Safranal Alleviated OVA-Induced Asthma Model and Inhibits Mast Cell Activation

Abstract

Introduction: Asthma is a chronic and recurring airway disease, which related to mast cell activation. Many compounds derived from Chinese herbal medicine has promising effects on stabilizing mast cells and decreasing inflammatory mediator production. Safranal, one of the active compounds from Crocus sativus, shows many anti-inflammatory properties. In this study, we evaluated the effect of safranal in ovalbumin (OVA)-induced asthma model. Furthermore, we investigate the effectiveness of safranal on stabilizing mast cell and inhibiting the production of inflammatory mediators in passive systemic anaphylaxis (PSA) model.

Methods: OVA-induced asthma and PSA model were used to evaluate the effect of safranal in vivo. Lung tissues were collected for H&E, TB, IHC, and PAS staining. ELISA were used to determine level of IgE and chemokines (IL-4, IL-5, TNF-α, and IFN-γ). RNA sequencing was used to uncovers genes that safranal regulate. Bone marrow-derived mast cells (BMMCs) were used to investigate the inhibitory effect and mechanism of safranal. Cytokine production (IL-6, TNF-α, and LTC₄) and NF-κB and MAPKs signaling pathway were assessed.

Results: Safranal reduced the level of serum IgE, the number of mast cells in lung tissue were decreased and Th1/Th2 cytokine levels were normalized in OVA-induced asthma model. Furthermore, safranal inhibited BMMCs degranulation and inhibited the production of LTC₄, IL-6, and TNF-α. Safranal inhibits NF-κB and MAPKs pathway protein phosphorylation and decreases NF-κB p65, AP-1 nuclear translocation. In the PSA model, safranal reduced the levels of histamine and LTC₄ in serum.

Conclusions: Safranal alleviates OVA-induced asthma, inhibits mast cell activation and PSA reaction. The possible mechanism occurs through the inhibition of the MAPKs and NF-κB pathways.

Keywords: BMMCs; Crocus sativus; Safranal; asthma; passive systemic anaphylaxis.

Figure 1

Safranal alleviates OVA-induced asthma. (A) OVA-induced asthma model. Mice were sensitized with OVA on days 1 and 14 before oral administration of vehicle, Safranal or Dexamethasone. On days 22, 23, and 24 Mice were challenged with OVA. Blood samples were collected by cardiac puncture and level of serum IgE was determined (B). The data are presented as the means ± S.E.M. of n=10. Ns=no significance ^### p < 0.001 compared to nontreated group, *p < 0.05, ***p < 0.001 compared to OVA treated group.

Figure 2

Histological of lung tissue. (A, B) H&E and PAS staining showed safranal suppressed infiltration of inflammatory cells, amount of mucus secretion. (C, D) Toluidine blue staining of lung tissue showed the number of mast cell in lung tissue. Numbers of mast cells within the lung were quantified in 200×200µm. (E, F) IHC staining of c-kit showed marker of the mast cells. The data are presented as the means ± S.E.M. of n=10. ^## p<0.01 compared to nontreated group. *p<0.05 compared to OVA treated group. The scale labels shown are 50 μm.

Figure 3

Effect of Safranal on Th1/Th2 cytokines and T cells in lung tissues. Safranal decrease the level of IL-4, IL-5, IL-13 (A–C) in lung tissues, while IFN-γ level increased (D). Effect of safranal on T cell in lung tissues. (E, F) IHC staining of CD4 and Foxp3. The result showed decreased of CD4 and Foxp3 in safranal treated group. The data are presented as the means ± S.E.M. of n = 10. ^# p <0.05, ^## p <0.01, ^### p <0.001 compared to nontreated group. **p <0.01, ***p <0.001 compared to OVA treated group. The scale labels shown are 50μm.

Figure 4

Effect of Safranal on NF-κB and MAPKs pathways in splenocyte. Safranal suppressed the activation of the NF-κB and MAPKs pathways proteins in splenocyte (A–E). The data are presented as the means ± S.E.M. of n = 10. ^# p <0.05, ^## p <0.01, ^### p <0.001 compared to nontreated group. *p <0.05 compared to OVA treated group.

Figure 5

RNA-sequencing of safranal and DNP-HSA treated BMMCs. BMMCs were treated with safranal before stimulating with DNP-HSA. (A) Heatmap of genes expression shows effect of safranal treated BMMC on representative differentially expressed genes comparing to DNP-HSA treated. False Discovery Rate (FDR) ≤ 0.05 and |Log2Ratio| ≥ 1. (B) KEGG pathway database analysis found 13 differentially expressed genes that were related to the immune systems. (C) The protein interactions of these genes were evaluated, Ccl7 and Cxcl10 were found to be most related among the differentially expressed genes.

Figure 6

Safranal inhibited degranulation and decreased inflammatory mediator production in BMMCs. (A) Cytotoxicity of safranal in BMMC. Cells were treated with various concentrations of safranal, cell viability was measured using the MTT assay. (B–E) BMMCs were incubated with safranal and stimulation with DNP-HSA. Supernatant were collected and analyzed for β-hexosaminidase, IL-6, TNF-α, and LTC₄ production. (F) Level of 5-LO and p-cPLA₂ in cytosol and nucleus were analyzed by western blot analysis. The data shown are representative of three independent experiments. Data are the means ± S.E.M. of three independent experiments. ^### p <0.001 compared to nontreated group, *p <0.05, **p <0.01, ***p <0.001 compared to DNP-HSA.

Figure 7

Safranal inhibited the MAPKs and NF-κB signaling pathways. Cells were pretreated with Safranal for 1 h prior to DNP-HSA treatment. The levels of phosphorylation of proteins in MAPKs pathways ERK, JNK and p38 (A–D) and NF-κB pathway IKK and IκBα (E–G) were measured by western blot and normalized to β-actin. The data shown are representative of three independent experiments and indicate the means ± S.E.M. ^##p < 0.01, ^### p <0.001 compared to nontreated group, *p <0.05, **p <0.01compared to DNP-HSA.

Figure 8

Safranal inhibited the nuclear translocation of NF-κB, AP-1. (A) BMMCs were collected after incubation with safranal and stimulation of DNP-HSA. Transcriptional factors p65, p-c-jun, and c-Fos in cytoplasmic or nuclear extracts were determined using western blot. (B, C) Nuclear transcriptional factors binding activity of p65, and AP-1 were determined by Trans AM kit, the result present in OD value. The data shown are representative of three independent experiments and indicate the means ± S.E.M. ^## p <0.01 compared to nontreated group, *p <0.05, **p <0.01 compared to DNP-HSA.

Figure 9

Safranal alleviated PSA reaction. (A) Mice were sensitized with DNP-IgE 24 h before oral administration of PBS or safranal. After 1 h mice were i.v. injected with DNP-HSA for 5 min. Blood samples were collected by cardiac puncture. (B) Serum was collected and analyzed for LTC₄ and histamine production. All data shown are the means ± S.E.M. of n=7. ^### p <0.001 compared to nontreated group, **p <0.01, ***p <0.001 compared to DNP-HSA treated group.

Figure 10

Safranal alleviated OVA-induced asthma model and inhibits mast cell activation.