Attenuation of doxorubicin-induced cardiotoxicity by cryptotanshinone detected through association analysis of transcriptomic profiling and KEGG pathway

Effects of CPT on cardiomyocyte viability

CPT at different concentrations did not decrease the viability of myocardial H9c2 cells at all the time points examined (Figure 1B). DOX treatment significantly reduced H9c2 cell viability, which was ameliorated by 10μM CPT treatment for 12 h (P<0.05, Figure 1C).

Effects of CPT on the surface area of cardiomyocytes

H9c2 cells were stained with F-actin, and the changes in the cell surface area were examined to determine the effects of CPT on DOX-induced changes in H9c2 cells in vitro. No significant difference was found in the surface area of cardiomyocytes between the control (Ct) group and the Ct + CPT group (Figure 1D). Treating H9c2 cells with DOX for 12h significantly reduced the surface area of cardiomyocytes (P<0.05, Figure 1D). However, the cells treated with 10μM CPT for 12h had a significantly increased cardiomyocyte surface area compared with the cells treated with DOX alone (P<0.05, Figure 1D).

Effects of CPT on DOX-induced H9c2 apoptosis, ROS production, and decline in mitochondrial membrane potential

Apoptosis, ROS production and mitochondrial membrane potential (MMP) in H9c2 cardiomyocytes were assessed using flow cytometry. Treating H9c2 cells with DOX for 12 h significantly increased ROS production, reduced MMP, and induced apoptosis (P<0.05; Figure 1E–1G). Further, 10 μM CPT significantly suppressed DOX-induced H9c2 apoptosis, ROS production, and MMP reduction. Moreover, CPT had no obvious effect on the apoptosis rate, level of ROS production, and MMP in H9c2 cardiomyocytes (Figure 1E–1G).

CPT alleviated DOX-induced cardiac dysfunction in rats

In this study, echocardiography and hemodynamic examination were conducted to evaluate cardiac function in rats. The cardiac function of the Wistar rats significantly reduced after six injections of DOX within 2 weeks. The ejection fraction (EF) (%) and shortening fraction (FS) (%) of the rats in the DOX group significantly reduced (P<0.05; Figure 2A and 2B). Moreover, the hemodynamic examination also showed that DOX induced a left shift of the pressure-volume (PV) loop. Importantly, the EF (%) and FS (%) significantly increased in rats orally administered with CPT and DOX injection compared with those treated with DOX alone (P<0.05; Figure 2A and 2B). Meanwhile, CPT also induced a significant right shift of the PV loop along the horizontal axis (Figure 2C). These results indicated that CPT treatment alleviated DOX-induced rat cardiac insufficiency. Further, the rat cardiac function was neither upregulated nor downregulated by the oral administration of CPT in the Ct + CPT group compared with the Ct group (Figure 2).

Figure 2. Oral administration of cryptotanshinone (CPT) ameliorated cardiac dysfunction in doxorubicin-induced injury. Evaluation of cardiac function by echocardiography with/without CPT treatment, indicated by ejection fraction (EF, %) of LV and fractional shortening (FS, %) of LV, in the doxorubicin-induced rat model (A, B). Measurement of invasive hemodynamic examinations for analyzing both LV pressure and volume (pressure-volume loop) simultaneously, also in the rats model, indicated by end-systolic pressure (Pes), end-diastolic pressure (Ped), maximal rates of the increase in ventricular pressure (dp/dt_max) and maximal rates of decline in ventricular pressure (dp/dt_min) (C). ^*P<0.05 vs control, ^#P<0.05 vs DOX.

Effects of CPT on DOX-induced rat cardiac histology

The cardiac histology was assessed using hematoxylin-eosin (HE), Masson’s trichrome stain (MTS) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to evaluate the effects of CPT on DOX-induced changes in rat cardiac histological morphology. At the end of the observation after the injection of Wistar rats with DOX, cavities were found in the nucleus; also, the cardiomyocytes were loosely aligned, and the surface areas of the cardiomyocytes significantly reduced (P<0.05; Figure 3A). However, following 6 weeks of CPT treatment, the number of hollow nuclei reduced, the cardiomyocytes were more tightly aligned, and the surface areas of the cardiomyocytes significantly increased (P<0.05; Figure 3A). DOX induced significant collagen deposition in the hearts of rats, and the level of cardiac collagen significantly increased compared with that in control Wistar rats (P<0.05; Figure 3B). Further, TUNEL assay showed that CPT significantly reduced the DOX- induced cardiomyocyte apoptosis rate in the hearts of rats (P<0.05; Figure 3C). Moreover, consistent with the previous findings, CPT treatment did not cause collagen deposition and apoptosis in the hearts of rats compared with the control group (Figure 3).

