PTZ, MMP-9 and myricetin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). PTZ was dissolved in freshly prepared saline prior to treatment (100 mg/ml). Antibodies against cleaved caspase-3 (cat. no. 9661; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), BDNF (cat. no. PA5-15198: 1:2,000; Invitrogen; Thermo Fishes Scientific, Inc., Waltham, MA, USA), TrkB (cat. no. 4606; 1:1,000; Cell Signaling Technology, Inc.), GABA_AR (cat. no. PA5-78386; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.), MMP-9 (cat. no. 3852; 1:1,000; Cell Signaling Technology, Inc.) and GAD65 (cat. no. PA5-22260; 1:2,000; Invitrogen; Thermo Fisher Scientific, Inc.) were obtained and Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) provided antibodies against apoptosis regulator B-cell lymphoma-2 (Bcl-2; cat. no. sc509; 1:1,000), apoptosis regulator Bcl-2-like protein 4 (Bax; cat. no. sc-7480; 1:1,000), Bcl2-associated agonist of cell death (Bad; cat. no. sc-8004; 1:1,000), anti-apoptotic regulator Bcl extra large (Bcl-xL; cat. no. sc-136132; 1:1,000) and β-actin (cat. no. sc-47778; 1:1,000). All other standard chemicals used in the current study were purchased from Sigma-Aldrich; Merck KGaA.

C57BL/6 male mice (n=9; weight, 20±2 g; age, 4–6 weeks) were obtained from the animal house of Hedong People's Hospital (Linyi, China) and housed under standard laboratory conditions (temperature, 23±1°C; humidity 50–60%; 12-h light/dark cycle). Mice had access to standard pellet diet and water ad libitum, and were allowed to acclimate to the laboratory conditions for 4–5 days prior to the experiments. The current study was approved by the Animal Studies Ethics Committee of Hedong People's Hospital and the protocols and handling of animals were in compliance with National Institutes of Health guidelines for the care and use of laboratory animals and the National Animal Welfare Law of China (34–36).

Kindling is an experimental technique used to study epileptogenesis and epilepsy-associated cognitive and behavioral alterations, and to assess the efficacy of novel antiepileptic drugs (37,38). In the current study, the animals were given a total of 13 intraperitoneal (i.p.) injections of sub-effective dose of PTZ (35 mg/kg) on alternate days (9). The animals were observed for 30 min following each PTZ injection. Separate treatment groups (n=18/group) received myricetin at 100 or 200 mg/kg body weight, orally for 26 days, 30 min prior to each PTZ injection; the concentrations used in the study were based on the results of previous experiments using various concentrations of myricetin (data not shown). Control animals were treated with an equal volume of saline instead of administration of PTZ or myricetin. Epileptic control mice were treated with PTZ, but not treated with myricetin. Standard drug control mice were treated with PTZ and received valproate (100 mg/kg; i.p.) (6). The mice were anesthetized with isoflurane and perfused transcardially with 0.9% saline (100 ml), followed by 4% paraformaldehyde (200 ml) and sacrificed 24 h after the last PTZ challenge. The brain tissues were immediately collected and analyzed.

The mice were monitored for 30 min after each PTZ injection. Seizures were classified and scored according to the modified Racine scale (39): 0, no response; phase 1, hyperactivity and vibrissae twitching; phase 2, head nodding, head clonus and myoclonic jerk; phase 3, unilateral forelimb clonus; phase 4, rearing with bilateral forelimb clonus; phase 5, generalized tonic-clonic seizure with loss of postural control; and phase 6, absence of movement and loss of function of vital organs/mortality. Mortality rate was determined using the following formula: Mortality count/total number of animals in the particular experimental group ×100.

Nissl staining was performed to assess the effect of myricetin on the alterations in brain architecture following PTZ challenge. Coronal sections (7-µm-thick) were subjected to Nissl staining with toluidine blue (1%), as previously described (40). Viable cells in the CA1 and CA3 regions of the hippocampus per 1-mm length of bilateral hemisphere were counted under high magnification (×400) using a light microscope (Nikon Corporation, Tokyo, Japan).

