CT (cat. no. 447-43-0) was provided by the PUSH Bio-Technology. Fetal bovine serum and Dulbecco's modified Eagle's medium (DMEM) were offered by Gibco (Thermo Fisher Scientific, Inc.). Streptomycin and penicillin were purchased from HyClone (GE Healthcare Life Sciences). Nimodipine injection (cat. no. 12301323) was provided by Bayer Schering Pharma AG. Lactate dehydrogenase (LDH) assay kit (cat. no. 20090723), total protein extraction (cat. no. 20150323) and BCA protein quantification kits (kit no. 20150323) were provided by Nanjing Jiancheng Bioengineering Institute. ZSGB-BIO supplied horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat. no. ZB-2301). Primary antibodies against α-tubulin (cat. no. 2148), Apaf-1 (cat. no. 8723), Bcl-2 (cat. no. 2876) and Bax (cat. no. 2772) were provided by Cell Signaling Technology, Inc. Antibodies against procaspase-9 (cat. no. ab2013), procaspase-7 (cat. no. ab25900), cleaved-caspase-3 (cat. no. ab32042) and procaspase-3 (cat. no. ab44976) were provided by Abcam. Cleaved-caspase-7 (cat. no. AF4203), cleaved-caspase-9 (cat. no. AF5240) were supplied by Affinity Biosciences.

The mol2, a molecular structure recording format designed by Sybyl molecular simulation software (version no. 2.1.1; Certara), the mol2 format of CT was downloaded from PubChem (http://pubchem.ncbi.nlm.nih.gov). CT and its interacting protein crystals (the structure is a protein complex with an inhibitor) were introduced into the Maestro 11.1 software ligprep module (Schrödinger, Inc.). The energy optimization of CT in the first field (OPLS-2005) was minimized. The structures of caspase-9, caspase-3 and caspase-7 with endogenous ligands were obtained from the Protein Data Bank. Using the Maestro 11.1 software Glide module, the target protein is modified, dehydrated and hydrogenated under default parameters. The active site of docking is generated by centering on the original ligand. Finally, CT is molecularly docked with the target protein (Fig. 2).

The PC12 cells were obtained from the rat adrenal medulla of pheochromocytoma and have been widely used as an in vitro cellular model of neurological diseases and cell signal transduction pathways due to its sympathetic neuron's physiological characteristics. PC12 cells were provided by the Chinese Academy of Sciences Shanghai Cell Biology Institute. PC12 cells were cultured in high-glucose DMEM supplemented with 5% fetal calf serum, streptomycin (100 µg/ml), penicillin (100 U/ml) and 5% CO₂ at 37°C (19,20).

A stock solution was prepared using dimethyl sulfoxide, CT concentrations were set at 2.5, 5 and 10 µM according to our preliminary experimental results. Nimodipine is a calcium channel blocker, and it was used as a positive control drug in this experiment at a concentration of 5 µM (21). Prior to the investigation, the cells were inoculated in culture plates at a density of 1.0×10⁵ cells/ml. After 12 h of adhering, to initiate oxygen-glucose deprivation (OGD) by replacing the cell culture medium with the glucose-free medium, the cells were incubated for 2 h in an oxygen-free chamber (5% CO₂; 95% N₂) at 37°C. At the end of the OGD period, the cells were incubated under normal growth conditions (5% CO₂ and 95% O₂) for 24 h to achieve reperfusion (R) (22). Cells in the experimental groups were treated with CT (2.5, 5 or 10 µM) and Nimodipine (5 µM) during the entire period of OGD/R.

Following OGD/R, the cells were washed three times with PBS, and the inverted microscope (Olympus Corporation; TH4-200; magnification, ×200) was used to capture the images.

A CCK-8 assay measured the cell viability, according to the manufacturer's protocols. PC12 cells were cultured in 96-well plates (1×10⁴ cells/well) and, following OGD/R, CCK-8 (10 µl per well) was added, and cells were incubated for 4 h at 37°C. The OD value at the wavelength of 450 nm was detected by an enzyme labeling instrument.

Cytotoxicity was assessed by measuring the level of LDH in the culture medium. Following reperfusion, the medium was collected for LDH level measurement. The LDH level was measured by spectrophotometry determination at 440 nm, according to the manufacturer's protocol. The release of LDH reflects the degree of cell damage.

