Fig 1.
Effects of IBMX and Vinp in an OVA-induced asthma model.
(A) Schedule for inducing asthma in mice model. (B) The methacholine test was performed to measure airway resistance. Mice in the OVA, OVA + IBMX, OVA + Vinp, and OVA + Dex groups were exposed to methacholine (4, 8, 16 mg/ml). After methacholine exposure, the airway resistance of the lungs was measured by plethysmography. Inflammatory cells in BALF were stained with Kwik-Diff. (C) Stained inflammatory cells were counted by a hemocytometer. (D) Differentiated cells (macrophages, eosinophils, lymphocytes, and neutrophils) were analyzed based on standard morphological criteria. (E) Stained cells were observed using a microscope (red arrows, eosinophils; black arrows, macrophages; magnification, 63×; scale bar, 10 μm). Data are expressed as mean ± SEM. Statistical analysis was performed using the Student’s t-test, one-way ANOVA, and two-way ANOVA (Data were considered significant at *P<0.05, **P<0.01, and ***P<0.001 compared with the control group and #P<0.05, ##P<0.01, and ###P<0.001 compared with the OVA group). IBMX, 3-isobutyl-1-methylxanthine; BALF, broncho-alveolar lavage fluid; OVA, ovalbumin; Vinp, vinpocetine; Dex, dexamethasone.
Table 1.
The primers used for RT-qPCR in this study.
Fig 2.
Inhibition of ovalbumin-induced histological changes by IBMX and Vinp.
(A) H&E, (B) PAS, and (C) Congo red staining were performed to investigate the histological changes. H&E and PAS staining (left-hand side: magnification 10×; scale bar, 100 μm; right-hand side: magnification 40×; scale bar, 30 μm) show infiltration of inflammatory cells (yellow arrows), damage to epithelial cells (black arrows), and mucus stained magenta (red arrows). Congo red staining (magnification 63×, scale bar, 20 μm) shows the eosinophils (white arrows). (D) The inflammation scores were determined based on criteria. (E) PAS-stained areas were analyzed using ImageJ software. (F) Congo red-stained eosinophils were counted in a 20,000-μm2 area. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (Data were considered significant at **P<0.01 and ***P<0.001 compared with the control group and #P<0.05, ##P<0.01, and ###P<0.001 compared with the OVA group). IBMX, 3-isobutyl-1-methylxanthine; H&E, hematoxylin–eosin; PAS, periodic acid–Schiff; OVA, ovalbumin; Vinp, vinpocetine; Dex, dexamethasone.
Fig 3.
Effects of IBMX and Vinp on OVA-induced increase in allergic inflammatory mediators.
Plasma samples obtained from mice were analyzed by ELISA to investigate systemic allergic inflammation. (A) Anti-OVA IgE in serum and (B) the release of IL-13 in BALF were measured by ELISA. The mRNA expression levels of (C) IL-13 in lung tissues were measured by RT-qPCR. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (Data were considered significant at **P<0.01 compared with the control group and #P<0.05 and ##P<0.01 compared with the OVA group). IBMX, 3-isobutyl-1-methylxanthine; BALF, broncho-alveolar lavage fluid; OVA, ovalbumin; Vinp, vinpocetine; Dex, dexamethasone.
Fig 4.
Effects of IBMX and Vinp on MIP-1β expression and release in eosinophilic lung inflammation.
(A) MIP-1β in BALF was measured by ELISA. (B) The mRNA expression level of MIP-1β in lung tissues was measured by RT-qPCR. (C) MIP-1β expression in lung tissue was detected by immunofluorescence staining (magnification, 63×; scale bar, 20 μm). (D) In immunofluorescence-stained tissue, the green fluorescence intensity of stained images was measured using ImageJ. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (Data were considered significant at **P<0.01 compared with the control group and #P<0.05 compared with the OVA group). IBMX, 3-isobutyl-1-methylxanthine; BALF, broncho-alveolar lavage fluid; Vinp, vinpocetine; MIP-1β, macrophage inflammatory protein-1β; OVA, ovalbumin; Dex, dexamethasone.
Fig 5.
PDE1 expression levels in lung tissues were selectively increased by OVA exposure.
(A) PDE1A, PDE1B, and PDE1C protein expression levels in the lung tissues were analyzed by western blot. (B) PDE1A, PDE1B, and PDE1C mRNA expression levels in the lung tissues were measured by RT-qPCR. Data are expressed as mean ± SEM. Statistical analysis was performed using the Student’s t-test (*P<0.05 and **P<0.01 were compared with the control). OVA; ovalbumin.