Pu-Erh tea and GABA attenuates oxidative stress in kainic acid-induced status epilepticus
Correspondence: Chien-Wei Hou firstname.lastname@example.org
Department of Biotechnology, Yuanpei University, Hsinchu, Taiwan
Pu-Erh tea is one of the most-consumed beverages due to its taste and the anti-anxiety-producing effect of the gamma-aminobutyric acid (GABA) if contains. However the protective effects of Pu-Erh tea and its constituent, GABA to kainic acid (KA)-induced seizure have not been fully investigated.
We analyzed the effect of Pu-Erh tea leaf (PETL) and GABA on KA-induced neuronal injury in vivo and in vitro.
PETL and GABA reduced the maximal seizure classes, predominant behavioral seizure patterns, and lipid peroxidation in male FVB mice with status epilepticus. PETL extracts and GABA were effective in protecting KA-treated PC12 cells in a dose-dependent manner and they decreased Ca2+ release, ROS production and lipid peroxidation from KA-stressed PC12 cells. Western blot results revealed that mitogen-activated protein kinases (MAPKs), RhoA and cyclo-oxygenase-2 (COX-2) expression were increased in PC12 cells under KA stress, and PETL and GABA significantly reduced COX-2 and p38 MAPK expression, but not that of RhoA. Furthermore, PETL and GABA reduced PGE2 production from KA-induced PC12 cells.
Taken together, PETL and GABA have neuroprotective effects against excitotoxins that may have clinical applications in epilepsy.
Pu-erh tea is one of the most widely consumed beverages in the Orient. In recent years, studies the possible investigating health benefits of Pu-erh tea have shown salutary effects on oxidative stress, cancer, cholesterol levels, blood pressure, and blood sugar, and the bacterial flora of the intestines [1-6]. Soluble ingredients in Pu-erh tea fermented with S. bacillaris or S. cinereus enhance the content of gamma-aminobutyric acid (GABA) and statin [7,8]. GABA metabolism in substantia nigra (SN) plays a key role in seizure arrest. When seizures stop, a major increase in GABA synthesis in postictal SN. GABA synthesis in SN may be reduced in status epilepticus . Studies have shown that tea and its bioactive constituents may decrease the incidence of dementia, Alzheimer's disease and Parkinson's disease [10,11]; however, its effect on epilepsy has not been thoroughly investigated.
Status epilepticus (SE) is defined as a period of continuous seizure activity and has been implicated as a major predisposing factor for the development of mesial temporal sclerosis and temporal lobe epilepsy . This emergency condition requires prompt and appropriate treatment to prevent brain damage and eventual death. Animal studies have shown that SE causes recurrent spontaneous seizures; i.e., epilepsy . and releases free radicals from experimental models of kainic acid toxicity [14,15].
Kainic acid (KA), a glutamate-related compond, increases nerve excitability, and is widely used to induce limbic epilepsy in animal models . KA causes neuron epilepticus and excitotoxicity with the increased production of reactive oxygen species (ROS) and lipid peroxidation [17-19]. Mitogen-activated protein kinases (MAPKs) and Rho kinases are associated with seizures, inflammation and apoptosis [20-22]. KA triggers neurons membrane depolarization by the release of calcium ions which are involved in nerve impulse transmission as the calcium action potential reaches the synapse . A apoptosis of nerve cells can result in the release of calcium ions, and activation of calcium ion-dependent enzymes, resulting in break DNA fragments of the nerve cells with death .
More than one third of brain neurons use GABA for synaptic communication and the concentration of brain GABA regulates the mental and the physical health of humans . GABA has been implicated in many human disease states, including anxiety and sleep disorders, epilepsy and seizures, learning and memory disorders [24-27]. Since GABA is abundant in short-term fermented Pu-erh tea  and has a strong antioxidant activity , it might protect human cells from injury by scavenging of free radicals. Therefore, the aim of this study was to investigate the protective mechanisms of GABA and Pu-erh tea leaf extract on KA-induced injury in neuronal cells in vivo and in vitro.
GABA and kainic acid (KA) were obtained from Sigma-Aldrich (Steinem, Germany) and Cayman Chemical (Ann Arbor, MI, USA), 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) was obtained from Molecular Probes (Eugene, OR, USA).
Pu-Erh tea leaf extract
Pu-Erh tea leaves were prepared as described by Hou et al . Briefly, Pu-Erh tea leaves were ground to a fine powder with the aid of a stainless-steel mill and stored and dried to constant weight in a vacuum desiccator. With regard to the extraction procedure, triplicate one-gram samples of Pu-Erh powder from each site was mixed with 20 ml of reverse osmosis water, vortexed vigorously for 5 min, and then centrifuged at 2,000 Ã g for 10 min. The tea extracts were sterilized by filtration through a 0.25 Î¼ m Millipore membrane filter (Millipore, Bedford, USA).
