A total of 24 male BALB/c mice (age, 5 weeks; weight, 18-20 g) were supplied by RaonBio, Inc. and maintained at 22±2˚C and 50±10% humidity in a 12-h light/dark cycle and were freely provided with tap water and commercial food. The animals' weight was measured once a week to monitoring their conditions. SHO [1-[4-Hydroxy-3-methoxyphenyl]-4-decen-3-one; C17H24O3; purity, >98% as detected via high-performance liquid chromatography (HPLC); cat. no. 555-66-8] and GIN ([5S]-5-Hydroxy-1-[4-hydroxy-3-methoxy-phenyl]decan-3-one; [S]-[6]-Gingerol; and 3-Decanone, 5-hydroxy-1-[4-hydroxy-3-methoxyphenyl]; purity, >98% as detected by HPLC; cat. no. 23513-14-6) were purchased from Chengdu Biopurify Phytochemicals Ltd.

The mice were divided into four groups: Control, OVA, OVA + SHO and OVA + GIN, with n=6 per group. The induction of asthma in the mice treated with OVA was performed as described previously (17). The asthma mouse model was established by sensitizing the mice with an intraperitoneal (i.p.) injection of 50 µg OVA (cat. no. A5503; Sigma-Aldrich; Merck KGaA) and 1 mg aluminum hydroxide (cat. no. 239186; Sigma-Aldrich; Merck KGaA) in 200 µl PBS on days 0 and 14 (Fig. 1). The control group was treated with 200 µl PBS.

On days 28, 29 and 30, the mice received an i.p. injection of SHO at 10 mg/kg or GIN at 10 mg/kg 2 h before they were challenged with OVA. SHO and GIN were dissolved in 0.5 µl DMSO, and 89.5 µl PBS with 10 µl 50% Tween-20 was added to the 100-µl final dose. The mice were then challenged with 2% OVA in sterile saline through a whole-body exposure system with a nebulizer on days 28, 29 and 30 for 20 min. The control group inhaled sterile saline. The mice were sacrificed at 24 h after the final OVA challenge by cervical dislocation.

BALF was obtained from the mice at 24 h after the final OVA challenge by flushing 0.5 ml cold PBS through the lungs three times with a tracheal catheter. The BALF was centrifuged at 318 x g for 10 min at 4˚C to collect the pellet, which was resuspended with PBS and stored at 4˚C for a differential cell count. The BALF cells in the pellet were stained with the Diff-Quik stain kit (cat. no. 38721; Sysmex Corporation), and 5 µl BALF cells were smeared onto one end of a glass slide according to the manufacturer's instructions. The cells were stained with Diff quick solution II and I for 30 sec at room temperature each, and rinsed with tap water. Stained cells were counted with a hematocytometer (HSU-0650030; Paul Marienfeld GmbH & Co. KG). The differential cells in the BALFs were classified as lymphocytes, neutrophils, macrophages or eosinophils. The distribution of the cells in the BALFs was expressed as a percentage.

The right lung lobes of the mice were fixed in 4% formaldehyde at 4˚C for 48 h, embedded in paraffin and sliced to 4-µm sections (6 slides/mouse). The sections were stained with hematoxylin at room temperature for 5 min and then with eosin at room temperature for 3 min to analyze the degree of inflammation in the lung tissues. The degree of inflammation was assigned an arbitrary score of 0 (normal = no inflammation), 1 (minimal = perivascular, peribronchial or patchy interstitial inflammation involving <10% of lung volume), 2 (mild = perivascular, peribronchial or patchy interstitial inflammation involving 10-20% of lung volume), 3 (moderate = perivascular, peribronchial, patchy interstitial or diffuse inflammation involving 20-50% of lung volume) and 4 (severe = diffuse inflammation involving >50% of lung volume). The average inflammatory score was analyzed using a total of 18 slides (3 slides/mouse). In addition, the thickness of the lung epithelial cells was measured, calculated as the average value of the top, bottom, left and right of three random alveoli per slide using a light microscope (Leica Microsystems GmbH) with Leica AF6000 modular systems.

