As expected, Western blot analysis showed that levels of HIF-1α protein in nuclear protein extracts of tracheal epithelial cells from OVA-treated mice were increased significantly, as compared with the levels

in tracheal epithelial cells from the control mice (Fig. 2C and D). Treatment with the PI3K-δ inhibitor IC87114 reduced significantly the increased HIF-1α levels in tracheal epithelial cells from OVA-treated mice. Involvement of HIF-1α in VEGF expression was evaluated using their respective inhibitors. Levels of VEGF protein in lung tissues and BALF were 3-MA cell line significantly increased 48 h after the last challenge of OVA, as compared with the levels in the control mice, and administration of 2ME2 (HIF-1α translation inhibitor) or CBO-P11 (VEGF receptor inhibitor) substantially reduced the increased VEGF protein levels in lung tissues (Fig. 3A and B) and BALF (Fig. 3C). In addition, Evans blue dye assay revealed that plasma extravasation was significantly increased 48 h after the last challenge of OVA (Fig. 3D). The increase in plasma extravasation was significantly reduced by administration

of 2ME2 or CBO-P11. To determine whether inhibition of HIF-1α and VEGF suppresses Th2 inflammation in lungs of OVA-treated mice, we measured levels of Th2 cytokines. As shown in Fig. 4, the levels of IL-4, IL-5, and IL-13 in lung tissues and BALF were significantly increased 48 h after the last challenge of OVA, as compared with the selleck chemical Histone demethylase levels in the control mice. The increased IL-4, IL-5, and IL-13 levels after the OVA inhalation were decreased significantly by administration of 2ME2 or CBO-P11. Numbers of total cells, lymphocytes, neutrophils, and eosinophils in BALF were increased significantly 48 h after OVA inhalation, as compared with the numbers in BALF of the control mice (Fig. 5A). The increased numbers

of total cells, lymphocytes, neutrophils, and eosinophils were significantly reduced by administration of 2ME2 or CBO-P11. Effects of the inhibitors of HIF-1α and VEGF receptor on airway responsiveness were evaluated by measuring methacholine-mediated respiratory system resistance (Rrs). As presented in Fig. 5, at dose of 50 mg/mL of methacholine, percent Rrs increased significantly in the OVA-treated mice, as compared with the controls. Administration of 2ME2 or CBO-P11 to OVA-treated mice significantly reduced the levels of Rrs at 50 mg/mL of methacholine inhalation, as compared with the untreated mice. These results suggest that administration of 2ME2 or CBO-P11 reduces OVA-induced airway hyperresponsiveness. Histologic analysis revealed that numerous inflammatory cells as well as eosinophils infiltrated tissue around the bronchioles, the airway epithelium was thickened, and mucus and debris had accumulated in the lumen of bronchioles (Fig. 5D and E), as compared to the control (Fig. 5C). Mice treated with 2ME2 (Fig. 5F) or CBO-P11 (Fig.