The Ethanol Fraction of White Rose Petal Extract Abrogates Excitotoxicity-Induced Neuronal Damage In Vivo and In Vitro through Inhibition of Oxidative Stress and Proinflammation

Since oxidative stress and inflammation are involved in seizure-related neurotoxicity, the neuroprotective effect of a white rose (Rosa hybrida) petal extract (WRPE) in mice that are challenged with kainic acid (KA) were examined using behavioral epileptiform seizures as well as biochemical and morphological parameters of oxidative stress and inflammation. WRPE (50–200 mg/kg) was orally administered to male ICR mice for 15 days, and intraperitoneally challenged with KA (30 mg/kg). Seizure activity, lipid peroxidation, inflammatory cytokines, and related enzymes were analyzed in the brain tissue, in addition to the morphological alterations in the hippocampal pyramidal neurons. Separately, antioxidant ingredients in WRPE were analyzed, and antioxidant, anti-inflammatory, and neuroprotective activities of WRPE were investigated in HB1.F3 human neural stem cells (NSCs) to elucidate underlying mechanisms. Total polyphenol and flavonoid contents in WRPE were 303.3 ± 15.3 mg gallic acid equivalent/g extract and 18.5 ± 2.2 mg catechin/g extract, respectively. WRPE exhibited strong radical-scavenging activities and inhibited lipid peroxidation in vitro, and protected glutamate-induced cytotoxicity in NSCs by suppressing inflammatory process. Treatment with WRPE attenuated epileptiform seizure scores to a half level in KA-challenged mice, and decreased hippocampal pyramidal neuronal injury and loss (cresyl violet and DAPI staining) as well as astrocyte activation (GFAP immunostaining). Lipid peroxidation was inhibited, and mRNA expression of antioxidant enzymes (GPx, PHGPx, SOD1, and SOD2) were recovered in the brain tissues. Inflammatory parameters (cytokines and enzymes) including NF-kB, IL-1β, TNF-α, IL-6, HMGB1, TGF-β, iNOS, COX2, and GFAP mRNAs and proteins were also down-regulated by WRPE treatment. Taken together, the results indicate that WRPE could attenuate KA-induced brain injury through antioxidative and anti-inflammatory activities.


Introduction
Seizures induce neuronal death through over-activation of glutamate receptors [1], which might be mediated by excessive calcium ion influx into neurons and related enzymatic as well as non-enzymatic neuronal injuries [2]. Glutamate receptor-mediated excitotoxicity is well known to be a key causative factor in diverse neurological diseases [3].
Over the past 10 years, several studies have confirmed that inflammatory processes in the brain might constitute a crucial mechanism of seizures and epilepsy [4][5][6]. For example, steroid and other 40 • C (N-1000, Eyela, Tokyo, Japan), dissolved in methanol, and filtered through a 0.2-µm syringe filter (Millipore, Billerica, MA, USA). An HPLC series system (ACME 9000 system, Younglin, Anyang, Korea) with a UV detector was used, and separation was achieved using a Mightysil RP-18 GP column (4.6 × 250 mm, 5 µm, Kanto Chemical, Tokyo, Japan). The absorbance was measured at 280 nm. The mobile phases were 0.1% acetic acid in acetonitrile (solvent A) and 0.1% acetic acid in water (solvent B). The injection volume was 20 µL and the gradient was as described in Table 1. The run time was 70 min at a flow rate of 1 mL/min. Table 1. High-performance liquid chromatographic (HPLC) conditions for the analysis of phenolic acid.

Determination of Polyphenol and Flavonoid Contents
In order to measure total phenolic compounds, the sample (100 µL) was mixed with 2% Na 2 CO 3 (2 mL). At least 3 min later, 50% Folin-Ciocalteu reagent (100 mL) was added, and the absorbance was measured at 750 nm while using a spectrophotometer (UV-1650, Shimadzu Corporation, Kyoto, Japan). The amount of phenolic ingredients was quantified using a calibration curve prepared with standard gallic acid solutions (0.1-0.5 mg/mL).
Lipid peroxidation was measured by determining the formation of thiobarbituric acid (TBA)-reactive substances (TBARS) as previously described [12].

