Lactic Acid Bacteria Fermented Cordyceps militaris (GRC-SC11) Suppresses IgE Mediated Mast Cell Activation and Type I Hypersensitive Allergic Murine Model

Abstract

Cordyceps militaris (C. militaris) has various biomedical applications in traditional oriental medicine for different diseases including inflammatory and immune-dysregulated diseases. It is a reservoir of nutritional components such as cordycepin, polysaccharides, and antioxidants. To improve its bioactivity, we fermented C. militaris with a Pediococcus pentosaceus strain isolated from a salted small octopus (SC11). The current study aimed to evaluate whether P. pentosaceus (SC11) fermentation could enhance the anti-allergic potential of C. militaris cultured on germinated Rhynchosia nulubilis (GRC) against a type I hypersensitive reaction in in vitro and in vivo studies. Total antioxidant capacity and cordycepin content were significantly increased in GRC after SC11 fermentation. GRC-SC11 showed significantly enhanced anti-allergic responses by inhibiting immunoglobulin E (IgE)/antigen-induced degranulation in RBL-2H3 cells, compared to GRC. The results demonstrated the significant inhibition of phosphorylated spleen tyrosine kinase (Syk)/ p38/GRB2-associated binding protein 2 (Gab2)/c-jun in IgE/Ag-triggered RBL-2H3 cells. Furthermore, suppressed mRNA levels of interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α) in IgE/Ag-activated RBL-2H3 cells were observed. GRC-SC11 significantly ameliorated IgE-induced allergic reactions by suppressing the ear swelling, vascular permeability, and inflammatory cell infiltration in passive cutaneous anaphylaxis (PCA) BALB/c mice. In conclusion, GRC fermented with P.pentosaceus exerted enhanced anti-allergic effects, and increased the cordycepin content and antioxidants potential compared to GRC. It can be used as bio-functional food in the prevention and management of type I allergic diseases.

Keywords: Cordyceps militaris; anti-allergy; fermentation.

Figure 1

Total antioxidant capacity (TAC) and cordycepin content of C. militaris grown on germinated R. nulubilis (GRC) and GRC fermented by P. pentosaceus isolated from the salted small octopus (SC11) (GRC-SC11). The experiments were repeated three times and results are presented as mean ± SD. TAC was expressed as mg ascorbic acid equivalent (mgAAE). Results were considered significant at the p value of ## p < 0.01. Data comparisons between the two groups were analyzed using the Student’s t-test.

Figure 2

Effects of GRC and GRC-SC11 on degranulation and cell viability of RBL-2H3 cells. (A) The release of β-hexosaminidase was calculated from the degranulation of RBL-2H3 cells. RBL-2H3 cells were treated with the indicated concentrations of GRC and GRC-SC11 and 10 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) for 30 min. Control (−) is the untreated control, while control (+) is stimulated with immunoglobulin E (IgE)/Ag. PP2 is an Src family kinase inhibitor that was used as the positive control. Different letters indicate the significant differences between groups. Results were compared for statistical significance through a one-way analysis of variance (ANOVA) followed by Duncan’s t-test (p < 0.05). (B) RBL-2H3 cells were exposed to different concentrations (10, 100, and 300 µg/mL) of GRC-SC11 for 24 h and cell viability was investigated. The experiments were repeated three times and results are presented as the representative or mean ± SD. Results were compared for statistical significance through a one-way analysis of variance (ANOVA followed by Duncan’s t-test (p < 0.05)).

Figure 3

Effect of GRC-SC11 on the production of pro-inflammatory cytokines in IgE/Ag-stimulated RBL-2H3 mast cells. The RBL-2H3 cells were untreated or treated with indicated concentrations of GRC-SC11 in the absence or presence of the 200 ng/mL of IgE/Ag or 10 µM of PP2. GRC-SC11 reduced the expressions of (A) tumor necrosis factor (TNF)- α and (B) interleukin (IL)-4 in IgE/Ag-stimulated RBL-2H3 mast cells. The expression study was carried out by Western blotting method using GAPDH as the loading control. The relative amounts of TNF- α, IL-4 were analyzed by using the Image J software. The results are presented as the representative or mean ± SD of three independent experiments. Results were analyzed for statistical significance through a one-way ANOVA followed by Dunnett’s t-test. The probability value of ## p < 0.01 was considered significant compared to the non-treated cells and *** p < 0.01 compared to the IgE/Ag-treated cells.

Figure 4

Effects of GRC-SC11 on the signaling molecules IgE/Ag-activated RBL-2H3 cells. RBL-2H3 cells were sensitized with dinitrophenol (DNP)-IgE and then stimulated with antigen for 15 min. (A) Western blotting assay of phospho-spleen tyrosine kinase (p-Syk), Syk, phospho-GRB2-associated binding protein 2 (p-Gab2), Gab2, p-p38, and p-c-Jun. (B) Expression levels of GRC-SC11. The levels were normalized to β-actin levels. The experiments were repeated three times and results are presented as the representative or mean ± SD. The probability values of # p < 0.05 and ## p < 0.01 were considered to be significant compared to the non-treated cells, and * p < 0.05 and *** p < 0.001 compared to the IgE/Ag treated cells. One-way ANOVA was used for the comparison of group means, followed by Dunnett’s t-test.

Figure 5

Effect of GRC-SC11 on the IgE/Ag-stimulated passive cutaneous anaphylaxis (PCA) BALB/c mice model. (A) Scheme of the PCA design. PCA was induced by subcutaneous injection of 0.5 μg of DNP-specific IgE into the ear of male BALB/c mice for 24 h and followed by the intravenous administration of 200 μg of DNP-bovine serum albumin (BSA) antigen having 2% Evans blue dye in the tail region. Anti-allergic activity of GRC-SC11 was assessed at the concentration of 100 mg/kg, while cetirizine was used as the standard drug (10 mg/kg). Group I: Normal control (non-treated). Group II: PCA disease control (0.5 µg IgE, anti-dinitrophenyl (DNP) IgE sensitized and challenged with 200 µg DNP-HAS + 2% Evans blue solution). Group III: PCA mice treated with GRC-SC11 (100 mg/kg). Group IV: PCA mice treated with the reference drug control (10 mg/kg). (B) Representative image of murine ear (n = 7 mice/group). (C) The mean±SD of extravasated dye (n = 7 mice/group). Differences were significant at the level of ### p < 0.001 (compared with normal animals) and *** p < 0.001 (compared with normal PCA animals). Statistical analysis of results was performed through one-way ANOVA followed by Dunnett’s t-test.

Figure 6

Effect of GRC-SC11 on IgE/Ag-induced ear swelling response in the BALB/c mice models. Histopathological examination of ear tissues of the IgE/Ag-triggered PCA BALB/c mice models. (A) Representative images of ear tissues stained with hematoxylin and eosin (n = 7/group; magnifications, 100× and 200×) of group-I: normal control, group-II: PCA disease control (IgE/Ag, anti-DNP IgE sensitized and DNP-HAS challenged), group-III: GRC-SC11 (100 mg/kg), and group-IV: drug control (10 mg/Kg). (B) A dial thickness gauge was used to measure ear thickness. Data are expressed as mean ± SD (n = 7/group). (C) Infiltrated immune cells were counted in the ear of mice. Data are expressed as mean ± SD (n = 7/group). Results were considered significant at the level of ## p < 0.01 (compared with normal animals) and ** p < 0.01 (compared with normal PCA model control). Statistical analysis of results was performed through one-way ANOVA followed by Dunnett’s t-test.