Determination of GABA content
The quantity of GABA in extracts of Pu-Erh tea was determined using the method described by Zhang and Bown . Tea liquor was prepared as described above with 200 mg of dry tea powder. Samples of standard tea liquor (1 mL each) were placed in glass tubes to which was added 0.6 mL of 0.1 M lysis buffer and 1 mL of 0.3% 2-hydroxynaphthaldehyde (the derivatizing reagent) (TCI, Japan). The tubes were placed in a water bath for 10 min maintained at 80Â°C and then cooled to room temperature. Sufficient methanol was then added to give a final volume of 5 mL. The guard and analytical column used in HPLC analysis was Merck LiChrosper100 RP18 (5 Î¼ m, 4.0 mm i.d. Ã 15 cm). The mobile phase was comprised of methanol and H2O (62:38), the flow speed was 1.0 mL/min, the detection wavelength was 330 nm, and the injection amount was 20 Î¼ L. GABA standard liquor was prepared by diluting GABA with pure water to different strengths (10, 50, 100, 150, and 200 Î¼ g/mL) to obtain different chroma values. The derivatization reaction was observed with GABA liquor at five values of chroma. Each sample was tested three times, and the average value of the absorbance at different values of concentration was calculated.
Oxidative stress in mice
Adult male FVB mice, body weight 30-35 g, were used for this experiment. SE was induced by KA (10 mg/ml in phosphate-buffered saline (PBS), 10 mg/kg, subcutaneous injection). Pu-Erh tea leaf (PETL) powder and GABA was separately diluted in normal saline 10 mg/ml and 1 mg/ml. The animals were fed with PETL (10 mg/kg) and GABA by gavage for 3 days before the KA experiment. The control group was fed with an equal volume of vehicle (normal saline). The procedures were conducted in accordance with the Taichung Veterans General Hospital Animal Care and Use Committee, Taichung, Taiwan (IACUC Approval No. LA-99741) and all possible steps were taken to avoid animals' suffering at each stage of the experiment. Diazepam at lethal dosage, 60 mg/kg i.p., was given to stop seizures 2 h after KA injection and the animals were sacrificed by decapitation under CO2 asphyxia. The whole brain was immediately removed and frozen in liquid nitrogen and stored at -70Â°C until use.
Malondialdehyde (MDA), a thiobarbituric acid reacting substance (TBARS) was used as an indicator of lipid peroxidation. To estimate oxidative stress, the amount of TBARS in the brain from each group was measured. Manual homogenization of brains was carried out at 4Â°C using cold lysis buffer. Protein concentration of the homogenate was determined by BCA protein assay using bovine serum albumin as a standard. For TBARS assay , the sample (0.2 ml) was mixed with the same volume of 20% (w/v) trichloroacetic acid (TCA) and 1% (w/v) thiobarbituric acid in 0.3% (w/v) NaOH. The mixture was heated in a water bath at 95Â°C for 40 min, cooled to room temperature and centrifugated at 5000 rpm for 5 min at 4Â°C. The fluorescence of the supernatant was determined by spectrophotometry with excitation at 544 nm and emission at 590 nm.
Mortality and behavior
Mice were fed with and without PETL extract or GABA for 3 days before the SE experiment was conducted. The control group was treated with the vehicle (normal saline). SE was induced with kainic acid (KA, 10 mg/kg, s.c.). Each behavioral seizure was recorded according to a modification of the classification from Racine : 0, exploring; 1, immobility 2, rigid posture; 3, head nodding; 4, bilateral forelimb clonus and falling; 5, continued clonus and falling; 6, generalized tonus. Three behavioral patterns of SE could be recognized: I, initial (class 1-2), M, middle (class 3) and C, critical (class 4-6). Diazepam, 25 mg/kg i.p., was given to stop seizures at 5 hours of SE and the 10-h mortality rate was recorded.
Adult male FVB mice were observed and recorded the behavior of status epilepticus severity induced by KA stress. After recovery for 24 h, mice were injected with a lethal intraperitoneal injection of pentobarbital (120 mg/kg), and brain tissue sections were perfused with 4% paraformaldehyde for fixation. Coronal paraffin sections were prepared with Hematoxylin and Eosin (H&E) staining for cells damage and TUNEL staining to assess apoptosis study. After fixation for 1 h, mice brain sections were added with freshly prepared permeabilisation solution (0.1% (v/v) Triton X-100 in 0.1% sodium citrate) and then washed with cold PBS and added with TUNEL stain mixture (Roche, Mannheim, Germany), at 37Â°C in the dark, for 1 h. The apoptosis of neuronal cells was quantified by fluorescence microscopy with excitation at 450-500 nm and detection wavelength at 515-565 nm.
The Rat pheochromacytoma cell line PC12 was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 5% horse serum, 100 U/ml penicillin and 100 Î¼ g/ml streptomycin at 37Â°C in a humidified incubator under 5% CO2. Confluent cultures were passaged by trypsinization. Cells were washed twice with warm DMEM (without phenol red), then treated in serum-free medium. In all experiments, cells were treated with GABA and/or KA-stress for the indicated times.
Preparation of cell extracts
Test medium was removed from culture dishes and cells were washed twice with ice-cold phosphate-buffered saline, scraped off with the aid of a rubber policeman, and centrifuged at 200 Ã g for 10 min at 4Â°C. The cell pellets were resuspended in an appropriate volume (4 Ã 107 cells/ml) of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10 Î¼ g/ml aprotinin, and 5 Î¼ g/ml pepstain A. The suspension was then sonicated. Protein concentration was determined by Bradford assay (Bio-Rad, Hemel, Hempstead, UK) after cells were suspended to 2 mg/ml with in lysis buffer.
