Human nasal mucosal epithelial cells (HNEpCs) were purchased from Shanghai Huzhen Industrial Co., Ltd. at passage 4. IPA, PA and ovalbumin (OVA) were purchased from Sigma-Aldrich; Merck KGaA. The solvent used for the aluminum hydroxide solution was saline.

The experimental procedures followed the guidelines set by the Ethical Review Committee for Laboratory Animal Welfare of The First Hospital of Shanxi Medical University (approval no. DWLL-2024-015; Taiyuan, China). The experiment followed the Guide for the Care and Use of Laboratory Animals (24). Female BALB/c mice (n=30; weight, 20 g; age, 6–8 weeks) were obtained from Nanjing Junke Bioengineering Co., Ltd. The mice were housed in a controlled environment with a temperature of 25±1°C and a relative humidity of 45–55%, under a 12-h light-dark cycle, with ad libitum access to food and water. Mice were thoroughly examined and observed twice daily at 8:30 a.m. and 5:30 p.m. to ensure that they received adequate attention and care during the experiment. Prior to the commencement of the experiment, the mice were acclimated for 1 week following standard procedures to ensure that they were familiar with and adapted to the new living environment. Subsequently, a 29-day experimental intervention phase was initiated. During this period, the mice were provided with ample feed and maintained a comfortable living environment to ensure the smooth progression of the experiment and data reliability. The entire experimental procedure, including the feeding adaptation period and the experimental intervention period, spanned a total of 36 days.

The literature relevant to OVA-sensitized mice was extensively reviewed (25,26) and our expertise was drawn upon in developing the mouse models of AR. The mice were randomly assigned to the following three groups: i) A blank control group (Control group), ii) an OVA sensitization group (OVA group) and iii) an IPA treatment group (OVA + IPA group). Sensitization involved intraperitoneal injections of OVA (100 µg/mouse in 0.1 ml 1 mg/ml aluminum hydroxide solution) on days 0, 7 and 14, followed by daily nasal administration of OVA (20 µl/nostril, 10 mg/ml) between days 21 and 28. The concentration of IPA and the intervention duration were established based on the preliminary investigation by Zhou et al (14) and our pre-experiment. The OVA + IPA group was also administered a daily oral gavage of IPA (20 mg/kg) throughout the same timeframe. The control group received saline injections and nasal stimulation. The volume of saline used for the control group during both the sensitization and challenge phases was identical to that used for the mice in the OVA group. Furthermore, during the nasal stimulation experiment, a pre-prepared OVA solution was employed. For the specific procedure, mice were placed in a comfortable position, and a 20-µl pipette was used to slowly administer the solution intranasally, to minimize the discomfort of the mice and prevent asphyxiation (Fig. 1). On the final day, after deeply anesthetizing the mice via intraperitoneal injection with 50 mg/kg sodium pentobarbital, the mice were euthanized by cervical dislocation, and nasal mucosa and blood samples were collected. Animal euthanasia was confirmed by observing respiration and heartbeat, examining pupil reactions and using the touch method, which specifically entailed checking the skin temperature by touching, and assessing heartbeat and pulse through palpation. Throughout the experiment, the health status of the mice was closely monitored and no unexpected deaths occurred. When the mice appeared to have seriously deteriorated in health, were untreatable or exhibited abnormal behavior, or when the purpose of the experiment had been achieved and no further valuable data could be obtained, the principles of the ethics review were strictly followed and the animals were euthanized. This was done to minimize the pain and discomfort suffered by the mice. No mice were sacrificed early due to the aforementioned circumstances. All mice died as a result of the euthanasia procedure required by the experimental design, in full compliance with ethical review requirements and animal welfare standards.

During the 21–28-day post-sensitization period, an observer blinded to the groupings recorded the frequency of sneezing and nose scratching, and the intensity of rhinorrhea in the mice. The symptoms were assessed using a standardized scale after OVA exposure, with a total score >5 indicating effective sensitization (27). The score for AR (SFAR) was calculated as the sum of the scores for sneezing, nasal scratching and rhinorrhea; and the total scores were analyzed using a non-parametric test. The specific scoring details are summarized in Table I.

