Male Sprague-Dawley (SD) rats (age, 6–7 weeks; weight, 200–250 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and were housed under controlled standard conditions at 65±5% humidity and 21±2°C, a 12 h light/dark cycle and with food and water available. The rats were housed under these conditions for one week prior to treatment. Experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University, according to international standards.

Animals were divided randomly into 6 groups: Sham group (treated with saline as a control, n=21); EP group (treated with pilocarpine; n=78); small interfering RNA (siRNA) group (treated with pilocarpine plus siRNA; n=24); pyrrolidinedithiocarbamic acid (PDTC) group (treated only with PDTC; n=12); EP + PDTC group (treated with pilocarpine plus PDTC; n=12); scrambled group (treated with pilocarpine plus scrambled siRNA; n=4).

The purpose of the operation was to inject siRNA. SD rats were anesthetized with 10% chloral hydrate (3 ml/kg) and placed on stereotactic apparatus. A stainless steel single guide cannula (PlasticsOne Inc., Roanoke, VA, USA) was implanted into the right lateral ventricle with coordinates of 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline and 4.5 mm depth to the surface of the skull (22). The cannula was fixed with dental cement (Heraeus Kulzer GmbH, Wehrheim, Germany).

Rats were administrated with lithium chloride (127 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) by intraperitoneal (IP) injection 24 h before pilocarpine treatment. Methyl scopolamine (1 mg/kg, IP; Sigma-Aldrich) was delivered 30 min before pilocarpine to reduce the peripheral effects and to enhance survival. A single dose of pilocarpine (30 mg/kg, IP) (Sigma-Aldrich) was administered. Subsequently, rats received repeated injections of pilocarpine (10 mg/kg, IP) every 30 min until they developed convulsive seizures. The maximum number of pilocarpine injections was 5 per animal (23). Diazepam (10 mg/kg; Sigma-Aldrich) was utilized to terminate seizure activity 90 min after the onset of SE. Severity of the convulsions were evaluated by Racine's classification and denoted the following stages: 0, behavioral arrest; 1, face clonus; 2, head nodding; 3, forelimb clonus; 4, forelimb clonus and rearing, 5, forelimb clonus with rearing and falling (24). Animals classified as less than Racine's stage 4 were excluded from the experiment. The sham groups were injected with normal saline instead of pilocarpine.

siRNAs targeting HMGB1 and RAGE mRNA were designed and synthesized by Shanghai GenePhama Co., Ltd. (Shanghai, China). siRNA sequences are listed in Table I. As described previously, the rats of the siRNA group were treated with 10 µl siRNA (5 µmol/l) (25) by a microinjection pump via a pre-implanted cannula 30 min prior to pilocarpine injection. Following each injection, the needle was left in place for 5 min to allow for drug diffusion. The sham group was treated with 10 µl saline. The Scrambled group was administrated with 10 µl scrambled siRNA (5 µmol/l) and served as the negative control.

Rats in the EP + PDTC group were treated with PDTC (100 mg/kg, IP; BioVision, Inc., Milpitas, CA, USA) at 14.5 h and 15 min prior to the first pilocarpine injection and at 90 min after onset of SE. A further two injections of PDTC at the same dose was administrated 5 h after termination of SE and 23.5 h after the first injection of pilocarpine (26). The sham group was treated with the same volume of saline and the PDTC group was treated IP with 100 mg/kg PDTC alone.

Total RNA was extracted from the hippocampus of the rats using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The concentration and purity of the RNA was measured spectrophotometrically at 260 and 280 nm. Total RNA (500 ng) was subjected to DNase I digestion (Takara Bio, Inc., Otsu, Japan), and was subsequently utilized for cDNA synthesis using the PrimeScript™ RT Master Mix (Takara Bio, Inc.). qPCR was then performed using the SYBR® Premix Ex Taq™ kit (Takara Bio, Inc.) with 1 µl 0.5 µM forward and reverse primers (final concentration, 0.5 µm; Sangon Biotech Co., Ltd., Shanghai, China; Table II) and 8 µl diluted cDNA on a 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cycling conditions were as follows: Initial predenaturation step at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 31 sec. The relative mRNA expression of the target gene was normalized to GAPDH and was calculated using the 2^−ΔΔCq method (27).

