Sixteen adult female BALB/c mice, approximately 8 weeks of age, were purchased from the Laboratory Animal Center of Chongqing Medical University and maintained under specific pathogen-free conditions. The animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 8023, revised 1978). Mice were randomly divided into 2 groups: the control group and the asthma group. Mice in the asthma group were sensitized by intraperitoneal injection of 0.2 ml of sensitizing agents (in PBS as a solvent) on days 0 and 7. The sensitizing agents were 25 μg of ovalbumin (OVA) (A5503-5G, grade V, Sigma-Aldrich, Pudong, Shanghai, China) and 1 mg of aluminium hydroxide. Mice in the control group were intraperitoneally injected with an equal volume of PBS. Following intraperitoneal injection of sensitizing agents, mice in the asthma group received 10 ml of aerosolized stimulant twice a day on days 21–28. The aerosolization agent was 1% OVA formulated in PBS (0.1 g of OVA + 10 ml of PBS). Mice in the control group were aerosolized with 10 ml of PBS twice a day on days 21–28. Mice in both the control group and asthma group were sacrificed 24 h after the last OVA challenge.

The lungs were harvested from the mice and immediately fixed with 4% paraformaldehyde for 24 h. Then, the lung tissues were embedded in paraffin, cut into sections and prepared for immunohistochemistry according to the instructions of an SP-kit. After quenching endogenous peroxidase with 3% H₂O₂, tissue slides were incubated with 1% normal rabbit serum to minimize non-specific binding. Next, the slides were incubated with rabbit polyclonal anti-piezo1 antibody (1:200, Abcam, Pudong, Shanghai, China) at room temperature for 1 h in a dark, humidified box. After washing with PBS 3 times, slides were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, ZSGB-BIO, Beijing, China) for 30 min. After washing with PBS, slides were then incubated with 100 ng/ml DAPI for 10 min. After washing with PBS 3 times, the slides were embedded in 50% glycerol. Tissues were visualized using a confocal microscope (TCS SP2, Leica, Germany). Representative images were obtained with a digital camera and then processed with ImageJ 1.5.

Primary human small airway epithelial cells (HSAECs, PCS-301-010) were purchased from the American Type Culture Collection (Manassas, VA, United States). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS (Thermo Fisher Scientific, Inc., Waltham, MA, United States), penicillin (100 IU/ml), streptomycin (100 IU/ml), and bronchial epithelial cell growth agent (PCS-300040, ATCC, Manassas, VA, United States) at 37°C in a 5% CO₂ incubator and passaged when the cells were 70-80% confluent. Before experimental grouping, cells were counted and transferred to microporous membrane (aperture of 0.4 μm) inserts at the air-liquid interface (ALI) in equal amounts. The upper chamber containing the microporous membrane was then inserted into a 24-well plate. Each 24-well plate well contained 600 μl of the culture medium mentioned above. Cells were maintained at 37°C in a 5% CO₂ incubator, and the culture medium was changed every 2 days until a complete cell layer was formed. For further experiments, 24-well plates were separately placed in a homemade incubation chamber. A simple incubation chamber with a piston at the top for pressure regulation and a small pressure detection device was designed. The sides of the chamber were designed to contain pipes that connected a unidirectional valve for gas exchange. Cells were divided into 2 groups: the control (CTL) group and the pressure culture (PC) group. Cells in the CTL group were placed in the homemade incubation chamber without additional pressure on the piston above the chamber. Cells in the PC group were placed in the chamber with additional pressure on the piston above the chamber to maintain the pressure in the chamber at 10 cmH₂O (A schematic diagram of the chamber is shown in Supplementary Figure 1). Both chambers containing cells in the CTL group or PC group were placed in an incubator containing 5% CO₂ and 95% air.

The pGC-silencer-U6/Neo/GFP vector was purchased from Santa Cruz Biotechnology (Shanghai) Co., Ltd. (Pudong, Shanghai, China). siRNA against human piezo-1 and a control (CTL) siRNA were designed and chemically synthesized by Shanghai GeneChem Co., Ltd. (Shanghai, China). The piezo-1 siRNA sequence was 5′-GCCUCGUGGUCUACAAGAUTT-3′ (for the sense strand) and 5′-AUCUUGUAGACCACGAGGCTT-3′ (for the antisense strand). CTL siRNA was scrambled siRNA containing a GU content similar to that of piezo-1 siRNA. Prior to transfection, cells in exponential growth phase were plated in 6-well cell culture plates and incubated at 37°C for 12 h. Following 3 washes with PBS to avoid any interference caused by antibiotics or serum, the cells were transfected by FuGENE^® HD reagent (E2311, Promega Corporation, Madison, WI, United States) with piezo-1-specific siRNA or CTL siRNA (20 μg of DNA:60 μl of transfection reagent) at 22°C for 15 min according to the manufacturer’s protocol. Following transfection, cells were washed with PBS 3 times and incubated in complete culture medium for 24 h at 37°C prior to western blotting and subsequent experiments.

