Deguelin Attenuates Allergic Airway Inflammation via Inhibition of NF-κb Pathway in Mice

Abstract

Asthma is a chronic respiratory disease characterized by airway inflammation and remodeling, resulting in a substantial economic burden on both patients and society. Deguelin, a constituent of the Leguminosae family, exhibits anti-proliferative and anti-inflammatory activities in cancer mice models via inhibiting phosphatidylinositol 3-kinases and the NF-κB pathway. We demonstrated that deguelin effectively reduced OVA-induced inflammatory cell recruitment, decreased lung tissue inflammation and mucus production, suppressed airway hyperresponsiveness, and inhibited serum immunoglobulin and Th2 cytokine levels in a dose-dependent manner in asthmatic mice. In addition, we found that deguelin reduced inflammatory gene expressions both in vivo and in vitro, which were closely associated with activation of the NF-κB signaling pathway. Thus, we further explored the underlying mechanisms of deguelin in normal human bronchial epithelial cells (BEAS-2B). Our results suggested that deguelin inhibited NF-κB binding activity by enhancing the ability of IκBα to maintain NF-κB in an inactive form in the cytoplasm and preventing the TNF-α induced translocation of p65 to the nucleus. In conclusion, our research indicates that deguelin attenuates allergic airway inflammation via inhibition of NF-κB pathway in mice model and may act as a potential therapeutic agent for patients with allergic airway inflammation.

Keywords: Asthma; Deguelin; Inflammation; NF-κB pathway; TSLP..

Figure 1

Experimental protocol for the development of allergic asthma and treatment with deguelin or dexamethasone (DXM). The mice were divided into five groups (n = 12 in each group) and sensitized to OVA on days 0 and 14. Subsequently, mice were intraperitoneally injected with deguelin or DXM 1h before challenged by 2% OVA for 20 min in PBS on days 21-23. Mice were sacrificed on day 24.

Figure 2

Deguelin suppressed OVA-induced inflammatory cell recruitment in a dose-dependent manner. Inflammatory cell counts in BALF were obtained from sensitized mice 24 h after the last aerosol challenge. The BALF was centrifuged, and the cell pellets were resuspended and applied to a slide to obtain differential cell counts by Wright staining. Values are shown as mean ± SEM (n = 6 for each group). DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group; ^△P<0.05, ^△△P<0.01 vs. Deguelin (1mg/kg) group.

Figure 3

Deguelin inhibited OVA-induced airway inflammation and mucus production. (A, B) 24 h after the final challenge, lung tissues were obtained from controlled mice, OVA-sensitized and challenged mice, deguelin (4mg/kg, 1mg/kg) and dexamethasone (1mg/kg) treated mice. Representative photomicrographs of hematoxylin-eosin (H&E) stained lung sections from each group. (C, D) Alcian blue/periodic acid-schiff (AB/PAS) stained lung sections from each group. (E) The percentage of positively stained epithelial areas (AB/PAS). Arrowed in B2 showed eosinophil. A1-4, magnification ×100; B1-4, magnification ×400; C1-4, magnification ×100; D1-4, magnification ×200. Data are presented as the mean ± SEM (n = 6 for each group). ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group; ^△P<0.05, ^△△P<0.01 vs. Deguelin (4mg/kg) group.

Figure 4

Effects of deguelin on AHR to methacholine in OVA-induced mice. Airway responsiveness of mice in response to aerosolized methacholine was measured 24 h after the last challenge with pretreatment of either deguelin (4mg/kg) or dexamethasone (1mg/kg). AHR was assessed and shown as Penh values. The results were expressed as percentage change in Penh above baseline. Values are shown as mean ± SEM (n = 12 for each group). DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group.

Figure 5

Effects of deguelin treatment on systemic immunoglobulin production in the serum of OVA-sensitized asthmatic mice. Serum was collected 24 h after the last OVA aerosol challenge. The levels of total IgE, IgG1 and IgG2a were analyzed using ELISA. Values are shown as mean ± SEM (n = 6 for each group). Deg 1 = Deguelin 1mg/kg; Deg 4 = Deguelin 4mg/kg; DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group; ^△P<0.05, ^△△P<0.01 vs. Deguelin (4mg/kg) group.

