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Abstract

Enterococcus faecalis is a facultative anaerobic gram-positive commensal bacterium common in the gastrointestinal tract of animals and humans. This study aimed to investigate the protective effects of heat-killed E. faecalis EF-2001 (EF-2001) on acute gastric ulcer using a murine model of ethanol (EtOH)-induced acute gastric injury. EF-2001 (20, 40, and 80 mg/kg/day) was administered by oral gavage for 5 days before EtOH treatment (10 mL/kg body weight). EF-2001 effectively attenuated EtOH-induced gastric mucosal injury with reduced gastric mucosal ulcer and histological damage score. Pretreatment of EF-2001 markedly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38MAPK). In addition, EF-2001 significantly inhibited phosphorylation of nuclear factor kappa B (NF-κB) and subsequently suppressed the upregulation of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 in gastric tissues. Taken together, these results suggest that EF-2001 exerts a gastroprotective effect against acute gastric injury, and the underlying mechanism might be associated with the suppression of MAPKs and NF-κB signaling and consequent reduction of pro-inflammatory mediators or cytokines.

Keywords

Ethanol acute gastric ulcer anti-inflammatory activity MAPK/NF-κB pathway

Introduction

Gastric ulcer is the most common gastrointestinal disorder with multiple etiologies, affecting quite a number of people worldwide.¹ The pathogenesis of gastric ulcer is complicated and multifactorial manner, usually caused by an imbalance between mucosal defensive factors and gastric aggressive factors.¹ Many etiologies are associated with increased incidence of gastric ulcer include stress, Helicobacter pylori infection, and over ingestion of nonsteroidal anti-inflammatory drugs and alcohol.^1,2 Excessive consumption of alcohol is one of the greatest contributors to gastric mucosal injury, characterized by inflammation, hemorrhage, and erosive lesions.^3,4 The clinical approach for the management of gastric ulcer focuses on the use of H₂ receptor antagonist or proton pump inhibitors. However, the usage of chemical medicines has been limited by its adverse effects.⁵ Thus, the search for a natural resource or alternative medicine still attracts a lot of researchers.⁶

The ethanol (EtOH)-induced gastric injury model simulate conditions to several features of the developing acute gastric ulcer which occurs as a result of physiological stress in humans. Thus, the EtOH-induced gastric injury model provides a mean for assessing anti-ulcerogenic activities along with their implicated mechanisms.^7,8 The mechanisms underlying the EtOH-induced gastric injury have not been fully defined. Nuclear factor kappa B (NF-κB) is the pro-inflammatory transcription factor consisting of p50/p65 and IκBα. Many stimuli can lead to the activation of NF-κB and subsequent translocation of freed NF-κB into the nucleus. The activation of NF-κB triggers the transcription of various inflammatory mediators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin 6 (IL-6).^9
–11 These pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 are key mediators in the pathophysiology of gastric inflammation or damage.^6,12
–14 The mitogen-activated protein kinase (MAPK) signaling pathway is involved in various toxic substance-induced injuries and inflammation in response to various external stimuli.⁶ These MAPK pathways were found to be activated in acute gastric ulcer caused by EtOH ingestion.^6,15 Thus, suppression of NF-κB and MAPK pathway are able to alleviate the gastric damage caused by EtOH.

Several probiotic bacteria have been reported as favorable candidates for the treatment of diseases through the modulation of the host immune system.^16,17 Enterococcus faecalis is a facultative anaerobic gram-positive commensal bacterium common in the gastrointestinal tract of animals and humans. Enterococcus faecalis have been used as probiotics for a variety of beneficial purposes.¹⁸ Enterococcus faecalis FK-23 is well-known to have immune-stimulatory or regulatory activities.^19,20 Heat-killed E. faecalis was reported to have a radioprotective effect and antitumor activity in vivo.^21,22 Recently, heat-killed E. faecalis EF-2001 (EF-2001) ameliorated atopic dermatitis and inflammatory bowel disease in murine model due to its immunomodulatory effect,^18,23 implying that EF-2001 may be developed to a potential strategy in treatment for inflammatory diseases. However, to our knowledge, direct evidence for the effect of EF-2001 on EtOH-induced acute gastric ulcer has not been elucidated yet. Therefore, the aim of this study was to investigate the protective effects of heat-inactivated EF-2001 on EtOH-induced acute gastric ulcer using a murine model.