Total protein was extracted from the hippocampus of rats using a protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Samples were homogenized and centrifuged at 14,000 × g for 15 min, and the supernatant was harvested and diluted to 6.7 µg/µl in SDS/bromophenol blue loading buffer, prior to heating for 5 min at 100°C. Samples were loaded onto 10% gels, separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h and subsequently incubated at 4°C overnight with the following primary antibodies: Rabbit anti-HMGB1 (1:1,000; catalog no. ab18256; Abcam, Cambridge, UK); rabbit anti-RAGE (1:1,000; catalog no. ab3611; Abcam); mouse anti-P-gp (1:50, catalog no. 517310; Calbiochem; EMD Millipore); rabbit anti-phosphorylated-NF-κB p65 (p-p65) (1:1,000, catalog no. 3033; Cell Signaling Technology, Inc., Danvers, MA, USA). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; catalog no. ZB230; ZSGB-BIO, Beijing, China) or anti-mouse secondary antibodies (1:5,000; catalog no. ZB2305; ZSGB-BIO) for 1.5 h at room temperature, proteins were detected using electrochemiluminescence (catalog no. WBKLS0500; EMD Millipore) according to the manufacturer's instructions, with Quantity One software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The optical density of each sample was measured using Scion Image software (version 1.47; Scion Corporation, Frederick, MD, USA). The optical density of each sample was normalized to the mouse anti-β-actin (1:500; catalog no. BM0627; Wuhan Boster Biological Technology, Ltd., Wuhan, China) loading control.

Rat brains were fixed in 4% paraformaldehyde following trans-cardiac perfusion overnight at 4°C, and subsequently soaked in 20 and 30% sucrose for 2 to 3 days, respectively. The brains were then embedded into optimal cutting temperature compound (catalog no. C1400; Applygen Technologies, Inc., Beijing, China). Sections (8-µm thick) were prepared using a vibrating slicer and staining was performed using a standard procedure (28). Briefly, sections were blocked in 0.01 mol/l serum (either rabbit or mouse according to the secondary antibody used; Wuhan Boster Biological Technology, Ltd.) at room temperature for 2 h. The redundant serum was removed then sections were incubated with the following primary antibodies: Rabbit anti-HMGB1 (1:500; catalog no. ab18256; Abcam), rabbit anti-RAGE (1:200; catalog no. ab3611; Abcam) and mouse anti-P-gp (1:50; catalog no. 517310; EMD Millipore) at 4°C overnight. Goat anti-rabbit FITC-conjugated IgG (1:1,000 PBS dilution; catalog no. BA1105; Wuhan Boster Biological Technology, Ltd.) or goat anti-mouse cyanine 3-conjugated IgG (1:1,000 PBS dilution; catalog no. BA1101; Wuhan Boster Biological Technology, Ltd.) secondary antibodies were added at room temperature for 1 h, followed by DAPI (catalog no. AR1177; Wuhan Boster Biological Technology, Ltd.) nuclear stain. Sections were visualized using a Leica fluorescence microscope (DM4000; Leica Microsystems GmbH, Wetzlar, Germany) and the Leica QWin version 3 software (Leica Microsystems GmbH) was used for analysis. Different cell types were identified by analyzing cell morphology.

Data was analyzed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA) and are presented as the mean ± standard deviation. Significant differences were analyzed using a one-tailed Student's t-test, or by one-way analysis of variance followed by the Least Significant Difference post-hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The concentration of pilocarpine (35.50±9.12 mg/kg) utilized for inducing seizures was equal to that of previous experiments (26). Following pilocarpine administration, hypersalivation, piloerection, licking, chromodacryorrhea, swallowing and chewing occurred immediately. Subsequently, clonic movements of forelimbs, head bobbing and motor limbic seizures were observed. The average latency of first seizures (29) and from first pilocarpine injection to SE were 35.02±24.43 and 37.93±24.78 min, respectively. Induction success refers to the percentage of rats who had seizures, however, the level of which may not be suitable for further experiments; this also includes rats who had seizures however, they succumbed within 90 min following SE. Model success refers to the percentage of rats whose seizure level reached the experimental requirements; these rats survived the 90 min period following SE. The rates of induction and model success in rats in the present study were 91.28 and 82.05%, respectively. The rate of failure, the percentage of rats that were not model successful, was 8.21% and the rate of total mortality, which included anesthesia-associated mortality, rats succumbing during the induction of SE and mortality prior to the scheduled time of specimen collection, was 18.46%.

