Korean J Physiol Pharmacol.  2020 Jul;24(4):329-338. 10.4196/kjpp.2020.24.4.329.

Luteolin reduces fluid hypersecretion by inhibiting TMEM16A in interleukin-4 treated Calu-3 airway epithelial cells

• Kim HJ
• Woo J
• Nam YR
• Seo Y
• Namkung W
• Nam JH
• Kim WK

• KMID: 2503326
• DOI: http://doi.org/10.4196/kjpp.2020.24.4.329

Abstract

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl– and HCO₃ – is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calciumactivated Cl– channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SP_EtOH) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SP_EtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SP_EtOH, significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SP_EtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SP_EtOH and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SP_EtOH- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

Keyword

Airway hypersecretion; Allergic rhinitis; Anoctamin-1; Luteolin

Figure

• 

  Fig. 1 Ethanolic (30%) extract of Spirodela polyrhiza (SPEtOH) inhibits anoctamin-1 (ANO1)-mediated Cl– current (IANO1) in ANO1-overexpressing HEK293T (HEKTANO1) cells. (A) Representative chart trace recordings showing the effects of 30, 100, and 300 μg/ml SPEtOH on IANO1. At 300 μg/ml, SPEtOH almost fully inhibited IANO1, similar to the effects of 30 μM T16Ainh-A01 (AO-1). (B) Typical current (I)/voltage (V) relationship curves for IANO1, showing the inhibition of IANO1 by 30, 100, and 300 μg/ml SPEtOH. (C) Summary of IANO1 inhibition at +100 mV. Normalized amplitudes of currents (IExt/Ictrl × 100%) were measured at a clamp voltage of +100 mV. Data are presented as the mean ± standard error of mean. Ext, SPEtOH; ctrl, control. ****p < 0.0001 compared to the control (n = 6).

• 

  Fig. 2 High-performance liquid chromatography analysis of flavonoids in the ethanolic (30%) extract of Spirodela polyrhiza (SPEtOH). (A) Five flavonoid compounds were found in SPEtOH and (B) identified as orientin (1), vitexin (2), luteolin 7-glucoside (3), apigenin 7-glucoside (4), luteolin (5), and apigenin (6). (C) The chemical structures of the identified compounds.

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  Fig. 3 Effects of orientin, vitexin, luteolin 7-glucoside (Lu 7-G), apigenin 7-glucoside (Api 7-G), and luteolin on anoctamin-1-mediated Cl– current (IANO1). (A) Representative current (I)/voltage (V) relationship curves showing the effects of the five compounds on IANO1. Luteolin and Lu 7-G significantly inhibited IANO1. (B) Changes (%) in remaining current at +100 mV caused by the five identified compounds. Luteolin and Lu 7-G had a significant effect on IANO1 at a concentration of 100 μM. ****p < 0.0001, *p < 0.05 compared to the control (n = 4).

• 

  Fig. 4 Ethanolic (30%) extract of Spirodela polyrhiza (SPEtOH) specifically inhibits the calcium-activated Cl– current (ICaCC) but not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl– current (ICFTR) in Calu-3 cells. (A) Representative traces of ICaCC following activation by 600 nM intracellular free calcium and inhibition by SPEtOH (0.3, 1, and 3 mg/ml) and 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9, 1 μM). The holding potential was –60 mV and 4-s step pulses (each 10 mV, –100 to +100 mV) were applied every second. (B) Bar graph showing normalized ICaCC and its inhibition by SPEtOH, which was calculated as IExt/Ictrl × 100% at +100 mV. Data are presented as the mean ± standard error of mean. ****p < 0.0001 compared to the control (n = 10). (C) A typical trace showing the amplitudes of ICFTR and the effects of serially treating Calu-3 cells with 1 mg/ml SPEtOH and 10 μM CFTRinh-172. The results show that ICFTR was not modulated by SPEtOH; however, it was completely inhibited by CFTRinh-172. (D) Representative current (I)/voltage (V) curves for ICFTR after treatment with SPEtOH and 10 μM CFTRinh-172. (E) Normalized current amplitude of ICFTR at 100 mV. Data are presented as the mean ± SEM. ****p < 0.0001 compared to the control (n = 6).

• 

  Fig. 5 Luteolin inhibits calcium-activated Cl– channel current (ICaCC), but slightly activates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl– current (ICFTR) at a high concentration (> 100 μM) in Calu-3 cells. (A) Representative traces of ICaCC, obtained by applying step voltage pulses and inhibiting ICaCC with various concentrations of luteolin and 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9). The holding potential was –60 mV and 4-s step pulses (each 10 mV, –100 to +100 mV) were applied every second. (B) Dose-response curve for luteolin-induced inhibition of ICaCC. The half-maximal inhibitory concentration (IC50) was 27.91 ± 1.61 μM. (C) Changes (%) in remaining current at +100 mV caused by vitexin, luteolin 7-glucoside (Lu 7-G), apigenin 7-glucoside (Api 7-G), orientin (each at a concentration of 100 μM), and a mixture of the five flavonoid compounds at their respective concentrations in the ethanolic (30%) extract of Spirodela polyrhiza (1 mg/ml). Data are presented as the mean ± standard error of mean. *p < 0.05 compared to the control (n = 4). (D) Representative current (I)/voltage (V) curve for ICFTR. The curves show the effects of luteolin and CFTRinh-172 (inh-172) on ICFTR. Luteolin slightly potentiated ICFTR at a concentration of 100 μM. (E) Normalized current amplitude following the treatment of Calu-3 cells with 100 μM luteolin and inh-172. Data are presented as the mean ± SEM. *p < 0.05 compared to the control (n = 5).

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  Fig. 6 Effects of luteolin on ISC in interleukin-4 (IL-4)-treated Calu-3 cells. (A) Calu-3 cells were pretreated with IL-4 (10 ng/ml) for 24 h; CFTRinh172 was then added to the apical chamber and cells were incubated for 10 min prior to the treatment with adenosine triphosphate (ATP). Apical membrane currents were recorded for Calu-3 cells expressing anoctamin-1 (ANO1). (B) Representative current traces showing luteolin-mediated inhibition of ANO1 at the indicated concentrations. ANO1 was activated by 100 μM ATP. (C) Summary of dose-responses (mean ± standard error of mean, n = 4).

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  Fig. 7 Diagram illustrating the inhibitory effect of Spirodela polyrhiza (SPEtOH) and its chemical constituents, luteolin and luteolin 7-glucoside, in electrolyte secretion via anoctamin-1 (ANO1) inhibition in the airway epithelium. CFTR, cystic fibrosis transmembrane conductance regulator.

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