The percentage of the apoptotic cells was determined using an Annexin V-FITC/PI kit (Nanjing KeyGen Biotech Co., Ltd.). The PC12 cells were cultured in 6-well plates (1×10⁶ cells/well), washed twice with ice-cold PBS following treatment, collected by trypsinization without EDTA. Next, cells were suspended in 400 µl 1X Annexin V and then Annexin V-FITC staining fluid 5 µl was added in the dark, at 4°C for 15 min, prior to 10 µl PI staining fluid being added at 4°C for 5 min. Finally, the cells were analyzed by flow cytometry (BD Biosciences) using BD Accuri C6 software (version no. 1.0.264.21; BD Biosciences) The total apoptosis rate is equal to the early apoptotic rate plus the dead cells rate.

Disruption of the MMP is one of the earliest intracellular changes following apoptosis induction (19). It has been reported that the decrease in MMP is associated with the apoptosis of numerous types of cells (23). JC-1 is a probe commonly used to detect the MMP of cells. When the MMP is high, JC-1 may accumulate in the mitochondrial matrix and form polymers to generate red fluorescence. When the MMP is low, the monomer state glows green. Following reperfusion, the cells were washed twice with PBS. Next, the cells were pretreated and stained according to the method of the MMP detection kit (Nanjing KeyGen Biotech Co., Ltd.). The fluorescence microscope (IX-73; Olympus Corporation; magnification, ×200) detected the intensity of fluorescence in each group.

Fluo-3 AM is the most commonly used for the detection of intracellular [Ca²⁺]. Following reperfusion, the PC12 cells were washed twice with PBS and incubated with 5 µM/l Fluo-3/AM staining solution at 37°C for 30 min in the dark. Subsequently, the cells were washed twice with PBS. Next, with a fluorescence microscope (IX-73; Olympus Corporation; magnification, ×200) to observe the fluorescence intensity.

The protein expression of Apaf-1, cleaved-caspase-7, procaspase-7, cleaved-caspase-3, procaspase-3, cleaved-caspase-9, procaspase-9, Bcl-2 and Bax in the cells following OGD/R injury were detected by Western blotting. Following treatment, cells were collected and cell lysates were prepared by incubation in RIPA buffer containing a protease inhibitor cocktail (Beijing Leagene Biotech Co., Ltd.) according to the manufacturer's protocols. Following protein concentration estimation using a BCA protein quantitation assay kit (Nanjing KeyGen Biotech Co., Ltd.), equal amounts (30 µg) of proteins were separated via 10% SDS-PAGE, prior to being transferred to a PVDF membrane. The membranes were blocked with 5% skimmed dry milk in tris-buffer solution (TBST) for 2 h at room temperature. Membranes were incubated with primary antibodies for overnight at 4°C, and the primary antibodies used were as follows: Bcl-2 (dilution, 1:1,000), Bax (dilution, 1:300), Apaf-1 (dilution, 1:300), procaspase-9 (dilution, 1:1,000), procaspase-7 (dilution, 1:500), procaspase-3 (dilution, 1:500), cleaved-caspase-3 (dilution, 1:500), cleaved-caspase-7 (dilution, 1:500), cleaved-caspase-9 (dilution, 1:500) and α-tubulin (dilution, 1:1,000). Next, labelled membranes were washed with TBST three times, and the membrane was probed with an HRP-labeled secondary antibody (dilution, 1:2,000; BIOSS) for 1 h at room temperature. The bands were then visualized using the Amersham Imager 600 imaging system (GE Healthcare Life Sciences).

Immunofluorescence was detected to investigate the expression of Bcl-2 and Bax. Following reperfusion, the cells were washed 3 times with PBS, then post-fixed in 4% paraformaldehyde at room temperature for 20 min and incubated in 0.3% Triton X-100 for a further 20 min. Cells were washed with PBS and blocked with 1% BSA at 37°C for 2 h. Next, Bcl-2 (dilution, 1:100) and Bax (dilution, 1:100) antibodies were added and incubated overnight. Cells were washed with PBS, fluorescent second antibody (dilution, 1:500) was added and incubated for 2 h in the dark at room temperature, the cells were washed 3 times with PBS, and DAPI (dilution, 1:500) was added for 30 min. Finally, cells were viewed using a fluorescence microscope (Olympus IX71; Olympus Corporation; magnification, ×100).