Figure 3. Cryptotanshinone (CPT) gavage attenuated myocardial fibrosis and apoptosis in rats treated with doxorubicin. Hematoxylin and eosin staining for paraffin section (A). Masson’s trichrome staining for fibrosis (B). TUNEL assay for apoptotic cardiac cells (C). Values are expressed as mean ± standard error of the mean. ^*P<0.05 vs control, ^#P<0.05 vs DOX. Scale bar: 50 μm.

CPT reversed DOX-induced MMP reduction and suppressed ROS production in rat cardiomyocytes

DOX significantly downregulated MMP levels (Figure 4A) and drastically increased ROS levels in rat cardiomyocytes (Figure 4B). After 6-week CPT treatment, the DOX-induced reduction in cardiac MMP levels in rats was significantly attenuated, and ROS levels were suppressed (Figure 4A and 4B). However, its intervention did not upregulate the levels of MMP and ROS in cardiomyocytes of the Ct + CPT group compared with of the Ct group (Figure 4A and 4B). Also, the levels of antioxidants and pro-oxidants in the cardiomyocytes of rats were measured, revealing that CPT significantly elevated the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) levels but reduced the malondialdehyde (MDA) level (P<0.05; Figure 4C). Consistent with the aforementioned results, no significant difference was found in the levels of antioxidants and oxidants between the Ct and the Ct + CPT groups (Figure 4C). These results indicated that CPT suppressed DOX-induced ROS production and reversed DOX-induced MMP reduction. Meanwhile, CPT improved the antioxidant capacity of the hearts of the rats.

Figure 4. Cryptotanshinone (CPT) alleviated myocardial mitochondrial membrane potential (MMP) decline and oxidative stress in rats treated with doxorubicin. JC-1 fluorescent mitochondrial depolarization of LV cardiomyocytes for evaluating MMP (A). Detection of intracellular ROS by observing the fluorescence intensity of DCF in the LV of the rats (B). The levels of SOD, MDA, CAT, and GSH-Px in LV tissues of the rats induced by doxorubicin (C). Values are expressed as mean ± standard error of the mean. ^*P<0.05 vs control, ^#P<0.05 vs DOX. CAT, Catalase; GSH-Px, glutathinone peroxidase; MDA, malonydialdehyde; SOD, superoxide dismutase. Scale bar: 50 μm.

Transcriptomic profiling and KEGG pathway analysis

The changes in cardiac gene expression was examined using transcriptomic profiling to investigate the mechanism underlying the cardioprotective function of CPT in the DOX-induced cardiotoxicity rat model (Figure 5A and 5B). The results indicated that 589 differentially expressed genes were closely involved in cell growth and death (comparison between the DOX + CPT and DOX groups, with P<0.05 indicating a significant difference in gene expression levels between the two groups, Figure 5C). Considering the cardiotoxicity of DOX and that KEGG analysis of the differentially expressed genes showed a difference in the p53 signaling pathway, the following experiments focused on the effects of CPT and DOX on the p53 signaling pathway that is involved in the apoptosis of cardiomyocytes of rats (Figure 6A–6C).

Figure 5. Effects of the different regulated genes and pathways of cryptotanshinone (CPT) on the heart tissues of the DOX-treated rats using transcriptomic profiling. Changed genes in the heart tissues of the two groups displayed by a heat map (D1-3 = DOX group, DC1-3 = DOX + CPT group) (A). The volcano map exhibits the differentially regulated genes by comparison of the two groups (DOX and DOX + CPT groups); the gray scatter denotes the non-significantly differentially expressed genes; both red and green scatters denote the significantly differentially expressed genes; the x-axis represents log₂ fold change, and the y-axis represents the –log₁₀P value (B). KEGG pathway classification; the horizontal axis represents the ratio (%) of the total number of genes (differentially expressed genes) annotated with each level 2 metabolic pathway and the genes (differentiated genes) annotated to the KEGG pathway, and the vertical axis represents the level 2 pathway term; the number on the right side of the column represents the annotation of the number of differentially expressed genes in the level 2 pathway term (C).

