Houttuyniae Herba Attenuates Kainic Acid-Induced Neurotoxicity via Calcium Response Modulation in the Mouse Hippocampus

• Introduction
• Results and Discussion
• Materials and Methods
• Plant material
• Other materials
• Cultures of rat hippocampal neuronal cells
• Measurement of cell viability
• Measurement of the intracellular calcium level
• Measurement of the intracellular reactive oxygen species level
• Measurement of the mitochondrial membrane potential
• Animals, surgery procedure, and treatment
• Estimation of epilepsy status
• Novel object recognition test
• Brain tissue preparation
• Immunohistochemistry
• Fluoro-jade B staining
• Statistical analysis
• Supporting information
• Acknowledgements
• References

Abstract

Epilepsy is a complex neurological disorder characterized by the repeated occurrence of electrical activity known as seizures. This activity induces increased intracellular calcium, which ultimately leads to neuronal damage. Houttuyniae Herba, the aerial part of Houttuynia cordata, has various pharmacological effects and is widely used as a traditional herb. In the present study, we evaluated the protective effects of Houttuyniae Herba water extract on kainic acid-induced neurotoxicity. Kainic acid directly acts on calcium release, resulting in seizure behavior, neuronal damage, and cognitive impairment. In a rat primary hippocampal culture system, Houttuyniae Herba water extract significantly protected neuronal cells from kainic acid toxicity. In a seizure model where mice received intracerebellar kainic acid injections, Houttuyniae Herba water extract treatment resulted in a lower seizure stage score, ameliorated cognitive impairment, protected neuronal cells against kainic acid-induced toxicity, and suppressed neuronal degeneration in the hippocampus. In addition, Houttuyniae Herba water extract regulated increases in the intracellular calcium level, its related downstream pathways (reactive oxygen species production and mitochondrial dysfunction), and calcium/calmodulin complex kinase type II immunoreactivity in the mouse hippocampus, which resulted from calcium influx stimulation induced by kainic acid. These results demonstrate the neuroprotective effects of Houttuyniae Herba water extract through inhibition of calcium generation in a kainic acid-induced epileptic model.

Introduction

Epilepsy is a common chronic neurological disorder characterized by the repeated occurrence of sudden and transitory episodes called seizures [1]. Worldwide, 1.5 million patients suffer from epilepsy and the morbidity rate is continually increasing [2]. In 30 % of patients, epilepsy is caused by traumatic injury, encephalitis, or brain tumors, which can generate external neuronal damage. In 70 % of epilepsy patients, the reason for epilepsy onset is unknown [3]. Regardless of cause, excessively high neuronal activity exists in epilepsy patients, and it is triggered by the glutamate receptors N-methyl-D-aspartate and AMPA, and kainate receptors [4]. Thus, antiepileptic drugs inhibit glutamate receptors with the intention to reduce further neuronal damage and seizures [5], as seizures occur due to an imbalance between excitatory and inhibitory transmission [1]. However, glutamate receptor inhibition has many adverse effects, such as short-term memory disorders, paresthesia, sudden sleep, and diarrhea [6]. Thus, the search for new drugs is ongoing.

Houttuynia cordata Thunb. (Saururaceae) is a well-known traditional oriental medicine used to treat diuresis and for detoxification [7] that may also be beneficial for preventing or treating inflammatory disease [8], [9]. It is associated with a broad range of pharmacological activities, including antiviral, antileukemic, antioxidative, anti-pulmonary fibrosis, antiobesity, anti-severe acute respiratory syndrome, and anticancer effects [10], [11], [12], [13], [14]. Moreover, H. cordata contains a variety of compounds including volatile oils, alkaloids, fatty acids, sterols, flavonoids, and polyphenolic acids (e.g., rutin, hyperin, and quercetin) and those phytochemicals have shown beneficial neuroprotective actions in various experimental models [15], [16], [17], [18]. However, a few studies have demonstrated the effect of H. cordata on neuro-associated models; H. cordata has protective effects against amyloid beta or scopolamine-induced neurotoxicity [18], [19].

Considering the effects of H. cordata on neuroprotection, calcium regulation, and apoptosis inhibition, we evaluated the protective effects of standardized HCW against excitotoxicity. We further explored its possible mechanisms by performing calcium-related assays in primary rat hippocampal cells and in a mouse model of epilepsy induced by kainic acid.

Results and Discussion

We evaluated the protective effects of a standardized HCW against kainic acid-induced excitotoxicity in models of epilepsy. In addition, the underlying mechanism of action (i.e., calcium regulation) was investigated.

To evaluate whether HCW protects neuronal cells against kainic acid, rat primary hippocampal cell viability was examined using the MTT assay. Glutamatergic or cholinergic agonist administration is routinely used to induce excitotoxicity in experimental models of epilepsy [1]. Agonist-induced excitotoxicity resulting from AMPA or kainite receptor overstimulation leads to increased intracellular calcium, which directly triggers mitochondrial dysfunction and caspase cascades, ultimately damaging neurons in the brain [20]. Thus, we used kainic acid, originally isolated from “Kaininsou” seaweed. Kainic acid is an ionotropic glutamate receptor subtype agonist that induces the influx of cellular calcium, mitochondrial dysfunction, neuronal apoptosis, and degeneration [21]. Previous studies have suggested that kainic acid injection can induce epilepsy in mice, and that the symptoms are similar to epilepsy in humans, particularly in the hippocampus [22]. In this study, treatment at several HCW concentrations (1, 10, and 100 µg/mL) for 24 h had no influence on cell proliferation and did not result in cell toxicity. Hippocampal neurons were damaged by 200 µM kainic acid (55.06 ± 1.12 % compared to the control), but treatment with LEV, a positive control, the active compounds in HCW (CA and CGA), and HCW at 10 and 100 µg/mL significantly protected cells from this excitotoxicity, and 100 µg/mL HCW showed a similar effect to those of LEV and CA ([Fig. 1]).