Protein samples containing 50 Î¼ g of protein were separated on 12% sodium dodecyl sulfate polyacrylamide gels and transferred to Immobile polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were incubated for 1 h with 5% dry skim milk in TBST buffer (0.1 M Tris-HCl, pH 7.4, 0.9% NaCl, 0.1% Tween-20) to block nonspecific binding, and then incubated with rabbit anti-COX-2, Rho A (1:1000; Cayman chemical; Cell Signaling, USA), and anti-phospho-MAPKs. Subsequently, membranes were incubated with secondary antibody streptavidin-horseradish peroxidase conjugated affinity goat anti-rabbit IgG (Jackson, West Grove, PA, USA).
Reactive oxygen species generation
Intracellular accumulation of ROS was determined using H2DCF-DA, which is a nonfluorescent compound that accumulates in cells following deacetylation. H2DCF then reacts with ROS to form fluorescent dichlorofluorescein (DCF). PC12 cells were plated in 96-well plates and grown for 24 h before addition of DMEM plus 10 Î¼M H2DCF-DA, incubaed for 60 min at 37Â°C, and treated with 150 Î¼M KA for 60 or 120 min. Cells were then washed twice at room temperature with Hank's balanced salt solution (HBSS without phenol red). Cellular fluorescence was monitored on a Fluoroskan Ascent fluorometer (Labsystems Oy, Helsinki, Finland) using an excitation wavelength of 485 nm and emission wavelength of 538 nm.
MTT reduction assay for cell viability
Cell viability was measured using blue formazan that was metabolized from colorless 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial dehydrogenases, which are active only in live cells. PC12 cells were preincubated in 24-well plates at a density of 5 Ã 105 cells per well for 24 h. Cells incubated with various concentrations of GABA were treated with 150 Î¼M KA for 24 h, and grown in 0.5 mg/ml MTT at 37Â°C. One hour later, 200 Î¼ l of solubilization solution was added to each well and absorption values read at 540 nm on microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). Data were expressed as the mean percent of viable cells vs. control.
Lactate dehydrogenase (LDH) release assay
Cytotoxicity was determined by measuring the release of LDH. PC12 cells treated with various concentrations of GABA were incubated with 150 Î¼M KA for 24 h and the supernatant was then assayed for LDH activity. A absorbance was read at 490/630 nm using a microtiter plate reader. Data were expressed as the mean percent of viable cells vs. 150 Î¼M KA control.
Calcium release assay
PC12 cells with various concentrations of GABA were treated with 150 Î¼M KA for 24 h and the supernatant was used to assay the release of Ca2+. The 10 Î¼ l supernatant was added to 1 ml Ca2+ reagent (Diagnostic Systems, Holzheim, Germany) and mixed well, allowed to stand for 5 min, then transferred the 100 Î¼ l supernatant to 96 well. Calcium concentration was determined using a microplate reader with a 620 nm absorbance and quantified with a 10 mg/ml Ca2+ standard solution.
Measurement of lipid peroxidation
Lipid peroxidation was assessed by measuring malondialdehyde (MDA) in extracts of PC12 cells using a lipid peroxidation assay kit (Cayman Chemical, Ann Arbor, MI, USA). This kit works on the principle of condensation of one molecule of either malondialdehyde (MDA) or 4-hydroxyalkenals with two molecules of N-methyl-2-phenylindole to yield a stable chromophore. MDA levels were assayed by measuring the amount produced by 5 Ã 105 cells. A absorbance at 500 nm was determined using an ELISA reader (spectraMAX 340, Molecular Devices, Sunnyvale, CA, USA).
Assay of PGE2 concentration and Caspase-3 Activation
PGE2 release and caspase-3 activity were measured by ELISA assay. PC12 cells (5 Ã 105) were added to 0.5 ml homogenization buffer (0.1 M phosphate pH 7.4, 1 mM EDTA) and homogenized. The lysate was then centrifuged at 12,000 Ã g for 15 min at 4Â°C. The supernatant was transferred to a clean test tube, and its total protein content was analyzed using the Bradford assay (Bio-Rad, Hemel, Hempstead, UK). PGE2 concentration and caspase-3 activity were determined using PGE2 and caspase-3 ELISA kits (R&D Systems, Minneapolis, MN, USA). A absorbance at 450 nm was determined using a microplate reader (spectraMAX 340, Molecular Devices, Sunnyvale, CA, USA).
All data were expressed as the mean SEM. For single variable comparisons, Student's test was used. For multiple variable comparisons, data were analyzed by one-way analysis of variance (ANOVA) followed by Scheffe's test. P values less than 0.05 were considered significant.
Results and discussion
We analyzed short-term fermented Pu-erh tea samples processed with tea-leaf extract for the content of GABA . The amount of the bioactive component GABA in the Pu-erh tea leaf was 177 Â± 35 Î¼ g/g.
Effect on mortality and behavior
Treatment of FVB mice with PETL or GABA on KA-induced SE did not affect mortality (Table 1). However, PETL and GABA both significantly attenuated the maximal seizure classes and the predominant behavioral seizure patterns in the SE mice compared with the vehicle (Table 1, GTL and GABA, p < 0.001,).
