Jong Hee Choi,Tae Woo Kwon,Hyo Sung Jo,Yujeong Ha,Ik-Hyun Cho
Gintonin, a Panax ginseng-derived LPA receptor ligand, attenuates kainic acid-induced seizures and neuronal cell death in the hippocampus via anti-inflammatory and anti-oxidant activities
Gintonin,Kainic acid-induced epilepsy,Anti-inflammation,Antioxidant,Lysophosphatidic acid receptor
Background Gintonin (GT), a Panax ginseng-derived lysophosphatidic acid receptor (LPAR) ligand, has positive effects in cultured or animal models for Parkinson's disease, Huntington's disease, and so on. However, the potential therapeutic value of GT in treating epilepsy has not yet been reported. Methods Effects of GT on epileptic seizure (seizure) in kainic acid [KA, 55mg/kg, intraperitoneal (i.p.)]-induced model of mice, excitotoxic (hippocampal) cell death in KA [0.2 μg, intracerebroventricular (i.c.v.)]-induced model of mice, and levels of proinflammatory mediators in lipopolysaccharide (LPS)-induced BV2 cells were investigated. Results An i.p. injection of KA into mice produced typical seizure. However, it was significantly alleviated by oral administration of GT in a dose-dependent manner. An i.c.v. injection of KA produced typical hippocampal cell death, whereas it was significantly ameliorated by administration of GT, which was related to reduced levels of neuroglial (microglia and astrocyte) activation and proinflammatory cytokines/enzymes expression as well as increased level of the Nrf2-antioxidant response via the upregulation of LPAR 1/3 in the hippocampus. However, these positive effects of GT were neutralized by an i.p. injection of Ki16425, an antagonist of LPA1-3. GT also reduced protein expression level of inducible nitric-oxide synthase, a representative proinflammatory enzyme, in LPS-induced BV2 cells. Treatment with conditioned medium clearly reduced cultured HT-22 cell death. Conclusion Taken together, these results suggest that GT may suppress KA-induced seizures and excitotoxic events in the hippocampus through its anti-inflammatory and antioxidant activities by activating LPA signaling. Thus, GT has a therapeutic potential to treat epilepsy.
Gintonin, a Panax ginseng-derived LPA receptor ligand, attenuates kainic acid-induced seizures and neuronal cell death in the hippocampus via anti-inflammatory and anti-oxidant activities

Gintonin (GT), a Panax ginseng-derived lysophosphatidic acid receptor (LPAR) ligand, has positive effects in cultured or animal models for Parkinson's disease, Huntington's disease, and so on. However, the potential therapeutic value of GT in treating epilepsy has not yet been reported.

Effects of GT on epileptic seizure (seizure) in kainic acid [KA, 55mg/kg, intraperitoneal (i.p.)]-induced model of mice, excitotoxic (hippocampal) cell death in KA [0.2 μg, intracerebroventricular (i.c.v.)]-induced model of mice, and levels of proinflammatory mediators in lipopolysaccharide (LPS)-induced BV2 cells were investigated.

An i.p. injection of KA into mice produced typical seizure. However, it was significantly alleviated by oral administration of GT in a dose-dependent manner. An i.c.v. injection of KA produced typical hippocampal cell death, whereas it was significantly ameliorated by administration of GT, which was related to reduced levels of neuroglial (microglia and astrocyte) activation and proinflammatory cytokines/enzymes expression as well as increased level of the Nrf2-antioxidant response via the upregulation of LPAR 1/3 in the hippocampus. However, these positive effects of GT were neutralized by an i.p. injection of Ki16425, an antagonist of LPA1-3. GT also reduced protein expression level of inducible nitric-oxide synthase, a representative proinflammatory enzyme, in LPS-induced BV2 cells. Treatment with conditioned medium clearly reduced cultured HT-22 cell death.

Taken together, these results suggest that GT may suppress KA-induced seizures and excitotoxic events in the hippocampus through its anti-inflammatory and antioxidant activities by activating LPA signaling. Thus, GT has a therapeutic potential to treat epilepsy.

