Male BALB/c mice that were 6–8 weeks old and 20–25 g (specific pathogen-free grade) were obtained from the Experimental Animal Center of Nanchang University (Nanchang, China). Mice were bred in a clean environment at 22–24°C under a 12-h light/dark cycle and fed with an ovalbumin (OVA)-free diet. The present study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee of Nanchang University.

In total, 24 BALB/c mice were randomly divided into four groups (n=6 per group): Group I, no-treatment control (control); group II, PBS treatment control (PBS control); group III, scramble small interfering RNA (siRNA) treatment (off-target); and group IV, CCR3 siRNA treatment (pLVX-shRNA-mCCR3).

The mice in groups II to IV were intranasally administered with 8 µl PBS, control siRNA or CCR3 siRNA, respectively, twice a day on days 0 and 14. Additionally, these mice were intraperitoneally injected with an OVA/aluminum hydroxide [Al(OH)₃] mixture [containing 10 µg OVA and 4 mg Al(OH)₃] for allergization twice a day on days 2 and 16 and continuously intranasally administered with 600 µg/ml OVA twice a day from day 21 to 27 for excitation. At 5 h before excitation, the mice were administered intranasal treatments as described above. The control group was administered the same dose of saline. Samples of the bone marrow, peripheral blood and nasal mucosa were obtained 24 h after administration of the final treatment.

BALB/c mice were sacrificed and their femurs were isolated. Femoral marrow was rinsed with RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with streptomycin and penicillin (HyClone), 20% fetal bovine serum (HyClone), Fms-related tyrosine kinase 3 ligand (FLT3-L; PeproTech, Inc., Rocky Hill, NJ, USA), stem cell factor (SCF; PeproTech, Inc.) and recombinant mouse IL-5 (rmIL-5; PeproTech, Inc.). A total of 10⁷ cells/ml were incubated at 37°C in 5% CO₂.

Untreated cells (1×10⁶) were collected from culture wells and rinsed with PBS, and the supernatant was discarded. Anti-IL-5 receptor (alpha) PE antibody (eBioscience, Inc., San Diego, CA, USA) and anti-CD34 fluorescein isothiocyanate (FITC) antibodies (eBioscience, Inc.) were added to untreated cells, single cell suspensions were prepared and double-immunopositive cells were sorted by flow cytometry.

Lentivirus transduction was performed on the tenth day of EOS culture. One day before transduction, EOS were seeded at a density of 5×10⁵ cells/well into a 12-well culture plate, which were divided into three groups when performing transductions. Group I was treated with shRNA-mCCR3 virus mixed with medium to 100 µl (with MOI=10); group II was treated with the same amount of control virus; and group III was treated with 0.1% PBS-medium mixture. Cells were cultured for 48 h after transduction; subsequently, the medium was aspirated, cells were washed with 0.1% PBS and then treated according to the following methods.

EOS were resuspended in TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China), and RNA was extracted according to the manufacturer's instructions. RNA was converted to cDNA using a reverse transcription kit(Takara Biotechnology Co., Ltd.), and primers were designed for CCR3 (IDNM_009914.4) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ID: NM_017008.4, as the control, synthesized by Oligo). Reverse transcription-polymerase chain reaction RT-PCR reactions used to amplify cDNA were set up in accordance with the appropriate annealing temperature and cycles. Furthermore, the expression of mRNA for each group was detected by electrophoresis. Samples of the bone marrow, peripheral blood and nasal mucosa were tested according to the procedures described above.

EOS were homogenized in radioimmunoprecipitation assay lysis buffer (Pierce Protein Biology; Thermo Fisher Scientific, Inc., Rockford, IL, USA). The homogenate was cooled on ice for 20 min and centrifuged at 4°C and 14,000 rpm to remove the insoluble material. The supernatant was mixed with 4% SDS sample buffer, boiled for 5 min, and resolved using 10% SDS-PAGE (Ready Gel J; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA), blocked with 5% skimmed milk diluted in 1X Tris-buffered saline with Tween 20 (TBST) buffer for 2 h, and incubated with the primary antibody (Abcam, Cambridge, UK) overnight at 4°C. The membranes were rinsed with TBST three times, incubated with the secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc., Beijing, China) for 2 h, and washed again with TBST three times. An anti-GAPDH antibody was used as an internal reference protein to normalize protein loading (Anbo Biotechnology Co., Ltd., Nanjing, China) and concentrations of each target protein were normalized against GAPDH. The membranes were evenly mixed with chemiluminescence reagent, incubated in the dark at room temperature for 3–5 min, then exposed and developed using X-ray films. The intensity levels of bands from the electrophoretic gels and photographic films were measured using a dedicated image analysis software (Band Leader 3.0; http://en.bio-soft.net/chip/BandLeader.html).Samples of the bone marrow, peripheral blood and nasal mucosa were tested according to the procedures described above.

