Montelukast sodium, OVA (grade V) and bovine serum albumin (BSA) were purchased from MilliporeSigma. Aluminum hydroxide gels were purchased from Thermo Fisher Scientific, Inc. The alcian blue & periodic acid-Schiff (AB-PAS) staining kit and TUNEL cell apoptosis kit were purchased from Solarbio Science & Technology Co., Ltd. The Masson's trichrome staining kit was purchased from Maxim Biotech, Inc. The hematoxylin & eosin (H&E) staining kit and Wright-Giemsa stain kit were purchased from Abcam. Primary antibodies targeting CysLTR1 (cat. no. 27372-1-AP), Nrf2 (cat. no. 16396-1-AP) and GAPDH (cat. no. 60004-1-Ig) were purchased from Proteintech Group. Inc. The RIPA lysis buffer was purchased from Wuhan Boster Biological Technology, Ltd. The ECL reagent was purchased from Tanon Science and Technology Co., Ltd. and the BCA reagent was purchased from Thermo Fisher Scientific, Inc. The Rat IgE ELISA kit (cat. no. EKF58258) was purchased from Biomatik. The Rat IL-17 (cat. no. KTE9005) and IL-4 (cat. no. KTE9003) ELISA kits as well as HRP-conjugated secondary antibodies (cat. nos. A21020 and A21010) were purchased from Abbkine Scientific Co., Ltd. The reduced glutathione (GSH)/oxidized glutathione (GSSG) Ratio Fluorometric Detection Assay Kit (cat. no. 50120ES70) was purchased from Shanghai Yeasen Biotechnology Co., Ltd. The total RNA extraction kit (cat. no. LS1040) was purchased from Promega Corporation. The First Strand Kit and QuantiFast SYBR^® Green PCR Kit were purchased from Qiagen GmbH.

A total of 30 Sprague-Dawley male rats (age, 8-10 weeks) weighing 240±5 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All rats were housed in specific pathogen free cages under standard laboratory conditions, which included a temperature of 22-25˚C, a relative humidity of 40-55% and a 12/12 h light/dark cycle with free access to water and food. Following acclimation, the rats were randomly assigned to five treatment groups: i) The control (Group I); ii) model (Group II); iii) low-dose (1 mg/kg) montelukast sodium (Group III); iv) medium-dose (4 mg/kg) montelukast sodium (Group IV); and v) high-dose (30 mg/kg) montelukast sodium (Group V) groups, with 6 rats/group. OVA was dissolved in 4% aluminum hydroxide gels to prepare an OVA solution (2 mg/ml). The OVA-induced asthmatic rat model was established as previously described (31,32) with some amendments. On days 0 and 14, rats in Groups II-V were sensitized with an intraperitoneal injection of OVA solution (0.5 ml/rat). Rats in Group I were administered an equal volume of saline. From the 15th day, all rats apart from the control group, were administered with inhaled OVA aerosol (10 mg/ml; dissolved in saline; 30 min/day) for 3 weeks. Before the OVA challenge (from the 15th day onwards), rats in Group I and II were given 10 ml/kg/day of saline by gavage, while rats in Groups III-V were given montelukast sodium by gavage at doses of 1, 4 and 30 mg/kg/day, respectively. The gavage procedures were continuously conducted until sample collection. The experiment duration was 5 weeks. The heart rate of the animals was monitored each day using a polyethylene cannula (PE 50) filled with heparinized saline (100 IU/ml) inserted into the right carotid artery. The cannula was connected to a transducer, and the signal was amplified by bioamplifier and an acquisition data system (AD Instruments Pvt. Ltd. with software LabChart 7.3; AD Instrument Pvt. Ltd). Body weight was monitored each week. Throughout the experiment, the aim was to minimize the utilization of animals and alleviate their distress as much as possible. According to the analgesic methods described in previous studies, intraperitoneal injection of 5 mg/kg tramadol has been reported to be a safe and effective analgesic in rats and mice (33,34). In preliminary experiments, 5 mg/kg tramadol was found to effectively alleviate pain in rats without any side effects or mortality (data not shown). Therefore, 5 weeks later, all animals received an intraperitoneal injection of tramadol (5 mg/kg) as an analgesic method to minimize pain, suffering and distress. The rats were housed individually in a polycarbonate cage and allowed to recover on a heating pad to maintain a body temperature of 37.5±0.5˚C. In addition, the rats were monitored for any signs of fatigue and stress. Researchers were trained to apply the humane endpoints, if any animal exhibited features of a compromised welfare. The humane endpoints included rapid weight loss (>20% of normal body weight) and/or rapid or labored breathing. No animals died naturally during the experiments and all of the rats were euthanatized by an intraperitoneal injection of pentobarbital sodium overdose (200 mg/kg) on day 35. Death was confirmed by cardiac and respiratory arrest and a lack of response to tail clamping. The bronchoalveolar fluid lavage (BALF) was obtained by washing the lungs and subsequent analysis involved the collection of lung tissues and airway tissues. To analyze the level of OVA-specific IgE in serum, blood samples (300 µl) were collected from rats by cardiac puncture. Whole blood was collected and left to coagulate at room temperature for at least 30 min, and then centrifuged at 1,000 x g for 10 min at 4˚C. The serum samples were stored at -20˚C until use. The experiments in the present study were carried out by three skilled technicians who were unaware of the experimental design and purpose. Animal experiments followed the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and received approval from the Ethics Committee of Jinhua Polytechnic (approval no. 20221221; Jinhua, China).