Epilepsy is a common chronic neurological disorder of the central nervous system characterized by recurrent and unprovoked focal seizures due to an acute systemic or neurologic insult [1,2]. The epileptic seizures (seizure) not only adversely affect a patient's quality of life, but also damage the brain, causing memory decline and cognitive impairment [1,2]. Although cellular and molecular mechanisms of epilepsy remain unclear, genetic predisposition, developmental dysfunction, and pathological over-release of excitatory neurotransmitters have been suggested to play a role [1,2]. Recently, accumulating evidences strongly suggest that focal or systemic unregulated inflammatory processes (activation of microglia and astrocytes; synthesis of pro-inflammatory cytokines, chemokines, and enzymes; activation of transcriptional pathways; and so on) and oxidative stress (reactive oxygen species, reactive nitrite species, and so on) can lead to aberrant neural connectivity and a hyper-excitable neuronal network, which can mediate the onset of epilepsy [3,4]. Additionally, anti-inflammatory and antioxidant agents can positively regulate epileptogenesis and seizure [[1], [2], [3], [4]]. Although anti-seizures drugs (AEDs) such as phenytoin, carbamazepine, felbamate, and lamotrigine have good effects on seizure control worldwide, they might produce numerous side effects including memory impairment, ataxia, and drug resistance in about 30% of patients unfortunately [1,5]. Therefore, it is necessary to explore new AEDs that are safe and effective in attenuating epileptogenesis and seizure. Lysophosphatidic acid (LPA) is an important bioactive lipid species that can regulate a broad range of cellular and molecular effects in various cell and tissue types. LPA is primarily involved in intercellular signal transduction via activation of six cognate G protein-coupled receptors, LPA receptors (LPARs) 1-6 [6]. Signal transduction via LPARs can control various cellular functions such as cell mobility, cell migration, neurogenesis, neuroplasticity, and angiogenesis in healthy and pathological status of nervous systems [6]. LPAR1 specifically labels seizure-induced hippocampal reactive neural stem cells and regulates their division [7]. Mouse lacking LPA1 shows defects in cortical layer width, reduced proliferation, and neurogenesis in adult dentate gyrus [8,9]. Absence of LPAR2 fully reverts changes to hippocampal network excitability, increase of glutamatergic transmission, and seizure activity [10]. Inhibition of LPAR1–3 can deteriorate spinal demyelination by inducing inflammation and oxidative stress [11] as well as LPAR1–3 highly express in rodent microglia [6] and human microglial cell lines [6]. Although complexities of LPAR signaling in neuronal degeneration have been demonstrated, they are well appreciated yet in epileptic seizure. Gintonin (GT) is a novel glycolipoprotein fraction isolated from Panax (P.) ginseng [12]. GT consists of a complex of LPAs, ginseng proteins such as ginseng major latex-like protein 151 (GLP151), and ginseng ribonuclease-like storage protein [13]. GT can play as an exogenous LPAR ligand which elicits in vitro [Ca2+]i transients in neuronal and non-neuronal cells [14,15]. GT has various pharmacological effects in cultured and animal models of neurodegenerative and inflammatory disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and multiple sclerosis (MS) due to its antineuronal, anti-inflammatory, or antioxidant activities by regulating LPAs signaling [[16], [17], [18]]. Although these studies have raised the possibility that GT might also be an effective therapeutic for epilepsy, a multifaceted neurological disease, its roles in the context of epileptic seizure and mechanism are presently unknown. Thus, the objective of this study was to determine whether GT could mitigate seizure and hippocampal excitotoxicity in a kainic acid (KA; an analog of glutamate)-induced epilepsy mouse model. An intraperitoneal (i.p.), intrahippocampal or intracerebroventricular (i.c.v.) injection of KA can induce seizures and excitotoxicity in specific brain regions including the hippocampus of animals [[19], [20], [21], [22], [23], [24]]. KA has been widely used as a tool to experimentally mimic human temporal lobe epilepsy, the most common type of epilepsy [19,20]. Our results demonstrated that GT might have therapeutic benefits for seizure and excitotoxicity due to its anti-inflammatory and antioxidative activities via LPAR1/3.

