DISCUSSION

This study demonstrates that OE, Quer, YE, and HSP have protective effects against ischemia/hypoxia-induced BBB dysfunction by inhibiting redistribution of ZO-1 and FoxO3a-mediated degradation of claudin-5.

The BBB is the important system that shields the brain from harmful compounds in the blood (Persidsky et al., 2006). The primary function of the BBB is the strict regulation of permeability (Sandoval and Witt, 2008). It has been reported that the majority of cerebral ischemia-induced brain injuries are related to the disruption of the BBB, which results in edema and secondary damage such as dementia and herniation (Asahi et al., 2001; Pluta, 2005). Indeed, maintenance of the BBB integrity has become an important objective to protect brain from edema (Qu et al., 2009). Recently, it has been reported that the protective effects of natural dietary antioxidants on ischemic brain damage may be attributable to the prevention of BBB disruption (Zhao et al., 2007). In this study, in in vivo model, OE, Quer, YE, and HSP reduced brain edema and BBB hyperpermeability after MCAO, as evaluated by brain water content and Evans blue extravasation. In in vitro model, they also attenuated BBB hyperpermeability after hypoxia, as measured by a value of TEER and a concentration of Evans blue and NaF. The first-line of the defense between blood circulation and brain tissue is the endothelium (Sandoval and Witt, 2008). The endothelium of BBB is strictly limited via physical barrier (paracellular pathway) and transport and metabolic barrier (transcellular pathway) (Persidsky et al., 2006; Abbott et al., 2010). Several studies reported that vasogenic edema is occurred due to paracellular permeability of BBB in cerebral ischemia (Rosenberg and Yang, 2007; Witt et al., 2008; Tu et al., 2011), and thereby it was

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focused on paracellular permeability of BBB in later experiments. TJs among endothelial cells of BBB form the basic structure of BBB and regulate paracellular permeability (Liu et al., 2012b). TJs are multiprotein complexes that consist of integral transmembrane proteins (occludin and claudins) and cytoplasmic accessory proteins (ZO), and are necessary to maintain BBB integrity (Hyun et al., 2013). Claudins are principal barrier-forming proteins and consist of at least 24 members, with each showing a specific organ and tissue distribution (Niessen, 2007). Especially, brain endothelial cells mainly possess the claudin-5 and possibly some other claudins (Stamatovic et al., 2008). Importantly, claudin-5 deletion resulted in a size-selective increase in permeability in endothelial cells (Stamatovic et al., 2008). The claudin-5-deficient mice also present an altered BBB with higher permeability (Luissint et al., 2012). ZO-1 molecules are located at the cytoplasmic side of the brain microvascular endothelial cell plasma membranes connecting transmembranous TJs such as claudin-5 with the actin cytoskeleton (Niessen, 2007). ZO-1 also serves as a recognition protein for TJs placement and as a support structure for signal transduction proteins (Huber et al., 2001). In the recent study, in addition, hyperpermeability and disruption of BBB are related to ZO-1 during hypoxia (Fischer et al., 2005). In this study, TJs in the ischemic hemisphere of the mouse brain was especially disrupted after MCAO, as demonstrated by the lack of immunoreactivity for ZO-1 and claudin-5 within vessels stained with UEA-1, and that the loss of immunoreactivity was significantly prevented by treatment with OE, Quer, YE, and HSP. The four materials also reduced hypoxia-induced disruption of ZO-1 and claudin-5 when detected immunocytochemistry. Thus, the preventive effect of OE, Quer, YE, and HSP against hyperpermeability of BBB is largely due to its ameliorating effect on

42 structural disruption of TJs during hypoxia.

It is reported that the hypoxia-induced disruption of TJs can be caused by degradation or redistribution of TJs in brain endothelial cells (Kim et al., 2010). Cerebral ischemia induced two parallel processes, degradation of occludin and redistribution of claudin-5 (Liu et al., 2012a). In addition, BBB integrity is paralleled by redistribution and degradation of TJs such as claudin-5 and occludin (Selvakumar et al., 2013). Therefore, it was hypothesized that TJs are redistributed and degraded in parallel during hypoxia. Present study showed that ZO-1 was redistributed in parallel with degradation of claudin-5 at 4 h of hypoxia, consistent with this hypothesis. In addition, OE, Quer, YE, and HSP inhibited the redistribution of ZO-1 and degradation of claudin-5 induced by hypoxia.

