For all experiments reported in our study, we used adult male C57BL/6 J mice weighing 20 ± 2 g provided from Chengdu Dossy Experimental Animal Co., Chengdu, China. The animals were maintained under standard husbandry conditions with free access to water and food. The light/dark cycle was set to 12 h (light from 7 a.m. to 7 p.m.), and the ambient temperature was maintained at 22 ± 2 °C. The animal study was reviewed and approved by the Institutional Review Board of Center for Brain Disorders, TongRen Municipal People's Hospital, Tongren, China (Approval NO.: ZMU21-2403–068). All experiments were carried out in accordance with relevant guidelines and regulations.

Moxi—7 days group: After binding and fixing the mice, moxibustion treatment was performed at a distance of 1 cm from the bilateral Zusanli (ST36) and Dazhui (GV14) acupoints, once daily for 20 min, for 7 consecutive days. The pretreatment methods for the Moxi—14 days group and Moxi—21 days group were the same as those for Moxi -7 days group, but were conducted for 14 and 21 days, respectively. The fixation method and duration for the KA group were the same as those for the Moxi group. For the Moxi + BzATP group, the moxibustion pretreatment method was the same as for the Moxi—14 days group. The other three groups (Veh, A438079 and BzATP) used the same fixation method and duration as the Moxi—14 days group but did not receive moxibustion intervention.

Mice were anesthetized using an inhalation anesthesia machine (isoflurane concentration: 1–1.5%, oxygen concentration: 0.4 l/min) and fixed on a stereotactic device (RWD Life Science Co., Ltd., China). Stereotactic implantation of cannulas (1.6 mm in length and 3.7 mm in diameter) was performed into the hippocampal CA1 area (coordinates from bregma: anteroposterior, -1.85 mm; mediolateral, ± 2.15 mm; dorsoventral, -1.6 mm) and fixed to the skull with dental cement. After surgery, the mice were allowed to recover for two weeks before conducting experimental pretreatment.

Before treating mice with KA, mice were placed individually in transparent acrylic boxes in a quiet environment for 30 min at room temperature (22 ± 2 °C). The Vehicle (Veh) group received an injection of 2 µl phosphate-buffered saline (PBS; Hyclone Labs., UT, Logan, USA) into the bilateral hippocampal CA1 region. The BzATP group and the Moxi + BzATP group were both injected with Bz-ATP; 10.5 nmol BzATP = 0.00215 g + 1 ml PBS), while the A438079 group was injected with 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride (A-438079; 1.75 nmol A438079 = 0.34261 g + 1 ml PBS) using the same method and dosage as the Veh group. After drug injections, all mice were injected with i.p. KA (30 mg/kg). Based on previous studies [17, 18], all epileptic seizure models of mice were immediately videotaped after intraperitoneal injection of KA (30 mg/kg) to observe behavioral changes within 60 min starting from the time of KA injections. The Racine scale was used to evaluate the severity of seizures [19]. Behavior was scored as follows: 0, behavioral arrest (motionless), piloerection, excitement and rapid breathing; 1, movement of the mouth, lips tongue and vibrissae, salivation; 2, head and eye clonus; 3, forelimb clonus, “wet dog shakes”; 4, clonic rearing; 5, clonic rearing with loss of postural control and uncontrolled jumping. The complete experimental process including Racine score every 10 min, the highest Racine score during 60 min and the duration of 4–5 Racine score time is summarized in Fig. 1.

Schematic of experimental design. A Mice were pre-treated with moxibustion for different lengths of time (i.e., 7 days, 14 days or 21 days). Mice were then injected with KA (i.p. 30 mg/kg) and seizures video recorded for 60 min. Behaviour seizures were analyzed using the Racine scale. B A subset of mice, subjected to 14 days of moxibustion treatment, was also recorded with cortical EEG electrodes implanted 2 weeks prior to i.p. KA injection. C Another subset of mice, subjected to 14 days of moxibustion treatment, was also injected BzATP or A438079 prior to i.p. KA injection

Mice had three electrodes implanted and secured with dental cement. This procedure included placing two electrodes over each temporal cortex and one reference electrode in the cerebellum. A period of 2 weeks was allowed to pass before starting experimental measurement. EEG signals were recorded using a telemetric apparatus (LabChart 8; AD Instruments, Colorado Springs, CO, USA), during KA-induced status epilepticus and at time points matching the 14-day moxibustions pretreatments. The EEG signals were recorded for a duration of 1 h, starting 1 h after the injection of KA. EEG total power (µV2), a function of EEG amplitude over time, was analyzed by integrating frequency bands from 0 to 100 Hz. Power spectral density bands were generated within the LabChart 8 (spectral view), with the frequency domain filtered from 0 to 50 Hz and the amplitude domain filtered from 0 to 50 mV. For the calculation of the n-fold total power, LabChart 8 was used, and for greater uniformity, the period between 29 and 39 min was chosen.