Seizure activity was measured to investigate the protective effects of HCW against kainic acid, which is known to induce typical epileptic behavior in a dose-dependent manner in a mouse model [23]. Seizures are a characteristic of epilepsy and can disturb the daily life of epilepsy patients [24]. Mice that were administered kainic acid had higher seizure scores using a previously described scale [25]. LEV-treated mice had significantly lower seizure behavior over 120 min, and HCW-treated mice also exhibited significantly decreased seizure activity after 60 min ([Fig. 2]). In addition, cognitive decline was measured to examine the inhibitory effect of HCW on memory deficits induced by kainic acid, as cognitive deficits represent a serious symptom in epilepsy patients [26] and intracellular cascades activated by excitotoxicity overlap in epileptogenic brain areas, resulting in learning and memory failure [1]. NORT was employed to investigate whether HCW improved memory impairment in mice after kainic acid toxicity. During the test session on the next day, sham mice spent more time exploring the novel object than the familiar object by 74.51 ± 1.51 %. However, the kainic acid-injected mice spent similar amounts of time exploring the two objects at 48.45 ± 0.36 %, representing impaired learning and memory, similar to previous reports [26], [27]. Treatment with LEV at 20 mg/kg/day and HCW at 200 mg/kg/day significantly improved kainic acid-induced memory deficits by 60.13 ± 2.17 % and 59.77 ± 2.14 %, respectively ([Fig. 3]).

To control epilepsy, a reduction in necrosis and apoptosis and subsequent neuronal death are generally required [28]. Thus, we measured survival and degenerating neurons in the hippocampus. Kainic acid activates presynaptic kainate receptors, which are located on glutamatergic terminals in the hippocampus [29]; thus, an intracerebellar injection of kainic acid causes neuronal degeneration in CA1 and CA3 [30]. In the present study, kainic acid-administered mice had significantly fewer NeuN-positive neurons in CA3 compared to the sham. In addition, degenerating neuron fluorescence intensity was increased in the CA1 and CA3 of the hippocampus in the kainic acid group. However, LEV and HCW treatment inhibited neuronal damage in the CA3 compared to the sham (35.59 ± 3.28 % and 37.03 ± 2.05 %, respectively; [Fig. 4]). In addition, LEV- and HCW-treated mice had significantly fewer degenerating neurons in CA1 (54.31 ± 18.73 % and 63.21 ± 4.25 %, respectively) and CA3 (72.34 ± 1.91 % and 54.49 ± 9.34 %, respectively) compared to the kainic acid-injected mice ([Fig. 5]). These results indicate that HCW suppresses kainic acid-induced neuronal damage in the mouse hippocampus.

The exact molecular mechanisms of neurodegeneration induced by kainic acid excitotoxicity remains unclear, but both in vitro and in vivo studies have demonstrated that kainic acid induces cell death via activation of proinflammatory cytokines and accumulation of intracellular calcium, which stimulates ROS production and mitochondrial dysfunction, thereby leading to neuronal cell death [29]. In the present study, significant increases in the levels of intracellular calcium and ROS as well as potential depolarization in the mitochondrial membrane were observed in the kainic acid group. However, LEV and HCW treatment significantly inhibited these phenomena. The intracellular calcium level was increased to 204.04 ± 8.36 % of the control in the kainic acid-treated group, whereas HCW at 10 and 100 µg/mL significantly decreased the calcium level to 140.27 ± 5.23 % and 118.35 ± 3.01 % of the control, respectively ([Fig. 6 A]). ROS production was also increased to 260.08 ± 8.04 % of the control in the kainic acid-treated group, whereas HCW at 10 and 100 µg/mL significantly decreased the calcium level to 184.90 ± 8.32 % and 163.50 ± 9.04 % of the control, respectively ([Fig. 6 B]). In addition, we measured red and green fluorescnences, which represent normal and depolarized mitochondria, respectively. Kainic acid induced a decrease in the ratio of red to green fluorescence (46.64 ± 3.46 % compared to the control), however HCW attenuated this depolarization, showing ratios of 73.59 ± 7.13 % and 82.32 ± 6.32 % at 10 and 100 µg/mL, respectively.

Moreover, the effect of HCW on calcium regulation was confirmed in a mouse model of epilepsy. CaMK II plays an important role in calcium homeostasis by binding to the calcium/calmodulin complex [31], and an activated form of CaMK II, p-CaMK II, is increased in the hippocampus during kainic acid-induced neuronal cell death [32]. A recent study also revealed that increased p-CaMK II may lead to cell apoptosis, caspase-3 activation, and cytochrome c release [33]. The kainic acid-treated group exhibited increased p-CaMK II-immunoreactivity (160.70 ± 10.33 %) in the CA3 compared to the sham. However, LEV and HCW treatment decreased the immunoreactivity to 105.59 ± 12.50 and 114.47 ± 11.29 % in the CA3, respectively, compared to the sham ([Fig. 7]). This finding indicates that HCW might regulate calcium homeostasis, leading to the inhibition of ROS production and mitochondrial dysfunction, finally resulting in neuroprotection.

Current antiepileptic drugs have various adverse effects (e.g., sudden sleepiness and motor disturbances) observed during clinical treatment [34]. Therefore, there are limits to using these agents. Herbal therapy has historically been used to treat epilepsy. For example, Kava (Piper methysticum Roxb.; Piperaceae) enhances γ-aminobutyric acid transmission and acts as a voltage-gated ion channel blocker; both of these actions are relevant to seizure disorders by controlling endogenous glutamate release [35]. H. cordata possesses potential bioactive components that may prevent or treat excitotoxicity-induced neuronal damage: quercetin and kaempferol. Quercetin is a calcium antagonist. This compound may block excessive calcium influx into the cell membrane, which might be helpful for maintaining calcium homeostasis [36]. Quercetin also has a modulatory effect on the calcium/calmodulin complex [37], which could reduce CaMK activity. Kaempferol is a type 1 DNA topoisomerase inhibitor [38], which helps reduce seizure activity [39]. Moreover, phenolic compounds in H. cordata (e.g., CGA, CA, and rutin) have been shown to protect against neurotoxin-induced damage in several experimental models by inhibiting apoptosis, oxidative stress, and inflammation, underlying pathways of excitotoxicity-induced neuronal damage [18], [40], [41], [42], [43], [44], [45], [46]. Furthermore, in a previous study, we demonstrated that H. cordata treatment exerted its neuroprotective effect via regulation of calcium and apoptosis in an in vitro and an in vivo model of Alzheimerʼs disease [18], [19]. Collectively, these results indicate that H. cordata possesses various capabilities that contribute to the neuroprotective effects observed in this epileptic model induced by kainic acid.

