Blumberg, 2000). Tight junctions create a passive paracellular transport pathway between cells, while the transcellular pathway is both active and passive, and primarily operates by nutrient transporters located on the basal, lateral, and apical sides of the cell (Anderson, 2001). Furthermore, IECs are also involved in the absorption process. Nutrient transporters are essential for gut homeostasis and proper absorption of nutrients. B0 system neutral amino acid co-transporter 1 (B0AT1), system ASC sodium-dependent neutral amino acid exchanger 2 (ASCT2), sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1), and peptide transporter 1 (PepT1) are main nutrient transporters and chemosensing systems, which are located on the apical side of IECs, and they play important roles in the transport of glucose, amino acids, and peptides (Omonijo et al., 2019). Paracellular permeability is mainly regulated by tight junctions localized at the apical-lateral membranes to prevent the leakage of macromolecules from the gut lumen (Laukoetter et al., 2008). However, when piglets are experiencing a weaning period, they are prone to be subjected to the disrupted gut barrier, which cannot prevent piglets from the harmful microorganism, toxins, and antigens in the gut (Campbell et al., 2013). Inflammation is a common immune response to invasive pathogens, and it is one of the consequences of the “leaky gut” and compromised gut barrier function (Groschwitz and Hogan, 2009; Moeser et al., 2017). This causes malfunctioning of epithelial cells and breaks tight junctions, leading to barrier malfunction and damaged GIT health.

Antibiotics have been used at both low and high levels in the swine industry for over 50 yr. They have been proven to be an effective growth promoter when they are used at low levels (subtherapeutic) in swine diets and at high levels (therapeutic) to treat swine diseases (Cromwell, 2002). Antibiotic growth promoter (AGP) can improve growth performance, feed utilization efficiency, and decrease morbidity and mortality. Despite these benefits, there is an increasing concern that the widespread use of antibiotics in the feed may lead to antibiotic resistance in both animals and humans (Barton, 2000). Thus, many countries have banned or placed strict restrictions on antibiotic use in animals (Ben Lagha et al., 2017). Consequently, AGP alternatives are required and urgently needed.

Eugenol (4-allyl-2-methoxyphenol) is a major phenolic constituent of essential oils extracted from clove, nutmeg, and cinnamon. It exhibits antimicrobial, anti-inflammatory, and antioxidative properties (Rathinam et al., 2017). It has also been widely used in flavoring, as well as in the cosmetic, pharmaceutical, and dentistry industries (Mohammadi Nejad et al., 2017). Nowadays, the application of eugenol in the agricultural industry, such as in animal feeds, is increasing. Previous studies have reported that the administration of eugenol improved the health status and growth performance in beef cattle (Compiani et al., 2013). Additionally, eugenol showed antimicrobial activity against pig gut flora (Michiels et al., 2007). Moreover, eugenol has been found to possess anti-inflammatory properties by decreasing the production of proinflammatory cytokines through regulating inflammation and redox status in lipopolysaccharide (LPS)-induced inflammation in acute lung injury of mice (Huang et al., 2015). Eugenol suppressed cyclooxygenase-2 expression in LPS-stimulated mouse macrophage RAW264.7 cells (Kim et al., 2003). Therefore, eugenol has the potential to be an alternative to AGP, by preventing and alleviating the inflammatory gut diseases caused by weaning stress. Additionally, the flavor of eugenol in feeds positively affects postweaning performance and feeding behavior of piglets, which helped piglets adapt to a novel solid food from liquid (Blavi et al., 2016).

To our knowledge, no one has investigated the anti-inflammatory effect of eugenol on porcine intestinal epithelial cells. We hypothesized that a low dose of eugenol can suppress LPS-induced inflammation and improve the barrier and absorption function in porcine intestinal epithelial cells. Therefore, this study was to investigate whether eugenol has anti-inflammatory effects and if eugenol can improve barrier and absorption function using an in vitro LPS-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2).

Materials and Methods

Animal care and use committee approval was not needed because no animal trial was involved in this study. All the experiments were conducted using a commercial cell line (IPEC-J2).

Materials and reagents

Eugenol (99%), LPS from Salmonella enterica serotype Typhimurium (L6143), water-soluble tetrazolium salts-1 (WST-1) cell proliferation reagent, 2′, 7′-dichlorofluorescein diacetate (DCF-DA), four KDa fluorescein isothiocyanate-dextran (FITC-dextran 4), radioimmunoprecipitation assay (RIPA) buffer, paraformaldehyde (PFA), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol reagent, Halt protease, phosphatase inhibitor cocktail, and Pierce bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Scientific (Waltham, MA, USA); 4 × Laemmli sample buffer was purchased from Bio-Rad (Hercules, CA, USA).

