(a) Total and differential cell counts in BALF were enumerated on slide preparations that were stained with the Kwik-Diff staining set. The numbers of (b) total inflammatory cells and (c) eosinophils in the AO-treated groups were dramatically reduced, compared to the numbers in OVA-challenged, vehicle-treated mice. A, vehicle control; B, asthma induction; C, dexamethasone; D, 50 mg/kg/day AO; E, 200 mg/kg/day AO; F, 400 mg/kg/day AO. Each bar represents the mean ± SEM (n = 6). *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day; ^¥p<0.05 vs. 400 mg/kg/day; ^¥¥p<0.001 vs. 400 mg/kg/day.Bar = 50 μm.

https://doi.org/10.1371/journal.pone.0130510.g001

The serum levels of IgE were significantly reduced by AO (p<0.01) in a dose-dependent manner, compared with that in OVA-challenged, vehicle-treated mice. A, vehicle control; B, asthma induction; C, dexamethasone; D, 50 mg/kg/day AO; E, 200 mg/kg/day AO; F, 400 mg/kg/day AO. Each bar represents the mean ± SEM (n = 6). *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day; ^¥p<0.05 vs. 400 mg/kg/day; ^¥¥p<0.001 vs. 400 mg/kg/day.

https://doi.org/10.1371/journal.pone.0130510.g002

(a) As observed using the hematoxylin and eosin stain, AO dose-dependently decreased inflammatory cell (eosinophil) infiltration around the vessels and bronchioles, mucus secretion, and goblet cell hyperplasia in the lungs. (b) AO reduced glycoprotein (mucus) secretion in the bronchioles in a dose-dependent manner, as detected by the Periodic acid-Schiff stain. Bar = 10 μm; Arrow = eosinophil infiltration. Br, bronchiole; M, mucus secretion; V, vessel. A, vehicle control; B, asthma induction; C, dexamethasone; D, 50 mg/kg/day AO; E, 200 mg/kg/day AO; F, 400 mg/kg/day AO.

https://doi.org/10.1371/journal.pone.0130510.g003

AO (a) inhibited the expression of CD3⁺ T-cell co-receptors in a dose-dependent manner, (b) significantly suppressed the upregulation of CD4⁺ Th cells, (c) downregulated the expression of CD8⁺ cytotoxic T cells, and (d) decreased CD68⁺ macrophasges. (e) AO also inhibited the expression of MHC class II. Immunopositive cells were counted in five randomly selected non-overlapping fields (×200 magnification) of three separately immunostained lung sections per animal. A, vehicle control; B, asthma induction; C, dexamethasone; D, 25 mg/kg/day ACA; E 50 mg/kg/day ACA. CD3: T-cell co-receptor; CD4: Th cell; CD8: cytotoxic T cell; CD68: macrophage; MHC class II: major histocompatibility complex class II molecules. *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day; ^¥p<0.05 vs. 400 mg/kg/day; ^¥¥p<0.001 vs. 400 mg/kg/day.

https://doi.org/10.1371/journal.pone.0130510.g004

AO reduced not only the expression of transcription factor, GATA-3 (b), to control Th2 cells proliferation but also the expression of transcription factor, T-bet (a), to do Th1 cells proliferation. Immunopositive cells were counted in five randomly selected non-overlapping fields (×200 magnification) of three separately immunostained lung sections per animal. A, vehicle control; B, asthma induction; C, dexamethasone; D, 50 mg/kg/day AO; E, 200 mg/kg/day AO; F, 400 mg/kg/day. *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day; ^¥p<0.05 vs. 400 mg/kg/day; ^¥¥p<0.001 vs. 400 mg/kg/day.

https://doi.org/10.1371/journal.pone.0130510.g005

Treatment with 400 mg/kg/day AO for 5 days suppressed the expression of IL-1β mRNA. AO also decreased the mRNA levels of Th2-related cytokines (TNF-α, IL-4, IL-5, IL-6, and IL-13) and Th1-related cytokines (IFN-) via dose-dependent manners, and it significantly inhibited the expression of IL-5, IL-6, and IL-13 mRNA. AO slightly increased the mRNA level of IL-12α compared to that of control, and treatment with dexamethasone inhibited IL-12α mRNA expression, which was recovered by treatment with 40 mg/kg/day or 200 mg/kg/day AO. At 400 mg/kg/day, AO down-regulated IL-12α mRNA expression. *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day.

https://doi.org/10.1371/journal.pone.0130510.g006

OVA induced the expression of Th1- and Th2-related cytokines. AO reduced the expression of Th1-related cytokines, such as (a) IFN-γ and (b) IL-12α, and Th2-related cytokines, such as (c) IL-4, (d) IL-5, and (e) IL-13, in the lungs. Immunopositive cells were counted in five randomly selected non-overlapping fields (×200 magnification) of three separately immunostained lung sections per animal. A, vehicle control; B, asthma induction; C, dexamethasone; D, 50 mg/kg/day AO; E, 200 mg/kg/day AO; F, 400 mg/kg/day. *p<0.05 vs. control; **p<0.001 vs. control; ^$p<0.05 vs. asthma induction; ^$$p<0.001 vs. asthma induction; ^#p<0.05 vs. dexamethasone; ^##p<0.001 vs. dexamethasone; ^sp<0.05 vs. 50 mg/kg/day; ^ssp<0.001 vs. 50 mg/kg/day; ^pp<0.05 vs. 200 mg/kg/day; ^ppp<0.001 vs. 200 mg/kg/day; ^¥p<0.05 vs. 400 mg/kg/day; ^¥¥p<0.001 vs. 400 mg/kg/day.

https://doi.org/10.1371/journal.pone.0130510.g007

https://doi.org/10.1371/journal.pone.0130510.g008