Nigella sativa (Black Cumin) Seed Extract Alleviates Symptoms of Allergic Diarrhea in Mice, Involving Opioid Receptors

The incidence of food hypersensitivity and food allergies is on the rise and new treatment approaches are needed. We investigated whether N. sativa, one of its components, thymoquinone, or synthetic opioid receptor (OR)-agonists can alleviate food allergy. Hence, ovalbumin (OVA) -sensitized BALB/c-mice were pre-treated either with a hexanic N. sativa seed extract, thymoquinone, kappa- (U50'4889) or mu-OR-agonists (DAMGO) and subsequently challenged intra-gastrically with OVA. All 4 treatments significantly decreased clinical scores of OVA-induced diarrhea. N. sativa seed extract, thymoquinone, and U50'488 also decreased intestinal mast cell numbers and plasma mouse mast cell protease-1 (MMCP-1). DAMGO, in contrast, had no effect on mast cell parameters but decreased IFNγ, IL-4, IL-5, and IL-10 concentration after ex vivo re-stimulation of mesenteric lymphocytes. The effects on allergy symptoms were reversible by OR-antagonist pre-treatment, whereas most of the effects on immunological parameter were not. We demonstrate that N. sativa seed extract significantly improves symptoms and immune parameters in murine OVA-induced allergic diarrhea; this effect is at least partially mediated by thymoquinone. ORs may also be involved and could be a new target for intestinal allergy symptom alleviation. N. sativa seed extract seems to be a promising candidate for nutritional interventions in humans with food allergy

<i>In vitro</i> opioid receptor-displacement assay.

<p>(Abbr.: CHO = Chinese hamster Ovary, HEK = Human embryonic kidney, DOP = delta opioid; KOP = kappa opioid, MOP = mu opioid, recomb. = recombinant).</p

Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = −0.95; −0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p &lt; 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers

<i>In vitro</i> ligand displacement by thymoquinone.

a)<p>20–50% = moderate displacement (Abbr.: MOR = μ-opioid receptor, KOR = ê-opioid receptor, DOR = δ-opioid receptor).</p

KOR-agonist (U50'488) and MOR-agonist (DAMGO) alleviate allergy related immune markers in OVA-allergic mice.

<p>OVA-sensitized mice were challenged with saline (Saline) or OVA (OVA) with or without pre-treatment. The graph shows the concentration of total plasma MMCP-1 (<b>A</b>) at sacrifice after subcutaneous treatment with 5 mg/kgBW KOR-agonist U50'488 (U50'488), with 20 mg/kgBW NorBNI (U50'488+NorBNI), or with NorBNI alone (NorBNI), and the concentration of plasma IL-4 (<b>B</b>) and IFN-gamma (<b>C</b>) after subcutaneous treatment with 5 mg/kgBW MOR-agonist (DAMGO) and naloxone-methiodide (DAMGO+N-M 1 mg), or naloxone-methiodide alone (N-M 1 mg). MMCP-1 levels were unchanged with MOR-agonist and IL-4 and IFN-gamma levels were unchanged with KOR-agonist respectively (data not shown). Each dot represents the corresponding value for one animal and the bars represent the median and interquartile range, * <i>p</i><0.05; n = 5–10.</p

Experimental set up for OVA-induced allergic diarrhea.

<p>Intervention corresponds to either intragastric <i>N. sativa</i> or thymoquinone (with or without sub-cutaneous OR-antagonist pre-treatment) or subcutaneous OR-agonists (with or without sub-cutaneous OR-antagonist pre-treatment). (Abbreviations: i.p. = intraperitoneal, OR = opioid receptor, OVA = ovalbumin, BW = body weight, † = sacrifice).</p

Thymoquinone decreases clinical symptoms, plasma MMCP-1 levels, and mast cell numbers in OVA-allergic mice.

<p>OVA-sensitized mice were challenged with saline (Saline), OVA (OVA), or with OVA after intragastric treatment with 13 mg/kgBW thymoquinone (Thq) and with thymoquinone after 30 mg/kgBW naloxone-methiodide pre-treatment (Thq N-M). Panel <b>A</b> represent the median of clinical macroscopic scores at sacrifice. Panel <b>B</b> shows the plasma concentrations of MMCP-1 and Panel <b>C</b> the numbers of mast cells per intestinal villus at sacrifice. The dots correspond to results from individual animals and the bars represent the median with interquartile range, * = p<0.05, panels show the results from two pooled experiments, n = 10–20. Panel <b>D–G</b> are representative histological pictures of mast cells in the jejunum of OVA-sensitized mice challenged with saline (<b>D</b>), OVA (<b>E</b>), or with OVA after intragastric treatment with 13 mg/kgBW thymoquinone (<b>F</b>) and with thymoquinone after 30 mg/kgBW naloxone-methiodide pre-treatment (<b>G</b>). Mast cells appear in bright red.</p

<i>N. sativa</i> seed extract decreases clinical macroscopic scores and immune parameters in OVA-allergic mice.

<p>OVA-sensitized mice were challenged with saline (Saline), OVA (OVA), or with OVA after intragastric administration of <i>N. sativa</i> seed extract (Ns) with the OR antagonist naloxone-methiodide pre-treatment at the dose indicated (Ns N-M), or with naloxone-methiodide alone (N-M). Panels <b>A</b> and <b>B</b> represent the median of clinical macroscopic scores at sacrifice. Panels <b>C</b> and <b>D</b> show the plasma concentration of MMCP-1 (<b>C</b>) and the numbers of mast cells per intestinal villus (<b>D</b>) at sacrifice. Each dot represents the corresponding value for one animal and the bars represent the median with interquartile range, * = <i>p</i><0.05; n = 5–19.</p

KOR-agonist (U50'488) and MOR-agonist (DAMGO) decrease symptoms in OVA-allergic mice.

<p>OVA-sensitized mice were challenged with saline (Saline) or OVA (OVA) with or without pre-treatment. Panel <b>A</b> shows the sum of clinical symptoms after subcutaneous treatment with 5 mg/kgBW KOR-agonist U50'488 (U50'488) and 20 mg/kgBW NorBNI (U50'488+NorBNI), or with NorBNI alone (NorBNI). Panel <b>B</b> shows the sum of clinical symptoms after subcutaneous treatment with 5 mg/kgBW MOR-agonist (DAMGO) and naloxone-methiodide (DAMGO+N-M 1 mg), or naloxone-methiodide alone (N-M 1 mg). Each dot represents the corresponding value for one animal and the bars represent the median with interquartile range, * = <i>p</i><0.05; n = 5–10. For both panels the table shows the median clinical score for each challenge from the 3^rd to 6^th challenge.</p