In summary, this study is the first to report a protective effect of H. cordata in kainic acid-induced epileptic models. H. cordata protected hippocampal cells against kainic acid-induced excitotoxicity by inhibiting a calcium increase and its related downstream pathways, ROS production, and mitochondrial dysfunction, resulting in reduced seizure behavior and improved memory. These results suggest that H. cordata may be a useful agent for preventing and treating epilepsy.

Materials and Methods

Plant material

A dried Houttuyniae Herba was purchased from Jung Do herbal Drug Co. (lot #30 202 245B) and the voucher specimen (KHUOPS-MH022) was deposited in the herbarium of the College of Pharmacy, Kyung Hee University. HCW was the same as that used in a previous study [18], [19] in which chemical profiling and standardization of HCW using CA and CGA had been performed [18].

Other materials

Neurobasal medium and B27 supplement were purchased from Gibco. Penicillin and streptomycin were purchased from Hyclone Lab Inc. A Fluo-4 NW calcium assay kit was purchased from Molecular Probes Inc. Fluoro-jade B staining stock and mouse monoclonal anti-NeuN antibody were purchased from Millopore Bioscience Research. Rabbit polyclonal anti-p-CaMK II antibody was purchased from Santa Cruz Biotechnology. Biotinylated goat anti-rabbit and goat anti-mouse antibodies, normal goat serum, and avidin-biotin complex were purchased from Vector Labs. Zoletil50® and Rompun® were purchased from Virbac and Bayer Korea, respectively. Kainic acid monohydrate (≥ 98 %, from Digenea simplex), LEV (≥ 98 %), CGA, CA, MTT, H₂DCF-DA, JC-1, L-glutamine, DMSO, paraformaldehyde, poly-L-lysine, 3,3-diaminobenzidine, sucrose, ethanol, and PBS were purchased from Sigma-Aldrich.

Cultures of rat hippocampal neuronal cells

Cell cultures were prepared from the hippocampus of 18-day embryos of timed pregnant Sprague-Dawley rats (Daehanbiolink Co. Ltd.). The hippocampus was dissected, collected, dissociated, and plated in a poly-L-lysine precoated 96-well plate at a density of 1.2 × 10⁴ cells/well. Cultures were maintained in a humidified incubator of 5 % CO₂ at 37 °C in a neurobasal medium with 2 mM glutamine, 2 % B27, and 1 % penicillin/streptomycin. The medium was replaced with a new medium every 3 days. On in vitro day 10 (DIV 10), the cells were treated with HCW and stressed with 200 µM kainic acid for a further 2 h, 12 h, or 24 h.

Measurement of cell viability

Treated cells with HCW, CA, CGA, or LEV and kainic acid for 24 h were incubated with 1 mg/mL of MTT as described [18] using a spectrophotometer (Versamax microplate reader; Molecular Device) at a wavelength of 570 nm and then expressed as a percentage of the control value.

Measurement of the intracellular calcium level

The intracellular calcium level was measured using a Fluo-4 NW calcium assay kit as previously described [18]. The cells were incubated at 37 °C for 15 min followed by 15 min of incubation at room temperature before the addition of HCW or LEV and kainic acid for 2 h. The fluorescence intensity of Fluo-4 was measured at an excitation wavelength of 494 nm and an emission wavelength of 516 nm using a fluorescence microplate reader (SpectraMax Gemini EM; Molecular Device). The fluorescence intensity was expressed as a percentage of the control value.

Measurement of the intracellular reactive oxygen species level

The intracellular ROS level was measured using a fluorescent probe, H₂DCF-DA, as previously described [18]. The cells were treated with HCW or LEV and kainic acid for 2 h, then they were incubated with 10 mM H₂DCF-DA at 37 °C for 30 min. The fluorescence intensity of DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a fluorescence microplate reader. The fluorescence intensity was expressed as a percentage of the control value.

Measurement of the mitochondrial membrane potential

The mitochondrial membrane potential was measured using a fluorescent dye, JC-1, as previously described [18]. The cells were treated with HCW or LEV and kainic acid for 12 h, then they were incubated with JC-1 at 37 °C for 15 min. The red and green fluorescence intensities were measured at an excitation wavelength of 585 and 510 nm and an emission wavelength of 590 and 527 nm, respectively, using a fluorescence microplate reader. The ratio of red-to-green fluorescence intensity was expressed as a percentage of the control value.

