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.