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Introduction

Ischemic stroke is one of the most important causes of death and disability worldwide (1,2). In China, stroke became the top leading cause of mortality in 2017. Approximately 1.5 million people die from strokes each year (3). In ischemic stroke, the insufficient supply of oxygen and glucose causes brain damage within the subsequent few hours (4,5). The primary method is to restore the blood supply of ischemic brain tissue as soon as possible. However, the reopening of the occluded cerebrovascular usually leads to pathological damage in the ischemic tissue, which may further aggravate or potentially make the damage irreversible. Neuronal injury caused by ischemia/reperfusion is a complex process involving various mechanisms, including apoptosis, oxygen-free radicals, glutamic acid toxicity and [Ca2+] overload (6). It remains an area of interest to perform drug research on stroke treatment (7).

Apoptosis is an essential mechanism of ischemic stroke (8,9). Apoptosis is regulated through the death receptor pathway, mitochondrial pathway and endoplasmic reticulum pathway. In the majority of vertebrates, apoptosis is regulated by the mitochondrial pathway (10,11). Mitochondria are the active centers of apoptosis regulation and the executors (12). The pro-apoptotic factor causes the mitochondrial permeability transition pore (MPTP) to open excessively, decreasing the membrane potential, releasing cytochrome c from the mitochondria to the cytosol. Next, caspase is activated and cells undergo apoptosis (13,14).

Mu-Xiang-You prescription is a classic prescription of Hui medicine, which affects ischemic stroke (15). Aucklandia lappa Decne is the main drug in the prescription (45% by weight); therefore, we hypothesized that Aucklandia lappa Decne is an essential drug in treating ischemic stroke. CT (C15H20O2; Fig. 1), an active sesquiterpene lactone, is one of the main useful components of Aucklandia lappa Decne. CT is used to control the quality of Aucklandia lappa Decne in Chinese Pharmacopoeia. We hypothesized that the effect of this prescription in the treatment of ischemic stroke is associated with CT. CT has multiple pharmacological activities, including inhibiting tumour cell proliferation (16) and decreasing the inflammatory response (17,18). However, it is unclear whether CT may attenuate cerebral ischemia/reperfusion injury and its anti-apoptosis mechanism. Therefore, the present study aimed to investigate the effects of CT and mechanisms on OGD/R-induced PC12 cell injury.

Figure 1. Chemical structure of CT. The molecular formula for CT is C15 H20 O2 and the molecular weight is 232.32 kD. CT, costunolide.

Materials and methods

Drugs and reagents

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.

Molecular docking

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).

Figure 2. Molecular binding mode in the protein domains of CT docking. (A) Caspase-9, (B) Caspase-7 and (C) Caspase-3. Droplet shapes represent amino acid groups that interact with CT in 4Å. Capital letters represent the abbreviation for amino acids. The numbers represent the numbering of amino acids in proteins. The line represents the CT in the protein cavity. The arrow mark indicates hydrogen bonding interactions between CT and protein domain residues. CT, costunolide.

Cell culture

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).

Drug treatment

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.

Morphological changes in cells

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.

CCK-8 assay and LDH release assay

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.

Flow cytometric apoptosis assay

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.

Mitochondrial membrane potential (MMP) measurement

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.

Intracellular [Ca2+] measurement

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.

Western blot analysis

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 detection

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).

Statistical analysis

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.

Results

Results of molecular docking

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.

Effect of CT on PC12 cell morphological changes

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.

Figure 3. Effect of CT on the morphology of the OGD/R injury model in PC12 cells (magnification, ×200). The arrow points to the cells with morphological changes. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

Effect of CT on PC12 cell viability and cell cytotoxicity

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.

Figure 4. Effects of CT on (A) cell survival and (B) LDH release in PC12 cells following OGD/R. Values are presented as the mean ± standard deviation (n=10). #P<0.05, compared with the control group; *P<0.05 and **P<0.01, compared with the OGD/R group. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion; OD, optical density; LDH, lactate dehydrogenase.

Effects of CT on apoptosis in PC12 cells

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.

Figure 5. Effect of CT on the apoptotic rate following OGD/R in PC12 cells. (A) Cell apoptosis was determined by flow cytometry. (B) Quantitative analysis of apoptotic rate. Values are presented as the mean ± standard deviation (n=3). #P<0.05, compared with control group; *P<0.05, compared with the OGD/R group. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

Effects of CT on MMP in PC12 cells

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).

Figure 6. Effect of CT on mitochondrial membrane potential in PC12 cells following OGD/R injury (magnification, ×200). The left image shows the JC-1 aggregate form, the middle image shows the JC-1 monomer form, and the right image shows the merge of the left and central images. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

Effects of CT on intracellular [Ca2+] in PC12 cells

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).

Figure 7. Effects of CT on intracellular [Ca2+] concentration in PC12 cells following OGD/R injury (magnification, ×200). The CT (2.5, 5 and 10 µM) decreased the intracellular [Ca2+] concentration. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

Effects of CT on the expression of Apaf-1, cleaved-Caspase-3, Caspase-3, cleaved-Caspase-7, Caspase-7, cleaved-Caspase-9, Caspase-9, Bax and Bcl-2 in PC12 cells following OGD/R

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).

