Glycyrrhizin (GL), a triterpene that is present in the roots and rhizomes of licorice (Glycyrrhiza glabra), has been reported to have anti-inflammatory and anti-viral effects. Recently, we demonstrated that GL produced the neuroprotective effects with the suppression of microglia activation and proinflammatory cytokine induction in the postischemic brain with middle cerebral artery occlusion (MCAO) in rats and improved motor impairment and neurological deficits. In the present study, we investigated whether GL has a beneficial effect in kainic acid (KA)-induced neuronal death model. Intracerebroventricular (i.c.v.) injection of 0.94 nmole (0.2 µg) of KA produced typical neuronal death in both CA1 and CA3 regions of the hippocampus. In contrast, administration of GL (10 mg/kg, i.p.) 30 min before KA administration significantly suppressed the neuronal death, and this protective effect was more stronger at 50 mg/kg. Moreover, the GL-mediated neuroprotection was accompanied with the suppression of gliosis and induction of proinflammatory markers (COX-2, iNOS, and TNF-α). The anti-inflammatory and anti-excitotoxic effects of GL were verified in LPS-treated primary microglial cultures and in NMDA- or KA-treated primary cortical cultures. Together these results suggest that GL confers the neuroprotection through the mechanism of anti-inflammatory and anti-excitotoxic effects in KA-treated brain.

Glycyrrhizin (GL), a triterpene present in the roots and rhizomes of licorice (Glycyrrhiza glabra), has been shown to have anti-inflammatory and anti-viral effects. In our previous reports, we demonstrated the neuroprotective effects of GL in the postischemic brain and in kainic acid (KA)-induced seizure animal model. In this KA-induced seizure model, the systemic administration of GL 30 min before KA administration significantly suppressed neuronal cell death and markedly suppressed gliosis and proinflammatory marker inductions. In the present study, we showed that high-mobility group box 1 (HMGB1), an endogenous danger signal, was induced in hippocampal CA1 and CA3 regions of the same KA-induced model, and peaked at ~3 h and at 6 days post-KA. HMGB1 was transiently induced in neurons and astrocyte at 3 h post-KA, and it was released from dying neurons and accumulated in serum at 12 h post-KA. Furthermore, after ~4 days of almost undetectable levels in the hippocampus, delayed and marked HMGB1 induction was detected at 6 days post-KA, mainly in astrocytes and endothelial cells, in which HMGB1 was localized in nuclei, and not secreted into serum. Interestingly, GL suppressed HMGB1 inductions in hippocampus and also suppressed its release into serum in KA-treated mice. Since we established previously that GL has anti-inflammatory and anti-excitotoxic effects in this KA-induced seizure model, these results indicate that the neuroprotective effect of GL in the KA-injected mouse brain might be attributable to the inhibitions of HMGB1 induction and release, which in turn, mitigates the inflammatory process.

Drugs are currently being developed to attenuate oxidative stress as a treatment for brain injuries. C-phycocyanin (C-Pc) is an antioxidant protein of green microalgae known to exert neuroprotective effects against oxidative brain injury. Astrocytes, which compose many portions of the brain, exert various functions to overcome oxidative stress; however, little is known about how C-Pc mediates the antioxidative effects of astrocytes. In this study, we revealed that C-Pc intranasal administration to the middle cerebral artery occlusion (MCAO) rats ensures neuroprotection of ischemic brain by reducing infarct size and improving behavioral deficits. C-Pc also enhanced viability and proliferation but attenuated apoptosis and reactive oxygen species (ROS) of oxidized astrocytes, without cytotoxicity to normal astrocytes and neurons. To elucidate how C-Pc leads astrocytes to enhance neuroprotection and repair of ischemia brain, we firstly developed 3D oxidized astrocyte model. C-Pc had astrocytes upregulate antioxidant enzymes such as SOD and catalase and neurotrophic factors BDNF and NGF, while alleviating inflammatory factors IL-6 and IL-1β and glial scar. Additionally, C-Pc improved viability of 3D oxidized neurons. In summary, C-Pc was concluded to activate oxidized astrocytes to protect and repair the ischemic brain with the combinatorial effects of improved antioxidative, neurotrophic, and anti-inflammatory mechanisms.

It has been reported that the innate immune response plays important roles in brain ischemia and that the infiltration of blood-derived immune cells is a key initiator of this response. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In the present study, we investigated the proinflammatory and neuroprotective effects of Ninj1 and a dodecamer peptide harboring Ninj1 N-terminal adhesion motif (N-NAM, Pro26~Asn37) in a rat middle cerebral artery occlusion (MCAO) model of stroke. Ninj1 was predominantly induced in neutrophils and endothelial cells in the ischemic hemispheres around 12 h to 1 day post-MCAO, which coincided with a massive neutrophil influx. We demonstrated that intranasal administration of Ninj1 small interfering RNA (siRNA) or N-NAM significantly blocked neutrophil infiltration in postischemic brains. In addition, intranasal administration of Ninj1 siRNA or N-NAM reduced the mean infarct volume to 46.5 ± 9.2 or 30.6 ± 11.7% of that of the PBS-treated MCAO controls, respectively, which was accompanied by significant amelioration of neurological and motor deficits. We showed that N-NAM or Ninj1 siRNA effectively blocked the adhesion and transendothelial migration of TNF-α-stimulated human myelocytic leukemia cells to human umbilical vein endothelial cells and similarly suppressed adhesion and migration of monocytes. Activations of phosphoinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1 are involved in these Ninj1-mediated processes and can be inhibited by N-NAM or Ninj1 siRNA. These results indicate that Ninj1 plays an important role in neutrophil infiltration in the postischemic brain and N-NAM confers robust neuroprotective and anti-inflammatory effects by inhibiting Ninj1-mediated infiltration of neutrophils.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata Blume (GEB), a traditional herbal medicine used in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in the brain, and in particular, its anti-inflammatory, anti-oxidative, and anti-zinc-toxic effects have been implicated in the postischemic brain. Here, the authors investigated the anti-oxidative effect of 4-HBA on astrocytes and sought to identify the underlying molecular mechanisms involved. 4-HBA dose-dependently suppressed H2O2-induced astrocyte cell death. More specifically, pre-incubation of C6 cells (an astrocyte cell line) with 100 μM 4-HBA for 6 hrs increased survival when cells were treated with H2O2 (100 μM, 1 hr) from 54.2±0.7% to 85.9±1.5%. In addition, 4-HBA was found to up-regulate and activate Nrf2, and subsequently, to induce the expressions of several anti-oxidative genes, such as, HO-1, NQO1, and GCLM. Notably, HO-1 was induced by 3.4-fold in 4-HBA-treated C6 cells, and siRNA-mediated HO-1 knockdown demonstrated that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of 4-HBA. ERK and Akt signaling pathways were activated by 4-HBA in C6 cells, suggesting their involvements in protective effect of 4-HBA. In addition, 4-HBA-conditioned astrocyte culture medium was found to have neuroprotective effects on primary neuronal cultures or fresh C6 cells exposed to oxidative stress, and these effects seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), which both accumulated in 4-HBA-treated astrocyte culture media. Thus, the 4-HBA-mediated activation of Nrf2 and induction of HO-1 in astrocytes were found to act via autocrine and paracrine mechanisms to confer protective effects. Furthermore, given the pleiotropic effects of 4-HBA with respect to its targeting of various brain cell types and functions, it would appear that 4-HBA has therapeutic potential for the prevention and amelioration of various brain diseases.