Pilocarpine hydrochloride was purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany); ghrelin was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA); NF-κB, TNF-α and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. [Dallas, TX, USA; cat. nos. NF-κB P-65 (sc-109), TNF-α (sc-52791); TRIzol reagent was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA); reverse transcription kit (NF-κB p65, sc-8008), Taq DNA Polymerase and DNA Marker DL2000 were purchased from Takara Bio. Inc. (Otsu, Japan); and SP-9000 Histostain-Plus kits were obtained from OriGene Technologies, Inc. (Rockville, MD, USA)].

Healthy male Wistar rats (60 in total; ~3 weeks old; 40–50 g) were obtained from Shengjing Hospital Laboratory Animal Centre of China Medical University (Shenyang, China). The present study was approved by the ethics committee Qingdao Municipal Hospital. Individuals were kept at 19±2°C in a quiet environment on a diurnal cycle, and were breast-fed. Unweaned rats and young rats were bred together with 12-h light/dark cycle. Rats were divided at random into the normal control group (n=8), pilocarpine group (n=8), pilocarpine + saline group (n=8) and pilocarpine + ghrelin group (n=8).

At 9:00 a.m., 3 mg/kg lithium chloride was injected into the peritoneal cavity. The control group rats were administered with physiological saline only. The following day at 8:30 a.m., 80 µg/kg ghrelin or saline was injected intraperitoneally, then 30 min later 30 mg/kg pilocarpine was injected to SE. The behavioral manifestations were observed and a resultant epileptic seizure of Racine stage IV or V was used as the inclusion criteria for further analysis (3). Seizure severity was ranked using Racine's scale: 1, seizure consisted of immobility and occasional facial clonus; 2, head nodding; 3, bilateral forelimb clonus; 4, rearing; 5, rearing and falling. In addition to the four groups, the experiments were performed with young mice, a group of young mice, and their mothers could continue to have mice. These mice as well as rats were kept at 19±2°C in a quiet environment on a diurnal cycle, and were breast-fed. Unweaned rats and young rats were kept together with 12-h light/dark cycle. Diazepam (10 mg/kg) was injected 60 min following the epileptic seizure. Epileptic rats were administered diazepam intraperitoneally to reduce the mortality of these rats when undergoing an epileptic seizure. Lithium chloride combined with pilocarpine was employed to produce the epileptic rat model.

After 24 h following the onset of epileptic seizure, under 2% lidocaine (~4.0–4.5 mg/kg, intraperitoneal injection) abdominal anesthesia, the chest was opened and the heart exposed. The ascending aorta of the left ventricle was intubated and 50 ml cold saline followed by 50 ml 4% paraformaldehyde PBS was perfused to fix the brain tissue at 4°C overnight. Fixed brain tissue was placed in 20% sucrose for 12 h, and 30% sucrose for a further 12 h. Following washing with PBS, structural parts of the cerebral cortex were cut into 5-µm coronal sections, dried at room temperature for 6 min and then preserved at −20°C. A total of 9 sections were cut from each rat cortex in each group (n=8). Brain tissue fixed in 4% paraformaldehyde for 24 h, conventional materials and 20% sucrose solution for 48 h and then embedded in paraffin. Sections of samples were cleaned with 0.01 mol/l PBS 100 ml three times for 5 min. Paraffin-embedded sections were treated with a solution of methanol and 0.3% hydrogen peroxide (methanol + 0.01 mol/l PBS 100 ml 80 ml + 30% hydrogen peroxide) for 30 min at room temperature after three washes with 0.01 mol/l PBS for 5 min in room temperature. Subsequently, paraffin-embedded sections were treated with 0.3% Triton X-100 (30% Triton X-100 + 100 ml 0.01 mol/l PBS) at 20°C for 30 min, followed by three washes with 0.01 mol/l PBS for 5 min. Primary antibodies were used at the following dilutions: NF-κB P-65 (cat. no. SC-372:1), 1:200 and TNF-α, (cat. no. SC-8301:1) 1:300 and sections were incubated in a wet box at room temperature for 1 h, then 4°C overnight; subsequently, the temperature was increased to 37°C after 45 min out of the fridge. Following incubation, the sections were incubated with secondary antibodies NF-κB P-65 (cat. no. SC-8008:2), 1:250 and TNF-α, (cat. no. SC-358919:2), 1:300 at 37°C at room temperature for 2 h then washed with 0.01 mol/l PBS, three times, three min each time. Immunohistochemical staining reagent. (Vector Laboratories, Inc., Burlingame, CA, USA; cat. no. PK-6103) was used. Immunohistochemical staining reagent was added, incubated at room temperature for 30 min, and washed with PBS three times, each time for 3 min. Then washed with distilled water for three times of 2 min each. Dehydrate: Put the slices in 50, 70, 80, 90, 95 and, 100% ethanol for two min. Transparent: Place the slices in 100% xylene for 10 min. Neutral resin 50 µl seal, room temperature preservation.

Primary and secondary antibodies were replaced with PBS for a blank control. NF-κB and TNF-α immunohistochemically-positive cells were counted using dark-field microscopy (CX31-lv320; Olympus Corporation, Tokyo, Japan) and quantified using Image-Pro Plus image analysis software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA; Table I). A total of 5 views per field were analyzed.