Cells were treated with lentivirus for 48 h and control cells were incubated with antibodies provided in an apoptosis kit (PeproTech, Inc.), and single cell suspensions were prepared with PBS following the manufacturer's instructions. Apoptosis was detected with flow cytometry.

Samples of the bone marrow and peripheral blood were collected, and cells were isolated with lymphocyte isolation liquid (GE Healthcare Life Sciences) to prepare the single cell suspension. The cells were labeled with anti-CD34 FITC primary antibody and detected with flow cytometry following resuspension.

SPSS software, version 18 (SPSS, Inc., Chicago, IL, USA) was used for analysis of variance in order to determine intergroup differences, which were expressed as the mean ± standard deviation. Each experiment was repeated three times. The error bars indicate the standard error of the samples. P<0.05 was used to indicate a statistically significant difference.

Under the induction of the growth factors, FLT3-L and SCF, the majority of the primary bone marrow cells differentiated into mature EOS. Due to the effect of rmIL-5, EOS survived and apoptosis was delayed. Moreover, an optical microscope was used to observe cell morphology after incubation with the indicated factors. HE and Wright staining of EOS are shown in Fig. 1.

As shown in Fig. 2, the mRNA expression of CCR3 in the control that did not receive any treatment and the control siRNA treatment group was not significantly different (Fig. 2A). However, the mRNA expression of CCR3 in the CCR3 siRNA treatment group was significantly lower than that in the other two groups (P<0.05) (Table I).

As shown in Fig. 2B, the protein expression of CCR3 in the control group that did not receive any treatment and the control siRNA treatment group was not significantly different, whereas the protein expression of CCR3 in the CCR3 siRNA treatment group was significantly lower than that in the other two groups (P<0.05). Furthermore, the experiment was repeated three times.

EOS express both CD34 and IL-5; therefore, it is possible to identify EOS by the specific labeling of these proteins (Fig. 3) using anti-IL-5 PE and CD34 FITC antibodies. The CD34 epitope could be detected in early-differentiated EOS, whereas its expression was not detected significantly in apoptotic EOS. Moreover, the IL-5 epitope could be detected during both stages of EOS.

Using flow cytometry, the percentage of cultured cells labeled with the anti-IL-5 PE and CD34 FITC antibodies were found to be 65.7±3.25 and 17.5±2.27%, respectively, which were also significantly different (P<0.05). These percentages can be used to estimate the percentage of EOS in the cultured cells.

The apoptotic rates of EOS were measured for the following groups: 24.52±4.56% for the control that did not receive any treatment, 20.7±2.32% for the control siRNA and 67.5±5.88% for the CCR3 siRNA groups. The apoptotic rate of the CCR3 siRNA group was significantly higher than the apoptotic rates of the other two groups (P<0.05). However, there was no significant difference between the control that did not receive any treatment and the control siRNA groups (P>0.05; Fig. 4).

The mRNA expression levels of CCR3 in the PBS and control siRNA groups were not significantly different, whereas the mRNA expression of CCR3 in the CCR3 siRNA group was significantly lower than that in the other two groups (P<0.05; Fig. 5A). In addition, the experiment was repeated three times.

Moreover, the protein expression of CCR3 in the PBS and the control siRNA group was not significantly different, whereas the protein expression of CCR3 in the CCR3 siRNA group was significantly lower than that in the other two groups (P<0.05) (Table II and Fig. 5B). The experiment was repeated three times.

Edema, inflammatory cell infiltration, epithelial shedding and quantity of exudates and EOS in the nasal mucosa were worse in the PBS and control siRNA groups compared with the control group, indicating that the histological results of these animal models met the pathological characteristics of AR. Moreover, in the CCR3 siRNA group IV, the number of EOS was decreased and mucosal edema was alleviated, indicating that silencing of CCR3 reduced the pathology of AR (Fig. 6).

No significant difference was detected in the number of CD34⁺ cells from the bone marrow, peripheral blood and CCR3 siRNA groups (P>0.05; Fig. 7). Moreover, the experiment was repeated three times.