Male C57BL/6 mice, weighing 18-22 g, were obtained from the Animal Center of Wenzhou Medical University. All animals were housed at constant room temperature with a 12:12-h light: Dark cycle, and fed a standard rodent diet and water. All animals were allowed to acclimatize for at least 3 days before the experiments. Animal care and experimental protocols were approved by the Committee on Animal Care of Wenzhou Medical University (approval document no. wydw2017-0027).

A total of 35 mice were randomly divided into five groups (n=7 per group) as follows: Mice sensitized with phosphate-buffered saline (PBS) and administered equal volumes of 0.5% carboxymethylcellulose sodium (CMCNa; CON group) or 10 mg/kg PL (PL10 group) intragastrically; mice sensitized with OVA and administered equal volume of 0.5% CMCNa (OVA group); mice sensitized with OVA and treated with 5 or 10 mg/kg PL (PL5 + OVA or PL10 + OVA groups, respectively). The flowchart of the experimental design is shown in Fig. 1B. Mice were sensitized on days 0 and 14 with an intraperitoneal (i.p.) injection of 20 μg OVA (Sigma-Aldrich; Merck KGaA) emulsified with 2 mg aluminum hydroxide (Sigma-Aldrich; Merck KGaA) in 200 μl PBS (pH 7.4). On days 25-31, the mice were subjected to an airway challenge with 1% OVA or PBS for 30 min delivered with a PARI TurboBOY N medical compressed air nebulizer (PARI GmbH). At 30 min prior to the OVA inhalation challenge, the mice were intragastrically administered PL dissolved in 0.5% CMCNa. One day after the last challenge, the mice were anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg) and blood was collected for further analysis. The mice were then sacrificed by i.p. injection of 200 mg/kg sodium pentobarbital, followed by collection of bronchoalveolar lavage fluid (BALF) and lung tissue.

Blood was collected from the orbital vascular plexus and centrifuged at 15,777 × g for 10 min. The serum was collected and stored at −80°C. To obtain BALF, PBS (200 μl) was infused into the left lung four times (total volume, 800 μl). The collected BALF was centrifuged at 110 × g for 10 min at 4°C. The supernatant was collected and stored at −80°C and the precipitate was resuspended in 40 μl PBS. The total number of cells in BALF was determined with a cell counting instrument. The number of eosinophils in BALF was examined by Wright-Giemsa staining.

The middle lobe of the right lung was collected and fixed in 4% paraformaldehyde, then embedded in paraffin and cut into 5-μm sections. The areas of inflammation, severe fibrosis and mucus secretion were analyzed by hematoxylin and eosin (H&E), Sirius Red and periodic acid-Schiff (PAS) staining, respectively. The sections were stained using standard protocols for light microscopy examination. A pathologist blindly determined the histopathological score according to the following two categories: Peribronchial inflammation and perivascular inflammation. A value of 0-3 per criterion was assigned to each tissue section as follows: 0, no inflammatory cells; 1, occasional cuffing with inflammatory cells; 2, most bronchi or vessels surrounded by a thin layer (1-5) of inflammatory cells; 3, most bronchi or vessels surrounded by a thick layer (>5 cells). Subepithelial fibrosis was estimated by dividing the area of Sirius Red staining by the length of the bronchiolar basement membrane. PAS-staining was identified using Image J software (National Institutes of Health), and the pixel intensities of each color channel (red, blue and green) were averaged. The mucus index was determined using the following equation: Mucus index = [(area of PAS staining) × (mean intensity of PAS staining)]/[total area of airway epithelium (including PAS staining)] (20). The final indices were the results of a mean of 5 images per lung, encompassing large and small bronchial airways.

AHR was determined by measuring the changes in airway resistance (Rn) after the mice were subsequently exposed to increasing concentrations of methacholine (Mch; 3.125-50 mg/ml; Sigma-Aldrich; Merck KGaA) in PBS using the flexiVent system (SCIREQ). After the mice were anesthetized with 1% pentobarbital sodium (50 mg/kg), tracheostomy was performed and the tracheostomized mice were mechanically ventilated at 200 breaths/min with a tidal volume of 10 ml/kg under appropriate anesthesia. Mch aerosol was challenged for 10 sec, after which time airway resistance was continuously monitored and recorded.

Lung sections (5 μm) were prepared, deparaffinized in xylene, and rehydrated using an ethanol gradient. A pressure cooker was used for antigen retrieval (10 mM sodium citrate buffer, pH 6.0). After using 3% hydrogen peroxide to remove catalase, all sections were blocked in 1% bovine serum albumin (BSA) for 30 min, then incubated with TNF-α (mouse monoclonal, dilution: 1:200, cat. no. ab1793, Abcam) or α-smooth muscle actin (SMA; mouse monoclonal, dilution: 1:200, cat. no. ab7817, Abcam) antibody overnight at 4°C, followed by incubation with secondary antibody for 60 min at 37°C. After the sections were incubated with 3,3-diaminobenzidine tetrahydrochloride for color development, they were counterstained with hematoxylin and visualized under a light microscope. The percentage expression of α-SMA or TNF-α in 10 fields of view was analyzed by Image J software (National Institutes of Health).