Cell culture and treatments

Porcine intestinal epithelial cells (IPEC-J2, ACC 701, RRID: CVCL_2246) were purchased from DSMZ-German Collection of Microorganisms and Cell Cultures and maintained in the Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (Gibco/Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco/Life Technologies) and 50 U/mL penicillin/streptomycin (Gibco/Life Technologies) at 37 °C with a 5% CO2 atmosphere. Fresh medium was replaced every 2 to 3 d. Cells were seeded into different types of multi-well plates depending on different experimental requirements. Eugenol solution was prepared in ethanol as a 100-mM stock solution and diluted with the culture medium right before using. For optimizing eugenol concentration, cells were treated with eugenol at different concentrations (0, 10, 20, 50, 100, 200, 500, 1,000, 1,500, 2,000 and 2,500 µM) for 24 h, and cell viability was measured (Figure 1). According to the result, 100 µM was set as a working concentration for formal experiments. There were three treatment groups: Control: 0 μM eugenol dilution pretreatment for 2 h and complete medium culture for 6 h (without LPS stimulation); LPS: cells were pretreated with 0 μM eugenol for 2 h and then stimulated with 10 μg/mL LPS stimulation for 6 h (Omonijo et al., 2019); LPS + Eugenol: cells were pretreated with 100 μM (16.42 mg/L) eugenol for 2 h and then stimulated with LPS (10 μg/mL) for 1 h (for reactive oxygen species (ROS) assay and transepithelial electrical resistance [TEER]) or 6 h (for other assays).

Figure 1.

Figure 1.

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Dose–effect of eugenol on the viability of IPEC-J2 cells. Cells were treated with eugenol at the indicated concentrations for 24 h. The data were presented as mean ± SEM, n = 6. *represents a significant difference (P < 0.05) compared with 0 μM eugenol.

Cell viability

WST-1 cell proliferation reagent was used to measure cell viability according to the manufacturer’s protocol. IPEC-J2 cells were seeded into a 96-well plate (Corning Costar) at a density of 5 × 104 cells/mL and cultured for at least 1 wk. After different treatments, WST-1 with 20 times dilution ratio in 5% FBS medium was added to cells followed by washing with phosphate-buffered saline (PBS). The absorbance was measured at 450 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek) after 1 h incubation at 37 °C. Cell viability was presented as a percentage of untreated control cells.

Proinflammatory cytokine assay

After different treatments, the cell supernatants were collected for interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) measurement using a porcine IL-8 and TNF-α enzyme-linked immunosorbent assay(ELISA) kit (Thermo Scientific) according to the manufacturer’s protocol. The absorbance was read at the endpoint at 450 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek). The IL-8 concentrations were calculated from the IL-8 standard calibration curve. The IL-8 level was expressed as pg/mL.

ROS assay

Cellular ROS was measured using a fluorescent dye, DCF-DA, according to the method of Omonijo et al. (2019) with some modifications. Cells were cultured in a 96-well plate for 1 wk. After a eugenol pretreatment, cells were stimulated with LPS for 1 h. DCF-DA dissolved in PBS was added into wells at 10 µM in PBS. Cells were incubated for 30 min and then the fluorescence of DCF (Ex/Em = 485/528 nm) was detected by a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek).

RNA extraction and gene expression analysis by real-time quantitative polymerase chain reaction

Cells were cultured in a 6-well plate. After treatments, total RNA extraction using TRIzol reagent was conducted as described by the manufacturer’s instructions. The quality and integrity of RNA were checked by Nanodrop-2000 spectrophotometer (Thermo Scientific) and RNA electrophoresis in agarose gel. Next, the first-strand complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis kit (Bio-Rad) according to the corresponding manufacturer’s protocol. The relative mRNA abundances of TNF-α, IL-8, zonula occludens-1 (ZO-1), occludin (OCLN), claudin (CLDN)-1, CLDN-3, PepT1, B0AT1, ASCT2, SGLT1, and EAAC1 were measured using SYBR Green Supermix (Bio-Rad) by a CFX Connectreal-time PCR detection system (Bio-Rad). Polymerase chain reaction amplification was performed at a setting of 3 min at 95 °C, then 40 cycles of 20 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. At the end of each cycle, the fluorescence was monitored for 10 s. Each reaction was completed with a melting curve analysis to ensure the specificity of the reaction. Primers used in this study are listed in Table 1 (Hyland et al., 2006; Omonijo et al., 2019). Relative gene expression was calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Cyclophilin A (CypA) was used as the reference gene (Hyland et al., 2006).

Table 1.

