Tuberc Respir Dis.  2013 Nov;75(5):205-209.

Effect of Prunetin on TNF-alpha-Induced MUC5AC Mucin Gene Expression, Production, Degradation of IkappaB and Translocation of NF-kappaB p65 in Human Airway Epithelial Cells

• Ryu J
• Lee HJ
• Park SH
• Sikder MA
• Kim JO
• Hong JH
• Seok JH
• Lee CJ

Abstract

BACKGROUND
We investigated whether prunetin significantly affects tumor necrosis factor-alpha (TNF-alpha)-induced MUC5AC mucin gene expression, production, inhibitory kappa B (IkappaB) degradation and nuclear factor kappa B (NF-kappaB) p65 translocation in human airway epithelial cells.
METHODS
Confluent NCI-H292 cells were pretreated with prunetin for 30 minutes and then stimulated with TNF-alpha for 24 hours or the indicated periods. MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The effect of prunetin on TNF-alpha-induced degradation of IkappaB and translocation of NF-kappaB p65 was investigated by western blot analysis.
RESULTS
We found that incubation of NCI-H292 cells with prunetin significantly inhibited mucin production and down-regulated the MUC5AC gene expression induced by TNF-alpha. Prunetin inhibited TNF-alpha-induced degradation of IkappaB and translocation of NF-kappaB p65.
CONCLUSION
This result suggests that prunetin inhibits the NF-kappaB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production regulated by the NF-kappaB signaling pathway.

Keyword

Mucins; MUC5AC Protein, Human; Prunetin

MeSH Terms

Blotting, Western
Enzyme-Linked Immunosorbent Assay
Epithelial Cells*
Gene Expression*
Humans*
Isoflavones
Mucin 5AC
Mucins*
NF-kappa B*
Polymerase Chain Reaction
Reverse Transcription
Tumor Necrosis Factor-alpha
Isoflavones
Mucin 5AC
Mucins
NF-kappa B
Tumor Necrosis Factor-alpha

Figure

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  Figure 1 Effect of prunetin on tumor necrosis factor-α (TNF-α)-induced MUC5AC mucin production. NCI-H292 cells were pretreated with various concentrations of prunetin (1, 10, 50, and 100 µM) for 30 minutes and then stimulated with TNF-α (10 ng/mL) for 24 hours. Cell lysates were collected for measurement of MUC5AC mucin production by enzyme-linked immunosorbent assay. Three independent experiments were performed and the representative data were shown. Each bar represents the mean±SEM of three culture wells in comparison with that of the control, set at 100%. *Significantly different from control (p<0.05). †Significantly different from TNF-α alone (p<0.05). Concentration unit is µM. Pru: prunetin.

• 

  Figure 2 Effect of prunetin on tumor necrosis factor-α (TNF-α)-induced MUC5AC mucin gene expression. NCI-H292 cells were pretreated with 1, 10, 50, and 100 µM of prunetin for 30 minutes and then stimulated with TNF-α (10 ng/mL) for 24 hours, respectively. MUC5AC mucin gene expression was measured by reverse transcription polymerase chain reaction. As a quantitative control, Rig/S15 rRNA, which encodes a small ribosomal subunit protein, a housekeeping gene that was constitutively expressed, was used. Three independent experiments were performed and the representative data were shown. Concentration unit is µM. Pru: prunetin.

• 

  Figure 3 Effect of prunetin on tumor necrosis factor-α (TNF-α)-induced degradation of inhibitory kappa B alpha (IκBα). NCI-H292 cells were incubated with 50 µM prunetin for 24 hours and treated with TNF-α (50 ng/mL) for the indicated periods. Cytoplasmic extracts were prepared and analyzed by western blot using the antibody against anti-IκBα. The results shown are representative of three independent experiments. Equal protein loading was evaluated by β-actin levels.

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  Figure 4 Effect of prunetin on tumor necrosis factor-α (TNF-α)-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65. NCI-H292 cells were either untreated or pretreated with 50 µM prunetin for 24 hours at 37℃ and then stimulated with TNF-α (50 ng/mL) for the indicated periods. Nuclear protein extracts were prepared and resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and probed with an antibody against p65. The results shown are the representative of three independent experiments. To ensure equal protein loading, the membrane was re-probed with the anti-poly(ADP-ribose) polymerase (PARP) antibody.

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