2.1. Preparation of GEAT decoction
GEAT decoction in our study was composed of G. elata (“Tianma” in Chinese) and A.tatarinowii (“Shichangpu” in Chinese). Herbs were purchased from Beijing Tongrentang pharmaceutical chain Co., Ltd. Briefly, G. elata (30 g) and A.tatarinowii (15 g) were soaked in 500 mL of distilled water under normal temperature for 60 min before being boiled for 0.5 h. Filter and collect the filter liquor, and then add 250 mL of distilled water to the residue and continue to boil for 25 min. Afterwards, combined the filter liquor and then concentrated using a rotary evaporator (model: Heidolph Hei-VAP). The concentrated solution was transferred to a glass bottle and then reserved at 4 °C in an ice box.
2.2. Animals
SPF adult male Kunming mice (Scxk (Guangdong) 2020-0051) weighing between 24 and 28 g were obtained from the BesTest Bio-Tech Co., Ltd. They were housed in the regulated environmental (23±2 ℃; 50±10% humidity, 12 h light/dark cycle) with free access to pellet food and water. All experiments complied following the guidance of the management regulations of Guangdong Medical Laboratory Animal Center (Guangdong, China), and carried out in accordance with the NIH guidelines. All experimental protocols were approved by the Animal Care Committee of Zunyi Medical University (Zhuhai, China) (ZYLS-[2020] No. 2-081).
2.3. Drugs and reagents
Pentylenetetrazol (PTZ) was purchased from Alfa Aesar, Shanghai, China. Lot: 10180463; Trimercaptopropionic acid (3-MP, Lot: LD50Q10), and reference drug carbamazepine (CBZ, Lot: LLA0P07) was purchased from J&K Scientific Ltd., Beijing, China. Both GEAT decoction and CBZ were dissolved in saline containing 0.5% Poloxamer.
2.4. Treatment processes
2.4.1. Acute seizure test
The mice were divided randomly into five groups, 12 mice in each group. The model control mice received 0.9 % sodium chloride (NaCl) containing 0.5% Poloxamer. The mice of the positive control group received CBZ (a most commonly used antiepileptic drug), at a dose of 50 mg/kg. The mice of the treated groups received three different doses of GEAT decoction at 50, 100, and 200 mg/kg, respectively. The different dose of GEAT decoction, normal saline, and positive drugs were treated to mice in a double-blind way, and the mice were orally administrated doses of NaCl, CBZ or GEAT decoction once a day for consecutive 7 days.
2.4.2. Chronic seizure test
The mice were divided randomly into 6 groups, 12 mice in each group. The different dose of GEAT decoction, normal saline, and positive drugs were treated to mice in a double-blind way, and the mice were orally administrated test doses of NaCl, CBZ or GEAT decoction once a day for consecutive 28 days. The mice in the control group received 0.9 % sodium chloride (NaCl) containing 0.5% Poloxamer. Except for the mice in the normal control group, all mice in the other groups administered PTZ in a dose of 35 mg/kg for 14 days on alternate days. The mice in the control group received normal saline injections.
2.5. MES test
MES tests were carried out according to the previously described methods [11, 23]. Thirty mice were randomly divided into five groups and administered with double-blind method as mentioned earlier. 0.5, 1, 2, and 4 h after the last administration, mice were stimulated with a 0.25 s, 64 Hz, 50 mA stimulus by ear-clip electrodes using an electronic generator (Rodent Shocker). Mice was considered “protected” when full hind-limb tonic extension (HLTE) was absent from them [11, 24]. The number of mice protected from HLTE induced by electrical stimulation was recorded after the last drug administered 0.5, 1, 2, and 4 h.
2.6. PTZ-induced seizure test
2.6.1. Acute PTZ-induced mouse seizure model
Mice from each group were treated with the drug doses described in the experimental groups (0.9 % NaCl, CBZ, 50 mg/kg, GEAT decoction, 50 mg/kg, GEAT decoction 100 mg/kg, or GEAT decoction 200 mg/kg), for 7 days. One hour after the last dose, 85 mg/kg of freshly prepared solution of PTZ was administered subcutaneously to all mice. Then, the tested mice were placed immediately in a transparent plastic square box for observation for 20 min. Mice was considered “protected” when there is an absence of a single 5 sec episode of clonic spasm [23]. Latent time for the onset, the number of animals with generalized tonic-clonic seizures (GTCS), clonic seizures (CS), as well as mortality were recorded for 20 min after PTZ injection. In addition, seizure severity was evaluated primarily based on Racine scale with minor modifications. Briefly, stage 0: no response; 1: facial and ears twitching; stage 2: hyperactivity, vibrissae twitching, and myoclonic jerks; stage 3: unilateral forelimb clonus; stage 4: clonic convulsions with preservation of righting refex; stage 5: generalized GTCS loss of postural control [25].