Materials and methods

Chemicals and reagents

Absolute EtOH (CAS No. 64-17-5) was purchased from Merck Millipore (Darmstadt, Germany). EF-2001 was a commercially available probiotic and provided from KOREA BeRM Co., Ltd (Wonju, Republic of Korea). It was supplied as a heat-killed and dried powder. One gram of dried EF-2001 was comparable to 7.5 × 10⁸ CFU of cells prior to being heat-killed. Omeprazole was purchased from Tokyo Chemical Industry (Tokyo, Japan). Lipopolysaccharide (LPS) of Escherichia coli (O111:B4) was purchased from Sigma-Aldrich (St Louis, Missouri, USA). In the case of the primary antibodies, phosphor-JNK1/2 (p-JNK1/2), total JNK1/2 (t-JNK1/2), p-Erk1/2, t-Erk1/2, p-p38MAPK, t-p38MAPK, and p-p65NF-κB antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA) and iNOS, COX-2, and TNF-α antibodies were obtained from Abcam (Cambridge, UK). Commercial enzyme-linked immunosorbent assay (ELISA) kit of TNF-α, IL-6, and IL-1β was purchased from R&D system (Minneapolis, Minnesota, USA). All other chemicals were of the highest grade commercially available.

Cell culture and cell viability assay

RAW264.7 cells (murine macrophage cell line) were maintained at 1 × 10⁵ cells/mL in Dulbecco’s modified Eagle’s medium (Gibco, UK) supplemented 10% heat-inactivated fetal bovine serum (Gibco) and 1% (weigt/volume) of antibiotic–antimycotic solution (Invitrogen, Grand Island, New York, USA) in 5% CO₂ humidified atmosphere at 37°C. In cell viability assay, RAW264.7 cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well and incubated for 24 h. The cells were treated with various concentrations of EF-2001 (0, 25, 50, 100, and 200 μg/mL) for 24 h. After 24 h incubation, the effect of EF-2001 on the cell viability was measured using water-soluble tetrazolium salt 1 (WST-1; EZ-CyTox, Dogen, Republic of Korea), according to the manufacturer’s instructions. Ten percent total volume of WST-1 solution was added to each well and incubated for 1 h, and absorbance was measured using a microplate reader (iMark™, Bio-Rad Laboratories, Richmond, California, USA) at 450 nm. The optical density of control cells (untreated) was taken as 100% viability.

Determination of NO production in the culture medium and gastric tissues

The contents of nitric oxide (NO) in culture medium and gastric tissues were determined using the Griess reaction. RAW264.7 cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well and incubated for 24 h. The cells were incubated with EF-2001 at 25, 50, and 100 μg/mL at 2 h before LPS (1 μg/mL; Sigma-Aldrich) treatment and incubated at 37°C for 24 h after LPS treatment. After 24 h incubation, 100 μL of collected culture medium and lysate of gastric tissue were dispensed into 96-well plate and mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, and 5% phosphoric acid) and incubated at room temperature for 10 min. Using sodium nitrite to generate a standard curve, the concentration of nitrite was measured for absorbance at 540 nm. The optical density of LPS-stimulated cells was taken as 100% production.

Animals and environmental conditions

ICR mice (7 weeks old) were obtained from a specific pathogen-free colony at Samtako Co. (Osan, Republic of Korea) and used after 1 week of quarantine and acclimatization. The mice were maintained with food and water ad libitum in an animal facility with a temperature of 22 ± 2°C and a relative humidity of 50 ± 10% with a 12-h light/12-h dark cycle and with 13–18 air changes per hour. All experiment procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (approved number: KRIBB-AEC-18125).