Female BALB/c mice were anesthetized intraperitoneally with midazolam before siRNA transfection in vivo. A fine catheter was placed into the nostril. Through the catheter, 60 μl RNAse-free water (containing 60 μg siRNA) were perfused at a rate of 6 μl/min using a micro syringe pump. Oxygen was supplied during the whole procedure in order to avoid hypoxia. The mice were perfused thrice (every second day). Seventy -two hours after the last transfection, bronchoalveolar lavage fluid (BALF) was collected, and centrifuged. The supernatant was prepared for IgM detection according to the instructions of mice IgM ELISA kits (Westang, Shanghai, China). The left lung was digested for western blotting.

Transepithelial electrical resistance (TER) was measured with a Millicell-ERS system (Millipore Co.) For measurements, the basal electrode was covered with equilibrated ALI medium. Then, 50 μL of ALI medium was added to cover the microporous membrane surface. The apical electrode was placed into the apical liquid. The measurements were performed immediately after positioning of the apical electrodes. The TER values (Ω × cm²) were calculated with the following equation: (TER_sample – TER_blank) × surface area.

The paracellular permeability was measured using a fluorescein isothiocyanate (FITC)-dextran (4 kD, Sigma-Aldrich) permeation test. Briefly, the upper chamber containing a microporous membrane was washed with PBS 3 times. Phenol red (0.2 ml) and serum-free DMEM (containing 0.5 mg/L FITC-dextran) were added to the upper chamber. Phenol red (1.0 ml) and serum-free DMEM (containing 0.5 mg/L dextran without FITC conjunction) were added to the lower chamber. Then, the cells were incubated in a dark incubator with 5% CO₂ and 95% air for 2 h. Liquid samples (0.1 ml and 0.5 ml) were collected from the upper and lower chambers, respectively, for fluorescence measurement. The excitation and emission wavelengths were 488 and 525 nm, respectively. The concentrations of FITC-dextran in the upper and lower chambers were calculated based on a fluorescence standard curve. The permeability coefficient of dextran (Pd) was calculated with the following formula: Pd(cm/s) = ([A]/T) × (1/A) × (V/[L]), where [A] is the concentration of FITC-dextran in the lower chamber, T is the time(s), A is the area of the bottom of the upper chamber (cm²), V is the volume of liquid in the lower chamber, and [L] is the concentration of FITC-dextran in the upper chamber.

The microporous membrane covered with HSAECs was cut from the upper chamber and placed into a 24-well plate. Each microporous membrane was loaded with 5 μM Fura 2-AM (Invitrogen, Carlsbad, CA, United States) Cells were then incubated in the dark for 1 h at 37°C and washed with PBS for 20 min to remove residual dye. Fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Data were acquired and are expressed as fluorescence ratios (F340/F380).

The expression levels of proteins of interest in the cellular lysate supernatants were detected by western blotting. Generally, cells were washed with PBS 3 times and lysed on ice for 20 min with IP lysis buffer (P0013, Beyotime Institute of Biotechnology, Songjiang, Shanghai, China). The lysates were then centrifuged at 20,000 × g for 15 min at 4°C to remove nuclei and intact cells. Supernatants were standardized for equal protein concentration (5 μg/μl) using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Songjiang, Shanghai, China). Following separation by SDS-PAGE, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% skimmed milk for 1 h at room temperature. PVDF membranes were then incubated with primary rabbit antibodies against ZO-1 (Abcam, ab96587) at a 1:1000 dilution, piezo-1 (Abcam, ab128245) at a 1:500 dilution, claudin-18 (Abcam, ab203563) at a 1:2000 dilution, occludin (Abcam, ab235986) at a 1:2000 dilution, or β-actin (ZSGB-BIO, Beijing China) at a 1:1000 dilution at 4°C overnight. The PVDF membranes were washed with TBST 3 times and subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (ZSGB-BIO, Beijing China) at a 1:1000 dilution for 2 h. Blots were visualized using enhanced chemiluminescence following the manufacturer’s specifications (KeyGen, Nanjing, China). The intensity of each band was measured using a Fluor-S multi-imager and Quantity One software (Bio-Rad, Hercules, CA, United States). The expression levels of the proteins of interest were normalized to that of β-actin.

