Glycyrrhiza uralensis flavonoids present in anti-asthma formula, ASHMI™, inhibit memory Th2 responses in vitro and in vivo

Abstract

Allergic asthma is associated with Th2-mediated inflammation. Several flavonoids were isolated from Glycyrrhiza uralensis, one of the herbs in the anti-asthma herbal medicine intervention. The aim of this investigation was to determine whether Glycyrrhiza uralensis flavonoids have inhibitory effects on memory Th2 responses in vitro and antigen-induced Th2 inflammation in vivo. The effects of three Glycyrrhiza uralensis flavonoids on effector memory Th2 cells, D10.G4.1 (D10 cells), were determined by measuring Th2 cytokine production. Isoliquiritigenin, 7, 4'-dihydroxyflavone (7, 4'-DHF) and liquiritigenin significantly suppressed IL-4 and IL-5 production in a dose-dependent manner, 7, 4'-DHF being most potent. It was also evaluated for effects on D10 cell proliferation, GATA-3 expression and IL-4 mRNA expression, which were suppressed, with no loss of cell viability. Chronic treatment with 7, 4'-DHF in a murine model of allergic asthma not only significantly reduced eosinophilic pulmonary inflammation, serum IgE levels, IL-4 and IL-13 levels, but also increased IFN-γ production in lung cell cultures in response to antigen stimulation.

Keywords: D10. G 4.1; GATA-3; Glycyrrhiza uralensis; Th2 cytokines; flavonoids; murine model of asthma.

Figure 1. Mouse model protocol

The protocol of sensitization, challenge, and treatment with 7, 4’-DHF is diagrammed. Mice were sensitized twice intraperitoneally (i.p.) and challenged intratracheally (i.t.) on day 14, 21, 28, 45 and 46 with 100μg OVA in 0.05mL of phosphate-buffered saline. Twenty-four hours after the first i.t. challenge, mice were treated with 6.0 μg of 7, 4’-DHF intragastrically or 0.1 % DMSO (Sham group) (i.g. twice daily).

Figure 2. The presence in ASHMI of flavonoids isolated from Glycyrrhiza uralensis

A. Chemical structures of flavonoids (isoliquiritigenin, 7, 4’-DHF, and liquiritigenin) isolated from Glycyrrhiza uralensis. B. Chromatogram of the peak intensities of each compound present in ASHMI and in ASHMI mixed with 100 μg of each compound. Purple peaks, each flavonoid in ASHMI; Red peaks, each flavonoid in the mixture of ASHMI and 100 μg pure flavonoid.

Figure 3. IL-4 and IL-5 levels in culture supernatants of D10 cells treated with Glycyrrhiza uralensis flavonoids

D10 cells (0.25 × 10⁶) and APC (1.25 × 10⁶) were stimulated with CA and incubated in the presence of either isoliquiritigenin, 7, 4’-DHF, or liquiritigenin at the concentrations shown. Supernatants were collected and analyzed by ELISA for IL-4 (A) and IL-5 (B) production. Cell viability was analyzed using trypan blue exclusion assay (C). ** p<0.05, *** p<0.001.

Figure 4. D10 cells proliferation, cell viability, GATA-3 protein and IL-4 mRNA expression in response to 7, 4’-DHF in vitro treatment

A. Cell proliferation ([³H] thymidine incorporation) assay in D10 cells with or without 7, 4’-DHF at 6.25 μg/mL. Experimental data are expressed as the mean ± SEM of four independent experiments; B. Cell viability assay of 7, 4’-DHF at 6.25 μg/mL was assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Results are expressed as optical density (OD); C. Western blot of GATA-3 in D10 cells in the presence of different concentrations of 7, 4’-DHF. GATA-3 expression was determined using monoclonal antibodies specific for GATA-3. D. Relative mRNA expression of IL-4 by D10 cells with treatment of 7, 4’-DHF. ** p<0.05.

Figure 5. Lung histology

A. PAS and H&E stained sections of mouse airways. Bars=100μm. B. Percentage of PAS positive goblet cells in mouse airways. (*** p<0.001).

Figure 6. The immunomodulatory effect of 7, 4’-DHF in vivo treatment (6.0 μg/day) on lung cell culture cytokines and serum IgE

Pooled lung cells were cultured in complete RPMI media either with media alone, OVA or Con A and incubated for 72 hours at 37°C. The supernatants were analyzed for cytokines by ELISA. A. IL-4 (treated group (n=8) :63.98±1.75 pg/mL; sham group (n=6): 155.61±1.9 pg/mL); B. IL-5 (treated group: 2433.02 ± 349 pg/mL; sham group: 2975.66 ± 381 pg/mL); C. IL-13 (treated group: 721.25 ± 142 pg/mL; sham group: 2345.12 ± 104 pg/mL); D. IFN-γ (treated group: 78.91±13.35 pg/mL; sham group: 11.52±2.63pg/mL); E. OVA-specific IgE levels were significantly reduced in treated mice (n=4) compared to sham (n=5). ** p<0.05, *** p<0.001.

Figure 7. Effect of 7, 4’-DHF treatment (6.0 μg/day) on airway inflammation and AHR in a murine model of allergic asthma

A. Total leukocytes in BALF; B. Eosinophil percent in BALF. In cytospun slides of BALF stained with Hema 3 kit, percent eosinophils were significantly reduced in treated group (n=8) compared to sham (n=6) (p= 0.016); C. Invasive Airway Pressure Time Index (APTI) values. APTI with acetylcholine challenge showed possible suppression of AHR in the treated group (n=8) (p= 0.059). **p<0.05, *** p<0.001.