Fig 1.
Chemical structure of Methylophiopogonanone A (MO-A).
Fig 2.
MO-A protects against cerebral injury after MCAO in rats.
(A) Infarct images using TTC staining at day 7 after MCAO; (B) Determination of infarct volume at day7 after MCAO (n: sham = 14, model = 9, MO-A 1.25mg/Kg group = 8, 2.50 mg/Kg group = 7, 5.00 mg/Kg group = 8); (C) Neurological deficit was evaluated using a five-point scale (n = 6–14); (D) The changes of animal body weights at day 3, 5 and 7, subtracted the body weights of day 1 (n = 6–14); (E) Animal survival times, rats were examined once every 3 hours at day 1 and once every 12 hours at day 2 to day 7 (n = 6–14). “Low mobility” and “animals incapable of feeding” were used as humane endpoints; (F) Determination of brain water contents in transient MCAO rats (n = 6–14); Data are given as mean ± S.D.; # P < 0.05 and ## P < 0.01 vs. sham group by Student’s t-test statistical analysis; *P < 0.05 and **P < 0.01 vs. MCAO model group by Kruskal-Wallis H test in 7 day neurological deficit (C), weitht changes of 7 day (D) and water contents (F), and others by one-way ANOVA statistical analysis.
Fig 3.
Effect of MO-A on BBB disruption.
(A) cell viability of OGD/R-induced bEnd.3 cell injury was determined with MTT assay; (B) Effect of MO-A on hypoxic BBB damage in vitro; Data are given as mean ± S.D. (n = 3); # P < 0.05 and ## P < 0.01 vs. sham group by Student’s t-test statistical analysis; *P < 0.05 and **P < 0.01 vs. MCAO model group by one-way ANOVA statistical analysis.
Fig 4.
Effect of MO-A on the expression of MMP-9 and tight junction proteins after MCAO in rats.
(A) Representative blots of MMP-9 determined by Western blotting; (B) Quantitative analysis of the ratio of MMP-9; (C) Representative blots of claudin-3 and claudin-5 determined by Western blotting; (D) Quantitative analysis of the ratio of claudin-3 and claudin-5; Data are given as mean ± S.D. (n = 3); # P < 0.05 and ## P < 0.01 vs. sham group by Student’s t-test statistical analysis; *P < 0.05 and **P < 0.01 vs. MCAO model group by one-way ANOVA statistical analysis.
Fig 5.
Effects of MO-A on OGD/R-induced ROS generation in bEnd.3 cells.
(A) DCF images indicate ROS generation in bEnd.3 cells; (B) graph represents quantification of fluorescence intensity in the images; Data are given as Mean ± S.D. (n = 6), # P < 0.05 and ## P < 0.01 vs. sham group by Student’s t-test statistical analysis; *P < 0.05 and **P < 0.01 vs. MCAO model group by one-way ANOVA statistical analysis.
Fig 6.
Direct effects of MO-A on the activation of endothelial cells and leukocytes.
(A) Expression of ICAM-1 in OGD/R-induced bEnd.3 cells using ELISA, data are shown as Mean ± S.D. (n = 6); (B) Expression of VCAM-1 in OGD/R-induced bEnd.3 cells using ELISA, data are shown as Mean ± S.D. (n = 6); (C) Density of THP-1 cell adhesion to bEnd.3 following stimulation by TNF-α (10 ng/mL) or IL-1β (10 ng/ml), data are shown as Mean ± S.D. (n = 3); (D) MMP-9 secretion in undifferentiated and differentiated THP-1 cells stimulated by PMA (200 ng/mL) as detected by ELISA, data are shown as Mean ± S.D. (n = 6); # P < 0.05 and ## P < 0.01 vs. sham group by Student’s t-test statistical analysis; *P < 0.05 and **P < 0.01 vs. MCAO model group by one-way ANOVA statistical analysis.