AST (purity>95%) was obtained from the National Institutes for Food and Drug Control (Beijing, China) and was suspended in a 1% carboxymethylcellulose solution. Dexamethasone (DEX) was purchased from Shenyang Comeboard Technology Co., Ltd (Shenyang, China). Ovalbumin (OVA, fraction V) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Aluminium hydroxide [Al (OH)₃] was obtained from Pierce; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Immunoglobulin (Ig)E (cat no. SEA545Ra) and IgG (cat no. HEA544Ra) ELISA kits were purchased from Cloud-Clone Corp. (Houston, TX, USA). Anti-GATA-3 (cat no. 5852T), anti-T-box protein expressed in T cells (T-bet; cat no. 13232T), anti-signal transducer and activator of transcription (STAT)-3 (cat no. 12640S) and anti-β-actin antibody (cat no. 3700S) monoclonal antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). An anti-forkhead box protein 3 (Foxp3; cat no. ab20034) antibody was purchased from Abcam (Cambridge, MA, USA).

A total of 25 male and 25 female BALB/c mice (age: 6–8 weeks; weight, 15–20 g) were purchased from the Laboratory Animal Center of Chongqing Medical University (Chongqing, China), and were maintained in horizontal laminar flow cabinets and provided with free access to sterile food and water in a specific pathogen-free facility (temperature, 18–29°C; 12/12 h light/dark cycles). Mice were randomly assigned to the following five groups (n=10 mice/group): Normal control (saline only), OVA-induced allergic rhinitis (OVA), AST (25 mg/kg and 50 mg/kg; n=10 mice/sub-group) and DEX (3 mg/kg). The 25 and 50 mg/kg doses of AST were selected based on previous studies (17).

The present study was conducted in accordance with the internationally accepted principles for laboratory animal use and care, as outlined in the European Commission guidelines (EEC Directive of 1986; 86/609/EEC). The experimental procedures were approved by the Animal Ethics Committee of Chongqing Medical University.

Mice in the OVA, AST and DEX groups were administered intraperitoneally with 0.5 mg/ml OVA and 20 mg/ml Al(OH)₃ in saline, at a dose of 0.2 ml/mouse (sensitization), as previously described (18). Sensitization was repeated three times at weekly intervals (days 0, 7 and 14), and was followed by daily administration of OVA solution (40 mg/ml in saline) into the nostrils (0.02 ml/mouse), on days 21–42 (OVA challenge). Mice in the AST and DEX treatment groups were administered intragastrically with AST (25 or 50 mg/kg) or DEX (3 mg/kg) daily for 3 weeks, 30 min prior to the OVA challenge. Mice in the normal group were administered only with the last intranasal OVA challenge on day 42. Nasal symptoms were evaluated via counting the number of sneezes for 10 min immediately following the last OVA intranasal challenge.

The weight of each mouse was measured weekly using an electronic scale. A total of 48 h following the last treatment, all mice were weighed, anesthetized with ether and sacrificed. The spleen and liver were harvested and used for further analysis. The spleen and hepatic indices were calculated according to the following formula: Spleen/hepatic index=spleen/liver weight (µg)/body weight (g) × 10, as previously described (19).

A total of 48 h following the last treatment, mice were anesthetized with ether and sacrificed. Blood samples were collected from the orbital sinus. Samples were kept standing for 30 min at room temperature, were centrifuged at 3,000 × g for 10 min at room temperature and the serum was isolated. Concentrations of OVA-specific IgE and total IgG were evaluated using the appropriate ELISA kits.

