Allergic rhinitis (AR) is defined as an allergic inflammation of the nasal mucosa. Approximately 10%–40% of the global population suffers from AR [1]. Although AR is an upper respiratory tract inflammatory disease, people with AR may also suffer from allergic reactions in the lower respiratory tract, which can result in the development of asthma. Moreover, increases in the levels of airborne allergens and air pollutants, which are caused by climate change and air pollution, have led to an increase in the number of patients with AR and asthma [2].

Currently, AR is treated using oral antihistamines, intranasal corticosteroids, and leukotriene receptor antagonists [1,3-5]. However, these drugs cause side effects, including drowsiness, prolonged QT interval, throat irritation, epistaxis, stinging, burning, and nasal dryness [4-6]. Allergen immunotherapy is currently emerging as a new treatment for AR; however, it is not yet widely used because its clinical efficacy and safety have not been fully demonstrated [7]. For example, it has been associated with a potential risk of causing anaphylaxis. Therefore, the development of treatment strategies for AR that are safe and effective is needed.

The clinical signs and symptoms of AR include nasal congestion, sneezing, and rhinorrhea. Rhinorrhea, which is also known as nasal hypersecretion, is caused by the excessive secretion of mucin (inflammatory secretion) and liquid (water efflux) from the nasal submucosal glands (SMGs) [8,9]. It results in an uncontrollable liquid outpour from the nose and contributes to difficulty in breathing, which is uncomfortable and disturbs cognitive functions of patients [8,10,11]. The secretion of water and salts from the serous cells in nasal submucosal glands is mainly driven by the active secretion of Cl^– and HCO₃^–, which is stimulated by cyclic AMP or Ca²⁺ [9]. The accumulation of these anions on the apical side of the lumen results in a local increase in the osmolarity, thereby allowing water molecules to move from the basolateral side via the solvent drag effect [9].

In previous studies, it was reported that the secretion of Cl^– and HCO₃^– from the apical membranes of the submucosal gland serous cells is mainly regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. However, a recent study suggested that the calcium-activated Cl^– channel (CaCC) contributes more than CFTR to the secretion of Cl^– via cholinergic stimulation [9]. Moreover, the hypersecretion of fluid and mucus from the SMGs during AR also involves CaCC. It was recently reported that TMEM16A (ANO1), one of the major CaCCs, is expressed in nasal epithelial cells. Interleukin (IL)-4 and IL-13 induce goblet cell metaplasia and increase the expression of ANO1 [12-14]. In AR, increased ANO1 expression significantly increases mucin and fluid secretion via the release of histamine and the stimulation of protease-activated receptor 2 or cholinergic receptors [15-17]. ANO1 expression is significantly increased in patients with AR [17]. Moreover, IL-4 augments Cl^– and mucin secretion in primary cultured human nasal epithelial cells; however, this is suppressed by ANO1 inhibitors [17,18]. ANO1 mediates the hypersecretion of mucus, electrolytes, and water via the release of histamine, which is an important mediator of AR [9,17,19]. Therefore, ANO1 has potential as a unique therapeutic target for the management of AR. In addition, ANO1 channel inhibitors can potentially serve as dual-acting agents by treating mucin and fluid hypersecretion.

Spirodela polyrhiza (L.) Schleid. (SP) is widely used to relieve inflammation, urticaria, and symptoms of skin irritation, including pruritus, eczema, and rash [20,21]. According to a recent report, SP exerts a potential inhibitory effect on the calcium release-activated calcium channel, which is encoded by the ORAI1 gene and is responsible for Th2 activation and mast cell degranulation [22-24]. In addition, formulations containing SP or its ethanolic extract have been shown to alleviate allergic inflammation and pruritus in mice with atopic dermatitis [21,25].

