The intestinal epithelium serves as a primary physical barrier against invading bacteria, toxins, and various environmental pollutants; however, it also plays an active role in the maintenance of absorption, regulation of barrier function, and immune homeostasis [1]. Gut-derived infections may be triggered by lipopolysaccharide (LPS) release from Gram-negative bacteria such as Salmonella spp. and E. coli. LPS can induce inflammatory responses, predominantly mediated by the activation of the NF-κB pathway, contributing to proinflammatory cytokine (e.g., IL-6, IL-8, and TNF-α) overproduction, and it triggers the production of reactive oxygen species as well [2]. Infections caused by pathogenic bacteria and consequent inflammatory responses can cause human disease and can result in significant economic losses in animal industry. In human healthcare, it is essential to promote prudent antibiotic use by preventing nosocomial infections and reduce overprescribing of antimicrobials. Most of the classes of antibiotics used for the treatment of bacterial infections in humans are also used in animals [3]. Misuse and overuse of antimicrobials in the treatment of human diseases and in animal husbandry can lead to bacterial resistance against clinically relevant drugs [4]. 

The intestinal epithelium serves as a primary physical barrier against invading bacteria, toxins, and various environmental pollutants; however, it also plays an active role in the maintenance of absorption, regulation of barrier function, and immune homeostasis [1]. Gut-derived infections may be triggered by lipopolysaccharide (LPS) release from Gram-negative bacteria such as Salmonella spp. and E. coli. LPS can induce inflammatory responses, predominantly mediated by the activation of the NF-κB pathway, contributing to proinflammatory cytokine (e.g., IL-6, IL-8, and TNF-α) overproduction, and it triggers the production of reactive oxygen species as well [2]. Infections caused by pathogenic bacteria and consequent inflammatory responses can cause human disease and can result in significant economic losses in animal industry. In human healthcare, it is essential to promote prudent antibiotic use by preventing nosocomial infections and reduce overprescribing of antimicrobials. Most of the classes of antibiotics used for the treatment of bacterial infections in humans are also used in animals [3]. Misuse and overuse of antimicrobials in the treatment of human diseases and in animal husbandry can lead to bacterial resistance against clinically relevant drugs [4]. 

Therefore, the use of natural ingredients to improve gut health is of high importance. Gut health and intestinal barrier integrity could be improved and maintained with proper selection of feed additives which prevent pathogen invasion and inflammation, furthermore stimulating growth of useful microorganisms. Food and feed supplements derived from plants often demonstrate various beneficial effects—they could have significant anti-inflammatory and antibacterial activity. A fermented wheat germ extract (FWGE) is a natural nontoxic substance which is already used in the human medicine as a supportive therapy in cancer patients under radiotherapy and chemotherapy [5]. The two major components to which beneficial effects are related, namely, 2-methoxy-benzoquinone and 2,6-dimethoxy-benzoquinone [6], are found in high concentrations in these extracts. FWGE has immunomodulatory [7], antiproliferative, antimetastatic, and antiangiogenic effects, and also, it can induce apoptosis in certain tumour cells like breast, colon, lung, and prostate cancer cells [8]. Lee et al. found that FWGE can promote apoptosis in different types of cancer cell lines such as SNU-5, MKN-45, SNU-620, SNU-1, and SNU-16. FWGE has significant antiproliferative effects and kills tumour cells by the induction of apoptosis via the caspase-poly (ADP-ribose) polymerase pathway [9]. Combined treatment of FWGE and 5-fluorouracil or dacarbazine resulted in a synergistic effect in the HCR-25 human cancer cell line and B16 murine melanoma cell line [6]. 