A total of 48 h following the last treatment, mice were sacrificed and their heads and spleens were harvested and immersed in 4% neutral-buffered formalin at room temperature for 7 days. Following fixation, heads were decalcified in 5% formic acid at room temperature for 6 days. Subsequently, the heads and spleens were trimmed, embedded in paraffin and sectioned coronally at a thickness of 4 µm for HE and immunohistochemical staining. For the immunohistochemical detection of GATA-3, T-bet and Foxp3, slides were placed in 0.01 M citric buffer (pH 6.0) and autoclaved at 121°C for 20 min. Sections were blocked with mountain goat serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at room temperature for 20 min and incubated with anti-GATA-3 (1:1,00), anti-T-bet (1:1,00) and anti-Foxp3 (1:2,00) polyclonal antibodies at 4°C for 12 h. Subsequently, sections were washed, incubated with an anti-mouse horseradish peroxidase (HRP) -conjugated IgG secondary antibody (1:1,000; Beyotime Institute of Biotechnology, Haimen, China; cat no. A0216) at 37°C for 1 h and stained with 3,3′-diaminobenzidine. Numbers of eosinophils infiltrating the nasal mucosa were counted in HE-stained sections in 3 randomly selected fields under a ×400 magnification. Stained sections were observed under a Leica DM2500 optical microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and photomicrographs were captured.

Total RNA was extracted from spleen tissue samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) and an oligo (dT) primer (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The reaction volume was 8 µl (5X reaction buffer 4 µl, dNTP mix 1 µl, Rnase Inhibitor 0.5 µl, Rnase free H₂O 0.5 µl, M-MLV enzyme 2 µl). Amplification conditions were as follows: at 30°C for 10 min, at 42°C for 60 min and at 70°C for 15 min. Primer sequences for murine GATA-3, T-bet, retinoic acid receptor-related orphan nuclear receptor (ROR) γt, Foxp3 and β-actin, which served as the internal control, are presented in Table I. The cDNA products were used as templates for qPCR. The CFX96™ Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for analysis with SYBR® Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Dalian, China). The reaction was performed in a total volume of 25 µl (SYBR® Premix Ex Taq™ 2X 12.5 µl, forward primer (10 µmol/l) 1 µl, reverse primer (10 µmol/l) 1 µl, cDNA 2 µl, ddH₂O 8.5 µl) with the following thermocycling conditions: Initial denaturation at 5°C for 2 min, followed by 10 min at 95°C, 40 cycles of annealing at 95°C for 15 sec followed by extension at 60°C for 1 min. Gene expression was quantified using the comparative 2^−∆∆Cq method and normalized to β-actin (20).

Spleen tissue was dissected and sonicated in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) containing protease cocktail (Beyotime Institute of Biotechnology) on ice. The lysates were centrifuged at 10,000 × g for 10 min at 4°C. Protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology). Equal amounts of extracted protein samples (50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk at 37°C for 1 h and incubated overnight at 4°C with the following primary antibodies: Anti-GATA-3 (1:1,000), anti-T-bet (1:1,000), anti-STAT-3 (1:1,000), anti-Foxp3 (1:2,000) and anti-β-actin (1:5,000). Subsequently, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (cat no. A0208) or goat anti-mouse IgG (cat no. A0258) secondary antibodies (1:2,000; Beyotime Institute of Biotechnology) at 37°C for 2 h. Protein bands were visualized using the Immun-Star™ HRP Chemiluminescent kit (Bio-Rad Laboratories, Inc.) and the Molecular Imager® VersaDoc™ MP imaging system (Bio-Rad Laboratories, Inc.). β-actin served as the loading control.

The statistical significance of the difference between groups was assessed by one-way analysis of variance followed by a post hoc Dunnett's test for multiple comparisons. Data are expressed as the mean ± standard deviation of three independent experiments. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using the SPSS software version 22.0 (IBM SPSS, Armonk, NY, USA).