Table 1. Effects of Pu-Erh tea leaf extract and GABA on the predominant behavior patterns/maximal seizure class (MSC) and 10-h mortality rate of the mice with 5-hour KA-induced SE
Protection from KA toxicity
We further evaluated H&E stained section of the brains of KA-stressed FVB mice. KA (10 mg/kg) caused epilepticus and neuronal damage. However, after PETL (10 mg/kg) or GABA (1 mg/kg) treatment, the damage in cortical neuronal cells was reduced (Figure 1). The TUNEL staining assay showed that PETL or GABA significantly reduced KA-induced apoptosis in hippocampus of the FVB mice as compared to the control (Figure 2). In order to understand the protective mechanism, KA-induced injury in neuronal PC12 cells were investigated using LDH and the MTT assay. As shown in Figure 3, PC12 cells were protected from the injury by the PETL extract (1, 10 Î¼ g/ml) and GABA (0.1, 1, 10 Î¼M). The reduction in LDH release and increase in cell viability caused by the PETL extract and GABA were consistent with the in vivo data.
Figure 1. H&E stain of KA-stressed FVB mice cortex. Kainic acid (KA, 10 mg/kg) caused neuronal damage. After 5 h KA-induced SE of FVB mice, the cortex was observed with cell shrinkage and long shape (B). PETL 10 mg/kg (C) or GABA 1 mg/kg (D) significantly reduced KA-induced neuronal damage in cortex of the FVB mice as compared to control (A). (20x)
Figure 2. DAPI and TUNEL staining of hippocampus form KA-stressed mice. KA induced apoptosis (green fluorescence) of hippocampus neurons on vehicle control mice (B). The TUNEL staining showed that 10 mg/kg PETL (C) and 1 mg/kg GABA (D) significantly reduced KA-induced apoptosis in hippocampus of the FVB mice brain as compared to control (A). (200x)
Figure 3. Effect of PETL extract and GABA on cell viability and cytotoxicity of KA-stressed PC12 cells. Cells were treated with KA (150 Î¼M) alone or with various concentrations of PETL extract (1, 10 Î¼ g/ml) or GABA (0.1, 1, 10 Î¼M) for 24 h. LDH (A) release was decreased and cell viability (B) was increased by PETL extract and GABA. *P < 0.01 as compared to KA control.
KA-induced calcium release
KA triggers neuronal membrane depolarization by releasing calcium ions from neuron cells . In the present study, KA induced calcium release from PC12 cells in a time-dependent manner (data not show). PETL extract and GABA significantly reduced KA-induced calcium release in PC12 cells (Figure 4).
Figure 4. Effect of PETL extract and GABA on Ca2+ generation from KA-treated PC12 cells. Cells were treated with KA (150 Î¼M) alone or with various concentrations of PETL extract or GABA for 24 h. PETL and GABA were effectively reducing the release of Ca2+ under KA stress. *P < 0.01 as compared to the KA control.
ROS and lipid peroxidation
ROS and lipid peroxidation can damage neuronal cells [16,18]. KA-treated cells increased DCF fluorescence by 80% after 120 min as compared with the control cells. Treatment with PETL extract or GABA protected cells against KA cytotoxicity by decreasing KA-induced ROS accumulation (Figure 5). Marked increases in MDA and 4-hydroxyalkenals levels were observed in KA-exposed cells, as compared with the control cells (Figure 6A). The PETL extract and GABA significantly protected cells against KA toxicity by lowering MDA levels (p < 0.01, as compared to the KA-treated cells). PETL and GABA were Consistently effective in reducing TBARS levels in the KA-induced SE mice (Figure 6B, P < 0.01 as compared to the KA control).
Figure 5. Effect of PETL extract and GABA on ROS generation in PC12 cells under KA stress. PETL extract (1, 10 (j,g/ml) and GABA (0.1, 1, 10 uM) were effectively reducing the ROS production from PC12 cells induced by KA stress (150 uM) at 120-min. *P < 0.01 as compared to the KA control.
Figure 6. In vitro and in vivo effect of PETL extract and GABA on the KA-induced oxidative stress. KA-induced lipid peroxidation of PC12 cells and brain neuron tissue of FVB mice were determined by ELISA and spectrophotometry, respectively. PETL or GABA was effectively reducing lipid peroxidation of PC12 cells by under 24-h KA stress (A) and in mice with 2-h KA-induced SE (B). *P < 0.01 as compared to the KA control.
Status epilepticus causes the death of nerve cells partly due to apoptosis. PETL and GABA significantly reduced KA-induced apoptosis in hippocampus cells of the mice (Figure 2). Therefore, we further evaluated whether the apoptotic signaling pathways was involved in the KA-treated PC12 cells. KA and GABA affected caspase-3 activation (Figure 7). Cells were treated with KA (150 Î¼M) alone or with PETL extract or GABA in various concentrations for 24 h. Both PETL and GABA decreased the caspase-3 activity significantly in KA-treated PC12 cells.
Figure 7. Kainic acid-induced caspase-3 activation. Cells were treated with KA (150 Î¼M) alone or with PETL extract and GABA in various concentrations for 24 h. Both PETL and GABA decreased the caspase-3 activity significantly, *P < 0.01 as compared to the KA control.
COX-2 and MAPKs activation
The effect of GABA or PETL extract on KA-induced signaling pathways in PC12 cells was evaluated by Western blot assay. KA induced the cell signal activation of MAP kinases (JNK, ERK. P38), COX-2, RhoA, and S100 in PC12 cells at 30 min. Only the activated COX-2 and MAPKs expression, but not RhoA were suppressed by GABA and PETL extract as compared to KA controls. GABA suppressed 50~80% COX-2 expression whereas GABA and PETL suppressed 80~90% S100-beta expression level as compared to KA controls (Figure 8).