Adult male BALB/c and C57BL/6 mice (Narabiotec Co. Ltd. Seoul, Republic of Korea; 7-8 weeks; weight, 23-25 g) were kept at a constant temperature of 23 ± 2 °C with a 12-hour light-dark cycle (light on 08:00 to 20:00) and were fed food and water ad libitum. The mice were allowed to habituate to the housing facilities for 1 week before the experiments. All experimental procedures were reviewed and were approved by the Institutional Animal Care and Use Committee or Ethics Committee at Kyung Hee University (Permit Number: KHUASP/SE-16-117). In this process, the proper randomization of laboratory animals and data handling were performed in a blind manner in accordance with recent recommendations from an NIH Workshop on preclinical models of neurological diseases [25].

To measure effects of GT on KA-induced seizure, BALB/c mice were randomly subdivided into the following five groups (n = 10 per group): 1) sham [normal control group; saline, i.p.) + saline, per oral (p.o.), 2] KA [KA-treated seizure group; 55 mg/kg of KA, i.p. + saline, p.o.], 3) KA + GT50 [KA and GT-treated group; 55 mg/kg of KA, i.p. + 50 mg/kg of GT, p.o.], 4) KA + GT100 [KA and GT-treated group; 55 mg/kg of KA, i.p. + 100 mg/kg of GT, p.o.], and 5) GT100 group (GT-alone treated group; saline, i.p. + 100 mg/kg of GT, p.o.]. To examine effect of Ki16425, an antagonist of LPAR1-3, on KA-induced seizure, the experimental group was randomly divided into the following six groups (n = 6 per group): 1) sham (saline, i.p. + saline, p.o.), 2) KA (55 mg/kg of KA, i.p. + saline, p.o.), 3) KA + GT100 [55 mg/kg of KA, i.p. + 100 mg/kg of GT, p.o.], 4) KA + GT100 + Ki30 [55 mg/kg of KA, i.p. + 100 mg/kg of GT, p.o. + 30 mg/kg of Ki16425, i.p.], 5) KA + Ki30 [55 mg/kg of KA, i.p. + 30 mg/kg of Ki16425, i.p.], and 6) GT100 group (saline, i.p. + 100 mg/kg of GT, p.o.].

To induce KA-induced seizure model [19,20], mice were i.p. injected with KA (Milestone Pharmtech USA Inc, New Brunswick, NJ, USA) as previously described [26]. KA was prepared in physiological saline. Concentration (55 mg/kg) of KA was determined based on a preliminary study (data not shown). GT was kindly provided by professor Seung Yeol Nah (Konkuk University, Seoul, Republic of Korea). GT was isolated from P. ginseng as previous described [12]. Characteristics of GT and bioactive lipids besides LPAs have been determined [27]. To examine positive effects of GT on KA-induced seizure (i.p.), mice were p.o. Administrated with 50 and 100 mg/kg of GT at 2 hours prior to KA-injection. Physiological saline was used as vehicle. Consecutively, to examine whether LPAR antagonist might neutralize positive effects of GT on KA-induced seizure (i.p.), Ki16425 (Tocris Bioscience, Bristol, UK), a representative LPAR antagonist, was i.p. inoculated 30 minutes before GT treatment.

Seizure was evaluated as previously described [26]. Seizure was measured in a 5-minute interval for 120 minutes with the following clinical scoring scale: grade 0, normal; grade 1, immobilization; grade 2, forelimb and/or tail extension, rigid posture; grade 3, head nodding, uni-/bilateral forelimb clonus, [rotation], frequent wet dog shakes; grade 4, generalized limbic seizures with rearing, falling, running, [rotation], and salivation; grade 5, continuous generalized seizures with tonic limbic extension; and grade 6, death.

To examine the therapeutic effect of GT for KA-induced cell death in the hippocampus, mice from the KA + GT group were randomly subdivided into the following groups (n = 6-7 per group): 1) sham [saline, intracerebroventricular (i.c.v.) + saline, p.o.)], 2) KA (0.2 μg of KA, i.c.v. + saline, p.o.), 3) KA + GT100 (0.2 μg of KA, i.c.v. + 100 mg/kg of GT, p.o.), and 4) GT100 group (0.2 μg of saline, i.c.v. + 100 mg/kg of GT, p.o.]

To order to explore the effect of GT on excitotoxic cell death in the hippocampus, an i.c.v. injection of KA (0.2 μg) was bilaterally performed under light isoflurane anesthesia (1-2%), as previously described [[21], [22], [23], [24],26,28]. Mice were p.o. Administrated with 50 or 100 mg/kg of GT at two hours before KA injection.