Lactate Dehydrogenase (LDH) Assay in NSCs
Cytotoxicity of glutamate and protective effect of WRPE were quantified by measuring LDH release from HB1.F3 NSCs. To determine median lethal concentration (50% LDH release), HB1.F3 cells (1 × 10 6 cells) were treated with various concentrations (0-50 mM) of sodium glutamate for 2 h. After washing with DMEM medium, the cell viability was determined 24 h later while using the LDH assay. The LDH content was determined using a commercial non-radioactive LDH assay kit (Promega, Madison, WI, USA) according to its protocol. In order to evaluate the protective effect of WRPE, HB1.F3 cells were treated with various concentrations (0-1250 µg/mL) of WRPE and, 30 min later, with 2.5 mM glutamate. After washing with fresh medium in 2 h, the cells were cultivated for 24 h further to measure LDH release. The experiments were performed in triplicate.

Western Blot Analysis in NSC
NSCs were homogenized in 10 volumes of RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Western blot analysis was conducted according to previously described methods [24]. The membranes were then immunoblotted with primary antibodies, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-goat secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies used in this study are summarized in Table 3. The band densities were measured using ImageJ (NIH, Bethesda, Maryland, USA) and normalized to the density of GAPDH.

Animals and Treatment
Seven-week-old male ICR mice were purchased from Daehan Biolink (Eumseong, Korea). They were housed in an environmentally-controlled room with constant temperature (23 ± 3 • C), relative humidity (50 ± 10%), and 12-h light/dark cycle. The animals were fed a standard rodent chow and purified water ad libitum.
After acclimation to the laboratory environment for one week, the mice (n = 15/group) were assigned to treatment groups. WRPE at 50, 100 or 200 mg/kg/day or its vehicle (purified water) was orally administered for 15 days, and KA was injected into the intraperitoneal cavity to induce epilepsy 30 min after the last WRPE treatment. Nine animals from each group were sacrificed 4 h after KA injection to analyze the oxidative and inflammatory reactions in the brain. The remained animals (n = 6/group) were sacrificed three days later for the analysis of histopathological alterations. All of the experimental procedures were carried out in accordance with the Standard Operating Procedures of the Laboratory Animal Center, Chungbuk National University (CBNU), Korea, and this protocol was approved by the Institutional Animal Care and Use Committee of CBNU (Approval #: CBNUA-469-13-02).

Scoring of KA-Induced Seizures
Following KA and/or WRPE administration, behavioral seizure activity was rated every 10 min for a total period of 120 min according to the scales devised by Racine [28]: facial clonus (stage 1), nodding (stage 2), forelimb clonus (stage 3), forelimb clonus with rearing (stage 4), and rearing, jumping, and falling (stage 5). Animals were scored after three consecutive seizures at each stage.

Analysis of Cytokines and Lipid Peroxidation in the Brain Tissue
The concentration of IL-1β and TNF-α in the brain tissue was determined in the prepared supernatant with an ELISA kit (RayBiotech, Norcross, GA, USA) according to the manufacturer's instructions. The protein concentration of the supernatants was analyzed using a Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, MA, USA). All of the samples were analyzed in duplicate and the data were expressed as pg/mg protein.
Lipid peroxidation in the brain homogenate was performed described above.

Quantitative Real-Time PCR in the Brain Tissue
Quantitative PCR in total RNA isolated from the brain homogenate was performed described above. The primer sets were used to amplify NF-κB, TNF-α, IL-6, iNOS, COX2, and SOD2 (Table 2).

Western Blot Analysis in the Brain Tissue
Western blot analysis in the brain homogenate was performed described above.

Nissl Staining of the Brain Tissue
In order to observe morphological alterations, brain tissues were removed and post-fixed overnight, followed by cryoprotection in a 30% sucrose solution for 48 h. Brain coronal cryosections (15 µm in thickness) including the hippocampus (−1.8 to −2.2 mm from the bregma) were obtained using a microtome (Leica, Solms, Germany) and stained with cresyl violet (Sigma-Aldrich, St. Louis, MO, USA). Neurons with normal appearance in the pyramidal cell layer of the CA3 region (mediolateral 1.5 mm, dorsoventral −1.0 mm) were counted.

