Malondialdehyde (MDA), superoxide dismutase (SOD), NO, NOS, calcium (Ca²⁺) and Na⁺-K⁺-ATP assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Bcl-2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA), while antibodies against β-actin were purchased from Tianjin Jinmai Gene Mapping Technology Co., Ltd. (Tianjin, China). The PQDS were obtained from Dr Yanping Chen and dissolved in physiological saline for use. All other chemicals were analytical reagents.

Male Sprague-Dawley rats (weight, 230–280 g) were purchased from the Experimental Animal Center of Jilin University (Changchun, China). All rats were allowed free access to food and water. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Jilin University, and approved by the Ethics Committee of Jilin University.

The rats were randomly divided into five groups (12 rats in each group): i) Sham surgery, where physiological saline was administered to the rats by intraperitoneal (i.p.) injection at a dose of 2 ml/kg; ii) model, where physiological saline was administered to the rats by i.p. injection at a dose of 2 ml/kg; iii) PQDS 12.5 mg/kg, where rats were treated with PQDS by i.p. injection at a dose of 12.5 mg/kg; iv) PQDS 25.0 mg/kg, where rats were treated with PQDS by i.p. injection at a dose of 25.0 mg/kg; and v) PQDS 50.0 mg/kg, where rats were treated with PQDS by i.p. injection at a dose of 50.0 mg/kg. The drug or saline treatment was administered once a day for three consecutive days. Thirty minutes following the final treatment, the rats were anesthetized for the induction of cerebral ischemia.

The MCAO was performed with a modification, as described previously (14). Briefly, the rats in the model and PQDS treatment groups were anesthetized with a 10% i.p. injection of 10% choral hydrate (350 mg/kg). The right common carotid artery, external carotid artery and internal carotid were exposed through a neck incision. The external carotid artery was cut and a monofilament nylon suture with a heated-rounded tip was inserted into the external carotid artery and gently advanced into the internal carotid artery, until a slight resistance was felt. The sham-surgery rats underwent the same surgical procedure with the exception of the MCAO ligation. The suture was tightened around the intraluminal filament to prevent bleeding and the suture was left in place until the rats were sacrificed. The body temperature of the rats was monitored with a rectal probe and was maintained at 37±0.5ºC by a heating pad throughout the surgery.

Twenty-four hours subsequent to the induction of the ischemia, the neurological deficits in each rat were assigned a score, using a scale as previously described (15): 0, no observable deficit; 1, difficulty in fully extending the contralateral forelimb; 2, unable to extend the contralateral forelimb; 3, mild circling to the contralateral side; 4, severe circling; and 5, falling to the contralateral side.

The infarct volume was assessed using the 2,3,5-triphenyltetrazolium chloride (TTC) method, as described previously (16). The rats were sacrificed and the brains were extracted following the measurement of the neurological deficit score. Each brain was cut into five coronal slices with a blade, and the brain slices were stained with 2% TTC solution at 37ºC for 30 min. Following staining, the areas of cerebral infarction were identified using the different color-tones (white for ischemic cerebral tissue and red for non-ischemic cerebral tissue). The infarct size was calculated as a percentage fraction of the ischemic cerebral tissue in the whole brain.

The brain water content was measured using the wet-dry method (17). Following the measurement of the neurological deficit score, the rats were anesthetized using chloral hydrate (350 mg/kg, i.p.) and then sacrificed, prior to the rat brains being rapidly extracted. The pons and olfactory bulb were removed and the wet weight of the brains was measured using an electronic balance. Subsequently, the brains were dried for 24 h at 100ºC in order to obtain the dry weight. The brain water content (BW) was calculated as follows: BW = [(wet weight - dry weight) / wet weight] × 100, and used as an index for brain edema.

Following the collection of the blood samples, the brains were removed, weighed and homogenized in ice-cold phosphate-buffered saline (PBS). The homogenate was centrifuged (2,500 × g, 15 min) and the supernatant was obtained to measure the activities of SOD and Na⁺-K⁺-ATPase and the levels of MDA and Ca²⁺ using assay kits and a spectrophotometer (7202B; Unico (Shanghai) Instrument Co., Ltd., China), in accordance with the kit manufacturer's instructions.

Having measured the neurological deficit score, the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.). Blood samples were collected from the abdominal artery into non-heparinized tubes, allowed to clot for 2 h at room temperature and then centrifuged at 2,000 × g for 15 min. The NO level and the activity of NOS were measured using diagnostic kits according to the manufacturer's instructions.