In this study, we aimed to: (i) investigate whether ANO1 is a suitable therapeutic target to inhibit nasal hypersecretion induced by allergic rhinitis; and (ii) determine whether substances that inhibit ANO1 and are found in natural extracts are potential therapeutic candidates. Herein, we report whether SP extract and its chemical constituents, in particular, luteolin, alleviate electrolyte secretion by inhibiting ANO1 activity in human airway epithelial cells.

Cell culture

HEK293T (human embryonic kidney) cells (catalog no. CRL-3216) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene Inc., Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) (Welgene Inc.) and 1% penicillin/streptomycin (P/S) (Life Technologies, Carlsbad, CA, USA). Calu-3 cells were purchased from ATCC and cultured in minimum essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% P/S. All cells were cultured at 37°C in a humidified incubator. HEK293T cells were cultured under 10% CO₂ conditions, whereas Calu-3 cells were cultured in 5% CO₂ conditions.

Transient transfection

To measure the ANO1-mediated current, HEK293T cells were transfected with human ANO1 (hANO1) expression vector using TurboFect transfection reagents (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The ratio of green fluorescent protein (pEGFP-N1, Life Technologies) to hANO1 vector (10:1) was used to confirm the successful transfection of the cells. HEK293T cells were prepared the previous day in a 35-mm culture dish (Thermo Fisher Scientific). Then, 1.8 mg of hANO1 vector, 0.2 mg pEGFP, and 4 ml of TurboFect were added to 200 μl of DMEM (without FBS and P/S), followed by incubation at 25°C for 15 min. Lastly, 2 ml of complete medium (DMEM, 10% FBS, and 1% P/S) was added to the mixture and cultured with the prepared HEK293T cells for 24 h.

Electrophysiology

The ANO1-mediated current in the hANO1-transfected HEK293T cells and Calu-3 cells was measured using the patch clamp technique. The CFTR-mediated chloride current (I_CFTR) in the Calu-3 cells was also measured. The current was recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and a Digidata 1440A interface (Molecular Devices). Sampling was performed at 10 kHz with a low-pass filter set at 5 kHz. pCLAMP 10.4 software (Molecular Devices) was used for the analysis. The recorded currents were analyzed using pCLAMP 10.4, GraphPad Prism 6 (GraphPad, La Jolla, CA, USA), and Origin 8 (MicroCal LLC, Northampton, MA, USA). The cells were transferred into a perfusion chamber with a glass bottom and allowed to settle for 5–10 min. Patch pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL, USA) using a flaming/brown micropipette puller (p-1000; Sutter Instrument, Novato, CA, USA) and fire-polished to a pipette resistance of 2-7 MW (Narishige, East Meadow, NY, USA). The bath solution contained 140 mM Tris-Cl, 10 mM HEPES, 5 mM glucose, 1 mM MgCl₂, and 2 mM CaCl₂ (pH 7.4, Tris-base). The pipette solution for hANO1 comprised 140 mM N-methyl-d-glucosamine-Cl (NMDG-Cl), 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 7.85 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES (pH 7.2, Tris-base). In order to activate ANO1, the intracellular calcium concentration was set at 600 nM and calculated using WEBMAXC (Stanford University, https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). The pipette solution for CFTR contained 140 mM NMDG-Cl, 10 mM HEPES, 10 mM EGTA, 3.3 mM CaCl₂, and 1 mM MgCl₂ (pH 7.2, NMDG-base). The intracellular calcium concentration was set at 600 nM and calculated using WEBMAXC. To obtain the current/voltage relationship (I/V curve) in hANO1, we applied ramp-like pulses from –100 mV to +100 mV for 1 s every 20 s at a holding potential of –60 mV. The I_CFTR was determined at a holding potential of 0 mV. In the Calu-3 cells, step pulse was fixed at a potential of –60 mV. Voltage was increased by 20 mV from –100 mV to 100 mV. The pulse was maintained for 3 s during each voltage change.