The microporous membrane covered with HSAECs was cut from the upper chamber and placed into a 24-well plate. After 3 washes with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and washed again with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then washed 3 times with PBS again. The cells were then blocked in 5% goat serum for 60 min and incubated with rabbit primary antibodies against ZO-1 (Abcam, ab96587) at a 1:200 dilution, claudin-18 (Abcam, ab203563) at a 1:200 dilution, and occludin (Abcam, ab235986) at a 1:100 dilution at 4°C overnight. After washing with PBS, the cells were incubated for 60 min with Alexa Fluor^® 594-conjugated goat anti-rabbit IgG (ZF-0516, ZSGB-BIO, Beijing China) at a 1:200 dilution in the dark. After washing with PBS, membranes were embedded in 50% glycerol. Cells were visualized using a confocal microscope (TCS SP2, Leica, Germany). Representative images were obtained with a digital camera and then processed with Adobe Photoshop CS2.

Normally distributed data are described using the mean and standard deviation (SD), and differences between two groups were analyzed using Student’s t-test. More than two groups were compared using one-way analysis of variance (ANOVA), followed by Bonferroni analysis. Non-normally distributed data are described using the median and interquartile range (IQR), and differences between groups were analysed using the Mann-Whitney test. All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) version 17 software (SPSS, Inc.). All statistical graphs were drawn using GraphPad Prism 8.

Immunochemistry and confocal microscopy were applied to visualize Piezo-1 in bronchial epithelial cells from model mice. Piezo-1 was expressed in the bronchial epithelium of normal and asthmatic mice. The Piezo-1 levels found in the immunohistochemical experiment in the bronchial epithelium are expressed as the integrated optical density/area (IOD/area). The IOD/area for Piezo-1 in the bronchial epithelium was slightly higher in asthmatic mice than in normal control mice (21.6 ± 5.5 vs. 14.5 ± 3.6; P < 0.05) (Figure 1). To determine the effect of siRNA transfection in lung by airway perfusion, lung tissue was digested for western blot detecting for piezo-1. We found a reduction in the piezo-1 protein content to about 32.1% 72 h after transfection with piezo-1siRNA (Figure 2A). Later, we examined the degree of airway permeability using the detection of large protein IgM (Frampton et al., 1999) in bronchoalveolar lavage fluid (BALF). The level of IgM in the BALF was markedly elevated in the asthmatic mice compared to the normal control mice. Piezo-1siRNA airway transduction partially abolished the increased IgM in the BALF in asthmatic mice (Figure 2B).

In an in vitro study, a conventional MTT assay was performed to evaluate cell viability after exposure to pressure for different durations. According to the MTT assay, an extra 10 cmH₂O of pressure for 16 h caused a significant reduction in HSAEC cell viability. Thus, 12 h was selected as the appropriate exposure time for the following experiments (Figure 3).

Airway epithelial function was estimated by TER (Ω × cm²) and FITC-dextran permeability tests as described in the experimental section. Exposure to 10 cmH₂O of additional pressure caused a time-dependent reduction in TER in HSAECs. The TER value began to decrease significantly after 4 h of exposure to additional pressure. In the first 4 h, the TER value decreased to 77.3% of the initial level. Meanwhile, the Pd increased by 27.7% of the initial level. At 12 h of exposure, the TER value had decreased to approximately 30.9% of the initial level, and the Pd had increased to 3.3-fold the original value (Figure 4).

The additional 10 cmH₂O of pressure evoked an increase in [Ca²⁺]_i in HSAECs. Following exposure to high-pressure culture for 12 h, the F340/F380 ratio increased from 1.23 ± 0.15 at baseline to 1.75 ± 0.25 (P < 0.05 vs. baseline). Pretreatment with the piezo-1 inhibitor GsMTx4 partially attenuated the increase in F340/F380 after high-pressure culture. The F340/F380 ratio increased to 1.47 ± 0.14 in GsMTx4-pretreated cells. To further demonstrate the crucial role of piezo-1 in pressure-induced calcium influx in HSAECs, we constructed piezo-1-knockdown (piezo-1 KD) HSAECs with piezo-1-specific siRNA transfection (as mentioned in the ‘Materials and Methods’ section). The efficiency of piezo-1 downregulation was verified by western blotting, as shown in the ‘Supplementary Materials’ section (Supplementary Figure 2). In piezo-1-specific siRNA-transfected cells, the F340/F380 value increased to 1.39 ± 0.14 after culture under extra pressure (Figure 5). These results indicate that extra pressure triggers a piezo-1-dependent increase in [Ca²⁺]_i manner.