Fluoro-Jade C (FJC) Staining of the Brain Tissue
To visualize degenerative neurons, brain sections were stained with an FJC kit (Biosensis, Thebarton, Australia), according to the manufacturer's instructions with some modifications. In brief, after immersing in DW for 2 min, brain cryosections were incubated in potassium permanganate solution for 10 min, rinsed with DW for 2 min, and incubated in FJC solution for 10 min. The slides were then washed and mounted on coverslips with Vectashield mounting medium (Vector, Vector Laboratories, CA, USA). All of the sections were observed and photographed under a fluorescence microscope with a blue excitation light (LSM710, Zeiss, Thornwood, NY, USA).

Glial Fibrillary Acidic Protein (GFAP) Immunostaining of the Brain Tissue
For immunohistochemical staining of astrocytic GFAP, brain cryosections were rinsed in TBS and treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. After washing with TBS and blocking with 5% BSA, the sections were incubated overnight at 4 • C with an antibody specific to GFAP (1:1000; rabbit polyclonal, Chemicon, Temecula, CA, USA) and with a secondary antibody being conjugated with Alexa Fluor-594 (1:1000; Molecular Probes, Eugene, OR, USA). The sections were counterstained with 4 ,6-diamino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) to identify cellular nuclei and viewed under a laser-scanning confocal microscope (LSM710; Zeiss, Oberkochen, Germany).

Statistical Analysis
Statistical comparisons between the groups were performed using one-way analysis of variance followed by a Tukey's multiple comparison test. All analyses were conducted while using the Statistical Package for Social Sciences for Windows software, version 12.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was assessed at p < 0.05. All data were expressed as the mean ± SD.

Neuroprotective and Anti-Inflammatory Activities of WRPE in NSCs
Following exposure to increasing concentrations (0-50 mM) of glutamate for 2 h, cell death (LDH release) increased in a concentration-dependent manner between 0.8 and 12.5 mM ( Figure 3A). Higher concentrations (>12.5 mM) of glutamate did not induce further increase in LDH release, which is indicative of complete cytotoxicity at around 12.5 mM. Therefore, median (50%) lethal concentration of glutamate in NSCs was estimated to be 2.5 mM, which was used for the neuroprotective effects of WRPE in our subsequent studies.

Neuroprotective and Anti-Inflammatory Activities of WRPE in NSCs
Following exposure to increasing concentrations (0-50 mM) of glutamate for 2 h, cell death (LDH release) increased in a concentration-dependent manner between 0.8 and 12.5 mM ( Figure 3A). Higher concentrations (>12.5 mM) of glutamate did not induce further increase in LDH release, which is indicative of complete cytotoxicity at around 12.5 mM. Therefore, median (50%) lethal concentration of glutamate in NSCs was estimated to be 2.5 mM, which was used for the neuroprotective effects of WRPE in our subsequent studies.
Exposure of NSCs to 2.5 mM glutamate resulted in 176% increase in LDH release ( Figure 3B). Notably, such a cytotoxicity induced by glutamate was significantly attenuated by 30 min pretreatment with WRPE (≥78 μg/mL). Although 1250 μg/mL WRPE fully inhibited the LDH release to a level lower than that in normal cells, direct cytotoxicity of WRPE was not excluded. Thus, we analyzed the anti-inflammatory effects of WRPE at doses of 50, 100, and 200 μg/mL.  Exposure of NSCs to 2.5 mM glutamate resulted in 176% increase in LDH release ( Figure 3B). Notably, such a cytotoxicity induced by glutamate was significantly attenuated by 30 min pretreatment with WRPE (≥78 µg/mL). Although 1250 µg/mL WRPE fully inhibited the LDH release to a level lower than that in normal cells, direct cytotoxicity of WRPE was not excluded. Thus, we analyzed the anti-inflammatory effects of WRPE at doses of 50, 100, and 200 µg/mL. Treatment of NSCs with glutamate (2.5 mM) significantly up-regulated the NF-κB mRNA expression ( Figure 3C). Interestingly, however, the NF-κB expression was markedly attenuated by treatment with WRPE (50-200 µg/mL), leading to decrease to normal level at ≥100 µg/mL. In parallel with the change in NF-κB, the expression of TNF-α and IL-6 was also remarkably increased by glutamate, which was fully blocked by treatment with WRPE (50-200 µg/mL) ( Figure 3D,E). According to the up-regulation of NF-κB and inflammatory cytokines, the gene expression of inflammatory enzymes, iNOS and COX2, was increased following exposure to glutamate ( Figure 3F,G), which is actively involved in the regulation of proinflammatory proteins such as iNOS and COX2. Notably, pretreatment with WRPE significantly inhibited the enzyme expression in a concentration-dependent manner.
In the western blot analysis, in addition to iNOS and COX2, the production of proteins of proinflammatory cytokines such as TGF-β and high-mobility group box 1 (HMGB1) was increased by glutamate ( Figure 3H,I). However, the glutamate-induced over-production of the proteins was markedly attenuated by treatment with WRPE, although there were differences in sensitivity.