Histological evaluations were carried out at three days after KA or KA + GT injection as previously described [22,23]. After taking a photograph, the neuronal loss at the pyramidal layer of the CA1 and CA3 regions was assessed using the 0 - 6 rating scale that was previously described [23]: 0, no observable damage; +1, ≤10%; +2, 11–25%; +3, 26–50%; +4, 51–75%; +5, 76–90%; +6, ≥91% of observable cell loss.

Immunohistochemistry was carried out at three days after KA injection as previously described [[29], [30], [31]] using rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:2,000; WAKO, Osaka, Japan) or rabbit anti-glial fibrillary acidic protein (GFAP) (1:1,000; Millipore, Bedford, MA, USA) antibodies. A semiquantitative histological evaluation for Iba-1 antibody (40 × magnified microscope field) was conducted and scored blindly by 2 observers using the following scale: resting microglia, mildly activated microglia, and moderately activated microglia [32].

Mice were sacrificed under light isoflurane (1-2%) anesthesia at three days after KA injection, the brains were rapidly removed from the skull. Coronal brain slices (3-mm thickness) were prepared on ice-cold subbed slide glass using a brain matrix device (ASI instrument, Warren, MI, USA). CA1 and CA3 regions of bilateral hippocampus were sampled using micro scissors and blade on ice-cold slide glass under a dissection microscope. RT-PCR was carried out as previously described [23,24]. Briefly, primer sequences of mouse interleukin (IL)-1β, IL-6, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were previously described [29,30]. Expression levels of each gene were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The bilateral hippocampus was dissected from mice under light isoflurane (1-2%) anesthesia at three days after KA injection. The hippocampus was then homogenized. Proteins were then used for Western blot analysis as previously described [[29], [30], [31]]. Briefly, the membranes were blocked and probed overnight with rabbit anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-cleaved caspase-9 (1:1,000; Cell signaling Technology), rabbit anti-Iba-1 (1:1,000; WAKO), mouse anti-GFAP (1:5,000; Millipore), mouse anti-iNOS (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-COX-2 (1:500; Santa Cruz), mouse-anti-nuclear factor erythroid-2-related factor 2 (Nrf2) (1:500; Santa Cruz), mouse anti-NAD(P)H: quinone oxidoreductase 1 (NQO1) (1:1,000; Cell signaling Technology), mouse anti-heme oxygenase-1 (HO-1) (1:1,000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-LPAR1 and LPAR3 (1:1,000; Abcam, Cambridge, UK), rabbit anti-interleukin 1 beta (IL-1β) (1:1,000; Cell signaling Technology), mouse anti-IL-6 (1:1,000; Cell signaling Technology), and mouse anti-neuronal nuclear protein (NeuN) (1:2,000; Millipore) at 4°C and were then incubated with the appropriate secondary antibody conjugated to HRP at RT for 1 hour. A signal was developed using an ECL kit (Santa Cruz Biotechnology) and a densitometry analysis was performed using the ImageJ software.

A statistical analysis was performed with the SPSS 21.0 package (SPSS Inc, Chicago, USA) for Windows. The data were presented as mean ± standard error of the mean (SEM) and were compared using the one-way ANOVA with a Tukey post hoc test to compare multiple groups. Data from seizure evaluation were compared at each time point after KA injection and P-values of less than 0.05 were accepted as statistically significant.

To explore positive effects of GT on KA-induced seizure, mice were orally pre-administrated with 50 or 100 mg/kg of GT at 2 hours before KA injection (55 mg/kg, i.p.). The seizure was first observed within 5 minutes after KA injection, reaching a peak (4.3 ± 0.1) at 30-60 minutes. It then decreased. However, the severity level of the seizure was significantly alleviated in the time course (Fig. 1A) and the sum of seizure score (Fig. 1B) in a dose-dependent manner by pre-administration of GT at 50 or 100 mg/kg. Mice from the sham control group and GT treated group did not show any neurologic dysfunction (Fig. 1A and B). Since100 mg/kg of GT was more effective, we applied 100 mg/kg of GT in further assays unless otherwise described. These results suggest that GT could prevent KA-induced seizures.