Primers used in this study

Genes Accession No. Primer sequences
TNF-α NM_214022.1 F: 5′-ATGGATGGGTGGATGAGAAA-3′
R: 5′-TGGAAACTGTTGGGGAGAAG-3′
IL-8 NM_213867.1 F: 5′-CACCTGTCTGTCCACGTTGT-3′
R: 5′-AGAGGTCTGCCTGGACCCCA-3′
SGLT1 NM_001012297.1 F: 5′-GGCTGGACGAAGTATGGTGT-3′
R: 5′-GAGCTGGATGAGGTTCCAAA-3′
ASCT2 XM_003355984.4 F: 5′-GCCAGCAAGATTGTGGAGAT-3′
R: 5′-GAGCTGGATGAGGTTCCAAA-3′
EAAC1 NM_001164649.1 F: 5′-CCAAGGTCCAGGTTTTGGGT-3′
R: 5′-GGGCAGCAACACCTGTAATC-3′
B 0 AT1 XM_003359855.4 F: 5′-AAGGCCCAGTACATGCTCAC-3′
R: 5′-CATAAATGCCCCTCCACCGT-3′
PepT1 NM_214347.1 F: 5′-CATCGCCATACCCTTCTG-3′
R: 5′-TTCCCATCCATCGTGACATT-3′
ZO-1 XM003353439.2 F: 5′-GATCCTGACCCGGTGTCTGA-3′
R: 5′-TTGGTGGGTTTGGTGGGTT-3′
OCLN NM_001163647.2 F: 5′-GAGAGAGTGGACAGCCCCAT-3′
R: 5′-TGCTGCTGTAATGAGGCTGC-3′
CLDN-1 NM_001244539.1 F: 5′-CTGTGGATGTCCTGCGTGT-3′
R: 5′-GGTTGCTTGCAAAGTGGTGTT-3′
CLDN-3 NM_001160075.1 F: 5′-CTACGACCGCAAGGACTACG-3′
R: 5′-TAGCATCTGGGTGGACTGGT-3′
CypA JX523419.1 F: 5′-GCGTCTCCTTCGAGCTGTT-3′
R: 5′-CCATTATGGCGTGTGAAGTC-3′
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Western blot analysis

The expression of ZO-1 was determined by Western blot analysis. Cells were cultured in a 6-well plate. After treatments, cells were washed with PBS twice and lysed using precooled RIPA buffer containing Halt protease and phosphatase inhibitor cocktail on ice. Cell lysates were vortexed for 40 s and placed on ice for a 5-min period. This cycle was repeated five times, and then the cell lysates were centrifuged at 12,500 × g for 15 min. Total protein concentration was determined using the Pierce BCA protein assay kit as described by the manufacturer’s instructions. Protein was then denatured using 4 × Laemmli sample buffer with 0.1 M DTT at 95 °C for 9 min; the denatured protein samples (30 μg) were loaded and separated by 4% to 15% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), and then transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked using 5% skim milk powder dissolved in tris-buffered saline, 0.1% Tween 20, and incubated with the ZO-1 polyclonal primary antibody (Cat# 61-7300, 1:2,000 dilution, Thermo Scientific) and β-actin monoclonal primary antibody (Cat# AM4302, 1:5,000 dilution, Thermo Scientific) at 4 °C overnight. Secondary antibody incubation used horseradish peroxidase-conjugated goat anti-rabbit IgG (Cat# 324300, 1:10,000 dilution, Thermo Scientific) and goat anti-mouse IgG (Cat# 31430, 1:1,000 dilution, Thermo Scientific). After enhanced chemiluminescent reaction using Clarity Western ECL Substrate (Bio-Rad), the images were detected by ChemiDoc Imaging Systems (Bio-Rad). The protein determination was normalized to β-actin, and the quantitative results were presented relative to the control.

Immunofluorescent staining

Immunofluorescent staining was conducted as described previously Omonijo et al. (2019). Briefly, cells were cultured onto coverslips (Thermo Scientific) in 12-well plates for at least 1 wk and then fixed with 4% PFA. The cells were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and then incubated with a ZO-1 polyclonal antibody (Cat# 61-7300, 1:100 dilution, Thermo Scientific), followed by an Alexa fluor 488 goat anti-rabbit antibody (Cat# A-11034, 1:1,000 dilution, Thermo Scientific) incubation. Rinsed cells were mounted with Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). For F-actin staining, fixed cells were incubated with phalloidin, CF488A (Cat# 00042-T, 1:100 dilution, Biotium, Fremont, CA, USA), and mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories). Images were captured by a Zeiss fluorescence microscope (Zeiss Group, Oberkochen, Germany).

