Houttuynia cordata Thunb inhibits the production of pro-inflammatory cytokines through inhibition of the NFκB signaling pathway in HMC-1 human mast cells

Abstract

Houttuynia cordata Thunb (HCT) is widely used in oriental medicine as a remedy for inflammation. However, at present there is no explanation for the mechanism by which HCT affects the production of inflammatory cytokines. The current study aimed to determine the effect of an essence extracted from HCT on mast cell-mediated inflammatory responses. Inflammatory cytokine production induced by phorbol myristate acetate (PMA) plus a calcium ionophore, A23187, was measured in the human mast cell line, HMC-1, incubated with various concentrations of HCT. TNF-α, IL-6 and IL-8 secreted protein levels were measured using an ELISA assay. TNF-α, IL-6 and IL-8 mRNA levels were measured using RT-PCR analysis. Nuclear and cytoplasmic proteins were examined by western blot analysis. The NF-κB promoter activity was examined by luciferase assay. It was observed that HCT inhibited PMA plus A23187-induced TNF-α and IL-6 secretion and reduced the mRNA levels of TNF-α, IL-6 and IL-8. It was also noted that HCT suppressed the induction of NF-κB activity, inhibited nuclear translocation of NF-κB and blocked the phosphorylation of IκBα in stimulated HMC-1 cells. It was concluded that HCT is an inhibitor of NF-κB and cytokines blocking mast cell-mediated inflammatory responses. These results indicate that HCT may be used for the treatment of mast cell-derived allergic inflammatory diseases.

Figure 1

Effect of HCT on cell viability in activated mast cells. HMC-1 cells (1×10⁶ cells/ml) were pretreated with the indicated concentrations of HCT (0.05 −0.4 mg/ml) for 1 h and incubated with PMA and A23187 for 24 h. Cell viability was determined by MTS assay. Data are presented as the mean ± SD of three independent experiments. C, induced control; CD, induced control with DMSO treatment; HCT, Houttuynia cordata Thunb.

Figure 2

Effect of HCT on PMA plus A23187-stimulated TNF-α expression. HMC-1 cells (1×10⁶ cells/ml) were pre-incubated with various concentrations of HCT (0.05–0.2 mg/ml) for 1 h and treated with PMA plus A23187 for 4 h. (A) TNF-α secreted protein levels in the supernatant were measured by ELISA assay. (B) TNF-α mRNA levels were measured by RT-PCR. Lanes 1, normal cells; 2, control cells; 3, DMSO control cells; 4, HCT (0.05 mg/ml) + PMA plus A23187; 5, HCT (0.1 mg/ml) + PMA plus A23187; 6, HCT (0.2 mg/ml) + PMA plus A23187. Data are presented as the mean ± SD of three independent experiments (^*P<0.05 and ^**P<0.01, vs. control). N, no treatment; C, induced control; CD, induced control with DMSO treatment; HCT, Houttuynia cordata Thunb; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription polymerase chain reaction.

Figure 3

Effect of HCT on PMA plus A23187-stimulated IL-6 expression. HMC-1 cells (1×10⁶ cells/ml) were preincubated with various concentrations of HCT (0.05–0.2 mg/ml) for 1 h and treated with PMA plus A23187 for 4 h (A and B). (A) IL-6 secreted protein levels in the supernatant were measured by ELISA assay. (B) IL-6 mRNA levels were measured by RT-PCR. Lanes 1, normal cells; 2, control cells; 3, DMSO control cells; 4, HCT (0.05 mg/ml) + PMA plus A23187; 5. HCT (0.1 mg/ml) + PMA plus A23187; 6, HCT (0.2 mg/ml) + PMA plus A23187. Data are presented as the mean ± SD of three independent experiments (^*P<0.05 and ^**P<0.01 vs. control). N, no treatment; C, induced control; CD, induced control with DMSO treatment; HCT, Houttuynia cordata Thunb; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription polymerase chain reaction.

Figure 4

Effect of HCT on PMA plus A23187-stimulated IL-8 expression. HMC-1 cells (1×10⁶ cells/ml) were preincubated with various concentrations of HCT (0.05–0.2 mg/ml) for 1 h and treated with PMA plus A23187 for 4 h (A and B). (A) IL-8 secreted protein levels in the supernatant were measured by ELISA assay. (B) IL-8 mRNA levels were measured by RT-PCR. Lanes 1, normal cells; 2, control cells; 3, DMSO control cells; 4, HCT (0.05 mg/ml) + PMA plus A23187; 5, HCT (0.1 mg/ml) + PMA plus A23187; 6, HCT (0.2 mg/ml) + PMA plus A23187. Data are presented as the mean ± SD of three independent experiments. N, no treatment; C, induced control; CD, induced control with DMSO treatment; HCT, Houttuynia cordata Thunb ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription polymerase chain reaction.

Figure 5

Effect of HCT on PMA plus A23187-stimulated NF-κB activation and IκBα phosphorylation. HMC-1 cells (1×10⁶ cells/ml) were incubated with HCT (0.05–0.2 mg/ml) for 1 h and stimulated with PMA plus A23187 for 2 h. Nuclear and cytoplasmic proteins were isolated by lysis buffer and examined for NF-κB and p-IκBα by western blot analysis. N, nuclear extract; C, cytosol extract; HCT, Houttuynia cordata Thunb.