We subsequently examined whether the beneficial effect of GT for KA i.p.-induced seizures was associated with protective effects in terms of excitotoxic (hippocampal) cell death. However, an i.p. Treatment of KA (55 mg/kg) did not produce clear hippocampal cell death [26]. Therefore, we used a KA (0.2 μg)-i.c.v. injection model as previous described [[22], [23], [24],26]. Three days after the i.c.v. injection of KA, levels of cell death in CA1 and CA3 regions of the hippocampus were evaluated using a 0 to 6 rating scale as previous described [23]. Mean cell death scores in CA1 and CA3 regions of the hippocampus were 2.7 ± 0.7 (n = 3) and 5.7 ± 0.3 (n = 3), respectively, in the KA-injected group (Fig. 1C–E). Interestingly, mean cell death scores significantly decreased by 1.0 ± 0.6 (n = 3) and 1.0 ± 0.6 (n = 3), respectively, due to pretreatment with 100 mg/kg of GT (Fig. 1C–E). Since apoptotic cell death was clearly observed in the hippocampus of KA-injected mice [[22], [23], [24],26] with a similar pattern of cell death by the Nissl staining [26], we measured protein expression levels of cleaved caspase-3, a representative apoptosis marker, by Western blot analysis (Fig. 1F and G). Protein expression level of cleaved caspase-3 was increased in the hippocampus from the KA group, while its expression was decreased in the group with GT pretreatment (Fig. 1F and G). GT pretreatment alone did not induce any clear cell death or apoptosis in the hippocampus (Fig. 1C–G). These results suggest that GT could prevent hippocampal cell death caused by KA i.c.v. injection, corresponding to an ability to mitigate seizures.

To measure whether GT could mediate the inflammatory process in the hippocampus of KA-induced excitotoxicity model, levels of microglial and astroglial activation were investigated by performing immunohistochemical staining for Iba-1 (a marker for microglia/macrophage lineage cells) and GFAP (a marker for astrocyte), respectively (Fig. 2A–F). In CA1 and CA3 regions from the sham group, Iba-1-positive cells generally displayed small cell bodies with thin processes (Fig. 2A), a typical morphology of resting microglia [23,24]. However, in the KA group, Iba-1-positive cells clearly showed alterations in the number and shape of Iba-1-positive cells, forming an enlarged cell body with short and thick processes (Fig. 2A), a typical morphology of activated microglia [[22], [23], [24]]. Impressively, pre-administration with 100 mg/kg of GT remarkably reduced the level of Iba-1-positive microglial activation relative to that of the KA group (Fig. 2A–C). However, pre-administration with GT alone did not induce significant alteration in microglial activation (Fig. 2A–C). These results were consistent with the expression pattern of Iba-1 by Western blot assay (Fig. 2G and H). Since microglia activation may influence astrocyte activation (Cho et al, 2008), its level was investigated using GFAP antibody. Similar to microglia, in CA1 and CA3 regions from the KA group, GFAP-positive astrocytes were clearly activated. However, their activation level was significantly lowered in the KA + GT group (Fig. 2D–F), corresponding to the expression pattern of GFAP protein by Western blot assay (Fig. 2G and I).

We then evaluated whether down-regulation in the activation of microglia and astrocyte due to GT (Fig. 2) was associated with altered expression of inflammatory mediators in the hippocampus by Western blot and RT-PCR assays. Results showed that mRNA expression levels of representative proinflammatory cytokines (IL-1β and IL-6) and enzymes (COX-2 and iNOS) were clearly increased in CA1 and CA3 regions from the KA i.v.c. Group compared to those in the sham group. However, up-regulation in their mRNA expression was significantly blocked in the GT (100 mg/kg)-treated group (Fig. 3A–J). Protein levels of COX-2 and iNOS were also markedly increased in the hippocampus from the KA group, while their elevation was inhibited in the GT-treated group (Fig. 3K–M). No alteration in the level of GAPDH was observed in the hippocampus after KA injection regardless of whether specimens were pre-treated with GT (Fig. 3A–M). These suggest that GT can reduce KA i.c.v-induced excitotoxic (hippocampal cell death) by inhibiting the expression of inflammatory mediators in the hippocampus.