Short-circuit current measurement

For short-circuit current measurement, Calu-3 cells were plated onto collagen precoated 12-mm permeable Snapwell inserts (Corning-Costar, Cambridge, MA, USA) in DMEM supplemented with 10% FBS and 1% P/S. For the IL-4 treatment experiments, Calu-3 cells were cultured in DMEM supplemented with 10% FBS, 1% P/S, and 10 ng/ml IL-4 (Peprotech, Rocky Hill, NJ, USA) for 24 h.

Snapwell inserts containing Calu-3 cells were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA, USA). The basolateral and luminal side chamber was filled with HCO₃^–-buffered solution. The HCO₃^–-buffered solution was composed of 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM D-glucose, 5 mM HEPES, and 25 mM NaHCO₃ (pH 7.4). The cells were allowed to stabilize for 20 min at 95% O₂/5% CO₂ conditions at 37°C. Subsequently, luteolin was added in the apical chamber, and ATP was added to the basolateral chamber to induce cytosolic calcium increase. Apical membrane currents were measured with an EVC4000 Multi-Channel V/I Clamp (World Precision Instruments, Sarasota, FL, USA) and recorded using PowerLab 4/35 (AD Instruments, Castle Hill, Australia). The sampling rate was 4 Hz. Data were collected and analyzed using Labchart Pro 7 software (AD Instruments).

Preparation of 30% ethanolic extract of SP (SP_EtOH)

The dried whole aquatic parts of SP cultivated in South Korea were purchased from Kwangmyungdang Medicinal Herbs Co. (Ulsan, Korea). The dried plant (200 g) was cut into small pieces and macerated in 2 L of 30% ethanol. Extraction was performed under reflux for 3 h under heated conditions. The resulting extract was filtered using filter paper (no. 1 filter paper; Whatman plc, Maidstone, UK). The extract was evaporated to dryness under reduced pressure to obtain SP_EtOH at a yield of 9.3%. The extract was stored at –20°C until use.

Analysis of SP_EtOH

SP_EtOH and the relevant standard compounds were analyzed by high-performance liquid chromatography (HPLC) (1290 system; Agilent, Santa Clara, CA, USA) at the Korea Basic Science Institute (Seoul, Korea). Chromatographic separation of SP_EtOH (10 mg/ml) was performed on a Poroshell 120 SB-C18 column (3.0 × 100 mm, 2.7 μm; Agilent) at a detection wavelength of 340 nm. The mobile phase (A, 0.05% formic acid; B, 0.05% formic acid in acetonitrile) was set a flow rate of 0.5 ml/min and run in gradient mode as follows: 5%–10% B for 3 min, 10%–30% B for 2 min, 30%–40% B for 5 min, 40%–90% B for 2.5 min, and equilibration with 5% B, successively.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 6.0 and Origin 8.0. All results are expressed as the mean ± standard error of mean. Data were analyzed using one-way analysis of variance (ANOVA) and Bonferroni’s post-hoc test. In case of the isolated chemical compound experiments, data were analyzed using paired Student’s t-test for comparisons between two groups (control vs. chemical treatment). p-values < 0.05 were considered statistically significant.

Effect of SP extract on human ANO1

To elucidate whether ANO1-mediated calcium-activated Cl^– current (I_ANO1) is inhibited by SP_EtOH, a whole-cell patch clamp experiment was performed using hANO1-overexpressing HEK293T (HEKT_ANO1) cells. Whole-cell configuration using a pipette solution containing 600 nM free Ca²⁺ generates outward rectifying Cl^– currents, which is a typical biophysical property of ANO1 [26]. After I_ANO1 reached a steady state, the bath solution was serially treated with 30, 100, and 300 μg/ml SP_EtOH. At the end of experiment, T16Ainh-A01, which is a potent small-molecule inhibitor of ANO1 [27], was applied to confirm its basal current. As shown in Fig. 1A and B, I_ANO1 was effectively inhibited by SP_EtOH by about 34.13% ± 2.14%, 57.1% ± 3.34%, and 87.96% ± 1.02% at concentrations of 30, 100, and 300 μg/ml, respectively. The normalized data are summarized in Fig. 1C.

