Table 1.
Ingredients and chemical composition of the experimental diet.
Table 2.
Guidelines on the recognition of pain, distress and discomfort.
Table 3.
Oligonucleotide sequences used to determine target gene expression.
Fig 1.
Accumulated feed consumption (kg/pig) and body weight gain (kg) of pigs challenged chronically with LPS and fed an Olive-oil Bioactive Extract (OBE).
(A) Feed consumption (kg/pig) of pigs chronically challenged with LPS and fed a commercial pre-starter diet untreated (C-, C+) or supplemented with an olive-oil extract (OBE; 500 mg/kg diet). On d 20, 23, 26 and 29, OBE and positive control (C+) pigs received E.coli-derived LPS injections at increasing doses (60, 66, 72 and 78 μg/kg). Negative control animals (C-) were injected with saline. (B) Body weight gain (kg) and (C) efficiency of feed conversion (kg of feed consumed/kg of BW gain during the experiment) of the pigs treated as described in A. Bars are least squares means ± SEM (n = 10–11). Different letters indicate significant differences among groups (p < 0.05).
Fig 2.
Immune-inflammatory response of pigs and macrophages treated with an Olive-oil Bioactive Extract (OBE) and challenged with LPS.
(A, B) Concentration of pigMAP and ILB1 in peripheral circulation of pigs. Piglets were fed a commercial diet untreated (C-, C+) or supplemented with an olive-oil extract (OBE; 500 mg/kg diet). On d 20, 23, 26 and 29, OBE and positive control (C+) pigs received E.coli-derived LPS injections at increasing doses (60, 66, 72 and 78 μg/kg). Negative control animals (C-) were injected with saline. Plasma samples were collected 3 h after final LPS administration and pigMAP and cytokine levels were determined by sandwich ELISA. Bars are least squares means ± SEM (pigMAP, n = 10–11, IL1B, n = 3–6). (C, D, E) Expression of IL1B, iNOS and Mip1a genes in RAW 264.7 macrophages. Cells were treated with OBE (50 and 25 μg/mL) and DMSO (0.1% v/v) for 24 h and subsequently challenged with LPS (10 ng/mL) for 6 h. The concentration of mRNA was measured via qRT-PCR. Bars represent means ± SEM of 3 independent experiments performed in duplicate. Values are normalized to the sham-treated control. Different letters indicate significant differences among groups (p < 0.05).
Fig 3.
Relative concentrations of IL1B (A), TNF-α (B), and iNOS (C) mRNA in ileal mucosa of pigs challenged chronically with LPS and fed an Olive-oil Bioactive Extract (OBE).
Piglets were fed a commercial diet either untreated (C-, C+) or supplemented with an olive-oil extract (OBE; 500 mg/kg diet). On d 20, 23, 26 and 29, OBE and positive control (C+) pigs received E.coli-derived LPS injections at increasing doses (60, 66, 72 and 78 μg/kg). Negative control animals (C-) were injected with saline. Mucosa samples were collected 3 h after final LPS administration and mRNA levels of the abovementioned markers were measured via qRT-PCR. Bars are means ± SEM (n = 9–10).
Fig 4.
Concentrations of permeability markers (A-C) in plasma and relative concentrations of CDH1; (D), OCLN (E), and ZO-1 (F) in the ileal mucosa of pigs challenged chronically with LPS and fed an Olive-oil Bioactive Extract (OBE).
Piglets were fed a standard diet either untreated (C-, C+) or supplemented with an olive-oil extract (OBE; 500 mg/kg diet). On d 20, 23, 26 and 29, OBE and positive control (C+) pigs received E.coli-derived LPS injections at increasing doses (60, 66, 72 and 78 μg/kg). Negative control animals (C-) were injected with saline. Plasma samples were collected via jugular venipuncture 1 h after intragastric infusion of the marker solution containing 0.15 g mannitol/kg BW and 0.1 g cobalt-EDTA/kg BW. Mucosa samples were collected 3 h after final LPS administration and mRNA levels of junctional proteins were measured via qRT-PCR. Bars are least squares means ± SEM (n = 9–11). Different letters indicate significant differences among groups (p < 0.05).
Fig 5.
Effect of an olive-oil bioactive extract on basal TEER (A) and TNF-α induced decrease in TEER (B) in vitro.
Caco-2/TC-7 cells were allowed to differentiate on permeable filters in the presence of an olive-oil bioactive extract (OBE; 100 μg/mL), genistein (50 μmol/L; positive control), and DMSO (0.1% v/v; vehicle control [C]) for 5 d. (A) Barrier integrity was assessed by measuring the TEER over time. To investigate potential protective effects of OBE on Caco-2/TC-7 barrier integrity, TNF-α (100 ng/mL) was added to the basolateral side for 24 h on d 5 to induce monolayer disruption. (B) TEER was measured before and after TNF-α administration and values were normalized to the untreated control. Bars represent means of 3 individual experiments ± SEM. Different letters indicate significant differences among treatments (p < 0.05).
Fig 6.
Gut microbial composition and predicted functions in pigs chronically challenged with LPS and fed an Olive-oil Bioactive Extract (OBE).
(A) Percent reads at the phylum level in colonic contents resulting from the 16S rRNA gene sequencing analysis. (B) Diversity of colonic microbiota within groups of pigs (alpha diversity; C- vs. C+ p < 0.12, C- vs. OBE p < 1.0, C+ vs. OBE p < 1.0). (C) Linear discriminant analysis (LDA) scores for microbial functions predicted by PICRUSt (α = 0.05, LDA score > 3.0). Piglets were fed a standard diet either untreated (C-, C+) or supplemented with an olive-oil extract (OBE; 500 mg/kg diet). On d 20, 23, 26 and 29, OBE and positive control (C+) pigs received E.coli-derived LPS injections at increasing doses (60, 66, 72 and 78 μg/kg). Negative control animals (C-) were injected with saline. Samples of colonic content were collected 3 h after final LPS administration and analyzed via massive sequencing of the V1-V2 hypervariable regions of the 16S rRNA gene (n = 9–11).
Fig 7.
Supposed metabolic targets involved in OBE mediated effects.
Repeated LPS injection stimulates the systemic secretion of pro-inflammatory IL1B and simultaneously suppresses feed intake and growth in challenged animals. OBE is capable of counteracting LPS stimulated IL1B secretion most likely through interaction with NF-κB signal cascade. OBE treatment further increases the concentration of junctional mRNA (OCLN, CDH1, ZO-1) and promotes TJ-functionality as indicated by decreased ion flux (TEER) and improved cobalt to mannitol ratio. Increased gene expression of junctional proteins as well as an improved TJ-functionality can be linked to enhanced gut integrity, further supporting animal growth and performance. Particular importance is devoted to the finding that the growth promoting effect of OBE is mediated independent of changes in gut microbial composition and diversity. Plain connections represent observed (solid) and supposed (interrupted) metabolic effects of chronic LPS challenge. Colour-filled arrows indicate effects of OBE treatment.