Anticonvulsant and Antioxidant Activities of WRPE in KA-Challenged Mice
Seizure activity increased up to 60 min after KA injection, reaching mean score of 4.78 ± 0.14, and then gradually disappeared, lasting to 110-120 min ( Figure 4A). Notably, pretreatment of WRPE not only significantly attenuated the KA-induced seizure intensity, but also shortened the duration. Especially, the seizure intensity and duration were decreased to a half level and 80 min, respectively, by 200 mg/kg WRPE.
Intensive seizures that were induced by KA challenge increased the TBARS concentration up to 3 times the control level ( Figure 4B). However, such lipid peroxidation was reduced by treatment with WRPE in dose-dependent manner. Such antioxidant effect of WRPE led us to investigate the relationship with antioxidant enzyme activities. Although GPx1, PHGPx, SOD1, and SOD2 mRNA levels were down-regulated by KA, WRPE treatment markedly enhanced the gene expression of all the enzymes above their control levels ( Figure 4C-F).

Anti-Inflammatory Activity of WRPE in KA-Challenged Mice
The concentration of IL-1β and TNF-α significantly increased in the brain tissue of KA-challenged mice ( Figure 5A,B). As inferred from the cytokine levels, the expression of NF-κB and TNF-α mRNA was also markedly increased by KA exposure ( Figure 5C,D). In addition, the mRNA expression of IL-6 as well as inflammatory enzymes iNOS and COX2 was greatly up-regulated, too ( Figure 5E-G). It is of interest to note that treatment with WRPE potently lowered all of the inflammatory cytokines and enzymes, although there were differences in dose-response relationship.
In the western blot analysis, the production of proteins of inflammatory enzymes iNOS and COX2 as well as proinflammatory cytokines TGF-β and HMGB1 was markedly increased by KA-induced seizures ( Figure 5H,I). Notably, however, the KA-induced over-production of the proteins was inhibited by treatment with WRPE in a dose-dependent manner. Nutrients 2018, 10, x FOR PEER REVIEW 10 of 19

Neuroprotective Effects of WRPE in KA-Challenged Mice
In cresyl violet staining, KA-induced neuronal death exhibiting many halos around shrunk cells was mainly observed in the CA3 pyramidal cell layer (Figure 6(B-1)-(B-3)). Cresyl violet-stained intact neurons in the CA3 pyramidal cell layer decreased to one-third level ( Figure 6F). However, the pyramidal neurons were significantly preserved in the mouse brain pretreated with WRPE, displaying a dose-dependent effect ( Figure 6C-F).

Neuroprotective Effects of WRPE in KA-Challenged Mice
In cresyl violet staining, KA-induced neuronal death exhibiting many halos around shrunk cells was mainly observed in the CA3 pyramidal cell layer (Figure 6(B-1)-(B-3)). Cresyl violet-stained intact neurons in the CA3 pyramidal cell layer decreased to one-third level ( Figure 6F). However, the pyramidal neurons were significantly preserved in the mouse brain pretreated with WRPE, displaying a dose-dependent effect ( Figure 6C-F Many FJC-positive (green colored) cells were observed in the hippocampal pyramidal cell layer of KA-challenged mice ( Figure 7B,F). Such degenerating neurons disappeared when WRPE was pretreated, in a dose-dependent manner ( Figure 7F).
Inflammation-activated CNS astrocytes are characterized by hypertrophy and proliferation, along with an up-regulation of their cytoskeletal GFAP. There were intensive GFAPimmunoreactivities (red-colored) of activated astrocytes in the subventricular zone and striatum of the KA-challenged mice ( Figure 8B,F). However, astrocytic activation was significantly inhibited by pretreatment with WRPE (100-200 mg/kg) to the normal level in the subventricular zone ( Figure 8D-F). Interestingly, there were decreased DAPI-stained neurons in the CA3 pyramidal cell layer in a reversed relationship with GFAP content (Figure 8, column 1), in parallel with the neuronal death in cresyl violet staining ( Figure 6). Such neuronal loss and astrocytic activation were ameliorated by pretreatment with WRPE. Many FJC-positive (green colored) cells were observed in the hippocampal pyramidal cell layer of KA-challenged mice ( Figure 7B,F). Such degenerating neurons disappeared when WRPE was pretreated, in a dose-dependent manner ( Figure 7F).
Inflammation-activated CNS astrocytes are characterized by hypertrophy and proliferation, along with an up-regulation of their cytoskeletal GFAP. There were intensive GFAP-immunoreactivities (red-colored) of activated astrocytes in the subventricular zone and striatum of the KA-challenged mice ( Figure 8B,F). However, astrocytic activation was significantly inhibited by pretreatment with WRPE (100-200 mg/kg) to the normal level in the subventricular zone ( Figure 8D-F). Interestingly, there were decreased DAPI-stained neurons in the CA3 pyramidal cell layer in a reversed relationship with GFAP content (Figure 8, column 1), in parallel with the neuronal death in cresyl violet staining ( Figure 6). Such neuronal loss and astrocytic activation were ameliorated by pretreatment with WRPE. Nutrients 2018, 10, x FOR PEER REVIEW 13 of 19