2.6.2. Chronic PTZ-induced kindling mouse model
The mice were randomly divided into six groups: normal group, in which each mouse was daily oral administration of NaCl; model group (NaCl+PTZ), in which each mouse was daily oral administration of NaCl 30 min before administering a subconvulsive dose of PTZ (25 mg/kg); CBZ+PTZ group, in which each mouse were daily treated with CBZ (50 mg/kg) 30 min before PTZ injection; GEAT decoction (50, 100 and 200 mg/kg)+PTZ group, in which each mouse were daily treated with corresponding dose of GEAT decoction 30 min before PTZ injection. All groups were treated for 28 days. The Racine Scale was used to record and assess seizure severity of mice within 20 min after each PTZ injection [25]. 24 h after completion of the kindling test, behavioral assessment models were carried out to evaluate the ameliorative effects of GEAT decoction on anxiety and cognitive function in the kindled mice. After completion of the behavioral testing, all mice were immediately executed. Blood from the heart was collected and centrifuged at 1000 g for 5 min and collected plasma for standby. The brain tissues were removed and the hippocampal was collected and immediately stored at −20 ºC. The pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 levels was tested using enzyme-linked immunosorbent assay (ELISA, Elabscience Biotechnology Co., Ltd) with the sensitivity of 18.75 pg/mL, 4.688 pg/mL and 9.375 pg/mL, respectively. In addition, the biomarkers of oxidative stress including SOD, MDA, GSH and CAT content in the hippocampus was also detected using corresponding assays (Nanjing Jiancheng Reagent Co., Ltd). The performance of the biochemical tests was strictly following the instructions of each assay. Besides, all mice in the study underwent a battery of behavioral tests, in the following order: high plus maze (29th day after the induction of status seizures) and open field test (30th day after the induction of status seizures).
2.7. 3-MP-induced seizures test
3-MP-induced seizure tests were carried out according to previously described methods [11, 13]. Mice grouping and treatment in this test were similar to that of PTZ-induced acute seizure test. 30 min after the last treatment, 60 mg/kg of freshly prepared solution of 3-MP was administered subcutaneously to all mice. The latency to myoclonic jerk was noted along with the occurrence of generalized tonic seizures and CS. The mouse death was also monitored. The observation period was 20 min for an individual mice.
2.8. Elevated plus maze test
The elevated plus maze (EPM) test is a simple method to assess anxiety-like behaviors in mice by estimating contradictory and conflicting behavior between the exploring characteristics of animals in new/different environments and the fear of hanging open arms forms [26]. The maze (Shanghai xinruan Information Technology Co., Ltd, XR-XG201) consists of a plus-shaped platform 50 cm above the floor with two open (35 cm long ×5 cm wide) arms, a central square (5 cm long × 10 wide), and two closed (35 cm long × 5 cm wide ×15 cm height) arms. On the basis of a standard type pre-experiment, the high plus maze test was performed at 29 th day when PTZ was administered 24 h later to mice in PTZ-induced chronic seizure model. Each mouse was placed in the central area of the maze and monitored for 10 min, and the time spent and residence time of mice entering the open arm within 10 min were recorded by the software monitored during the test.
2.9. Open field test
The open field test (OFT) was mainly and commonly used to observe the locomotor activity, exploratory behavior, and neuropsychiatric changes of experimental animals in new and different environments [27]. The opening box inner of the floor divided into 9 equal quadrants (Shanghai, XR-XZ301) is 50 cm in diameter and 40 cm in height. On the basis of a standard pre-experiment, the OFT was performed at the 30th day when PTZ was administered 48 h later to mice in PTZ-induced chronic seizure model. The mice were placed in the opening box inner, and the video analysis system was used to analyze the total distance and time spent of mice entry into the central zone within 5 min.
2.10. Statistical analysis
Data in this study were presented as mean ± SEM. One-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test was performed to analyze the data. Kruskal-Wallis ANOVA was applied for Racine score. Chi square test was used for counting data. Values of P <0.05 were considered statistically significant. The statistical analyses were conducted using GraphPad Prism 7 software.