Therapeutic Effect of Dipsacus asperoides C. Y. Cheng et T. M. Ai in Ovalbumin-Induced Murine Model of Asthma

Abstract

Dipsacus asperoides C. Y. Cheng et T. M. Ai (DA) has been used in China as a traditional medicine to treat lumbar and knee pain, liver dysfunction, and fractures. We explored the suppressive effect of DA on allergic asthma using an ovalbumin (OVA)-induced asthma model. In the asthma model, female Balb/c mice were sensitized to OVA on day 0 and 14 to boost immune responses and then exposed to OVA solution by using an ultrasonic nebulizer on days 21 to 23. DA (20 and 40 mg/kg) was administered to mice by oral gavage on days 18 to 23. Methacholine responsiveness was determined on day 24 using a plethysmography. On day 25, we collected bronchoalveolar lavage fluid, serum, and lung tissue from animals under anesthesia. DA treatment effectively inhibited methacholine responsiveness, inflammatory cell infiltration, proinflammatory cytokines such as interleukin (IL)-5 and IL-13, and immunoglobulin (Ig) E in OVA-induced asthma model. Reductions in airway inflammation and mucus hypersecretion, accompanied by decreases in the expression of inducible nitric oxide synthase (iNOS) and the phosphorylation of nuclear factor kappa B (NF-κB), were also observed. Our results indicated that DA attenuated the asthmatic response, and that this attenuation was closely linked to NF-κB suppression. Thus, this study suggests that DA is a potential therapeutic for allergic asthma.

Keywords: Dipsacus asperoides C. Y. Cheng et T. M. Ai; allergic asthma; inducible nitric oxide synthase; nuclear factor kappa B; pro-inflammatory cytokine.

Figure 1

Chromatogram of Dipsacus asperoides C. Y. Cheng et T. M. Ai (DA) detected at 237 (a) and 198 nm (b).

Figure 2

DA decreased methacholine responsiveness in ovalbumin (OVA)-induced asthma model. DA-treated mice exhibited a significant reduction in methacholine responsiveness after OVA exposure. NC: normal controls, PBS treatment, and inhalation; OVA: asthma model, PBS treatment, and OVA sensitization and inhalation; Mon: montelukast-treated mice, montelukast (10 mg/kg, oral gavage), and OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) and OVA sensitization and inhalation. The values shown are the mean ± SD. ## p < 0.01 vs. NC; * p < 0.05, ** p < 0.01, respectively.

Figure 3

DA decreased the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) of OVA-induced asthma model. DA-treated mice exhibited a marked decline in inflammatory cell counts compared with OVA-induced asthma model. NC: normal controls, PBS treatment and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. The values shown are the mean ± SD. ## p < 0.01 vs. NC; * p < 0.05, ** p < 0.01, respectively.

Figure 4

DA suppressed inflammatory cell infiltration in the lung tissue of OVA-induced asthma model. The DA-treated mice exhibited lower inflammatory cell infiltration into the lung tissue than OVA-induced asthma model. NC: normal controls, PBS treatment and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. Scale bar = 50 µm. The values shown are the mean ± SD. ## p < 0.01 vs. NC; * p < 0.05, ** p < 0.01, respectively.

Figure 5

DA decreased the level of Th2 cytokines and IgE in OVA-induced asthma model. (A) IL-5, (B) IL-13, (C) Eotaxin, (D) Total IgE, (E) OVA-specific IgE, and (F) MUC5AC. NC: normal controls, PBS and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. The values shown as the mean ± SD. ## p < 0.01 vs. NC; * p < 0.05, ** p < 0.01, respectively.

Figure 6

DA inhibited the expression of iNOS and the phosphorylation of NF-κB in OVA-induced asthma model. Densitometric band value was determined using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression value is expressed as iNOS vs. β-actin and p-p65 vs p65. (A) Expression of iNOS; (B) expression of phosphorylation of NF-κB. Relative expression value NC: normal controls, PBS treatment and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. The values shown are the means ± SD. ## p < 0.01 vs. NC; * p < 0.05, ** p < 0.01, respectively.

Figure 7

DA decreased iNOS expression in the lung tissue of OVA-induced asthma model. DA-treated mice exhibited a reduction in iNOS expression in the lung tissue compared with OVA-induced asthma model. NC: normal controls, PBS treatment and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. Scale bar = 50 µm.

Figure 8

DA reduced mucus production in the lung tissue of OVA-induced asthma model. Lung tissue was stained with PAS solution. NC: normal controls, PBS treatment and inhalation; OVA: PBS treatment with OVA sensitization and inhalation; Mon: montelukast-treated mice (10 mg/kg, oral gavage) with OVA sensitization and inhalation; DA20 and 40: Dipsacus asperoides C. Y. Cheng et T. M. Ai-treated mice (20 and 40 mg/kg, respectively, oral gavage) with OVA sensitization and inhalation. Scale bar = 50 µm. The values shown as the mean ± SD. ## p < 0.01 vs. NC; * p < 0.05.

Figure 9

Experimental procedure.