The data used to support the findings of this study are available from the corresponding author upon request.

Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS), is a severe inflammatory pulmonary process triggered by varieties of pathophysiological factors, among which endothelial barrier disruption plays a critical role in the progression of ALI/ARDS. As an inhibitor of myosin II, blebbistatin inhibits endothelial barrier damage. This study aimed to investigate the effect of blebbistatin on lung endothelial barrier dysfunction in LPS induced acute lung injury and its potential mechanism. Mice were challenged with LPS (5 mg/kg) by intratracheal instillation for 6 h to disrupt the pulmonary endothelial barrier in the model group. Blebbistatin (5 mg/kg, ip) was administrated 1 h before LPS challenge. The results showed that blebbistatin could significantly attenuate LPS-induced lung injury and pulmonary endothelial barrier dysfunction. And we observed that blebbistatin inhibited the activation of NMMHC IIA/Wnt5a/β-catenin pathway in pulmonary endothelium after LPS treatment. In murine lung vascular endothelial cells (MLECs) and human umbilical vein endothelial cells (HUVECs), we further confirmed that Blebbistatin (1 μmol/L) markedly ameliorated endothelial barrier dysfunction in MLECs and HUVECs by modulating NMMHC IIA/Wnt5a/β-catenin pathway. Our data demonstrated that blebbistatin could inhibit the development of pulmonary endothelial barrier dysfunction and ALI via NMMHC IIA/Wnt5a/β-catenin signaling pathway.

Acute lung injury (ALI) is a severe inflammatory disease of the lungs with high morbidity and mortality, characterized by respiratory distress, progressive hypoxemia, and pulmonary edema. At present, there are no effective therapeutic drugs and preventive strategies for ARDS in clinical practice. Although mechanical ventilation and restrictive fluid management have been widely used in lung protection with the deepening of the study on the pathological mechanism of ARDS, the mortality rate of ARDS is still up to 50% (Villar et al., 2016). The membrane barrier formed by endothelial cells, epithelial cells, and their fused basal layer in lung tissue maintains the rapid and effective exchange of gaseous substances between alveoli and blood vessels. Under physiological conditions, the barrier protects against the invasion of harmful substances such as particulate matter and microorganisms inhaled from the outside, playing a significant role in the body (Müller Redetzky et al., 2014). Under pathological conditions, the dysfunction of the pulmonary endothelial barrier can lead to increased vascular permeability, pulmonary edema, and the accumulation of a large number of inflammatory cells, thus affecting the exchange of gas and blood, resulting in hypoxemia and respiratory failure, which eventually induces acute respiratory distress syndrome (ARDS) (Thompson et al., 2017). Therefore, pulmonary vascular endothelial barrier dysfunction is the most critical pathophysiological feature in the early stage of ALI/ARDS (Millar et al., 2016; Bhattacharya and Matthay, 2013). It is an essential strategy for the clinical prevention and treatment of ALI/ARDS that maintains the integrity of pulmonary vascular endothelial barrier.

It is well known that adherens junction (AJs) is a significant component of the pulmonary endothelial junction, and VE-cadherin, as one of the crucial components of AJs, plays a vital role in maintaining the integrity of pulmonary endothelial barrier (Gong et al., 2015; Dong et al., 2018). Meanwhile, β-catenin involves regulating microvascular endothelial cell hyperpermeability, which is positively associated with endothelial VE-cadherin stability (Rho et al., 2017; Tharakan et al., 2012). Therein, the extracellular region of VE-cadherin is clustered between adjacent endothelial cells by homologous binding, and the intracellular part of VE-cadherin is mainly bound to the catenin family. The C-terminal area of VE-cadherin intracellular tail anchors α-catenin by binding to β-catenin, thereby promoting the interaction between VE-cadherin and the cytoskeleton. In the process of endothelial barrier dysfunction caused by stimulating factors, phosphorylation of the binding site of VE-cadherin and β-catenin leads to degradation of VE-cadherin and β-catenin, resulting in reduced expression of adhesive connexins on endothelial cell membranes. For example, in the process of LPS or TNF-α-induced lung endothelial barrier damage, the expression of β-catenin and VE-cadherin in lung tissue is significantly reduced (Liu et al., 2012; Gong et al., 2008; Xiao et al., 2005). These drugs can dramatically protect the pulmonary vascular endothelial barrier function and improve the survival rate of ARDS mice by inhibiting the expression of β-catenin and VE-cadherin (Yu et al., 2016; Chen et al., 2018). The Wnt/β-catenin signaling pathway is involved in regulating various physiological and biochemical functions. Meanwhile, the relationship between Wnt/β-catenin and ALI has been studied early (Hii et al., 2015). Among them, studies have shown that the expression of Wnt5a is elevated in the ALI model, and inhibiting Wnt5a attenuates the progress of ALI (Villar et al., 2014). In summary, the Wnt5a/β-catenin signaling pathway in endothelial cells is closely related to the pulmonary endothelial barrier.