The airway tissues were fixed using 4% paraformaldehyde (48 h; 4˚C), embedded in paraffin and cut into 5-µm sections. Subsequently, sections were stained with hematoxylin for 5 min and eosin for 2 min at room temperature using a H&E staining kit. Masson's staining was performed using a Masson's trichrome staining kit in accordance with the manufacturer's protocol to observe collagen deposition at room temperature for a total of 15 min (Wiegert's iron hematoxylin, 8 min; Biebrich scarlet, 5 min; aniline blue, 2 min) at room temperature. Based on the manufacturer's protocol of the AB-PAS staining kit, sections were stained with alcian blue for 30 min and periodic acid Schiff for 15 min at room temperature. The pathological structure of airway tissues was observed using a BX53 light microscope (Olympus Corporation).

According to the manufacturer's instructions, the GSH/GSSG ratio, levels of IL-4 and IL-17 in lung tissues and serum IgE concentration were determined using corresponding commercial kits (35).

Total RNA from lung tissues was extracted using a total RNA extraction kit. The concentration of total RNA was measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Total RNA (500 ng) was reverse transcribed into cDNA at 42˚C for 45 min using a First Strand Kit and RT-qPCR was performed using the QuantiFast SYBR^® Green PCR Kit, according to the manufacturer's instructions. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 3 min; followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 30 sec and elongation at 72˚C for 1 min, as well as a final extension at 72˚C for 5 min. To determine gene expression levels, the 2^-ΔΔCq method was used (36) and results were normalized to GAPDH as a reference gene (37). The primer sequences used are presented in Table I.

The BALF samples were stained using a Wright-Giemsa stain kit, and the eosinophil, lymphocyte and macrophage counts were recorded under a light microscope (Olympus Corporation) and analyzed using Image-Pro-Plus (version 6.0; Media Cybernetics). The inhibitory activity (%) was evaluated as the following formula: (1-A/B) x100%. A represents the number of inflammatory cells in different groups of montelukast sodium; B represents the number of inflammatory cells in the model group.

Lung tissue sections (5 µm) underwent deparaffinization in xylene for 10 min at room temperature and rehydration with descending concentrations of ethanol (100, 95 and 70% for 3-5 min each), followed by antigen retrieval in heated citrate buffer (10 mM; pH 6.0) at 80˚C for 25 min. Subsequently, the samples were washed three times with PBS before being permeabilized using 0.5% TritonX-100 in PBS. The sections were then blocked with 5% BSA at room temperature for 1 h. Next, the samples were incubated with primary antibodies against Nrf2 (1:50) overnight at 4˚C and the corresponding secondary antibodies (1:200) for 2 h at room temperature. Finally, after staining with DAPI (1 µg/ml) at room temperature for 15 min, the samples were imaged using a fluorescence microscope (Olympus Corporation). Image-Pro-Plus (version 6.0; Media Cybernetics) was adopted to analyze the fluorescence intensity.