It is known that antioxidants can delay disease progression and mitigate pathogenesis and symptoms of neurological disorders [33] and that GT has an antioxidant activity in cultured cells or animal models of neurological disorders such as MS and HD [16,17]. Therefore, we further studied whether GT could stimulate the Nrf2 signaling pathway, a pivotal mechanism of the endogenous antioxidant activity, using Western blot assay [16,17]. Transcription factor Nrf2 and representative antioxidant enzymes NQO1 and HO-1 [11,18] were slightly but significantly upregulated in the hippocampus of the KA i.c.v. Group (137.0%, 290.0%, and 135.6% respectively) as compared to those in the sham group. Their expression levels were significantly increased in the KA i.c.v. + GT group (32.1%, 28.6%, and 31.6%, respectively), as compared to those in the KA i.c.v. Group (Fig. 4A–D). These results suggest that the protective activity of GT on KA-induced seizure and hippocampal cell death might be closely associated with its antioxidant activity.

Malfunction of LPA signaling may produce various neurological disorders such as AD and pain [6]. And GT is a ligand of LPARs [14,15]. Thus, we studied whether expression levels of LPARs were changed in the hippocampus of KA-induced excitotoxicity mice and whether their expression was regulated by GT pre-administration using Western blot assay. Impressively, expression levels of representative LPAR1 and LPAR3 were significantly increased in hippocampus of the KA group (294.0% and 1,137.7%, respectively) as compared to those in the sham group. Their expression levels were greater in the hippocampus of the KA + GT group (39.3% and 36.0%, respectively) as compared to those in the KA group (Fig. 5A–C). These results suggest that GT might have positive effects on KA-induced hippocampal cell death by stimulating LPAR1 and LPAR3.

Since GT, an exogenous ligand of LPA receptor [14,15], mitigated KA i.p.-induced seizure (Fig. 1A and B) and activated the LPA pathway in the hippocampus after KA i.v.c. Injection (Fig. 6), we next investigated whether GT's anti-seizure effects could be neutralized by pretreatment with Ki16425, an LPARs antagonist. Mice were i.p. Inoculated Ki16425 30 minutes before GT treatment. As expected, the mitigating effect of GT on the exacerbation in seizure was significantly offset by pre-inhibition of LPA signaling with Ki16425 (Fig. 5D and E).

GT exerted anti-seizure and anti-neuronal cell death effects by inhibiting microglial activation in the hippocampus from KA induced seizure model (Fig. 1). To further investigate GT's action mechanism, cultured BV2 cells (cell line for microglia) were stimulated with GT (0.01-1.0 μg/ml) at 1 hour before LPS stimulation. As a result, protein expressions of IL-1β, IL-6, and iNOS were increased by LPS treatment. However, these increases were significantly inhibited by GT in a dose-dependent manner (Fig. 6A–D).

Subsequently, to examine whether conditioned medium from GT/LPS-treated BV2 cells (CM-GT/LPS) could reduce degeneration of cultured HT-22 cells (mouse hippocampal neuronal cell line) compared to conditioned medium from LPS-treated BV2 cells (CM-LPS), cells were treated with CM-GT/LPS or CM-LPS. As a result, protein expression level of NeuN was significantly reduced by LPS stimulation. However, such reduction was clearly blocked by CM-GT/LPS in a dose-dependent manner compared to treatment with CM-LPS (Fig. 6E and F). Levels of cleaved caspase-3 and cleaved caspase-9 increased by LPS stimulation were also inhibited by CM-GT/LPS in a dose-dependent manner (Fig. 6E–H).