TEER and cell permeability measurements

The integrity of the IPEC-J2 cells monolayer seeded onto Millicell membrane cell inserts (24 wells, Corning Costar) was measured by monitoring the TEER across the monolayer using a Millicell ERS-2 Voltohmmeter (MilliporeSigma). TEER value was measured referring to the method of Omonijo et al. (2019). The TEER was monitored and measured every 2 d until a monolayer of cells was completely differentiated and the TEER values were above 4,000 Ω, and then the cells can be pretreated with eugenol for 2 h. TEER was measured before and after 1 h LPS treatments. The data were presented as a percentage of initial values. The paracellular permeability of cell monolayers was measured using 1 mg/mL of FITC-dextran 4. FITC-dextran 4 was added to the apical side of Millicell membrane cell inserts (24 wells, Corning Costar). The fluorescence intensity (Ex/Em = 485/528 nm) of basolateral medium aliquots was measured after 6 h and 16 h incubation by a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek).

Statistical analysis

Each well from a multi-well plate was used as the experimental unit for all analyses. The data from one of the three independent experiments were expressed as mean ± SEM with at least three biological repeats and at least two technical repeats. Statistical significance was determined by t-test or one-way ANOVA followed by Tukey’s multiple-comparison test using GraphPad Prism 6 software (San Diego, CA, USA). P-value < 0.05 was considered statistically significant, and P-value between 0.05 and 0.1 was recognized as a tendency.

Results

Dose–effect of eugenol on the viability of IPEC-J2 cells

As shown in Figure 1, concentrations of eugenol lower than 100 μM did not have significant differences in the cell viability of IPEC-J2 cells, but concentrations above 100 μM significantly decreased the cell viability compared with 0 μM (P < 0.05). Based on the results, 100 μM was chosen as the working concentration of eugenol throughout all experiments.

Effect of eugenol on cell viability and IL-8 secretion

As shown in Figure 2A, the LPS treatment and LPS + Eugenol treatment had no significant effects on cell viability compared with the control. LPS stimulation significantly increased IL-8 secretion indicating that it was a successful LPS-induced inflammation model (P < 0.05), and eugenol pretreatment has a significant effect on returning IL-8 secretion back to similar levels as the control (P < 0.05, Figure 2B). However, TNF-α concentration was under the detection limit (data not shown).

Figure 2.

Figure 2.

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Effect of eugenol on the cell viability and IL-8 secretion during LPS-induced inflammation. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h. The data were presented as mean ± SEM, n = 6. Different letters represent a significant difference (P < 0.05).

Effect of eugenol on ROS production

ROS production has been shown to be involved in LPS-induced inflammatory responses. As shown in Figure 3, cells with LPS-induced inflammation significantly increased the fluorescent intensity of ROS compared with the control (P < 0.05). Eugenol pretreatment did not significantly suppress the ROS production caused by LPS (P > 0.1).

Figure 3.

Figure 3.

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Effect of eugenol on ROS production. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 1 h. The data were presented as mean ± SEM, n = 3. Different letters represent significant differences (P < 0.05).

Effect of eugenol on the gene expression of proinflammatory cytokines

Cells treated with LPS significantly upregulated the relative mRNA abundance of TNF-α compared with the control (P < 0.05) and eugenol pretreatment significantly suppressed this effect (Figure 4A, P < 0.05). Both LPS and eugenol treatment had no significant effect on the relative mRNA abundance of IL-8 (P > 0.05) compared with the control, though LPS tended to upregulate the relative mRNA abundance of IL-8 (P = 0.0889, Figure 4B).

Figure 4.

Figure 4.

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Effect of eugenol on the gene expression of proinflammatory cytokines TNF-α (A) and IL-8 (B) during LPS-induced inflammation. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h. Data were presented as mean ± SEM, n = 3. Different letters a and b represent a significant difference (P < 0.05), and x and y mean a trend (0.05 < P < 0.1).

Effect of eugenol on the gene expression of tight junction proteins

Tight junction proteins, such as ZO-1, OCLN, CLDN-1, and CLDN-3, are very important and help maintain barrier function. As shown in Figure 5, LPS treatment significantly downregulated the relative mRNA abundance of ZO-1 (P < 0.05, Figure 5A) and OCLN (P < 0.05, Figure 5B) but had no effect on CLDN-1 (Figure 5C) and CLDN-3 (Figure 5D) compared with the control (P > 0.1). Cells pretreated with eugenol significantly recovered the downregulation of OCLN mRNA abundance by LPS stimulation (Figure 5B, P < 0.05). Eugenol pretreatment also significantly upregulated the relative mRNA abundance of CLDN-3 compared with the control or the LPS treatment (Figure 5D, P < 0.05).

Figure 5.

Figure 5.