EtOH-induced acute gastric injury and treatment regimen

Male ICR mice (specific pathogen-free, 7 weeks old, 20–25 g) were randomly divided into six groups (n = 7 per group). Omeprazole served as a positive control drug and the dose of omeprazole was 20 mg/kg/day by oral gavage for 5 days based on the earlier studies.^24,25 The mice of the EtOH + EF-2001 groups received EF-2001 20, 40, and 80 mg/kg/day by oral gavage for 5 days, respectively. The animals were fasted for 12 h and allowed to water access before the last treatment of EF-2001. The mice were administered orally with a single dose of absolute EtOH (10 mL/kg body weight) to induce acute gastric mucosal injury at 2 h after EF-2001 treatment.^6,26 The doses of EF-2001 were selected based on our preliminary study. In the preliminary study, EF-2001 at dose levels of 20 and 80 mg/kg/day (n = 4) was administered by oral gavage for 3 days. In gross findings of gastric mucosa, we confirmed the ameliorative effects of EF-2001 against EtOH-induced acute gastric ulcer (data not shown). Thus, the dose levels of EF-2001 were selected as 20, 40, and 80 mg/kg/day in the present study. The normal control group provided with distilled water in equivalent volume. All animals were killed at 2 h after receiving the absolute EtOH treatment.

Necropsy and gastric mucosal ulcer scoring

All animals were anesthetized by CO₂ inhalation 2 h after absolute EtOH treatment. Stomach was removed, opened along the greater curvature, and rinsed with cold phosphate-buffered saline (1 × PBS, 4°C). The stomach was stretched on a clean paper with the mucosal surface facing upward. Photographs of gastric mucosal ulcer of the stomach were taken with a photometric digital camera (Cannon EOS 5Ds, Melville, New York, USA). The degree of gastric mucosal damage was graded according to ulcer score scales as described previously²⁷: 0, no lesions; 1, one hemorrhagic ulcer with length <5 mm and thin; 2, one hemorrhagic ulcer with length >5 mm and thin; 3, more than one ulcer grade 2; 4, one ulcer with length >5 mm and width >2 mm; 5, two or three ulcers of grade 4; 6, from four to five ulcers of grade 4; 7, more than six ulcers of grade 4; and 8, complete lesion of the mucosa. The mean value for each group was calculated and expressed as the mean ± standard deviation (SD). After gross inspection, the stomach tissue was cut in half and stored at −80°C for biochemical analysis.

Gastric histological damage scoring

The extent of EtOH-induced gastric mucosal injury and the effects of EF-2001 were evaluated by assessing histological changes in gastric tissue sections staining with hematoxylin and eosin (H&E). The remaining half of the gastric tissue was fixed in 10% neutralized formalin solution for 1 week. The fixed gastric tissues were processed routinely, embedded in paraffin, and sectioned to 4 µm thickness. The sectioned samples were deparaffinized and rehydrated, and stained with H&E. All lesions were examined manually with a light microscope with 20× and 40× objective lens in a totally blind manner. The degree of gastric histological damage was graded according to score scales as described previously²⁸: 0, no lesions; 1, slight damage of surface gastric mucosa; 2, damage greater than that of score 1 and involving <50% of the thickness of gastric mucosa; and 3, damage involving >50% of the thickness of the gastric mucosa. The mean value for each group was calculated and expressed as the mean ± SD.

Immunoblotting analysis

The gastric tissues were homogenized in lysis buffer (1:10 w/v; Thermo Scientific, Waltham, Massachusetts, USA) with protease/phosphatase inhibitors. Equal amounts of protein (40 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Thermo Scientific). The membranes were blocked with blocking buffer (5% skim milk) for 1 h at room temperature followed by overnight incubation at 4°C with appropriate primary antibodies. The following primary antibodies and dilutions were used: p-JNK1/2, t-JNK1/2, p-Erk1/2, t-Erk1/2, p-p38MAPK, t-p38MAPK, and p-p65NF-κB (1:1000 dilution; Cell Signaling Technology), iNOS, COX-2, and TNF-α (1:500 dilution; Abcam). Membranes were washed three times with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody (Thermo Scientific) for 1 h at room temperature. Membranes were again washed three times with TBST and then developed using an enhanced chemiluminescence kit (Thermo Scientific). Protein concentration was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, Illinois, USA). Protein expression was quantified based on band density using ImageJ software (National Institute of Health, Bethesda, Maryland, USA). Relative intensity was calculated by dividing the densities of respective loading control density value.