The mouse nasal mucosa epithelium was excised and fixed in 4% paraformaldehyde for 24 h at 4°C. Following fixation, the samples underwent dehydration using a tissue dehydrator and were embedded in paraffin. Each paraffin-embedded block was pre-frozen at −20°C for 30 min, sectioned to a thickness of 5 µm and then baked for 2 h. Subsequently, the sections were deparaffinized using xylene and ethanol gradients, and were subjected to hematoxylin and eosin (H&E) staining, periodic acid-Schiff staining (PAS) and toluidine blue staining (TBS).

H&E, PAS and TBS (1%) (cat. nos. G1120, G1281 and G3668; all from Beijing Solarbio Science & Technology Co., Ltd.), were used at their original concentrations as produced by the manufacturer. The staining procedures were performed at room temperature. For H&E staining, the sections were dewaxed in 100% xylene twice (10 min each), rehydrated through a graded series of ethanol (100, 95, 85 and 75%; 3 min each) and then immersed in distilled water for 2 min. After staining with hematoxylin solution for 5 min and rinsing with distilled water to remove excess stain, the sections were differentiated with differentiation solution for 30 sec, followed by two 5-min rinses with tap water. Eosin staining was then performed for 1 min, after which excess stain was removed and sections were rapidly dehydrated. For dehydration, clearing and mounting, sections were sequentially immersed in graded ethanol (75, 85, 95 and 100% ethanol, 3 sec each; 100% ethanol for 1 min; and 100% xylene twice, 1 min each), before being mounted with neutral gum.

For PAS, the sections were rinsed with tap water for 3 min and then with distilled water twice, before applying periodic acid and incubating the sections at room temperature for 5 min. After another rinse with tap water and two rinses with distilled water, Schiff staining solution was applied and the sections were stained in the dark for 10 min. After a 10-min rinse with distilled water, the sections were immersed in hematoxylin staining solution for 1 min to stain the nuclei, differentiated with acidic differentiation solution for 5 sec and then blued in tap water for 10 min. Finally, sections were dehydrated through a graded series of ethanol, cleared in 100% xylene, and mounted with neutral gum.

For TBS, paraffin-embedded sections were dewaxed in 100% xylene twice (15 min each), rehydrated through a series of ethanol solutions (1 min each) and then rinsed with distilled water for 2 min. TBS solution was then applied for 10 min, followed by a 1-min rinse with distilled water and blotting dry with filter paper. Sections were differentiated with 95% ethanol until mast cells appeared purple-blue and the background light blue, rapidly dehydrated in 95% ethanol for 5 sec and then immersed in absolute ethanol twice (1 min each). Finally, the sections were cleared in 100% xylene and mounted with neutral gum. Pathological changes were examined under a light microscope (Pannoramic Scan II; 3DHISTECH, Ltd.) and image analysis was performed using CaseViewer 2.4.0 (https://www.lumingrj.com/2024/12/18/WIN/CaseViewer-2.4.0).

Mouse blood was collected from the eyeballs of mice after sacrifice and incubated at room temperature for 2–3 h. The whole blood was centrifuged at 1,800 × g for 15 min at room temperature to obtain the serum. Subsequently, the levels of the inflammatory factors IL-4, IL-5, IL-10, IL-13 and IgE were quantified using ELISA-specific kits (cat. nos. JM-02448M1, JM-02447M1, JM-02459M1, JM-02456M1 and JM-02339M1, respectively; Jiangsu Jingmei Biological Technology Co., Ltd.) according to the manufacturer's protocol.