Effects of CPT on the expressions of signaling proteins of p53 pathway

The effects of CPT on the expression of some critical signaling proteins in this pathway were examined by Western blot analysis to confirm the changes in the expressions of proteins related to the p53 signaling pathway identified in using transcriptome sequencing and bioinformatics analysis. After the injection of DOX into Wistar rats, the expression levels of myocardial 14-3-3σ and p-c-Jun N-terminal kinase (JNK) were significantly elevated in the rats of the DOX group (P<0.05; Figures 7A and 8A). Meanwhile, the expression levels of PI3 kinase p85 and p-AKT were all significantly downregulated (P<0.05; Figures 6A and 7A). However, after treatment of DOX-injected rats with CPT for 6 weeks, the expression levels of PI3 kinase p85 and p-AKT were all significantly upregulated compared with those in the DOX group (P<0.05; Figures 6A and 7A). CPT further suppressed the DOX-induced 14-3-3σ expression and p-JNK phosphorylation (P<0.05; Figures 6A and 7A). Moreover, CPT intervention alone did not affect the expression level and the phosphorylation level of the aforementioned proteins, including 14-3-3σ, p85, p-JNK and p-AKT, in the hearts of Wistar rats (P<0.05; Figures 6A and 7A).

Figure 6. Top 20 of pathway enrichment statistics based on the differentially expressed gene in the rat hearts after cryptotanshinone (CPT) treatment. Scatter plot of KEGG pathway enrichment statistics (the total number of differentially expressed genes) (A). Scatter plot of KEGG pathway enrichment statistics (the number of differentially expressed genes, which were downregulated (B). p53 signaling pathway in a KEGG map (C). Enrichment score is the ratio of the number of differentially expressed genes to the number of all genes in this pathway term.

Figure 7. Cryptotanshinone (CPT) treatment adjusted p53 signaling pathway in the rat heart induced by doxorubicin. Representative blots of 14-3-3σ, p-JNK, JNK, PI3K p85, p-AKT, AKT, and GAPDH (A). Representative blots of Bax, Bak, Bim, PUMA, Bcl-2, Bcl-xl, and GAPDH (B). Representative blots of pro-caspase-3/cleaved caspase-3, pro-caspase-9/cleaved caspase-9, and GAPDH (C). Representative blots of Foxo1, p53, PCNA, and β-tubulin (D). GAPDH, β-tubulin and PCNA were used as internal reference.

Additionally, the DOX-induced downregulation of protein expression of B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) in rat cardiomyocytes was attenuated by CPT treatment (P<0.05; Figures 7B and 8B). Meanwhile, the expressions of cardiac Bcl-2 associated x (Bax), Bcl-2-antagonist/killer (Bak), Bcl-2 interacting mediator of cell death (Bim), and P53 up-regulated modulator of apoptosis (PUMA) in the rats of the DOX and DOX + CPT groups. CPT significantly suppressed the expressions of these proteins (P<0.05; Figures 7B and 8B). Cleaved caspase-9 and cleaved caspase-3, related to mitochondria apoptosis, were significantly upregulated. However, CPT significantly suppressed the cleavage of caspase-9 and caspase-3 (P<0.05; Figures 7C and 8C).

Figure 8. Statistical analysis of the relative protein expression of 14-3-3σ, PI3K p85, p-JNK, p-AKT, cleaved caspase-9, cleaved caspase-3, Bax, Bak, Bim, PUMA, Bcl-xl, and Bcl-2. Semi-qualification of 14-3-3σ, PI3K p85, p-JNK and p-AKT (A). Semi-qualification of Bax, Bak, Bim, PUMA, Bcl-xl, and Bcl-2 (B). Semi-qualification of cleaved caspase-9 and cleaved caspase-3 (C). Both GAPDH and PCNA were used as internal reference. ^*P<0.05 vs control, ^#P<0.05 vs DOX.

