Korean J Physiol Pharmacol.  2020 Mar;24(2):157-163. 10.4196/kjpp.2020.24.2.157.

Eupatilin downregulates phorbol 12-myristate 13-acetate-induced MUC5AC expression via inhibition of p38/ERK/JNK MAPKs signal pathway in human airway epithelial cells

• Cheon YH
• Kim MS
• Kim JY
• Kim DH
• Han SY
• Lee JH

• KMID: 2471037
• DOI: http://doi.org/10.4196/kjpp.2020.24.2.157

Abstract

Chronic inflammatory airway diseases, such as chronic rhinosinusitis, chronic obstructive pulmonary disease, and asthma, are associated with excessive mucus production. Hence, the regulation of mucus production is important for the treatment of upper and lower airway diseases. Eupatilin is a pharmacologically active ingredient obtained from Artemisia asiatica Nakai (Asteraceae) and exerts potent anti-inflammatory, anti-allergic, and anti-tumor activities. In the present study, we investigated the effect of eupatilin on phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC and MUC5B expression in human airway epithelial cells. We found that eupatilin treatment significantly inhibited PMA-induced mucus secretion in PAS staining. In addition, qRT-PCR results showed that eupatilin dose-dependently decreased the mRNA expression of MUC5AC in human airway epithelial cells. Western blot and immunofluorescence assay also showed that PMA-induced protein expression of MUC5AC was inhibited by eupatilin treatment. Finally, we investigated MAPKs activity after stimulation with PMA using western blot analysis in human airway epithelial cells. The results showed that eupatilin downregulated the levels of phosphorylated p38, ERK, and JNK. In summary, the anti-inflammatory activities of eupatilin, characterized as the suppression of MUC5AC expression and secretion in human airway epithelial cells, were found to be associated with the inhibition of p38/ERK/JNK MAPKs signaling pathway of MUC5AC secretion.

Keyword

Eupatilin; MAPKs; MUC5AC; Periodic acid Schiff's staining; Phorbol 12-myristate 13-acetate

MeSH Terms

Artemisia
Asthma
Blotting, Western
Epithelial Cells*
Fluorescent Antibody Technique
Humans*
Mucus
Pulmonary Disease, Chronic Obstructive
RNA, Messenger
Signal Transduction*
RNA, Messenger

Figure

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  Fig. 1 The structure and effect of eupatilin on NCI-H292 cell viability. (A) The chemical structure of eupatilin. (B) NCI-H292 cells were treated with indicated doses of eupatilin or vehicle (DMSO, control). Each well was treated with cell viability assay solution for 4 h and the viability of cells was determined with absorbance at 450 nm. ns, not significant.

• 

  Fig. 2 Effects of eupatilin on the phorbol 12-myristate 13-acetate (PMA)-induced mRNA expression levels of MUC5AC and MUC5B in NCI-H292 cells. (A, B) NCI-H292 cells were stimulated with indicated doses of PMA or vehicle (DMSO, control) for 8 h. The mRNA expression levels of PMA-stimulated MUC5AC (A) and MUC5B (B) was determined via real-time RT-PCR. (C, D) NCI-H292 cells were pre-treated with varying concentrations of eupatilin for 1 h, and then, stimulated with 20 nM PMA for 8 h. MUC5AC (C) and MUC5B (D) gene expressions were determined by real-time RT-PCR. ns, not significant compared with PMA alone; *p < 0.05, **p < 0.01 compared with control; ##p < 0.01 compared with PMA alone.

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  Fig. 3 Effects of eupatilin on the phorbol 12-myristate 13-acetate (PMA)-induced protein expression level of MUC5AC in NCI-H292 cells. (A) Serum-starved NCI-H292 cells were pre-treated with or without eupatilin for 1 h, and then, stimulated with PMA for indicated time periods. The protein expression level of MUC5AC was determined by western blot analysis. (B) MUC5AC protein expression in PMA-induced NCI-H292 cells stimulated with or without eupatilin was visualized by immunofluorescence staining. Cells were stained with anti-MUC5AC antibody (Alex 488, green) and DAPI (nuclear stain, blue). Cells were stained with anti-MUC5AC antibody (Alex 488, green) and DAPI (nuclear stain, blue) and were visualized with a 40× objective.

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  Fig. 4 Effects of eupatilin on the phorbol 12-myristate 13-acetate (PMA)-induced MAPK signal in NCI-H292 cells. Serum-starved NCI-H292 cells were pre-treated with or without eupatilin for 1 h, and then, stimulated with PMA for indicated time periods. The protein levels of ERK, p-38, and JNK were analyzed by western blotting.

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  Fig. 5 Effect of eupatilin on the phorbol 12-myristate 13-acetate (PMA)-induced the production of mucus. Serum-starved NCI-H292 cells were pre-treated with or without eupatilin and stimulated with PMA. The cells were stained by PAS and incorporated with hematoxylin counterstaining the nuclei. The images were acquired with a 10× objective. EUP, eupatilin.

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