Animals, surgery procedure, and treatment

Male ICR mice (9 weeks, 30–32 g), were purchased from Orient Bio. Animals were housed 6 per cage, had free access to water and food, and were maintained under a constant temperature (23 ± 1 °C), humidity (60 ± 10 %), and a 12-h light/dark cycle. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85–23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea (KHP-2013-08-01). The animals were anesthetized with a mixture of oxygen (0.6 L/min at 1.0 bar, 21 °C), nitrous oxide (0.3 L/min at 1.0 bar, 21 °C), and 0.8–2.0 % vaporized isoflurane (Forane; Choongwae) using an anesthesia vaporizer (Model 100 Vaporizer, SurgiVet, Inc.) and mounted in a stereotaxic apparatus (myNeuroLab). Each mouse was unilaterally injected (at rate 0.5 µL/min) with 3 µL of kainic acid (0.1 µg/µL in saline) into the right lateral ventricle (coordinate with respect to bregma in mm: AP − 2.0, ML 3.0, DV − 3.5), according to the stereotaxic atlas of the mouse brain [47]. The sham-operated mice were injected with the same volume of saline alone. Then, the mice were divided into four groups (n = 6 in each group); (1) Sham group (sham-operated group), (2) kainic acid group (kainic acid + intraorally saline-treated group), (3) LEV group (kainic acid + intraorally LEV 20 mg/kg/day-treated group), and (4) HCW group (kainic acid + intraorally HCW 200 mg/kg/day-treated group). LEV and HCW dissolved in saline were administrated once per day during 4 days and the sham group and kainic acid group were treated with the same volume of vehicle.

Estimation of epilepsy status

After kainic acid injection, the animals were carefully monitored for 2 h and seizure activity was scored by following the rating scale as described previously [25]: (+ 1) arrest of motion, (+ 2) myoclonic jerk of head and neck with brief twitching movements, (+ 3) unilateral clonic activity, ipsilateral turning, frequent focal convulsions, (+ 4) bilateral forelimb tonic and clonic activity, sudden running, and (+ 5) continuous generalized limbic seizures with loss of postural tone, and death within 2 h.

Novel object recognition test

NORT was carried out in a grey open field box (45 cm × 45 cm × 50 cm) as previously described [19]. Results are expressed as the percentage of novel object recognition time: time percentage = time (t) of exploring novel object/(t of exploring novel object + t of the familiar object) × 100.

Brain tissue preparation

After the last groupʼs behavioral test, the mice were immediately anesthetized and perfused transcardially with 0.05 M PBS, and then fixed with cold 4 % paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and post-fixed in 0.1 M phosphate buffer containing 4 % paraformaldehyde overnight at 4 °C and then immersed in a solution containing 30 % sucrose in 0.05 M PBS for cryoprotection. Serial 30 µm thick coronal sections were cut on a freezing microtome (Leica) and stored in cryoprotectant (25 % ethylene glycol, 25 % glycerol, 0.05 M phosphate buffer) at 4 °C until use for immunohistochemistry.

Immunohistochemistry

Brain sections were briefly rinsed in PBS buffer and treated with 1 % hydrogen peroxide for 15 min to remove endogenous peroxidase activity. Then, the brain sections were incubated overnight at room temperature with a rabbit anti-p-CaMK II antibody (1 : 1000 dilution) or mouse anti-NeuN antibody (1 : 1000 dilutions). They were then incubated with a biotinylated anti-rabbit or mouse IgG for 90 min, followed by incubation in an avidin-biotin complex solution for 1 h at room temperature. The peroxidase activity was visualized with 3,3-diaminobenzidine for 3 min. After every incubation step, the cells or tissues were washed three times with PBS. Finally, the free-floating brain tissues were mounted on gelatin-coated glass slides, dehydrated, cleared with xylene, coverslipped using histomount medium, and photographed with a research microscope (BX51 T-32F01; Olympus Corporation). The number of NeuN-positive cells in the CA3 of the hippocampus was estimated by measuring at 200× magnification using AnalySIS LS Research (Soft Imaging System Ltd.). The optical density of p-CaMK II immunoreactivity in the CA3 of the hippocampus at 200× was analyzed with ImageJ software. Data are presented as percentages of the sham value.

Fluoro-jade B staining

The brain sections were mounted onto gelatin-coated slides, dried on a slide warmer, and immersed in an alcohol series consisting of absolute alcohol for 3 min, 70 % ethanol for 1 min, 50 % ethanol for 1 min, 30 % ethanol for 1 min, and distilled water for 1 min. They were then transferred to 0.1 % potassium permanganate for 15 min and gently agitated on a rotating platform. After rinsing in distilled water for 2 min, they were incubated for 30 min in a 0.0015 % solution of fluoro-jade B that had been dissolved in 0.09 % acetic acid in the dark, rinsed with distilled water three times, dried overnight, dehydrated in xylene for 15 min, and covered with a coverslip after the addition of DPX media. The images in the CA1 and CA3 of the hippocampus were examined using an epifluorescent microscope equipped with a filter cube designed for visualizing FITC. Data are presented as percentages of the KA value.

Statistical analysis

All statistical parameters were calculated using Graphpad Prism 4.0 software. Values are expressed as the mean ± S. E. M. Results were analyzed by one-way ANOVA analysis followed by the Tukeyʼs post hoc test. Differences with a p value less than 0.05 were considered statistically significant.

Supporting information

The specifications of standardized HCW are available as Supporting Information.

Acknowledgements

This research was supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048795) of the Ministry of Science, ICT, and Future Planning through the National Research Foundation.

Conflict of Interest

There are no conflicts of interest to declare.

