Clin Exp Otorhinolaryngol.  2014 Sep;7(3):198-204. 10.3342/ceo.2014.7.3.198.

Delphinidin Inhibits LPS-Induced MUC8 and MUC5B Expression Through Toll-like Receptor 4-Mediated ERK1/2 and p38 MAPK in Human Airway Epithelial Cells

• Bae CH
• Jeon BS
• Choi YS
• Song SY
• Kim YD

• KMID: 1973470
• DOI: http://doi.org/10.3342/ceo.2014.7.3.198

Abstract

OBJECTIVES
Delphinidin is one of the anthocyanidins. It is believed to have anti-inflammatory property including antioxidant, antiangiogenic, and anti-cancer properties. However, the anti-inflammatory effect of delphinidin in mucin-producing human airway epithelial cells has not been determined. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of delphinidin in lipopolysaccharide (LPS)-induced MUC8 and MUC5B expression in human airway epithelial cells.
METHODS
In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay were used for investigating the expressions of MUC8, MUC5, and Toll-like receptor 4 (TLR4), after LPS treatment and delphinidin treatment. And the signaling pathway of delphinidin on LPS-induced MUC8 and MUC5B expression was investigated using the RT-PCR, and immunoblot analysis. To confirm the involvement of TLR4 in LPS-induced MUC8 and MU5B expression, the cells were transfected with TLR4 siRNA.
RESULTS
In NCI-H292 airway epithelial cells, LPS (100 ng/mL) significantly induced TLR4, MUC8, and MUC5B expression. TLR4 siRNA significantly blocked LPS-induced MUC8 and MUC5B mRNA expression. LPS (100 ng/mL) significantly activated the phosphorylation of extracellular signal related kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK). Delphinidin (50 and 100 microM) inhibited LPS-induced TLR4, MUC8, and MUC5B expression and LPS-induced phosphorylation of ERK1/2 and p38 MAPK. In the primary cultures of normal nasal epithelial cells, delphinidin (50 and 100 microM) significantly inhibited LPS-induced TLR4, MUC8, and MUC5B gene expression.
CONCLUSION
These results suggest that delphinidin attenuates LPS-induced MUC8 and MUC5B expression through the TLR4-mediated ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells. These findings indicated that delphinidin may be a therapeutic agent for control of inflammatory airway diseases.

Keyword

Delphinidin; Lipopolysaccharides; Toll-like receptor 4; Human MUC8 protein; Human MUC5B protein; Airway epithelial cell

MeSH Terms

Anthocyanins
Epithelial Cells*
Gene Expression
Humans
Immunoenzyme Techniques
Lipopolysaccharides
p38 Mitogen-Activated Protein Kinases*
Phosphorylation
Phosphotransferases
Protein Kinases
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Toll-Like Receptor 4
Toll-Like Receptors*
Anthocyanins
Lipopolysaccharides
Phosphotransferases
Protein Kinases
RNA, Messenger
RNA, Small Interfering
Toll-Like Receptor 4
Toll-Like Receptors
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 The effects of LPS on MUC8, MUC5B, and TLR4 expression in human NCI-H292 cells. (A) Results of reverse transcriptase-polymerase chain reaction (RT-PCR) showed that MUC8 and MUC5B mRNA expressions were significantly increased by treatment with all dosages of LPS. (B) Results of RT-PCR showed TLR4 mRNA expression was significantly increased by treatment with LPS in a time-dependent manner. (C) Results of RT-PCR showed that LPS-induced MUC8 and MUC5B expressions were significantly attenuated by knockdown of TLR. LPS, lipopolysaccharide; TLR, toll like receptor; Con, control (not treated); NC, negative control (treated with lipofectamine 2000 only). *P<0.05 compared with zero value of LPS. **P<0.05 compared with LPS (100 ng/mL) only.

• 

  Fig. 2 The effects of delphinidin on LPS-induced TLR4, MUC8, and MUC5B expression in NCI-H292 cells. (A) Results of reverse transcriptase-polymerase chain reaction (RT-PCR) showed that delphindin significantly attenuated LPS-induced TLR4, MUC8 and MUC5B mRNA expression. (B) Results of enzyme-linked immunosorbent assay (ELISA) showed that delphinidin significantly attenuated LPS-induced MUC8 and MUC5B protein production. LPS, lipopolysaccharide; TLR, toll like receptor. *P<0.05 compared with zero value of LPS. **P<0.05 compared with LPS (100 ng/mL) only.

• 

  Fig. 3 The effects of delphinidin on LPS-induced phosphorylation of ERK1/2 and p38 MAPK in NCI-H292 cells. (A) Results of Western blot showed that LPS significantly activated the phosphorylation of ERK1/2 and p38 MAPK. (B) Results of Western blot showed that LPS-induced phosphorylation of ERK1/2 and p38 MAPK were significantly blocked by pretreatment with delphinidin for 60 minutes before being incubated with LPS for 60 minutes. ERK1/2, extracellular signal related kinase 1/2; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide. *P<0.05 compared with zero value of LPS. **P<0.05 compared with LPS (100 ng/mL) only.

• 

  Fig. 4 The effects of delphinidin on LPS-induced TLR4, MUC8, and MUC5B in human nasal epithelial cells. (A) Results of real-time polymerase chain reaction (PCR) showed that MUC8 and MUC5B mRNA expression was significantly increased by treatment of LPS and peaked at 100 ng/mL of LPS. (B) Results of reverse transcriptase-PCR showed that LPS-induced TLR4, MUC8 and MUC5B mRNA expression was significantly attenuated by delphinidin. TLR, toll like receptor; LPS, lipopolysaccharide. *P<0.05 compared with zero value of LPS. **P<0.05 compared with LPS (100 ng/mL) only.

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