Figure 1. Ethanolic (30%) extract of Spirodela polyrhiza (SP_EtOH) inhibits anoctamin-1 (ANO1)-mediated Cl^– current (I_ANO1) in ANO1-overexpressing HEK293T (HEKT_ANO1) cells. (A) Representative chart trace recordings showing the effects of 30, 100, and 300 μg/ml SP_EtOH on I_ANO1. At 300 μg/ml, SP_EtOH almost fully inhibited I_ANO1, similar to the effects of 30 μM T16Ainh-A01 (AO-1). (B) Typical current (I)/voltage (V) relationship curves for I_ANO1, showing the inhibition of I_ANO1 by 30, 100, and 300 μg/ml SP_EtOH. (C) Summary of I_ANO1 inhibition at +100 mV. Normalized amplitudes of currents (I_Ext/I_ctrl × 100%) were measured at a clamp voltage of +100 mV. Data are presented as the mean ± standard error of mean. Ext, SP_EtOH; ctrl, control. ****p < 0.0001 compared to the control (n = 6).

The chemical components of SP_EtOH

According to previous reports, the ethanolic extract of SP contains the following flavonoids: orientin, vitexin, luteolin 7-O-glucoside (Lu 7-G), apigenin 7-O-glucoside (Api 7-G), luteolin, and apigenin [20]. Chromatograms from the HPLC analysis of SP_EtOH showed five peaks eluting at 5.7 min (1), 5.91 min (2), 6.008 min (3), 6.334 min (4), and 7.915 min (5) (Fig. 2A). By comparing the chromatograms with those of six standard compounds obtained from previous studies conducted under the same HPLC conditions (Fig. 2B), we confirmed the presence of five of these flavonoids in SP_EtOH. Only apigenin was not detected in the extract (Fig. 2A). In addition, we estimated the concentrations of the compounds in 1 mg/ml SP_EtOH by comparing the peak areas with those of known amounts of analytical standard compounds (Table 1). We found that 1 mg/ml SP_EtOH contains 77.99 μg/mg (173 μM) orientin, 12.61 μg/mg (29.1 μM) vitexin, 46.87 μg/mg (104.5 μM) Lu 7-G, 3.94 μg/mg (9.12 μM) Api 7-G, and 1.027 μg/mg (3.58 μM) luteolin. The chemical structures of the five compounds are presented in Fig. 2C.

Table 1 . Data obtained from high-performance liquid chromatography analysis of the five flavonoid compounds in the ethanolic (30%) extract of Spirodela polyrhiza.

Compounds	RT (min)	Average area	Average amount (μg/ml)	Calculated amounta (μg/mg)
Orientin	5.7	329.2	7.799	77.992
Vitexin	5.91	56.1	1.261	12.605
Luteolin 7-glucoside	6.008	250.9	4.687	46.871
Apigenin 7-glucoside	6.334	19.7	0.394	3.944
Luteolin	7.915	5.1	0.103	1.027

RT, retention time. ^aCalculated amount = average amount × 50 (dilution factor)/5 mg.

Figure 2. High-performance liquid chromatography analysis of flavonoids in the ethanolic (30%) extract of Spirodela polyrhiza (SP_EtOH). (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.

Effects of SP_EtOH constituents on I_ANO1

We also investigated whether the five aforementioned compounds inhibit I_ANO1 in HEKT_ANO1 cells. Each compound was tested at a concentration of 100 μM. As shown in Fig. 3A, luteolin and Lu 7-G significantly inhibited I_ANO1 by 62.14% ± 4.3% and 18.51% ± 5.93%, respectively. The other constituents inhibited I_ANO1 by < 15% (orientin, 5.49% ± 10.21%; vitexin, 8.29% ± 3.47%; and Api 7-G, 15.48% ± 5.39%). We also identified the constituents that contribute to the inhibitory effect of SP_EtOH on ANO1. Although 1 mg/ml SP inhibited I_ANO1 by > 90%, a mixture of all five components inhibited I_ANO1 by only 20.1% ± 4.71%. The normalized I_ANO1 data are presented in Fig. 3B.

