J Korean Acad Oral Health.  2020 Dec;44(4):214-221. 10.11149/jkaoh.2020.44.4.214.

Studies on inhibition of gingival fibroblast proliferation by nicotine concentration

• Han KU
• Lee CH
• Lee JH
• Jeon YM
• Yoo HJ

• KMID: 2510253
• DOI: http://doi.org/10.11149/jkaoh.2020.44.4.214

Abstract

Objectives
To investigate the effect of nicotine on the healing of an oral cavity wound, high and low concentrations of nicotine were administered on human gingival fibroblasts.
Methods
Nicotine at concentrations of 0.1, 1, 5, and 10 mM were administered to gingival fibroblasts to evaluate the survival capability of the cells. Nicotine at 0.1 mM, a nonapoptotic concentration, was administered to evaluate apoptosis using Annexin V-FITC/Propidium Iodide cell staining. Nicotine at 1, 10, and 100 mM were administered to measure the expression of inflammatory cytokines, which was measured by RT-PCR and ELISA. FGF was treated with an additional 1, 10, or 100 mM of nicotine to evaluate cell proliferation and wound healing.
Results
As the concentration of nicotine increased (0.1, 1, 5, and 10 mM), the survival capability of the cells reduced. When cells were exposed to low nicotine concentration (0.1 mM) for 24 h, apoptosis occurred. Moreover, if the cell was exposed for 48 h, cell apoptosis occurred with necrosis. As the concentration of nicotine increased (1, 10, and 100 mM), more inflammatory cytokines were expressed. When EC LPS and TF LPS were combined with a low concentration of nicotine (1 and 10 mM), the expression of inflammatory cytokines was suppressed. The FGF level decreased as the nicotine concentration increased (1, 10, and 100 mM).
Conclusions
Nicotine interferes with the wound healing process of gingival fibroblasts. To maintain the wound healing process after a surgery or dental procedure, cessation of smoking is recommended.

Keyword

Fibroblasts; Necrosis; Nicotine

Figure

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  Fig. 1 Observation of cell viability. The cell viability was measured by phase-contrast microscope.

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  Fig. 2 Investigation of apoptosis and necrosis. Nicotine-treated HGF for 24 h and 48 h was treated with 2 mM ethylenediaminetetraacetic acid in PBS to detach from cell culture dish. The cell was washed twice with cold PBS. The cells were reacted 1.25 µl of Annexin V-FITC in Binding buffer (200 µl; 0.1 M HEPES, pH7.4; 1.4 M NaCl; 25 mM CaCl2) for 15 min at RT in the dark and then add 4 µl of propidium iodine (0.05 mg/ml). The cells were analyzed by a fluorescence-activated cell sorter.

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  Fig. 3 Investigation of inflammation cytokines by real-time RT PCR. HGF was treated in the presence or the absence of nicotine. The cell was used to RT-PCR. *Statistically significant difference compared with E. coli LPS (P<0.05). EC LPS, E. coli LPS.

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  Fig. 4 Investigation of inflammation cytokines by T. forsythia LPS. HGF was treated with T. forsythia LPS in the presence or the absence of nicotine. The cell was used to RT-PCR. *Statistically significant difference compared with T. forsythia LPS (P<0.05). TF LPS, T. forsythia LPS.

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  Fig. 5 Investigation of inflammation cytokines by ELISA. HGF was treated in the presence or the absence of nicotine. The cell was used to RT-PCR, and the conditioned media was used to ELISA. *Statistically significant difference compared with E. coli LPS (P<0.05).

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  Fig. 6 Investigation of inflammation cytokines by T. forsythia LPS. HGF was treated with T. forsythia LPS in the presence or the absence of nicotine. The cell was used to RT-PCR, and the conditioned media was used to ELISA. *Statistically significant difference compared with T. forsythia LPS (P<0.05).

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  Fig. 7 Inhibitory effect of nicotine on cell proliferation. The gingival fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum, and cells were dropped using trypsin-EDTA and dispensed into a 96-well polystyrene culture dish at 1×103 cells/ml at a concentration of 10 ng/ml was added to the culture medium, Of nicotine was added to the culture medium to remove the existing culture medium. After incubation in a 5% CO2 incubator for 12 hours, cell growth was examined using MTT. *Statistically significant difference compared with fibroblast growth factor (P<0.05). FGF, fibroblast growth factor.

Reference

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