Mouse neuroblastoma cell line N2a was purchased from BeNa Culture Collection (Jiangsu, China) and incubated in culture medium (Dulbecco's modified Eagle's medium and opti-Minimum Essential Medium 1:1, both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) in a 5% CO₂ incubator at 37°C. The medium was replaced with fresh medium every two days. The cells were used for propagation or experimental use when 70–80% confluent.

The cells were divided into five groups; Negative control (NC) group, IR injury group, PSP low dose treatment group (PSP1; l 10 mg/ml), PSP middle dose treatment group (PSP2; 30 mg/ml) and PSP high dose treatment group (PSP3; 50 mg/ml).

When the cells were cultured to 80% confluence, the cells in the NC group were continually cultured in complete culture solution, while the cells in the IR and PSP groups were treated with an equal volume of equilibrated medium, containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 1 mM NaH₂PO₄, 0.9 mM CaCl₂ and 10 mg/l phenol red. The cells were incubated in 5% CO₂ and 95% N₂ for 30 min at 37°C, and subsequently the equilibrated medium was replaced with complete culture solution. Different concentrations of PSP (Xi'an Tianrui Biological Technology Co., Ltd. Xi'an, China) were added to the PSP groups.

The cells were seeded on a 96-well plate at a density of 10⁵ cells/well and incubated in 5% CO₂ at 37°C. Following cell growth to 80%, different concentrations of PSP were added to the wells. After 24 h, 10 µl (1 g/l) of MTT solution (Gibco; Thermo Fisher Scientific, Inc.) was added into each well and cultured for 4 h at 37°C. The supernatant was removed and 150 µl dimethyl sulfoxide was added. The optical (OD) was measured with the multifunctional microplate reader SpectraMax M5 (Molecular Devices, LLC, Sunnyvale, CA, USA) at 490 nm. Each group was conducted in six parallel experiments.

The medium was collected and centrifuged at 4°C and 380 × g for 5 min to obtain the supernatant. The LDH activity was determined by ELISA. The experimental process was completely performed according to the protocols of an LDH kit (AB102526; Abcam, Cambridge, UK). The OD value of LDH protein was measured using a microplate reader at 450 nm. The standard curve was established, according to the concentration and OD value. The experiment was repeated three times.

The cells were harvested and lysed in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 20 min. Following centrifugation at 44°C and 380 × g for 5 min, the supernatant was collected and the total proteins were quantified, according to a caspase-3 activity assay kit (Jiancheng Bioengineering Institute, Nanjing, China). The measurement of the caspase-3 activity was processed in protein analytic buffer (20 nmol/l HEPES, 2 mmol/l dithiothreitol, 10% glycerol and 20 µmol/l DEVD-pNA) at 37°C for 2 h. The OD value of the sample was measured at 405 nm using a microplate reader. The content of caspase-3 was calculated using the standard curve. The experiment was repeated three times.

A total of 75 healthy male SD rats (10–12 weeks; 200–250 g) were purchased from The Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China). All rats were kept in controlled conditions at 25±3°C and 35–60% relative humidity. A 12-h light/dark cycle was maintained. All the animals were allowed to acclimate to laboratory conditions for ≥1 week prior to the experiment and had free access to water and food. All the animal works were performed stringently, according to the regulation of the Guide for the Care and Use of Laboratory Animals in China (22). The protocol was approved by the Committee of the Ethics of Animal Experiments of Shanghai Jiaotong University Affiliated Sixth Hospital Provincial Centre for Disease Control and Prevention (proposal authorization no. SYXK/2016/050122). All surgeries were conducted using appropriate anesthesia and all efforts were made to reduce the pain and suffering of the animals.

The rats were randomly divided into five groups; sham operation group (sham; n=15), IR injury group (n=15), PSP1 (150 mg/kg; n=15), PSP2 (200 mg/kg; n=15) and PSP3 (250 mg/kg; n=15). First, 10% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was injected intraperitoneally into rats at a dose of 300–350 mg/kg. There were no significant signs of peritonitis. Following anesthesia, the model of the right middle cerebral artery occlusion was selected for further experimental use. In the sham group, the right common carotid artery, external carotid artery and internal carotid artery were separated without a plug bolt. The plug was pulled out in the other groups after 2 h of ischemia, followed by reperfusion for 24 h. The low, middle and high dose group rats received 150, 250, 250 mg/kg, respectively, via intraperitoneal injection at 2 h after ischemia; the sham group and IR group were intraperitoneally injected with an equal volume of saline solution at the same time point.

The neurological assessment was processed, according to the Zea Longa method (23). The neurological deficiency was assessed consistently according to a 5-point scale system: 0, no neurological deficit; 1, the contralateral torso and forelimb may not be thoroughly stretched; 2, when the tail was held, the animal may be turned to the ipsilateral side; 3, no spontaneous motor activity or falling to the left side; 4, unable to walk or loss of consciousness. Each experiment was repeated three times.