Figure 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 (I_ANO1). (A) Representative current (I)/voltage (V) relationship curves showing the effects of the five compounds on I_ANO1. Luteolin and Lu 7-G significantly inhibited I_ANO1. (B) Changes (%) in remaining current at +100 mV caused by the five identified compounds. Luteolin and Lu 7-G had a significant effect on I_ANO1 at a concentration of 100 μM. ****p < 0.0001, *p < 0.05 compared to the control (n = 4).

Effects of SP_EtOH on calcium-activated and cyclic adenosine monophosphate (cAMP)-activated Cl^– currents in human airway epithelial cells (Calu-3)

Airway epithelial glands secrete Cl^– ions, which are responsible for the secretion of water and mucus. Cl^– efflux across the apical membrane occurs via the cAMP-activated Cl^– channel CFTR and the CaCC ANO1 [9]. Therefore, we investigated whether SP_EtOH inhibits the two Cl^– channels, or only CaCC in Calu-3 cells. Using the whole-cell patch clamp technique, we generated a CaCC current (I_CaCC) using a pipette solution containing 600 nM free Ca²⁺, followed by serial treatment with 0.3, 1, and 3 mg/ml SP_EtOH. As shown in Fig. 4A, SP_EtOH inhibited I_CaCC by 36.9% ± 0.11%, 64.47% ± 0.04%, and 84.98% ± 0.04% at concentrations of 0.3, 1, and 3 mg/ml, respectively. Therefore, we investigated whether the activated current resulted from ANO1 expression by treating the cells with the ANO1-specific inhibitor 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) (Fig. 4A). As a result, Ani9 was found to completely inhibit I_CaCC, which confirmed that I_CaCC resulted from ANO1 expression in Calu-3 cells. The normalized I_CaCC data are presented in Fig. 4B.

Figure 4. Ethanolic (30%) extract of Spirodela polyrhiza (SP_EtOH) specifically inhibits the calcium-activated Cl^– current (I_CaCC) but not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^– current (I_CFTR) in Calu-3 cells. (A) Representative traces of I_CaCC following activation by 600 nM intracellular free calcium and inhibition by SP_EtOH (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 I_CaCC and its inhibition by SP_EtOH, which was calculated as I_Ext/I_ctrl × 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 I_CFTR and the effects of serially treating Calu-3 cells with 1 mg/ml SP_EtOH and 10 μM CFTRinh-172. The results show that I_CFTR was not modulated by SP_EtOH; however, it was completely inhibited by CFTRinh-172. (D) Representative current (I)/voltage (V) curves for I_CFTR after treatment with SP_EtOH and 10 μM CFTRinh-172. (E) Normalized current amplitude of I_CFTR at 100 mV. Data are presented as the mean ± SEM. ****p < 0.0001 compared to the control (n = 6).

We observed that cAMP-activated Cl^– current (I_CFTR), which was mediated by CFTR, was not inhibited by SP_EtOH (Fig. 4C–E). Further, I_CFTR was induced by adding 10 μM forskolin, an adenylate cyclase activator, to the bath solution. We also confirmed the presence of I_CFTR by treating the cells with CFTRinh-172, which is a CFTR-specific inhibitor (Fig. 4C–E).

Luteolin inhibits I_CaCC in Calu-3 cells

Next, we investigated whether the five constituents of SP_EtOH modulate I_CaCC in Calu-3 cells. As observed in the HEKT_ANO1 cells, luteolin inhibited I_CaCC Sequential treatments with various concentrations of luteolin also resulted in the dose-dependent inhibition of I_CaCC (Fig. 5A). The half-maximal inhibitory concentration (IC₅₀) of luteolin against I_CaCC was found to be 27.91 ± 1.61 μM (Fig. 5B). However, the other flavonoids simply inhibited I_CaCC (Fig. 5C).