The human bronchial epithelial cell line Beas-2B was obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal bovine serum (HyClone; GE Healthcare Life Sciences) and 1% penicillin/streptomycin in a humidified atmosphere at 37°C with 5% CO₂. Beas-2B cells were treated with DMSO or PL (5 or 10 μM) for 30 min, and then treated with or without 5 ng/ml TNF-α for an additional 24 h to collect medium, 12 h to collect mRNA, and 30 min to collect protein.

Lung tissue homogenate (100 μg) and cell protein (30 μg) samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% milk, the membranes were probed with a specific primary antibody against IκBα (rabbit monoclonal, dilution 1:1,000, cat. no. 4812, Cell Signaling Technology Inc) and β-actin (mouse monoclonal, dilution: 1:1,000, cat. no. ab8226, Abcam) overnight at 4°C. The membranes were then washed and incubated with corresponding secondary antibody (Yeasen). The blots were visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories Inc.). The density of the immunoreactivity bands was analyzed using Image J software (National Institutes of Health).

Beas-2B cells were seeded into 96-well plates at a density of 3,000 cells per well. PL was dissolved in DMSO and diluted with RPMI-1640. The cells were incubated with different concentrations of PL (1.25, 2.5, 5, 10, 20 and 40 μM) for 24 h prior to the MTT assay. A fresh solution of MTT (5 mg/ml) prepared in PBS was added to each well. The plate was then incubated in a CO₂ incubator for 4 h, formazan crystals were dissolved with 150 μl DMSO, and analyzed in a multi-well-plate reader at 490 nm.

Total RNA was prepared using TRIzol reagent (Invitrogen; Thermo Fisher Scientific. Inc.) and quantified by ultraviolet absorption at 260 and 280 nm. Complementary DNA (cDNA) was generated from 2 μg total RNA using a cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). Then cDNA gene expression was assayed by qPCR with SYBR-Green qPCR Super Mix-UDG kit on a Mastercycler^® ep realplex detection system (Eppendorf). The relative mRNA levels were calculated with the 2^−ΔΔCq method (21) using β-actin as an internal control. The sequences of the sense and antisense primers are listed in Table I.

The levels of IL-4, IL-5 and IL-13 in BALF supernatants and lung homogenates and the IL-1β level in cell culture supernatants were detected using ELISA kits (eBioScience) according to the manufacturer's instructions.

Data collected from the experiments were analyzed using Graphpad Prism 7.0 software (GraphPad Software, Inc.). Values are expressed as mean ± standard error of the mean. One-way analysis of variance followed by Dunnett's post hoc test was employed to analyze the differences between sets of data. A P-value of <0.05 was considered to indicate statistically significant differences.

C57BL/6 mice were sensitized and challenged with OVA for 31 days to induce asthma (Fig. 1B). On the 32nd day, the mice were sacrificed, and lung tissues and BALF were collected 24 h after the last challenge with OVA. The histological sections revealed that the lung tissues from both the CON and PL10 groups had normal morphology, with thin-lined alveolar septa and well-organized alveolar spaces (Fig. 1C). Administration of OVA induced severe alveolar injury, with thickened intra-alveolar septa, collapsed alveolar spaces, edema and inflammatory cell infiltration. Treatment with PL significantly mitigated OVA-induced lung injury. The quantification of lung injury score is shown in Fig. 1D. Furthermore, AHR was established with Mch to determine whether PL exerts any effects on AHR. As shown in Fig. 1E, OVA sensitization and challenge led to an increased Rn value in response to increasing concentrations of Mch. However, the PL (5 and 10 mg/kg) groups exhibited significantly lower Rn values compared with the OVA-treated group. Moreover, the total BALF protein concentration was markedly increased in OVA-challenged mice, and treatment with PL dose-dependently lowered the protein levels, suggesting that treatment with PL reduces pulmonary permeability (Fig. 1F). Furthermore, the total IgE levels in the serum were higher in OVA-sensitized mice compared with those in CON group mice (Fig. 1G). Treatment with PL effectively reduced the increased serum IgE levels induced by OVA.

