Immunostimulatory Activity of Cordyceps militaris Fermented with Pediococcus pentosaceus SC11 Isolated from a Salted Small Octopus in Cyclophosphamide-Induced Immunocompromised Mice and Its Inhibitory Activity against SARS-CoV 3CL Protease

Abstract

In this study, we investigated the immune-enhancing and anti-viral effects of germinated Rhynchosia nulubilis (GRC) fermented with Pediococcus pentosaceus SC11 (GRC-SC11) isolated from a salted small octopus. The cordycepin, β-glucan, and total flavonoid contents increased in GRC after SC11 fermentation. GRC-SC11 inhibits 3CL protease activity in severe acute respiratory syndrome-associated coronavirus (SARS-CoV). GRC-SC11 significantly increased thymus and spleen indices in immunocompromised mice. The rate of splenocyte proliferation was higher in GRC-SC11-treated immunocompromised mice than that in GRC-treated immunocompromised mice in the presence or absence of concanavalin A. In addition, GRC-SC11 increased the phagocytic activity and nitric oxide production in immunocompromised mice. The mRNA expression of interferon-gamma (IFN-γ), interferon-alpha (IFN-α), and interferon-stimulated gene 15 (ISG15) was up-regulated in GRC-SC11 treated RAW 264.7 macrophages, compared to GRC. Our study indicates that GRC-SC11 might be a potential therapeutic agent for immunocompromised patients who are vulnerable to SARS-CoV infection.

Keywords: Cordyceps militaris grown on germinated Rhychosia nulubilis fermented with Pediococcus pentosaceus SC11 (GRC-SC11); SARS-CoV 3CL protease; cyclophosphamide; immune stimulatory; macrophage; phagocytic activity; splenocyte proliferation.

Figure 1

Effect of GRC fermented with different lactic acid bacteria stains on nitric oxide (NO) production (A) RAW264.7 cells were treated with GRC-SC11, GRC-SC64, GRC-Bro17, GRC-ON188, GRC-S. pum8, GRC. Nitrite levels in the culture media were determined using Griess reagent and were presumed to reflect NO levels. (B) RAW264.7 cells were treated with various concentrations (0.25%, 0.50%, and 1.00%) of GRC-SC11, GRC-ON188, GRC-Bro17, GRC-S. pum8, GRC-SC64, GRC for 24 h. RAW264.7 cell viability was assessed using CCK-8 assay. One-way analysis of variance was used for the comparison of group means, followed by Dunnett’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (con)).

Figure 2

Inhibition of 3CL protease activity of GRC-SC11 and GRC. The results are shown as the mean ± SD (n ≥ 8 animals in each group). GC376 is a 3CL inhibitor used as a positive control. Significance of the results was determined using one-way analysis of variance followed by Dunnett’s t-test (*** p < 0.001 and ** p < 0.01 vs. con).

Figure 3

Effects of GRC-SC11 on immune organs in CY-treated immunocompromised mice. Both GRC and GRC-SC11 were orally administered for 20 days. Distilled water was provided to control group. (A) The thymic and splenic indices of normal and CY-treated immunocompromised mice. (B) The body weights of normal and CY-treated immunocompromised mice. The results are shown as the mean ± SD (n ≥ 8 animals in each group). Significance of the results was determined using one-way analysis of variance followed by Dunnett’s t-test (# p < 0.05 vs. normal control, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CY-control).

Figure 4

The primary proliferation of splenocyte isolated from CY-treated immunocompromised mice. Con A is concanavalin A as a T-cell mitogen. The results are shown as the mean ± SD (n ≥ 8 animals in each group). Data comparisons among groups were analyzed using one-way analysis of variance, followed by Dunnett’s t-test (** p < 0.01 vs. con).

Figure 5

Effects of GRC-SC11 on the phagocytic activity of peritoneal macrophages isolated from CY-treated immunocompromised mice. (A) Immunofluorescence images (left panel) of primary peritoneal macrophages from CY-treated immunocompromised mice orally administrated with distilled water, 1% GRC-SC11, and 1% GRC. Quantification of fluorescent beads up-taken by primary peritoneal macrophages from CY-treated immunocompromised mice (exposure for 48 h) (right panel). (B) Nitric oxide (NO) production of primary peritoneal macrophages isolated from CY-treated immunocompromised mice. Nitrite levels in the culture media were determined using Griess reagent and were presumed to reflect NO levels. (C) Cell proliferation of primary peritoneal macrophages was measured using a CCK-8 assay. (D) The levels of INF-γ, IFN-α, and ISG15 mRNA expression in RAW 264.7 cells. One-way analysis of variance was used to compare group means, followed by Dunnett’s t-test for the significance of individual comparisons (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control group). Each figure is representative of three independent experiments: Scale bars = 50 µm.