Discussion
In the present study, we demonstrated that WRPE containing polyphenols and flavonoids exerted neuroprotective effects via antioxidative and anti-inflammatory activities in glutamatetreated NSCs and KA-challenged mice.

Discussion
In the present study, we demonstrated that WRPE containing polyphenols and flavonoids exerted neuroprotective effects via antioxidative and anti-inflammatory activities in glutamate-treated NSCs and KA-challenged mice.
Generally, over-stimulation of glutamatergic neurons is major risk factor of excitotoxicity [29]. The mechanism of KA-induced seizure-mediated neuronal damage involves over-activation of glutamate receptors, which trigger excessive calcium influx into the neurons, eventually leading to necrotic and apoptotic neuronal deaths [1]. In our cresyl violet-and DAPI-stained findings, there was extensive injury and the loss of hippocampal pyramidal neurons, in addition to degenerative functional change in FJC staining, in the mice experienced severe epileptiform seizures.
Since oxidative stress plays a critical role in excitotoxicity [30], antioxidants such as resveratrol, vineatrol, and curcumin have been demonstrated to exhibit neuroprotective effects [7,8,29]. In our previous study, WRPE was found to have a potent antioxidant activity [31]. In the present study, the antioxidant potentials of WRPE were also confirmed via ABTS and DPPH radical-scavenging and lipid peroxidation assays. Furthermore, WRPE exerted a neuroprotective effect in human neural stem cells that were exposed to glutamate, a major excitotoxin in CNS.
It is well known that phenolic and flavonoid compounds are secondary metabolites with high antioxidative and anti-inflammatory properties [32,33]. The activities of phenolic compounds in vitro and in vivo are related to the number of hydroxyl functional groups in their structures [34]. As active ingredients that are responsible for the antioxidative and neuroprotective effects of WRPE, GC-MS analysis of the butanol fraction of WRPE revealed that it contained 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (4.95%), 5-(hydroxymethyl)-2-furancarboxaldehyde (23.73%), and 1,2,3-benzenetriol (pyrogallol) (43.60%) composing 72.30% of the total peak area [24]. Pyrogallol is especially known as an antioxidant and anti-allergic component of WRPE. In addition, it was confirmed in the present study that an ethanolic (50%) extract of WRPE contain seven phenolic compounds including gallic acid, gentisic acid, (+)-catechinic acid, caffeic acid, veratric acid, hesperidin, and cinnamic acid. Based on these characteristics of ingredients, WRPE was inferred to have potent antioxidant and neuroprotective activities, which were confirmed in the ABTS-and DPPH-scavenging, lipid peroxidation, and glutamate-mediated cytotoxicity studies.
In our previous studies, WRPE has been shown to improve allergic dermatitis, skin aging, and especially ischemic stroke via its antioxidant and anti-inflammatory properties [21][22][23][24]. Therefore, we extended the activity of WRPE to excitotoxic brain injury, in which oxidative and inflammatory processes play an important role. KA injection induces epileptiform seizures, and enhances oxidative parameters in the blood and brain [13]. Similarly, in the present study, KA-induced seizures increased the brain TBARS level following diminished antioxidant enzymes (GPx1, PHGPx, SOD1, and SOD2), indicating that oxidative stress is involved in KA-induced brain damage, as previously suggested [35]. Notably, such seizures and alterations in antioxidant enzymes and TBARS were potently reversed by pretreatment with WRPE. These results suggest that WRPE can prevent the ROS-mediated detrimental effects that are caused by KA in the mouse brain.
