Triterpenoids and Polysaccharide Fractions of Ganoderma tsugae Exert Different Effects on Antiallergic Activities

Abstract

This study was to investigate antiallergic effects of triterpenoids (Gt-TRE) and polysaccharide (Gt-PS) extracts from Ganoderma tsugae, using mast cell line RBL-2H3, T cell line EL4, primary T cells, and transfected RAW264.7 macrophage cells. The results showed that histamine secreted from activated RBL-2H3 mast cells was significantly suppressed by Gt-TRE but not Gt-PS. Interleukin- (IL-) 4 secreted from activated EL4 cells was significantly suppressed by Gt-TRE but not Gt-PS. Further primary CD4(+) T cells cultures also confirmed that Gt-TRE (5 ~ 50 µg/mL) significantly suppressed Th2 cytokines IL-4 and IL-5 secretions but had no effect on Th1 cytokines IL-2 and interferon (IFN)-γ. Gt-PS did not affect IL-4 and IL-5 secretions until higher doses (400, 500 µg/mL) and significantly suppressed IFNγ secretions but enhanced IL-2 at these high doses. The reporter gene assay indicated that Gt-TRE inhibited but Gt-PS enhanced the transcriptional activity of NF-κB in activated transfected RAW264.7 cells and transfected EL4 cells. IL-4 secreted by this transfected EL-4 cells was also significantly decreased by Gt-TRE but not by Gt-PS, suggesting that these two fractions may exert different effects on NF-κB related cytokines expression. These data suggested that triterpenoids fraction of Ganoderma tsugae might be the main constituents to alleviate allergic asthma.

Figure 1

The histamine secretion from ionomycin-stimulated RBL-2H3 mast cells cultured with different doses of triterpenoids and polysaccharide extracts from G. tsugae. The RBL-2H3 mast cells (5 × 10⁴ cells/mL) were pretreated with Gt-TRE or Gt-PS extracts from G. tsugae for 24 hr, and then stimulated with 500 ng/mL ionomycin for 5 hr. The supernatants were collected and assayed by histamine ELISA kit. The data are representative for three independent experiments. ^∗ P < 0.05 as compared to the 0 μg/mL control cells.

Figure 2

The IL-4 and IL-2 productions of activated EL4 T cells cultured with different doses of triterpenoids and polysaccharide extracts from G. tsugae. EL4 murine T cells (2 × 10⁵ cells/mL) were cultured with Gt-TRE or Gt-PS extracts from G. tsugae and activated by 50 ng/mL PMA plus 1 μg/mL ionomycin for 24 hrs. The supernatants were collected and cytokine levels were determined by ELISA method. The data are representative for five independent experiments. ^∗ P < 0.05 as compared to the 0 μg/mL control cells.

Figure 3

The relative Th2 cytokines (a) and Th1 cytokines (b) productions from primary T cells cultured with different doses of triterpenoids and polysaccharide extracts from G. tsugae. Isolated CD4⁺ helper T cells (5 × 10⁵ cells/mL) from the DO11.10 transgenic mice with high percentage of OVA peptide-specific T cell receptor were cultured with different doses of Gt-TRE or Gt-PS in the presence of OVA peptide and APC for 72 hrs. The supernatants were collected and cytokine levels were determined by ELISA method. The results are expressed as the mean ± SD of four independent experiments. ^∗ P < 0.05 as compared to the 0 μg/mL control cells.

Figure 4

The NF-κB transcriptional activity of transfected RAW264.7 cells cultured with different doses of triterpenoid and polysaccharide extracts from G. tsugae. RAW264.7 macrophages (1 × 10⁵ cells/mL) transiently transfected with NF-κB-promoted luciferase reporter plasmid were treated with Gt-TRE or Gt-PS and stimulated with LPS (100 ng/mL) plus IFNγ (1000 units/mL) for 8 hr. Mocks are the RAW264.7 cells transfected with empty vector as the negative control without Gt-TRE or Gt-PS treatment. The NF-κB responsive reporter luciferase activity was assayed as described in Materials and Methods. The data were representative for five independent experiments. ^∗ P < 0.05 as compared to the 0 μg/mL control cells.

Figure 5

The NF-κB transcriptional activity and IL-4 production by activated transfected EL4 T cells cultured with different doses of triterpenoids and polysaccharide extracts from G. tsugae. EL4 T cells (2 × 10⁵ cells/mL) transiently transfected with NF-κB-promoted luciferase reporter plasmid were treated with Gt-TRE or Gt-PS and stimulated with 50 ng/mL PMA plus 1 μg/mL ionomycin for 24 hrs. Mocks are the EL4 cells transfected with empty vector as the negative control without Gt-TRE or Gt-PS treatment. The data are representative for five independent experiments. ^#0.05 < P < 0.1, ^∗ P < 0.05 as compared to the 0 μg/mL control cells.