Apoptosis of lung tissues was determined in accordance with the experimental procedures outlined in the manufacturer's guidelines for the TUNEL kit. In brief, the lung tissues were fixed using 4% paraformaldehyde (48 h; 4˚C), embedded in paraffin and cut into 5-µm sections. The deparaffinized tissue sections were incubated with 3% hydrogen peroxide in methanol for 10 min at 25˚C in the dark, washed three times with PBS and incubated with 0.1% Triton X-100 in freshly prepared 0.01% sodium citrate for 8 min at 25˚C. Tissue sections were then incubated with proteinase K working solution for 25 min at 37˚C and washed three times with PBS (pH 7.4) for 5 min each. A total of 50 µl TUNEL reagent was added to each sample and incubated at 37˚C for 60 min. The sections were washed three times with PBS (pH 7.4) and then cell nuclei were counterstained with 2 µg/ml DAPI solution at room temperature for 10 min in the dark and mounted with 50 µl anti-fade mounting medium. TUNEL-positive cells were observed in five randomly-selected fields using a fluorescence microscope (Olympus Corporation) and analyzed using Image-Pro-Plus (version 6.0; Media Cybernetics).

The RIPA lysis buffer was employed for the extraction of total proteins from lung tissues, followed by quantification using a BCA kit. Subsequently, a total of 50 µg of protein/lane was separated by 10% SDS-PAGE and proteins then were transferred to polyvinylidene fluoride membranes. Following blocking with 5% nonfat milk for 2 h at 25˚C, membranes were incubated with primary antibodies against CysLTR1 (1:4,000), Nrf2 (1:7,000) and GAPDH (1:50,000) overnight at 4˚C. Then, tris-buffered saline with 0.05% Tween-20 was used to wash the membranes three times. Subsequently, at room temperature, the HRP-conjugated secondary antibodies (1:10,000) were incubated with samples for 1 h. The signals were detected using an ECL kit (Beyotime Institute of Biotechnology) and blots were quantified under a Gel-Proanalyzer (version 4.0; Media Cybernetics). GAPDH was used as the loading control (38).

Data were analyzed using one-way ANOVA, followed by Tukey's post-hoc test. Data analysis was performed using SPSS software (version 22.0; IBM Corp.). The data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The heart rate and body weight of rats were monitored throughout the study. The results demonstrated a significant decrease in the heart rate of the model group compared with that in the control group (Fig. 1A; P<0.001). Administration of montelukast sodium significantly increased the heart rate of rats at all doses tested, compared with that in the model group (P<0.001). From day 21, a significant decrease in body weight was observed in the model group compared with the control group (Fig. 1B; P<0.05); however, administration of montelukast sodium significantly restored the body weight of asthmatic rats from the 28th day onwards (P<0.05).

Following administration of different doses of montelukast sodium, the mRNA and protein expression levels of CysLTR1 in lung tissues were measured. The model group demonstrated a significant increase in both the mRNA and protein expression levels of CysLTR1 compared with that of the control group (Fig. 1C; P<0.001). Moreover, it was demonstrated that, compared with the model group, administration of montelukast sodium significantly reduced the mRNA expression level of CysLTR1 to varying degrees (P<0.05); however, only administration of the medium-dose montelukast sodium decreased the protein expression level of CysLTR1 significantly compared with that in the model group (Fig. 1D; P<0.05). On the contrary, a significant decrease in the protein expression level of Nrf2 was observed in the model group compared with that in the control group (P<0.001), while all doses of montelukast sodium significantly increased the Nrf2 protein expression level, with the higher increase being observed at a dosage of 4 mg/kg (P<0.001).