The area of cerebral infarction was identified using triphenyl tetrazolium chloride (TTC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining. Following the neurological assessment grading, five rats in each group were intraperitoneally injected with 10% chloral hydrate. The brains were collected following anesthesia and decapitation, and were subsequently placed in −20°C for 15 min. The tissue slices were processed to ~1 mm thickness using a cryostat microtome. The slices were placed in 2% TTC solution, incubated at 37°C for 30 min, and were subsequently fixed in 4% paraformaldehyde at 4°C for 24 h. Images of the slices were captured, the infarct area in each section was calculated using an image analyzer (Image-Pro Plus version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). and the cerebral infarction volume percentage was calculated using the formula:

The infarcted volume was calculated as a percentage of the total brain area.

Male Sprague-Dawley rats were injected intraperitoneally with 10% chloral hydrate (300–350 mg/kg body weight; Sinopharm, Chemical Reagent Co., Ltd.), and the hearts were exposed through thoracotomy following anesthesia. The needle was inserted from the apical position, the right atrial appendage vein was cut open and 0.9% saline solution was rapidly perfused in the heart until the clear liquid outflowed from right atrial appendage, subsequently 4% paraformaldehyde (Sinopharm, Chemical Reagent Co., Ltd.) was perfused to the heart followed by quick decapitation and collection of the brain, which was fixed at 4°C for 24 h with 4% paraformaldehyde. The coronal brain tissue was cut into slices of 3 mm thickness followed by routine embedding in paraffin. The brains were subsequently sliced to 5 µm thickness, dewaxed with xylene and hydrated using a series of graded concentrations of ethanol (100% ethanol, 5 min; 95% ethanol, 1 min; 80% ethanol, 5 min; 75% ethanol, 5 min; distilled water, 2 min). Hematoxylin and eosin (H&E) staining was performed using the routine method at room temperature for 12 min. Following dehydration, sections were treated with xylene at room temperature for 10 min twice. The tissue sections was examined using a light microscope (OriGene Technologies, Inc.) to examine the histopathological morphology at a magnification of ×100.

Western blotting was performed to determine the protein expression of silent information regulator protein (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax), according to a standard protocol. The brain tissues were lysed on ice and centrifuged at 4°C and 13,680 × g for 20 min. The supernatant was extracted using RIPA buffer and the protein concentration was measured using a bicinchoninic acid protein quantification kit. Total proteins (20 µg) were loaded and separated by 12% SDS-PAGE, and transferred to polyvinylidine difluoride membranes (GE Healthcare). The membranes were blocked at 25°C with 5% skimmed milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h and subsequently incubated overnight at 4°C with the individual primary antibodies, rabbit-anti-SIRT1 (9475; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), PGC-1α (#2178, 1:1,000; Cell Signaling Technology, Inc.), Bax (2772; 1:1,000; Cell Signaling Technology, Inc.), Bcl-2 (15071; 1:1,000; Cell Signaling Technology, Inc.), caspase-3 (9662; 1:1,000; IDUN Pharmaceuticals, Inc., San Diego. CA, USA), β-actin (4970; 1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA). Following washing for three times with TBS-Tween for 5 min every time, the membrane was incubated with secondary antibodies of horseradish peroxidase-conjugated immunoglobulin G (Sigma-Aldrich; Merck KGaA; 1:5,000) for 1 h at room temperature. Following washing three times for 5 min, the proteins were visualized with enhanced chemiluminescent substrates (GE Healthcare), and the image of each band was further quantified using Multigauge computer software version 3.0 (Berthold Australia Pty Ltd, Bundoora, Australia).

All statistical analyses were performed with SPSS software version 13.0 for windows (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. One-way analysis of variance with Bonferroni's correction was used to compare the statistical difference between multiple groups. The Least Significant Difference test was used for comparison between groups. P<0.05 was considered to indicate a statistically significant difference.

Following simulation of IR conditions, the N2a cell viability was analyzed in an MTT assay (Fig. 1). Compared with the NC, the cell viability significantly decreased following IR with a survival rate of ~60% (Fig. 1; P<0.05). Following treatment with PSP, the cell activity was significantly improved compared with the IR group and exhibited a dose-dependent manner (P<0.05). The cell viabilities of the PSP1, PSP2 and PSP3 groups were ~84, 90 and 95%, respectively. The data indicated that treatment with higher doses of PSP resulted in significantly higher cell viability (P<0.05).