Occludin and ZO-1 were detected as they play an indispensable role in regulating barrier tightness and a critical role in the establishment of airway belt-like TJs, respectively (Furuse, 2010; Matter and Balda, 2014). Claudin-18, a lung-specific claudin that is highly associated with airway epithelial barrier dysfunction in asthma, was also detected (Sweerus et al., 2017; Yang et al., 2019). To further assess whether the disruption in occludin, ZO-1, and claudin-18 occurred because of additional pressure exposure, we first investigated cellular occludin, ZO-1, and claudin-18 by western blot assay. The results indicated that an additional 10 cmH₂O of pressure promoted the degradation of occludin, ZO-1, and claudin-18 in HSAECs (Figure 6, vertical panels 1 and 2, ‘CTL’ vs. ‘PC’). To visualize the distribution of TJ proteins in epithelial HSEAC cells, cellular immunofluorescence and confocal microscopy were performed. High-pressure exposure decreased intracellular TJs and caused discontinuous occludin, ZO-1, and claudin-18 staining in HSAECs (Figure 7, ‘CTL’ vs. ‘PC’). These findings were consistent with our western blot findings.

According to the results of western blot assessment, pretreatment with GsMTx4, a piezo-1 inhibitor, or Piezo-1 siRNA transfection partially attenuated the degradation of occludin, ZO-1 and claudin-18 (Figure 6). As visualized by cellular immunochemistry, GsMTx4 treatment or Piezo-1-specific siRNA transfection attenuated intracellular occludin, ZO-1 or claudin-18 degradation and partially preserved the continuity of intercellular TJ bands (Figure 7). These results indicated a crucial role of Piezo-1 in the degradation of small airway TJ proteins under additional pressure stimulation. Since Ca²⁺ is an important second messenger with crucial roles in the regulation of TJs and because activated piezo-1 allows Ca²⁺ influx (Gangwar et al., 2017; Du et al., 2020), we detected the influence of additional pressure on TJs following pretreatment with the Ca²⁺ chelator BAPTA-AM (10 μM). Similarly, pretreatment with the Ca²⁺ chelator partially reduced the pressure-induced degradation of occludin, ZO-1, and claudin-18 (Figure 6) and preserved the continuity of TJ bands in HSAECs (Figure 7). As calpain, a calcium-dependent protease, has been demonstrated to play an essential role in cytoskeletal degradation and tight junction impairment (Wang et al., 2012; Alluri et al., 2016), we inhibited calpain protease activity with ALLN (30 μM). ALLN partially abolished the degradation of occludin, ZO-1, and claudin-18 induced by additional pressure (Figure 6). As visualized by cellular immunochemistry, HSAECs pretreated with ALLN retained several TJ bands under high-pressure exposure, which indicated that calpain also participated in the degradation of occludin, ZO-1, and claudin-18 (Figure 7).

As mentioned above, the TJ function in HSAECs was estimated by TER and FITC-dextran permeability tests. Exposure to extra pressure for 12 h caused a reduction in TER (59.1 ± 15.7 Ω⋅cm²) as mentioned above. Pretreatment with GsMTx4 or piezo-1 siRNA to inhibit activation of the piezo-1 channel attenuated the reduction in TER by extra-pressure exposure. The TER of HSAECs pretreated with the Ca²⁺ chelator BAPTA-AM decreased to 139.1 ± 30.3 Ω⋅cm², which was significantly higher than that of untreated cells exposed to extra pressure. Pretreatment with the calpain protease inhibitor ALLN also partially reduced the decrease in TER in HSAECs under high-pressure exposure (Figure 8A). A further FITC-dextran permeability test was performed to estimate paracellular permeability. The results were highly consistent with the TER outcomes (Figure 8B). These results strongly indicate that a mechanism involving Piezo-1/[Ca²⁺]_i/calpain participates in the disruption of small airway epithelial cells in vitro under extra pressure.