Non-muscle myosin heavy chain IIA (NMMHC IIA), the primary subtype of type-II non-muscle myosin (NMM II), is a crucial protein regulating body growth and barrier function (Wang et al., 2020a, Wang et al., 2020b; Zhai et al., 2017). Previous studies showed that NMM II knockdown improves survival and therapeutic effects of implanted bone marrow-derived mesenchymal stem cells in lipopolysaccharide-induced acute lung injury (Wu et al., 2021). Meanwhile, konckout non-muscle myosin light kinase (nmMLCK),a factor closely related to NMMHC IIA, can improve lung endothelial cell inflammation and lung PMN infiltration stimulated by a variety of factors (Sun et al., 2021; Fazal et al., 2013). Furthermore, in NMMHC IIA knockout mice, endodermal abnormalities were observed during embryonic development, during which E-cadherin and β-catenin levels were significantly decreased, and the embryos died on day 7.5 (Conti et al., 2004). At the same time, it has been reported that the loss of NMMHC IIA in epithelial cells can spontaneously cause an increase in intestinal epithelial barrier permeability, which is mainly manifested as increased infiltration of lymphocytes, increased release of inflammatory factors, and decreased content of β-catenin, p120-catenin, and occludin protein-7. And in the experimental colitis model, intestinal barrier and mucosal damage were aggravated in the epithelial NMMHC IIA deficient mice compared with the control mice (Naydenov et al., 2016). These findings suggest that NMMHC IIA regulates the expression of β-catenin, thereby affecting barrier function. Moreover, NMMHC IIA is involved significantly in endothelial barrier regulation, and its overexpression leads to endothelial barrier dysfunction (Gong et al., 2021; Wu et al., 2021). Meanwhile, NMMHC IIA targeted drugs and endothelium-specific allelic knockout of NMMHC IIA can improve the LPS-induced decrease of VE-cadherin expression in lung tissues and play a role in protecting the lung endothelial barrier function.

Previous studies have shown that blebbistatin (a non-specific inhibitor of NMMHC IIA) ameliorates endothelial barrier-injury induced by OGD/R in vitro and inhibits cerebral I/R injury-induced BBB disruption in vivo. However, whether and how blebbistatin regulates pulmonary endothelial barrier disruption to inhibit LPS-induced ALI.

In the present study, we observed the effect of blebbistatin on LPS-induced pulmonary endothelial barrier dysfunction through modulating NMMHC IIA/Wnt5a/β-catenin signaling pathway in vivo and in vitro. These findings suggest that blebbistatin may have potential clinical application in the prevention and treatment of ALI and explain the underlying mechanism by which blebbistatin protects endothelial barrier function.

Lipopolysaccharide LPS L2880 was obtained from Sigma-Aldrich St. Louis, MO, USA. A myeloperoxidase (MPO) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-CD31 (AF3628) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-MYH9 (11128–1-AP) and anti-IgG (B900610) were purchased from Proteintech Group (Chicago, IL, USA). Anti-Wnt5a (sc-365,370), Anti-β-catenin (sc-7963), Anti-TLR4 (sc293072), and Protein A/G PLUS-Agarose (sc-2003) were obtained from Santa Cruz Biotechnology Inc. (Texas, CA, USA). Anti-VE-cadherin (ab33168) was provided by Abcam (Cambridge, MA, USA). Duolink®In Situ PLA® Probe Anti-Rabbit PLUS (DUO92002), Duolink® In Situ PLA® Probe Anti-Mouse MINUS (DUO92004), and Duolink®In Situ Detection Reagents Red (DUO92008) were obtained from Sigma–Aldrich (St. Louis, MO, USA).