The effect of FWGE , which is a commercial dietary FWGE supplement for cancer patients, was also studied. Apoptosis induction in many Jurkat T leukaemia tumour cells [10], HL-60 human promyelocytic leukaemia cells [11], and tumour B and T cells [12] was also observed. FWGE has been also studied in autoimmune diseases. The anti-inflammatory effect of the FWGE extract was examined in rat adjuvant arthritis. It has been found that the FWGE treatment regulated inflammation-induced COX-1 and COX-2 expression and showed an additive effect with diclofenac [13]. FWGE can mitigate the clinical signs of systemic lupus erythematosus in mice via inhibiting Th2 response [14]. Although several studies have been performed on tumour and immune cells, the effect of this natural extract in the healthy gastrointestinal system is poorly examined. IPEC-J2 cells are isolated from the jejunum of neonatal nonsuckled piglets [15]. These intestinal columnar porcine epithelial cells are noncancerous; therefore, they mimic the human physiological circumstances more closely compared to other transformed or tumorigenic cell lines. Using IPEC-J2 cells is a great tool to investigate epithelial integration, status of the antioxidant defense system, inflammation, and effects of pro- and prebiotics and other nutrients [16–18]. These cells can express and produce different types of inflammatory proteins (e.g., IL-6 and IL-8), cytokines (GM-CSF, TNF-α), receptors (toll-like receptors, F4 fimbrial receptors), and mucins [15]. Because swine and human intestinal functions are closely related, investigations performed on IPEC-J2 can provide reliable information regarding the pathogenesis of human intestinal infections [19]. 

In this study, our aim was to examine the anti-inflammatory and antioxidant effect of FWGE in jejunal epithelial cells under lipopolysaccharide stimulation. Moreover, the impact on the intestinal paracellular permeability of FWGE was also observed. To our knowledge, this is the first study, where an effect on reactive oxygen species and influence on the intestinal epithelial barrier of FWGE are studied on healthy intestinal epithelial cells. Go to: 2. Materials and Methods 2.1. Chemicals FWGE was manufactured by Biropharma (Kunfehértó, Hungary) under the trade name FWGE, and lipopolysaccharides (LPS) (derived from Salmonella enterica ser. Typhimurium, Escherichia coli O55:B5, E. coli O111:B4, and E. coli O127:B8, suitable for cell culture) were purchased from Sigma-Aldrich–Merck (Darmstadt, Germany). 2.2. Cell Line and Culture Conditions The IPEC-J2 cell line was derived from the jejunum of a healthy neonatal piglet [20]. The cell culture was grown in the 1 : 1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) (plain medium) augmented with 5% foetal bovine serum (FBS), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (ITS), 5 ng/ml EGF, and 1% penicillin-streptomycin (Sigma-Aldrich–Merck).

 IPEC-J2 cells were grown at 37°C in a humidified atmosphere of 5% CO2. For the experiments, IPEC-J2 cells between passages 48 and 52 were seeded onto six-well polystyrene cell culture plates (Corning Inc., Corning, NY, USA), at a density of 1.5 × 105 cells/ml (the volume of medium was 2 ml in each well according to the manufacturer's prescription). Cells were fed every second day until confluence was achieved. 2.3. Cell Viability Measurement by the Neutral Red Uptake Assay Influence of FWGE on the viability of IPEC-J2 cells at different concentrations (1%, 2%, and 4%) and different durations of treatment (1 h, 2 h, and 24 h) was tested. The viability of the cells was also investigated at different concentrations (1, 10, and 20 μg/ml) of the Salmonella LPS strains. FWGE was dissolved in distilled water, filtered with a 0.22 μm membrane filter (Sigma-Aldrich–Merck, Darmstadt, Germany), and diluted in cell culture medium. LPS were freshly dissolved in plain medium. IPEC-J2 cells were seeded in a 96-well plate and incubated with FWGE for 1, 2, and 24 h, respectively. LPS strains were tested for a 1 h treatment period. The percentage of living cells was determined after 24 h of treatment by neutral red assay following the method of Repetto et al. [21]. 2.4. Incubation of Enterocytes with LPS and FWGE After IPEC-J2 monolayers have reached confluency, they were washed twice with plain medium. LPS derived from different bacterial strains (Salmonella enterica ser. Typhimurium, Escherichia coli O55:B5, E. coli O111:B4, and E. coli O127:B8) was used to evoke oxidative stress and inflammation. Control samples were treated with DMEM/F12 plain medium. LPS solutions were added to the plain medium at 10 μg/ml concentration [17].

