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 transfer to a glass bottle, and then reserved at 4 °C in ice box.
Animals
SPF adult 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 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).
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.
Treatment processes
The mice were divided randomly into 6 groups, 6 mice in each group. The normal control and model control mice received 0.9 % sodium chloride (NaCl) containing 0.5% Poloxamer. The mice of positive control group received CBZ (a most commonly used antiepileptic drugs), at a dose of 50 mg/kg. The mice of 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 treated to mice in a double-blind way, and the mice were administrated daily doses of NaCl, CBZ or GEAT decoction once by way of stomach for 14 days.
MES test
MES test were carried out according to previously described method (He et al. 2018). 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 there were no mice deaths and full hindlimb extension was absent from the MES-stimulated seizures (He et al. 2018; Goerl et al. 2021). Before the drug administration, all mice were preliminarily screened under the electrical stimulation conditions set in the experiment in order to ensure that each experimental mouse has a positive reaction (i.e. full tonic extension) to the MES test. Then, these qualified mice were randomly divided into six groups and administered with double-blind method as per item 2.4 shown. The number of protected mice after electrical stimulation was recorded after drug administered 0.5, 1, 2 and 4 h.
PTZ-induced seizures
PTZ-induced acute seizure model
Mice from each group treated 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 100 mg/kg), for 14 days. After the last dose of the drugs, 85 mg/kg of freshly prepared solution of PTZ was administered subcutaneously to all the mice. Then, the tested mice were placed immediately in a transparent plastic square box for observation for 20 min. Mice was considered “protected” when the duration of generalized tonic seizure was less than 5 s. Latent time for the onset, the number of animals of tonic and clonic seizures as well as the mortality were recorded for 20 min after PTZ injection.
PTZ-induced chronic seizure 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 administered 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 14 days. The Racine Scale was used to recorded and assess seizure severity of mice within 20 min after PTZ injection. After the last drug administration, except for the normal control group, the mice were observed for 20 min and then 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 tissue were removed and 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). In addition, the biomarkers of oxidative stress including SOD, MDA, GSH and CAT content in hippocampus was also detected using corresponding assays (Nanjing Jiancheng Reagent Co., Ltd). The performance of the biochemical tests was strictly follow the instructions of the each assay. Besides, all of the mice in the study underwent a battery of behavioral tests, in the following order: high plus maze (10 days after the induction of status seizure) and open field test (10 days after the induction of status seizure).
3-MP-induced seizures
3-MP-induced seizures test were carried out according to previously described methods (He et al. 2018; Bai et al. 2019). Mice grouping and treatment of mice 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 the mice. Latent time for the onset, the number of animals of tonic and clonic seizures as well as the mortality were recorded for 20 min after 3-MP injection.
Elevated plus maze
The elevated plus maze (EPM) is a simple method to assess anxiety-like behaviors in mice by estimating contradictory and conflicting behavior between the exploring characteristics of animals to new/different environments and the fear of hanging open arms forms (Guillén-Ruiz et al. 2021). 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. In this study, the high plus maze test was performed at 10 days when PTZ was administered 2 h 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 times and residence time of mice entering the open arm within 10 min were recorded by software monitored during the test.
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 (Flores-Fuentes et al. 2021). The opening box inner with the floor divided into 9 equal quadrants (Shanghai, XR-XZ301) is 50 cm in diameter and 40 cm in height. In this study, the OFT was performed at 10 days when PTZ was administered 2 h 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 movement time of mice in the central area within 5 min.
Statistical analysis
Data in this study were presented as mean ± SDE. One-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test was performed to analyze the data, while Chi square test was used for counting data. Values of p < 0.05 were considered statistically significant. The statistical analyses were conducted using Prism 5 software.