Reagents and chemicals

Gadolinium (G7532-5G), pyrazole 3 (Pyr3, P0032-5MG), tetraethylammonium chloride (TEA-Cl, T2265-100G), nifedipine (C11875500BT), niflumic acid (NA, N0613-10G) and paxilline (PAX, P2928-10MG) were purchased from Sigma (St. Louis, MO, USA). Dexamethasone (Dex, MB1434) was purchased from Meilunbio (Dalian, China). PP121 (V0200) was purchased from InvivoChem (Guangzhou, China). Acetylcholine chloride (ACh, S30170-5 g), and ovalbumin (OVA, S28623-25 mg) were purchased from Yuanye Bio-Technology (Shanghai, China), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, G4000) was purchased from Promega (Beijing, China). All chemicals were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China) unless stated otherwise. The antibody for β-actin (AA128) was purchased from Beyotime (Shanghai, China). The antibodies for p-VEGFR2 (AP0382) and VEGFR2 (A11127) were purchased from ABclonal (Wuhan, China). Antibodies against p-Akt (#4058S), Akt (#4685S), p-p38 MAPK (#4631S) and p38 MAPK (#9212) were purchased from Cell Signaling Technology (Beverly, MA, USA). A human bronchial epithelial cell line (16HBE, ZQ0001) was purchased from Zhongqiaoxinzhou (Shanghai, China).

Establishment of an asthmatic mouse model

The establishment of the asthma mouse model was performed as previously described (Shi et al. 2020). Briefly, male BALB/c mice (6–8 weeks old, sexually mature, Zikeheng Biotech, Wuhan, China) were housed in the Experimental Animal Center of South-Central Minzu University, which provided a specific pathogen-free (SPF)-grade environment. All animal procedures were designed and performed under the supervision of the Animal Care and Ethics Committee of South-Central Minzu University. Briefly, sexually mature mice were randomly divided into a control group (n = 6 mice), an asthma group (n = 6 mice), a Dex group (n = 6 mice) and a PP121 group (n = 6 mice). For establishment of the asthma mouse models, the asthma group, Dex group and PP121 group were OVA-sensitized by intraperitoneal (IP) injections of 3 mg/mL OVA (200 µL per 20 g) on Days 0, 7 and 14, while the control group was injected with physiological saline. From Day 21 to Day 34, the asthma group, Dex group and PP121 group were stimulated with the intranasal instillation of 3 mg/mL OVA (20 μL per 20 g, once per day), supplemented with daily gavage of Dex (3 mg/kg) in the Dex group or PP121 (20 mg/kg and 50 mg/kg, respectively) in the PP121 group. The control group was intranasally instilled and gavaged with physiological saline.

Measurement of mouse airway smooth muscle tension

The measurement of mouse airway smooth muscle tension was performed as previously described with subtle modifications (Luo, et al. 2018). Tracheae (n = 6/6 mice) were dissected from euthanized mice and transferred to ice-cold physiological salt solution (PSS: 2 mM CaCl2, 10 mM glucose, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2·6H2O, 135 mM NaCl, pH adjusted to 7.4 with NaOH). Then, 5–7 mm mTRs were isolated and mounted in a 6 mL organ bath (Techman, Chengdu, China) filled with PSS bubbled with 95% O2 and 5% CO2 at 37 °C. The mTRs were equilibrated for 60 min, and then, the experiments were conducted with 80 mM K+ or 100 μM ACh. For analysis of the participation of NCXs, PSS was replaced with Li-PSS (2 mM CaCl2, 10 mM glucose, 10 mM HEPES, 5 mM KCl, 135 mM LiCl, 1 mM MgCl2·6H2O, pH adjusted to 7.4 with Tris-base).

For the cross-sectional area measurement of the bronchus, tracheae and lungs (n = 6/6 mice) were isolated from euthanized mice. The lung tissues were filled with 41 °C low melting agarose through the cannulated tracheae. Then, the tracheae and lungs were transferred to Hanks’ balanced salt solution (HBSS: 1.26 mM CaCl2, 5.56 mM glucose, 20 mM HEPES, 5.33 mM KCl, 0.44 mM KH2PO4, 0.49 mM MgCl2·6H2O, 0.41 mM MgSO4, 0.34 mM Na2HPO4, 137.93 mM NaCl, 4.17 mM NaHCO3, pH adjusted to 7.4 with NaOH) and incubated on ice for 30 min at 4 °C. The agarose-inflated lungs were sectioned into 350 μm thick slices with a vibratome (VT1000S, Leica, Nussloch, Germany). The lung slices were incubated in HBSS, and the lumen area of the bronchus under different stimuli was photographed and analyzed with LightTools (Optical Research Associates, CA, USA).