• References

• 1 Bozzi Y, Dunleavy M, Henshall DC. Cell signaling underlying epileptic behavior. Front Behav Neurosci 2011; 5: 45
  CrossrefPubMedSearch in Google Scholar
• 2 Strine TW, Kobau R, Chapman DP, Thurman DJ, Price P, Balluz LS. Psychological distress, comorbidities, and health behaviors among US adults with seizures: results from the 2002 National Health Interview Survey. Epilepsia 2005; 46: 1133-1139
  CrossrefPubMedSearch in Google Scholar
• 3 Henry TR, Babb TL, Engel jr. J, Mazziotta JC, Phelps ME, Crandall PH. Hippocampal neuronal loss and regional hypometabolism in temporal lobe epilepsy. Ann Neurol 1994; 36: 925-927
  CrossrefPubMedSearch in Google Scholar
• 4 Mayer ML. Glutamate receptor ion channels. Curr Opin Neurobiol 2005; 15: 282-288
  CrossrefPubMedSearch in Google Scholar
• 5 Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004; 5: 553-564
  CrossrefPubMedSearch in Google Scholar
• 6 Loring DW, Meador KJ. Cognitive side effects of antiepileptic drugs in children. Neurology 2004; 62: 872-877
  CrossrefPubMedSearch in Google Scholar
• 7 Toda S. Antioxidative effects of polyphenols in leaves of Houttuynia cordata on protein fragmentation by copper-hydrogen peroxide in vitro . J Med Food 2005; 8: 266-268
  CrossrefPubMedSearch in Google Scholar
• 8 Lu HM, Liang YZ, Yi LZ, Wu XJ. Anti-inflammatory effect of Houttuynia cordata injection. J Ethnopharmacol 2006; 104: 245-249
  CrossrefPubMedSearch in Google Scholar
• 9 Cai DS, Zhou H, Liu WW, Pei L. Protective effects of bone marrow-derived endothelial progenitor cells and Houttuynia cordata in lipopolysaccharide-induced acute lung injury in rats. Cell Physiol Biochem 2013; 32: 1577-1586
  PubMedSearch in Google Scholar
• 10 Chang JS, Chiang LC, Chen CC, Liu LT, Wang KC, Lin CC. Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. Am J Chin Med 2001; 29: 303-312
  CrossrefPubMedSearch in Google Scholar
• 11 Chen YY, Liu JF, Chen CM, Chao PY, Chang TJ. A study of the antioxidative and antimutagenic effects of Houttuynia cordata Thunb. using an oxidized frying oil-fed model. J Nutr Sci Vitaminol (Tokyo) 2003; 49: 327-333
  CrossrefPubMedSearch in Google Scholar
• 12 Ng LT, Yen FL, Liao CW, Lin CC. Protective effect of Houttuynia cordata extract on bleomycin-induced pulmonary fibrosis in rats. Am J Chin Med 2007; 35: 465-475
  CrossrefPubMedSearch in Google Scholar
• 13 Tang YJ, Yang JS, Lin CF, Shyu WC, Tsuzuki M, Lu CC, Chen YF, Lai KC. Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells. Oncol Rep 2009; 22: 1051-1056
  PubMedSearch in Google Scholar
• 14 Miyata M, Koyama T, Yazawa K. Water extract of Houttuynia cordata Thunb. leaves exerts anti-obesity effects by inhibiting fatty acid and glycerol absorption. J Nutr Sci Vitaminol (Tokyo) 2010; 56: 150-156
  CrossrefPubMedSearch in Google Scholar
• 15 Ch MI, Wen YF, Cheng Y. Gas chromatographic/mass spectrometric analysis of the essential oil of Houttuynia cordata Thunb by using on-column methylation with tetramethylammonium acetate. J AOAC Int 2007; 90: 60-67
  PubMedSearch in Google Scholar
• 16 Meng J, Leung KS, Dong XP, Zhou YS, Jiang ZH, Zhao ZZ. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromatography-diode array detector-electrospray mass spectrometry. Fitoterapia 2009; 80: 468-474
  CrossrefPubMedSearch in Google Scholar
• 17 Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflammatory cell function. Inflamm Allergy Drug Targets 2010; 9: 263-285
  CrossrefPubMedSearch in Google Scholar
• 18 Park H, Oh M. Houttuyniae Herba protects rat primary cortical cells from Aβ(25–35)-induced neurotoxicity via regulation of calcium influx and mitochondria-mediated apoptosis. Hum Exp Toxicol 2012; 3: 698-709
  PubMedSearch in Google Scholar
• 19 Huh E, Kim HG, Park H, Kang MS, Lee B, Oh MS. Houttuynia cordata improves cognitive deficits in cholinergic dysfunction Alzheimerʼs disease-like models. Biomol Ther (Seoul) 2014; 22: 176-183
  CrossrefPubMedSearch in Google Scholar
• 20 Lin TY, Huang WJ, Wu CC, Lu CW, Wang S. Acacetin inhibits glutamate release and prevents kainic acid-induced neurotoxicity in rats. PLoS One 2014; 9: e88644
  CrossrefPubMedSearch in Google Scholar
• 21 Cheng Y, Sun AY. Oxidative mechanisms involved in kainate-induced cytotoxicity in cortical neurons. Neurochem Res 1994; 19: 1557-1564
  CrossrefPubMedSearch in Google Scholar
• 22 Candelario-Jalil E, Al-Dalain SM, Castillo R, Martínez G, Fernández OS. Selective vulnerability to kainate‐induced oxidative damage in different rat brain regions. J Appl Toxicol 2001; 21: 403-407
  CrossrefPubMedSearch in Google Scholar
• 23 Luo L, Jin Y, Kim ID, Lee JK. Glycyrrhizin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus. Exp Neurobiol 2013; 22: 107-115
  CrossrefPubMedSearch in Google Scholar
• 24 Stasiukyniene V, Pilvinis V, Reingardiene D, Janauskaite L. Epileptic seizures in critically ill patients. Medicina (Kaunas) 2009; 45: 501-507
  PubMedSearch in Google Scholar
• 25 Sperk G, Lassmann H, Baran H, Seitelberger F, Hornykiewicz O. Kainic acid-induced seizures: dose-relationship of behavioural, neurochemical and histopathological changes. Brain Res 1985; 338: 289-295