Figure 8. Effect of PETL extract and GABA on KA-activated signaling pathway. COX-2, JNK, ERK, p38 MAP kinases, and RhoA in PC12 cell under KA stress for 30-min was determined by Western blot assay. Values represent the mean from three independent experiments. *P < 0.05 as compared to the KA control.
Effect of GABA on PGE2 production in PC12 cells
Since COX-2 controls PGE2 production, we inquired whether KA-induced COX-2 would affect PGE2 production. We found that PETL extracts and GABA significantly reduced the PGE2 production in KA-induced PC12 cells as predicted. PETL extracts and GABA reduced 30~40% PGE2 production as compared with the KA control cells. (Figure 9).
Figure 9. Effect of PETL extract and GABA on PGE2 production. PETL extract and GABA, significantly reduced the PGE2 production of KA-induced PC12 cells. *P < 0.01 as compared to the KA control.
The main result of the present study is the finding PETL and GABA protected animals from KA-induced brain injury. MDA and apoptosis were significantly reduced in the GABA and PETL-treated animals as compared with the vehicle control (Figure 2 and Figure 6). This effect was confirmed by the in vitro effects of GABA and PETL: decreased LDH release, ROS generation, lipid peroxidation, caspase-3 activation, and the increased cell viability of KA-stimulated PC12 cells. GABA appears to be a well bioactive component in the extract of Pu-Erh tea leaves. GABA has long been advocated for the treatment of cancer, oxidative stress, inflammation and diabetes, but few studies have evaluated modes of action in these processes. The present study demonstrates that GABA was effective in protecting PC12 cells from KA-induced injury in a dose-dependent manner. GABA and PETL extract decreased KA-induced Ca2+ and ROS release and lipid peroxidation in PC12 cells and FVB mice. Western blot analysis revealed that MAPKs, COX-2, RhoA and S-100 expression were increased in PC12 cells under KA stress. However, MAPKJNK2/1, MAPKERK1/2, COX-2 and RhoA expression but not MAPK p38 were significantly reduced by GABA (10 Î¼M). Furthermore, GABA and PETL treatment reduced PGE2 production by PC12 cell under KA stress.
PC12 cells derived from rat pheochromacytoma have been widely used for neurological studies [33,34]. Increases in ROS accumulation and lipid peroxidation were observed in KA-treated PC12 cells. KA-induced ROS accumulation was significantly reduced by PETL extract or GABA (Figure 4). These observations agree with earlier reports that shown that kainate induces lipid peroxidation in the rat neurons [14,35]. Lipid peroxidation is essential to assess the role of oxidative injury in pathophysiological disorders [36,37]. Lipid peroxidation results in the formation of highly reactive and unstable hydroperoxides of saturated or unsaturated lipids. We found that KA induced the activation of MAP kinases (JNK, ERK, p38), RhoA, S100, and COX-2 in PC12 cells. It is noteworthy that KA-activated COX-2 and MAPKs were reduced by GABA and PETL extract. In particular, GABA suppressed KA-activated S100, COX-2 and MAPKs expression. This result is in accord with observation that administration of tea extract (TF3) to rats with cerebral ischemia-reperfusion reduced mRNA and protein expression of COX-2, iNOS and NF-ÎºB activation in treated animals . In vitro studies showed that antioxidants suppress PGE2 production and COX-2 activity in lipopolysaccharide (LPS)-activated macrophages and microglia cells [39,40]. Consistently, Icariin attenuates lipopolysaccharide-induced microglial activation and resultant death of neurons by inhibiting TAK1/IKK/NF-ÎºB and JNK/p38 MAPK pathways . The present results are consistent with previous reports which show that KA-induced neuronal death can be prevented either by inhibiting xanthine oxidase, a cellular source of superoxide anions, or by the addition of free radical scavengers to the culture medium . ROS generation is correlated with KA induced-excitotoxicity [16,18,41,42]. The ability of kainate to induce lipid peroxidation is also related to the exposure of excitotoxin to the brain . It is widely accepted that neuronal degeneration after KA administration is associated with a depletion of AT P and accumulation of [Ca2+]i in neuron. The increase in [Ca2+]i can trigger Ca2+-activated free radicals formation . Thus, our data showing suppression of ROS and Ca2+ release by PETL are consistent with the proposed role of GABA and PETL extract in neuronal protection.
Cytokines and chemokines play key roles in the inflammatory response and its perpetuation [43,44]. It is conceivable that besides factors canonically implicated in the inflammatory response, other factors, including members of the S100 protein family [45,46], act to sustain the inflammatory response or to determine direct effects on neurons and/or microglia, thus switching the inflammatory response to neuronal death. The Ca2+-modulated protein of S100B is thought to be one factor that plays such a dual role [45,46]. A role of cerebral COX-2 mRNA and protein in KA toxicity has also been postulated [47-49]. KA-induced COX-2 expression parallels the appearance of neuronal apoptotic features . The KA-inducted COX-2 is also involved with free radicals formation . Several protease families have been implicated in apoptosis, the most prominent being caspases . However, we did find that KA affected the caspase-3 activation in PC12 cells. Since S100 and COX-2 may be involved in pathways leading to neuronal death, these additional effects of GABA could account for its neuroprotective properties, such as inhibition of KA-induced inflammatory mediators . Since PGE2 was synthesised in response to activation of COX-2 expressing cells, directly hyperpolarises GABA-induced neurons . GABA and PETL extract, as predicted, reduced PGE2 production dose-dependently, and S100, and COX-2 activation in KA-induced PC12 cells. Taken together, these results indicate that antioxidant and anti-inflammatory effects might account for the protective mechanisms of gallic acid on KA-induced PC12 cell injury.
