Effects of anticholinergic agent on miRNA profiles and transcriptomes in a murine model of allergic rhinitis

Abstract

Anticholinergic agent, ipratropium bromide (IB) ameliorates symptoms of allergic rhinitis (AR) using neuroimmunologic mechanisms. However, the underlying molecular mechanism remains largely unclear. In the present study, 27 mice with AR induced by ovalbumin were randomly allocated to one of three groups: Model group, model group with IB treatment for 2 weeks, and model group with IB treatment for 4 weeks. Allergic symptoms were evaluated according to symptoms scores. Differentially expressed genes [microRNAs (miRNAs) and messenger RNAs (mRNAs)] of nasal mucosa were identified by microarray analysis. The expression levels of candidate genes were measured by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The data indicates that the symptoms scores in allergic mice were significantly reduced by IB treatment. In the nasal mucosa of allergic mice with IB treatment, 207 mRNAs and 87 miRNAs were differentially expressed, when compared with the sham group. IB treatment significantly downregulated the expression levels of interleukin‑4Rα and prostaglandin D2 synthase, whereas the leukemia inhibitory factor, A20 and nuclear receptor subfamily 4, group A, member 1 expression levels were upregulated. Similarly, the expression levels of mmu‑miR‑124‑3p/5p, ‑133b‑5p, ‑133a‑3p/5p, ‑384‑3p, ‑181a‑5p, ‑378a‑5p and ‑3071‑5p were significantly increased. RT‑qPCR data further validated these mRNA and miRNA expression levels. Thus, IB treatment regulated expression of allergic immune‑associated mRNAs and miRNAs of the nasal mucosa in allergic mice, which may be associated with ameliorated nasal allergic symptoms.

Figure 1.

Flowchart demonstrating the establishment of the AR model and treatment protocol. Stage I, systemic sensitization by OVA; stage II, nasal mucosa local challenge with OVA; Stage III, IB treatment with a concomitant OVA challenge. Control: Groups A and B. Model: Groups C-E. AR, allergic rhinitis; OVA, ovalbumin; IB, ipratropium bromide; NS, normal saline.

Figure 2.

Symptom scores in different groups before and after IB treatment. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). Data are expressed as the mean ± standard deviation. ^※P<0.05 vs. group C before IB treatment; *P<0.05 vs. group C after IB treatment; ^#P<0.05. IB, ipratropium bromide.

Figure 3.

Venn diagram of differentially expressed (A) mRNAs and (B) miRNAs. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). When mRNAs or miRNAs exhibit similar characteristics, they appear in the overlapping boxes. mRNA, messenger RNA; miRNA, microRNA.

Figure 4.

IB treatment regulated mRNA expression levels of allergic-associated genes. (A-M) The mRNA expression level in nasal mucosa was determined by reverse transcription-quantitative polymerase chain reaction with β-actin as the reference gene. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). Data are presented as the mean ± standard deviation. *P<0.05 vs. group C following IB treatment; ^#P<0.05. IB, ipratropium bromide; mRNA, messenger RNA; IL, interleukin; PGDS, prostaglandin D2 synthase; LIF, leukemia inhibitory factor; A20, tumor necrosis factor α-induced protein 3; NR4A1, nuclear receptor subfamily 4, group A, member 1; IFN-γ, interferon-gamma; FoxP3, forkhead box P3.

Figure 5.

PPI network for differentially expressed proteins. The gene network was constructed using Cytoscape software based on the STRING database. The circular node represents a single differentially expressed gene, and edges between nodes indicate the confidence score between them. The size of the node is correlated with the positivity degree, namely the number of genes interacting with it, and the width of the edge is positively associated with the confidence score of PPI. The colors of the nodes gradually change from green to red, reflecting the clustering coefficient depicting the positive connectivity of the nodes. PPI, protein-protein interaction.

Figure 6.

IB treatment regulated allergic-associated miRNA expression levels. (A-I) The miRNA expression level in nasal mucosa was determined by reverse transcription-quantitative polymerase chain reaction with U6 as the reference gene. Control: Group A (n=7) and group B (n=8); model: Group C (n=8), group D (n=10) and group E (n=9). Data are presented as the mean ± standard deviation. *P<0.05 vs. group C after IB treatment; ^#P<0.05. IB, ipratropium bromide; miRNA, microRNA; Mmu: Mus musculus.