Intravenous administration is generally the preferred clinical approach for the treatment of acute ischemia stroke. Kaempferol-3-O
-glucoside (KGS) and kaempferol-3-O
-glucoside (KGS) have relatively higher water solubility than kaempferol without a sugar moiety. The previous study showed that kaempferol inhibited lipopolysaccharide-induced inflammatory response in cultured BV2 microglial cells 
. Herein, we studied the effects of intravenous injection of KRS or KGS on neuroinflammation and brain injury in tMCAO rats.
Middle cerebral artery infarction is found in nearly 60% of clinical patients with cerebral infarction. Therefore, cerebral injury induced by tMCAO is the most relevant model of human stroke 
. Intravenous KRS administration after the onset of ischemia reduced cerebral infarct volume and neurological deficit scores in tMCAO rats 
. In the present study, we further evaluated the neuroprotection induced by KRS administered after the onset of reperfusion following ischemia in tMCAO rats. We then compared the efficacy of KRS to another flavonoid glycoside, KGS. At the end of 2 h MCAO, the rats with neurological scores ≥2 were randomly divided into the different treatment groups to ensure the comparability of cerebral postischemic outcome. We found that treatment with equimolar doses of KRS (10 mg/kg) and KGS (7.5 mg/kg) similarly and significantly ameliorated cerebral infarction and neurological deficits compared with the vehicle-treated group. To our knowledge, this is the first report of the neuroprotective effect of KGS against ischemic brain damage.
To further determine the neuroprotective role of KRS and KGS in acute ischemic stroke, we examined the effects of these flavonoid glycosides on ischemia-reperfusion injury in both gray matter neurons and white matter axons using immunohistochemistry. NeuN, a specific marker of mature neurons, has been widely used to determine neuronal death or survival in ischemic brain tissue 
. APP, a marker of disrupted axonal flow marker, is regarded as a key predictor of axonal injury in central nervous system diseases 
. Consistent with previous studies, we found that cerebral ischemia-reperfusion significantly induced neuron loss in the cerebral cortex and axonal damage in the corpus callosum and striatum in the ipsilateral ischemic hemisphere 
. With KRS and KGS treatment, however, the number of NeuN-positive neurons was markedly increased, and APP expression was significantly decreased. Our results showed that postischemic treatment with these kaempferol glycosides prevented the neuron and axon damage after cerebral ischemia-reperfusion.
Numerous studies have shown that cerebral ischemia-reperfusion not only damages neurons and axons but also affects all types of glial cells, including microglia, astrocytes, and oligodendrocytes. The glial response to cerebral ischemia-reperfusion is involved in the evolution of ischemic brain damage [26~28]. Therefore, we examined the effects of KRS and KGS on specific markers of glial cells following ischemia-reperfusion using immunohistochemistry (i.e., OX-42 for activated microglia; GFAP, an astrocyte-specific cytoskeletal protein, for reactive astrocytes). We found that postischemic administration of KRS and KGS significantly reduced the expression of OX-42 and GFAP in the ipsilateral cortex. Our findings suggest that the neuroprotective effects of these kaempferol glycosides are associated with inhibitory effects on the glial response to cerebral ischemia-reperfusion.
In the brain, glial cells bimodally contribute to the pathophysiological events of neuroinflammation. Microglial activation, for example, is normally necessary for scavenging necrotic debris and other exogenous substances, but the overactivation of microglia and reactive astrocytes after ischemia-reperfusion may promote the activation of proinflammatory transcription factors and subsequent production of many proinflammatory mediators [3, 5, 28). Of these, the activation of STAT3 and NF-κB plays a predominant role in the neuroinflammatory cascades and secondary cerebral injury after ischemic stroke by controlling the expression of many proinflammatory genes 
. STAT proteins were identified as critical transcription factors that mediate virtually all cytokine-driven signaling 
. Of the various STAT isoforms, STAT3 is the most-conserved isoform, and phosphorylation at Tyr705 is required for STAT3 activity. After phosphorylation at Tyr705, dimerization, and nuclear translocation, STAT3 binds to DNA to induce the expression of many genes that contribute to neuroinflammation and brain damage after focal ischemia 
. Additionally, NF-κB comprises a family of transcription factors that consists of five different proteins–p50, RelA/p65, c-Rel, RelB, and p52–that can combine to form active dimers in response to ischemic insult. NF-κB activation is mainly mediated by the IκB degradation-dependent pathway and a novel IκB degradation-independent pathway that involves the phosphorylation of p65 at Ser536 
. By inducing the degradation of IκB proteins, the classic pathway results in the liberation of the p65/p50 heterodimer and translocation of this protein into the nucleus, where the p65/p50 complexes bind to target sites and induce the transcription of proinflammatory mediators. Consistent with previous data, our study showed that the phosphorylation of STAT3 at Tyr705 and NF-κB p65 at Ser536 was significantly increased in the ischemic cortical penumbra. Moreover, immunoblotting revealed an increase in the nuclear content of NF-κB p65 and STAT3 phosphorylation in the tissue extracts of the ischemic cortex. Therefore, cerebral ischemia-reperfusion induced the activation of STAT3 and NF-κB, including both the independent and dependent pathways of IκB degradation. Accordingly, the expression of main proinflammatory genes, including TNF-α, IL-1β, iNOS, MMP-9, and ICAM-1, was significantly increased in the ipsilateral cortical penumbra. However, treatment with KRS and KGS strongly inhibited the activation of STAT3 and NF-κB in the ipsilateral ischemic region, thereby repressing the subsequent expression of proinflammatory mediators.
Furthermore, neutrophil infiltration in the ischemic brain plays an important role in secondary inflammatory injury after stroke. A previous study found that neutrophils were the first inflammatory cells to arrive at ischemic brain tissue, as early as hours after reperfusion 
. After the upregulation of adhesion molecule expression (e.g. ICAM-1) at the vascular endothelium and breakdown of BBB integrity, with dysfunction of the reconstruction of the extracellular matrix following increased MMP-9 expression, neutrophils may migrate from blood to brain parenchyma where they may further increase the degree of cerebral ischemia by obstructing microvessels and releasing inflammatory mediators 
. To explore whether the neuroprotective effect of kaempferol glycosides is related to the inhibition of neutrophil infiltration after cerebral ischemia-reperfusion, we examined the effects of KRS and KGS on the expression of MPO, a specific neutrophil marker. We observed a very high level of MPO immunoreactivity in cerebral ischemic penumbra in tMCAO rats, whereas treatment with KRS and KGS strongly inhibited MPO expression. Considering a previous report in which KRS induced the production of NO by upregulating eNOS activity and maintaining regional cerebral blood flow (CBF) in tMCAO rats 
, the present study further suggests that KRS and KGS likely prevented neuroinflammatory injury by improving neurovascular dysfunction after cerebral ischemia-reperfusion.
In summary, postischemic treatment with KRS and KGS prevented the neurobehavioral deficits, cerebral infract volume, neuron and axon damage, and neuropathological response of glial cells (i.e., microglia, astrocytes, and oligodendrocytes) induced by transient focal cerebral ischemia in rats. The mechanism of these kaempferol flavonoid effects appears to be at least partially associated with antineuroinflammatory effects by inhibiting the activation of STAT3 and NF-κB p65, including independent and dependent pathways of IκB degradation, and subsequent expression of proinflammatory mediators. Our findings suggest that postischemic treatment with KRS or KGS attenuates cerebral ischemia-reperfusion injury and neuroinflammation by inhibiting the activation of STAT3 and NF-κB and has the therapeutic potential for the neuroinflammation-related diseases, such as ischemic stroke.