Measurement of channel currents

For isolation of single mouse airway smooth muscle cells (mASMCs) for current measurement, the mouse tracheal muscles were removed from the trachea in mASMC dissociation buffer (0.1 mM CaCl2, 11 mM glucose, 10 mM HEPES, 5.2 mM KCl, 0.6 mM KH2PO4, 1.2 mM MgCl2, 120 mM NaCl, and 25 mM NaHCO3, pH adjusted to 7.0 with NaOH). The dissected smooth muscles were incubated and digested for 22–24 min at 37 °C in digest solution I, which contained 2–3 mg/mL papain, 1 mg/mL bovine serum albumin (BSA), and 0.15 mg/mL dithiothreitol (DTT), and then dissolved in mASMC dissociation buffer. Then, the tissues were transferred to digest solution II (1 mg/mL collagenase H, 1 mg/mL BSA, dissolved in mASMC dissociation buffer) for 3–5 min at 37 °C. The tissues were washed with 1 mg/mL BSA and gently flicked to yield single mASMCs. mASMCs were kept on ice for further experiments.

The measurement of channel currents was conducted as previously described (Shi et al. 2020). For measurement of L-VDCC currents, mASMCs (n = 5 cells/5 mice) were patched and held in bath solution I (27.5 mM BaCl2, 10 mM HEPES, 11 mM glucose, 1 mM MgCl2, 107 mM NaCl, 10 mM TEA-Cl, pH adjusted to 7.2 with NaOH) at − 70 mV. The pipette was filled with intracellular solution I (130 mM CsCl, 10 mM EGTA, 10 mM HEPES, 4 mM MgATP, 4 mM MgCl2, pH adjusted to 7.3 with CsOH). L-VDCC currents were recorded with an EPC-10 patch-clamp amplifier (HEKA, Lambrecht, Germany) under a stepped voltage ranging from − 70 to + 40 mV in 10 mV increments every 50 ms.

For measurement of NSCC currents, mASMCs (n = 5 cells/5 mice) were patched and held in bath solution II (1.5 mM CaCl2, 11 mM glucose, 10 mM HEPES, 126 mM NaCl, pH adjusted to 7.2 with NaOH) at − 60 mV. The pipette was filled with intracellular solution II (1 mM CaCl2, 108 mM cesium acetate, 18 mM CsCl, 3 mM EGTA, 10 mM HEPES, 1.2 mM MgCl2, pH adjusted to 7.2 with Tris-base). NSCC currents were recorded with a 500 ms ramp from − 80 to + 60 mV, and the data at 70 mV were used to construct current–time curves.

For measurement of BKCa channel currents, mASMCs (n = 5 cells/5 mice) were patched and held in bath solution III (5.4 mM CaCl2, 10 mM HEPES, 5.4 mM KCl, 0.8 mM MgCl2, 150 mM NaCl, pH adjusted to 7.2 with KOH). The pipette was filled with intracellular solution III (10 mM EGTA, 10 mM HEPES, 125 mM KCl, 6.2 mM MgCl2, 10 mM NaCl, pH adjusted to 7.2 with KOH). BK currents were recorded from a holding potential of − 80 to + 80 mV in 10 mV increments.

Measurement of cell viability in 16HBE cells

MTT assays were performed on 16HBE cells to analyze the effect of PP121 on cell viability. Approximately 2 × 103 16HBE cells (per well) were seeded in 96-well plates with 3.16 μM, 10 μM and 56.23 μM PP121. After 24 h or 48 h of incubation, 100 µg MTT reagent was added to each well and incubated for 4 h. Then, 150 µL of dimethyl sulfoxide (DMSO) was added, and the absorbance at 490 nm was recorded.

