A total of 50 adult male Sprague Dawley rats (280±20 g, 12–14 weeks old) were purchased from the Animal Center of Hebei Cangzhou Central Hospital (Cangzhou, China). Animal experiments were approved by the Ethics Committee of Cangzhou Central Hospital (Cangzhou, China) and were performed in accordance with the National Institute of Health Guidelines. Rats were housed at 22–25°C with 55–60% humidity, were subjected to a 12-h light/dark cycle and had free access to food and water.

Rats were randomly divided into the following groups (n=10): Negative control group, cerebral I/R injury group and cerebral I/R injury with chicoric acid treatment group (1, 10 or 100 mg/kg). Rats were injected with 35 mg/kg pentobarbital sodium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A cerebral I/R rat model was induced by transient middle cerebral artery occlusion (MCAO) for 1 h as described previously (14). In the negative control group, surgery was performed in the same way without inserting threads. In the cerebral I/R damage group, the rats were injected with 200 µl normal saline. In the cerebral I/R damage with chicoric acid treatment group, the rats were injected with 1, 10 or 100 mg/kg/day of chicoric acid (Sigma-Aldrich; Merck KGaA) for 1 week.

Neurological deficits were evaluated following 1 day of treatment post-surgery and were scored on a 5-point scale. Scoring was performed as follows: 0, No deficit; 1, mild deficit-failure of left forepaw extension; 2, moderate deficit-circling to the left; 3, severe deficit-falling to the left; 4, critical deficit-depressed consciousness, no spontaneous walking (15).

Rats were sacrificed and brain tissue samples were harvested. Brain tissues was fixed with 4% paraformaldehyde for 24 h at room temperature, and tissue samples were sliced into 2 mm sections and stained with 2% 2,3,5-triphenyltetrazolium chloride in the dark for 30 min at 37°C. The infarct area was demarcated as follows: Infarct volume (%) = (volume_ipsilateral - volume_{contralateral})/volume_{contralateral} × 100.

Brains tissue samples were harvested, fixed with 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin. Hippocampal tissues were separated and the hippocampal embedded tissue samples were cut into 6 µm sections and stained with hematoxylin and eosin (H&E) at 37°C for 15 min. H&E tissue samples were examined under a light microscope (BX51; magnification, ×10; Olympus Corp., Tokyo, Japan) and images were captured using a DP72 camera (Olympus Corp.).

Brain tissue samples were harvested, homogenated and fractionated by centrifugation at 2,000 × g for 5 min at 4°C. The separated upper layer was incubated with 2′,7′-dichlorodihydrofluorescein diacetate (Nanjing Jiancheng Biology Engineering Institute Co., Ltd., Nanjing, China) in buffer solution (Nanjing Jiancheng Biology Engineering Institute Co., Ltd.) at 37°C for 30 min. ROS production was measured using an Olympus fluorescence microscope (magnification, ×10; Olympus Corp.).

A total of 100 µl of venous blood was collected and fractionated by centrifugation at 2,000 × g for 5 min at 4°C. The separated upper plasma layer was collected and the levels of TNF-α (cat. no. PT516) and IL-1β (cat. no. PI303) were analyzed using an ELISA microplate reader at an absorbance of 450 nm.

Brains tissue samples were quickly separated, homogenated with Griess reagent (Sigma-Aldrich; Merck KGaA) and 100 µl of bronchoalveolar lavage fluid obtained from rats, and subsequently incubated for 10 min at room temperature. The concentration of NO was determined at 540 nm using an ELISA microplate reader and NO kit (cat. no. A012-1; Nanjing Jiancheng Biology Engineering Institute). The PGE2 concentrations were determined using a prostaglandin E2 assay kit (cat. no. H099; Nanjing Jiancheng Biology Engineering Institute).

Brain tissue samples were separated and homogenated with liquid nitrogen. Protein content was determined using a BCA assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (50 µg) were separated by SDS-PAGE (10–12%) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with PBS containing 1% Tween-20 and 5% non-fat dried milk for 1 h at room temperature, and subsequently incubated with the following primary antibodies: Anti-cyclooxygenase (COX)-2 (sc-7951, 1:1,000), anti-phosphorylated (p)-c-Jun (sc-7980-R, 1:1,000), anti-AKT (sc-8312, 1:2,000), anti-p-AKT (sc-135651, 1:1,500; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-actin (1:1,000; Sangon Biotech Co., Ltd., Shanghai, China) overnight at 4°C. Membranes were subsequently incubated with goat anti-rabbit IgG-horserasish peroxidase secondary antibody (sc-2004, 1:15,000; Santa Cruz Biotechnology, Inc.) for 2 h at 37°C. β-actin was used as an internal control.