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Effect of eugenol on the gene expression of tight junction protein ZO-1 (A), OCLN (B), CLDN-1 (C), and CLDN-3 (D) during LPS-induced inflammation. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h. Data were presented as mean ± SEM, n = 3. Different letters represent a significant difference (P < 0.05).

Effect of eugenol on the gene expression of nutrient transporters

The LPS treatment significantly downregulated the relative mRNA abundance of PepT1 (Figure 6A) and ASCT2 (Figure 6C) compared with the control (P < 0.05), and eugenol significantly reversed the effects caused by LPS stimulation (P < 0.05). Meanwhile, LPS treatment tended to downregulate the relative mRNA abundance of B0AT1 (P = 0.0606, Figure 6B) and SGLT1 (P = 0.0558, Figure 6D) compared with the control, respectively. However, the LPS + eugenol treatment significantly upregulated these gene expressions compared with the LPS treatment (P < 0.05, Figure 6B and D). As shown in Figure 6E, the relative mRNA abundance of EAAC1 in the LPS treatment group did not significantly differ compared with the control (P > 0.1), but eugenol significantly upregulated the mRNA abundance of EAAC1 compared with the LPS treatment (P < 0.05).

Figure 6.

Figure 6.

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Effect of eugenol on the gene expression of nutrient transporters PepT1 (A), B0AT1 (B), ASCT2 (C), SGLT1 (D), and EAAC1 (E) during LPS-induced inflammation. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h. Data were presented as mean ± SEM, n = 3. Different letters represent a significant difference (P < 0.05).

Effect of eugenol on tight junction ZO-1 expression

The result of Western blotting shows that the protein expression of ZO-1 in IPEC-J2 cells stimulated with LPS was decreased compared with the control, but this effect was recovered by eugenol pretreatment (Figure 7A). The relative quantitative result further consolidated the above conclusion (Figure 7B).

Figure 7.

Figure 7.

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Effect of eugenol on tight junction ZO-1 expression during LPS-induced inflammation. Representative Western blot images (A) and relative expression level (B) of ZO-1 protein are shown. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h. Data were presented as mean ± SEM, n = 3. Different letters represent a significant difference (P < 0.05).

Effect of eugenol on the morphological changes of tight junction protein

Morphology of tight junction was visualized by ZO-1 immunofluorescent staining. As shown in Figure 8, in control cells, the paracellular distribution of ZO-1 was continuous, and the staining delineated the points of cell–cell contact around the periphery of the cells at the apical surface. LPS stimulation resulted in some loss of continuity of staining, suggesting the damage of the tight junction. However, eugenol pretreatment can offset the negative changes to some extent. ZO-1 staining was more intense, indicating that the even redistribution of ZO-1 was facilitated. The cytoskeletal structure of the actin fiber in IPEC-J2 cells was disorganized due to LPS stimulation compared with the control, but eugenol pretreatment restored the morphology of actin fiber.

Figure 8.

Figure 8.

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Effect of eugenol on the morphological changes of tight junction protein ZO-1 and actin fiber during LPS-induced inflammation. IPEC-J2 cells were treated with eugenol (100 μM) for 2 h prior to LPS (10 μg/mL) stimulation for 6 h.

Effect of eugenol on TEER and cell permeability

As shown in Figure 9A, IPEC-J2 cells pretreated with eugenol tended to promote the TEER value compared with the control (P = 0.0874). Figure 9B shows that there were no significant differences in relative TEER value among the control, LPS, and LPS + Eugenol treatments (P > 0.1). The leakage of FITC-dextran from the apical side to the basolateral side represents the cell permeability of the barrier formed by the IPEC-J2 cells. As shown in Figure 9C, the relative fluorescent intensity did not significantly differ between LPS and LPS + Eugenol after 6 h incubation, whereas 16 h eugenol pretreatment significantly decreased the relative fluorescent intensity compared with the LPS treatment (P < 0.05).

Figure 9.

Figure 9.

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Effect of eugenol on TEER and permeability during LPS-induced inflammation. The relative TEER value measured after 2 h eugenol pretreatment in IPEC-J2 cells is shown (A); the relative TEER value for 2 h eugenol pretreatment, followed by 1 h LPS (10 μg/mL) stimulation in IPEC-J2 cells, was measured (B); permeability was tested by FITC-dextran 4, and the relative fluorescence intensity is shown (C). Data for TEER value were presented as a percentage of initial TEER value and given as means ± SEM, n = 4. Different letters a and b represent a significant difference (P < 0.05), and x and y mean a trend (0.05 < P < 0.1).

