Effects of 4-DAMP on allergic rhinitis in guinea pigs and its potential mechanism

Abstract

Background: Allergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mainly mediated by immunoglobulin E (IgE), which seriously affects the life quality of affected patients. This study aimed to observe the effects of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a selective M3 receptor antagonist) on ovalbumin (OVA)-induced AR guinea pigs, and to explore its potential mechanism of involvement.

Methods: An AR model was established by inducing male guinea pigs (4-6 weeks of age) with OVA. AR guinea pigs were randomly divided into a model group, 0.6 mg/kg ipratropium bromide (IB) group, 0.12 and 0.6 mg/kg 4-DAMP group (n=18). The 0.6 mg/kg IB group, 0.12 and 0.6 mg/kg 4-DAMP group animals were treated with IB (0.6 mg/kg) and 4-DAMP (0.12 or 0.6 mg/kg) by intranasal instillation per nostril daily. Animals in the model group and normal group were treated with saline as control. The AR symptom scores were counted and nasal secretion weights were measured. Histopathological methods were used to observe nasal mucosa. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of histamine and cytokines. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expressions of mucin 5AC (MUC5AC), matrix metalloproteinase 9 (MMP9), and epidermal growth factor receptor (EGFR).

Results: Compared with model group animals, the AR symptom scores and nasal secretion weights of animals treated with 4-DAMP were reduced significantly, goblet cell metaplasia was reversed, and eosinophil infiltration was visibly alleviated. The levels of histamine and cytokines in nasal lavage fluid (NLF) were decreased, and the protein and messenger RNA (mRNA) expressions of MUC5AC, MMP9, and EGFR were inhibited.

Conclusions: Treatment with 4-DAMP has a certain effect on AR, especially for mucus hypersecretion, which provides a new idea for clinical treatment of AR.

Keywords: 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP); M3 receptor; allergic rhinitis (AR); chronic inflammatory; hypersecretion.

Figure 1

Experimental protocol of modeling and administration. OVA, ovalbumin; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; qd, quaque die; qod, quaque omni die.

Figure 2

Effects of 4-DAMP on the total symptom scores of AR in guinea pigs. *, P<0.05; **, P<0.01; ***, P<0.001, vs. model group (n=6). AR, allergic rhinitis; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 3

Effects of 4-DAMP on the weights of nasal secretions in AR guinea pigs. ***, P<0.001, vs. model group (n=6). AR, allergic rhinitis; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 4

Effects of 4-DAMP on the histopathology of nasal mucosa in AR guinea pigs (original magnification ×400). AR, allergic rhinitis; HE, hematoxylin and eosin; AB-PAS, Alcian blue periodic acid-Schiff; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 5

Effects of 4-DAMP on the numbers of eosinophils and goblet cells in the nasal mucosa of AR guinea pigs. **, P<0.01; ***, P<0.001, vs. model group (n=6). AR, allergic rhinitis; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 6

Effects of 4-DAMP on MUC5AC in the nasal mucosa. (A) MUC5AC protein level in the nasal mucosa was measured by Western-blot analysis (n=3). (B) MUC5AC mRNA levels in the nasal mucosa were measured by RT-PCR (n=4). **, P<0.01; ***, P<0.001, vs. model group. mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction; MUC5AC, mucin 5AC; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 7

Effects of 4-DAMP on the levels of histamine and cytokines IL-4, IL-13, and TNF-α in guinea pig NLF. The levels of histamine (A) and cytokines IL-4 (B), IL-13 (C), and TNF-α (D) were measured by ELISA (n=6). ***, P<0.001, vs. model group. IL-4, interleukin 4; IL-13, interleukin 13; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 8

Effects of 4-DAMP on MMP9 in the nasal mucosa. (A) MMP9 protein level in the nasal mucosa was measured by Western-blot analysis (n=3). (B) MMP9 mRNA level in the nasal mucosa was measured by RT-PCR (n=5). *, P<0.05; **, P<0.01; ***, P<0.001, vs. model group. mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide; MMP9, matrix metalloproteinase 9.

Figure 9

Immunohistochemistry of MMP9 and EGFR in nasal mucosa (original magnification ×400). EGFR, epidermal growth factor receptor; MMP9, matrix metalloproteinase 9; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.

Figure 10

Effects of 4-DAMP on EGFR in the nasal mucosa. (A) EGFR mRNA level in the nasal mucosa was measured by RT-PCR (n=5). (B) EGFR protein level in the nasal mucosa was measured by Western-blot analysis (n=3). *, P<0.05; **, P<0.01; ***, P<0.001, vs. model group. mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction; EGFR, epidermal growth factor receptor; 4-DAMP, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; IB, ipratropium bromide.