Present data also showed that GABA or PETL could decrease the severity of seizure behavior. Further studies are needed to confirm whether GABA has direct effects on the seizure behavior and the related molecular mechanism in this issue. The present results are consistent with previous reports which show that antioxidants such as resveratrol  and vitamin E  are also protective against various animal models of SE in terms of the oxidative stress or convulsions. Resveratrol protects against KA-induced neuronal damage and subsequent epilepsy . Stopping seizure activity promptly is the best way to prevent SE-induced free radical formation and neuronal damage. However, clinical experience shows that SE can be refractory to the commonly used medications. Therefore, intervention by antioxidants can be a potential beneficial approach in the treatment of SE.
In conclusion, we found that Pu-Erh tea leaves had abundant content of GABA as bioactive components. The metabolites of GABA are also potent antioxidants and anti-inflammatory agents. This suggests that natural antioxidants play an important role in neuroprotection under excitotoxins and GABA in the Pu-Erh tea was responsible for this protection. Pu-Erh leaf extract and GABA ameliorates oxidative stress in KA-induced status epilepticus. The molecular mechanisms of PETL extract and GABA on SE-induced excitotoxicity warrants further study for their therapeutic potential.
The author has no competing interests in this manuscript.
We would like to thank Dr. Robert. H. Glew (University of New Mexico, USA) for critical proof reading and assistance of this manuscript.
1. Duh PD, Yen GC, Yen WJ, Wang BS, Chang LW: Effects of Pu-erh tea on oxidative damage and nitric oxide scavenging.
J Agric Food Chem 2004, 52:8169-8176. PubMed Abstract | Publisher Full Text OpenURL
2. Jie G, Lin Z, Zhang L, Lv H, He P, Zhao B: Free radical scavenging effect of Pu-erh tea extracts and their protective effect on oxidative damage in human fibroblast cells.
J Agric Food Chem 2006, 54:8058-8064. PubMed Abstract | Publisher Full Text OpenURL
3. Hayakawa S, Kimura T, Saeki K, Koyama Y, Aoyagi Y, Noro T, Nakamura Y, Isemura M: Apoptosis-inducing activity of high molecular weight fractions of tea extracts.
Biosci Biotechnol Biochem 2001, 65:459-462. PubMed Abstract | Publisher Full Text OpenURL
4. Chiang CT, Weng MS, Lin-Shiau SY, Kuo KL, Tsai YJ, Lin JK: Pu-erh tea supplementation suppresses fatty acid synthase expression in the rat liver through downregulating Akt and JNK signalings as demonstrated in human hepatoma HepG2 cells.
Oncol Res 2005, 16:119-128. PubMed Abstract OpenURL
5. Anderson RA, Polansky MM: Tea enhances insulin activity.
J Agric Food Chem 2002, 50:7182-7186. PubMed Abstract | Publisher Full Text OpenURL
6. Weisburger JH: Tea and health: the underlying mechanisms.
Proc Soc Exp Biol Med 1999, 220:271-275. PubMed Abstract | Publisher Full Text OpenURL
7. Jeng KC, Chen CS, Fang YP, Hou CW, Chen YS: Effect of microbial fermentation on content of statin, GABA, and polyphenols in Pu-Erh tea.
J Agric Food Chem 2007, 55:8787-92. PubMed Abstract | Publisher Full Text OpenURL
8. Hou CW, Jeng KC, Chen YS: Enhancement of Fermentation Process in Pu-Erh Tea by Tea-Leaf Extract.
J Food Science 2010, 75:H44-48. Publisher Full Text OpenURL
9. Wasterlain CG, Fujikawa DG, Penix L, Sankar R: pathophysiological mechanisms of brain damage from status epilepticus.
Epilepsia 1993, 1:S37-53. OpenURL
10. Kandinov B, Giladi N, Korczyn AD: Smoking and tea consumption delay onset of Parkinson's disease.
Parkinsonism Relat Disord 2009, 15:41-46. PubMed Abstract | Publisher Full Text OpenURL
11. Mandel SA, Amit T, Weinreb O, Reznichenko L, Youdim MB: Simultaneous manipulation of multiple brain targets by green tea catechins: a potential neu-roprotective strategy for Alzheimer and Parkinson diseases.
CNS Neurosci Ther 2008, 14:352-365. PubMed Abstract | Publisher Full Text OpenURL
12. Wasterlain CG, Fujikawa DG, Penix L, Sankar R: Pathophysiological mechanisms of brain damage from status epilepticus.
Epilepsia 1993, 34:S37-53. PubMed Abstract | Publisher Full Text OpenURL
13. Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA: Review: cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy.