Neuroapoptosis was assessed using the TUNEL assay, as previously described by Li et al (41). Brain tissue sections (5-µm-thick) were sliced in the same plane of the hippocampal region of each mouse and subjected to analysis using the DeadEnd™ fluorometric TUNEL system (Promega Corporation, Madison, WI, USA). Tissue sections were protected from direct light during the assay. TUNEL-positive cells in the CA1 and CA3 regions of the hippocampus were observed and analyzed using NIS-Elements Basic Research microscope imaging software (version, 4.0; Nikon Corporation).

The mice were sacrificed as described above 50 min after the last PTZ injection. Brain tissues were isolated immediately, weighed and homogenized in 0.1 M PBS, and GABA and glutamate contents in the tissues were analyzed by high performance liquid chromatography with reference standards as described previously (42).

An RT-PCR analysis was performed to determine the effects of myricetin on MMP-9, BDNF and TrkB mRNA expression. Total RNA was extracted from the hippocampal tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). First-strand cDNA was synthesized using the Revert Aid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc., Waltham, MA, USA). PCR reactions were performed using the 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following primers were used: Forward, 5′-CGAAGAGCTGCTGGATGAG-3′, and reverse, 5′-ATGGGATTACACTTGGTCTCG-3′, for BDNF; forward, 5′-CCTCCACGGATGTTGCTGA-3′, and reverse, 5′-GGCTGTTGGTGATACCGAAGTA-3′ for TrkB; forward, 5′-GTCTTCCCCTTCGTCTTCCT-3′, and reverse, 5′-GCTGGATGCCTTTTATGTCG-3′ for MMP-9; forward, 5′-CCGTATCGGACGCCTGGTTA-3′, and reverse, 5′-GGCTGTTGGTGATACCGAAGTA-3′, for GAPDH. GAPDH was used as the internal control. PCR products were separated by 1% agarose gel electrophoresis and visualized using ethidium bromide (0.05%) staining. The band intensities of the products were analyzed with the Bio-Gel imagery apparatus with Quantity One^® 1-D analysis software (4.3.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Gel zymography was performed to evaluate MMP-9 activity. The samples were prepared as previously described by Mizoguchi et al (43). The samples were subjected to SDS-PAGE using 10% gels containing 0.1% gelatin under non-reducing conditions. Triton X-100 gels (2.5%) were washed to remove the SDS, and further washed in incubation buffer (50 mM Tris-HCl, pH 7.4; 5 mM CaCl₂; 200 mM NaCl and 2 µM ZnCl₂) at 25±2°C for 30 min and subsequently incubated for 24 h at 37°C. Following the incubation, the gels were stained with Coomassie Brilliant Blue solution (1% Coomassie Brilliant Blue G-250; 30% methanol and 10% acetic acid) for 3 h at room temperature and destined using 7% acetic acid and 40% methanol until clear bands representing gelatinolysis were observed against a dark background. Total MMP-9 activity was determined using the ATTO Densitograph Software Library Lane Analyser (ATTO Corporation, Tokyo, Japan). Purified MMP-9 (Sigma-Aldrich; Merck KGaA) was used as the internal standard.

Western blotting was performed to detect the protein expression levels. The hippocampal tissues were homogenized in lysis buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA, 150 mM NaCl and 0.5% Nonidet P-40) containing protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin and 1 mg/ml pepstatin A) and incubated for 30 min on ice. Total protein contents of the tissues were determined using the bicinchoninic acid assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of protein (60 µg/lane) from each group were separated by SDS-PAGE (10% gel), and the bands were transferred onto polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The membranes were blocked in 3% bovine serum albumin in TBS containing Tween-20 (0.1%; TBST) at room temperature for 40 min and incubated with specific primary antibodies, as described above, at 4°C overnight. The membranes were incubated for 2 h at room temperature with goat anti-mouse horseradish peroxidase-labelled secondary antibody (IgG; cat. no. ab97240; 1:2,000; Abcam, Cambridge, MA, USA) prior to washing with TBST. The enhanced chemiluminescence method (GE Healthcare, Chicago, IL, USA) was used to detect and analyze the immunoreactive bands.

