A blend of selected botanicals maintains intestinal epithelial integrity and reduces susceptibility to Escherichia coli F4 infection by modulating acute and chronic inflammation in vitro

Abstract

In the pig production cycle, the most delicate phase is weaning, a sudden and early change that requires a quick adaptation, at the cost of developing inflammation and oxidation, especially at the intestinal level. In this period, pathogens like enterotoxigenic Escherichia coli (ETEC) contribute to the establishment of diarrhea, with long-lasting detrimental effects. Botanicals and their single bioactive components represent sustainable well-recognized tools in animal nutrition thanks to their wide-ranging beneficial functions. The aim of this study was to investigate the in vitro mechanism of action of a blend of botanicals (BOT), composed of thymol, grapeseed extract, and capsicum oleoresin, in supporting intestinal cell health during inflammatory challenges and ETEC infections. To reach this, we performed inflammatory and ETEC challenges on Caco-2 cells treated with BOT, measuring epithelial integrity, cellular oxidative stress, bacterial translocation and adhesion, gene expression levels, and examining tight junction distribution. BOT protected enterocytes against acute inflammation: while the challenge reduced epithelial tightness by 40%, BOT significantly limited its drop to 30%, also allowing faster recovery rates. In the case of chronic inflammation, BOT systematically improved by an average of 25% the integrity of challenged cells (p < 0.05). Moreover, when cells were infected with ETEC, BOT maintained epithelial integrity at the same level as an effective antibiotic and significantly reduced bacterial translocation by 1 log average. The mode of action of BOT was strictly related to the modulation of the inflammatory response, protecting tight junctions' expression and structure. In addition, BOT influenced ETEC adhesion to intestinal cells (-4%, p < 0.05), also thanks to the reduction of enterocytes' susceptibility to pathogens. Finally, BOT effectively scavenged reactive oxygen species generated by inflammatory and H₂O₂ challenges, thus alleviating oxidative stress by 40% compared to challenge (p < 0.05). These results support the employment of BOT in piglets at weaning to help manage bacterial infections and relieve transient or prolonged stressful states thanks to the modulation of host-pathogen interaction and the fine-tuning activity on the inflammatory tone.

Keywords: botanicals; enterotoxigenic Escherichia coli; inflammation; intestinal health; oxidative stress.

Figure 1

TER results during an acute LPS + cytomix challenge (24 h) on Caco-2 cells treated with a blend of botanicals (BOT). Panel (A) displays the TER of the challenge at D0, applied simultaneously to the treatment, while panel (B) shows the TER of the challenge at D6, applied on the last day of BOT treatment. Negative control (CTR-) was shared between the two challenges. Experimental groups with a “+” in the name were challenged. Data in graphs are represented as means ± SEM. Different letters indicate significant differences with p < 0.05 at each time point.

Figure 2

TER results during a chronic LPS + cytomix challenge (7 days) on Caco-2 cells treated with a blend of botanicals (BOT) for 7 days. Experimental groups with a “+” in the name were challenged. Data in graphs are represented as means ± SEM. Different letters indicate significant differences with p < 0.05 at each time point.

Figure 3

Reactive oxygen species levels in Caco-2 cells treated with a blend of botanicals (BOT) and challenged with LPS + cytomix for 24 h (A) or with hydrogen peroxide (H₂O₂) for 1 h (B). Groups with the challenge are represented with a “+” in the name. Data in the graphs are reported as means ± SEM. Different letters denote significant differences with p < 0.05.

Figure 4

Gene expression analysis of Caco-2 cells treated with a blend of botanicals (BOT) and challenged with LPS + cytomix for 7 days. Panel (A) displays the inflammatory cytokines (TNFα, IL-1β, IL-6, IL-8), while panel (B) reports the data for tight-junction markers (ZO-1, ZO-2, CLD-1, OCCL). Groups with the inflammatory challenge are represented with a “+” in the name. Data in the graphs are reported as means ± SEM. Different letters denote significant differences with p < 0.05; tendencies (p < 0.1) are highlighted by reporting their respective p values and indicated by a line spanning the two evaluated groups.

Figure 5

Immunofluorescence staining of Caco-2 cells treated with a blend of botanicals (BOT) and challenged with LPS + cytomix for 24 h. Groups with the inflammatory challenge are represented with a “+” in the name. The left pictures show the DAPI staining of cell nuclei, the central column depicts ZO-1 staining with FITC, while the right column displays the merge of the first two images. White arrows identify areas with anomalies of ZO-1 disposition, or loss of cells. Each row displays a different experimental group.

Figure 6

TER (A), bacterial translocation (B), and ETEC adhesion (C) assay results after an enterotoxigenic E. coli infection on Caco-2 cells treated with a blend of botanicals (BOT). Experimental groups with a “+” in the name were infected. Data in graphs are represented as means ± SEM. Different letters indicate significant differences with p < 0.05 at each timepoint.

Figure 7

Gene expression analysis of Caco-2 cells treated with a blend of botanicals (BOT) and challenged with enterotoxigenic E. coli for 4 h. Panel (A) displays the inflammatory cytokines results (TNFα, IL-1β, IL-6, IL-8), panel (B) reports the data for tight-junction markers (ZO-1, ZO-2, CLD-1, OCCL), while panel C shows MUC13, BD1, and GUCY2C, three genes related to cellular response to bacterial infection. Groups with bacterial infection are represented with a “+” in the name. Data in the graphs are represented as means ± SEM. Different letters denote significant differences with p < 0.05; tendencies (p < 0.1) are highlighted by reporting their respective p values and indicated by a line spanning the two evaluated groups.

Figure 8

Immunofluorescence staining of Caco-2 cells treated with a blend of botanicals (BOT) and challenged with enterotoxigenic E. coli for 2 h. Groups with bacterial infection are represented with a “+” in the name. Left pictures show the DAPI staining of cell nuclei, the central column depicts ZO-1 staining with FITC, while the right column displays the merge of the first two images. White arrows identify areas with anomalies in ZO-1 disposition, or loss of cells. Each row displays a different experimental group.