Antimicrobial resistance is one of the biggest health challenges nowadays. Probiotics are promising candidates as feed additives contributing to the health of the gastrointestinal tract. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. We established a co-culture model, in which gastrointestinal infection was modeled using Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). Using intestinal porcine epithelial cells (IPEC-J2), the effects of pre-, co-, and post-treatment with Lactobacillus (L.) rhamnosus on the barrier function, intracellular (IC) reactive oxygen species (ROS) production, proinflammatory cytokine (IL-6 and IL-8) response, and adhesion inhibition were tested. E. coli- and S. Typhimurium-induced barrier impairment and increased ROS production could be counteracted using L. rhamnosus (p < 0.01). S. Typhimurium-induced IL-6 production was reduced via pre-treatment (p < 0.05) and post-treatment (p < 0.01); increased IL-8 secretion was decreased via pre-, co-, and post-treatment (p < 0.01) with L. rhamnosus. L. rhamnosus demonstrated significant inhibition of adhesion for both S. Typhimurium (p < 0.001) and E. coli (p < 0.001 in both pre-treatment and post-treatment; p < 0.05 in co-treatment). This study makes a substantial contribution to the understanding of the specific benefits of L. rhamnosus. Our findings can serve as a basis for further in vivo studies carried out in pigs and humans.

Intestinal infections caused by Escherichia coli and Salmonella enterica pose a huge economic burden on the swine industry that is exacerbated by the development of antimicrobial resistance in these pathogens, thus raising the need for alternative prevention and treatment methods. Our aim was to test the beneficial effects of the flavonoid luteolin in an in vitro model of porcine intestinal infections. We infected the porcine intestinal epithelial cell line IPEC-J2 with E. coli and S. enterica subsp. enterica serovar Typhimurium (10⁶ CFU/mL) with or without previous, concurrent, or subsequent treatment with luteolin (25 or 50 µg/mL), and measured the changes in the reactive oxygen species and interleukin-6 and -8 levels of cells. We also tested the ability of luteolin to inhibit the adhesion of bacteria to the cell layer, and to counteract the barrier integrity damage caused by the pathogens. Luteolin was able to alleviate oxidative stress, inflammation, and barrier integrity damage, but it could not inhibit the adhesion of bacteria to IPEC-J2 cells. Luteolin is a promising candidate to be used in intestinal infections of pigs, however, further studies are needed to confirm its efficacy. The use of luteolin in the future could ultimately lead to a reduced need for antibiotics in pig production.

Ochratoxin A (OTA) and lipopolysaccharide (LPS) intake can cause gastrointestinal disorders. Polyphenolic chrysin (CHR) and luteolin (LUT) display anti-inflammatory and antioxidant properties. Porcine intestinal epithelial (jejunal) IPEC-J2 cells were treated with OTA (1 µM, 5 µM and 20 µM), E. coli LPS (10 µg/mL), CHR (1 µM) and LUT (8.7 µM) alone and in their combinations. Cell viabilities (MTS assay) and extracellular (EC) hydrogen-peroxide (H₂O₂) production (Amplex red method) were evaluated. Intracellular (IC) reactive oxygen species (ROS) were assessed using a 2′-7′dichlorodihydrofluorescein diacetate (DCFH-DA) procedure. ELISA assay was used to evaluate IL-6 and IL-8 secretion. OTA decreased cell viabilities (p < 0.001) which could not be alleviated by LUT or CHR (p > 0.05); however, EC H₂O₂ production was successfully suppressed by LUT in IPEC-J2 cells (p < 0.001). OTA with LPS elevated the IC ROS which was counteracted by CHR and LUT (p < 0.001). IL-6 and IL-8 secretion was elevated by LPS + OTA (p < 0.001) which could be inhibited by LUT (p < 0.01 for IL-6; p < 0.001 for IL-8). Based on our results, CHR and LUT exerted beneficial effects on IC ROS levels and on cytokine secretion (LUT) in vitro; thus, they might be used as dietary and feed supplements to avoid OTA- and LPS-related health risks.

