No abstract

Naringenin (Nar) is a flavonoid derived from plant foods. It has been shown to have anti-inflammatory properties. Many studies have shown that overexpression of reactive oxygen species (ROS) and nuclear factor-κB (NF-κB) leads to increased mucin (MUC) 5AC expression in chronic inflammation of the airway. In addition, some studies have reported that naringenin inhibits NF-κB activity in a murine model of asthma. We speculated that naringenin might be associated with mucous hypersecretion, but the molecular mechanisms remain to be defined. Our study has also investigated whether naringenin could inhibit production of ROS and the activity of NF-κB on the inflammatory pulmonary diseases induced by human neutrophil elastase (HNE) and reduce the mRNA and protein levels of MUC5AC as shown by reverse transcriptase-polymerase chain reaction and real-time PCR (RT-PCR). Serum total MUC5AC protein was detected by enzyme-linked immunosorbent assay (ELISA), the protein morphological changes of MUC5AC were also observed by immunofluorescence and confocal laser technology. Hyperactivation of epidermal growth factor receptor (EGFR) signaling is commonly involved in the mucous hypersecretion process and initiates both the activation of extracellular signal-related kinases 1/2 (ERK1/2) and of phosphatidylinositol 3-kinase (PI3K) and Akt kinase. NF-κB is a key factor downstream of PI3K/Akt signaling, which induces overexpression of the MUC5AC gene. Our data revealed that naringenin inhibited the activation of EGFR resulting in the downregulation of the enzyme activities. Naringenin also reduced the protein expressions of p-EGFR, PI3K, p-Akt, p-ERK1/2, and NF-κB as shown by western blotting. Furthermore, naringenin significantly inhibited PI3K/Akt and ERK MAPKinase signaling with a concurrent reduction in production of ROS and NF-κB activities. These results suggest that naringenin may play a protective role by minimizing mucous production during airway inflammation by down-regulating ROS production and inhibiting the NF-κB activity via EGFR-PI3K-Akt/ERK MAPKinase signaling pathway.

Mucus hypersecretion is commonly observed in many chronic airway infl ammatory diseases. Mucin 5AC (MUC5AC) is a major airway mucin because of its high expression in goblet cells. Here, the authors identifi ed a gene called SAM domain -containing prostate-derived Ets factor (SPDEF) that was induced by interleukin (IL)-13. Their results showed that specifi c knockdown of SPDEF reduced IL-13-induced MUC5AC expression in human airway epithelial cells. This fi nding was associated with decreased expression of anterior gradient 2 (AGR2) and Ca 2 ϩ -activated Cl Ϫ channel (CLCA1), which regulate IL-13-mediated MUC5AC overproduction. Furthermore, transfection with SPDEF siRNA enhanced expression of forkhead box a2 (Foxa2), a key transcription factor that is known to prevent mucus production. The authors also demonstrated that the repression of STAT6 inhibited expression of SPDEF and MUC5AC induced by IL-13. These results show that SPDEF plays a critical role in regulating a transcriptional network mediating IL-13-induced MUC5AC synthesis dependent on STAT6.

Aims: To explore the signaling mechanism associated with the inhibitory effect of nicotine on tumor necrosis factor (TNF)- α expression in human airway epithelial cells. Methods: HBE16 airway epithelial cells were cultured and incubated with either nicotine or cigarette smoke extract (CE). Cells were then transfected with α1, α5, or α7 nicotinic acetylcholine receptor (nAChR)-specific small interfering RNAs (siRNAs). The effects of nicotine on the production of proinflammatory factors TNF-α, in transfected cells were analyzed. Furthermore, we assayed the expression levels of myeloid differentiation primary response gene 88 (MyD88) protein, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) p65 protein, NF-ĸB activity and NF-ĸB inhibitor alpha (I-ĸBα) expression in cells after treatment with nicotine or α7 nAChR inhibitor, α -bungarotoxin (α-BTX). Results: The production of TNF-α was lower in cells pretreated with nicotine before lipopolysaccharide (LPS) stimulation, compared with LPS-only-treated cells. In contrast, in α7 siRNA-transfected cells incubated with nicotine and LPS, TNF-α expression was higher than that in non-transfected cells or in α1 or α5 siRNA-transfected cells. Addition of MyD88 siRNA or the NF-ĸB inhibitor pyridine-2,6-dithiocarboxylic acid (PDTC) also reduced TNF-α expression. Furthermore, we found that nicotine decreased MyD88 protein, NF-ĸB p65 protein, NF-ĸB activity and phospho-I-ĸBα expression induced by CE or LPS. The inhibitor α-BTX could reverse these effects. Conclusion: Nicotine reduces TNF-α expression in HBE16 airway epithelial cells, mainly through an α7 nAChR/MyD88/NF-ĸB pathway.

Cigarette smoking is strongly implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Mucus hypersecretion is the key manifestation in patients with COPD and mucin 5AC (MUC5AC) is a major component of airway mucus. Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor which can be stimulated to bind to the MUC5AC promoter and induce MUC5AC promoter activation. Previous studies have reported that activation of HIF-1α pathways by cigarette smoke contributes to the development of COPD. We hypothesize that cigarette smoke up-regulates HIF-1α production and HIF-1 activity through epidermal growth factor receptor (EGFR)-activated signal cascades pathways, leading to mucin production in human airway epithelial cells (16HBE). We show that cigarette smoke increases HIF-1α production, HIF-1 activity and MUC5AC expression. These effects are prevented by small interfering RNA (siRNA) for HIF-1α, indicating that cigarette smoke-induced mucin production is HIF-1α-dependent. Cigarette smoke activates extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) signal pathways, both of which are inhibited by gefitinib (an inhibitor of EGFR), suggesting that cigarette smoke-activated signal pathways are mediated by EGFR in 16HBE cells. Furthermore, pretreatment with gefitinib and the pharmacological inhibitors of PI3K (LY294002) and ERK1/2 (PD98059) prevented cigarette smoke-mediated Akt and ERK1/2 phosphorylation responses, HIF-1α production, HIF-1 activity and MUC5AC expression. These observations demonstrate an important role for EGFR-mediated signaling pathways in regulating cigarette smoke-induced HIF-1 activation and MUC5AC expression. Our results suggest that cigarette smoke activates EGFR-mediated signaling pathways, leading to HIF-1α production and HIF-1 activation, resulting in mucin expression in human airway epithelial cells.