This study shows for the first time that wogonin administered 10 min after CCI reduces neuronal damage and cerebral edema and improves long-term sensory-motor recovery after TBI in mice. These beneficial effects are associated with a reduction in mRNA transcripts and protein expression of TLR4/NF-κB pathway-related mediators. Our results demonstrate that wogonin treatment is effective at attenuating the severity of TBI in mice, both clinically and neuropathologically.
TBI induces neurological deficits and can result in functional impairment as well as dependence. In clinical studies, the functional outcome following a traumatic insult is one of the most important parameters. We observed that wogonin treatment caused a significant reduction in both sensory-motor deficits (assessed by mNSS) and motor dysfunction (assessed by rotarod and beam walk tests) up to 4 weeks post-injury, indicating that wogonin was highly effective in providing long-lasting protection to the traumatized brain. We utilized rotarod and beam walk tests to measure motor coordination and balance 
, and mNSS to evaluate overall neurological deficits 
. Notably, we found that a single dose of wogonin reduced functional deficits caused by experimental TBI in mice as early as 1 day post-injury and promoted sustained improvement of motor-sensory functions over a 4-week post-injury period. The protection afforded by wogonin was associated with a decrease in neuronal damage and apoptotic cell death at day 1, and a reduction in contusion volume at both day 1 and day 28 in the cortical regions involved in motor and sensory functions. These findings suggest that wogonin effectively attenuated the pathological events leading to post-traumatic deficits during the first 24 h, which consequently led to a better prolonged recovery of neurologic function.
TLR4-mediated NF-κB signaling plays a vital role in the initiation of cerebral inflammation in several central nervous system (CNS) diseases, such as inflammatory or autoimmune CNS diseases, and cerebral ischemic injury 
. The activation of NF-κB leads to transcription of many pro-inflammatory genes that encode cytokines, chemokines, and enzymes such as COX-2 and MMP-9, mediators that are involved in the development of secondary brain injury following TBI 
. The upregulation of cytokines and chemokines, e.g., IL-1β, IL-6, and MIP-2, may activate microglia, initiate the infiltration of inflammatory cells into the brain, and trigger a series of events ultimately leading to neuronal death 
. In addition, MMP-9 can degrade the neurovascular matrix, leading to edema and tissue injury 
. Induction of COX-2 up-regulates prostaglandin production, generates free radical species, and contributes to edema and neuronal death 
. Here, we showed that wogonin treatment could attenuate TBI-induced reduced TLR4/NF-κB signaling pathway activation in the injured brain. It is possible that wogonin suppressed microglia activation and neutrophil infiltration and reduced injury-induced IL-1β, IL-6, MIP-2, MMP-9, and COX-2 expression, thereby ameliorating brain damage. Our results are in agreement with previous studies demonstrating that suppression of TLR4/NF-κB signaling by anti-inflammatory agents, including statins and progesterone, reduced TBI-induced brain damage 
. There is abundant evidence showing that wogonin attenuates several inflammatory processes known to be important during TBI. For example, wogonin has been shown to alleviate inflammatory processes by decreasing prostaglandin E2 production and COX-2 expression in macrophages 
. Wogonin inhibited MCP-1 gene expression in human endothelial cells and suppressed MMP-9 expression in human aortic smooth muscle cells 
. It also ameliorated myocardial ischemia/reperfusion injury, suppressed activation of NF-κB and p38 mitogen-activated protein kinase and inhibited MCP-1 expression in vivo 
. In the CNS, wogonin attenuated LPS-induced production of NO and pro-inflammatory cytokines in cultured microglia and astrocytes 
and suppressed microglial cell migration via inhibition of NF-κB activity 
. Additionally, it exhibited inhibitory effects on inducible nitric oxide synthase (iNOS
) protein expression and MMP-9 enzyme activity in glioma cells 
. These properties of wogonin may contribute to its neuroprotective effects. Indeed, inhibition
of microglia by wogonin reduced cytotoxicity
. In vivo
experiments also showed that wogonin treatment protected against brain damage by reducing the production of inflammatory mediators, e.g., tumor necrosis factor (TNF)-α and iNOS
, preventing the death of hippocampal neurons in cerebral ischemia 