The putative protective effects of AST against allergic rhinitis were examined in allergic rhinitis mice, following intragastric administration of AST (25 or 50 mg/kg), DEX (3 mg/kg) or saline (control). Mice in the OVA model group exhibited a marked increase in the number of sneezes (31.5±2.04 counts/10 min) compared with in the normal, non-sensitized group that received only the last challenge immediately prior to assessment (8.0±2.7 counts/10 min; P<0.05; Fig. 1A). Sneeze counts of AST-treated allergic (17.0±2.6 counts/10 min, 25 mg/kg AST; 17.3±3.5 counts/10 min, 50 mg/kg AST) and DEX-treated mice (18.5±3.0 counts/10 min) were significantly reduced compared with mice in the OVA model group (P<0.05; Fig. 1A). These results suggested that treatment with AST may ameliorate the symptoms associated with allergic rhinitis.

OVA-specific IgE and IgG levels in mouse serum samples were examined by ELISA. OVA-specific IgE serum levels were significantly increased in mice with OVA-induced allergic rhinitis compared with control mice (Fig. 1B). However, treatment with 50 mg/kg AST reversed this effect. Conversely, IgG levels in mouse serum samples remained unaffected, regardless of the treatment administered (Fig. 1C).

Morphological alterations in nasal mucosal and spleen tissue samples of allergic mice were investigated using HE staining. Nasal mucosal tissue isolated from allergic mice exhibited prominent alterations, including eosinophil infiltration, blood vessel sprouting, gland hyperplasia and enlargement, and mucosal remodeling. In addition, the nasal epithelium became multilayered (Fig. 2A). Spleen tissue isolated from allergic mice was characterized by splenic corpuscle proliferation (Fig. 2B). Notably, the administration of AST or DEX appeared to prevent the infiltration of eosinophils in the submucosa, the acidophilic alterations and the development of fibrosis. Furthermore, AST and DEX appeared to attenuate splenic corpuscle proliferation (Fig. 2B). These findings suggested that AST administration may alleviate allergic responses in OVA-challenged mice.

It has previously been reported that Th2-type cytokines, as well as the GATA-3/T-bet expression ratio in lung tissue isolated from allergic mice were affected following Astragalus membranaceus administration (21) Therefore, it may be hypothesized that the mechanisms underlying the antiallergic effects of AST involve the regulation of transcription factors, including T-bet and GATA-3, during Th1 and Th2 cellular differentiation. The present study used immunohistochemistry, RT-qPCR and western blot analysis to investigate the expression of GATA-3 and T-bet in nasal mucosal and spleen tissue of allergic mice. Immunohistochemical results demonstrated that GATA-3 expression levels were downregulated in nasal mucosal (Fig. 3A) and spleen (Fig. 3B) tissue samples following AST or DEX administration, whereas T-bet expression levels were upregulated (Fig. 4). Similar expression alterations for GATA-3 and T-bet were observed at the protein (Fig. 5A) and mRNA (Fig. 5B) level, as determined by western blotting and RT-qPCR, respectively. These findings suggested that treatment with AST may downregulate the expression of GATA-3 and upregulate the expression of T-bet during allergic responses in vivo.

The expression of the inflammation-associated proteins Foxp3, RORγt and STAT-3 in mouse spleen tissue was altered following AST and DEX administration. Mice in the OVA model group demonstrated decreased Foxp3 protein (Fig. 5A) and mRNA (Fig. 5B) expression levels compared with the normal group. Notably, AST and DEX administration upregulated Foxp3 protein and mRNA expression levels compared with the OVA model group (Fig. 5A and B, respectively). Similar effects were observed via immunohistochemical staining (Fig. 6). Conversely, protein expression levels of STAT-3 and mRNA expression levels of RORγt appeared to be upregulated in allergic mice; however, treatment with AST and DEX ameliorated this effect (Fig. 5A and B, respectively).

The body weight of mice from all groups was measured from days 1–42. The results demonstrated that mice in the DEX group started to lose weight from day 29 (Fig. 7A). Following long-term therapy with DEX (42 days), the weight and spleen index of mice in the DEX group were significantly reduced (P<0.05 vs. the normal group; Fig. 7B and C, respectively). However, DEX administration exerted no effects on the liver index (Fig. 7D).