Fig. 1. Chemical structure (A) and LC-MS chromatogram (B) of L-BS. L-BS was synthesized from BS as thoroughly described in the Materials and methods section.

Fig. 2. Schematic diagram of an experimental protocol in mice. The mice were divided into five groups, and airway inflammation was induced in four groups. They were immunized through intraperitoneal (i.p.) injection with 20 μg of chicken OVA and 1 mg of aluminum hydroxide on days 1 and 14. The mice were exposed to a 5% ovalbumin solution aerosolized using an ultrasonic nebulizer for 1 h per day from days 21 to 27 after the second sensitization. Three of the asthma control groups were treated through intraperitoneal (i.p.) injection with 20 μg of lactose-β-sitosterol (L-BS), β-sitosterol (BS) or dexamethasone (Dex) between days 14 and 27. The negative control group was sensitized and challenged with PBS without drug administration.

Fig. 3. Effect of L-BS and BS on the total and differential leukocytes in BAL fluid. BAL fluid was collected from ovalbumin-induced mice untreated (Con; closed bar) or treated with 20 μg of lactose-β-sitosterol (L-BS; thick hatched bar), β-sitosterol (BS; thin hatched bar) or dexamethasone (Dex; dotted bar) between the sensitization and inhalation period. The negative control group (Normal; open bar) was sensitized and challenged with PBS without drug administration. The number of cells in the fluid was totally and differentially counted (A, B, C) following the centrifugation of the BAL fluid or the cells were incubated for 10 min at room temperature with PBS containing 3.3 μM 2′,7′-dichlorofluorescein diacetate (DCFDA) to measure the ROS level and were then immediately observed by fluorescence-activated cell sorting analysis (D). The results from the leukocyte counting in the cytospined BAL fluid were expressed as the means ± S.E.M. of five independent experiments (A, B, C). p < 0.05 and p < 0.01 are assessed as a significant difference between the normal group and asthma control group or between the asthma control group and drug-treated group.

Fig. 4. Effect of L-BS and BS on airway inflammation and mucus hypersecretion in lung tissues. Lung tissues were excised from ovalbumin-induced mice untreated (Con) or treated with 20 μg of lactose-β-sitosterol (L-BS), β-sitosterol (BS) or dexamethasone (Dex) between the sensitization and inhalation period. The negative control group (Normal) was sensitized and challenged with PBS without drug administration. The pathologic alternation of lung tissues was analyzed by histochemistry using H&E staining (A) or alcian blue & P.A.S staining (B) as described in the Methods section. Scoring of inflammatory cell infiltration (closed bar) and mucus production (open bar) in the lung tissues was performed as described in the Methods section (C). The scoring data are expressed as means ± S.E.M. of five independent experiments. p < 0.05 and p < 0.01 are assessed as a significant difference between the normal group and asthma control group or between the asthma control group and drug-treated group. Magnification (A and B) × 200.

Fig. 5. Effect of L-BS and BS on the expression of IL-4, IL-5, IL-13, IFN-γ and eotaxin in lung tissues and BAL fluid. (A) Lung tissues were excised from ovalbumin-induced mice untreated (Con; closed bar) or treated with 20 μg of lactose-β-sitosterol (L-BS; thick hatched bar), β-sitosterol (BS; thin hatched bar) or dexamethasone (Dex; dotted bar) between the sensitization and inhalation period. The negative control group (Normal; open bar) was sensitized and challenged with PBS without drug administration. The total RNA was extracted from the tissues. The mRNA levels of IL-4, IL-5, IL-13, eotaxin and IFN-γ were analyzed by RT-PCR as described in the Methods section. The bands were normalized with β-actin. The data are expressed as representative of five independent experiments. (B) The densitometric data are expressed as the means ± S.E.M. of five independent experiments, in relation to the negative group, which was set at 100%. (C) BAL fluid was collected from ovalbumin-induced mice untreated (Con; closed bar) or treated with 20 μg of lactose-β-sitosterol (L-BS; thick hatched bar), β-sitosterol (BS; thin hatched bar) or dexamethasone (Dex; dotted bar) between the sensitization and inhalation period. The negative control group (Normal; open bar) was sensitized and challenged with PBS without drug administration. The protein expression of IL-4, IL-5, IL-13, eotaxin and IFN-γ in BAL fluid was analyzed by ELISA as described in the Methods section. The data are expressed as the means ± S.E.M. of five independent experiments. p < 0.05 and p < 0.01 are assessed as a significant difference between the normal group and asthma control group or between the asthma control group and drug-treated group.

Fig. 6. Effect of L-BS and BS on the total IgE and ovalbumin-specific IgE levels in the serum and BAL fluid. Serum and BAL fluid were collected from ovalbumin-induced mice untreated (Con; closed bar) or treated with 20 μg of lactose-β-sitosterol (L-BS; thick hatched bar), β-sitosterol (BS; thin hatched bar) or dexamethasone (Dex; dotted bar) between the sensitization and inhalation period. The negative control group (Normal; open bar) was sensitized and challenged with PBS without drug administration. The total IgE and ovalbumin (OVA)-specific IgE levels in the serum (A, B) and BAL fluid (C, D) were analyzed by ELISA as described in the Materials and methods section. The data are expressed as the means ± S.E.M. of five independent experiments. p < 0.05 and p < 0.01 are assessed as a significant difference between the normal group and asthma control group or between the asthma control group and drug-treated group.

Fig. 7. Effect of L-BS and BS on the survival rate of the splenocytes. The splenocytes were isolated from the spleens of normal (open bar) and asthmatic control mice (closed bar). 2 × 10⁵ splenocytes in 100 μl of DMEM medium containing 10% FBS were seeded onto a 96-well culture plate. L-BS, BS or dexamethasone (1 μg/ml) in the absence or presence of 100 μg/ml ovalbumin was added to the individual well and then the plate was incubated for 48 h at 37 °C in a CO₂ incubator. For MTT assay (A), the cells were incubated with 10 μl of MTT solution for 4 h and then 100 μl of solubilization solution was added to the each well. After 24 h incubation, the absorbance was measured by using an ELISA reader at 550 nm. For the apoptosis assay (B), the cells were incubated with annexin V-FITC and PI and analyzed by FACSort cytofluorimeter using CellQuest software. Negative cells against annexin V and PI staining were considered as viable or non-apoptotic cells. The data are expressed as the means ± S.E.M. of three independent experiments, in relation to the negative group, which was set at 100%. p < 0.05 and p < 0.01 are assessed as a significant difference between the untreated group and drug-treated group or between ovalbumin-treated group and ovalbumin plus drug-treated group.