The nuclear translocation of p53 and Foxo1 in the myocardium was examined. The findings revealed that cytoplasmic p53 and Foxo1 were significantly translocated to the nucleus in the cardiomyocytes of DOX-treated rats. Importantly, 6 weeks of oral CPT administration significantly suppressed p53 nuclear translocation and enhanced Foxo1 nuclear retention (Figure 7D).

Drugs and chemical reagents

Doxorubicin hydrochloride for injection was produced by Haizheng-Pfizer Pharmaceutical Ltd. (Shanghai, China). The DOX used for in vitro experiments was a product of Solarbio (Beijing, China). CPT was purchased from the Desite Biotechnology Co., Ltd. (Chengdu, China) and MedChemExpress LLC (Shanghai, China).

Cell culture and experimental design

The rat H9c2 cardiomyocyte cell line was acquired from the Cell Biology Laboratory of the Advanced Research Center in Central South University. Cardiomyocytes were cultured in the Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Shanghai, China) containing 15% fetal bovine serum, 200 U/mL penicillin (Gibco) and 200 μg/mL streptomycin (Gibco) in an incubator supplemented with 5% CO₂ at 37°C. The cells were plated in cell culture plates at a certain density and synchronized in serum-free RPMI 1640 medium for 6 h after culturing for 18 h. After synchronization, the RPMI 1640 complete culture medium containing 2, 5 and 10μM CPT was added to each well, with each concentration having three replicates. The H9c2 cell viability was assessed at 0, 3, 6 and 12 h after CPT treatment using the CCK-8 kit (Beyotime, Shanghai, China). Additionally, 1.0x10⁴ cells were plated onto a 96-well plate and divided into 5 groups: control group (Ct) treated with phosphate buffer saline (PBS), DOX (2μM) group, DOX (2μM) + CPT (2μM) group, DOX (2μM) + CPT (5μM) group, and DOX (2μM) + CPT (10μM) group. The cell viability was measured at 3, 6 and 12 h after the treatment, with each group having three replicates.

F-actin staining and flow cytometry

H9c2 cardiomyocytes were plated onto a six-well glass-bottom cell culture plate (NEST, Wuxi, China). When the cells reached a confluence of approximately 60%, PBS, culture medium containing CPT (10μM) and DOX (2μM), and culture medium containing DOX (2μM) + CPT (10μM) were added and cultured for 12 h. The cells were then subjected to F-actin staining using phalloidin (Abcam, Shanghai, China). Images were taken from 10 random fields using a Zeiss fluorescence microscope, and the cell surface area was analyzed using Image-Pro Plus 6.2 software. The MMP level, ROS level and apoptosis of the rat H9c2 cardiomyocytes in each group were assessed using a JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide, JC-1) kit (Beyotime), a ROS detection kit (DOJINDO, Shanghai, China) and an Annexin V-FITC (fluorescein isothiocyanate, FITC) apoptosis detection kit (BD Bioscience, CA, USA) respectively, according to the manufacturers’ protocols.

Animal model and experimental design

The animals used in this study were male Wistar rats, weighing 220−260 g, acquired from the SJA Laboratory Animal Co., Ltd. (Hunan, China). Housing and experimental procedures were performed in accordance with the guidelines of the Experimental Animal Center of Hunan Province, Department of Laboratory Animal Sciences of Xiangya School of Medicine, Central South University and Hunan Provincial Laboratory Animal Public Service Center. All rats were housed in a specific pathogen free (SPF) environment under a light−dark cycle with free access to food and water.

A total of 28 Wistar rats were randomly divided into the healthy control group (Ct), healthy control with oral administration of CPT group (Ct + CPT), DOX treatment group (DOX), and DOX + CPT treatment group (DOX + CPT), with 7 rats in each group. The detailed treatments were as follows:

1. The rats in the Ct group (n=7) were orally treated with 1 mL of saline (0.9% sodium chloride solution) every day.

2. The rats in the Ct + CPT group (n=7) were orally administered with CPT at a dose of 50mg/kg once every day for a total of 6 weeks.

3. The rats in the DOX group (n=7) were injected with doxorubicin hydrochloride at a dose of 2 mg/kg, with three injections each week and a total of six injections.