The sections also were stained with periodic acid-Schiff (PAS) at room temperature for 15 min to visualize the goblet cells to determine the extent of mucus production. A point-counting method was performed to quantify the number of cells stained positively in three random fields from each slide. The average of the cells was analyzed with a total of 18 slides (3 slides/mouse). These lung sections were observed using light microscope (magnification, x100) and analyzed with LAS AF Ink (Leica Microsystems GmbH) and ImageJ software (v1.53e; National Institutes of Health).

Total RNA was isolated from the mouse lung tissues using the TRI-Solution (cat. no. TS200-001; Bio Science Technology). The total RNA was synthesized to cDNA using a PrimeScript™ 1st strand cDNA Synthesis kit (cat. no. 6110; Takara Biotechnology Co., Ltd.) with oligo-dT primers. The qPCR reaction mixture contained 8 µl cDNA, 10 µl Power SYBR^®-Green PCR Master mix (cat. no. 4367659; Applied Biosystems; Thermo Fisher Scientific, Inc.), 1 µl 0.2-pmol forward primer and 1 µl 0.2-pmol reverse primer. The forward and reverse primers for β-actin (5'-GGCTCTTTTCCAGCCTTCCT-3' and 5'-GTCTTTACGG ATGTCAACGTCACA-3', respectively), IL-4 (5'-CCACGGAT GCGACAAAAATC-3' and 5'-GACGTTTGGCACATCCAT CTC-3'), IL-5 (5'-GATGGACGCAGGAGGATCAC-3' and 5'-GTGTGGCATCCCTCAGCAA-3'), IL-13 (5'-GGCCAG CCCACAGTTCTACA-3' and 5'-ACCACCAAGGCAAGCAA GAG-3'), superoxide dismutase 1 (SOD1; 5'-GACTTGGGC AAAGGTGGAAA-3' and 5'-CAGGGAATGTTTACTGCGC AAT-3'), SOD2 (5'-TGCTCTTGATTGAACATTTTCGTTA-3' and 5'-GCCCCCCAAAACAGAGATG-3'), catalase (5'-CGA CCAGGGCATCAAAAACT-3' and 5'-ATTGGCGATGGCAT TGAAA-3') and glutathione peroxidase-1 (GPx-1; 5'-AGAAAG CGATGCCACGTGAT-3' and 5'-GGAGATGTTGGGACTC AAACG-3') were used. The qPCR was run on the Applied Biosystems real-time PCR program (StepOnePlus™ Real-Time PCR System) at 95˚C for 10 min, followed by 40 cycles of a cycling stage at 95˚C for 15 sec and 60˚C for 1 min, and a melt curve stage at 95˚C for 15 sec, 60˚C for 1 min and 95˚C for 15 sec. All data were analyzed using the 2^-ΔΔCq method (23) and expressed as fold change relative to controls. The relative expression levels of IL-4, IL-5, IL-13, SOD1, SOD2, catalase and GPx-1 were normalized to those of β-actin.

Lung tissues were lysed using the PRO-PREP for Cell/Tissue Protein Extraction Solution kit (cat. no. 17081; Intron Biotechnology, Inc.) with the Protease Inhibitor Cocktail (cat. no. P3100; GenDEPOT, LLC). The protein concentrations of the tissue lysates were determined using the BCA protein assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). A total of 30 µg protein from each sample was separated via 10-12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in a 1X TBS-Tween-20 (TBST; containing 0.05% Tween-20) at room temperature for 1 h and incubated with primary antibodies against IL-4 (monoclonal; rat anti-mouse; 1:1,000; cat. no. ab11524; Abcam), IL-5 (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-398334; Santa Cruz Biotechnology, Inc.), IL-13 (polyclonal; rabbit anti-mouse; 1:1,000; cat. no. ab106732; Abcam), SOD1 (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-101523; Santa Cruz Biotechnology, Inc.), SOD2 (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-133134; Santa Cruz Biotechnology, Inc.), catalase (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-271803; Santa Cruz Biotechnology, Inc.), GPx-1/2 (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-133160; Santa Cruz Biotechnology, Inc.) or β-actin (monoclonal; mouse anti-mouse; 1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) diluted with 3% skim milk in a 1X TBST buffer at 4˚C overnight. Then, the blots were washed three times with 1X TBST buffer and incubated with anti-mouse HRP-conjugated secondary antibodies (goat anti-mouse IgG-HRP; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) diluted at 1:5,000 with 3% skim milk in 1X TBST buffer at room temperature for 1 h. Next, the membranes were washed three times with 1X TBST buffer, and the protein bands were detected with the ECL detection reagents (cat. no. 34580; Thermo Fisher Scientific, Inc.) and semi-quantified with ImageQuant LAS 500 (Cytiva). The protein expression level of β-actin was used as the loading control.