Measurement of respiratory system resistance

The respiratory system resistance (Rrs) was measured with a forced oscillation technique as previously described (Shi et al. 2020; Chen et al. 2019). In brief, mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (10 mg/kg). Then, the anesthetized mouse was tracheostomized and connected with the flexiVent system (SCIREQ, Montreal, PQ, Canada). Aerosolized ACh was gradually added to the system at increasing doses (3.125, 6.25, 12.5, 25 and 50 mg/mL). Then, dose‒response curves were generated, and the results were analyzed in Flexiware 8 software (SCIREQ, Montreal, PQ, Canada).

Histological analysis

Trachea and left lung specimens from 3 mice per group (control, asthma, Dex and PP121) were dissected and fixed in 4% paraformaldehyde (PFA, Servicebio, Wuhan, China). Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining were conducted by Servicebio, Wuhan, China. Stained sections (3 slides/3 mice) were photographed for further analysis.

Reverse transcription and quantitative real-time PCR

The right lung specimens of mice were cut into small pieces (< 1 mm3) and then homogenized in TRIzol® reagent with a hand-held homogenizer. Total RNA was extracted with an RNA extraction kit (TaKaRa, Otsu, Japan), and template cDNA was synthesized with a cDNA synthesis kit (TaKaRa, Otsu, Japan) according to the manufacturer’s protocol. The primer sequences were as follows:

Actin-F: 5′-AGAGGGAAATCGTGCGTGAC-3′

Actin-R: 5′-CAATAGTGATGACCTGGCCGT-3′

IL-4-F: 5′-AACGAAGAACACCACAGAGAGTG-3′

IL-4-R: 5′-CGATGAATCCAGGCATCGAAAAG-3′

IL-5-F: 5′-CGCTCACCGAGCTCTGTTG-3′

IL-5-R: 5′-CCAATGCATAGCTGGTGATTTTT-3′

MUC5AC-F: 5′-ATGGGCTGTGTTCCTGTGTC-3′

MUC5AC-R: 5′-CAGAACATGTGTTGGTGTGCAGTC-3′

MUC5B-F: 5′-GTGAGGAGGACTCCTGTCAAGT-3′

MUC5B-R: 5′-CCTCGCAGAAGGTGATGTTG-3′

TNF-ɑ-F: 5′-TGGAAGACTCCTCCCAGGTA-3′.

TNF-ɑ-R: 5′-ACGGCATGGATCTCAAAGAC-3′.

VEGF-F: 5′-ATGGATGTCTACCAGCGAAGCTACTG-3′

VEGF-R: 5′-GGTTTGATCCGCATGATCTGCA-3′

VEGFR2-F: 5′-CACCTGCCAGGCCTGCAA-3′

VEGFR2-R: 5′-GCTTGGTGCAGGCGCCTA-3′

Real-time PCR and melting curve analysis were carried out with SYBR Green qPCR Mix (Biosharp, Hefei, China) using the Applied Biosystems 7500 Fast Real-Time PCR System® (Applied Biosystems, Foster City, CA, USA) with the default program according to the manufacturer’s instructions. The mRNA expression levels of related genes were calculated utilizing the 2−ΔΔCt method and normalized to the expression levels of actin.

Western blotting

The lung tissues of mice were homogenized and lysed for 30 min in ice-cold RIPA buffer (Beyotime, Shanghai, China) with 1% PMSF (Beyotime, Shanghai, China). Then, the lysates were centrifuged at 16,000 ×g for 15 min at 4 °C. The supernatant was collected and quantified. Total proteins (20 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The membrane was blocked in Tris-buffered saline tween-20 (TBST, 8 g/L NaCl, 2.42 g/L Tris-base, 0.1% Tween 20) with 5% (wt/vol) powdered milk for 90 min and then incubated with primary antibodies (1:1000 dilution) at 4 °C overnight. After 3 washes with TBST, the proteins were blotted with horseradish peroxidase (HRP)-goat anti-rabbit IgG (CW0103S, CWBIO, Beijing, China) or HRP-goat anti-mouse IgG (EO32210-02, EARTHOX, Burlingame, CA, USA). The signal was detected by an ECL plus kit (Yeasen, Shanghai, China) and analyzed with Image Lab 3.0 (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. β-Actin was used as an internal control.

Statistical analysis

All the experimental results were analyzed using Student’s t test with Origin 8.0 (OriginLab, Northampton, MA, USA). All statistical data are presented as the means ± standard deviations (SD). p < 0.05 was considered statistically significant.