MMPs mediate brain ischemia-induced vasogenic edema and BBB dysfunction by degrading the basal lamina proteins, extracellular matrix, and TJs around the BBB (Rosell and Lo, 2008). MMPs are also the final common pathway for disruption of the BBB (Yang and Rosenberg, 2011). The major inducible MMPs are MMP-3 and MMP-9. When these are released, MMP-3 induces an increase of MMP-9 activity, there by result in degradation of TJs such as claudin-5 (Yang and Rosenberg, 2011). Among MMPs, MMP-2 was increased in early ischemic stroke stage, whereas induction of MMP-3 and MMP-9 was increased in late phase (Yang and Rosenberg, 2011). During hypoxia, in this study, MMP-2 mRNA level did not change, whereas MMP-3/9 levels were increased, compared with control group. In addition, several studies reported that among MMPs, especially MMP-9 disrupts the BBB by degrading the TJs (Jin et al., 2011; Lee et al., 2011; Tu et al., 2011).

It is known that FoxO1/β-catenin caused claudin-5 repression in IL-1β-mediated barrier

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dysfunction (Beard et al., 2014). FoxO1 also increased caveolae-mediated transcytosis across the BBB (Wang et al., 2010). In addition, FoxO4 regulated intestinal permeability through TJs, including ZO-1 and claudin-1 (Zhou et al., 2009). However, little is known that FoxO3a protein has a role in regulation of brain endothelial cell permeability. Lee and colleagues reported that the functional activation of MMP-9 via FoxO3a-mediated MMP-3 activation is involved in regulating EC survival and degrading extracellular matrix (Lee et al., 2008).

Emerging data indicated that FoxO3a may play an important role during injuries that involve cerebral ischemia and oxidative stresses (Maiese et al., 2007; Fukunaga and Shioda, 2009).

In the results from this study, hypoxia-induced degradation of claudin-5 attenuated by transfection of siFosO3a, thereby it was suggested that FoxO3a related to hypoxia-induced degradation of claudin-5, in part. However, transfection of siFoxO3a did not suppress induced redistribution of ZO-1 in brain endothelial cells, indicating that hypoxia-induced redistribution of ZO-1 does not require FoxO3a. In addition, Lee and colleagues reported that enzymatic activities of MMP-9 increased after FoxO3a activation in endothelial cells but their regulation involves indirect mechanism, MMP-3 activation (Lee et al., 2008).

The mRNA and protein levels of MMP-9 were not changed by FoxO3a activation (Lee et al., 2008). FoxO3a-induced MMP-3 activation contributes to increased enzymatic activity of MMP-9 (Lee et al., 2008). The consensus binding site for the forkhead transcription factors was not found in the promoter sequences of MMP-9 (Lee et al., 2008). In the present study, the knocking down FoxO3a significantly prevented hypoxia-induced BBB hyperpermeability and mRNA level of MMP-3. Therefore, it was suggested that FoxO3a regulates indirectly the activity of MMP-9 via MMP-3, and then increases the BBB

44 stress-induced translocation of FoxO3a causes BBB dysfunction though MMPs during hypoxia. In the present study, OE, Quer, YE, and HSP attenuated hypoxia-induced expression of MMPs mRNA and inhibited translocation of FoxO3a into nucleus. Trolox and NAC also reduced translocation of FoxO3a transcription factor and prevented BBB hyperpermeability and claudin-5 degradation during hypoxia in this study. Indeed, it was reported that oxidative stress is an upstream mediator of ZO-1 redistribution during hypoxia in brain endothelial cells (Park, 2011). In response to oxidative stress, FoxO transcription factors are regulated by several post-transcriptional modifications, including phosphorylation, acetylation, ubiqitination, and methylation (Xie et al., 2012a; Xie et al., 2012b). Among them, phosphorylation of FoxO3a at Thr32, Ser253, and Ser315 increases the association with 14-3-3 protein, which results in translocation of FoxO3a into nucleus (Xie et al., 2012a). Recent

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study reported that hesperidin inhibited phosphorylation of FoxO3a possibly by increasing Akt pathway (Rong et al., 2013). Therefore, it was hypothesized that OE, Quer, YE, and HSP may suppress translocation of FoxO3a through inhibiting phosphorylation of FoxO3a induced by oxidative stress.

This study is the first to show that OE, Quer, YE, and HSP attenuates ischemia/hypoxia-induced BBB hyperpermeability. This BBB protection involves inhibition of TJs disruption including redistribution of ZO-1 and FoxO3a-mediated degradation of claudin-5. Oxidative stress may play an important role in the regulation of TJs integrity during hypoxia as upstream signaling pathways of redistribution of ZO-1 and FoxO3a-mediated degradation of claudin-5.

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