Measurements of mRNA expression of pro-inflammatory cytokines in the gastric tissues

Total RNA was isolated from the gastric tissue using TRIzol™ reagent (Invitrogen, Carlsbad, California, USA) as instructed by the manufacturer. RNA concentration was quantified using a NanoDrop ND-1000 (Thermo Scientific) at 260 nm. Complementary DNAs (cDNAs) were synthesized from 1 μg of total RNA using PrimeScript™ first strand cDNA synthesis kit (Takara, Shiga, Japan). Quantitative real-time polymerase chain reaction was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, USA) using a CFX96™ Real Time System (Bio-Rad Laboratories). The sequences of forward and reverse primers for target genes, TNF-α, IL-1β, IL-6, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (housekeeping gene) are presented in Table 1. Samples were run in triplicate to ensure amplification integrity. The standard PCR conditions were 95°C for 3 min, 50°C for 20 s, and 72°C for 20 s, with 40 cycles as recommended by the primer manufacturer. The threshold cycle (C_t, the cycle number at which the amount of amplified gene of interest reached a fixed threshold) was determined subsequently. Relative quantitation of each messenger RNA (mRNA) expression was calculated by the comparative C_t method. The relative quantitation values of targets were normalized to the endogenous GAPDH control gene and were expressed as $2^{- Δ Δ C_{t}}$ (fold), where ^ΔC_t = C_t of target gene − C_t of endogenous control gene and ^ΔΔC_t = ^ΔC_t of samples for target gene − ^ΔC_t of the calibrator for the target gene.

Table 1.

Primer sequences used in quantitative real-time PCR.

Genes	Primer (5′–3′)
TNF-α
Forward	GAA AGC ATG ATC CGG GAC GTG
Reverse	GAT GGC AGA GAG GAG GTT GAC
IL-6
Forward	ATG CAA TAA CCA CCC CTG AC
Reverse	ATC TGA GGT GCC CAT GCT AC
IL-1β
Forward	AGC CAG GAC AGT CAG CTC TC
Reverse	ACT TCT TGC CCC CTT TGA AT
GAPDH
Forward	CAA AAG GGT CAT CAT CTC TG
Reverse	CCT GCT TCA CCA CCT TCT TG

TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; IL-1β: interleukin 1 beta; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Determination of pro-inflammatory cytokines in the gastric tissues

The gastric tissues were homogenized (1/10; w/v) in a cold 1 × PBS (4°C) and centrifuged at 8000 r/min for 10 min at 4°C. The supernatants were collected and stored at −70°C before cytokines and NO analysis. The levels of TNF-α and IL-6 in the gastric tissues were quantified using a competitive ELISA kit (R&D system) according to the manufacturer’s protocols. The absorbance of each sample was measured at 450 nm in a microplate reader (Bio-Rad Laboratories). The absolute concentrations were calculated by running standard curves on identical ELISA plates.

Statistical analysis

The numerical data were presented as mean ± SD, and all statistical comparisons were analyzed by one-way analysis of variance, followed by Dunnett’s multiple comparison test. A p value of <0.05 was considered significant. Statistical analyses were performed using the GraphPad InStat v.3.0 (GraphPad Software, Inc., California, USA).

Results

Effect of EF-2001 on cell viability and NO production in LPS-stimulated RAW264.7 cells

We evaluated the effects of EF-2001 on cell viability and NO production using murine macrophage RAW264.7 cells. The cell viability of RAW264.7 cells were not affected by EF-2001 at the tested concentrations (0, 25, 50, 100, and 200 μg/mL; Figure 1a). As shown in Figure 1(b), LPS-stimulated RAW264.7 cells showed increased NO production. In contrast, EF-2001 treatment exhibited significant reduction of NO production. EF-2001 treatment showed concentration-dependent suppression of NO production.

