Clin Exp Otorhinolaryngol.  2021 Aug;14(3):303-311. 10.21053/ceo.2020.01907.

Changes in Mucin Production in Human Airway Epithelial Cells After Exposure to Electronic Cigarette Vapor With or Without Nicotine

• Song SY
• Na HG
• Kwak SY
• Choi YS
• Bae CH
• Kim YD

• KMID: 2519185
• DOI: http://doi.org/10.21053/ceo.2020.01907

Abstract

Objectives
. The emergence of electronic cigarettes (e-cigarettes) has created new perceptions of the tobacco market. Unlike traditional tobacco, the greatest advantage of e-cigarettes is that they have less smell and are convenient and inexpensive. Most e-cigarette smokers believe that e-cigarette smoking is less harmful than traditional smoking. Information on the effects of e-cigarettes on human health is limited, and the issue remains controversial.
Methods
. We studied the effects of e-cigarette vapor on mucin (MUC5AC and MUC5B) and the change of MUC5AC and MUC5B from e-cigarette liquid with or without nicotine in respiratory epithelial cells. The effects of e-cigarette vapor with or without nicotine on mucin, along with the involved signaling pathways, were investigated using reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, enzyme immunoassays, and immunoblot analysis with several specific inhibitors and small interfering RNA.
Results
. E-cigarette vapor with or without nicotine stimulated MUC5AC, but not MUC5B, expression in respiratory epithelial cells. In addition, we showed that e-cigarette vapor with and without nicotine induced MUC5AC expression via activation of the mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase [ERK] 1/2 and p38) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways in human airway epithelial cells.
Conclusion
. E-cigarette vapor with and with nicotine significantly increased MUC5AC expression in human airway epithelial cells.

Keyword

E-Cigarette Vapor; MUC5AC; Mitogen-Activated Protein Kinase; NF-Kappa B; Epithelial Cell

Figure

• 

  Fig. 1. Effects of e-cigarette vapor (with or without nicotine) on NCI-H292 cells and human nasal epithelial cells. (A) Result of cytotoxicity testing of NCI-H292 cells with water-soluble tetrazolium salt-1 (WST-1) treatment, showing that e-cigarette vapor did not display visible cell killing activity with up to 2 mL of e-liquid vapor when incubated for 24 hours. (B) Result of cytotoxicity testing of human nasal epithelial cells with WST-1 treatment, showing that e-cigarette vapor did not display visible cell killing activity with up to 2 mL of e-liquid vapor when incubated for 24 hours. e-cigarette, electronic cigarettes.

• 

  Fig. 2. Effects of e-cigarette vapor (with or without nicotine) on airway mucin expression in NCI-H292 cells and human nasal epithelial cells. (A) Real-time polymerase chain reaction (RT-PCR) results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) meaningfully stimulated MUC5AC, but not MUC5B, mRNA expression in NCI-H292 cells. (B) Enzyme-linked immunosorbent assay (ELISA) results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) significantly stimulated MUC5AC, but not MUC5B, protein levels in NCI-H292 cells. (C) RT-PCR results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) meaningfully stimulated MUC5AC, but not MUC5B, mRNA expression in human nasal epithelial cells. (D) ELISA results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) significantly increased MUC5AC, but not MUC5B, protein levels in human nasal epithelial cells. Bars represent mean±standard deviation of three independent experiments performed in triplicate. PG, propylene glycol; VG, vegetable glycerin. a)P<0.05 vs. baseline.

• 

  Fig. 3. MUC5AC overexpression by e-cigarette vapor (with or without nicotine) in human airway NCI-H292 epithelial cells involves ERK1/2, p38, and NF-κB stimulation. (A, B) Western blot analysis showing that e-cigarette vapor with or without nicotine induced phosphorylation of ERK1/2, p38, and NF-κB. (C, D) Enzyme-linked immunosorbent assay (ELISA) results showing that U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), and BAY 11-7085 (an NF-κB inhibitor) treatment significantly attenuated the effect of e-cigarette vapor on MUC5AC protein levels. (E, F) ELISA results showing that knockdown of ERK1, ERK2, p38, and NF-κB by siRNA significantly blocked the effect of e-cigarette vapor on MUC5AC protein levels. Images are illustrative of three separate experiments performed in triplicate. Bars show mean±standard deviation of three independent experiments performed in triplicate. e-cigarette, electronic cigarettes; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; p-p38, phosphorylated p38; p-p65, phosphorylated p65; Con, control; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; siRNA, small interfering RNA. a)P<0.05 vs. baseline. b)P<0.05 vs. e-cigarette vapor alone.

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  Fig. 4. E-cigarette vapor (with or without nicotine)-induced MUC5AC expression in human nasal epithelial cells is associated with ERK1/2, p38, and NF-κB activation. (A) Immunofluorescence results showing that e-cigarette vapor without nicotine significantly induced MUC5AC expression, and this was significantly inhibited by pretreatment with U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or BAY 11-7085 (an NFκB inhibitor). E-cigarette vapor without nicotine also significantly increased MUC5AC expression in the cytoplasm. Each bar shows the mean± standard deviation of three independent experiments. (B) Immunofluorescence results showing that e-cigarette vapor with nicotine significantly increased MUC5AC expression, and this was significantly inhibited by pre-treatment with U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or BAY 11-7085 (an NF-κB inhibitor). Furthermore, e-cigarette vapor with nicotine significantly increased MUC5AC expression in the cytoplasm. Con, control; e-cigarette, electronic cigarettes; DAPI, 4ʹ,6-diamidino-2-phenylindole; ERK, extracellular signal-regulated kinase; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells.

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