Discussion

Weaning is a stressful period for piglets, where both physiological and physical changes might cause disturbances of the intestinal barrier, resulting in the translocation of luminal harmful bacteria, toxins, and antigens across IECs. The imbalanced GIT homeostasis is linked to gut disorders such as inflammatory bowel diseases and infectious diarrhea (Smith et al., 2010; Campbell et al., 2013). Gut inflammation is known as an immune response to noxious stimuli, such as endotoxins and bacterial pathogens, which can cause tissue and cell damage (Andrade et al., 2014). This damage can result in compromised mucosal integrity and barrier function of the gut, thereby negatively affecting the growth performance and leading to high morbidity in young piglets, especially weaned piglets Omonijo et al. (2019). The IPEC-J2 cell line is isolated from the jejunum of neonatal unsuckled piglets. These cells are ideal for in vitro studies, because they are neither transformed nor tumorigenic in nature (Vergauwen et al., 2015). Eugenol has been recognized as a functional major constituent of clove essential oil and possesses antimicrobial, antioxidative, anti-inflammatory, antifungal, anticancer, antipyretic, and analgesic properties (Rathinam et al., 2017). The U.S. Food and Drug Administration has stated that eugenol is generally recognized as safe when consumed orally (Sung et al., 2012).

In this study, 10 μg/mL of LPS derived from Salmonella enterica serotype Enteritidis was used to induce inflammation in porcine intestinal epithelial cells, in order to explore the potential protective effects of eugenol on LPS-induced inflammatory reactions in porcine intestinal epithelial cells and its underlying mechanisms. According to our results, eugenol at concentrations above 100 μM (16.42 mg/L) significantly inhibited the viability of IPEC-J2 cells. Therefore, the critical point 100 μM was chosen to pretreat cells in the following experiments.

Generally, the occurrence of acute inflammation is accompanied by oxidative damage. ROS are signaling molecules that can cause oxidative stress if found in elevated levels and ultimately produce tissue damage and inflammation (Mittal et al., 2014). After LPS treatment, ROS production increased immediately during 1 h, but the 2-h eugenol pretreatment did not help IPEC-J2 cells scavenge the ROS or prevent the production of ROS. A possible explanation for this might be that the LPS stimulation time was not long enough to detect the changes, or that eugenol had not been involved in mitochondrial electron transport to limit the generation of ROS (Kowaltowski et al., 2009).

Cytokine production is induced by invading pathogen-associated molecular patterns, such as bacterial endotoxin or LPS, which activate toll-like receptors on the cell surface, initiating a series of intracellular downstream signaling cascades and lead to the secretion of proinflammatory cytokines, such as TNF-α and IL-8 (Burkey et al., 2007). The secretion of IL-8, a typical proinflammatory biomarker, increased accordingly in IPEC-J2 cells treated with LPS for 6 h, and eugenol did exhibit the protective effect on the release of this cytokine associated with LPS-induced inflammation; meanwhile, the treatment did not have a negative effect on cell viability. These results indicated that this in vitro inflammation model was successful, which can be used to study the anti-inflammatory effects of eugenol in IPEC-J2. Similar results can be found in the relative mRNA abundance of TNF-α, where it was upregulated in LPS treatment and downregulated in LPS + Eugenol treatment. However, the variation trend of IL-8 mRNA abundance was not completely the same as its protein expression (eugenol significantly decreased the IL-8 production compared with the LPS treatment group, but eugenol just tended to downregulate the mRNA abundance of IL-8). A similar result was found in a study performed by Omonijo et al. (2019). There might be two reasons: 1) the transcription and translation occur at different time, thus they reached to the highest level of expression at different time after stimulation. For example, in the IPEC-J2 cells, after Salmonella enterica serovars Typhimurium invasion, the IL-8 protein expression pattern was different from the IL-8 gene expression pattern, and the IL-8 gene expression pattern was different from the TNF-α gene expression pattern as reported by Skjolaas et al. (2007) and 2) posttranslational modification may affect the innate inflammatory responses and the expression of proinflammatory cytokines (Liu et al., 2016). Many studies reported changes in the expression of inflammatory cytokines in the intestine of humans and animals during enteric infection and inflammatory bowel diseases (Turner, 2009). Both in vitro and in vivo investigations show that uncontrolled synthesis of proinflammatory cytokines can have a strong influence on gut integrity and epithelial barrier functions, including the permeability to macromolecules and transport of nutrients and ions (Pié et al., 2004).

