Minimum essential medium, fetal bovine serum, penicillin/streptomycin and trypsin were purchased from Gibco (Grand Island, NY, United States). Allicin was provided by Ailexin (Urumuqi, China). Amiloride, CFTRinh-172, DIDS, forskolin (FSK), isobutylmethylxanthine (IBMX) were purchased from Sigma-Aldrich (Missouri, United States). SQ22536 was purchased from MedChemExpress (New Jersey, United States). NaCl, KCl, MgSO₄, NaHCO₃, KH₂PO₄, CaCl₂, glucose, and gluconate were purchased from Guangzhou Chemical Pharmaceutical Factory (Guangzhou, China). HEPES was purchased from Mbchem Technology (Guangzhou, China). The direct cAMP enzyme immunoassay kit (KGE002B) was purchased from R&D Systems (Minneapolis, MN, United States). The bicinchoninic acid protein assay kit (kw0014) was purchased from KWBIO (Beijing, China).

Kunming mice weighing 25–30 g were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The mice were housed in specific pathogen-free conditions with a constant room temperature of 20°C and a 12 L:12 D photoperiod with food and water allowed ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (Guangzhou, China).

Human bronchial epithelial cell line 16HBE14o- and CF human bronchial epithelial cell line CFBE41o- were obtained as a kind gift from Prof. Wing-Hung Ko (School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China). The cells were cultured in minimum essential medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin−streptomycin at 37°C with 5% CO₂ in a humidified atmosphere.

The measurement of I _SC was performed following a modified procedure as previously described (Zhang et al., 2018) (Cottrill et al., 2021). In brief, 16HBE14o- or CFBE41o- cell monolayers grown on Millipore filter membranes (Millipore) with a pore diameter of 0.45 μm were clamped vertically between the two halves of an Ussing chamber (EM-CSYS-2 Ussing Chamber Systems, Physiologic Instruments, San Diego, United States). The confluent monolayer of cells was bathed in Krebs-Henseleit (K-H) solution (115 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, 25 mM NaHCO₃) at the basolateral side and with reduced Cl⁻ concentration (115 mM Na-gluconate, 5 mM KCl, 1 mM MgCl₂, 4 mM CaCl₂, 10 mM glucose, 25 mM NaHCO₃) at the apical side to generate a basolateral to apical Cl⁻ gradient. The solution was gassed with 95% O₂/5% CO₂ to obtain a constant pH of 7.4 and maintained at 37°C during the experiments. The epithelium exhibited a basal transepithelial potential difference (PD), which was measured by Ag/AgCl electrodes with KCl/agar bridges connected to the voltage-clamp amplifier (VCC MC6, Physiologic Instruments, San Diego, United States). For I _SC measurement, the transepithelial PD of the confluent monolayer was clamped at 0 mV. The change of I _SC (ΔI _SC), defined as the difference between the value at baseline and that at a peak following the stimulation, was synchronously displayed via a signal collection and analysis system (BL-420E+ system, Chengdu Technology & Market Co. Ltd., Chengdu, China) and normalized by the unit area of the preparation (ΔμA/cm²). At the beginning and the end of each experiment, 1 mV pulse was applied and the current change in response was used to estimate transepithelial resistance according to Ohm’s law. In the ion substitution experiments, HCO₃ ⁻ was substituted by HEPES, and Cl⁻ was substituted by gluconate. HCO₃ ⁻-free solution was ventilated in 100% O₂.

Intracellular cAMP content was measured using a direct cAMP enzyme immunoassay kit (R&D Systems, KGE002B, Minneapolis, MN, United States) according to the instructions. The assay was based on the competitive binding technique by using monoclonal antibodies specific for cAMP. The total protein content in the lysate was measured using the bicinchoninic acid protein assay kit (KWBIO, kw0014, Beijing, China).

The potent pro-secretory activity of allicin was evaluated by the phenol red secretion assay as previously described (Menezes et al., 2019). Briefly, the mice were treated with saline, allicin (100 μM, 200 μM, and 400 μM as low-dose, middle-dose and high-dose separately) or salbutamol (1 mg/ml) by aerosol for 30 min lasting for four consecutive days. The mice were intraperitoneally injected with 5% (w/v) phenol red dissolved in saline (50 μL/10 g body weight) or an aliquot of saline as the blank control 30 min after the last nebulization. Then, the mice were euthanized by cervical dislocation 30 min after the injection of phenol red and bronchoalveolar fluid (BALF) samples were collected. The BALF samples were centrifuged at 650 × g for 10 min and alkalinized with NaOH (0.1 mM). Then, the absorbance was measured at 565 nm wavelength in a spectrophotometer. The standard curve of phenol red was prepared with various concentrations of phenol red solutions (0, 0.5, 1, 5, 10, 50, 100 ng/ml). The calibration curve could be obtained by fitting the phenol red concentration corresponding to absorbance with the linear function “y = kx + b”. The concentration of phenol red (ng/ml) in BALF samples was then calculated.