4. The rats in the DOX + CPT group (n=7) were injected with doxorubicin hydrochloride at a dose of 2 mg/kg, with three injections each week and a total of six injections. Meanwhile, the rats in this group were orally administered with CPT at a dose of 50 mg/kg [14] once every 2 days for a total of 6 weeks.

After intervention, some rats were randomly selected from each group and subjected to echocardiography and hemodynamic examination. Then, all the rats were sacrificed according to the animal welfare regulations. Blood was extracted and treated with heparin for anticoagulation. Plasma was isolated after centrifugation and stored in a −80°C freezer for future use. The heart was isolated, and parts of the left ventricle were stored in liquid nitrogen or soaked in TRIzol reagent (Thermo Fisher Scientific, Shanghai, China) for the subsequent experiments. The remaining tissue was soaked in 10% paraformaldehyde and stored at room temperature.

Evaluation of cardiac function

At the end of the experiment, three rats were randomly selected from each group for echocardiography. First, the rats were anesthetized with 20% urethane solution. The cardiac function was then examined using the Vivid 7.0 Doppler ultrasound (GE, Illinois, USA); an ultrahigh-frequency probe was used (10 MHz). The LV function was assessed from the parasternal long- and short-axis views at the apical level of the mitral valve. End systole and diastole volumes were defined as the LV minimum and maximum capacities, respectively. The EF% and FS% were measured using the LV-M mode with a scanning speed of 50 mm/s.

Hemodynamic examination is an invasive means of assessing the changes in the LV pressure and volume in the animal being assessed. At the end of the observation, each rat was anesthetized with a 20% urethane solution at a dose of 5 mL/kg. A catheter (PowerLab, ADInstrument, New Zealand) with a pressure-responsive electrode (Millar, Texas, USA) was inserted at the proximal end through the left carotid artery of the rat. After inserting the head of the catheter with the pressure-responsive electrode into the LV of the rat, the PV-loop data was recorded when a smooth and stable pressure loop appeared.

Determination of oxidative and antioxidative stress markers

The supernatants of rat cardiac tissue homogenates were used to measure the activities of antioxidative stress-related enzymes and non-enzymatic substances. SOD, GSH-Px, CAT, and the nonenzymatic substance glutathione were measured using commercially available kits from Beyotime Biotechnology Inc. (Shanghai, China) and the Jiancheng Bioengineering Institute (Nanjing, China). The lipid peroxidation product MDA was measured using a kit from Solarbio Life Sciences (Beijing, China).

Histochemistry staining analysis

The rat heart fixed in paraformaldehyde was embedded in paraffin and sectioned at a thickness of 4−6 μm. The tissue sections were subjected to HE staining and MTS, as previously described. Images were taken by two independent investigators using inverted microscopy and analyzed using Image-Pro Plus 6.0.

Measurements of MMP and ROS

MMP was measured using the mitochondrial membrane potential assay kit with a JC-1 kit (Beyotime Biotechnology). Conversion of JC-1 from red fluorescence into green fluorescence is an early marker for apoptosis. The probe DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) (Beyotime Biotechnology) was used to determine the ROS levels in the frozen cardiac tissue sections. Intracellular ROS oxidizes nonfluorescent DCFH to form fluorescent DCF. The fluorescent signals of DCF were evaluated for intracellular ROS levels.

TUNEL assay

Apoptosis was assessed by TUNEL. Cardiomyocyte apoptosis in the 4-μm-thick frozen sections was examined using the TUNEL one-step fluorescence staining kit (Beyotime Biotechnology) according to the manufacturer’s protocol. Apoptotic cells were shown in green, and nucleus 4',6-diamidino-2-phenylindole (DAPI) staining was shown in blue. Some cardiomyocytes from eight randomly selected fields were counted from each animal heart tissue sections and the apoptosis rate were calculated using number of TUNEL-positive cells/number of total cells.