Figure 1.

Effect of EF-2001 on cell viability and NO production in LPS-stimulated RAW264.7 cells. The bar graphs show the (a) cell viability and (b) NO production in LPS-stimulated RAW264.7 cells. Values are presented as mean ± SD (n = 5). **p < 0.01: significantly different from NC; ^†p < 0.05: significantly different from EtOH; ^††p < 0.01: significantly different from EtOH. EF-2001: Enterococcus faecalis EF-2001; NO: nitric oxide; LPS: lipopolysaccharide; SD: standard deviation; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg).

Effect of EF-2001 on EtOH-induced gastric mucosal ulcer

The gastric mucosal ulcer was scored by gross inspection as shown in Figure 2. The normal control (NC) group showed stomach with normal appearance. In the EtOH-treated group, severe hemorrhagic lesions with hyperemia were observed in the entire gastric mucosa. The gastric mucosal ulcer score in the EtOH-treated groups also showed a significant increase compared with that in the NC group. The PC group (omeprazole-treated group) markedly attenuated gastric mucosal injuries. Moreover, EF-2001-treated groups showed a dose-dependent decrease in hemorrhage and hyperemia compared with the EtOH-treated group. In quantitative analysis, the EF-2001-treated groups indicated the dose-dependent and significant reduction in the gastric mucosal ulcer score compared with the EtOH-treated group (Figure 2b).

Figure 2.

Effect of EF-2001 on EtOH-induced gastric mucosal ulcer. (a) Representative photographs of gastric mucosa treated with EF-2001 (20, 40, and 80 mg/kg/day) and/or EtOH (10 mL/kg). (b) The bar graphs show gastric mucosal ulcer scoring index determined by morphological analysis. Values are presented as mean ± SD (n = 7). EF-2001: Enterococcus faecalis EF-2001; SD: standard deviation; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg); PC: positive control (omeprazole 20 mg/kg) + ethanol-treated mice; EF-L, EF-M, and EF-H: EF-2001 (20, 40, and 80 mg/kg, respectively) + ethanol-treated mice. **p < 0.01: significantly different from NC, ^††p < 0.01: significantly different from EtOH.

Effects of EF-2001 on EtOH-induced histological lesions in the gastric tissues

The effect of EF-2001 observed in gross inspection was confirmed by the histopathological analysis. As shown in Figure 3, the EtOH-treated mice showed severe hemorrhage (arrowheads) and loss of gastric mucosal epithelial cells (arrows) in the greater part of gastric mucosal layer. The histological damage score in the EtOH-treated group showed a significant increase compared with that in the NC group. In contrast, treatment with EF-2001 reduced the loss of epithelial cells and hemorrhage findings in the gastric mucosal layers and exhibited a dose-dependent decrease of histological damage score (Figure 3b).

Figure 3.

Effects of EF-2001 on EtOH-induced histological lesions in the gastric tissues. (a) Representative histological photographs of gastric mucosa treated with EF-2001 (20, 40, and 80 mg/kg/day) and/or EtOH (10 mL/kg). Ethanol-treated mice showed loss of epithelial cells with degeneration (arrows) and mucosal hemorrhage (arrowheads). (b) The bar graphs show histological damage scoring index determined by histological analysis. Values are presented as mean ± SD (n = 7). EF-2001: Enterococcus faecalis EF-2001; SD: standard deviation; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg); PC: positive control (omeprazole 20 mg/kg) + ethanol-treated mice; EF-L, EF-M, and EF-H: EF-2001 (20, 40, and 80 mg/kg, respectively) + ethanol-treated mice. **p < 0.01: significantly different from NC; ^††p < 0.01: significantly different from EtOH.