The immunohistochemical assay was employed to evaluate the expression levels of AKT, phosphorylated (P)-AKT, CEBPB and IL-10 in the epithelial tissues of the mouse nasal mucosa. Tissue sections underwent deparaffinization following H&E staining, followed by immersion in water for 10 min. Subsequently, the sections were treated with 0.01 M sodium citrate buffer (pH 6.0) and subjected to heat treatment in a pressure cooker. After the rotary valve popped up, which indicated that the water temperature had reached 100°C, the sections were heated for 2 min and then cooled to room temperature naturally. Endogenous peroxidase activity was blocked using a freshly prepared 3% H₂O₂ methanol solution for 10 min in the dark at room temperature, and the sections were subsequently washed with PBS. The sections were then incubated with sheep-derived blocking serum (cat. no. ZLI-9022; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 20 min. Primary antibodies (AKT: Cat. no. 10176-2-AP, 1:100; P-AKT (Ser473): Cat. no. 66444-1-Ig, 1:100; CEBPB: Cat. no. 23431-1-AP, 1:100; IL-10: Cat. no. 60269-1-Ig, 1:100; all from Wuhan Sanying Biotechnology) were then used to incubate the sections overnight at 4°C. On the following day, after the primary antibodies were removed, an appropriate quantity of secondary antibody (enzyme-labeled goat anti-mouse/rabbit IgG polymer: Cat. no. PV-6000, 1:100; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was applied, followed by color development using freshly prepared DAB solution (cat. no. ZLI-9017; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). The reaction was halted with tap water post-color development, and counterstaining was performed at room temperature with hematoxylin staining solution for 1 min, followed by rinsing with tap water for 10 min to reverse the blue coloration. Finally, the slides were sealed with resin and images were captured using an optical microscope. Finally, randomly selected regions were analyzed using ImageJ-win64 software (National Institutes of Health) to determine the expression levels of the target proteins.

The nasal mucosa tissue was removed from the −80°C freezer and thawed, TriQuick (cat. no. R1100, Beijing Solarbio Science & Technology Co., Ltd.) was added and the tissue underwent lysis in a homogenizer. Subsequently, chloroform was added, and the sample was mixed and centrifuged at 12,000 × g for 15 min at 4°C. The upper layer was then collected, mixed with isopropanol and incubated at −20°C overnight. The next day, the RNA precipitate was obtained by centrifugation at 12,000 × g for 10 min at 4°C, and RNA was reverse transcribed into cDNA using the RT kit (cat. no. RR036A; Takara Bio, Inc.) according to the manufacturer's instructions, as follows: 37°C for 15 min, 85°C for 15 sec and 16°C indefinitely. Subsequently, qPCR was performed using 2X M5 HiPer SYBR Premix EsTaq (with Tli RNaseH) (cat. no. MF787-01; Mei5 Biotechnology, Co., Ltd). The thermocycling conditions were as follows: Pre-denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec, and a final melt curve analysis. The relative mRNA expression levels were calculated using the 2^−ΔΔCq method (28) with GAPDH as an endogenous control. The primer sequences are listed in Table II.

HNEpCs were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 5% fetal bovine serum (cat. no. SA301.02, Cellmax Co., Ltd.) and 1% penicillin/streptomycin solution) (cat. no. PWL062; Dalian Meilun Biology Technology Co., Ltd.) at 37°C in a humidified atmosphere containing 5% CO₂. Upon reaching 80% confluence, the cells were passaged and seeded into 6-well plates. All groups were treated at 37°C in a humidified atmosphere containing 5% CO₂. The cells were then divided into four groups for treatment: Control group received standard medium as a control; OVA group was exposed to 0.1% OVA (dissolved in standard medium) for 24 h; OVA + PA group was treated with 20 µmol/l PA (dissolved in standard medium) for 24 h following 24 h of intervention with 0.1% OVA; and OVA + PA + AKT inhibitor VIII group was pretreated with 10 mmol/l AKT inhibitor VIII (cat. no. HY-10355; MedChemExpress) (dissolved in DMSO) for 5 h before the addition of 0.1% OVA for 24 h, followed by treatment with 20 µmol/l PA (dissolved in standard medium) for 24 h. The proteins were extracted at the end of the experiment and were subsequently subjected to western blot analysis to detect related markers.