Transcriptomic profiling and KEGG analysis

The total RNA from the cardiac tissue was extracted using the Trizol reagent (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s protocol. The total RNA was extracted from the sample. After DNA digestion using DNase, mRNA was enriched with oligo (dT)–coupled magnetic beads. The mRNA was broken into short fragments using the breaking reagent. Single-stranded cRNA was synthesized with the six-base random primers using the broken mRNA as templates. Double-stranded cDNA was then synthesized in the double-stranded reaction system and purified. Purified double-stranded cDNA was end-repaired, tagged with an A tail, ligated to the sequencing linker, subjected to fragment size selection and then subjected to PCR amplification. After the qualification analysis using the Agilent 2100 Bioanalyzer, the constructed cDNA library was sequenced using the Illumina HiSeq X Ten sequencer to generate double-ended data of 125 or 150 bp. The raw imaging data files from the high-throughput sequencing were converted into the original sequencing results by base calling, which were called raw data or raw reads. The results were stored in the FASTQ file format, which included the sequencing information of the reads and its corresponding sequencing quality information. A large number of sample double-ended sequencing data were acquired through the Illumina platform. Due to the impact of the data error rate on the results, Trimmonatic [37] was used to perform quality preprocessing on the raw data and statistically summarize the number of reads in the entire quality control process. HISAT2 [20] was used to compare the sequence of clean reads with the specified reference genome and acquired their locations on the reference genome or genes as well as the sequence characteristic information specific to the sequenced sample. The expression level of protein-coding genes was calculated by the fragments per kb per million reads (FPKM) method [38], which showed the number of fragments per kilobase length from a protein-encoding gene per million fragments. FPKM considered the effects of both the sequencing depth and the length of protein-coding genes on the counting of fragments. It is currently the most common method for evaluating the expression levels of protein-encoding genes. Differential expression analysis aimed to identify differentially expressed genes among different samples. After acquiring the differentially expressed genes, the gene ontology (GO) functional significance and KEGG pathway significance analyses were performed [39]. The transcriptomic profiling and bioinformatics analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Western blot analysis

The cardiac tissue from LV (approximately 20 mg) was homogenized in a homogenizer and lysed in radio immunoprecipitation assay (RIPA) buffer (Abcam, Shanghai, China) containing protease and phosphatase inhibitors (MedChemExpress LLC) for 5−10 min. The lysates were centrifuged at 12,000g for 10 min at 4°C, and the supernatants were collected for cardiac protein analysis. The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (BosterBio, Wuhan, China) according to the manufacturer’s protocols. Denatured proteins were separated by 6−12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane (Amersham, GE Healthcare, Germany). The membrane was blocked in a 5% bovine serum albumin (BSA) solution (BosterBio) for 2 h at room temperature and incubated with the primary antibody overnight at 4°C. The primary antibodies used in this study included 14-3-3σ (mouse, 1:1000 dilution; Santa Cruz Biotechnology, Shanghai, China), PI3Kinase p85 (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc., Shanghai, China), p-AKT (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), AKT (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), p-JNK (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), JNK (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), Bcl-2 (mouse, 1:1000 dilution; Santa Cruz Biotechnology), Bcl-xl (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), Bax (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), Bak (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), Bim (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), Puma (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), p53 (mouse, 1:1000 dilution; Cell Signaling Technology, Inc.), Foxo1 (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), caspase-9 (mouse, 1:1000 dilution; Santa Cruz Biotechnology), caspase-3 (rabbit, 1:1000 dilution; Cell Signaling Technology, Inc.), GAPDH (mouse, 1:1000 dilution; OriGene, Beijing, China), PCNA (mouse, 1:1000 dilution; OriGene) and β-tubulin (mouse, 1:1000 dilution; OriGene). The secondary antibodies used in this study were goat anti-mouse IgG (peroxidase conjugated, H+L; Millipore, MA, USA) diluted at 1:5000 and goat anti-rabbit IgG (peroxidase conjugated, H+L; BosterBio) diluted at 1:1000. The membrane was incubated with secondary antibodies for 2 h. The membrane was exposed using the chemiluminescent HRP substrate (Millipore), and the luminescence of the membrane was detected using a chemiluminescent imager (SmarChemi^TM, SAGECREAATION, Beijing, China).

Statistical analysis

SPSS 16.0 was used for statistical analysis. Data were subjected to one-way analysis of variance, chi-squared test and t-test. Quantitative data were represented by mean ± standard deviation. A P value less than 0.05 was considered statistically significant.