Surgical dislocation was used to remove head and brain 24 h following the onset of epileptic seizure. Seizures following 24 h, with 2% lidocaine (~4–4.5 mg/kg) abdomen anesthesia, then the cerebral cortex of the brain was isolated. Total RNA was extracted using TRIzol reagent. Following measurement of RNA concentration with a UV spectrophotometer, a 25 µl RT reaction was set up according to the kit instructions: 2 µg RNA was added to 5 µl 5× loading buffer, 2.5 µl MgSO₄ (40 mmol/l), 2.5 µl dNTP (2.5 mmol/l), 1 µl oligo (dT) primer, 40 µl RNase inhibitor, 30 µl UAMV and Rnase-free H₂O to 25 µl. Following mixing, cDNA was synthesized at 94°C for 60 sec, 37°C for 60 sec and 120 sec at 72°C; 25 cycles. PCR primers were designed to amplify TNF-α, NF-κB and β-actin cDNA by referring to previous literature (12) and are identified in Table II. Amplification products were electrophorised on 1.5% agarose gels (10 µl per lane) containing 20 g/l TAE buffer. UV camera systems were used to acquire images and analysis software (SPSS version 19.0, IBM Corp., Armonk, NY, USA) was used to multiply the size and strength of the strips as PCR product content. TNF-α and NF-κB mRNA expression levels were calculated relative to β-actin.

Data were analyzed using SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA). Each set of data obtained were the average values of 6 repeated experiments, and all data were expressed as the mean ± standard deviation. One-way analysis of variance was used to compare parameters between the different groups, and Fisher's Least Significant Difference method was used for pair comparison. P<0.05 was considered to indicate a statistically significant difference.

Compared with the cerebral cortex of young rats in the normal control group (Fig. 1A and Table II), TNF-α protein expression was significantly increased 24 h subsequent to induction of pilocarpine-induced SE (P<0.05; Fig. 1B and Table III). TNF-α protein expression was also significantly increased (compared with control) 24 h subsequent to induction of pilocarpine-induced SE when an i.p. injection of saline was administered 30 min prior to pilocarpine (P<0.05; Fig. 1C and Table III). However, in rats who received an i.p. injection of ghrelin 30 min prior to pilocarpine induction of SE, significantly fewer TNF-α positive cells were observed in the cytoplasm of cerebral cortical neurons compared with the pilocarpine and pilocarpine + saline groups (both P<0.05; Fig. 1D and Table III).

In addition, compared with the cerebral cortex of young rats in the normal control group (Fig. 2A and Table II), NF-κB protein expression was increased 24 h subsequent to induction of pilocarpine-induced SE (P<0.05; Fig. 2B and Table III). NF-κB protein expression was also increased (compared with control) 24 h subsequent to induction of pilocarpine-induced SE when an i.p. injection of saline was administered 30 min prior to pilocarpine (P<0.05; Fig. 2C and Table III). However, in rats who received an i.p. injection of ghrelin 30 min prior to pilocarpine induction of SE, significantly fewer NF-κB positive cells were observed in the cytoplasm of cerebral cortical neurons compared with the pilocarpine and pilocarpine + saline groups (both P<0.05; Fig. 2D and Table III).

A260/A280 nm of extracted RNA was 1.8–2.0, indicating that the total RNA purity was high, with no protein pollution. Compared with the cerebral cortex of young rats in the normal control group (Fig. 3, lane 1 and Table IV), TNF-α mRNA expression was significantly increased 24 h subsequent to induction of pilocarpine-induced SE (P<0.05; Fig. 3, lane 2 and Table IV). TNF-α mRNA expression was also significantly increased (compared with the control) 24 h subsequent to induction of pilocarpine-induced SE when an i.p. injection of saline was administered 30 min prior to pilocarpine (P<0.05; Fig. 3, lane 3 and Table IV). However, in rats who received an i.p. injection of ghrelin 30 min prior to pilocarpine induction of SE, TNF-α mRNA expression levels were significantly reduced in the cytoplasm of cerebral cortical neurons compared with the pilocarpine and pilocarpine + saline groups (P<0.05 and P<0.05, respectively; Fig. 3, lane 4 and Table IV).

In addition, compared with the cerebral cortex of young rats in the normal control group (Fig. 4, lane 1 and Table IV), NF-κB mRNA expression was also significantly increased 24 h subsequent to induction of pilocarpine-induced SE (P<0.05; Fig. 4, lane 2 and Table IV). NF-κB mRNA expression was also significantly increased (compared with control) 24 h subsequent to induction of pilocarpine-induced SE when an i.p. injection of saline was administered 30 min prior to pilocarpine (P<0.05; Fig. 4, lane 3 and Table IV). However, in rats who received an i.p. injection of ghrelin 30 min prior to pilocarpine induction of SE, NF-κB mRNA expression levels were significantly reduced in the cytoplasm of cerebral cortical neurons compared with the pilocarpine and pilocarpine + saline groups (both P<0.05; Fig. 4, lane 4 and Table IV).