Effects of EF-2001 on EtOH-induced MAPKs activation in the gastric tissues

We investigated the effects of EF-2001 on phosphorylation of MAPKs (JNK1/2, Erk1/2, and p38MAPK) in the gastric tissues of EtOH-treated mice. As presented in Figure 4, the EtOH-treated mice showed markedly increased phosphorylation of JNK1/2, Erk1/2, and p38MAPK in gastric tissue compared with the NC group. In contrast, the EF-2001-treated mice showed a dose-dependent decrease of phosphorylation of MAPKs compared with the EtOH-treated group.

Figure 4.

Effects of EF-2001 on EtOH-induced MAPKs activation in the gastric tissues. (a) Gel images show protein expression of MAPKs. The bar graphs show relative ratios of phosphorylation form/total form of (b) JNK1/2, (c) p38MAPK, and (d) Erk1/2 in the gastric tissues from mice treated with EF-2001 (20, 40, and 80 mg/kg/day) and/or EtOH (10 mL/kg) (loading control: β-actin). Values are presented as mean ± SD (n = 7). EF-2001: Enterococcus faecalis EF-2001; SD: standard deviation; MAPKs: mitogen-activated protein kinases; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg); PC: positive control (omeprazole 20 mg/kg) + ethanol-treated mice; EF-L, EF-M, and EF-H: EF-2001 (20, 40, and 80 mg/kg, respectively) + ethanol-treated mice. **p < 0.01: significantly different from NC; ^†p < 0.05: significantly different from EtOH; ^††p < 0.01: significantly different from EtOH.

Effects of EF-2001 on EtOH-induced NF-κB and pro-inflammatory mediators in the gastric tissues

To determine whether EF-2001 elicits its effects on NF-κB and pro-inflammatory mediators, we confirmed the protein expression levels of phosphorylated-p65NF-κB, TNF-α, iNOS, and COX-2. As shown in Figure 5, gastric tissues treated with EtOH showed a significant increase in the phosphorylated p65NF-κB protein level compared with that in the NC group. In addition, the protein levels of TNF-α, iNOS, and COX-2 in the EtOH-treated group were increased significantly compared to those in the NC group. In contrast, pretreatment with EF-2001 resulted in a significant decrease in the phosphorylation of p65NF-κB with concurrent decreases of TNF-α, iNOS, and COX-2 protein levels compared with that in the EtOH group in a dose-dependent manner.

Figure 5.

Effects of EF-2001 on EtOH-induced NF-κB and pro-inflammatory mediators in the gastric tissues. (a) Gel images show protein expression of phosphorylated p65NF-κB and pro-inflammatory mediators, iNOS, COX-2, and TNF-α. The bar graphs show relative expression levels of phosphorylation form of (b) p65NF-κB, (c) iNOS, (d) COX-2, and (e) TNF-α in the gastric tissues from mice treated with EF-2001 (20, 40, and 80 mg/kg/day) and/or EtOH (10 mL/kg) (loading control: β-actin). Values are presented as mean ± SD (n = 7). EF-2001: Enterococcus faecalis EF-2001; NF-κB: nuclear factor-kappa B; iNOS: inducible nitric oxide synthase; COX-2: cyclooxygenase-2; TNF-α: tumor necrosis factor-alpha; SD: standard deviation; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg); PC: positive control (omeprazole 20 mg/kg) + ethanol-treated mice; EF-L, EF-M, and EF-H: EF-2001 (20, 40, and 80 mg/kg, respectively) + ethanol-treated mice. **p < 0.01: significantly different from NC; ^†p < 0.05: significantly different from EtOH; ^††p < 0.01: significantly different from EtOH.

Effects of EF-2001 on EtOH-induced pro-inflammatory cytokines and NO in the gastric tissues

As shown in Figure 6, mRNA levels of TNF-α, IL-1β, and IL-6 in the EtOH group increased significantly compared with those in the NC group. In contrast, treatment with EF-2001 showed a significant decrease in TNF-α, IL-1β, and IL-6 mRNA levels compared with those in the EtOH group in a dose-dependent manner (Figure 6a-c). The TNF-α, IL-6, and NO levels in the gastric tissues were increased significantly compared with those in the NC group. However, the contents of TNF-α, IL-6, and NO in the gastric tissues of EF-2001-treated mice were decreased significantly compared with those in the EtOH group (Figure 6d-f).

