Korean J Physiol Pharmacol.  2018 Nov;22(6):671-677. 10.4196/kjpp.2018.22.6.671.

Tussilagone suppressed the production and gene expression of MUC5AC mucin via regulating nuclear factor-kappa B signaling pathway in airway epithelial cells

• Choi BS
• Kim YJ
• Yoon YP
• Lee HJ
• Lee CJ

• KMID: 2430089
• DOI: http://doi.org/10.4196/kjpp.2018.22.6.671

Abstract

In the present study, we investigated whether tussilagone, a natural product derived from Tussilago farfara, significantly affects the production and gene expression of airway MUC5AC mucin. Confluent NCI-H292 cells were pretreated with tussilagone for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. To elucidate the action mechanism of tussilagone, effect of tussilagone on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. Tussilagone significantly inhibited the production of MUC5AC mucin protein and down-regulated the expression of MUC5AC mucin gene, induced by EGF or PMA. Tussilagone inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Ba (IκBα). Tussilagone inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. These results suggest that tussilagone can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Keyword

Airway; Epithelium; Gene expression; Mucin; Tussilagone

MeSH Terms

Blotting, Western
Down-Regulation
Enzyme-Linked Immunosorbent Assay
Epidermal Growth Factor
Epithelial Cells*
Epithelium
Gene Expression*
Mucins*
NF-kappa B
Phosphorylation
Phosphotransferases
Tussilago
Epidermal Growth Factor
Mucins
NF-kappa B
Phosphotransferases

Figure

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  Fig. 1 Chemical structure of tussilagone.

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  Fig. 2 Effect of tussilagone on EGF-induced MUC5AC mucin production and gene expression from NCI-H292 cells. NCI-H292 cells were pretreated with varying concentrations of tussilagone for 30 min and then stimulated with EGF (25 ng/ml) for 24 h. Cell lysates were collected for measurement of MUC5AC mucin production by ELISA. Each bar represents a mean±S.E.M. of 3 culture wells in comparison with that of control set at 100% (B). MUC5AC gene expression was measured by RT-PCR (A). Three independent experiments were performed and the representative data were shown. *significantly different from control (p<0.05). †significantly different from EGF alone (p<0.05). cont: control, T: tussilagone, concentration unit is µM.

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  Fig. 3 Effect of tussilagone on PMA-induced MUC5AC mucin production and gene expression from NCI-H292 cells. NCI-H292 cells were pretreated with varying concentrations of tussilagone for 30 min and then stimulated with PMA (10 ng/ml) for 24 h. Cell lysates were collected for measurement of MUC5AC mucin production by ELISA. Each bar represents a mean±S.E.M. of 3 culture wells in comparison with that of control set at 100% (B). MUC5AC gene expression was measured by RT-PCR (A). Three independent experiments were performed and the representative data were shown. *significantly different from control (p<0.05). †significantly different from PMA alone (p<0.05). cont: control, T: tussilagone, concentration unit is µM.

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  Fig. 4 Effect of tussilagone on PMA-induced phosphorylation and translocation of NF-kB p65 in NCI-H292 cells. NCI-H292 cells were incubated with varying concentrations of tussilagone for 24 h and treated with 50 ng/ml PMA for 30 min. Nuclear protein extracts were prepared and subjected to western blot analysis using antibodies against p65 and phospho-p65. The result shown is a representative of three independent experiments. As a loading control, p84 levels were analyzed. cont: control, T: tussilagone, concentration unit is µM.

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  Fig. 5 Effect of tussilagone on PMA-induced phosphorylation of IκBα, degradation of IκBα and phosphorylation of IKKα/β in NCI-H292 cells. NCI-H292 cells were incubated with varying concentrations of tussilagone for 24 h and treated with 50 ng/ml PMA for 30 min. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody or antibody against anti-IκBα. Whole cell lysates (100 µg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody. The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. cont: control, T: tussilagone, concentration unit is µM.

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