Figure 1.
Structure of VBP15 and schematic of the OVA-induced model of acute allergic lung inflammation.
(A) Chemical structures of prednisone (left panel), prednisolone (mid panel), and VBP15 (right panel). VBP compounds include a delta-9,11 double bond and tail group modifications. (B) Diagram of OVA-induced mouse model of allergic lung inflammation.
Figure 2.
VBP15 reduces leukocyte infiltration in the OVA-induced model of acute allergic lung inflammation.
OVA-challenged mice were either left untreated or treated with oral doses of prednisone, VBP15 (20 mg/kg), or cherry syrup alone daily for 6 days. A group of non-challenged mice (naïve) was included in order to assess basal inflammatory parameters. Lung tissue and BAL cells underwent FACS analysis to determine the number of infiltrating eosinophils (A and C) and effector/memory CD4+T cells (B and D). Bar graphs represent mean (±SE) cell numbers. Percentages indicate percentage reduction compared to vehicle control. *p<0.05; **p<0.01; ***p<.001 compared to syrup group with n = 5 mice per group.
Figure 3.
VBP15 reduces acute allergic lung inflammation.
OVA-challenged mice were either left untreated or treated with oral doses of prednisone, VBP15 (20 mg/kg), or cherry syrup alone daily for 6 days. A group of non-challenged mice (naïve) was included in order to assess basal inflammatory parameters. Perfused whole lungs were processed for histological analysis and stained with H&E (A) or PAS (B). Images (10× magnification) represent areas of tissue surrounding bronchioles. Arrows on H&E sections indicate inflammatory foci. Percentage of PAS positive airways were counted via bright field microscopy (C). Bar graph represents mean (±SE)% PAS positive airways. *p<.05; **p<0.01 compared to the vehicle control group with n = 5 mice per group. IL-13 (D) and RANTES (E) were measured in BAL fluid by flow cytometric bead array. Bar graphs represent mean (±SE) cytokine concentration values. *p<.05; **p<0.01 compared to syrup group with n = 5 mice per group.
Figure 4.
A549 cells stably-transfected with a luciferase NFκB construct were exposed to increasing concentrations of VBP15 or prednisolone (3, 30, 300, 3000 nM) followed by TNFα stimulation before measuring luciferase activity. Bar graph represents mean (±SE) luciferase units. *p<.012 (due to the Bonferroni adjustment for multiple comparisons) compared to treatment with vehicle control. Data represents 4 biological replicates with assay performed in triplicate.
Figure 5.
VBP15 reduces leukocyte degranulation.
Anti-DNP-sensitized RBL-2H3 cells were treated with prednisolone (50 µM), VBP-15 (50 µM), or vehicle control (DMSO) for 7 minutes followed by addition of DNP to induce degranulation. The reaction was allowed to proceed for an additional 20 minutes before supernatant was removed and tested for β-hexosaminidase content. A well of untreated cells was lysed to gauge total β-hexosamindase content. Release percentage was determined using a formula described in Materials and Methods. Bar graph represents mean (±SE) release percentage. **p<0.01 compared to vehicle control. Data represents 3 biological replicates with assay performed in triplicate. N.S. = Not statistically significant.
Figure 6.
VBP15 reduces basolateral cytokine secretion from human bronchial epithelial cells obtained from asthmatic patients.
HBE cells from 3 separate human donors were pulse-treated with VBP15 (10 µM) or vehicle control (DMSO). Basolateral surface supernatant was tested for the presence of TGFβ1 (left panel) and IL-13 (right panel) by flow cytometric bead array. Bar graphs represent mean (±SE) concentration values. **, p<0.01 compared to vehicle control with n = 3 donors. N.D. = Not Detectible (lower limit of detection = 4.5 pg/ml).
Figure 7.
VBP15 does not induce tibia length shortening.
Wildtype outbred CD1 mice were treated daily for 5 weeks with VBP15 (30 and 45 mg/kg), prednisolone (10 mg/kg) or vehicle control starting at 12 days of age. At the end of the treatment, tibias were harvested and measured. Bar graph represents mean (±SE) tibia length values. *p<0.05 compared to vehicle control with n = 10 mice/group.