In dogs, chronic enteropathies, and impaired gut integrity, as well as microbiome imbalances, are a major problem. These conditions may represent a continuous low endotoxin load, which may result in the development of diseases that are attributable to chronic inflammation. Flavonoids are polyphenolic plant compounds with numerous beneficial properties such as antioxidant, anti-inflammatory and antimicrobial effects. For our experiments, we isolated primary white blood cells (peripheral blood mononuclear cells and polymorphonuclear leukocytes) from healthy dogs and induced inflammation and oxidative stress with Escherichia coli and Salmonella enterica serovar Enteritidis lipopolysaccharide (LPS). In parallel, we treated the cell cultures with various flavonoids luteolin, quercetin and grape seed extract oligomeric proanthocyanidins (GSOP) alone and also in combination with LPS treatments. Then, changes in viability, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) levels were measured in response to treatment with quercetin, luteolin and GSOP at 25 and 50 μg/mL concentrations. We found that ROS levels were significantly lower in groups which were treated by flavonoid and LPS at the same time compared to LPS-treated groups, whereas TNF-α levels were significantly reduced only by luteolin and quercetin treatment. In contrast, treatment with lower concentrations of GSOP caused an increase in TNF-α levels, while higher concentrations caused a significant decrease. These results suggest that the use of quercetin, luteolin and GSOP may be helpful in the management of chronic intestinal diseases in dogs with reduced intestinal barrier integrity or altered microbiome composition, or in the mitigation of chronic inflammatory processes maintained by endotoxemia. Further in vitro and in vivo studies are needed before clinical use.

Chronic gastrointestinal disorders in dogs are complex conditions often associated with oxidative stress, dysbiosis, and persistent immune activation, which can impair intestinal barrier integrity and promote bacterial translocation. Among intestinal bacteria, Escherichia coli may act as an opportunistic pathogen under inflammatory conditions and contribute to systemic inflammation. This study aimed to establish an in vitro model using primary canine peripheral blood mononuclear cells stimulated with canine-derived E. coli strains to investigate inflammatory and oxidative responses, and to evaluate the anti-inflammatory and antioxidant effects of selected flavonoids. Inflammatory activation was strongest following exposure to the enrofloxacin-inhibited E. coli 863 strain for 24 h. Treatments with quercetin, luteolin, and grape seed proanthocyanidins at concentrations of 25 and 50 µg/mL were then assessed. Minimum inhibitory and mutant prevention concentrations, intracellular reactive oxygen species, and tumor necrosis factor-α levels were determined. Although the tested flavonoids showed moderate antibacterial activity, all significantly reduced intracellular oxidative stress and inflammatory cytokine production compared to infected controls. These findings suggest that dietary flavonoids may exert beneficial immunomodulatory effects and could serve as potential adjuncts in managing canine chronic gastrointestinal inflammation. Further in vitro and in vivo validation is required to confirm their therapeutic relevance.

The intestinal barrier is essential for gastrointestinal and systemic homeostasis by enabling nutrient absorption while limiting the translocation of pathogens and toxins. When barrier function is impaired, bacterial components such as lipopolysaccharides (LPSs) may cross the epithelium and promote inflammatory signaling. In dogs, chronic inflammatory enteropathies are frequent disorders associated with barrier dysfunction, dysbiosis, and immune dysregulation, and may progress to protein-losing enteropathy or systemic inflammation. Humic substances, particularly humic acid (HA), are natural organic compounds with reported antioxidative, immunomodulatory, and barrier-supporting effects; however, the cellular mechanisms underlying these effects in intestinal and immune models remain insufficiently characterized. This study evaluated the effects of a commercially available HA-based supplement on epithelial barrier integrity and inflammatory responses using an in vitro system combining IPEC-J2 intestinal epithelial cells and primary canine peripheral blood mononuclear cells (PBMCs). Epithelial barrier integrity (FD4 paracellular flux), reactive oxygen species, and cytokine production (TNF-α, IL-6) were assessed under basal and LPS-stimulated conditions. HA treatment preserved epithelial barrier function and reduced LPS-induced pro-inflammatory cytokine production, supporting further investigation of HA as a nutraceutical adjunct for gut health support in dogs with chronic enteropathies.