Airway remodeling is a common characteristic of asthma, which is reflected by basement membrane thickening, subepithelial fibrosis, airway smooth muscle proliferation, goblet cell metaplasia and increased mucus secretion. PAS staining was performed to evaluate goblet cell metaplasia in the airway epithelium. As shown in Fig. 2A, marked goblet cell metaplasia was observed in OVA-induced asthmatic lung tissue, which was significantly reduced by either dose of PL. The quantified results revealed that PL effectively mitigated goblet cell metaplasia in a dose-dependent manner (Fig. 2B). To further assess the goblet cell response in asthmatic mice, the levels of Muc5AC and Muc5B were measured by RT-qPCR (Fig. 2C and D). OVA sensitization and challenge increased the mRNA levels of Muc5AC and Muc5B, while in mice treated with PL this increase was reversed. The degree of subepithelial fibrosis was evaluated by Sirius Red staining (Fig. 2E) and α-SMA IHC staining (Fig. 2G). As shown in Fig. 2E and G, OVA-sensitized mice exhibited high levels of subepithelial collagen and α-SMA, respectively. However, PL treatment reduced the OVA-induced increase in collagen and α-SMA levels in lung tissues. The quantified results also revealed that PL dose-dependently reduced the OVA-induced increase in collagen and α-SMA levels (Fig. 2F and H). Matrix metallopeptidase (MMP)-9 participated in airway remodeling, which contributes to airway narrowing. OVA upregulated the MMP-9 mRNA levels in lung tissue, and this effect was inhibited by PL treatment (Fig. 2I). Therefore, airway remodeling was more pronounced in OVA mice compared with control group mice, and PL treatment significantly inhibited airway remodeling, including subepithelial fibrosis, airway smooth muscle proliferation, goblet cell metaplasia and increased mucus secretion.

In addition to airway remodeling, airway Th2 inflammation is a major characteristic of asthma. IL-4, IL-5 and IL-13 are well-recognized inflammatory factors secreted by Th2 effector cells. Thus, the protein and mRNA levels of IL-4, IL-5 and IL-13 were measured by ELISA and RT-qPCR assay, respectively. As shown in Fig. 3A-F, the protein levels of IL-4, IL-5 and IL-13 in the BALF and lung tissue were upregulated by OVA sensitization, and this effect was inhibited by PL treatment. In addition, the mRNA levels of IL-4 (Fig. 3G), IL-5 (Fig. 3H) and IL-13 (Fig. 3I) in lung tissue were upregulated in the OVA group and downregulated in the PL treatment group. The results of the present study demonstrated that PL significantly decreased the OVA-induced upregulation of IL-4, IL-5 and IL-13 at the protein and mRNA levels.

OVA sensitization is associated with large numbers of inflammatory cells in the BALF, including eosinophils, while PL treatment reduced the total number of inflammatory cells (Fig. 4A) and eosinophils (Fig. 4B) in the BALF. TNF-α is a major pro-inflammatory cytokine that is considered to be crucial for the pathogenesis of asthma. OVA was found to increase the protein and mRNA levels of TNF-α in BALF and lung tissue, as detected by ELISA (Fig. 4C), IHC staining (Fig. 4D and E) and RT-qPCR assay (Fig. 4F), while these increases were significantly reduced by PL treatment. Another pro-inflammatory cytokine, IL-6 (Fig. 4G), and intracellular adhesion molecule (ICAM)-1 (Fig. 4H) were also found to be markedly increased in OVA-challenged mice compared with the CON group. However, treatment with PL inhibited the upregulation of IL-6 and ICAM-1. To elucidate the underlying mechanism through which PL reduces OVA-induced inflammatory response, NF-κB pathway activation was investigated. IκBα is an inhibitory protein of NF-κB, the degradation of which can activate the NF-κB signaling pathway. As shown in Fig. 4I, the protein level of IκBα was downregulated in the lung tissue of OVA group compared with CON group mice. However, PL treatment dose-dependently reversed the IκBα degradation. These results indicate that PL reduces OVA-induced inflammatory response by inhibiting the activation of the NF-κB pathway.

Our in vivo results verified that PL appears to relieve OVA-induced asthma and airway inflammation by inhibiting NF-κB activation. To further confirm the effects of PL on inflammatory response and the NF-κB pathway, TNF-α was used to induce inflammation in Beas-2B cells. First, the cytotoxicity of PL on Beas-2B cells was detected (Fig. 5A). Treatment with PL caused cell viability to decrease at concentrations of ≥10 μM. Therefore, PL was used at 2.5 and 5 μM for further experiments. TNF-α treatment significantly induced the protein (Fig. 5B) and mRNA (Fig. 5C) levels of IL-1β, IL-6 (Fig. 5D), the chemokine monocyte chemoattractant protein (MCP)-1 (Fig. 5E) and the adhesion molecule ICAM-1 (Fig. 5F) in Beas-2B cells, while pretreatment with PL dose-dependently inhibited this increase. In addition, TNF-α treatment downregulated the IκBα protein level, while pretreatment with PL reversed the degradation of IκBα (Fig. 5G and H).