HNEpCs were cultured on sterile coverslips in 12-well plates until reaching 50% confluence and were treated as aforementioned. Following fixation with 4% paraformaldehyde for 10 min at room temperature, the coverslips were subjected to immunofluorescence staining. The harvested cells were permeabilized using a 1% Triton X-100 solution (cat. no. T8200; Beijing Solarbio Science & Technology Co., Ltd.) and antigenic blocking was carried out using sheep-derived blocking serum (cat. no. ZLI-9022; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 30 min. The cells were then incubated overnight at 4°C with primary antibodies against AKT (cat. no. 10176-2-AP, 1:100), P-AKT (Ser473) (cat. no. 66444-1-Ig, 1:100), CEBPB (cat. no. 23431-1-AP, 1:100) and IL-10 (cat. no. 60269-1-Ig, 1:100) (all from Wuhan Sanying Biotechnology). Subsequently, the samples were incubated with the following secondary antibodies: CY3-conjugated avidin (cat. no. BA1037, 1:200) and DyLight^® 488-conjugated avidin (cat. no. BA1128, 1:200) (both from Boster Biological Technology), for 1 h at room temperature in the dark. After staining with DAPI solution (cat. no. AR1176; 1:000; Boster Biological Technology) for 10 min at room temperature and washing with PBS three times (2 min/wash), anti-fluorescence quencher (cat. no. AR0036; Boster Biological Technology) was applied to the slides. The immunofluorescence slides were carefully removed from the well plates and incubated overnight at 4°C prior to observation of the results using a fluorescence microscope. The fluorescence microscope revealed DAPI-labeled nuclei in blue, DyLight 488-labeled proteins in green and CY3-labeled proteins in red.

Total proteins were extracted from mouse nasal mucosa tissues and HNEpCs using RIPA lysis buffer (cat. no. AR0102; Boster Biological Technology), and the total protein concentration was quantified using a BCA kit (cat. no. AR1189; Boster Biological Technology) according to the manufacturer's instructions. The protein samples were then normalized, treated with 5X SDS-PAGE loading buffer and denatured at 100°C for 10 min. Subsequently, the denatured proteins were separated by SDS-PAGE on a 10% gel and transferred to a PVDF membrane within 1.5 h. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 2 h. Primary antibodies specific to AKT (cat. no. 10176-2-AP, 1:4,000), P-AKT (Ser473) (cat. no. 66444-1-Ig, 1:1,000), CEBPB (cat. no. 23431-1-AP, 1:6,000), IL-10 (cat. no. 60269-1-Ig, 1:4,000) and β-actin (cat. no. P60709, 1:10,000) from (all from Wuhan Sanying Biotechnology) were used to incubate the membranes overnight at 4°C. After washing the membrane with TBS-0.1% Tween, the membranes were incubated for 2 h at room temperature with HRP-goat anti-rabbit IgG (cat. no. AS014; 1:10,000; ABclonal Biological Technology) and HRP-goat anti-mouse IgG (cat. no. BA1050; 1:10,000; Boster Biological Technology), before proceeding with further washing steps. Finally, protein signals on the membranes were detected using an ultrasensitive ECL chemiluminescence kit (cat. no. AR1171; Boster Biological Technology), and protein analysis was performed based on optical density measurements using ImageJ-win64 software.

Data analysis was performed using GraphPad Prism 8.0 (Dotmatics). Data are presented as the mean ± standard deviation and were obtained from at least three independent experiments. For comparisons involving more than two groups, a one-way analysis of variance was performed, followed by Tukey's multiple comparisons test to evaluate pairwise distinctions. The non-parametric Kruskal-Wallis test and Dunn's post hoc test was employed to analyze the SFAR and rhinorrhea score, and these data are presented as the median (IQR). P<0.05 was considered to indicate a statistically significant difference.

On day 21 of sensitization, mice in the OVA group began showing visible signs of sneezing, nose scratching and rhinorrhea, with symptoms escalating as OVA stimulation increased. Analysis of behavioral recordings revealed that by day 8 of stimulation, scores for the blank control group remained <5, in contrast to the OVA group, where scores for nasal symptoms >5, confirming the successful model construction (Table III). Furthermore, mice in the OVA + IPA group exhibited a gradual alleviation of AR symptoms upon IPA drug intervention, with statistical significance (P<0.05) observed from day 5 onwards (Fig. 2A). By the final day of stimulation, significant reductions in sneezing, nose scratching and rhinorrhea symptoms were evident in the OVA + IPA group, with behavioral scores for nasal symptoms significantly decreased compared with those in the OVA group (P<0.05; Fig. 2B-D).

Histological examination using H&E staining revealed that the nasal mucosal epithelium in the control group exhibited structural integrity with uniformly arranged cilia, smooth submucosal blood vessels and the absence of scattered inflammatory cells (Fig. 3A). By contrast, the OVA group showed discontinuous and disorganized nasal mucosal epithelium, infiltration of numerous inflammatory cells, mucosal edema, congested and dilated arteriolar blood vessels, and varying degrees of nasal mucosa detachment. PAS and TBS staining indicated the presence of proliferating cup cells and mast cells in the OVA group, with cup cells stained purple-red and mast cells stained purple (Fig. 3B and C). Treatment with IPA resulted in a reduction in the aforementioned cell number of cup cells and mast cells.