The IPEC-J2 cells are nontransformed and nontumorigenic intestinal porcine enterocytes originally derived from the jejunum isolated from a neonatal unsuckled piglet, and the IPEC-J2 cell line has been characterized as a good model to study gut barrier function and nutrient absorption (Schierack et al., 2006; Vergauwen et al., 2015). IPEC-J2 cells were first used to investigate transepithelial ion transport and enterocyte differentiation (Berschneider, 1989). Numerous studies used IPEC-J2 cells to investigate feedstuffs and functional phytochemicals related to inflammation, oxidative stress, and intestinal permeability (Geens and Niewold, 2010; Hermes et al., 2011; Pan et al., 2013; Farkas et al., 2015; Vergauwen et al., 2015; Palócz et al., 2016; Gao et al., 2017; Tang et al., 2018; Xiao et al., 2018; Omonijo et al., 2019). The intestinal mucosal barrier provides a physical separation between the luminal contents and the body (Laukoetter et al., 2008), and is selectively permeable in order for cells to adequately absorb nutrients while still remaining protected from invasive molecules, such as LPS (Turner, 2009). Tight junction proteins are one of the most important protein complexes to regulate the selectively permeable barrier, because they are involved in both paracellular transport and transcellular transport pathways (Turner, 2009). The CLDN family is a very important group of transmembrane proteins, which are involved in several aspects of tight junction permeability (Turner, 2009). OCLN can directly interact with CLDNs and actin, and it was the first transmembrane tight junction protein to be identified (Turner, 2009). Peripheral membrane proteins such as ZO-1 are crucial to tight junction assembly and maintenance, due to their multiple domains linked with other tight junction proteins, including CLDNs, OCLN, and actin (Turner, 2009). The relative mRNA abundance of both CLDN-1 and CLDN-3 did not downregulate after LPS stimulation; however, significant downregulations were found in that of OCLN and ZO-1, which illustrated that the gene expression of different tight junction proteins had different responses to LPS stimulation, and the injury in tight junctions was induced selectively. The expressions of OCLN and ZO-1 were most likely regulated by posttranscriptional and posttranslational mechanisms (Robinson et al., 2015), thereby causing them to function differently during LPS-induced inflammation. A similar finding was reported by Chen et al. (2015) that after LPS treatment, the mRNA abundance of OCLN and ZO-1 significantly downregulated, but that of CLDN-3 did not decline. Moreover, the increased proinflammatory cytokine secretion might contribute to the declined expression of ZO-1 in turn after LPS stimulation because the increased permeability has been associated with damage to tight junction proteins.

Eugenol pretreatment upregulated the relative mRNA abundance of CLDN-1, CLDN-3, OCLN, and ZO-1 to varying degrees, suggesting that eugenol is beneficial at maintaining barrier function. In this study, the measurements of relative gene expression, protein expression, and immunofluorescent staining indicated that ZO-1 played an important role in regulating LPS-induced gut inflammation, because the eugenol pretreatment did enhance the expression of ZO-1 and its even redistribution, which has also been demonstrated by many other studies (Purohit et al., 2008; Vivinus-Nébot et al., 2014; Wang et al., 2015). Moreover, the gut integrity, tightness, and permeability can be reflected by TEER value and the FITC-dextran 4 flux result. The cell permeability was increased significantly with time. Especially at 16 h, eugenol pretreatment significantly decreased the post-effect caused by LPS, and it was consistent with the TEER value result that eugenol pretreatment tended to increase TEER value and maintained the barrier function accordingly. Nutrient transporters are essential for gut homeostasis and proper absorption of nutrients. The transcellular pathway primarily operates by nutrient transporters located on the basal, lateral, and apical sides of the cell (Anderson, 2001). Based on the results of mRNA abundance of five transporters, we found that B0AT1, ASCT2, SGLT1, EAAC1, and PepT1 were all upregulated after eugenol pretreatment, which means that absorption efficiency of energy-based nutrients, carbohydrates, amino acids, and proteins (in the form of glucose, amino acids, and di- and tripeptides) might be increased (Pan et al., 2004; Omonijo et al., 2019).

In this study, results indicated that eugenol maintained the barrier function and selective permeability of IPEC-J2 cells and had an excellent effect on attenuating inflammatory response caused by LPS. Similarly, studies using other essential oil components such as cinnamaldehyde to treat IPEC-J2 showed a positive effect on enhancing intestinal integrity and nutrient transport and absorption (Sun et al., 2017). There are many possible explanations associated with the positive effects of eugenol. Firstly, eugenol possesses a high absorption rate in the stomach and the proximal small intestine (Michiels et al., 2008), thereby allowing eugenol to play a role in the epithelial cells and affect the expression and distribution of tight junction proteins effectively, and further maintain the function of selectively permeable barrier. Secondly, eugenol is a typical phenolic compound that possesses antioxidative and anti-inflammatory effects (Pramod et al., 2010). Thirdly, eugenol might be a potential ligand or factor, which directly affects the inflammatory signaling pathways or indirectly modulates other receptors to alleviate inflammatory responses. For example, it is recognized that eugenol can suppress nuclear factor kappa-light-chain-enhancer of activated B cells and thus inhibit the production of TNF-α (Aggarwal et al., 2005). Our result of the mRNA abundance of TNF-α is also consistent with the Aggarwal et al. (2005) study. In another study, eugenol was reported to be involved in lipometabolism by modulating a transient receptor potential vanilloid-1 receptor and by increasing antioxidant enzymes (Harb et al., 2019). Overall, the underlying mechanism of eugenol is still worth being explored.