The bicinchoninic acid (BCA) protein assay kit and phenylmethanesulfonyl fluoride (PMSF) were procured from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 488- and 594-conjugated secondary antibodies and Dynabeads™ Sheep Anti-Rat IgG (11035) were from Thermo Fisher Scientific (Waltham, MA, USA).

C57BL/6 male mice (18–22 g) were provided by the Experimental Animal Center of Yangzhou University (Certificate number was SCXK 2020–0011). After 3 days of adjustable feeding, the mice were randomly divided into four groups (n = 6): control group, control +Ble (5 mg/kg), ALI group, and ALI + Ble (5 mg/kg) groups. After inhalation anesthesia with isoflurane, the mice in the ALI group were challenged with LPS (5 mg/kg) by intratracheal instillation for 6 h. At the same time, sterile saline was used in the control group. Blebbistatin was intraperitoneally injected 1 h before LPS treatment. All mice were euthanized after LPS induction, and lung tissues were collected for the subsequent experiments. All experimental animal procedures were carried out according to the National Institutes of Health guidelines, and the Animal Ethics Committee approved the protocols of China Pharmaceutical University.

Lung tissues fixed in 4% paraformaldehyde were embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin (H&E). Pathological images were examined using an optical microscope (Nikon, Japan), and pathologists scored the extent of the injury.

Mice were intravenously injected with EB-albumin (50 mg/kg) via the tail vein at 4 h after LPS exposure and perfused 2 h later with PBS containing 5 mM EDTA-2Na on the right ventricle to remove the intravascular dye from the lung. Lung tissues were collected, flash-frozen in liquid nitrogen, and homogenized in a ten-fold volume of formamide. The homogenate was incubated at 60 °C for 18 h. After centrifugation at 5000 ×g for 30 min, the absorption of EB in the supernatant was measured at 620 nm, and the EB content was measured with a standard curve.

The BALF was centrifuged at 4 °C, 1500 rpm for 10 min, and the precipitate was resuspended in 500 μL PBS. Cell amounts of total cells were calculated using a blood analyzer (ADVIA 2120, Siemens, Germany).

According to the corresponding manufacturer's instructions, the MPO content in the BALF and lung were determined using assay kits.

The frozen sections (10 μm) of lung tissues were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 5% bovine serum albumin (BSA) and probed with the following primary antibodies overnight at 4 °C: CD31 (1:100 dilution, R&D Systems, USA), VE-cadherin (1:100 dilution, Abcam, USA), MYH9 (1:200 dilution, Proteintech, USA), Wnt5a (1:50 dilution, Santa Cruz Biotechnology Inc., USA), β-catenin (1:50 dilution, Santa Cruz Biotechnology Inc., USA). The next day the appropriate fluorescence-conjugated secondary antibodies were used: Alexa Fluor 594-donkey anti-goat IgG (A11058), Alexa Fluor 488-goat anti-mouse IgG (A21202), and Alexa Fluor 488-donkey anti-rabbit IgG (A21206) (1:500 dilution, Thermo Fisher Scientific, USA). Fluorescent images were visualized using confocal laser scanning microscopy (CLSM, LSM700, Zeiss, Germany).

Human umbilical vein endothelial cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. HUVECs were grown in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, ScienCell, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.0 g/L sodium bicarbonate. Cells were maintained at 37 °C with 95% humidity and 5% CO2.

MLECs isolation and culture MLECs were isolated as described previously(Lim et al., 2003).

In brief, the lung lobes harvested from four mice were carefully dissected out from bronchus and mediastinal connective tissues. After being washed in 25 ml Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum FBS Gibco, USA, the organs were minced with sterile stainless steel blades for 10 min and digested in 25 ml of collagenase at 37 °C for 1 h. Then the digested tissue was filtered through a clean 70 μm cell strainer Corning, 352,350 and centrifuged at 1500 rpm for 10 min at 4 °C. The cell pellet was resuspended in 0.1% BSA-PBS and incubated with CD31-coated magnetic beads (Dynabeads™ Sheep Anti-Rat IgG, 11,035, Thermo Fisher Scientific, USA) at room temperature for 15 min with end-over-end rotation. The bead-bound cells were recovered by a magnetic separator and washed with DMEM-10% FBS. Then the recovered cells were suspended and cultured in DMEM supplemented with 20% FBS, 1%MEM Non-Essential Amino Acids, 2.5% HEPES buffer solution (1 M) (Gibco, USA), endothelial cell growth supplement (0.1 mg/ml) (Sigma, USA), 100 U/mL penicillin and 100 U/mL streptomycins (Gibco, USA) at 37 °C in a humidified incubator of 5% CO2 and 95% air.

