Reagents and experimental solutions
Phosphate-buffered saline (PBS) and Hank’s Balanced Salt Solution (HBSS) were purchased from Sigma Aldrich. BNO 1016, in a formulation brand named as Sinupret® ethanolic dry extract was obtained from Bionorica. It consists of gentian root (Gentianae radix), primrose flowers (Primulae flos), elder flowers (Sambuci flos), common sorrel herb (Rumicis herba) and vervain herb (verbenae herba) in a 1:3:3:3:3 ratio. This dry extract was initially dissolved in 100% DMSO to obtain a concentration of 100 mg/ml. The resulting suspension was sonicated in an ultrasonic bath (HF 35 kHz) at room temperature for 30 min, and then vortexed for an additional 2 min. The suspension was centrifuged for 10 min at 3000 g at room temperature, and the supernatant was removed to be used for all BNO 1016 preparations at the concentrations used in experiments.
Human tissue acquisition
Tissue was obtained with informed consent and Institutional Review Board approval. Patients undergoing sinonasal surgery at the Department of Otorhinolaryngology, Division of Rhinology at the University of Pennsylvania and the Philadelphia Veterans Affairs Medical Center were recruited and tissue was taken from residual clinical material at surgery and processed immediately. Patients were excluded if they had antibiotic, corticosteroid, or antibiologic use within 1 month of surgery, or a history of systemic disease such as Wegener’s or Cystic Fibrosis.
Air-liquid Interface (ALI) cultures
We have previously described the culture of human nasal epithelial cells at an ALI [14, 15]. Briefly, human sinonasal epithelial cells were enzymatically dissociated and grown with medium containing DMEM/Hem’s F-12 and bronchial epithelial based medium (Clonetics), in addition to 100 U/ml penicillin and 100 μg/ml streptomycin for 7 days. Following this, cells were trypsinized and placed on porous polyester membranes in transwell cell culture inserts (Transwell-clear, 12-mm diameter, 0.4-μm pores; Corning). These inserts were coated with 100 μl of coating solution (BSA 0.1 mg/ml; Sigma-Aldrich), type 1 bovine collagen (30 μg/ml; BD), and fibronectin (10 μg/ml; BD) in LHC basal medium (Invitrogen). After 5 days, the apical compartment was cleared and the epithelium was allowed to differentiate using a medium of 1:1 DMEM (Invitrogen) and BEBM (Clonetics, Cambrex), with the Clonetics complements for hEGF (0.5 ng/ml), epinephrine (5 μg/ml), hydrocortisone (0.5 μg/ml), BPE (0.13 mg/ml), insulin (5 μg/ml), triiodothyronine (6.5 μg/ml), and transferrin (0.5 μg/ml), supplemented with 100 UI/ml penicillin, 100 g/ml streptomycin, 0.1 nM retinoic acid (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich) in the basal compartment. For each experiment, three ALI cultures each obtained from three different human patients were utilized. Experiments were performed in paired fashion to control for inter-donor variability.
Bead transport velocity measurements
Mucociliary transport velocity was measured using in vitro fluorescent bead tracking using 2 μm polystyrene fluorescent microspheres (0.0025% by weight in 30 μl) that were added to the apical surface of the cultures after copious washing with PBS to remove mucus clumps as previously described . To obtain optimal imaging of the beads, the filters containing the ALI cultures were cut out of the support and placed directly on a cover slip. Test solutions were added to the apical side but as the filters were cut out, the solution in essence is in contact with both basoateral and apical surface of the cells. Beads were imaged using an inverted Nikon TE2000E epifluorescence microscope (20 × 0.5 NA PlanFluor objective) equipped with a 12-bit QImaging camera and computer running ImageJ (W Rasband, NIH) and μManager. A bead streak had to have a visible beginning and ending (see results), within the field of view, to be included in the statistical analysis of the data. The distances beads traveled were calculated using ImageJ within-application measurements and distance was divided by time to calculate a transport velocity for each bead. Either an ND4 or ND8 filter was used for bead-tracking experiments, depending on the thickness of the individual epithelial culture and resulting brightness of the beads.
Cell culture surface liquid (ASL) depth measurements
ASL was labeled with 10 μl of Texas red dextran (10,000 MW; 2 mg/ml) in PBS followed by 24 h incubation at 37 °C in a humidified incubator to allow ALIs to absorb excess fluid and reach ASL homeostasis as previously described . Upon removal from the incubator, cultures were immediately overlayed with 100 μL perfluorocarbon (PFC-77) to prevent ASL evaporation. The apical side of the cultures was otherwise unmodified; drug additions were made only basolaterally. Confocal images were then taken using a 60x (1.0 NA) water immersion objective with 0.33 μm step size.
All statistical analyses were perfomed using GraphPad Prism. All tests were two-tailed, and p < 0.05 was the curoff for statistical significance. Statistical analysis of the arithmetic means of the velocities or ASL height derived from each culture was performed using an ANOVA, followed by Dunnett’s test comparing each treatment group within a test item concentration range to the vehicle treated samples.