Gintonin is a non-saponin component of P. ginseng that consists of a group of glycolipoproteins, which contain a large amount of LPA, carbohydrates (glucose and glucosamine), and hydrophobic amino acids (linoleic, palmitic, oleic, and stearic acids) [12]. Although GT has positive effects in cell and animal models of neurological diseases including AD, PD, and HD, its pharmacological effects against epilepsy have not yet been experimentally demonstrated. We explored whether GT mighty produce a positive effect against epileptic seizure and seizure-related neuronal cell death by assessing a KA-induced model in mice leading to seizures (i.p.) and excitotoxic cell death in the hippocampus (i.c.v.). Interestingly, we verified that pre-administration with GT could clearly control KA-induced seizure (Fig. 1) in connection with reduced cell death (apoptosis), microglial activation, and pro-inflammatory cytokines (IL-1β and IL-6)/enzymes (COX-2 and iNOS) in CA1 and CA3 regions as well as enhanced antioxidant action (Fig. 1, Fig. 2, Fig. 3, Fig. 4). These GT's positive effects were related to enhanced LPAR1 and LPAR3 expression. GT's anti-seizure effects were dramatically neutralized by Ki16425, an antagonist for LPAR 1-3 (Fig. 5). These results suggest that GT might partially or indirectly control seizure and hippocampal cell death through its anti-inflammatory and anti-oxidant activities in the KA-induced model of mouse. In the present study, male BALB/c mice were i.p. Injected with KA (55 mg/kg) to evaluate the severity of seizure (Fig. 1), whereas male C57BL/6 mice were i.c.v. Injected with KA (0.2 μg per mouse) to measure the level of cell death and investigate its underlying mechanism (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6). In previous studies, we could not observe clear cell death in CA1 or CA3 regions from BALB/c mice at 3 days after i.p. injection with KA [26]. According to several studies, certain strains of mice, specifically C57BL/6 and BALB/c, are sensitive to KA-i.p. induced seizures but are resistant to KA-i.p. induced neuronal death in pyramidal cells of the hippocampus [34,35]. However, several strains of mice are sensitive to KA-i.c.v. induced neuronal death in the hippocampus [26,34,35]. These reports might explain why different injection routs (i.p. And i.c.v.) of KA into mice were used to test seizure and understand its underlying mechanism [26]. KA-induced excitotoxic cell death in the hippocampus displays characteristics of both acute and delayed cell death within/around lesion. Acute cell death begins to appear within the lesion from 3-5 hours after KA injection (i.p. Or i.c.v.) [[36], [37], [38], [39]]. Acute cell death is followed a little later by microglial activation at 6-48 hours after KA injection [[40], [41], [42]]. The activated microglia's secretions then induce an excessive neuronal death that can last over several days to weeks, called delayed cell death (Weise et al, 2005). Here, at 3 days after KA injection, GT-dependent suppression of the hippocampal cell death was accompanied by downregulation of microglial activation (Fig. 2) as well as pro-inflammatory cytokines (IL-1β and IL-6) and enzymes (COX-2 and iNOS) (Fig. 3). CM-GT/LPS also clearly inhibited neuronal cell death (NeuN) and apoptosis (cleaved caspase-3 and 9) in LPS-induced HT-22 cells (Fig. 6). These results suggest that pre-treatment with GT may exert protective effects on KA i.c.v.-induced delayed cell death (Fig. 1C–G) via its anti-inflammatory activity (Fig. 2, Fig. 3). Recently, GT's anti-inflammatory effects have been widely reported in cultured and animal models of neurodegenerative diseases (such as AD, PD, and HD) and autoimmune diseases such as MS [[16], [17], [18]]. Taken together, these findings suggest that GT might partially or indirectly inhibit KA i.c.v.-induced excitotoxic cell death (delay cell death) via its anti-inflammatory activity. LPARs are expressed in varying spatiotemporal patterns in various kinds of cells in the nervous and immune systems during the transition from fetal to adult life stages [6]. LPA signaling is responsible for diverse physiological and pathophysiological processes including proliferation, apoptosis, morphological changes, migration, and differentiation into cells with specialized functions within the nervous system [6,43]. Although LPA/LPAR signaling is pivotal to pathological events in various neurological disorders, the role of specific LPAR subtypes remains to be defined [6,44,45]. Among LPARs, LPAR1 and LPAR3 are abundantly expressed in monocyte-derived cells (microglia and macrophages) in rodent, human microglial cell lines [6,46], human peripheral blood mononuclear cells, and reactive murine spinal cord astrocytes [47]. LPAR1-deficit