All animal experiments were performed according to the Health Guide for the Care and Use of Laboratory Animals and were approved by the medical ethics committee of Shanxi Medical University. A total of 60 specific pathogen-free female BALB/c mice, aged 6-8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology. They housed in ventilated isolation cages in a climate-controlled room (20 ± 1°C and 50-70% humidity) with a 12 h light-dark cycle. Animals were acclimated for one week before use to monitor for abnormal behavior.

The 60 mice were randomly divided into seven groups: (i) a control group (C, n = 8) was mock-sensitized and mock-challenged with saline; (ii-iii) two identically treated model groups (M1 and M2, n = 8/group) were sensitized and challenged with OVA and mock-treated with saline; (iv-vi) three PEFF treatment groups (n = 9/group) were sensitized, challenged with OVA, and treated by i.n. instillation of PEFF at a low dose (LD, 0.01 g/kg), medium dose (MD, 0.02 g/kg), or high dose (HD, 0.04 g/kg); and (vii) a positive control group (n = 9) was sensitized, challenged with OVA, and treated by i.n. instillation of BUD (P, 0.052 mg/kg).

Establishment of the AR model and timing of the treatments are described in Fig. Fig.1.1. Briefly, the mice were sensitized by intraperitoneal (i.p.) injection of 200 μL saline containing 50 μg OVA and 2 mg aluminum hydroxide on days 0, 7, and 14. The mice were challenged i.n. with OVA (10 μL saline containing 100 μg/mL OVA) once daily from day 21 to day 34. The lyophilized PEFF was dissolved to the final concentration instilled into the nasal mucosa directly using 20% DMSO/saline. Between day 35 and day 55, the treatment groups were administered 20% DMSO/saline (M2), LD, MD, or HD PEFF, or BUD i.n. (10 μL per nostril) once daily, 1 h before the OVA challenge. The control mice received saline i.p. or i.n. on the same schedule. During the treatment period (days 35 to 55), the model (M2), PEFF, and BUD groups were challenged i.n. with 10 μL OVA (100 μg/mL) every other day to maintain the response. The data obtained from group M1 were used for evaluating the AR model, and data obtained from group M2 were used for evaluating the therapeutic effects of PEFF on AR.

Mouse model of AR. Groups of BALB/c mice were sensitized by intraperitoneal injection of OVA and aluminum hydroxide on days 0, 7, and 14, followed by intranasal challenge with OVA once daily from day 21 to day 34. Mice in the model and treatment groups were administered 20% DMSO/saline and PEFF or BUD, respectively, once daily from day 35 to day 55 by intranasal instillation. Every other day between days 35 and 55, the mice also received OVA intranasally 1 h before treatment to maintain AR. Mice in the control group were administered saline intraperitoneally or intranasally on the same schedule. All mice were sacrificed on day 56.

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