Figure 5. Luteolin inhibits calcium-activated Cl^– channel current (I_CaCC), but slightly activates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^– current (I_CFTR) at a high concentration (> 100 μM) in Calu-3 cells. (A) Representative traces of I_CaCC, obtained by applying step voltage pulses and inhibiting I_CaCC 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 I_CaCC. 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 I_CFTR. The curves show the effects of luteolin and CFTRinh-172 (inh-172) on I_CFTR. Luteolin slightly potentiated I_CFTR 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).

We tested the effect of a mixture of the five components at their respective concentrations in 1 mg/ml SP_EtOH; however, the mixture showed an inhibition rate of 37.02% ± 8.69%, which was half of the rate observed for 1 mg/ml SP_EtOH. Interestingly, luteolin showed different effects on I_CFTR. Treatment with 100 μM luteolin slightly potentiated I_CFTR, but significantly inhibited I_CaCC (Fig. 5D, E). Additionally, we confirmed the presence of I_CFTR by treating the cells with CFTRinh-172 (Fig. 5D, E). However, this phenomenon was only observed under a high concentration of luteolin (> 100 μM). Taken together, these results suggest that SP_EtOH and luteolin selectively inhibit ANO1 and may play critical roles in alleviating the hypersecretion of nasal mucus.

Luteolin suppresses electrolyte secretion in IL-4 treated Calu-3 cells

Since the patch clamp technique can only be used to measure the activity of ANO1, we performed Ussing chamber experiments to measure the total electrolyte transport across both ion channels and epithelial tissues. To this end, Calu-3 cells were cultured on transwell inserts to obtain an epithelial monolayer. The cells were then cultured with 10 ng/ml IL-4, which is one of the major cytokines causing allergic inflammation, for 24 days. Subsequently, the short-circuit currents (I_SC) of the treated cells were compared with those in case of the control cells. To evaluate the secretory function, calcium-dependent Cl^– secretion was stimulated by adenosine triphosphate (ATP). As expected, ATP treatment significantly increased I_SC in Calu-3 cells pretreated with IL-4 (Fig. 6A). As shown in Fig. 6B, treatment with luteolin significantly inhibited I_SC in a dose-dependent manner. Similar to the patch clamp experiments, the half-maximal inhibitory concentration (IC₅₀) of luteolin in Calu-3 cells was 23.13 ± 1.51 μM (Fig. 6C).

Figure 6. Effects of luteolin on I_SC 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).

In the present study, we examined the inhibitory effect on ANO1 of SP_EtOH, which is reported to have a potential therapeutic effect on atopic dermatitis [21,25]. As a result, SP_EtOH was found to potently inhibit hANO1 channel activity in hANO1-overexpressing HEK293T cells by 87.96% ± 1.02% at a concentration of 300 μg/ml (Fig. 1). Furthermore, using HPLC, we found that orientin, vitexin, Lu 7-G, Api 7-G, and luteolin are the major flavonoids in SP_EtOH (Fig. 2) [20]. Based on the results of this quantitative analysis, we assessed whether these compounds were able to inhibit I_ANO1 at individual concentrations of 100 μM or as a mixture. Luteolin and Lu 7-G inhibited I_ANO1 by 62.14% ± 4.3% and 18.51% ± 5.93%, respectively (Fig. 3)_. The mixture of the constituents inhibited I_ANO1 by only 20.1% ± 4.71%. This was much lower than the effect of SP_EtOH, which almost completely blocked I_ANO1 at a concentration of 300 μg/ml. This was most likely due to the effect of other compounds in SP_EtOH besides the flavonoids.