Data are presented as the mean ± standard deviation, representative of six replicates. SPSS software (version 22.0; IBM Corp., Armonk, NY, USA) was used for the statistical analysis. One-way analysis of variance followed by Duncan's Multiple Range Test were used for multiple group comparisons. P<0.05 was considered to indicate a statistically significant difference.

Repeated administration of sub-convulsive dose of PTZ (35 mg/kg) on alternating days (13 injections) resulted in an increase in the seizure score compared with the control group, eventually leading to widespread clonic-tonic seizures (Racine scale 5). Administration of myricetin (100 or 200 mg) prior to each PTZ injection decreased seizure severity, as evidenced by a decline in the seizure score compared with the PTZ control group (Fig. 1). Furthermore, the mortality rate following the PTZ injections decreased significantly (P<0.05) in response to pre-treatment with myricetin and the group treated with 200 mg myricetin exhibited 5% mortality rate (Fig. 2).

Mice were sacrificed 24 h after the last PTZ injection, and Nissl staining was used to determine seizure-induced neuronal loss in the CA1 and CA3 regions of the hippocampus. The viable cell count decreased significantly in the PTZ group compared with the normal control animals (P<0.05). Treatment with myricetin at both tested doses significantly attenuated seizure-induced neuronal loss (P<0.05) and the 200 mg dose exerted a higher protective effect compared with the 100 mg dose of myricetin (Fig. 3). Furthermore, negligible neuronal loss was observed among mice treated with valproate.

TUNEL-positive cell counts in the CA1 and CA3 hippocampal regions were determined following the PTZ injections. Pre-treatment with myricetin reduced the TUNEL-positive cell counts (P<0.05) compared with the PTZ group, demonstrating inhibition of neuronal apoptosis (Fig. 4). Apoptotic protein expression was measured to determine the molecular mechanism underlying the antiapoptotic effects of myricetin. Increased Bad and Bax expression levels were observed in the PTZ control mice, compared with the untreated control groups (Fig. 5). PTZ group mice pre-treated with myricetin (100 or 200 mg) exhibited decreased Bad, Bax and cleaved caspase-3 expression levels. The expression levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, increased in mice with epilepsy following pre-treatment with myricetin, indicating inhibited apoptosis. These observations suggest that myricetin regulates the expression of proteins associated with the apoptotic pathway and reduces neuronal cell apoptosis to induce neuroprotective effects.

The BDNF-TrkB signaling is pathway associated with epileptogenesis (19,44). BDNF potentiates glutamatergic neurotransmission and inhibits GABAergic transmission (45). In the current study, elevated BDNF and TrkB mRNA and protein expression levels were observed following the PTZ injections compared with the untreated control group, indicating sensitivity of the neurons and induction of epilepsy (Figs. 6 and 7). Pre-treatment with myricetin prior to the PTZ injections downregulated TrkB and BDNF protein expression in a dose-dependent manner and the 200 mg dose inhibited the BDNF-TrkB signaling pathway. This myricetin-mediated inhibition of the BDNF-TrkB signaling may contribute to the decreased seizure scores.

GAD65 and GABA_A expression levels decreased in the PTZ group compared with the untreated control group (Fig. 7). Pre-treatment with myricetin upregulated GAD65 and GABA_AR protein expression levels, suggesting that myricetin may exhibit an anti-epileptic potential.

A glutamate/GABA imbalance in the brain is associated with epileptogenesis (27). Myricetin (100 and 200 mg) significantly increased GABA levels while significantly decreasing glutamate levels compared with the PTZ group (both P<0.05), and normalized the glutamate/GABA ratio (Fig. 8).

MMP-2 and −9 serve roles in epilepsy (31,46). The effect of myricetin on the expression and activity of MMP-9 was assessed in the current study. The current study observed an increase in MMP-9 mRNA (Fig. 6) and protein (Fig. 7) levels at 24 h after the final PTZ injection, compared with the untreated control group. mRNA levels were unchanged following treatment with myricetin at both doses. The results of the gel zymography analysis revealed and increase in MMP-9 activity following PTZ kindling. MMP 9 activity was downregulated in a dose-dependent manner following treatment with myricetin (Fig. 9). These observations suggest that myricetin may regulate MMP-9 activity.