Data are presented as the mean ± standard error of the mean and were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Statistically significant differences were determined using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of chicoric acid is presented in Fig. 1. The neurological deficit score was significantly higher in the cerebral I/R injury group compared with the control group (P<0.01; Fig. 2). However, treatment with 10 or 100 mg/kg of chicoric acid significantly inhibited the cerebral I/R injury-induced neurological deficits in rats (P<0.01; Fig. 2). No significant difference was observed between the cerebral I/R injury and 1 mg/kg chicoric acid groups (Fig. 2).

The infarct volume of rats in the cerebral I/R injury group was significantly higher compared with the control group (P<0.01; Fig. 3). However, treatment with 10 or 100 mg/kg of chicoric acid significantly reduced infarct volume in rats with cerebral I/R injury (P<0.01; Fig. 3). No significant difference was observed between the cerebral I/R injury and 1 mg/kg chicoric acid groups (Fig. 3).

Cerebral I/R injury and the inhibition of the number of neuronal cells in the hippocampus were observed in the cerebral I/R injury group compared with the control group (Fig. 4). Treatment with 1 mg/kg chicoric acid had no marked effect on hippocampus injury and the number of neuronal cells compared with the cerebral I/R injury group (Fig. 4). However, treatment with 10 or 100 mg/kg chicoric acid markedly inhibited the extent of I/R injury and the number of neuronal cells in rats following MCAO (Fig. 4).

ROS production was significantly higher in the cerebral I/R injury group compared with the control group (P<0.01; Fig. 5). However, pretreatment with 10 or 100 mg/kg of chicoric acid significantly reduced ROS production in rats with cerebral I/R injury (P<0.01; Fig. 5). No significant difference was observed between the 1 mg/kg chicoric acid and cerebral I/R injury groups (Fig. 5).

Compared with the control group, TNF-α and IL-1β levels were significantly increased in the cerebral I/R injury group (P<0.01; Fig. 6). Treatment with 10 or 100 mg/kg of chicoric acid significantly suppressed the levels of TNF-α and IL-1β in cerebral I/R injury rats (P<0.01; Fig. 6). However, no significant differences in TNF-α or IL-1β levels were observed between the 1 mg/kg chicoric acid and cerebral I/R injury groups (Fig. 6).

NO levels in rats in the cerebral I/R injury group were significantly increased compared with control rats (P<0.01; Fig. 7). Treatment with 10 or 100 mg/kg of chicoric acid significantly reduced NO levels in rats with cerebral I/R injury (P<0.01; Fig. 7). No significant differences in NO levels were observed between the 1 mg/kg chicoric acid and cerebral I/R injury groups (Fig. 7).

COX-2 and p-p38-MAPK protein levels were significantly increased in the cerebral I/R injury group compared with control rats (P<0.01; Fig. 8). Treatment with 10 or 100 mg/kg chicoric acid significantly decreased COX-2 and p-p38-MAPK levels in rats with cerebral I/R injury (P<0.01; Fig. 8). No significant differences in COX-2 and p-p38-MAPK levels were observed between the 1 mg/kg chicoric acid and cerebral I/R injury groups (Fig. 8).

The results of the present study reveal that PGE2 levels were significantly increased in the cerebral I/R injury group compared with the control group (P<0.01; Fig. 9). Treatment with 10 or 100 mg/kg chicoric acid significantly attenuated the MCAO-induced upregulation of PGE2 in rats (P<0.01; Fig. 9). No significant differences were observed in PGE2 levels between the 1 mg/kg chicoric acid and cerebral I/R injury groups (Fig. 9).

The expression of p-c-Jun was upregulated in rats with cerebral I/R injury compared with control rats (P<0.01; Fig. 10A and B). Treatment with 10 or 100 mg/kg chicoric acid significantly attenuated the MCAO-induced increase in p-c-Jun (P<0.01; Fig. 10A and B), whereas no significant difference was observed in the 1 mg/kg chicoric acid group compared with the cerebral I/R injury group. Levels of p-AKT protein were significantly lower in the cerebral I/R group compared with the control group (P<0.01; Fig. 10A and C). Treatment with 10 or 100 mg/kg chicoric acid significantly increased p-AKT protein expression in cerebral I/R injury rats (P<0.01; Fig. 10A and C); however, no significant difference was observed between the 1 mg/kg chicoric acid and cerebral I/R injury groups.