H&E staining was performed to assess the impact of CysLTR1 blockade on inflammation in rat lung tissues. No apparent inflammatory cell infiltration was observed in the control group, while the model group demonstrated a noticeable infiltration of inflammatory cells and visible thickening of smooth muscle layers compared with the control group (Fig. 1E). Compared with the model group, the administration of montelukast sodium, particularly at a dosage of 4 mg/kg, reduced inflammatory infiltration and airway remodeling. Upon OVA challenge, a significant increase in IL-17 and IL-4 levels in the model group were observed compared with those in the control group, which was consistent with the H&E staining data (Fig. 1F; P<0.001). Furthermore, treatment with montelukast sodium significantly mitigated the proinflammatory effects induced by OVA challenge (P<0.05). IgE is reported to have evolved in mammals as a primary defense mechanism against pathogens, and increased IgE levels are considered indicative of an increased susceptibility to the development of asthma (39). It was demonstrated that IgE concentration was significantly increased in the model group compared with that in the control group (Fig. 1G; P<0.001), while the administration of montelukast sodium was effective in decreasing IgE levels (P<0.01). Additionally, due to the strong association of eosinophils, lymphocytes and macrophages with inflammatory processes in asthma (40), cell count analysis was performed on BALF samples from asthmatic rats. The results of Wright-Giemsa staining demonstrated that the model group exhibited a significant increase in the numbers of eosinophils, lymphocytes and macrophages, compared with those in the control group (Fig. 1H; P<0.001). Administration of montelukast sodium significantly decreased the elevated cell counts in the BALF of asthmatic rats induced by OVA challenge (P<0.05). The association between the dose of montelukast sodium and changes in the number of these inflammatory cells was then assessed. It was observed that varying dosages of montelukast sodium exhibited the lowest inhibitory activity on eosinophils and the highest inhibitory activity on macrophages. Moreover, among the three doses of montelukast sodium tested, the 4 mg/kg dose demonstrated the largest inhibitory efficacy across all three types of inflammatory cells.

The hyperplasia of goblet cells and the deposition of collagen in the lungs are crucial indicators for the progression of asthma (41,42). The model group of rats demonstrated marked goblet cell hyperplasia in comparison with the control group (Fig. 2A). However, the montelukast sodium-treated groups showed a decreased degree of goblet cell hyperplasia compared with that in the model group (Fig. 2A). Additionally, the asthmatic rat model exhibited an exacerbation of OVA-induced collagen deposition in the lung tissue; however, treatment with montelukast sodium mitigated these changes. A TUNEL assay was used to examine the impact of montelukast sodium on bronchial epithelial cell apoptosis. The number of TUNEL-positive cells in the model group was significantly increased compared with that in the control group (Fig. 2C; P<0.001), whereas the number of TUNEL-positive cells was significantly reduced by the administration of montelukast sodium, compared with that in the model group (P<0.001).

The expression levels of antioxidant genes NQO1 and HO-1 were measured to investigate the involvement of CysLTR1 blockade in oxidative stress. Additionally, the GSH/GSSG ratio was calculated, due to its reported role in scavenging free radicals (43). A significant decrease in the mRNA expression levels of NQO1 and HO-1 was demonstrated in the model group compared with those in the control group (Fig. 3A and B; P<0.001). Additionally, a significant reduction in the GSH/GSSG ratio was also demonstrated (Fig. 3C; P<0.001). These inhibitory effects induced by OVA challenge were reversed by treatment with montelukast sodium, particularly at a dosage of 4 mg/kg (Fig. 3A-C; P<0.001). The transcription factor Nrf2, which is responsible for regulating cellular redox balance and initiating protective antioxidant responses in mammals, has been reported to be an important therapeutic target for mitigating oxidative stress injury in asthma (44). Immunofluorescence microscopy demonstrated a significant decrease in Nrf2 fluorescence intensity in the model group compared with that in the control group (Fig. 3D; P<0.001), while there was a significant increase following treatment with 4 or 30 mg/kg montelukast sodium (Fig. 3D; P<0.05).