At 6, 12, 24, 48 and 72 h, and 5 and 7 days post-termination of seizures, HMGB1, RAGE, p-p65 and P-gp were measured in the hippocampus of rat brains by western blotting. Compared with the Sham group, the expression levels of HMGB1 were significantly enhanced following 48 h (P<0.05), 72 h (P<0.0001) and 5 days (P<0.01) SE termination, as were the expression levels of RAGE (P<0.01 and P<0.05) and P-gp (P<0.05 and P<0.0001) at 72 h and 5 days respectively, and p-p65 was significantly enhanced following 72 h (P<0.01; Fig. 1). However, the expression levels of HMGB1 were reduced at 24 h and 7 days compared with 12 h and 5 days SE termination, respectively (P<0.05; Fig. 1). The expression levels of RAGE (P<0.05) and p-p65 (P<0.01) were reduced following 7 days compared with 72 h, as were the expression levels of p-p65 and P-gp at 7 days compared with 5 days termination (P<0.05; Fig. 1). The greatest expression of HMGB1, RAGE and p-p65 was observed at 72 h, and declined at later time points (Fig. 1). However, expression levels of P-gp continued to increase at 5 days, which were subsequently reduced by 7 days (Fig. 1).

At 30 min prior to IP pilocarpine treatment, HMGB1 siRNA was administered to rats (ICV injection). At 3 days after SE termination, HMGB1, RAGE, p-p65 and P-gp expression levels were measured by western blotting. Protein levels of HMGB1 (P<0.01), RAGE (P<0.01), P-gp (P<0.01) and p-p65 (P<0.0001) in the EP group were significantly greater compared with expression in the sham group (Fig. 2A). Pre-treatment with HMGB1 siRNA significantly reduced the expression levels of all proteins compared with the EP and scrambled groups (P<0.01). The mRNA expression levels of HMGB1, RAGE, MDR1A and MDR1B supported the western blotting data (Fig. 2B). Immunofluorescence staining of tissue sections demonstrated that HMGB1 was localized to the nuclei and cytoplasm of neurons (Fig. 2C). P-gp was primarily localized to blood vessels. HMGB1 and P-gp were expressed in low levels in the Sham group; however, a greater number of cells expressed HMGB1 and P-gp in the hippocampal CA3 region of the EP group. Compared with the EP group, the number of cells expressing HMGB1 and P-gp was reduced in the group treated with HMGB1 siRNA (Fig. 2C).

Protein expression levels of RAGE, p-p65 and P-gp were significantly reduced in the siRNA group compared with the EP and scrambled group (P<0.01; Fig. 3A). Similarly, in the group treated with RAGE siRNA, mRNA expression of RAGE, MDR1A and MDR1B were significantly reduced compared with the EP and scrambled groups (Fig. 3B). Immunofluorescence staining demonstrated that RAGE and P-gp were upregulated in the EP group compared with the sham group. Pre-treatment with RAGE siRNA resulted in reduced numbers of P-gp positive cells compared with the scrambled group (Fig. 3C).

To determine whether NF-κB may regulate the expression of P-gp, PDTC, a specific inhibitor of NF-κB was utilized. As demonstrated in Fig. 4A, 72 h after SE termination, the protein expression levels of P-gp in the EP + PDTC group were significantly reduced compared with the EP group (P<0.01). This was supported by the mRNA expression levels of MDR1A/B (Fig. 4B), and the immunofluorescence staining of P-gp (Fig. 4C).