  CrossrefPubMedSearch in Google Scholar
• 26 Baños JH, LaGory J, Sawrie S, Faught E, Knowlton R, Prasad A, Kuzniecky R, Martin RC. Self-report of cognitive abilities in temporal lobe epilepsy: cognitive, psychosocial, and emotional factors. Epilepsy Behav 2004; 5: 575-579
  CrossrefPubMedSearch in Google Scholar
• 27 Milgram NW, Isen DA, Mandel D, Palantzas H, Pepkowski MJ. Deficits in spontaneous behavior and cognitive function following systemic administration of kainic acid. Neurotoxicology 1988; 9: 611-624
  PubMedSearch in Google Scholar
• 28 Henshall D. Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy. Biochem Soc Trans 2007; 35: 421-423
  CrossrefPubMedSearch in Google Scholar
• 29 Ullah I, Park HY, Kim MO. Anthocyanins protect against kainic acid-induced excitotoxicity and apoptosis via ROS-activated AMPK pathway in hippocampal neurons. CNS Neurosci Ther 2014; 20: 327-338
  CrossrefPubMedSearch in Google Scholar
• 30 Crepel V, Represa A, Beaudoin M, Ben-Ari Y. Hippocampal damage induced by ischemia and intra-amygdaloid kainate injection: effect on N-methyl-D-aspartate, N-(1-[2-thienyl]cyclohexyl)piperidine and glycine binding sites. Neuroscience 1989; 31: 605-612
  CrossrefPubMedSearch in Google Scholar
• 31 Anderson ME. Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. Pharmacol Ther 2005; 106: 39-55
  CrossrefPubMedSearch in Google Scholar
• 32 Suh HW, Lee HK, Seo YJ, Kwon MS, Shim EJ, Lee JY, Choi SS, Lee JH. Kainic acid (KA)-induced Ca2+/calmodulin-dependent protein kinase II (CaMK II) expression in the neurons, astrocytes and microglia of the mouse hippocampal CA3 region, and the phosphorylated CaMK II only in the hippocampal neurons. Neurosci Lett 2005; 381: 223-227
  CrossrefPubMedSearch in Google Scholar
• 33 Salas MA, Valverde CA, Sánchez G, Said M, Rodriguez JS, Portiansky EL, Kaetzel MA, Dedman JR, Donoso P, Kranias EG, Mattiazzi A. The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury. J Mol Cell Cardiol 2010; 48: 1298-1306
  CrossrefPubMedSearch in Google Scholar
• 34 Kwan P, Brodie MJ. Phenobarbital for the treatment of epilepsy in the 21st century: a critical review. Epilepsia 2004; 45: 1141-1149
  CrossrefPubMedSearch in Google Scholar
• 35 Jussofie A, Schmiz A, Hiemke C. Kavapyrone enriched extract from Piper methysticum as modulator of the GABA binding site in different regions of rat brain. Psychopharmacology (Berl) 1994; 116: 469-474
  CrossrefPubMedSearch in Google Scholar
• 36 Morales M, Lozoya X. Calcium-antagonist effects of quercetin on aortic smooth muscle. Planta Med 1994; 60: 313-317
  Thieme ConnectPubMedSearch in Google Scholar
• 37 Nishino H, Naitoh E, Iwashima A, Umezawa K. Quercetin interacts with calmodulin, a calcium regulatory protein. Experientia 1984; 40: 184-185
  CrossrefPubMedSearch in Google Scholar
• 38 Jang SY, Bae JS, Lee YH, Oh KY, Park KH, Bae YS. Caffeic acid and quercitrin purified from Houttuynia cordata inhibit DNA topoisomerase I activity. Nat Prod Res 2011; 25: 222-231
  CrossrefPubMedSearch in Google Scholar
• 39 Song J, Parker L, Hormozi L, Tanouye MA. DNA topoisomerase I inhibitors ameliorate seizure-like behaviors and paralysis in a Drosophila model of epilepsy. Neuroscience 2008; 156: 722-728
  CrossrefPubMedSearch in Google Scholar
• 40 Zhang L, Zhang WP, Chen KD, Qian XD, Fang SH, Wei EQ. Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury. Life Sci 2007; 80: 530-537
  CrossrefPubMedSearch in Google Scholar
• 41 Huang SM, Chuang HC, Wu CH, Yen GC. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Mol Nutr Food Res 2008; 52: 940-949
  CrossrefPubMedSearch in Google Scholar
• 42 Ansari MA, Abdul HM, Joshi G, Opii WO, Butterfield DA. Protective effect of quercetin in primary neurons against Aβ (1–42): relevance to Alzheimerʼs disease. J Nutr Biochem 2009; 20: 269-275
  CrossrefPubMedSearch in Google Scholar
• 43 dos Santos Sales ÍM, do Nascimento KG, Feitosa CM, Saldanha GB, Feng D, de Freitas RM. Caffeic acid effects on oxidative stress in rat hippocampus after pilocarpine-induced seizures. Neurol Sci 2011; 32: 375-380
  CrossrefPubMedSearch in Google Scholar
• 44 Joseph JA, Shukitt-Hale B, Brewer GJ, Weikel KA, Kalt W, Fisher DR. Differential protection among fractionated blueberry polyphenolic families against DA-, Abeta(42)- and LPS-induced decrements in Ca(2+) buffering in primary hippocampal cells. J Agric Food Chem 2010; 28: 8196-8204
  PubMedSearch in Google Scholar
• 45 Kumar A, Prakash A, Pahwa D. Galantamine potentiates the protective effect of rofecoxib and caffeic acid against intrahippocampal Kainic acid-induced cognitive dysfunction in rat. Brain Res Bull 2011; 30: 158-168
  PubMedSearch in Google Scholar
• 46 Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, Raza SS, Javed H, Vaibhav K, Khan A, Islam F. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009; 1292: 123-135
  CrossrefPubMedSearch in Google Scholar
• 47 Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd. edition. New York: Academic Press; 2001
  Search in Google Scholar