, and inhibiting the activation of microglia in excitotoxic brain injury 
We observed significant differences in brain water content and EB extravasation between wogonin-treated and vehicle-treated mice, providing evidence for the effects of wogonin on BBB permeability and brain edema after TBI. Global cerebral edema, or BBB dysfunction, has been reported as a major risk factor for poor outcomes after TBI 
. BBB dysfunction may potentially allow circulating cells and many blood-borne substances into the brain, thus augmenting cerebral inflammatory responses and leading to further neuronal damage and edema formation. Accumulating evidence shows that inflammation and activation of MMPs play key roles in the disruption of the BBB and brain edema formation after injury 
. Furthermore, multiple studies have shown that suppression of inflammation inhibited BBB disruption and edema 
. MMP-9 functions to degrade the extracellular matrix, including major components of the basal lamina and tight junctions as well as interendothelial tight junction proteins, causing BBB disruption after TBI 
. In addition, excessive accumulation of leukocytes causes the release of cytotoxic enzymes
, inflammatory mediators, and reactive oxygen species, thereby
potentially damaging the microvascular endothelium and leading to BBB disruption and edema 
. In the current study, we showed that wogonin treatment decreased the number of microglia, macrophages, and neutrophils recruited to the injured areas of the brain, reduced NF-κB activation and translocation to the nucleus, and interacted NF-κB binding activity, expression of inflammatory mediators (IL-1β, IL-6, MIP-2, and COX-2), and MMP-9 activity in the injured brain. This reduction in inflammation was associated with the protection of 2 tight junction proteins, ZO-1 and claudin-5, suggesting that wogonin may act to protect endothelial tight junctions, thereby keeping the BBB intact. Hence, the anti-edematous effect of wogonin observed in our study is likely to be related to the inhibition of inflammation. However, the beneficial effects of wogonin may be in part due to the prevention of free radical and oxidant formation since wogonin exerts potent antioxidant effects both in vitro 
and in vivo 
. Further investigations are needed to clarify the mechanisms underlying the anti-edematous effects of wogonin.
We found that a single post-injury injection of 40 mg·kg−1
wogonin protected against brain injury but did not induce renal or liver toxicity in mice. The neuroprotective potential of wogonin has been demonstrated in animal models of global and focal ischemia as well as excitotoxic injury by systemic kainate injection in rats. The effective doses ranged from 1 dose of 10 mg·kg−1
given 10 min after induction of global ischemia) to 2 doses of 20 mg·kg−1
given 30 min before and 4 h after induction of focal ischemia) 
. Nevertheless, all previous studies only tested the efficacy of wogonin at fairly early time points (1 to 7 days) after treatment. In contrast, our study investigated structural parameters (infarct size), functional outcomes, and physiological parameters (body weight, renal function, and liver function) over a period of up to 1 month. Furthermore, we found that post-injury wogonin treatment reduced cerebral edema, BBB permeability, and apoptotic cell death, which are major events that occur in the secondary injury phase of TBI. Wogonin has a good safety profile, and it is already used in humans in combination with other flavonoids extracted from S. baicalensis
Georgi for several different indications 
. These properties may facilitate clinical applications of wogonin to human TBI patients. Further investigations will be required to establish the optimal time window for treatment, route of administration, and therapeutic efficacy for different injury magnitudes and models.
In conclusion, our findings indicate that post-injury treatment with wogonin leads to improved long-term functional and histological outcomes and reduced brain edema in a clinically relevant model of TBI. This improvement was associated with attenuated expression of TLR4/NF-κB-pathway related mediators, including upstream factors (TLR4/NF-κB) and downstream factors (IL-1β, IL-6, MIP-2, and COX-2 expression, and MMP-9 activity), suggesting that the neuroprotective effects of wogonin following TBI may be mediated, in part, through modulation of the TLR4/NF-κB signaling pathway. Wogonin treatment may prove to be advantageous because chronic dosing is not required, wogonin has low toxicity, and it can be easily administered in emergency situations. Thus, wogonin could be a potential therapeutic in the treatment of TBI.