Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (1), 6884

Avenanthramide C From Germinated Oats Exhibits Anti-Allergic Inflammatory Effects in Mast Cells

Affiliations

Avenanthramide C From Germinated Oats Exhibits Anti-Allergic Inflammatory Effects in Mast Cells

Hima Dhakal et al. Sci Rep.

Abstract

Mast cells play a crucial role in allergic diseases via the release of inflammatory mediators, particularly histamine and pro-inflammatory cytokines. Avenanthramide (Avn) C, a polyphenol found mainly in oats, is known to exhibit various biological properties. In this study, we aimed to evaluate the effectiveness of Avn C from germinated oats against mast cell-mediated allergic inflammation. For the in vitro study, RBL-2H3, mouse bone marrow-derived mast cells and rat peritoneal mast cells were used. Avn C (1-100 nM) inhibited the immunoglobulin (Ig)E-stimulated mast cells degranulation by suppressing phosphorylation of phosphoinositide 3-kinase and phospholipase Cγ1 and decreasing intracellular calcium levels. It inhibited IgE-stimulated secretion of inflammatory cytokines via suppression of FcεRI-mediated signaling proteins Lyn, Syk, Akt, and nuclear factor-κB. To verify the effects of Avn C in vivo, ovalbumin-induced active systemic anaphylaxis (ASA) and IgE-mediated passive cutaneous anaphylaxis (PCA) models were used. Oral administration of Avn C dose-dependently attenuated the ASA reactions, as evidenced by the inhibition of hypothermia and reduction of elevated serum histamine, IgE, and interleukin-4 levels. Avn C also inhibited the PCA reactions, such as ear swelling and plasma extravasation. Our results suggested that Avn C from germinated oats might be a possible therapeutic candidate for mast cell-mediated allergic inflammation.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effects of Avn C on mast cell degranulation. (A) Chemical structure of Avn C. (BD) RBL-2H3, mBMMCs and RPMCs (3 × 104 cells/well) were pre-treated with or without Avn C, then incubated with MTT. The absorbance was detected using a spectrophotometer. For mast cell degranulation, RBL-2H3 and mBMMCs (5 × 105 cells/well), and RPMCs (3 × 104 cells/well) were sensitised with anti-DNP IgE (50 ng/mL). After incubation overnight, the cells were pre-treated with or without Avn C or Dexa for 1 h and then challenged with DNP-HSA (100 ng/mL). (E,G,I) The level of β-hexosaminidase was measured using β-hexosaminidase substrate buffer. (F,H,J) Histamine level was assayed using the o-phthaldialdehyde spectrofluorometric method. Each data presented as a graph represents the means ± SEM of three independent experiments. #Significantly different form the control group at p < 0.05. *Significantly different from the DNP-HSA-challenged group at p < 0.05. Dexa: dexamethasone.
Figure 2
Figure 2
Effects of Avn C on intracellular calcium levels in mast cells. RBL-2H3, mBMMCs, and RPMCs (3 × 104 cells/well) were pre-incubated with Fluo/3AM. Intracellular calcium was detected using a fluorescence plate reader. (AC) Intracellular calcium levels in RBL-2H3, mBMMCs, and RPMCs. Each data presented as a graph represents the means ± SEM of three independent experiments. (D) Representative fluorescence imaging of intracellular calcium levels in RBL-2H3 cells (original magnification × 200). Fluorescence images represent the 5 random cell sites. Scale bar: 100 μm. (E) RBL-2H3 (1.5 × 106 cells/well) were sensitised with anti-DNP IgE (50 ng/mL). After incubation overnight, the cells were pre-treated with or without Avn C or Dexa for 1 h and then challenged with DNP-HSA (100 ng/mL). The activation of signalling proteins (p-PI3K and p-PLCγ1) was assayed by Western blot analysis (p-: phosphorylated). Cropped blots are shown. Full-length blots are presented in Supplementary Fig. S2. The bands of the total form were used as loading controls. The bands shown are representative of three independent experiments. The Western band intensity was quantified using Image J software. The representative band intensity was digitized to the relative intensity. #Significantly different form the control group at p < 0.05. *Significantly different from the DNP-HSA-challenged group at p < 0.05. Dexa: dexamethasone.
Figure 3
Figure 3