Figure 6

Deguelin reduced OVA-induced Th2 cytokine expression in lung tissues. (A, B, C, D) The BALF was collected 24 h after the last OVA aerosol challenge and centrifuged. The supernatants were collected. The levels of IL-4, IL-5, IL-13, and IFN-γ were measured by ELISA. (E, F, G, H) Lung tissues were collected 24 h after the last OVA challenge. Total mRNA was extracted using TRIzol reagent. The gene expression of IL-4, IL-5, IL-13 and IFN-γ were detected by real-time RT-PCR. β-actin was used as an internal control. Values are shown as mean ± SEM (n = 6 for each group). Deg 1 = Deguelin 1mg/kg; Deg 4 = Deguelin 4mg/kg; DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group; ^△P<0.05, ^△△P<0.01 vs. Deguelin (4mg/kg) group.

Figure 7

Deguelin reduced OVA-induced inflammatory gene expression in lung tissues. (A, B, C, D) Lung tissues were collected 24 h after the last OVA challenge. Total mRNA was extracted using TRIzol reagent. The gene expression of E-selecitn, Muc5ac, iNOS and TSLP were detected by real-time RT-PCR. β-actin was used as an internal control. (E) Lung tissues were collected 24 h after the last OVA challenge. Single-cell suspensions prepared from lung tissues were double-stained with FITC-conjugated anti-CD4 and APC-conjugated anti-TSLP Receptor, and were analyzed by flow cytometry and software FlowJo 7.6. (F) Ratios of TSLP⁺/CD4⁺ T cells were presented. Values are shown as mean ± SEM (n = 6 for each group). Deg 1 = Deguelin 1mg/kg; Deg 4 = Deguelin 4mg/kg; DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group; ^△P<0.05, ^△△P<0.01 vs. Deguelin (4mg/kg) group.

Figure 8

Effects of deguelin on TNF-α induced inflammatory gene expression in BEAS-2B cells. (A) BEAS-2B cells were incubated in the presence or absence of different concentrations of deguelin for 24 h. Cell viability was measured with CCK-8 assay. (B, C, D, E) Cells were pretreated with 10 μM deguelin for 24 h and subsequently treated with 10 ng/ml TNF-α for 30 min. Total mRNA was extracted using Trizol reagent. The effects of deguelin on the mRNA expression of E-selectin, Muc5ac, TSLP, and RANTES were measured by real-time RT-PCR. DMSO was used as a negative control. β-actin was used as an internal control. Values are shown as mean ± SEM of three independent experiments. DMSO = dimethyl sulfoxide. ^#P<0.05, ^##P<0.01 vs. Basal group; ^*P<0.05, ^**P<0.01 vs. TNF-α-induced group.

Figure 9

Deguelin suppressed OVA-induced NF-κB activation in lung tissues. Total protein, cytosol protein and nuclear protein were separately extracted from lung tissues 24 h after the last OVA challenge. (A) Expressions of NF-κB p65, phospho-p65, IκBα, and phosph-IκBα were analyzed by western blotting analysis. β-actin was used as an internal control. (E) Expressions of cytosol p65 and nuclear p65 were analyzed by western blotting. β-actin and Lamin B were used as internal controls. (B, C, D, F, G, H) Grey values of the indicated proteins were measured by Quantity One software. Values are shown as mean ± SEM (n = 6 for each group). DXM = dexamethasone. ^#P<0.05, ^##P<0.01 vs. Control group; ^*P<0.05, ^**P<0.01 vs. OVA-challenged group.

Figure 10

Effects of deguelin on TNF-α induced NF-κB activation in BEAS-2B cells. BEAS-2B cells were pretreated with 10 μM deguelin for 24 h and then exposed to 10 ng/ml TNF-α for the indicated times. (A) Expressions of NF-κB p65, phospho-p65, IκBα, phospho-IκBα and nuclear p65 were analyzed by Western Blotting analysis. β-actin and Lamin B were used as internal controls. (B, C, D, E, F) Grey values of the indicated proteins were measured by Quantity One software. Values are shown as mean ± SEM of three independent experiments. ^#P<0.05, ^##P<0.01 vs. Medium plus TNF-α-10 min group; ^*P<0.05, ^**P<0.01 vs. Medium plus TNF-α-30 min group.