Calu-3 cells have characteristics of human airway serous cells. They are widely used as a model of airway serous cells, which are involved in the secretion of electrolytes and water to hydrate airway surfaces [27-30]. Calu-3 cells express two representative Cl^– channels in airway epithelial cells: CaCC and CFTR. Therefore, experiments were conducted to determine whether SP_EtOH affects the activity of the two Cl^– channels. It was interesting to note that SP_EtOH inhibited I_CaCC, which was activated by increasing the intracellular calcium level (600 nM); however, it showed a lesser activity in the HEKT_ANO1 cells (64.47% ± 4.28% inhibition rate at a concentration of 1 mg/ml, Fig. 4). It was recently discovered that a novel potent small-molecule inhibitor, Ani9, completely inhibits I_CaCC [31]. Although this phenomenon has not been fully explained, it is presumed that differences in the distribution of regulatory proteins that interact with ANO1 or co-expression of ANO1 subtypes may be the underlying cause [28]. SP_EtOH did not have any effect on CFTR activity (Fig. 4), which confirmed that SP_EtOH only affects ANO1 activity. We then used Calu-3 cells to determine whether the flavonoid constituents in SP_EtOH can modulate I_CaCC and I_CFTR. The results showed that luteolin inhibits I_CaCC in Calu-3 cells to the same level as that in HEKT_ANO1 cells (IC₅₀, 27.91 ± 1.61 μM; Fig. 5). Recently, it has been reported that luteolin suppresses ANO1 activity in the human prostate cancer cell line PC-3 and HEKT_ANO1 cells, with an IC₅₀ of about 9.5 μM [32,33], which indicates a higher sensitivity to these two cell types than to Calu-3 cells. This difference in IC₅₀ values can be attributed to the differences in the cells. A similar observation was made for SP_EtOH, which showed different sensitivities to HEKT_ANO1 and Calu-3 cells. It was also observed that luteolin only increased I_CFTR at high concentrations (> 100 μM).

Because Calu-3 cells form polarized monolayers when cultured on a transwell membrane filter [34], we performed Ussing chamber experiments on IL-4 (10 μg/ml)-treated Calu-3 cells to investigate the effect of luteolin on electrolyte transport through the apical membrane. I_SC activated by purinergic receptor stimulation was strongly inhibited by luteolin treatment; the IC₅₀ (27.91 ± 1.61 μM) was similar to that found for the ANO1 inhibition in the patch clamp study, indicating that luteolin inhibited the electrolytic secretion from the apical membrane by inhibiting ANO1 activity.

SP extract is used to treat allergic inflammatory diseases, such as atopic dermatitis. Furthermore, SP extract inhibits mast cell degranulation and suppresses IL-4 and IL-13 expression, which is mediated by Th2 cells [21,22,25]. Our study clearly showed that SP_EtOH and luteolin suppressed ANO1 activity, which is responsible for nasal hypersecretion in AR, in addition to alleviating electrolyte secretion via ANO1 inhibition in Calu-3 cells (Fig. 7). Therefore, our results demonstrated that ANO1 may serve as a potential therapeutic target for AR-induced rhinorrhea, whereby SP_EtOH and luteolin may be developed as agents for the prevention and treatment of AR. Moreover, our research can be used as a guide for the development of natural herbs as anti-allergic agents.

Figure 7. Diagram illustrating the inhibitory effect of Spirodela polyrhiza (SP_EtOH) 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.

This research was supported by the Convergence of Conventional Medicine and Traditional Korean Medicine R&D program funded by the Ministry of Health & Welfare (Korea) through the Korean Health Industry Development Institute (KHIDI) (grant number HI16C0766) and also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03023718).

Study conception and design: J.H.N. and W.K.K. Conducting experiments, acquisition of data, data analysis, and interpretation of data: H.J.K., J.W., Y-R.N., and Y.S. Drafting of the manuscript: H.J.K., J.W., Y-R.N., and J.H.N. Critical review of manuscript: W.N. and W.K.K. Final approval of manuscript for submission: J.H.N. and W.K.K.

The authors declare no conflicts of interest.