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
]. For example, steroid and other anti-inflammatory treatments display anti-convulsant activity in some pharmaco-resistant epileptic patients [6
]. Additionally, the level of proinflammatory cytokines has been observed to increase in febrile seizures [4
]. Particularly, the increased level of cytokines such as interleukin-6 (IL-6) [7
] and IL-1β [8
] in cerebrospinal fluid from epilepsy patients and the aggravating effect of IL-1β, one of the most important proinflammatory cytokines in experimental models of epilepsy [9
], suggest that the studies on inflammatory factors that are involved in epileptogenesis can bring new insights into the molecular mechanisms of epilepsy.
Kainic acid (KA), an analogue of glutamate, has been used as a model compound for the study of neurotoxicity of various excitatory amino acids (EAAs), because KA triggers temporal lobe epileptiform seizures that are known to be associated with the excessive release of glutamate that may underlie the pathogenesis of neuronal injury [10
]. Systemic administration of KA induces neuronal damage by over-activating glutamate receptors, resulting in necrosis and apoptosis of intracellular organelles following intracellular calcium overload [1
]. Moreover, KA mediates the enhancement of inflammatory cytokines such as IL-1β and tumor necrosis factor-α (TNF-α) and the generation of oxidative stress in the brain [13
]. KA may induce neuronal damage through the excessive production of reactive oxygen species (ROS) and lipid peroxidation [14
], as triggered by the activation of EAA receptors.
Although various antiepileptic drugs have been prescribed to prevent seizures, interfere with epileptogenesis, and eliminate neurodegeneration in most patients, the development of novel natural products is important for patients who experience drug resistance and deleterious side effects to traditional drugs [16
]. Thus, many studies have focused on natural products possessing anti-epileptogenic, anti-inflammatory, and antioxidative activities. Earlier, we demonstrated that an extract of rough aster exhibited a neuroprotective effect via antioxidative activity in mice that are challenged with KA [17
]. Vineatrol, a grape-derived polyphenolic fraction that is isolated from vine-shoot extracts, and curcumin, a dietary antioxidant, also protected against KA-induced seizures via an antioxidative effect [18
]. Resveratrol, well known with its anti-inflammatory, anti-cancerous, and neuroprotective effects, has been shown to have antiepileptic activity in KA-injected animals [20
]. Additionally, capsaicin prevented epileptogenesis through its anti-ictogenic, hypothermic, antioxidative, and anti-inflammatory effects [13
Previously, we reported that white rose petal extract (WRPE) displayed a broad spectrum of antioxidant [21
], anti-bacterial and anti-fungal [22
], anti-allergic, and anti-inflammatory properties [23
]. Notably, a butanol fraction of WRPE containing high amounts of gallic acid and volatile compounds reduced the infarction volume in an animal model of ischemic cerebral stroke [24
]. Such results led us to investigate the neuroprotective effects of WRPE in KA-induced temporal lobe epilepsy model by analyzing the neurobehavioral and morphological effectiveness in the mouse brain, in addition to antioxidative, anti-inflammatory, and neuroprotective potentials of WRPE in vitro and in vivo as underlying mechanisms.
2. Materials and Methods
2.1. Preparation of the WRPE
Fresh white flowers of Rosa hybrida Colorado were procured from a rose farm (Rose Rangs, Jincheon, Korea). The flowers were collected in May 2014, completely dried in the shade to obtain dried petals, and then ground in a rotor mill (Laval Lab Inc., Laval, QC, Canada). The dried petal powder was extracted in an ultrasonic bath with 50% ethyl alcohol three times at 60 °C, and then filtered to obtain WRPE. The resulting fractions were completely dried in a vacuum evaporator.
2.2. HPLC Analysis of Free Phenolic Acids
High-performance liquid chromatographic (HPLC) analysis was performed while using a slightly modified protocol of Kim et al. [25
]. WRPE was extracted with an ethyl acetate and ether mixture (1:1 v
) using a separatory funnel. The upper phase was evaporated using a rotatory evaporator at 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.
2.3. Determination of Polyphenol and Flavonoid Contents
In order to measure total phenolic compounds, the sample (100 μL) was mixed with 2% Na2CO3 (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).
To determine total flavonoid content, distilled water (DW, 1 mL) was added to the sample (250 μL) and mixed with 5% NaNO2 (75 μL). Five min later, 10% AlCl3 (150 μL) was added. After 6 min, 1 M NaOH (500 μL) was added, and the absorbance was measured at 510 nm using the spectrophotometer. A calibration curve was made using (+)-catechin hydrate solutions (0.1–0.5 mg/mL) under the same procedures.
2.4. Measurement of Antioxidant Activity
Antioxidant activities of WRPE were measured with radical-scavenging and lipid peroxidation assays in vitro. 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and determining the formation of thiobarbituric acid (TBA)-reactive substances (TBARS) were measured of WRPE as previously described [12
]. All samples were analyzed in triplicate.
Lipid peroxidation was measured by determining the formation of thiobarbituric acid (TBA)-reactive substances (TBARS) as previously described [12
2.5. Human Neural Stem Cell (NSC) Culture
The HB1.F3 human NSCs [26
] were cultivated in a high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Biowest, Nuaillé, Cholet, France) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, Cholet, France), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Cultures were maintained under 5% CO2
at 37 °C in tissue culture flasks. The cells were grown to 90% confluency and subjected to the cytotoxicity test.
2.6. 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 × 106 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.
2.7. Quantitative Real-Time PCR in NSCs
Total RNA was isolated from HB1.F3 cells using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR were measured according to previously described methods [27
]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard to normalize the expression of the target transcripts. The primer sets were used to amplify nuclear factor-κB (NF-κB), TNF-α, IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), glutathione peroxidase 1 (GPx1), phospholipid hydroperoxide GPx (PHGPx), superoxide dismutase 1 (SOD1), and SOD2 (Table 2
). Triplicate data were analyzed by six independent assays using a comparative C
t method [27
2.8. 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.
2.9. 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).
2.10. 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.
2.11. 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.
2.12. 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
2.13. Western Blot Analysis in the Brain Tissue
Western blot analysis in the brain homogenate was performed described above.
2.14. 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.
2.15. 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).
2.16. 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).
2.17. 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.
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
]. 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
]. 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
]. 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
]. 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.