Apigenin Inhibits IL-31 Cytokine in Human Mast Cell and Mouse Skin Tissues

Abstract

IL-31 is a recently discovered cytokine that is produced not only in T-cells but also in mast cells. It is strongly implicated to play a key role in inflammatory diseases and in the pathogenesis of itch in atopic dermatitis. Apigenin, a flavonoid of plant origin has numerous biological applications. In this study, we showed that apigenin modulates IL-31 mRNA, protein expression, and release in stimulated human mast (HMC-1) by inhibiting the phosphorylation activation of MAPK and NF-κB. To determine whether apigenin has similar effects in vivo, using Compound 48/80, we developed an atopic dermatitis itch model in mice and found an increase in IL-31 expression in the skin. We also revealed that apigenin prevents the infiltration and degranulation of mast cells and suppressed mRNA and protein expression of IL-31 in the skin of mice. These results provide a new suggestion of the potential applicability of apigenin for treatment of various inflammatory diseases and itch mediated by IL-31.

Keywords: IL-31; MAPK; NF-κB; apigenin; atopic dermatitis; mast cells.

Figure 1

Effects of apigenin on the human mast cells (HMC-1) viability and IL-31 production by HMC-1. Cells were pre-treated with apigenin at indicated concentrations for 24 h. Cell viability was determined by EZ-Cytox assay (A). Cells were pre-treated with apigenin at indicated concentrations for 1 h prior to PI stimulation for 12 h (B), 6 h (C), and 3 h (D). ELISA was used to measure IL-31 released in the cell culture media (B). Western blot analysis was used to determine the IL-31 protein expression levels in the cells and the bands were quantitated using Image J analysis software tool (C). Reverse-transcription (RT)-PCR was performed to determine the level of expression of IL-31 mRNA in the cells (D). The values presented are the means ± SD of three independent experiments. Bars with different small case letters indicated statistically significant difference at p < 0.05. PI, phorbol-12-myristate 13-acetate (50 nM) and ionomycin (1 μM).

Figure 2

Effects of apigenin on MAPK cascade signaling pathways in HMC-1 cells. Cells were pre-treated with apigenin at indicated concentrations for 1 h prior to PI stimulation for 30 min. Western blot analysis was performed to evaluate the expressions of molecules of the MAPK cascade signaling pathways in the cells. The bands were quantitated using Image J analysis software tool. The data presented are the means ± SD of three independent experiments. Bars with different small case letters indicated statistically significant difference at p < 0.05. PI, phorbol-12-myristate 13-acetate (50 nM) and ionomycin (1 μM).

Figure 3

Effects of apigenin on the NF-κB signaling pathway in HMC-1 cells. Cells were pre-treated with apigenin at indicated concentrations for 1 h prior to PI stimulation for 30 min. Western blot analysis was performed to evaluate the expressions of molecules of the NF-κB signaling pathway in the cells. The bands were quantitated using Image J analysis software tool. The data presented are the means ± SD of three independent experiments. Bars with different small case letters indicated statistically significant difference at p < 0.05. PI, phorbol-12-myristate 13-acetate (50 nM) and ionomycin (1 μM).

Figure 4

Effects of apigenin on compound 48/80 itch in mice. Five mice each were placed into four groups: Compound 48/80 negative control; compound 48/80 positive control; compound 48/80 with apigenin 75 mg/kg treatment; and compound 48/80 with apigenin 150 mg/kg treatment. Compound 48/80 (50 μg/site, 0.1 mL each) was injected into the dorsal dermis of the mice neck 60 min after administration of 2% geletin or apigenin at indicated concentrations. The number of scratches were monitored with micro-cameras for 30 min and counted in a double-blinded manner. Data are presented as the mean ± SEM of five mice per group. Bars with different small case letters indicated statistically significant difference at p < 0.05.

Figure 5

Effects of apigenin on compound 48/80-induced infiltration of mast cell and IL-31 expression in mice skin. Five mice each were placed into four groups: Compound 48/80 negative control; compound 48/80 positive control; compound 48/80 with apigenin 75 mg/kg treatment; and compound 48/80 with apigenin 150 mg/kg treatment. Compound 48/80 (50 μg/site, 0.1 mL each) was injected in the dorsal dermis of the mice neck 60 min after administration of 2% gelatin or apigenin at indicated concentrations. Skin tissue sections were stained with hematoxylin/eosin stains showing infiltration of polymorphonuclear leukocytes (A); toluidine blue showing mast cell infiltration into the skin (B); immunohistochemistry was done to demonstrate tryptase (C) and IL-31 (D) in the mice skin.

Figure 6

Western blot and RT-PCR analysis showing the effects of apigenin on compound 48/80-induced tryptase and IL-31 expression in the mice skin. Five mice each were placed into four groups: Compound 48/80 negative control; compound 48/80 positive control; compound 48/80 with apigenin 75 mg/kg treatment; and compound 48/80 with apigenin 150 mg/kg treatment. Compound 48/80 (50 μg/site, 0.1 mL each) was injected in the dorsal dermis of the mice neck 60 min after administration of 2% gelatin or apigenin at indicated concentrations. (A) Western blot analysis of tryptase and IL-31 protein expression. The bands were quantitated using Image J analysis software tool (B,C). (D) RT-PCR showing the expression of IL-31 mRNA in the mice skin. The data presented are the means ± SD of five mice per group. Bars with different small case letters indicated statistically significant difference at p < 0.05.