Synapse 1989, 3:154-171. PubMed Abstract | Publisher Full Text OpenURL
14. Gupta YK, Briyal S, Chaudhary G: Protective effect of trans-resveratrol against kainic acid-induced seizures and oxidative stress in rats.
Pharmacol Biochem Behav 2002, 71:245-249. PubMed Abstract | Publisher Full Text OpenURL
15. Miyamoto R, Shimakawa S, Suzuki S, Ogihara T, Tamai H: Edaravone prevents kainic acid-induced neuronal death.
Brain Res 2008, 1209:85-91. PubMed Abstract | Publisher Full Text OpenURL
16. Hasegawa T, Takano F, Takata T, Niiyama M, Ohta T: Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globules.
Phytochemistry 2008, 69:747-753. PubMed Abstract | Publisher Full Text OpenURL
17. Bruce AJ, Baudry M: Oxygen free radicals in rat limbic structures after kainate-induced seizures.
Free Radic Biol Med 1995, 18:993-1002. PubMed Abstract | Publisher Full Text OpenURL
18. D'Antuono M, Benini R, Biagini G, D'Arcangelo G, Barbarosie M, Tancredi V, Avoli M: Limbic network interactions leading to hyperexcitability in a model of temporal lobe epilepsy.
J Neurophysiol 2002, 87:634-639. PubMed Abstract | Publisher Full Text OpenURL
19. Sun AY, Cheng Y, Bu Q, Oldfield F: The biochemical mechanism of the excitotoxicity of kainic acid.
Mol Chem Neuropathol 1992, 17:51-63. PubMed Abstract | Publisher Full Text OpenURL
20. Dubreuil CI, Marklund N, Deschamps K, McIntosh TK, McKerracher L: Activation of Rho after traumatic brain injury and seizure in rats.
Exp Neurol 2006, 198:361-369. PubMed Abstract | Publisher Full Text OpenURL
21. Goodenough S, Davidson M, Chen M, Beckmann A, Pujic Z, Otsuki M, Matsumoto I: Immediate early gene expression and delayed cell death in limbic areas of the rat brain after kainic acid treatment and recovery in the cold.
Exp Neurol 1997, 145:451-461. PubMed Abstract | Publisher Full Text OpenURL
22. Matagne V, Lebrethon MC, GÃ©rard A, Bourguignon JP: Kainate/estrogen receptor involvement in rapid estradiol effects in vitro and intracellular signaling pathways.
Endocrinology 2005, 146:2313-2323. PubMed Abstract | Publisher Full Text OpenURL
23. Arispe N, Pollard HB, Rojas E: Î²-Amyloid Ca2+-channel hypothesis for neuronal death in Alzheimer disease.
Mol Cell Biochem 1994, 40:119-125. OpenURL
24. Roth T: A physiologic basis for the evolution of pharmacotherapy for insomnia.
J Clin Psychiatry 2007, 5:13-18. OpenURL
25. Clinckers R, Zgavc T, Vermoesen K, Meurs A, Michotte Y, Smolders I: Pharmacological and neurochemical characterization of the involvement of hippocampal adrenoreceptor subtypes in the modulation of acute limbic seizures.
J Neurochem 2010, 115:1595-1607. PubMed Abstract | Publisher Full Text OpenURL
26. Alreja M, Wu M, Liu W, Atkins JB, Leranth C, Shanabrough M: Muscarinic tone sustains impulse flow in the septohippocampal GABA but not cholinergic pathway: implications for learning and memory.
J Neurosci 2000, 20:8103-8110. PubMed Abstract | Publisher Full Text OpenURL
27. SaÅat K, Kulig K: GABA transporters as targets for new drugs.
Future Med Chem 2011, 3:211-222. PubMed Abstract | Publisher Full Text OpenURL
28. Liu C, Zhao L, Yu G: The Dominant Glutamic Acid Metabolic Flux to Produce Î³-Amino Butyric Acid over Proline in Nicotiana tabacum Leaves under Water Stress Relates to its Significant Role in Antioxidant Activity.
J Integr Plant Biol 2011, 53:608-618. PubMed Abstract | Publisher Full Text OpenURL
29. Zhang G, Bown AW: The rapid determination of Î³-aminobutyric acid.
Phytochemistry 1997, 44:1007-1009. Publisher Full Text OpenURL
30. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.
Anal Biochem 1979, 95:351-358. PubMed Abstract | Publisher Full Text OpenURL
31. Racine RJ: Modification of seizure activity by electrical stimulation. II. Motor seizure.
Electroencephalogr Clin Neurophysiol 1972, 32:281-294. PubMed Abstract | Publisher Full Text OpenURL
32. Kanada A, Nishimura Y, Yamaguchi JY, Kobayashi M: Extract of Ginkgo biloba leaves attenuates kainate-induced increase in intracellular Ca2+ concentration of rat cerebellar granule neurons.
Biol Pharm Bull 2005, 28:934-936. PubMed Abstract | Publisher Full Text OpenURL
33. Bhakar AL, Howell JL, Paul CE, Salehi AH, Becker EB, Said F, Bonni A, Barker PA: Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad.
J Neurosci 2003, 23:11373-11381. PubMed Abstract | Publisher Full Text OpenURL
34. Fuenzalida KM, Aguilera MC, Piderit DG, Ramos PC, Contador D, QuiÃ±ones V, Rigotti A, Bronfman FC, Bronfman M: Peroxisome proliferator-activated receptor gamma is a novel target of the nerve growth factor signaling pathway in PC12 cells.