The data were analyzed by Image Proplus and SPSS 17.0 software (SPSS, Inc.). Data are presented as the mean ± standard deviation, and the changes in variable parameters were analyzed by one-way analysis of variance, followed by the Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

Molecular docking was used to predict the probable targets of CT. The docking results revealed a high affinity of CT towards Caspase-9, Caspase-3 and Caspase-7 (Fig. 2). Caspase-9 showed interactions between CT and Gly306, Leu307, Arg308, Val292, Arg294, Asp297, Val298 and Lys299 forming a hydrogen bond effect. Specifically, CT and an active site with residues Arg294 forms a hydrogen bond effect. Caspase-7 was interacting with CT through Val226, Pro227, Tyr229, Lys160, Thr163, Ala164, Tyr223 and Phe221. Specifically, CT and an active site with residues, Asn148, forms a hydrogen bond effect. CT was interacting with the Lys82, Leu81, Asn80, Lbu223, Lys224, Gln225, Ala227 and Asp228 residues of Caspase-3 form a strong hydrophobic effect. Specifically, CT and an active site with residues, Lys229, form a hydrogen bond effect.

The results demonstrated that the number of PC12 cells significantly decreased following OGD/R treatment, certain cells became round or floating, and the cells exhibited typical swelling. Compared with the OGD/R group, in the OGD/R+CT group (2.5, 5 and 10 µM), the cell body was relatively full, the membrane was smooth and intact, and the adhesion was good (Fig. 3). The results indicated that CT has a protective effect on PC12 cell injury induced by OGD/R.

As shown in Fig. 4A, PC12 cell viability was significantly decreased following the cell being exposed to OGD for 2 h and reperfusion for 24 h. Following treatment with CT (2.5 and 5 µM), PC12 cell viability was significantly increased (P<0.05). LDH assays were performed as shown in Fig. 4B, and it was revealed that CT (2.5, 5 and 10 µM) significantly attenuated OGD/R-induced LDH leakage.

FITC-Annexin V/PI double staining was used with flow cytometry to analyze the anti-apoptotic capacity of CT under OGD/R conditions (Fig. 5). The results demonstrated that the cell apoptotic rate was significantly decreased following treatment with CT (2.5, 5 and 10 µM). The apoptotic cell rate with CT (10 µM) was higher than CT (5 µM), consistent with the effect of CT on the PC12 cell viability assay following OGD/R injury.

Fluorescence microscopy was used to detect MMP. Following treatment with the CT (2.5, 5 and 10 µM), red fluorescence increased in varying degrees, respectively, compared with the OGD/R group (Fig. 6).

As a second messenger, [Ca²⁺] regulates signal transduction and neurotransmitter release (24,25). Compared with the control group, the green fluorescence of the OGD/R group was very strong, which indicated that OGD/R increased the [Ca²⁺] concentration in PC12 cells. The green fluorescence of cells treated with CT (2.5, 5 and 10 µM) was decreased, suggesting that CT decreased intracellular [Ca²⁺] concentration. The results demonstrated that CT could inhibit the increase of intracellular [Ca²⁺] induced by OGD/R injury (Fig. 7).

The expression of Bax and Apaf-1 were significantly increased following OGD/R. CT (2.5, 5 and 10 µM) inhibited the expression of Bax and Apaf-1. Additionally, following treatment of PC12 cells with OGD/R, the expression of Bcl-2 was decreased, treatment with CT (2.5, 5 and 10 µM) significantly attenuated the decrease in the Bcl-2 expression (Fig. 8).

In the present study, the expression of cleaved-Caspase-3, cleaved-Caspase-7, cleaved-Caspase-9, procaspase-3, procaspase-7 and procaspase-9 was investigated. As shown in Fig. 8, compared with the control group, the expression of cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-7 were significantly enhanced following OGD/R. CT (2.5, 5 and 10 µM) may markedly inhibit the expression of these proteins.

The expression of Bcl-2 protein was significantly decreased following OGD/R. However, following the intervention with CT (5 µM), red fluorescence was enhanced, which indicated that CT may improve the expression of Bcl-2 (Fig. 9). As shown in Fig. 9, the expression of Bax was notably increased following OGD/R compared with the control group. Compared with the OGD/R group, red fluorescence became weaker, which indicated that CT may inhibit the expression of Bax.