Figure 8. Effects of CT on the expression of Apaf-1, Bcl-2, Bax, cleaved-Caspase-9, cleaved-Caspase-7, cleaved-Caspase-3 in PC12 cells following OGD/R. The stripe diagram represents Western blot analysis of Apaf-1, Bcl-2, Bax, cleaved-Caspase-9, cleaved-Caspase-7, cleaved-Caspase-3, procaspase-9, procaspase-7 and procaspase-3. α-tubulin was usedd as the loading control. The bar chart represents the quantitative analysis of Apaf-1, Bcl-2, Bax, cleaved-Caspase-9/procaspase-9, cleaved-Caspase-7/procaspase-7 and cleaved-Caspase-3/procaspase-3 expression. Values are presented as the mean ± standard deviation (n=3). #P<0.05, compared with the control group; *P<0.05, compared with OGD/R group. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

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.

Detection of Bcl-2 and Bax in PC12 cells following OGD/R by cellular immunofluorescence

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.

Figure 9. Effects of CT on the expression of Bcl-2 and Bax in PC12 cells following OGD/R (magnification, ×100). The left image shows immunofluorescent staining with specific markers. The middle image shows the staining of the nucleus with DAPI. The right image shows the merge of the left and middle images. CT, costunolide; OGD/R, oxygen-glucose deprivation/reperfusion.

Discussion

Molecular docking is a drug design method that follows the characteristics of the receptor, and the interaction between the receptor and the drug molecule (26). Proteins may undergo conformational transitions in physiological environments, and these transitions may stabilize following ligand binding (27). Drugs are small compounds that act by binding to or interacting with a protein kinase. The intermolecular interactions between protein and medicines may be due to hydrophobic interactions, electrostatic and van der Waals forces (28). The results demonstrated that CT contains binding sites with Caspase-9, Caspase-7 and Caspase-3. It suggested that these proteins may be the target of CT anti-apoptosis.

According to a previous study, the PC12 injury model induced by OGD/R is often used to mimic cerebral ischemia/reperfusion injury (29). This model was used in the present study to investigate the effect and mechanisms of CT. The results demonstrated that CT may protect PC12 cells from OGD/R-induce injury by enhancing cell viability and inhibiting LDH leakage.

Apoptosis plays an essential role in maintaining metabolic balance and controlling neuron damage. Available evidence has suggested that apoptosis serves a significant role in the cerebral ischemia/reperfusion process (30,31). The results of the present study demonstrated that the number of apoptotic cells increased significantly following OGD/R. CT (2.5, 5 and 10 µM) significantly decreased apoptosis cells. However, the anti-apoptotic effect of 10 µM CT was not as significant as that of 5 µM CT. In early experiments on the cytotoxicity of CT on PC12 cells, it was found that after the CT dose increased to 10 µM, it became cytotoxic, which led to an increase in apoptosis. Therefore, it may cause the apoptotic cell rate with 10 µM CT to be higher than 5 µM CT, but the toxicity of 10 µM CT is minimal, and it has a specific protective effect on PC12 cells following OGD/R injury. Therefore, 5 µM CT has a better anti-apoptotic effect than 10 µM CT. MMP is a sensitive indicator reflecting mitochondrial function. The decrease in MMP leads to instability of neuronal cell structure, thereby causing cell damage and ultimately results in cell death (22). A recent study has confirmed that OGD/R may cause intracellular [Ca2+] overload and lead to mitochondrial dysfunction, resulting in unstable neuronal cell structure and ultimately leading to neuronal apoptosis (32). Following treatment with OGD/R in PC12 cells, the MMP decreased, and the intracellular [Ca2+] increased. However, CT decreased intracellular [Ca2+] overload and enhanced MMP. These results indicated that CT has a protective role in PC12 cell injury induced by OGD/R.

A mitochondria-mediated apoptosis signalling pathway is one of the main pathways in the apoptotic of cerebral ischemia-reperfusion injury. The Bcl-2 protein family and its members form an incredibly complex network of interactions that regulate apoptosis. These proteins serve an essential role in regulating and controlling apoptosis (33). Bcl-2 is an essential anti-apoptotic protein that may stabilize mitochondrial membrane function and prevent mitochondria release Cyto-c (34). Bax is a pro-apoptotic protein in the cytoplasm under normal conditions. Bax increases the permeability of the mitochondrial membrane, which leads to the release of Cyto-c under certain stimulating conditions (35,36). Next, in the presence of dATP, Cyto-c binds to Apaf-1 to catalyze the activation of Caspase-9, and then activate downstream effector Caspases, including Caspase-3, Caspase-6 and Caspase-7, which in turn execute apoptosis by cleaving cellular proteins following specific Asp residues (37). In the mitochondria-mediated pathway, Caspase-3 is the main influencing factor in the process of apoptosis, and its activation is a sign that the cell is entering an irreversible stage of apoptosis (15). In the present study, the expression levels of Bcl-2 decreased, while the protein levels of Bax, Apaf-1, cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-Caspase-7 significantly increased in OGD/R-treated PC12 cells. These results were consistent with those reported in the literature (38,39). Reversal of these CT trends suggested that the protective effect of CT may be associated with the inhibition of mitochondrial-mediated apoptosis in PC12 cells.

Costunolide had a protective effect against OGD/R-induced PC12 cell injury, and the mechanism may be associated with the inhibition of mitochondrial-mediated apoptosis.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of Ningxia (grant no. 2020AAC02017) and the National Natural Science Foundation of China (grant no. 81660700).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LM, HM and JM established the PC12 cell injury model induced by OGD/R, and were responsible for detecting cell viability, LDH, [Ca2+] concentration, mitochondrial membrane potential and protein expression. TL and HM analyzed and interpreted the data. LM wrote the manuscript. YZ and QZ designed the study, supervised the research group and revised the manuscript critically for important intellectual content. YZ and QZ confirmed the authenticity of all the raw data. The final version of the manuscript was read and approved by all authors.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References