Mast cell degranulation was evaluated by the release of β-hexosaminidase from the mast cell line, RBL-2H3. Rat RBL-2H3 mast cells were obtained from the American Type Culture Collection and maintained in MEM medium (cat. no. 61100061; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (cat. no. A31604; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ incubator at 37˚C. RBL-2H3 cells were sensitized via an incubation with 0.2 µg/ml monoclonal anti-dinitrophenyl mouse IgE (cat. no. D8406; Sigma-Aldrich; Merck KGaA) diluted medium overnight at 37˚C. The cells were washed twice with a piperazine-N, N'bis (2-ethanesulfonic acid) (PIPES) buffer (pH 7.2) containing 25 mM PIPES, 0.05 mM NaOH, 110 mM DNP-IgE and 0.1% BSA (cat. no. A0100-010; GenDEPOT LLC). The cells were then incubated in PIPES buffer containing different concentrations of SHO and GIN (0, 10, 25, 50 and 100 nM) at 37˚C for 30 min. Next, the cells were incubated with 1 µg/ml human dinitrophenyl albumin (cat. no. A6661; Sigma-Aldrich; Merck KGaA) to induce degranulation for 15 min at 37˚C. After centrifugation at 125 x g for 5 min at 4˚C, 25 µl supernatant from each reaction was transferred to a 96-well microplate and incubated for 110 min with 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in a 0.1-M citrate buffer (pH 4.5; cat. no. N9376; Sigma-Aldrich; Merck KGaA). The reaction was terminated by adding 0.05 M sodium carbonate buffer (pH 10). The optical density of each reaction was measured at the absorbance wavelength of 405 nm using a microplate reader.

The concentrations of SHO and GIN were selected using a cell viability assay. Cell viability was evaluated to determine cytotoxicity of SHO and GIN using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.) assay, following the manufacturer's instructions. Jurkat cells (human T cell line; 1x10⁴ cells/well) were obtained from American Type Culture Collection and maintained in RPMI-1640 medium (cat. no. 31800022; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37˚C. Jurkat cells were incubated at 37 ˚C in 96-well plates with SHO and GIN at 0, 10, 50 and 100 nM for 0, 24, 48 and 72 h. RBL-2H3 cells (5x10³ cells/well) were incubated at 37˚C in 96-well plates with SHO (0, 1, 2.5 and 5 µM) and GIN (0, 0.5, 1 and 2 µM) for 0, 24, 48 and 72 h. Next, 10 µl CCK-8 reagent was added to each well, and cells were incubated at 37˚C for an additional 2 h. The absorbance was measured at 450 nm using a microplate reader.

Data were collected from ≥3 independent experiments. All results were expressed as the mean ± SD. All analyses were performed using SPSS software (version 20; IBM Corp.). Statistical significance between experimental groups was determined using one-way ANOVA for pair-wise comparisons with Bonferroni's multiple comparisons test. Ordinal data were analyzed using a Kruskal-Wallis test followed by Dunn's test. P<0.05 was considered to indicate a statistically significant difference.

In the OVA group, the eosinophil level was increased significantly compared with that of the control group. In the OVA + GIN group, this was decreased significantly compared with that of the OVA group (Fig. 2). The current study found no significant difference between the OVA + SHO and OVA groups, but did identify a decreasing trend. These data suggest that GIN strongly inhibited the recruitment of eosinophils in asthma; SHO also may have such a potential. Therefore, GIN and SHO likely have anti-inflammatory effects on asthma in a mouse model. Thus, additional experiments were conducted to confirm the anti-allergic effects of both using histological examination.

