Modulation of the Respiratory Epithelium Physiology by Flavonoids-Insights from 16HBEσcell Model

Abstract

Extensive evidence indicates that the compromise of airway epithelial barrier function is closely linked to the development of various diseases, posing a significant concern for global mortality and morbidity. Flavonoids, natural bioactive compounds, renowned for their antioxidant and anti-inflammatory properties, have been used for centuries to prevent and treat numerous ailments. Lately, a growing body of evidence suggests that flavonoids can enhance the integrity of the airway epithelial barrier. The objective of this study was to investigate the impact of selected flavonoids representing different subclasses, such as kaempferol (flavonol), luteolin (flavone), and naringenin (flavanone), on transepithelial electrical resistance (TEER), ionic currents, cells migration, and proliferation of a human bronchial epithelial cell line (16HBE14σ). To investigate the effect of selected flavonoids, MTT assay, trypan blue staining, and wound healing were assessed. Additionally, transepithelial resistance and Ussing chamber measurements were applied to investigate the impact of the flavonoids on the electrical properties of the epithelial barrier. This study showed that kaempferol, luteolin, and naringenin at micromolar concentrations were not cytotoxic to 16HBE14σ cells. Indeed, in MTT tests, a statistically significant change in cell metabolic activity for luteolin and naringenin was observed. However, our experiments showed that naringenin did not affect the proliferation of 16HBE14σ cells, while the effect of kaempferol and luteolin was inhibitory. Moreover, transepithelial electrical resistance measurements have shown that all of the flavonoids used in this study improved the epithelial integrity with the slightest effect of kaempferol and the significant impact of naringenin and luteolin. Finally, our observations suggest that luteolin increases the Cl- transport through cystic fibrosis transmembrane conductance regulator (CFTR) channel. Our findings reveal that flavonoids representing different subclasses exert distinct effects in the employed cellular model despite their similar chemical structures. In summary, our study sheds new light on the diverse effects of selected flavonoids on airway epithelial barrier function, underscoring the importance of further exploration into their potential therapeutic applications in respiratory health.

Keywords: airway epithelium; flavonoids; transepithelial electrical resistance; wound healing.

Figure 1

The effect of flavonoids on 16HBE14σ cells metabolic activity and cell viability. (A) changes in cell metabolic activity after 24 h incubation with flavonoids (kaempferol, luteolin, naringenin) at various concentrations (10, 30, and 50 μM). (B) Cell viability of cells incubated with flavonoids for 24 h was assessed by trypan blue staining. The results were normalized to control and presented as mean ± standard deviation (SD). Statistical analysis was evaluated by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

16HBE14σ cell migration in a scratch assay. Quantitative analyses of the migration assays are expressed as percentages relative to the area at time 0 h. after 3, 6, and 24 h after treatment with different concentrations of kaempferol (A), luteolin (B), and naringenin (C) at various concentrations. (D) Example images showing scratch closure process of control and 50 μM luteolin treated cells over time. The red scale indicates the distance of 100 μM. The data present mean values of scratch areas ± standard deviations (SD) (n = 3). Statistical analysis was assessed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Impact of flavonoids on transepithelial electrical resistance in 16HBE14σ cell monolayers. Tight and polarized cell monolayers were treated apically with kaempferol (A), luteolin (B), and naringenin (C) at concentrations of 10, 30, and 50 μM. TEER was measured after 24, 48, and 72 h of incubation. Results were normalized to the TEER value measured before treatment (time 0 h). Graphs display mean values ± standard deviations (SD). Statistical significance compared to control was determined using a one-way ANOVA (* p < 0.05).

Figure 4

Changes in basal chloride current flowing through 16HBE14σ cell monolayers treated with 50 μM luteolin for 72 h. Chloride current was measured in the Ussing chamber in basolateral to apical chloride gradient. The data are presented as mean ± standard deviations (SD).

Figure 5

The effect of specific ion channel modulators on short-circuit current. (A) short circuit current changes in non-treated 16HBE14σ cell monolayers. (B) short circuit current changes in luteolin treated 16HBE14σ cell monolayers. The modulators were added subsequently: amiloride (epithelial sodium channel ENaC inhibitor), forskolin (activator of cAMP-dependent CFTR current), Inh-172 (specific CFTR inhibitor), and ATP (to investigate purinergic calcium-dependent Cl⁻ transport). Data are presented as mean ± standard deviations (SD).