Fig 1.
Effects of YPL-001(YPL) and picroside II (PIC II) on House dust mite (HDM)-induced BALF composition.
(A) A timeline of allergen sensitization, exposure, and drug treatment in this study (DEX; dexamethasone, i.n; intranasal). The total cells (B) and differential cells (C) in BALF of mice were collected at 48h after the last HDM challenge, and quantified in DiffQuick-stained reagent. NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, YPL 15 and 30; YPL-001 (15 and 30 ㎎/㎏) + HDM-sensitized/challenged mice, PIC 15 and 30; picroside II (15 and 30 ㎎/㎏)+HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice. All data are representative of three independent experiments and represented as the mean ± SEM (n = 6 mice/group). #p<0.05, ##p<0.01, and ###p<0.001, compared with normal control (NC); *p<0.05, **p<0.01, and ***p<0.001, compared with model group.
Table 1.
Primer sequences for quantitative real-time PCR.
Fig 2.
Effects of picroside II on airway inflammation and mucus secretion in HDM-induced asthma model.
After the collection of BALF, lung tissue was fixed, sectioned at 4μm and stained with hematoxylin and eosin (H&E) (A) or periodic acid-Schiff (PAS) (B) solution (magnification 100x or 400x). NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, PIC 15 and 30; picroside II (15 and 30 ㎎/㎏) + HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice.
Fig 3.
Effects of picroside II on total IgE, HDM-specific IgE and HDM-specific IgG1 in serum.
Serum samples were collected 48h after the last HDM challenge. The levels of (A) total IgE, (B) HDM-specific IgE, and (C) HDM-specific IgG1 were measured using ELISA. NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, PIC 15 and 30; picroside II (15 and 30 ㎎/㎏) + HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice. All data are representative of three independent experiments and represented as the mean ± SEM (n = 6 mice/group). ###p<0.001, compared with normal control (NC); *p<0.05, **p<0.01, and ***p<0.001, compared with model group.
Fig 4.
Effects of picroside II on cytokine levels in BALF.
BALF samples were collected from mice 48h after the last HDM challenge. The levels of (A) Th2-related cytokines, (B) Th1-related cytokine, and (C) asthma-related cytokine were measured using ELISA. NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, PIC 15 and 30; picroside II (15 and 30 ㎎/㎏) + HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice. All data are representative of three independent experiments and represented as the mean ± SEM (n = 6 mice/group). ###p<0.001, compared with normal control (NC); *p<0.05, **p<0.01, and ***p<0.001, compared with model group.
Fig 5.
Effects of picroside II on mRNA expression of inflammatory cytokines/mediators in lung tissues.
The mRNA levels of (A) Th2-related cytokines, (B) Th1-related cytokine, (C) Th17-related cytokine, and (D) asthma-related mediators, were determined by real-time RT-PCR. The data were normalized to Gapdh gene expression. NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, PIC II; picroside II (30㎎/㎏) + HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice. All data are representative of three independent experiments and represented as the mean ± SEM (n = 6mice/group). ##p<0.01 and ###p<0.001, compared with normal control (NC); *p<0.05, **p<0.01, and ***p<0.001, compared with model group.
Fig 6.
Effects of picroside II on the expression of transcription factors, T-bet and GATA3 in lung tissues.
(A) T-bet and GATA3 mRNA expression were determined by real-time RT-PCR. The data were normalized to Gapdh gene expression. (B) T-bet and GATA3 protein were analyzed by western blot. (C) The western blot was quantitated by ImageJ. The levels of T-bet and GATA3 were calculated over GAPDH. NC; normal control mice treated with saline only, model; HDM-sensitized/challenged mice, PIC II; picroside II (30㎎/㎏) + HDM-sensitized/challenged mice, DEX; dexamethasone + HDM-sensitized/challenged mice. All data are representative of three independent experiments and represented as the mean ± SEM (n = 6mice/group). #p<0.05, compared with normal control (NC); *p<0.05 and ***p<0.001, compared with model group.
Fig 7.
Effects of picroside II on mRNA levels and protein expression of Th2-related cytokines and transcription factor in developing Th2 cells.
(A) Cytotoxicity of picroside II was assessed by CCK-8 assay. (B) The cytokine levels of IL-5 and IL-13 were measured using ELISA. (C) Th2-related cytokines (Il5, and Il13) and (D) gata3 were determined from the activated Th2 cells by real-time RT-PCR. (E) Western blotting of GATA3 and (F) STAT6 phosphorylation were analyzed from the activated Th2 cells. Each group was quantitated by ImageJ, the levels of GATA3 and p-STAT6 were calculated over GAPDH and STAT6, respectively. Data are presented as mean ± SEM of each group. *p<0.05, **p<0.01, and ***p<0.001 indicate statistically significant difference compared with the control (Th2 cells, alone).