All data were presented as the mean ± standard deviation (S.D.) with distinct dots for each measurement. The Student’s t-test (two-tailed) was used to compare the differences between two groups. For three or more groups, data were analyzed with one-way analysis of variance followed by Tukey’s multiple comparison tests (GraphPad Software, Inc, San Diego, CA, United States). Statistically significant differences between groups were defined as p < 0.05.

To investigate the effect of allicin on ion transport in airway epithelium, the I _SC was measured by using the Ussing chamber technique. In 16HBE14o- cell monolayers, the mean basal I _SC was 28.38 ± 8.32 μA/cm² (n = 37), with the transepithelial resistance of 2,837 ± 462.7 Ω cm² (n = 16) and the mean transepithelial PD of −6.55 ± 0.85 mV (n = 17). Apical application of allicin (200 μM) triggered a sustained increase in I _SC (Figure 1A) in a concentration-dependent manner (Figure 1D), with a half-maximal effective concentration at 213.6 μM. However, basolateral application of allicin induced a relatively blunted response (Figures 1B, C). Hence, 200 μM allicin was apically applied in the subsequent experiments to investigate its biological effect on airway epithelial cells.

To determine the ion composition involved in the allicin-induced I _SC response, a series of channel blocker tests and ion substitution experiments were carried out. As the epithelial Na⁺ channel (ENaC) plays an important role in pulmonary fluid regulation (Matalon et al., 2015), we initially investigated whether allicin induced the I _SC response by facilitating Na⁺ absorption via ENaC. Notably, the ENaC blocker amiloride (100 μM) had no significant effect on allicin-induced ΔI _SC (Figure 2B), ruling out the participation of Na⁺ transport in allicin-induced I _SC response. We then investigated whether anion secretion was involved in allicin-induced ΔI _SC. Removal of ambient HCO₃ ⁻ failed to affect the allicin-induced ΔI _SC (Figure 2D), while the I _SC response was significantly reduced in the solution without Cl⁻ (Figure 2C). Furthermore, when extracellular Cl⁻ and HCO₃ ⁻ were both removed, the allicin-induced ΔI _SC was abolished (Figure 2E). The above results demonstrated that the allicin-induced I _SC response in airway epithelial cells was primarily Cl⁻ secretion.

To verify which Cl⁻ channel is responsible for allicin-induced I _SC response, several Cl⁻ channel blockers were used. Application of the CFTR blocker CFTRinh-172 (20 μM), but not the Ca²⁺ activated Cl⁻ channel (CaCC) blocker DIDS (100 μM), significantly attenuated the allicin-induced ΔI _SC (Figures 3A–D), which revealed the involvement of CFTR in this process. We further performed the experiment in a CF cell line, CFBE41o-, which is homozygous for the most common CF mutation, the deletion of phenylalanine at position 508 (Bruscia et al., 2002). The allicin-induced ΔI _SC was abrogated in CFBE41o- cells, whereas the CaCC-mediated Cl⁻ secretion elicited by ATP (100 μM) remained detectable (Figures 3E, F). These observations demonstrated that allicin-induced Cl⁻ secretion was mediated by CFTR.

It is well known that the opening of CFTR is modulated by intracellular cAMP (Csanády et al., 2019). An increase in cAMP level can either be the result of the activation of adenylate cyclase (AC) or inhibition of phosphodiesterase (PDE) and vice versa. To verify whether allicin activated CFTR by increasing the intracellular cAMP level, FSK (10 μM), an AC activator, was used to stimulate the maximum AC activity while IBMX (100 μM), an inhibitor of PDE, was used to inhibit the degradation of cAMP. Pretreatment with FSK and IBMX abolished the I _SC response elicited by allicin (Figures 4B, D). Furthermore, the allicin-induced I _SC was significantly reduced by pretreatment of SQ22536 (50 μM), an inhibitor of AC (Figures 4C, D). Consistent with the I _SC results, the application of allicin triggered an elevation in intracellular cAMP level in an SQ22536-sensitive way (Figure 4E). The above results suggest that allicin modulates the activity of CFTR by increasing intracellular cAMP concentration via activation of AC activity rather than inhibition of PDE activity.

Transepithelial Cl⁻ secretion drives the water across the airway epithelium, leading to the hydration of the airway surface, which reflects the pharmacology of expectorant drugs. To investigate whether allicin harbors the mucosecretolytic activity by promoting ASL secretion, we evaluated the pro-secretory effect of allicin in murine models using the phenol red secretion assay. Similar to the mucokinetic agent salbutamol (also known as albuterol) (Menezes et al., 2019) (Rogers, 2007), inhaled allicin significantly increased the concentration of phenol red in BALF in a concentration-dependent manner (Figure 5A). These results indicated that allicin could facilitate ASL hydration by stimulating transepithelial Cl⁻ secretions in vivo.