Furthermore, many studies have recognized that eugenol essential oil exhibits an active antimicrobial property against a wide range of gram-negative and gram-positive bacteria and fungi (Marchese et al., 2017). The concentration of eugenol against the number of total anaerobic bacteria with a probability of 99.7% in small intestine simulations of pig gut flora was 223 mg/L (Michiels et al., 2007); 1,000 mg/kg eugenol significantly decreased fecal E. coli concentration in growing pigs (Yan and Kim, 2012). The minimal inhibitory concentration (MIC) of eugenol against enteroinvasive E. coli strain EcK 262/75 was 2.5 mM when incubated for 24 h in vitro (Dušan et al., 2006). Overall, previous studies indicate that the antimicrobial effect of eugenol is in a dose-dependent manner and that only a high dose of eugenol is effective (Marchese et al., 2017). No one has previously reported that a low dose of eugenol, such as 100 μM (16.42 mg/L), has shown an antimicrobial effect. However, in this study, a 100-μM dose of eugenol exhibited anti-inflammatory activity, which indicated that different levels of eugenol might serve in different roles. The low dose of eugenol could be a promising AGP alternative to prevent gut inflammation and maintain a healthy intestinal barrier function. In addition, 100 μM is far lower than the toxic dosage. The LD50 of eugenol is more than 2,000 mg/kg in a rat (shown in the safety data sheet of eugenol). According to previous MIC studies and in vivo intake studies of eugenol (Michiels et al., 2008; Marchese et al., 2017), the dose range of eugenol between 30 and 230 mg/L might be reasonable to investigate the anti-inflammatory effect of eugenol in piglets. In addition, encapsulation is an effective way to transport eugenol, as it has a volatile characteristic and it is easily degraded in the stomach and proximal small intestine (Michiels et al., 2008).

In conclusion, 100 μM (16.42 mg/L) eugenol pretreatment can attenuate inflammatory responses and enhance the selectively permeable barrier function in IPEC-J2 cells during LPS-induced inflammation by reducing the mRNA abundance and production of proinflammatory cytokines; increasing the expression and distribution of tight junctions; and upregulating the gene expression of five nutrient transporters involving glucose, amino acids, and protein transport. Eugenol has a promising potential to be used widely as an AGP alternative to prevent gut inflammation, to decrease the stress caused by weaning, and to boost the immune system of piglets. More in vivo studies still need to be conducted to further investigate the effect of eugenol on pigs. This study, using a low dose of eugenol, can provide support for practical applications in the swine industry.

Acknowledgments

This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) CRD Grant (C.Y., CRDPJ 503580-16), Manitoba Pork Council (C.Y., 47370), Jefo Nutrition Inc. (C.Y., 47369), the Start-Up Grant (C.Y., 46561) from the University of Manitoba, and Canada Foundation for Innovation (CFI) and Research Manitoba.

Glossary

Abbreviations

AGP

antibiotic growth promoter

ASCT2

system ASC sodium-dependent neutral amino acid exchanger2

BCA

bicinchoninic acid

B0AT1

B0 system-neutral amino acid co-transporter1

cDNA

complementary DNA

CLDN

claudin

CypA

cyclophilin A

DAPI

4′,6-diamidino-2-phenylindole

DCF-DA

2′, 7′ dichlorofluorescein diacetate

DTT

dithiothreitol

EAAC1

excitatory amino acid transporter1

FBS

fetal bovine serum

FITC-dextran 4

four KDa fluorescein isothiocyanate-dextran

GIT

gastrointestinal tract

IEC

intestinal epithelial cell

IL-8

interleukin-8

LPS

lipopolysaccharide

MIC

minimal inhibitory concentration

OCLN

occludin

PBS

phosphate-buffered saline

PepT1

peptide transporter1

PFA

paraformaldehyde

RIPA

radioimmunoprecipitation assay

ROS

reactive oxygen species

SGLT1

sodium-dependent glucose transporter1

TEER

transepithelial electrical resistance

TNF-α

tumor necrosis factor-α

WST-1

water-soluble tetrazolium salt-1

ZO-1

zonula occludens-1

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

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