Correspondence

• References

• 1 Bozzi Y, Dunleavy M, Henshall DC. Cell signaling underlying epileptic behavior. Front Behav Neurosci 2011; 5: 45
  CrossrefPubMedSearch in Google Scholar
• 2 Strine TW, Kobau R, Chapman DP, Thurman DJ, Price P, Balluz LS. Psychological distress, comorbidities, and health behaviors among US adults with seizures: results from the 2002 National Health Interview Survey. Epilepsia 2005; 46: 1133-1139
  CrossrefPubMedSearch in Google Scholar
• 3 Henry TR, Babb TL, Engel jr. J, Mazziotta JC, Phelps ME, Crandall PH. Hippocampal neuronal loss and regional hypometabolism in temporal lobe epilepsy. Ann Neurol 1994; 36: 925-927
  CrossrefPubMedSearch in Google Scholar
• 4 Mayer ML. Glutamate receptor ion channels. Curr Opin Neurobiol 2005; 15: 282-288
  CrossrefPubMedSearch in Google Scholar
• 5 Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 2004; 5: 553-564
  CrossrefPubMedSearch in Google Scholar
• 6 Loring DW, Meador KJ. Cognitive side effects of antiepileptic drugs in children. Neurology 2004; 62: 872-877
  CrossrefPubMedSearch in Google Scholar
• 7 Toda S. Antioxidative effects of polyphenols in leaves of Houttuynia cordata on protein fragmentation by copper-hydrogen peroxide in vitro . J Med Food 2005; 8: 266-268
  CrossrefPubMedSearch in Google Scholar
• 8 Lu HM, Liang YZ, Yi LZ, Wu XJ. Anti-inflammatory effect of Houttuynia cordata injection. J Ethnopharmacol 2006; 104: 245-249
  CrossrefPubMedSearch in Google Scholar
• 9 Cai DS, Zhou H, Liu WW, Pei L. Protective effects of bone marrow-derived endothelial progenitor cells and Houttuynia cordata in lipopolysaccharide-induced acute lung injury in rats. Cell Physiol Biochem 2013; 32: 1577-1586
  PubMedSearch in Google Scholar
• 10 Chang JS, Chiang LC, Chen CC, Liu LT, Wang KC, Lin CC. Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. Am J Chin Med 2001; 29: 303-312
  CrossrefPubMedSearch in Google Scholar
• 11 Chen YY, Liu JF, Chen CM, Chao PY, Chang TJ. A study of the antioxidative and antimutagenic effects of Houttuynia cordata Thunb. using an oxidized frying oil-fed model. J Nutr Sci Vitaminol (Tokyo) 2003; 49: 327-333
  CrossrefPubMedSearch in Google Scholar
• 12 Ng LT, Yen FL, Liao CW, Lin CC. Protective effect of Houttuynia cordata extract on bleomycin-induced pulmonary fibrosis in rats. Am J Chin Med 2007; 35: 465-475
  CrossrefPubMedSearch in Google Scholar
• 13 Tang YJ, Yang JS, Lin CF, Shyu WC, Tsuzuki M, Lu CC, Chen YF, Lai KC. Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells. Oncol Rep 2009; 22: 1051-1056
  PubMedSearch in Google Scholar
• 14 Miyata M, Koyama T, Yazawa K. Water extract of Houttuynia cordata Thunb. leaves exerts anti-obesity effects by inhibiting fatty acid and glycerol absorption. J Nutr Sci Vitaminol (Tokyo) 2010; 56: 150-156
  CrossrefPubMedSearch in Google Scholar
• 15 Ch MI, Wen YF, Cheng Y. Gas chromatographic/mass spectrometric analysis of the essential oil of Houttuynia cordata Thunb by using on-column methylation with tetramethylammonium acetate. J AOAC Int 2007; 90: 60-67
  PubMedSearch in Google Scholar
• 16 Meng J, Leung KS, Dong XP, Zhou YS, Jiang ZH, Zhao ZZ. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromatography-diode array detector-electrospray mass spectrometry. Fitoterapia 2009; 80: 468-474
  CrossrefPubMedSearch in Google Scholar
• 17 Chirumbolo S. The role of quercetin, flavonols and flavones in modulating inflammatory cell function. Inflamm Allergy Drug Targets 2010; 9: 263-285
  CrossrefPubMedSearch in Google Scholar
• 18 Park H, Oh M. Houttuyniae Herba protects rat primary cortical cells from Aβ(25–35)-induced neurotoxicity via regulation of calcium influx and mitochondria-mediated apoptosis. Hum Exp Toxicol 2012; 3: 698-709
  PubMedSearch in Google Scholar
• 19 Huh E, Kim HG, Park H, Kang MS, Lee B, Oh MS. Houttuynia cordata improves cognitive deficits in cholinergic dysfunction Alzheimerʼs disease-like models. Biomol Ther (Seoul) 2014; 22: 176-183
  CrossrefPubMedSearch in Google Scholar
• 20 Lin TY, Huang WJ, Wu CC, Lu CW, Wang S. Acacetin inhibits glutamate release and prevents kainic acid-induced neurotoxicity in rats. PLoS One 2014; 9: e88644
  CrossrefPubMedSearch in Google Scholar
• 21 Cheng Y, Sun AY. Oxidative mechanisms involved in kainate-induced cytotoxicity in cortical neurons. Neurochem Res 1994; 19: 1557-1564
  CrossrefPubMedSearch in Google Scholar
• 22 Candelario-Jalil E, Al-Dalain SM, Castillo R, Martínez G, Fernández OS. Selective vulnerability to kainate‐induced oxidative damage in different rat brain regions. J Appl Toxicol 2001; 21: 403-407
  CrossrefPubMedSearch in Google Scholar
• 23 Luo L, Jin Y, Kim ID, Lee JK. Glycyrrhizin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus. Exp Neurobiol 2013; 22: 107-115
  CrossrefPubMedSearch in Google Scholar
• 24 Stasiukyniene V, Pilvinis V, Reingardiene D, Janauskaite L. Epileptic seizures in critically ill patients. Medicina (Kaunas) 2009; 45: 501-507
  PubMedSearch in Google Scholar
• 25 Sperk G, Lassmann H, Baran H, Seitelberger F, Hornykiewicz O. Kainic acid-induced seizures: dose-relationship of behavioural, neurochemical and histopathological changes. Brain Res 1985; 338: 289-295
