Anti-MLCK monoclonal antibody (cat. no. ab34829), anti- α-SMA monoclonal antibody (mouse; cat. no. ab62736) and anti-collagen-I monoclonal antibody (mouse; cat. no. ab48262) were provided by Abcam (Cambridge, UK), while goat monoclonal GAPDH antibody (cat. no. AG019-1) was from Bioworld Technology Inc. (St Louis Park, MN, USA). Horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit cat. no. ZB-2301) were purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). ML-7 and ovalbumin (OVA) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). IL-4 (cat. no. ZC-23216), IL-5 (cat. no. ZC-23228), IL-13 (cat. no. ZC-23211), IL-25 (cat. no. ZC-23312) and IL-33 (cat. no. ZC-23142) ELISA kits were from Proteintech Group, Inc. (Chicago, IL, USA). Polyvinylidene fluoride (PVDF) membranes were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). The BX51T light microscope and High-Speed Centrifuge PIC017 were respectively provided by Olympus Corp. (Tokyo, Japan) and Heraeus Corp. (Berlin, Germany).

All animal experiments and surgical procedures were approved by the Institutional Animal Care and Use Committee of Shandong University (Shangdong China). A total of 45 healthy female BABL/c mice (weight, 20–28 g; age, 6–8 weeks) were obtained from the Animal Centre of Shandong University. The animals were bred in a temperature- and humidity-controlled room, and given ample food and tap water for the duration of the experimental session. Mice were allowed a week to adapt to the environment prior. The mice were randomly divided into three groups: The control group (PBS treatment), the OVA group (OVA challenge+PBS treatment) and the OVA+ML-7 group (OVA challenge+ML-7 treatment). The asthmatic models were established by challenge with OVA. Mice in the OVA and OVA+ML-7 groups were sensitized on days 1, 8 and 15 by intraperitoneal injection with 100 µg OVA adsorbed to 1 mg aluminum hydroxide. After the last sensitization, the mice were treated with 1% OVA aerosol inhalation for up to 30 min per day for 7 consecutive days. The mice in the control group were exposed to an equivalent amount of PBS instead. In the OVA+ML-7 group, mice were given a daily intraperitoneal injection of ML-7 (0.5 mg/kg in 0.5 ml PBS) prior to OVA inhalation challenge for the next 7 days. All of the mice were euthanized at 24 h after the final challenge.

After the mice were euthanized, collection of BALF was performed immediately with isotonic sterile PBS lavage for four times (0.5 ml each time). From each mouse, 2 ml BALF was collected separately. The cellular influx in the BALF was examined by using a cell counting plate. After centrifugation at 3,000 × g for 8 min at 37°C. After removing the supernatant, the precipitation cells were evenly spread on a slide and stained with 0.5 ml Wright-Giemsa reagent, which contains 0.5 g Wright pigment, 0.5 g giemsa pigment and 500 ml methanol for 1 min at 37°C. Cells were identified based on morphological features and counted in randomly selected areas of the slide using light microscopy. The levels of IL-4, IL-5, IL-13, IL-25 and IL-33 were measured in the supernatant of BALF using ELISA kits.

After the last challenge, the right lungs were removed from the chest cavity and immersed in 10% neutral buffered formalin at 25°C for 24 h. Lung samples were then embedded in paraffin and sectioned by using a microtome (5-µm). After dewaxing in xylene, rehydrating in graded ethanol, the sections were stained with haematoxylin at 25°C for 3 min and eosin at 25°C for 30 sec to assess the infiltration of inflammatory cells. All images were assessed at a magnification of ×20.

The left eye of the mice was removed for blood collection. After low-temperature centrifugation at 3,000 × g at 4°C for 8 min, the serum was obtained to determine the levels of OVA-specific (OVA-s) IgE using an ELISA kit (cat. no. YP-45821) purchased from Zhongshan Jinqiao Biotechnology Co., Ltd., within 24 h after final challenge. The levels of IL-4, −5, −13, −25 and −33 in the supernatant of BALF were assayed with ELISA kits according to the manufacturer's protocols.

The left lung tissue (10 mg) was minced and lysed in ice-cold radioimmunoprecipitation buffer containing1 mM phenylmethanesulfonyl fluoride, 1.5 M NaCl, 10% NP-40, 10 mM EDTA, 0.5 M Tris-HCl, (pH 7.4), 2.5% deoxycholic acid, and protease inhibitors (1:100 dilution) and protease inhibitors in order to isolate total protein, which was used to analyze the content of α-SMA and collagen-I. A total of 100 µg protein was suspended in 5X reducing sample buffer, followed by boiling for 3 min, and the proteins were centrifuged at 15,000 × g for 30 min at 4°C to obtain the supernatant for western blot analysis. The concentration of total protein was assessed by BCA protein assay, 10 µl protein loaded per lane for separation by 8% SDS-PAGE acrylamide gel and subsequent transfer to a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). After a blocking at 37°C for 2 h using 5% non-fat dried milk, and the membrane was incubated with rabbit polyclonal α-SMA (1:1,000 dilution in TBST), collagen-I (1:500 dilution in TBST) and GAPDH antibody (1:3,000 dilution) at 4°C overnight. After washing with Tris-buffered saline containing Tween-20, (TBST; pH 8.0) three times, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG as the secondary antibody (1:6,000 dilution in TBST) at room temperature for 2 h. The ECL method (ECL kit; Solarbio Science & Technology, Beijing, China) was used to develop the bands, and the gray value was determined with Image-Pro Plus 7.0 image analysis software (Media Cybernetics, Rockville, MD, USA). The expression levels of α-SMA and collagen-I were normalized to those of GAPDH.