Neuronal injury or death was assessed according to levels of LDH released into the extracellular medium as previously described (24). As demonstrated in Fig. 2, the IR group exhibited a significantly higher LDH activity (170%) compared with the NC (P<0.01). Following administration of PSP, the activity of LDH decreased with the concentration of PSP increasing between 10 and 50 mg/ml. A high dose of PSP was able to significantly decrease the activity of LDH with the expression percentage of 105% in culture medium compared with the NC (P<0.01; Fig. 2), which suggests that PSP is able to reduce the cell membrane permeability and consequently reduce injury to protect cells.

The activity of caspase-3 was determined by caspase-3 specific substrates. The results demonstrated that the activity of caspase-3 was significantly increased by up to ~90% following IR, and decreased markedly following treatment with PSP (P<0.01; Fig. 3). The activity of caspase-3 following treatment with PSP1, PSP2 and PSP3 was ~125, 120 and 110%, respectively. The results suggested that PSP may reduce the activity of caspase-3 to reduce apoptosis.

Neurological function assessment is a crucial procedure for the medical care of neurological patients, which may detect the presence of a neurological disease or injury and monitor its progression. In the present study, each group of rats was scored as 0–3 with no subarachnoid hemorrhage observed, suggestive of successful establishment of the model. As demonstrated in Table I, the mice in the sham group had no neurological deficit symptoms. In the IR group, the rats exhibited a neurological deficit with a higher score of 2.63 (P<0.01) compared with the sham group. Compared with the IR group, the neurological deficits following treatment with PSP were reduced (P<0.05), particularly in the PSP3 group (P<0.01). The neurological function scores of the low and middle-dose PSP groups were 2.17±0.25 and 1.53±0.33, respectively, which is higher compared with high-dose PSP (1.16±0.19). The neurological deficit score was the lowest in the PSP3 group.

To assess the protective effects of PSP against brain ischemia injury in vivo, a model of ischemia was established. Infarction size was assessed by the appearance of a white region following TTC staining (Fig. 4). The results demonstrated that there was no white region observed in brain tissue in the sham group, and the infarction was observed in the IR group and PSP groups. Compared with the IR group (42.93%), the infarction volume in the PSP treatment groups decreased significantly (P<0.01). Among the PSP administration groups, the score of PSP3 group (25.16%) was lower compared with PSP1 (36.17%; P<0.01) and PSP2 (33.53%; P<0.05). In addition, there was a significant difference between low- and middle-dose group (P<0.01). These results indicated that PSP may effectively reduce the infarction size in a dose-dependent manner.

H&E staining was used to examine the morphological alterations of the hippocampus area upon treatment with PSP following IR injury. As demonstrated in Fig. 5, the nerve cells in the sham group were structurally complete, arranged neatly, stained evenly and clearly. In the IR group, the number of nerve cells decreased, were arranged disorderly and the stain color deepened. Compared with the IR group, the number of cells in the PSP-treated groups increased, and the morphology of the nerve cells recovered well. These results indicated that PSP may reduce brain cell death in the hippocampus area caused by IR and reduce brain injury.

To verify whether the SIRT1 signaling pathway participates in the protection from IR injury mediated by PSP, the expression of SIRT1 and PGC-1α was examined by western blotting following IR injury. As demonstrated in Fig. 6, the expression of SIRT1 and PGC-1α was decreased in the IR group, with ~0.3 fold and ~0.25 fold change, respectively, compared with the sham group (P<0.01). Following intraperitoneal injection of different concentrations of PSP (150, 200 and 250 mg/kg), the expression of SIRT1 and PGC-1α was upregulated (P<0.05), particularly in the PSP3 group (~0.9 fold change and ~1.1 fold change, respectively) compared with the IR group. These results demonstrated that PSP may upregulate the expression of neuroprotective factors SIRT1 and PGC-1α, which may protect cerebral IR injury in rats.

The effect of PSP on Bax, Bcl-2 and caspase-3 was additionally examined by western blotting (Fig. 6). Compared with the sham group, the Bax/Bcl-2 protein expression level in the IR group was significantly increased at ~160% (P<0.01). Compared with the IR group, the Bax/Bcl-2 protein expression in the PSP group was downregulated to ~120, 90 and 68%, corresponding to PSP1, PSP2 and PSP3 (P<0.05). It is demonstrated in Fig. 6 that the caspase-3 protein expression level in the IR group was significantly increased (P<0.01) compared with the sham group. However, following treatment with PSP, the expression of caspase-3 protein was significantly decreased (P<0.05). A dose of 250 mg/kg PSP was able to downregulate the expression of caspase-3 more effectively. These data suggested that the protection of PSP against cerebral IR in rats may be due to an increase in Bcl-2 protein expression and a decrease in Bax and caspase-3 protein expression, thus reducing the apoptosis rate and reducing injury.