  CrossrefPubMedSearch in Google Scholar
• 26 Baños JH, LaGory J, Sawrie S, Faught E, Knowlton R, Prasad A, Kuzniecky R, Martin RC. Self-report of cognitive abilities in temporal lobe epilepsy: cognitive, psychosocial, and emotional factors. Epilepsy Behav 2004; 5: 575-579
  CrossrefPubMedSearch in Google Scholar
• 27 Milgram NW, Isen DA, Mandel D, Palantzas H, Pepkowski MJ. Deficits in spontaneous behavior and cognitive function following systemic administration of kainic acid. Neurotoxicology 1988; 9: 611-624
  PubMedSearch in Google Scholar
• 28 Henshall D. Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy. Biochem Soc Trans 2007; 35: 421-423
  CrossrefPubMedSearch in Google Scholar
• 29 Ullah I, Park HY, Kim MO. Anthocyanins protect against kainic acid-induced excitotoxicity and apoptosis via ROS-activated AMPK pathway in hippocampal neurons. CNS Neurosci Ther 2014; 20: 327-338
  CrossrefPubMedSearch in Google Scholar
• 30 Crepel V, Represa A, Beaudoin M, Ben-Ari Y. Hippocampal damage induced by ischemia and intra-amygdaloid kainate injection: effect on N-methyl-D-aspartate, N-(1-[2-thienyl]cyclohexyl)piperidine and glycine binding sites. Neuroscience 1989; 31: 605-612
  CrossrefPubMedSearch in Google Scholar
• 31 Anderson ME. Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. Pharmacol Ther 2005; 106: 39-55
  CrossrefPubMedSearch in Google Scholar
• 32 Suh HW, Lee HK, Seo YJ, Kwon MS, Shim EJ, Lee JY, Choi SS, Lee JH. Kainic acid (KA)-induced Ca2+/calmodulin-dependent protein kinase II (CaMK II) expression in the neurons, astrocytes and microglia of the mouse hippocampal CA3 region, and the phosphorylated CaMK II only in the hippocampal neurons. Neurosci Lett 2005; 381: 223-227
  CrossrefPubMedSearch in Google Scholar
• 33 Salas MA, Valverde CA, Sánchez G, Said M, Rodriguez JS, Portiansky EL, Kaetzel MA, Dedman JR, Donoso P, Kranias EG, Mattiazzi A. The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury. J Mol Cell Cardiol 2010; 48: 1298-1306
  CrossrefPubMedSearch in Google Scholar
• 34 Kwan P, Brodie MJ. Phenobarbital for the treatment of epilepsy in the 21st century: a critical review. Epilepsia 2004; 45: 1141-1149
  CrossrefPubMedSearch in Google Scholar
• 35 Jussofie A, Schmiz A, Hiemke C. Kavapyrone enriched extract from Piper methysticum as modulator of the GABA binding site in different regions of rat brain. Psychopharmacology (Berl) 1994; 116: 469-474
  CrossrefPubMedSearch in Google Scholar
• 36 Morales M, Lozoya X. Calcium-antagonist effects of quercetin on aortic smooth muscle. Planta Med 1994; 60: 313-317
  Thieme ConnectPubMedSearch in Google Scholar
• 37 Nishino H, Naitoh E, Iwashima A, Umezawa K. Quercetin interacts with calmodulin, a calcium regulatory protein. Experientia 1984; 40: 184-185
  CrossrefPubMedSearch in Google Scholar
• 38 Jang SY, Bae JS, Lee YH, Oh KY, Park KH, Bae YS. Caffeic acid and quercitrin purified from Houttuynia cordata inhibit DNA topoisomerase I activity. Nat Prod Res 2011; 25: 222-231
  CrossrefPubMedSearch in Google Scholar
• 39 Song J, Parker L, Hormozi L, Tanouye MA. DNA topoisomerase I inhibitors ameliorate seizure-like behaviors and paralysis in a Drosophila model of epilepsy. Neuroscience 2008; 156: 722-728
  CrossrefPubMedSearch in Google Scholar
• 40 Zhang L, Zhang WP, Chen KD, Qian XD, Fang SH, Wei EQ. Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury. Life Sci 2007; 80: 530-537
  CrossrefPubMedSearch in Google Scholar
• 41 Huang SM, Chuang HC, Wu CH, Yen GC. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Mol Nutr Food Res 2008; 52: 940-949
  CrossrefPubMedSearch in Google Scholar
• 42 Ansari MA, Abdul HM, Joshi G, Opii WO, Butterfield DA. Protective effect of quercetin in primary neurons against Aβ (1–42): relevance to Alzheimerʼs disease. J Nutr Biochem 2009; 20: 269-275
  CrossrefPubMedSearch in Google Scholar
• 43 dos Santos Sales ÍM, do Nascimento KG, Feitosa CM, Saldanha GB, Feng D, de Freitas RM. Caffeic acid effects on oxidative stress in rat hippocampus after pilocarpine-induced seizures. Neurol Sci 2011; 32: 375-380
  CrossrefPubMedSearch in Google Scholar
• 44 Joseph JA, Shukitt-Hale B, Brewer GJ, Weikel KA, Kalt W, Fisher DR. Differential protection among fractionated blueberry polyphenolic families against DA-, Abeta(42)- and LPS-induced decrements in Ca(2+) buffering in primary hippocampal cells. J Agric Food Chem 2010; 28: 8196-8204
  PubMedSearch in Google Scholar
• 45 Kumar A, Prakash A, Pahwa D. Galantamine potentiates the protective effect of rofecoxib and caffeic acid against intrahippocampal Kainic acid-induced cognitive dysfunction in rat. Brain Res Bull 2011; 30: 158-168
  PubMedSearch in Google Scholar
• 46 Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, Raza SS, Javed H, Vaibhav K, Khan A, Islam F. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009; 1292: 123-135
  CrossrefPubMedSearch in Google Scholar
• 47 Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd. edition. New York: Academic Press; 2001
  Search in Google Scholar

• Supplementary Material