 FWGE solutions were dissolved in distilled water and filtered with a 0.22 μm membrane filter; thereafter, the solutions were diluted in the plain medium in two different concentrations (1% and 2%). After 1 h incubation with LPS, FWGE test compounds, and their combinations, cells were rinsed with plain medium and cultured for additional 24 h for redox status and inflammation studies. 2.5. Measurement of Intracellular ROS and Extracellular H2O2 Levels in IPEC-J2 Cells IPEC-J2 cells were treated with the four different types of LPS (10 μg/ml), the FWGE (1%, 2%), and their combinations in phenol red-free medium on twenty-four-well culture plates. Extracellular measurement of H2O2 was performed using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Waltham, USA) following the manufacturer's instruction [22]. Fluorescence intensity was measured at an excitation wavelength of 560 nm and an emission wavelength at 590 nm (Victor X2 2030 fluorometer, PerkinElmer, Waltham, MA, USA). Change in the intracellular redox state of enterocytes was determined using a 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye (Sigma-Aldrich–Merck, Darmstadt, Germany). Intracellular ROS oxidize nonfluorescent DCFH-DA to the highly fluorescent dichlorofluorescein form (DCF) [23]. IPEC-J2 cells were treated with the LPS of different bacterial strains (10 μg/ml) and with the FWGE (1%, 2%) in phenol red-free DMEM/F12 for 1 h. DCFH-DA (10 μM) was added to IPEC-J2 cells for 30 minutes. Cells were rinsed with medium, scraped, and centrifuged for 10 minutes at 4500 rpm at 4°C. Fluorescence was determined with a Victor X2 2030 fluorometer at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

2.6. IL-6 ELISA IPEC-J2 cells were treated with the four different types of LPS (10 μg/ml), the FWGE (1%, 2%), and their combinations in phenol red-free medium on twenty-four-well culture plates for 1 h. After 1 h treatment, solutions were removed and cell culture medium was added to the IPEC-J2 cell. Supernatants were collected after 6 h, and IL-6 concentrations were measured from 100 μl of samples. Each sample was measured twice. The level of IL-6 secretion (pg/ml) was measured with porcine-specific IL-6 ELISA kits (Sigma-Aldrich–Merck, Darmstadt, Germany) following the manufacturer's guide. 2.7. Paracellular Permeability Measurement IPEC-J2 cells were seeded on 6-well polyester membrane inserts and were grown to confluent, differentiated monolayers. LPS derived from S. Typhimurium was added at 10 μg/ml concentration for 1 h to cells, and transepithelial electric resistance (TEER) values were measured prior to and 2, 4, and 24 h after LPS treatment. Simultaneously with LPS administration, cells were treated with 1 mg/ml fluorescein isothiocyanate-dextran 4 kDa (FD4) tracer dye (Sigma-Aldrich, Darmstadt, Germany) with different incubation times (2, 4, and 24 h). Medium samples from the basolateral chambers were collected, and the FD4 concentration was determined by a fluorescent method at excitation 485 nm and emission 535 nm (PerkinElmer, Victor X2 2030 fluorimeter). 2.8. Statistics Statistical analysis of our data was performed with R 3.3.2 (2016) software (R Foundation, Vienna, Austria).

 Differences between means were determined by two-way ANOVA, with data of normal distribution, and homogeneity of variances was also confirmed. To analyse treated groups to controls, the Dunnett post hoc test was applied and the Fisher LSD test was used to compare different treatments. Differences between treatment groups were considered proven if p values were <0.05. Go to: 3. Results 3.1. Viability of IPEC-J2 Cells To select the proper concentration of LPS for the experiments without reduction of cell viability, the neutral red uptake assay was performed. There was no significant reduction regarding cell viability after treatment with the highest LPS concentration (20 μg/ml) of E. coli O55:B5, O111:B4, and O127:B8 (data not shown). LPS from S. Typhimurium did not cause any significant alteration in cell viability at concentration of 10 μg/ml which was also true in the case of different E. coli strains (Figure 1). Based on the abovementioned results, 10 μg/ml LPS concentration was chosen for further experiments. 

FWGE showed no significant reduction in cell viability (Figure 2); moreover, in the case of the 2% FWGE 24 h treatment, the number of living IPEC-J2 cells was higher compared to that of the control. For further experiments, 1% and 2% FWGE was used for 1 h.3.2. ROS Production in IPEC-J2 Cells after Treatment with FWGE Stimulation of the IPEC-J2 cells with different LPS types caused significant increase in intracellular ROS level compared to the control group except for E. coli O55:B5 LPS treatment (Figure 3). FWGE per se in both applied concentrations (1% and 2%) significantly decreased the intracellular quantity of reactive oxygen species compared to the control group. Significant decrease in intracellular ROS level was also observed after the combined treatment of LPS from all E. coli types and FWGE. Only 1% FWGE did not cause significant reduction in the elevated ROS level after S. Typhimurium LPS treatment. 

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