Having measured the neurological deficit score, the rats were sacrificed by decapitation. The brains were rapidly removed and placed into 4% paraformaldehyde solution for one day and then embedded in paraffin. Five-micrometer sections were cut and the brain sections were stained with hematoxylin and eosin (H&E). The brain sections were subsequently examined under a microscope (Nikon ECLIPSE 80i; Nikon, Tokyo, Japan) and photomicrographs were taken.

The expression of Bcl-2 protein in the rats was analyzed using western blotting. Briefly, the brains were dissected and homogenized with a lysis buffer [1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and phenylmethylsulfonyl fluoride (PMSF)] to extract the cellular proteins, prior to being centrifuged at 1,200 × g for 15 min at 4ºC. The protein concentration was determined using the bicinchoninic acid (BCA) method. Equal quantities of protein were separated using 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequent to blocking with 5% non-fat milk in PBS-Tween-20 (PBST) for 1 h, the membranes were incubated overnight at 4ºC with anti-Bcl-2, and anti-β-actin antibodies. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Tianjin Jinmai Gene Mapping Technology Co., Ltd.) against rabbit for 1 h at room temperature. Immunoactive bands were visualized with an enhanced chemiluminescence (ECL) detection system using X-ray film. β-actin was used as an internal control.

All data are expressed as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett's test. In all cases, P<0.05 was considered to indicate a statistically significant difference.

Twenty-four hours subsequent to ischemia, the neurological deficits of the rats were assessed. The neurological deficit scores for the sham, MCAO and PQDS 12.5, 25.0 and 50.0 mg/kg groups were 0, 3.6±0.70, 3.2±0.63, 2.7±0.67 and 2.4±0.84, respectively. The behavioral abnormalities were particularly apparent in the MCAO group, while PQDS treatment (25.0 and 50.0 mg/kg) significantly suppressed the development of the behavioral abnormalities (P<0.05; Table I and Fig. 1).

To investigate the effect of PQDS on cerebral ischemia, the infarcted area and brain water content were measured. As shown in Fig. 2A, no infarcted area was observed in the sham group. The infarcted areas in the PQDS 12.5, 25.0 and 50.0 mg/kg groups were 27.5±3.78, 23.33±3.50 and 18.83±4.49%, respectively, which were lower than that in the MCAO group (31.17±5.56%). The infarcted area tended to be smaller following treatment with 12.5 mg/kg PQDS, although the reductions were not statistically significant.

The brain water content following 24 h ischemia is shown in Fig. 2B. In the sham group, the water content was 79.63±3.29%. Ischemia led to a significant increase in water content in the MCAO group compared with that in the sham group (83.09±2.57%, P<0.05). However, in the PQDS treatment groups, the water content was significantly decreased in a dose-dependent manner. The mean brain water contents were 80.71±1.08, 80.55±1.75 and 80.27±3.03% in the 12.5, 25.0 and 50.0 mg/kg PQDS groups, respectively.

To illustrate the effect of PQDS on the oxidative stress induced by MCAO, the levels of MDA and Ca²⁺ and the activities of SOD and Na⁺-K⁺-ATPase were measured. The levels of MDA and Ca²⁺ were increased in the MCAO group compared with those in the sham group (Fig. 3). PQDS treatment (25.0 and 50.0 mg/kg) significantly reduced the levels of MDA and Ca²⁺ in a dose-dependent manner compared with those in the MCAO group. Conversely, significant reductions in the activities of SOD and Na⁺-K⁺-ATPase were observed in the MCAO group, which were significantly attenuated by PQDS treatment (25.0 and 50.0 mg/kg).

The changes in the NO and NOS levels are shown in Fig. 4. The NO level and NOS activity were increased in the MCAO group compared with those in the sham group. PQDS treatment significantly reduced the NO level and NOS activity in a dose-dependent manner compared with those in the MCAO group.

To gain an insight into the apoptotic signaling, the expression of the antiapoptotic protein Bcl-2 was analyzed. The level of Bcl-2 protein expressed in the MCAO group was markedly decreased compared with that in the sham group, which was consistent with previous studies (18). Pretreatment with PQDS significantly increased the expression of Bcl-2 compared with that in the MCAO group, suggesting that the protective effects of PQDS may be mediated by the inhibition of apoptosis (Fig. 5).

As shown in Fig. 6, numerous pyramidal neurons were observed in the sham-surgery group, while marked morphological changes were observed in the model group, such as neuronal cell loss, nuclear shrinkage, neuronal vacuolization and dark staining of the neurons. Pretreatment with PQDS (25.0 and 50.0 mg/kg) markedly attenuated these pathological changes; however, 12.5 mg/kg PQDS exhibited no effect.