Fructus Amomi extract attenuates nasal inflammation by restoring Th1/Th2 balance and down-regulation of NF-κB phosphorylation in OVA-induced allergic rhinitis

Abstract

Fructus Amomi Cardamomi (FA) is the mature fruit of Amomum villosum Lour (family Zingiberaceae) and is commonly used in Chinese traditional medicine to treat various gastrointestinal disorders. FA's possible benefits as an allergic rhinitis (AR) treatment, however, have not been examined. We used an ovalbumin (OVA)-induced AR mouse model to identify any anti-allergic effects associated with the administration of 200 mg/kg FA or dexamethasone (Dex) 2.5 mg/kg by oral administration. The results of our testing confirm that FA ameliorated nasal symptoms and alleviated nasal epithelium swelling, reduced the goblet cell hyperplasia and eosinophil cell infiltration in the nasal epithelium, and inhibited lung tissue inflammation and Dex as well. Significantly decreased Th2 cytokine (interleukin (IL)-1β, IL-4, and IL-5) expression, and a correspondingly significant increase in Th1 cytokine (IL-12, interferon (IFN)-γ) production, was observed in nasal lavage fluid (NALF) taken from mice that received FA or Dex treatment. FA also reduced the presence of OVA-specific immunoglobulin (Ig) E, OVA-specific IgG1, and histamine levels in serum, and inhibited mast cell degranulation in vitro. In addition, these effects were involved with the reduction in NF-κB phosphorylation. These results suggest that FA restores Th1/Th2 balance and inhibits NF-κB phosphorylation and mast cell degranulation, thereby achieving a notable anti-inflammatory effect. Accordingly, it has the potential to be used as an efficacious therapeutic treatment for AR.

Keywords: Fructus Amomi; NF-κB phosphorylation; Th1/Th2 balance; allergic rhinitis; mast cell degranulation.

Figure 1. Effect of FA on nasal symptoms in the murine AR model

(A) Animal protocol. Mice were sensitized on days 0, 7, and 14; challenged between days 21 and 28. Mice in the OVA group were sensitized and challenged by OVA. Mice in the FA or Dex treatment groups were sensitized and challenged by OVA and administered 200 mg/kg FA or 2.5 mg/kg Dex between days 15 and 28 once a day by oral gavage (200 μl). (B) Rubbing and (C) sneezing scores. The values represent the mean ± S.E.M (n=6/group). Significant differences at *P<0.05 are compared with each group.

Figure 2. Effect of FA on levels of OVA-specific IgE, OVA-specific IgG₁, and histamine in serum of AR mice

The levels of (A) OVA-specific IgE, (B) OVA-specific IgG₁, and (C) histamine in the serum were measured by ELISA kit. The values represent the mean ± S.E.M (n=6/group). Significant differences at ***P<0.001, **P<0.01, *P<0.05 are compared with each group.

Figure 3. Effect of FA on nasal cavity and lung tissue of AR mice

(A) The inflammatory cells’ infiltration into NALF. (B) Lung histology. The NALF were isolated by cytospin and stained with Diff-Quick. An increase in the number of eosinophils (indicated by the red arrows) in the NALF of OVA group was observed. Lung histology of AR mice showed several inflammatory features: epithelial swelling, over-secreted mucus, and severely infiltrated inflammatory cell surround bronchia. FA treatment considerably alleviated these inflammatory features. Scale bar = 50 μm.

Figure 4. Effect of FA on nasal thickness, goblet cell hyperplasia, and eosinophil infiltration in AR nasal mucosa tissues

H&E staining results, the epithelia of the OVA group were significantly thicker than those of the Naive group; epithelial swelling was ameliorated by FA and Dex treatment. The number of goblet cells (purple arrows; PAS staining), the number of eosinophils (red arrows; Giemsa staining), and the number of mast cell (blue circles; Toluidine Blue) present in the OVA group was significantly higher than in the Naive group and was markedly decreased by FA and Dex treatment. The values represent the mean ± S.E.M (n=6/group). Significant differences at ***P<0.001, **P<0.01, *P<0.05 are compared with each group. Bar = 50 μm.

Figure 5. Effect of FA on the Th1 cytokines release in NALF

The level of (A) IL-12 and (B) IFN-γ in NALF were determined by ELISA kit. The values represent the mean ± S.E.M (n=6/group). Significant differences at ***P<0.001, **P<0.01 are compared with each group.

Figure 6. Effect of FA on the Th2 cytokines and pro-inflammatory cytokines release in NALF of AR mice

The levels of (A) IL-4, (B) IL-5, (C) IL-1β, (D) IL-6, (E) TNF-α in NALF were determined by ELISA kit. The values represent the mean ± S.E.M (n=6/group). Significant differences at ***P<0.001, **P<0.01, *P<0.05 are compared with each group.

Figure 7. Effect of FA on NF-κB signaling pathway of AR mice

(A) The expression of NF-κB signaling-related protein in lung tissue were quantified by Western blots. The ratios of (B) NF-κB/β-actin, (C) p-NF-κB/β-actin, (D) p-NF-κB/NF-κB, (E) I-κB/β-actin, (F) p-I-κB/β-actin, and (G) p-I-κB/I-κB were calculated via ImageJ program. Significant differences at ***P<0.001, *P<0.05 are compared with each group.

Figure 8. Effect of FA on the histamine derived-mast cell release

(A) RPMC morphology, (B) RPMC degranulation ratio, (C) level of histamine release. RPMCs were pretreated with FA (0.01, 0.1, 1 mg/ml) then stimulated with compound C48/80. FA significantly alleviated RPMCs degranulation and inhibited histamine release after stimulation with compound C48/80. The red arrows indicated degranulated mast cells. Significant differences at ***P<0.001, **P<0.01, *P<0.05 are compared with each group.

Figure 9. The mechanism of action FA in an OVA-induced AR mouse model

Exposing to allergens promotes Th0 cell to differentiate towards Th2 cells, stimulate B cells to produce IgE, IgE bind to mast cell and activate them. Once mast cells are triggered, it releases their pro-inflammatory cytokines and chemokines. Together with Th2 cell-released cytokines, the leukocytes are recruited and activated lead to the airway allergic inflammatory symptoms. NF-κB pathway is activated to more amplify the inflammatory situation. Here, FA showed their anti-allergic inflammatory effect by balancing the Th1/Th2 ratio, suppressing the NF-κB signaling, and inhibiting mast cell degranulation.