J Biol Chem 2005, 280:9604-9609. PubMed Abstract | Publisher Full Text OpenURL
35. Shin EJ, Jeong JH, Kim AY, Koh YH, Nah SY, Kim WK, Ko KH, Kim HJ, Wie MB, Kwon YS, Yoneda Y, Kim HC: Protection against kainate neurotoxicity by ginsenosides: attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress.
J Neurosci Res 2009, 87:710-722. PubMed Abstract | Publisher Full Text OpenURL
36. Halliwell B: Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans.
Free Rad Res 1996, 25:57-74. Publisher Full Text OpenURL
37. Porter NA, Mills KA, Caldwell SE: Mechanisms of free radical oxidation of unsaturated lipids.
Lipids 1995, 30:277-290. PubMed Abstract | Publisher Full Text OpenURL
38. Cai F, Li C, Wu J, Min Q, Ouyang C, Zheng M, Ma S, Yu W, Lin F: Modulation of the oxidative stress and nuclear factor kappaB activation by theaflavin 3,3'-gallate in the rats exposed to cerebral ischemia-reperfusion.
Folia-Biol-(Praha) 2007, 53:164-172. OpenURL
39. Lin CY, Shen YH, Wu SH, Lin CH, Hwang SM, Tsai YC: Effect of bismuth subgallate on nitric oxide and prostaglandin E2 production by macrophages.
Biochem-Biophys-Res-Commun 2004, 315:830-835. PubMed Abstract | Publisher Full Text OpenURL
40. Zeng KW, Fu H, Liu GX, Wang XM: Icariin attenuates lipopolysaccharide-induced microglial activation and resultant death of neurons by inhibiting TAK1/IKK/NF-ÎºB and JNK/p38 MAPK pathways.
Int Immunopharmacol 2010, 10:668-678. PubMed Abstract | Publisher Full Text OpenURL
41. Dykens JA, Stern A, Trenkner E: Mechanisms of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury.
J Neurochem 1987, 9:1222-1228. OpenURL
42. Puttfarcken PS, Getz RL, Coyle JT: Kainic acid-induced lipid peroxidation: protection with butylated hydroxytoluene and U7851F in primary cultures of cerebellar granule cells.
Brain Res 1993, 624:223-232. PubMed Abstract | Publisher Full Text OpenURL
43. Martiney JA, Cuff C, Litwak M, Berman J, Brosnan CF: Cytokine-induced inflammation in the central nervous system revisited.
Neurochem Res 1998, 23:349-359. PubMed Abstract | Publisher Full Text OpenURL
44. Cartier L, Hartley O, Dubois-Dauphin M, Krause KH: Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases.
Brain Res Brain Res Rev 2005, 48:16-42. PubMed Abstract | Publisher Full Text OpenURL
45. Donato R: S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.
Int J Biochem Cell Biol 2001, 33:637-668. PubMed Abstract | Publisher Full Text OpenURL
46. Van Eldik LJ, Wainwright MS: The Janus face of glial-derived S100B: beneficial and detrimental functions in the brain.
Restor Neurol Neurosci 2003, 21:97-108. PubMed Abstract | Publisher Full Text OpenURL
47. Hashimoto K, Watanabe K, Nishimura T, Iyo M, Shirayama Y, Minabe Y: Behavioral changes and expression of heat shock protein HSP-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid.
Brain Res 1998, 804:212-223. PubMed Abstract | Publisher Full Text OpenURL
48. Sandhya TL, Ong WY, Horrocks LA, Farooqui AA: A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainite lesions.
Brain Res 1998, 788:223-231. PubMed Abstract | Publisher Full Text OpenURL
Sanz O, Estrada A, Ferrer I, Planas AM: Differential cellular distribution and dynamics of HSP70, cyclooxygenase-2, and c-Fos in the rat brain after transient focal ischemia or kainic acid.
Neurosci 1997, 80:221-232. Publisher Full Text OpenURL
50. Candelario-Jalil E, Ajamieh HH, Sam S, Martinez G: Nimesulide limits kainate-induced oxidative damage in the rat hippocampus.
Eur J Pharmacol 2000, 90:295-298. OpenURL
51. Sarker KP, Nakata M, Kitajima I, Nakajima T, Maruyama I: Inhibition of caspase-3 activation by SB203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells.
J Mol Neurosci 2000, 15:243-250. PubMed Abstract | Publisher Full Text OpenURL
52. Ferri CC, Ferguson AV: Prostaglandin E2 mediates cellular effects of interleukin-1beta on parvocellular neurones in the paraventricular nucleus of the hypothalamus.
J Neuroendocrinol 2005, 17:498-508. PubMed Abstract | Publisher Full Text OpenURL
53. Tome AR, Feng D, Freitas RM: The effects of Î±-tocopherol on hippocampal oxidative stress prior to in pilocarpine-induced seizures.
Neurochem Res 2010, 35:580-587. PubMed Abstract | Publisher Full Text OpenURL
54. Wu Z, Xu Q, Zhang L, Kong D, Ma R, Wang L: Protective effect of resveratrol against kainate-induced temporal lobe epilepsy in rats.
Neurochem Res 2009, 34:1393-400. PubMed Abstract | Publisher Full Text OpenURL