Figure 6.

Effects of EF-2001 on EtOH-induced pro-inflammatory cytokines and NO in the gastric tissues. The bar graphs show the mRNA expression levels of (a) TNF-α, (b) IL-1β, (c) IL-6, and the contents of (d) TNF-α, (e) IL-6, and (f) NO in the gastric tissues of mice treated with EF-2001 (20, 40, and 80 mg/kg/day) and/or EtOH (10 mL/kg). Values are presented as mean ± SD (n = 7). EF-2001: Enterococcus faecalis EF-2001; NO: nitric oxide; mRNA: messenger RNA; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta; IL-6: interleukin-6; NC: normal control mice; EtOH: ethanol-treated mice (absolute ethanol 10 mL/kg); PC: positive control (omeprazole 20 mg/kg) + ethanol-treated mice; EF-L, EF-M, and EF-H: EF-2001 (20, 40, and 80 mg/kg, respectively) + ethanol-treated mice. **p < 0.01: significantly different from NC; ^†p < 0.05: significantly different from EtOH; ^††p < 0.01: significantly different from EtOH.

Discussion

Many probiotic bacteria have been described as therapeutic agents for various diseases by influencing the host’s immune system.²³ However, extensive use of live probiotics, shelf-life problems, and the risk of microbial translocation and infection have been led to emerging safety concerns.²⁹ In contrast, heat-killed lactic acid bacteria reduce the risks of microbial translocation and infection associated with live probiotics and provide the advantages of longer product shelf life.³⁰ Additionally, heat-killed lactic acid bacteria have been studied in humans and animals for its beneficial immunomodulatory effects in many diseases, including acute diarrhea, allergy, and influenza virus infection.^31
–33 Heat-killed EF-2001 has various pharmacological potentials, such as radioprotective, anticancer, and immunomodulatory activities. It has been also revealed that heat-killed EF-2001 has no toxic adverse effects in a single oral dose and a 13-week repeated oral toxicity study in mice with doses up to 5000 mg/kg.^18,21
–23 In this study, we explored the gastroprotective effects of heat-killed EF-2001 on EtOH-induced gastric mucosal ulcer in mice. Our study indicated that pretreatment of EF-2001 effectively decreased macroscopic and microscopic mucosal ulcer lesions. EF-2001 also suppressed phosphorylation of p65NF-κB and MAPKs, and production of NO and pro-inflammatory cytokines in EtOH-induced gastric mucosal ulcer model. These findings indicate that EF-2001 has potential anti-ulcerogenic activity against EtOH-induced acute gastric mucosal ulcer.

EtOH-induced acute gastric lesions are characterized by linear or fused hemorrhagic and hyperemic lesions on the glandular area of stomach with various pathological alterations including hemorrhage, edema, and loss of epithelial cells.^4,34 In this study, EtOH treatment to mice caused severe hemorrhagic and hyperemic lesions with a marked increase of gastric mucosal ulcer score. However, oral administration of EF-2001 significantly attenuated the gross lesions with decrease in the gastric mucosal ulcer score. These results were confirmed in histopathological changes showing decreased lesions including hemorrhage and loss of epithelial cells. These findings suggest that EF-2001 might have anti-ulcerogenic activity on EtOH-induced gastric mucosal injury.

The MAPK pathways are involved in diverse cellular responses, including inflammation-related pathways by receiving interior or exterior cell signals.³⁴ The main members of MAPKs are Erk1/2, JNK, p38 MAPK, and the activation of MAPKs can lead to inflammation, cell growth, or apoptosis.³⁵ The activation of major subgroups in the MAPK family regulates the expression of pro-inflammatory mediators, a phenomenon that has been reported in EtOH-induced gastric ulcer in mice.^6,14,34,36 In this study, we confirmed that the MAPKs were activated in gastric tissues of EtOH-treated mice. However, the pretreatment of EF-2001 dose dependently suppressed the phosphorylation of MAPKs in the gastric tissues. Thus, EF-2001 suppresses the activation of MAPK pathways and that might contributed to the protective effect against EtOH-induced gastric mucosal damages.

