Discussion
In this study, we demonstrated that GA has a neuroprotective effect by inhibiting the expression and activation of Jmjd3, thereby preventing BSCB disruption via down-regulating Jmjd3-mediaed activation of MMP-3 and -9 after injury. GA also alleviated the infiltration of blood cells such as neutrophils and macrophages after injury, thereby decreased the expression of cytokines and chemokines, resulting in reduced inflammatory responses. Furthermore, GA treatment inhibited apoptotic cell death of neurons and oligodendrocytes and improved functional recovery after SCI.
In the present study, GA (50 mg∙kg-1, i.p.) was administered immediately, 2 h and 8 h after injury and then was further injected once a day for 7 d with a same dose of GA. In previous studies, GA (10 mg∙kg-1, i.p.) treatment exerted protective effects against SCI-induced oxidative stress (Yang et al., 2015). Oral administration of GA (100 mg∙kg-1) also improved behavior, brain electrophysiology, and inflammation in a rat model of traumatic brain injury (Sarkaki et al., 2015). In addition, the report by Sun. J et al.(Sun et al., 2017) showed that GA (50 mg∙kg-1, 5 d, i.v.) exhibited the neuroprotective effect against cerebral ischemia/reperfusion injury. When we tested the efficacy of various concentrations of GA (25, 50, or 100 mg∙kg-1, i.p.), 50 mg/kg of GA was most effective for the reduction in BSCB disruption (See Fig. 4D). Furthermore, neither significant side effects nor increased mortality following GA treatment was observed. Therefore, we suspect that the dosage of GA (50 mg∙kg-1, i.p.) used in this study is the optimal concentration of GA.
The BSCB, which is a unique barrier between the circulating blood and the CNS, is composed of endothelial cells, astrocyte end-feet, pericytes, the basal lamina, and tight/adherence junction proteins (Hawkins & Davis, 2005). BSCB breakdown facilitates immune cell infiltration and triggers the posttraumatic inflammatory response (Hausmann, 2003; Lee et al., 2015; Noble et al., 2002). Recently, we reported that the regulation of Jmjd3 may serve as a new therapeutic intervention for preventing BSCB disruption after SCI, which is based on the experimental evidence that Jmjd3-mediated MMP activation is involved in BSCB disruption after SCI (Lee et al., 2014a; Lee et al., 2016). Thus, we showed in this study that GA, a natural compound, significantly inhibits Jmjd3 activity as a Jmjd3 inhibitor and expression after injury. Furthermore, the expression of MMP-3 and MMP-9 by ChIP assay in vitro OGD/reperfusion model using bEnd.3 cells was significantly inhibited by GA. Previously, we also showed that MMP-3 is involved in BSCB disruption after SCI (Lee et al., 2014a; Lee et al., 2016). Thus, these results suggest that the prevention of BSCB disruption by GA may be mediated by inhibiting Jmjd3 activity followed the inhibition of MMP3 and/or MMP-9 activation and expression after SCI. However, the effect of GA on MMP-3 activation and expression after SCI was not examined in this study and further study about this effect will be examined.
Accumulated studies have shown that GA has an anti-oxidant and anti-inflammatory activities (Huang et al., 2012; Mansouri et al., 2013b; Sarkaki et al., 2015). Reactive oxygen species (ROS) as well as reactive nitrogen species such as nitric oxide and peroxynitrite have been known to induce apoptotic cell death. Additionally, superoxide anion, one of the major ROS has been known to regulate the activation of inflammatory genes such as COX-2 and iNOS (Carroll et al., 2000; Kurtoglu et al., 2014). The report by Wang et al.(Yang et al., 2015) showed that GA exerts neuroprotective effect by mitigating SCI-induced oxidative stress and inflammatory response. Furthermore, ROS has been known to play an important role in the B-BB disruption and endothelial cell permeability (Schreibelt et al., 2007). However, the effect of GA on oxidative stress followed BSCB disruption after SCI was not examined in this study. Therefore, we can’t exclude the possibility that the prevention of BSCB disruption after SCI by GA may be mediated through the antioxidant and/or anti-inflammatory effect of GA and further study about this view is needed.
In previous our report, we showed that Jmjd3 up-regulation, in cooperation with NF-κB is required for Mmp-3 and Mmp-9 gene expressions in injured vascular endothelial cells results in the BSCB disruption after SCI (Lee et al., 2012c). Furthermore, we investigated and confirmed in this study that the neuroprotective effect and anti-inflammatory response of GA was mediated by inhibiting Jmjd3 activation and expression and thereby preventing BSCB disruption after SCI. However, it has also been known that Jmjd3 is involved in several physiological functions, including the inflammatory response and cell death (Tang et al., 2014; Yang et al., 2016). For example, the suppression of Jmjd3 mediates M1 microglial inflammatory responses by inhibiting M2 microglia polarization, leading to dopamine neuron death in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson’s disease (Tang et al., 2014). Also, it was reported that Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim (Yang et al., 2016). In addition, Jmjd3 is known to be up-regulated by many inflammatory mediators and stress inducers through different signaling pathways including STAT signaling. For example, Jmjd3 is activated by STAT-1 and STAT-3 in LPS-treated primary microglia cell and thereby enhances the transcription of crucial inflammatory genes (Przanowski et al., 2014). STAT-3 is also up-regulated and plays a critical role in regulating reactive astrogliosis and scar formation after SCI (Herrmann et al., 2008). Thus, we can’t rule out the possibility that Jmjd3 may influence on the various pathological events such as apoptotic cell death of neuron and oligodendrocyte, and microglial activation followed inflammatory response after SCI besides the regulation of BSCB integrity. Thus, further study will be needed to elucidate the precise role and underlying mechanisms of Jmjd3 in various pathological processes after SCI.
In summary, our findings showed that the neuroprotective effect of GA after SCI is mediated, in part, by inhibiting the expression and activation of Jmjd3, which thereby prevents BSCB disruption and subsequent blood cell infiltration by inhibiting the activation and expression of MMP-9 and/or MMP-3. Through the elucidation of the neuroprotective effect of GA by inhibiting Jmjd3 expression and activity, our study suggests that GA may be a potentially useful therapeutic agent for traumatic SCI.