Over the past decades, experimental and clinical findings have supported a crucial role of inflammatory processes in epilepsy, particularly in the mechanism underlying the generation of seizures [36]. Seizures induce high levels of inflammatory mediators such as IL-1β, TNF-α, TGF-β, and IL-6 in specific brain regions [37]. Upon excitotoxic brain injury, the prototypic inflammatory cytokines are up-regulated in activated microglia and astrocytes that trigger a cascade of downstream inflammatory events in neurons and endothelial cells of the blood-brain barrier (BBB) [30,36,38,39]. Strong IL-1β and IL-1R1 immunoreactivities were found in perivascular astrocytes and endothelial cells, which are associated albumin extravasation in the brain tissue reflecting BBB breakdown [40]. In turn, cytokines and other inflammatory mediators contribute to both excitotoxic and apoptotic neuronal deaths [41].
Interestingly, excitotoxicity also contributes to neuronal injury in diverse acute and chronic neurodegenerative diseases including cerebral infarction and traumatic brain injury (TBI) [42]. In particular, a rapid-onset inflammatory response is triggered in glia during seizures induced by chemo-convulsants [9]. The rapid release of HMGB1 from neurons, microglia, and astrocytes following epileptic injuries has been proposed as a crucial event for the initiation of brain inflammation and the reduction in the seizure threshold [43]. HMGB1 is considered a danger signal released from injured or stressed cells to alert the microenvironment of an immediate or ongoing injury. Its interaction with toll-like receptor 4 (TLR4) activates related inflammatory events [44]. Notably, NF-κB pathway is a prototypical signaling pathway governing the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. As inferred from the anti-epileptic activity, down-regulation of HMGB1 and NF-κB expressions by administration of WRPE might inhibit the occurrence of seizures and subsequent inflammatory processes, in a consistent manner as also observed in NSCs.
Notably, iNOS is an enzyme that is responsible for production of nitric oxide (NO) mediating oxidative and inflammatory responses during epileptogenesis [45]. Astrocytes are the most abundant cells in the brain and serve as an important source of inflammatory mediators during courses of neuroinflammation [46]. Activation of the TNF-α-iNOS-NO pathway occurred in parallel with the increased number of activated astrocytes, which were confirmed by up-regulation of the production of GFAP. Inducible COX2, producing prostaglandins, is expressed in excitatory neurons and up-regulated in many organs by a wide variety of inflammatory stimuli, and especially, it plays an important role in seizure-induced leukocyte infiltration, astrogliosis, microglial activation, and breakdown of the BBB [47]. Moreover, COX2 is up-regulated by seizure activity and produce large amounts of PGs [48] that induce deleterious effects during epilepsy in collaboration with NO [49]. Activated astrocytes are important source of inflammatory COX2, in addition to iNOS [50]. Importantly, excitatory brain injury triggers the over-activation of astrocytes and, in turn, inflammatory process aggravates the brain damage: i.e., the increased expression of GFAP, an astrocyte cytoskeletal protein, is a quantitative marker of neural tissue injury [51]. Our present study revealed that the GFAP immunoreactivity as well as iNOS and COX2 expressions in the brain tissue and NSCs were markedly suppressed by treatment with WRPE, suggesting the pivotal role of its anti-inflammatory activity.

Conclusions
WRPE containing polyphenols and flavonoids exerted neuroprotective effects via antioxidative and anti-inflammatory activities. Specifically, WRPE exhibited radical-scavenging and lipid peroxidation-inhibitory activities in vitro, and protected against glutamate-induced cytotoxicity in neural stem cells. Also, pretreatment with WRPE attenuated epileptiform seizures in KA-challenged mice, and reversed hippocampal pyramidal cell loss and astrocyte activation, in which the antioxidant and anti-inflammatory parameters were restored. Therefore, it is suggested that WRPE could be a good candidate as an anti-epileptic or neuroprotective agent for clinical trials to attenuate seizure-related brain injury.