The nuclear transcription factor NF-κB plays a vital role in regulating the immune responses and is known to be activated in EtOH-induced gastric ulcer in mice. Activated NF-κB mediates inflammatory mediators or cytokines, such as iNOS, COX-2, TNF-α, IL-1β, and IL-6.^1,14 The level of pro-inflammatory cytokines are associated with the severity of EtOH-induced gastric injury.⁶ NO plays a crucial role in various physiological processes.³⁷ NO dilates blood vessels, increases blood flow and stimulates gastric angiogenesis and cell proliferation in the healing process of gastric ulcer.³⁸ In the stomach, NO produced by constitutive NO synthase is cytoprotective, while excessive NO produced by iNOS is cytotoxic.³ Increased iNOS expression is found in chronic ulcerative colitis and peptic ulcer patients. Excessive NO generated by iNOS deleterious effects on the pathophysiological conditions of gastric ulcer.^3,34 In the present study, the EtOH-treated group showed an increased phosphorylation of p65NF-κB with upregulation of pro-inflammatory mediators, COX-2, TNF-α, and iNOS with increased NO levels in the gastric tissues. However, EF-2001-treated mice notably exhibited suppressed phosphorylation of p65NF-κB, with the accompanying downregulated expression of pro-inflammatory mediators and gastric NO levels in a dose-dependent manner. In addition, the pretreatment of EF-2001 effectively suppressed elevated levels of pro-inflammatory cytokines caused by EtOH treatment in the gastric tissues. We also found that EF-2001 did not significantly affect the cell viability in RAW264.7 cells until 24 h and reduced NO production in LPS-stimulated RAW264.7 cells. Heat-killed EF-2001 effectively inhibited an elevation of TNF-α, interferon-γ, IL-4, and IL-5 in atopic dermatitis model in mice.²³ Heat-killed EF-2001 has anti-inflammatory activity by inhibiting the phosphorylation of MAPKs and NF-κB in the LPS-stimulated RAW264.7 cells.³⁹ Thus, these results indicate that EF-2001 effectively attenuated EtOH-induced gastric mucosal injury by inhibiting NF-κB pathway and subsequently downregulated inflammatory mediators or cytokines.

In conclusion, the present study demonstrated that EF-2001 attenuated EtOH-induced gastric mucosal injury and these anti-ulcerogenic effects were closely related to the suppression of MAPKs and NF-κB phosphorylation with subsequent downregulation of inflammatory mediators or cytokines in gastric tissue. Thus, EF-2001 may effectively suppress EtOH-induced acute gastric ulcer as a potential prophylactic agent. However, it is yet known whether EF-2001 directly affects the gastric mucosal barrier or indirectly modulate the host immune system. In this study, there were no experimental groups for heat-killed or live EF-2001 without the administration of EtOH. Therefore, further studies are still needed to elucidate the potential effects of heat-killed or live EF-2001 on the host immune system and EtOH-induced acute gastric injury model.

Supplemental material

Supplemental Material, Graphic_abstract-Lee_11.16 - Effect of heat-killed Enterococcus faecalis EF-2001 on ethanol-induced acute gastric injury in mice: Protective effect of EF-2001 on acute gastric ulcer

Supplemental Material, Graphic_abstract-Lee_11.16 for Effect of heat-killed Enterococcus faecalis EF-2001 on ethanol-induced acute gastric injury in mice: Protective effect of EF-2001 on acute gastric ulcer by D-B Jeon, H-G Shin, B-W Lee, S-H Jeong, J-H Kim, J-H Ha, J-Y Park, H-J Kwon, W-J Kim, Y-B Ryu and I-C Lee in Human & Experimental Toxicology

Footnotes

Author contributions

D-BJ and H-GS have contributed equally to this work as co-first authors.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT [Grant No. NRF-2018R1C1B6004503] and grant from the KRIBB Research Initiative Program [KGM5241911].

ORCID iD

I-C Lee

Supplemental material

Supplemental material for this article is available online.

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