A Cocoa Peptide Protects <i>Caenorhabditis elegans</i> from Oxidative Stress and β-Amyloid Peptide Toxicity

<div><p>Background</p><p>Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases.</p><p>Methodology/Principal Findings</p><p>A bioactive peptide, 13L (DNYDNSAGKWWVT), was obtained from a hydrolyzed cocoa by-product by chromatography. The <i>in vitro</i> inhibition of prolyl endopeptidase (PEP) was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic <i>Caenorhabditis elegans</i> strain CL4176 expressing the human Aβ_1–42 peptide as a pre-clinical <i>in vivo</i> model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL) showed the highest antioxidant activity (<i>P</i>≤0.001) in the wild-type strain (N2). Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24–47 h period after Aβ_1–42 peptide induction (<i>P</i>≤0.0001). This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide.</p><p>Conclusions/Significance</p><p>These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a <i>C. elegans</i> model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.</p></div

Screening for antioxidant bacteria in <i>C. elegans</i>.

<p>Survival upon a 5 mM H₂O₂ treatment for 5 h of <i>C. elegans</i> BA 17 strain fed for 3 days with <i>Bifidobacteria, Lactobacilli</i> and <i>Streptococci</i> strains in liquid medium. Worms were fed with <i>E. coli</i> OP 50, for larvae 1^st stage synchronization. E. coli OP 50 strain was inhibited with antibiotics prior of LAB and <i>Bifidobacteria</i> feeding. Protection by <i>E. coli</i> OP50 is fixed at 100%.</p

<i>Lactobacillus rhamnosus</i> CNCM I-3690 has an anti-inflammatory effect <i>in vitro</i>.

<p>A. Relative IL-8, production and I-κB-luciferase detection from the HT-29-bacteria interaction assays. Each bar represent the mean value of three replicate samples and and error bars depict corresponding standard deviation. Black bars: I-kB-luciferase, grey bars: IL-8. **p-value ≤0.05. B. Phenotypical analysis of monocyte-derived DCs co-cultured with HT-29-NF-κB-luciferase cells. Cells were incubated with LPS or LPS+bacteria. CNCM I-3690 down-regulated the expression of HLA-DR and CD86 surface markers. Results were expressed according to the following equation [(bacteria+LPS)-LPS]/LPS-basal*100. Experiments were performed with two different donors. C. Cytokine ratios (IL-8/IL-10, IL-6/IL-10, IL-12/IL-10 and TNF-α/IL-10) of DCs from donor 2 in co-culture with HT-29-NF-κB-luciferase cells and bacteria w or w/o LPS. Experiments were performed with two different donors (data shown for donor 2). *p-value≤0.05.</p

Transcriptomic analysis of <i>C. elegans</i> N2 fed with CNCM I-3690 vs CNCM I-4317.

<p>Transcriptomic analysis of <i>C. elegans</i> N2 fed with CNCM I-3690 vs CNCM I-4317.</p

<i>Lactobacillus rhamnosus</i> CNCM I-3690 increases lifespan of <i>C. elegans</i> wild-type N2 strain and this effect is at least partially dependent of the DAF2-DAF16 pathway.

<p>A. <i>C. elegans</i> N2 wild-type strain was fed with <i>L. rhamnosus</i> CNCM I-3690, CNCM I-4317 and <i>E. coli</i> OP 50 and survival of worms was followed for 21 days. Mean lifespan, indicating the time in days where half of the worm population is still alive, is shown on the X-axis for the three situations. Curves comparisons are indicated (P-values) CNCM I-3690/CNCM I-4317 vs <i>E. coli</i> OP50 (red) and CNCM I-4317 vs <i>E. coli</i> OP50 (blue). NS: Non statistical difference. B, C, D. <i>C. elegans daf-16</i> (GR1307), <i>daf-2</i> (CB1370) and <i>skn-1</i> (LG333) loss-of-function mutant strains were fed with <i>L. </i><i>rhamnosus</i> CNCM I-3690 and <i>E. coli</i> OP 50. Survival of worms was followed up for 21 days. B. Curves comparisons vs <i>E. coli</i> OP50 are indicated in red (P-values).</p

Protective effect of <i>Lactobacillus rhamnosus</i> CNCM I-3690 in a TNBS-induced murine model of colitis.

<p>A. Design of the interventional animal study. B. 3-day post colitis body-weight losses (% of initial) for healthy control mice, vehicle-TNBS-treated animal and either <i>L. rhamnosus</i> CNCM I-3690-fed TNBS-treated mice or Prednisolone-TNBS-treated mice. Data represent the mean+/−SEM, (number of mice n = 10); ***: P<0.001. C: Individual inflammatory macroscopic (Wallace score, left panel), and histological damage scores (Ameho score, right panel) in Control, probiotic and drug treatment conditions respectively, means +/− SD are indicated, **: P<0.01, ***: P<0.001. D: Representative histological colon sections from mice treated in various conditions after induction of TNBS colitis; May-Grünwald and Giemsa-stainings of 5 µm paraffin sections, original magnification×40.</p

Secretory IgA content (A) and populations of <i>Clostridium difficile</i> (B), <i>Bacteroides</i> (C), <i>Lactobacillus</i> (D), and <i>Bifidobacterium</i> spp. (E) in the feces of healthy adults fed one daily probiotic capsule or placebo for 4 weeks as log CFU/g feces.

<p>Values are means ± SEM, n=20 per group. Labeled means without a common letter differ, <i>P</i><0.05. Time 1, first washout; Time 2, intervention; Time 3, second washout.</p

CONSORT flow diagram of the subjects in the SETOPROB study (NCT01479543).

<p>CONSORT flow diagram of the subjects in the SETOPROB study (NCT01479543).</p

Serum IL-4 (A), IL-10 (B), and IL-12 (C) concentrations and IL-10/TNF-α (D), and IL-10/IL-12 ratios (E) in healthy adults fed one daily capsule of probiotics or placebo for 4 weeks.

<p>Values are means ± SEM, n=20 per group. Labeled means without a common letter differ, <i>P</i><0.05. Time 1, first washout; Time 2, intervention.</p