The lung sections from the mice in different treatment groups were stained with H&E to confirm the inflammatory cell-counting results (Fig. 2). In chronic inflammation, inflammatory cells infiltrate the ASM cell layer, inducing eosinophilia in the lung tissues (24). The ASM cell thickness was increased in the OVA group compared with that in the control group, suggesting that a chronic inflammatory response, such as asthma, was induced in the OVA group (Fig. 3A and B). In the OVA + SHO and OVA + GIN groups, ASM thickness was significantly decreased compared to that of the OVA group (Fig. 3A and B). Moreover, the inflammatory score was significantly higher in the OVA group and was significantly lower in the OVA + SHO and OVA + GIN groups (Fig. 3C).

The mucins produced by the goblet cells were stained using PAS (red arrows; Fig. 4A). Mucus production was significantly increased in the OVA group compared with that in the control group; however, it was significantly reduced in the OVA + SHO and OVA + GIN groups as compared with the OVA group (Fig. 4B).

Th2 cytokines regulate the inflammatory response in allergic diseases such as asthma (25,26). The expression of genes encoding Th2 cell-mediated cytokines, including IL-4, IL-5 and IL-13, in the lung tissues after the final OVA challenge was examined using RT-qPCR (Fig. 5). The mRNA expression levels of IL-4, IL-5 and IL-13 were significantly increased in the OVA group compared with those in the control group (Fig. 5A-C). SHO and GIN significantly decreased the mRNA expression levels of IL-4 and IL-5 (Fig. 5A and B). In addition, both significantly reduced the expression level of IL-13 (Fig. 5C). The protein expression levels of IL-4, IL-5 and IL-13 were determined using a western blotting assay to confirm the mRNA expression data (Fig. 5D-G). The protein expression levels of IL-4, IL-5 and IL-13 were significantly increased in the OVA group. Furthermore, SHO and GIN decreased the protein expression levels of IL-4, IL-5 and IL-13.

Cells were treated with SHO or GIN at 0, 10, 25, 50 and 100 nM to determine their dose-dependent effects on the mast cells (Fig. 6). The OVA + SHO group showed a significant dose-dependent decrease in mast cell degranulation, to 61% in the group treated with 100 nM of SHO (Fig. 6A). GIN had the same tendency, decreasing degranulation to 70% in the group treated with 100 nM GIN (Fig. 6B). Additionally, the cytotoxicity of SHO and GIN in the RBL-2H3 and Jurkat cells were determined using CCK-8 assays, which demonstrated that SHO and GIN were not toxic (Fig. S1). The concentrations of SHO and GIN used for cell line treatment were selected based on the results in Fig. S1. These data suggested that both reagents effectively suppressed the allergic response in asthma by inhibiting mast cell degranulation.

Oxidative stress is an important pathological mechanism of asthma. SOD1, SOD2, catalase and GPx-1/2 are critical antioxidant enzymes to reduce oxidative stress (27-29). Total mRNA and proteins were isolated from the lung tissues from different treatment groups to determine the effects of SHO and GIN on the levels of various antioxidant enzymes. The mRNA expression level of SOD1 was significantly decreased in the OVA group compared with that in the control group, and the expression level of SOD1 was restored in the OVA + GIN group. In addition, the current study found no difference between the OVA + SHO and OVA groups, but observed an increasing tendency (Fig. 7A).

The mRNA expression levels of SOD2 and GPx-1 were increased significantly in the OVA group compared with the control group, but were decreased significantly in the OVA + SHO group as compared with the OVA group (Fig. 7B and C). Similarly, SOD2 expression was significantly decreased in the OVA + GIN group compared with the OVA group (Fig. 7B). However, no significant difference was detected in the expression level of GPx-1 between the OVA and OVA + GIN groups (Fig. 7C).

The mRNA expression levels of catalase were significantly decreased in the OVA group compared with those in the control group, and this expression was restored in the OVA + SHO group. However, no difference was observed in catalase expression between the OVA and OVA + GIN groups (Fig. 7D).

The expression patterns of the antioxidant genes were confirmed by examining the corresponding protein expression levels using western blot analysis (Fig. 8A). The protein expression levels of SOD1, SOD2 and catalase exhibited the same trend as those of the mRNA expression of the corresponding genes (Fig. 8B-D). However, GPx-1/2 expression showed the opposite tendency to the mRNA expression levels of GPx-1 (Fig. 8D). GPx-1/2 was decreased in the OVA group compared with the control group, was increased in the OVA + GIN group, but showed no change in the OVA + SHO group (Fig. 8D).