Transendothelial Electrical Resistance (TEER) Assays and EB-albumin leakage Assays.

Human umbilical vein endothelial cells were seeded on transwell inserts (0.4 μM pore, 6.5 mm diameter, Millipore, USA) for 7 days. The TEER of the monolayer was also measured daily with a Millicell-ERS voltmeter (Millipore, USA). Resistance values of multiple transwell inserts of an experimental group were calculated sequentially. The mean was expressed in the standard unit (Ω·cm2) after subtracting a blank cell-free filter value. The TEER of the monolayers was recorded when a stable resistance reading was achieved with triplicate measurements taken for each transwell. Blebbistatin (1 μmol/L) was added to the inner and outer chamber for 1 h, and 5 μg/mL LPS was added for 6 h. The inner chamber fluid was changed to 200 μL EB-albumin solution as the tracer, and the outer chamber fluid was changed to 600 μL 4% BSA solution and incubated in an incubator for 1 h. Collect the extracellular fluid, add 200 μL per well to the 96-well plate, measure the absorbance at 620 nm, and calculate the content of EB-albumin leakage according to the EB curve.

The second generation of lung endothelial cells was digested with trypsin solution and repeatedly blown to the cell suspension. After washing with PBS, cells were incubated with FcR blocking agent (Miltenyi, Germany) for 10 min at 4 °C. Then, cells were incubated with VE-cadherin (Miltenyi, Germany) for 30 min on ice, followed by 30 min secondary antibody incubation with PBS washing between the incubation periods. Samples were run on the FACScan flow cytometer(Miltenyi, Germany). Analyses were performed using FlowJo software. Surface antigen levels were expressed as the mean fluorescence intensity of cells.

The total protein extracted from HUVECs or lung tissues was quantified using a BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of total protein (30 μg) in each group were separated by SDS-PAGE gels and transferred to PVDF membranes. Then the membranes were blocked in 5% BSA for 2 h and incubated overnight at 4 °C with the following primary antibodies: VE-cadherin (1:1000 dilution, Abcam, USA), Wnt5a, and β-catenin (1:500 dilution, Santa Cruz Biotechnology, USA), MYH9 (1:8000 dilution, Proteintech, USA). After washing three times with TBST, the membranes were incubated with HRP anti-conjugated secondary antibodies for 1.5 h at room temperature. The bands were detected using an ECL kit (Vazyme Biotechnology, Nanjing, China) and analyzed with Image Lab™ Software.

To evaluate the amount of co-localization between two stains in the images, the plugin JaCoP in Image J (National Institutes of Health, Bethesda, MD, USA) was used in pixel matching co-localization analyses (Bolte and Cordelières, 2006). In brief, two different channels in one image were processed by Image J. Then all correlation-based co-localization were obtained, and information of microscope (CLSM, LSM700; Zeiss) and the emitted wavelength of fluorescence were entered into the JaCoP., After regulating the threshold, the co-localization was evaluated with the manders' co-localization coefficients.

Cell lysates were incubated with an equal amount of NMMHC IIA antibody or IgG antibody, followed by incubation with an equal amount of Protein A/G PLUS Agarose. Proteins were divided by SDS-PAGE followed by Western blot analysis.

A PLA kit (DUO92004, DUO92008, and DUO92002; Sigma–Aldrich) was employed to detect interactions between NMMHC IIA and TLR4 in MLECs, according to the manufacturer's protocols(Pierre and Scholich, 2010). Samples were incubated with primary antibodies for TLR4 and NMMHC IIA. Secondary antibodies, conjugated with PLA probes, were added to the reaction for subsequent ligation and rolling circle amplification. Images were observed under a CLSM (LSM700; Zeiss) and processed using ZEN imaging software.

All data were presented as mean ± standard deviations (SDs) and analyzed with GraphPad Prism software (version 8.0). Statistical significance was calculated with a one-way analysis of variance (ANOVA) followed by Dunnett's test. Differences were considered significant at P values