RT-qPCR was used to analyze the transcript levels of MLCK, α-SMA and collagen-I. The total RNA was isolated from the lung tissues using TRIzol reagent based on the manufacturer's protocol. Then RNA samples were treated with DNase I (Takara Bio Inc., Otsu, Japan) for 30 min at 37°C to remove genomic DNA contamination. Thereafter, complementary (c)DNA was generated via RT reaction by using a PrimeScript first-strand cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. PCR was performed with a Bio-Rad CFX96 Touch q-PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reaction mixture contained primers [1.0 µl of a 10 µmol/l stock including forward and reverse (Table I)], 0.15 µl 1 µg/µl Taq DNA polymerase, 5.0 µl 5X PCR buffer, 5.0 µl of a 1 µg/µl solution of cDNA template and 13.85 µl 0.1% diethylpyrocarbonate-treated water. The PCR process is as follows: Initial denaturation at 95°C for 60 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 65°C for 30 sec, and extension at 72°C for 120 sec. Fluorescence signals were collected at each annealing and elongation step. The relative transcriptional levels were finally calculated from quantification cycle values using the 2^−ΔΔCq method (29).

All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. The levels of cytokines and the quantity of MLCK were analyzed by one-way analysis of variance followed by Dunnett's test. Differences between groups were considered statistically significant at P<0.05. All experiments were repeated three times unless otherwise stated.

The murine model of airway inflammation was established through repetitive OVA sensitization. Thereby, an asthmatic phenotype similar to that observed in human asthma was established. Histological analysis of H&E-stained lung tissue indicated that OVA induced inflammatory cell infiltration and pathological transformation in the lung tissues. However, treatment of OVA-challenged mice with ML-7 significantly alleviated the degree of tissue inflammation and infiltration. Furthermore, the pathological changes were milder (Fig. 1). The serum levels of OVA-s IgE in the OVA group were significantly higher compared with those in the control group (131.46±10.72 vs. 25.37±4.89 ng/ml; P=0.004; Fig. 2A). Based on the histological and serological analysis, it was determined that the model was successfully established. The total number of inflammatory cells, including neutrophils, eosinophils, macrophages and lymphocytes in the BALF, are hallmarks of inflammation in the mice at the cellular level. Particularly eosinophils are generally considered the hallmark of the onset of asthma. Compared with the control group, the total number of cells and eosinophils increased in the OVA group (P<0.001 for each comparison; Fig. 2B and C). Of note, the percentage of eosinophils in the OVA+ML-7 group was significantly decreased compared with that in the OVA group (0.012±0.007 vs. 0.068±0.01; P=0.0075; Fig. 2C). The total number of cells and the percentage of neutrophils in the OVA + ML-7 group was also decreased compared with that in the OVA group (Fig. 2B and D). This partly reflected that ML-7 inhibited the accumulation of eosinophils in asthma-like airway inflammation.

MLCK has been reported to be a key mediator that promotes the production of other cytokines. To investigate the correlation among the mediators (IL-4, −5, −13, −25 and −33), their levels in the BALF were assessed. The levels of Th2 cytokines in the OVA group were significantly increased compared with those in the control group (P<0.05 for each comparison; Fig. 3). However, treatment with ML-7 attenuated the OVA-induced increases in the cytokines (P<0.05 for each comparison; Fig. 3). This proved that administration of ML-7 inhibited Th2-associated inflammatory cytokine release.

In asthmatic airway remodeling, α-SMA and collagen-I are significant pathogenic factors. The level of α-SMA and collagen-I in the lung tissue of mice in the OVA+ML-7 group was significantly reduced compared with that in the OVA group as demonstrated by western blot analysis (P<0.001 for each comparison; Fig. 4A and B). The changes in α-SMA and collagen-I expression levels were further confirmed by RT-qPCR analysis of lung tissue. The expression of mRNA of α-SMA and collagen-I in asthmatic mice was significantly higher than that in the control group (P<0.05 for each comparison; Fig. 4). In addition, the mRNA expression of α-SMA and collagen-I in the OVA+ML-7 group was significantly lower than that in the OVA group (P<0.05 for each comparison; Fig. 4). The above results demonstrated that inhibition of MLCK markedly inhibited airway remodeling in the OVA-induced asthma model.

MLCK has a key role in the regulation of smooth muscle contraction, and is mainly distributed in the bronchus where it stimulates smooth muscle cells. The expression of MLCK in the lung tissue of mice was examined using RT-qPCR. The results revealed that the expression of MLCK mRNA in mice in the OVA group was significantly higher than that in the control and OVA+ML-7 groups (5.05±0.72 vs. 1.21±0.37; P=0.001; Fig. 4C). Compared with that in the control group, the expression of MLCK in the OVA+ML-7 group was not significantly different (1.91±0.52 vs. 1.21±0.37; P=0.95; Fig. 4C), indicating that ML-7 treatment completely inhibited OVA-induced upregulation of MLCK.