The levels of cellular inflammatory factors (IL-4, IL-5, IL-13, IgE and IL-10) were detected in mouse serum samples using ELISA. Significantly higher levels of IL-4, IL-5, IL-13 and IgE were observed in the OVA group compared with those in the Control and OVA + IPA groups (P<0.05; Fig. 4A, B, D and E). Conversely, IL-10 levels were significantly higher in the Control and OVA + IPA groups than those in the OVA group (P<0.05; Fig. 4C).

The immunohistochemical analysis of AKT, P-AKT, CEBPB and IL-10 expression in mouse nasal mucosa tissues was conducted using a light microscope and analyzed with ImageJ software (Fig. 5). AKT and P-AKT were observed in the nucleus and cytoplasm; CEBPB was predominantly nuclear; and IL-10 was detected in the nucleus, cytoplasm and extracellularly, with positive staining appearing brownish-yellow (Fig. 5A). Significantly lower levels of P-AKT and IL-10 were detected in the OVA group compared with those in the Control group (P<0.05; Fig. 5C, D and F). Conversely, the OVA + IPA group exhibited significantly increased expression levels of P-AKT, CEBPB and IL-10 compared with those in the OVA group (P<0.05; Fig. 5B-F).

Based on the immunohistochemical findings, RT-qPCR was utilized to assess the mRNA expression levels of AKT, CEBPB, IL-10, IL-4, IL-5 and IL-13 in the nasal mucosa of mice across different experimental groups (Fig. 6). Statistical analysis revealed no significant differences in AKT levels among the various groups (P>0.05; Fig. 6A). Compared with those in the Control group, the OVA group exhibited notably decreased mRNA expression levels of CEBPB and IL-10, while showing significantly elevated levels of IL-4, IL-5 and IL-13 mRNA (P<0.05; Fig. 6B-F). Conversely, in contrast to the OVA group, the OVA + IPA group demonstrated significantly increased mRNA expression levels of CEBPB and IL-10, and significantly reduced levels of IL-4, IL-5 and IL-13 mRNA (P<0.05; Fig. 6B-F).

The protein expression levels of AKT, P-AKT, CEBPB and IL-10 in the nasal mucosa of mice from each group were assessed using western blotting (Fig. 7). Compared with those in the Control group, the OVA group exhibited a significant decrease in the protein expression levels of CEBPB and IL-10 (P<0.05; Fig. 7E and F). Conversely, the OVA + IPA group displayed notably elevated protein expression levels of P-AKT, CEBPB and IL-10 compared with those in the OVA group (P<0.05; Fig. 7C-F).

To elucidate the impact of PA on AR via the AKT/CEBPB/IL-10 signaling pathway, HNEpCs were treated with AKT inhibitor VIII, OVA and PA, followed by western blot analysis (Fig. 8). The findings revealed a significant decrease in the protein expression level of CEBPB in the OVA group compared with the control group, and a significant increase in the protein expression levels of P-AKT, CEBPB and IL-10 in the OVA + PA group compared with those in the OVA group (P<0.05; Fig. 8C-F). Conversely, following AKT inhibitor VIII intervention, there was a notable decrease in the protein expression levels of P-AKT, CEBPB and IL-10 (P<0.05).

Immunofluorescence staining was utilized to detect the protein expression levels of AKT, P-AKT, CEBPB and IL-10 in the nucleus and cytoplasm (Fig. 9). Significantly decreased fluorescence staining signals of P-AKT, CEBPB and IL-10 were observed in the HNEpCs of the OVA group compared with those in the control group, whereas significantly increased fluorescence staining signals of P-AKT, CEBPB and IL-10 were observed in the HNEpCs of the OVA + PA group compared with those in the OVA group (P<0.05; Fig. 9F-I). Conversely, intervention with AKT inhibitor VIII led to a notable decrease in the fluorescence signals of P-AKT, CEBPB and IL-10 (P<0.05).