Potential Suppressive Effect of Nicotine on the Inflammatory Response in Oral Epithelial Cells: An In Vitro Study

Abstract

Smoking is a well-recognized risk factor for oral mucosal and periodontal diseases. Nicotine is an important component of cigarette smoke. This study aims to investigate the impact of nicotine on the viability and inflammatory mediator production of an oral epithelial cell line in the presence of various inflammatory stimuli. Oral epithelial HSC-2 cells were challenged with nicotine (10^-8-10^-2 M) for 24 h in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS, 1 µg/mL) or tumor necrosis factor (TNF)-α (10^-7 M) for 24 h. The cell proliferation/viability was determined by MTT assay. Gene expression of interleukin (IL)-8, intercellular adhesion molecule (ICAM)-1, and β-defensin was assayed by qPCR. The production of IL-8 protein and cell surface expression of ICAM-1 was assessed by ELISA and flow cytometry, respectively. Proliferation/viability of HSC-2 cells was unaffected by nicotine at concentrations up to 10^-3 M and inhibited at 10^-2 M. Nicotine had no significant effect on the basal expression of IL-8, ICAM-1, and β-defensin. At the same time, it significantly diminished P. gingivalis LPS or the TNF-α-induced expression levels of these factors. Within the limitations of this study, the first evidence was provided in vitro that nicotine probably exerts a suppressive effect on the production of inflammatory mediators and antimicrobial peptides in human oral epithelial cells.

Keywords: epithelial cell; inflammation; lipopolysaccharide; nicotine.

Figure 1

Effect of nicotine on the proliferation/viability of oral epithelial cells. HSC-2 cells were stimulated with different concentrations of nicotine in the presence or in the absence of P. gingivalis LPS (Pg LPS, 1 µg/mL) or tumor necrosis factor (TNF)-α (10⁻⁷ M) for 24 h, and the proliferation/viability was measured using MTT assay. The y-axis shows optical densities (OD) values measured at 570 nm in five independent experiments. *—significantly different compared to the control.

Figure 2

Effect of nicotine on the expression of IL-8 in oral epithelial cells. HSC-2 cells were stimulated with different concentrations of nicotine in the presence or in the absence of P. gingivalis LPS (1 µg/mL) or TNF-α (10⁻⁷ M) for 24 h, and the gene expression (A) and the protein content of IL-8 (B) in conditioned media were determined by qPCR and ELISA, respectively. Changes in the gene expression levels were calculated using the 2^−ΔΔCT method taking GAPDH as an endogenous control gene and non-stimulated cells as a control. The content of IL-8 in the conditioned media after stimulation (B) was measured by commercially available ELISA. Each value represents the mean ± S.E.M. of four independent assays. *—significantly different compared to the control. #—significantly different compared to the group without nicotine.

Figure 3

Effect of P. gingivalis LPS on the ICAM-1 by oral epithelial cells. HSC-2 cells were stimulated with different concentrations of nicotine in the presence or in the absence of P. gingivalis LPS (1 µg/mL) or TNF-α (10⁻⁷ M) for 24 h, and the gene expression of ICAM-1 (A) and the protein content in conditioned media (B) were determined by qPCR and flow cytometry, respectively. Changes in the gene expression levels were calculated using the 2^−ΔΔCT method taking GAPDH as an endogenous control gene and non-stimulated cells as a control. The amount of ICAM-1 on the cell surface after stimulation (B) was measured by flow cytometry based on mean fluorescence intensity (MFI) of ICAM-1 stained cells. Each value represents the mean ± S.E.M. of four independent assays. *—significantly different compared to the control. #—significantly different compared to the group without nicotine.

Figure 4

Effect of nicotine on the expression of β-defensin in oral epithelial cells. HSC-2 cells were stimulated with different concentrations of nicotine in the presence or in the absence of P. gingivalis LPS (1 µg/mL) or TNF-α (10⁻⁷ M) for 24 h. Changes in the gene expression levels were calculated using the 2^−ΔΔCT method taking GAPDH as an endogenous control gene and non-stimulated cells as a control Data are presented as the mean ± S.E.M. of three independent experiments. *—significantly different compared to the control. #—significantly different compared to the group without nicotine.