GA also inhibits Jmjd3-mediated Mmp-3 and Mmp-9 gene activation
and the loss of TJ protein in bEnd.3 cells upon OGD/reperfusion injury.
Previously, we found that Jmjd3 regulates the expression and activation
of MMPs, which are involved in the disruption of TJ integrity in brain
microvessel endothelial bEnd.3 cells upon OGD/reperfusion injury
(Lee et al., 2012c). Thus, we next
examined whether GA suppresses the expression of MMP-3 and MMP-9 by ChIP
assay in in vitro OGD/reperfusion model using bEnd.3 cells,
thereby inhibits the loss of TJ molecules. The results show that the
gene expression of Mmp-3 and Mmp-9 by ChIP assay was
significantly increased in bEnd.3 cells that were subjected to 6 h OGD
followed by 1 h of reperfusion (Fig. 5A, +OGD). In contrast, GA
suppressed Jmjd3-mediated Mmp-3 and Mmp-9 gene activation
in bEnd.3 cells (Fig. 5A, +OGD/GA). By Western blot, the expression of
Jmjd3 was also increased in bEnd.3 cells subjected to 6 h OGD followed
by immediate, 1 h, and 3 h of reperfusion when compared to the control.
Moreover, the level of H3k27me3 was significantly reduced after
OGD/reperfusion injury (Fig. 5B and C +OGD). However, GA decreased the
protein level of Jmjd3 and the level of H3k27me3 was significantly
higher in GA-treated group at every time point after OGD/reperfusion
injury than in the vehicle control (Fig. 5B and C, +OGD/GA), indicating
that GA significantly inhibited the activity of the histone H3K27me3
demethylase Jmjd3 after OGD/reperfusion injury. In addition, the
expression of the TJ proteins, ZO-1 and occludin, was also decreased
(Fig. 5D and E, +OGD), whereas GA significantly attenuated the decrease
in ZO-1 and occludin expression in bEnd.3 cells upon OGD/reperfusion
injury (Fig. 5D and E, +OGD/GA).