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).