in murine can reduce apoptotic cell death of Schwann cells in sciatic nerves [48]. I.p. Treatment with Ki16425, an LPA1/3 antagonist, can mitigate LPA-stimulated nociception [49]. We have previously demonstrated that oral treatment with GT can improve motor disability by alleviating mutant huntingtin aggregation associated with reduction of microglial activation and inflammatory response in mice with adeno-associated viral vector serotype DJ-mediated overexpression of N171-82Q-mutant HTT in the striatum via LPAR1/3 [17]. GT can improve long-term memory impairment through reduction of amyloid plaque deposition via LPAR 1-5 activation in a transgenic mouse model of AD [14,15,50]. Impressively, pre-inhibition of LPAR1/3 by Ki16425 can neutralize the mitigating activity of GT on behavioral dysfunction related to spinal demyelination after EAE induction [18]. Here, expression levels of LPAR1 and LPAR3 proteins were slightly but significantly up-regulated in the hippocampus of KA i.c.v.-induced excitotoxicity model. Their expression levels were further increased by treatment with GT (Fig. 5). However, GT's positive activity was neutralized by pre-inhibition of LPAR1/3 through pre-treatment with Ki16425 (Fig. 5). Although the roles of GT and LPAR subtypes remain unclear, our results indicate that GT's activity as an LPA agonist may alleviate KA-induced seizures and hippocampal cell death. LPA signaling can stabilize Nrf2, an important molecule in a cell survival mechanism. The stabilized Nrf2 can stimulate transcription of antioxidant genes associated with oxidative stress [33]. Oxidative stress is critically engaged in the pathogenesis of various neurological dysfunction such as excitotoxic and demyelination [51,52]. However, anthocyanins can protect against KA i.c.v.-induced excitotoxicity and apoptosis by inhibiting ROS-activated AMPK pathway in hippocampal neurons [53]. In the same vein, Nrf2 KO mice produced more severe motor disability and excess inflammatory cells within the optic nerves with increased Th1-related immune response in the spleen in EAE mice (Larabee et al, 2016). Primary cortical neurons from Nrf2 knockout mice abolished neuroprotective effects of pioglitazone against oxidative and excitotoxic damage [54]. However, chalcone-derived Nrf2 activator [a novel chalcone analog, 1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone (Tak)] protects cognitive function by maintaining neuronal redox status [55]. Upregulation and activation of the Nrf-1 transcription factor were detected in the lesioned hippocampus [56]. These reports strongly suggest that Nrf2 is an attractive therapeutic target for epilepsy associated with excitotoxicity. Here, GT treatment upregulated Nrf2 protein expression and Nrf2 phase II enzymes (HO-1 and NQO-1) via stimulation of LPAR1 and LPAR3 in the hippocampus (Fig. 4, Fig. 5), in agreement with changes in seizures and delayed cell death after KA injection (Fig. 1). However, the beneficial effect of GT on KA-induced seizure was nullified by pre-treatment with Ki16425 (an LPAR1/3 antagonist, i.p.) (Fig. 5). Venkatraman et al (2015) have demonstrated that the stability of Nrf2 is reduced when LPA1 is knocked down using siRNA-LPA1 and Ki16425. We have recently reported that pre-stabilizing Nrf2 using Nrf2 pathway activators (DMF and AI-3) can alleviate striatal cell death-related motor disability and lethality [57] and that the beneficial effect of GT is nullified by pre-treatment with Nrf2 siRNA or an Nrf2 inhibitor (ML385) [17]. We have also reported that Ki16425, an LPAR1/3 antagonist, can neutralize the mitigating effect of GT on behavioral dysfunction related to spinal demyelination after EAE induction [18]. Taken together, our findings indicate that GT may be protective against motor disability and pathological abnormalities in the hippocampus following KA injection, strongly suggesting its potential therapeutic value in treating epilepsy via stabilization of Nrf2 through LPA/LPARs. In conclusion, GT, a P. ginseng-derived LPA receptor ligand, has a positive effect on seizures and hippocampal cell death through its anti-inflammatory and anti-oxidant activities via LPA-LPAR signaling pathway. Thus, GT could be used as a therapeutic for preventing and treating epilepsy.

JHC performed the behavioral experiments, immunohistochemistry, PCR analysis, and Western blots and also prepared the figures. WTK, HSJ, and YH assisted the behavioral and histological experiments. IHC conceived all experiments, analyzed the results, and wrote the manuscript. All authors have read and approved the final manuscript.

This work was supported by a grant (KHU- 20181292) from 10.13039/501100002597Kyung Hee University in 2018.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.