Adult C57B/J mice (6–8 weeks old) were randomized into the following groups: reference group (KA, n = 7), moxibustion—7 days group (Moxi—7 days, n = 7), moxibustion—14 days group (Moxi—14 days, n = 7), and moxibustion—21 days group (Moxi—21 days, n = 7) (Fig. 2). For our EEG recordings, the groups included the KA reference group (n = 5) and the moxibustion 14 days group (Moxi—14 days group, n = 5) (Fig. 3). The use of P2X7R agonists (3’-O-(4-Benzoyl)benzoyl-ATP (BzATP)) or antagonists (A-438079) was divided into four groups, all treated with i.p. KA: control group (Veh group, n = 7), P2X7R agonist group (BzATP group, n = 7), P2X7R antagonist group (A438079 group, n = 7), and P2X7R agonist group after 14 days of moxibustion pretreatment (Moxi + BzATP group,n = 7) (Fig. 4).

Behavioral effects of moxibustion pretreatment with different days on epileptic seizures mice (a) The seizure stage within 60 min in KA and Moxi—7 days groups. (b) The highest Racine score within 60 min in KA and Moxi—7 days groups. (c) The duration of status epilepticus (SE) in KA and Moxi—7 days groups. (d) The seizure stage within 60 min in KA and Moxi—14 days groups. (e) The highest Racine score within 60 min in KA and Moxi—14 days groups. (f) the duration 4–5 Racine score in KA and Moxi—7 days groups. (g) The seizure stage within 60 min in KA and Moxi—21 days groups. (h) The highest Racine score within 60 min in KA and Moxi—21 days groups. (i) the duration 4–5 Racine score in KA and Moxi—21 days groups. (j) The seizure stage within 60 min in Moxi—14 daysand Moxi—21 days groups. (k) The highest Racine score within 60 min in Moxi—14 days and Moxi—21 days groups. (l) the duration 4–5 Racine score in Moxi—14 days and Moxi—21 days groups. Bars represent the means ± Standard error of the mean deviation for each group (each group n = 7). ns: no significance, Mann–Whitney rank sum test, *p < 0.05, **p < 0.01

Moxibustion pretreatment 14 days reduced epileptic seizures the changes of EEG ((a) Graph showing representative EEG traces and heat maps of frequency and amplitude data starting at the time from 29—30 min after KA (i.p.). (b) Graph showing representative EEG traces and heat maps of frequency and amplitude data starting at the time of 10 min after Moxi—14 days. Of note, reduced seizure severity was seen in Moxi—14 days mouse. (c) Graphs showing total power on the EEG during KA (29—30 min EEG recording) and after Moxi—14 days (29—30 min EEG recording). Total power confirmed the significant reduction of seizure activity after moxibustion pretreatments 14 days mice compared to KA. Two-tailed unpaired t-test, ***p < 0.001

Moxibustion pretreatment 14 days affects epileptic seizures by mediation P2X7 receptor. A The seizure stage within 60 min in the Vel, A438079, BzATP and Moxi + BzATP groups. B The duration of status epilepticus (SE) in the Vel, A438079, BzATP and Moxi + BzATP groups. Bars represent the means ± Standard error of the mean deviation for each group (each group n = 7). One-way analysis of variance, *p < 0.05, **p < 0.01, and ***p < 0.001

All data were expressed as means ± Standard error of the mean (SEM) Data were statistically analyzed using GraphPad Prism 9.0. Normality was tested using the Shapiro–Wilk normality test. Two-tailed unpaired t-test and Mann–Whitney rank sum test were used for comparison of two groups. One-way analysis of variance was used for multiple comparisons. Post hoc tests were performed using the Holm-Siddak test and the Tukey test only when the F reached P < 0.05. P values less than 0.05 were considered statistically significant.