Effects of Avn C on secretion of pro-inflammatory cytokines in mast cells. RBL-2H3 (5 × 105 cells/well) were sensitised with anti-DNP IgE (50 ng/mL). After incubating overnight, the cells were pre-treated with or without drugs Avn C and Dexa for 1 h and then challenged with DNP-HSA (100 ng/mL). (A) The gene expression of inflammatory cytokines was determined by qPCR. (B) The release of inflammatory cytokines was measured by ELISA. The left Y-axis represents the release of IL-4 and right Y-axis represents the release of IL-6 and TNF-α. Each data presented as a graph represents the means ± SEM of three independent experiments. #Significantly different form the control group at p < 0.05. *Significantly different from the DNP-HSA-challenged group at p < 0.05. Dexa: dexamethasone.
Figure 4
Figure 4
Effects of Avn C on FcεRI-mediated signalling proteins in mast cells. RBL-2H3 (1.5 × 106 cells/well) were sensitised with anti-DNP IgE (50 ng/mL). After incubation overnight, the cells were pre-treated with or without Avn C or Dexa for 1 h and then challenged with DNP-HSA (100 ng/mL). The activation of signalling proteins was assayed by Western blot analysis (N-: nuclear, p-: phosphorylated). Cropped blots are shown. Full length blots are presented in Supplementary Fig. S2. The bands of β-actin, lamin B1, and total form were used as loading controls. The bands shown are representative of three independent experiments. The Western band intensity was quantified by using Image J software. The representative band intensity was digitized to the relative intensity. Dexa: dexamethasone.
Figure 5
Figure 5
Effects of Avn C on OVA-induced ASA. Each mouse (total n = 30, n = 5/group) was injected intraperitoneally with OVA mixture (100 μg of OVA and 2 mg of alum adjuvant) or PBS on days 0 and 7. Drugs, including Avn C (0.1–10 mg/kg) and Dexa (10 mg/kg), were orally administered on days 9, 11, and 13. On day 14, mice were challenged with an intraperitoneal injection of 200 μg of OVA, and then the rectal temperature was monitored and recorded every 10 min for 90 min. Mice were euthanised after 90 min, and blood samples were collected from the abdominal artery. (A) Rectal temperature was measured every 10 min for 90 min. (B) Rectal temperature of mice at 30 min. (CF) Serum histamine, total IgE, OVA-specific IgE, and IL-4 were detected by ELISA. Each data presented as a graph represents the means ± SEM (n = 5/group) of two independent experiments. #Significantly different form the control group at p < 0.05. *Significantly different from the DNP-HSA-challenged group at p < 0.05. Dexa: dexamethasone.
Figure 6
Figure 6
Effects of Avn C on IgE-mediated PCA. The ear skin of mice (total n = 30, n = 5/group) were sensitised with an intradermal injection of anti-DNP IgE (0.5 μg/site) or PBS for 48 h. Drugs, including Avn C (0.1–10 mg/kg) and Dexa (10 mg/kg) were administered per oral, 1 h before intravenous injection of DNP-HSA (1 mg/mouse) and 4% Evans blue (1:1) mixture. Thirty minutes later, the thickness of both ears was measured, and the ears were collected to measure the pigmentation. The extravasation of dye was detected using a spectrophotometer. (A) Representative photographs of ears. (B) Thickness of ears. (C) Absorbance representing dye extravasations. Each data presented as a graph represents the means ± SEM (n = 5/group) of two independent experiments. #Significantly different form the control group at p < 0.05. *Significantly different from the DNP-HSA-challenged group at p < 0.05. Dexa: dexamethasone.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Takano H, Inoue KI. Environmental pollution and allergies. J. Toxicol. Pathol. 2017;30:193–199. doi: 10.1293/tox.2017-0028. - DOI - PMC - PubMed
    1. Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nat. Med. 2012;18:693–704. doi: 10.1038/nm.2755. - DOI - PMC - PubMed
    1. Williams CM, Galli SJ. The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J. Allergy Clin. Immunol. 2000;105:847–859. doi: 10.1067/mai.2000.106485. - DOI - PubMed
    1. Krystel-Whittemore M, Dileepan KN, Wood JG. Mast Cell: A Multi-Functional Master Cell. Front. Immunol. 2015;6:620. - PMC - PubMed
    1. Ma HT, Beaven MA. Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases? Adv. Exp. Med. Biol. 2011;716:62–90. doi: 10.1007/978-1-4419-9533-9_5. - DOI - PubMed

Publication types

Feedback