Langendorff non-circulatory perfusion was employed to evaluate the protective effects of TP against I/R in rat cardiac tissues. Krebs-Henseleit (KH) buffer was equilibrated with 95% O₂ and 5% CO₂ at pH 7.4, and was flushed continually at 37°C. A total of 36 rats were randomly grouped into six and were anesthetized via intraperitoneal injection of heparin sodium (1,000 U/kg) and 10% chloral hydrate (350 mg/kg) for 20 min. The hearts were rapidly excised and placed in ice-cold KH buffer. The aorta of the control group was fixed with an infusion tube and perfused at a constant perfusion pressure of 75 mmHg using a Langendorff non-circulatory perfusion pump, for 170 min. The other groups underwent ischemia for 30 min following perfusion for 80 min, and were then reperfused for 60 min. A fluid-filled balloon was inserted into the left ventricle and attached to a pressure transducer. A cardiac pacemaker was used to generate a heart rate of 280 beats/min. The present study was approved by the Institutional Animal Care and Use Committee (IACUC-20130315-01).

Cardiac tissues were fixed in 10% formalin for 48 h. Tissues were dehydrated using ethanol and were cleared with xylene, after which they were embedded in paraffin and cut into 4-7 μm sections. Slides of the tissue sections were then deparaffinized, dehydrated and stained with hematoxylin and eosin (H&E). Infiltrating neutrophils were identified and visualized under a microscope.

For TUNEL staining, the slides were deparaffinized and dehydrated as aforementioned. The sections were digested for 40 min, and were then incubated with 50 μl TUNEL buffer at 37°C for 1 h, and 50 μl POD at 37°C for 30 min, respectively. Finally, the slides were stained with DAB for 3-10 min, sections were visualized under a microscope and apoptotic rate was calculated.

H9C2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 1% ×100 mycillin at 37°C in an atmosphere containing 5% CO₂. To study the effects of TP on I/R cells, H9C2 cells were then digested, seeded into 96-well plates (3×10³ cells/well) and divided into five groups in triplicate. The control group was incubated in normal culture medium. The I/R group was transferred into sugar- and serum-free DMEM, and was incubated at 37°C in an atmosphere containing 5% CO₂ and 1% O₂ for 2 h, after which, cells were transferred into normal DMEM and were cultured for 6 h. The TP groups were treated with various concentrations of TP for 1 h, and underwent hypoxia-reoxygenation as described in the I/R group. Furthermore, cultured cells were treated with N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC), in order to investigate the mechanisms underlying the protective effects of TP against I/R injury. To study the effects of TP on H₂O₂-treated cells, cells were pretreated with NAC, PDTC or TP for 1 h, and then treated with 100 μM H₂O₂ for 24 h. Cultured and treated cells were prepared for further experiments.

The ELISA method was used to measure the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in cardiac tissues and H9C2 cells by determining the absorbance at 450 nm. Briefly, ~10 mg cardiac tissues or 100 μl cultured cells were processed according to the manufacturers' protocols. TNF-α, IL-1β and IL-6 concentrations were calculated according to the standard curve, which was generated from a series of known concentrations of standard bovine serum albumin.

Cardiac antioxidant status was determined by measuring cardiac superoxide dismutase (SOD) content. Tissue samples were homogenized in 100 mmol/l Tris-HCl buffer and were centrifuged at 10,000 × g for 20 min. SOD, malondialdehyde (MDA) and catalase (CAT) levels were assessed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total protein concentration in heart homogenates was determined using the Coomassie blue method.

Cultured and treated H9C2 cells were treated with 100 μl serum-free DMEM containing 10% CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) at 37°C for 1 h in an atmosphere containing 5% CO₂. The optical density of cell suspensions was measured at 450nm using a spectrophotometer, in order to evaluate the viability of H9C2 cells.

Cultured and treated H9C2 cells were treated with 0.25% trypsin, in order to form a single cell suspension, and were washed with 10% PBS and centrifuged at 1,000 × g for 5 min. The supernatant was discarded and cells were then incubated with the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Diego, CA, USA) for 10 min at room temperature in the dark. Cell apoptotic rate was measured and data were obtained using flow cytometry (FACSCalibur; BD Biosciences).

Cultured cells were fixed with 3% paraformaldehyde and incubated at room temperature for 5 min. Subsequently, the cells were air-dried and stained with 10 ml Hoechst 33258 (Beyotime Institute of Biotechnology, Jiangsu, China) for 10 min, followed by the addition of 50% glycerol containing 20 mmol/l citric acid and 50 mmol/l orthophosphate. A fluorescence microscope was used to evaluate nuclear morphology.

Cultured cells were washed with PBS and digested using trypsin following treatment with PA for 24 h; followed by centrifugation at 1,500 rpm for 10 min. Cells were then collected and resuspended in staining buffer containing 50 μM dihydroethidium (Wegelasi Biotechnology Co., Ltd.). ROS was measured by flow cytometry.

Briefly, 20 mg tissue samples were cut into pieces and proteins were extracted using 250 μl radioimmunoprecipitation assay buffer (Shanghai Solarbio Science & Technology Co., Ltd., Shanghai, China) containing 0.01% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). In addition, cultured cells were washed twice with 1X PBS, and were lysed at 4°C. Furthermore, nuclear and plasma proteins were extracted from tissue samples using the NE-PER kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequently, samples were centrifuged at 12,000 × g for 15 min at 4°C and the supernatants were collected. The bicinchoninic acid protein quantification kit (Thermo Fisher Scientific, Inc.) was used to quantify protein contents. Tissue and cell proteins were separated by 15% (80 μg/well) or 10% (25 μg/well) SDS-PAGE, respectively, and proteins were electrophoretically transferred to a nitrocellulose filter membrane (EMD Millipore, Billerica, MA, USA). Blots were blocked with 5% skim milk at room temperature for 1 h, and were then incubated with anti-B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), caspase-3, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and inducible nitric oxide synthase (iNOS) (Abcam, Cambridge, UK), cyclooxygenase (COX)2, nuclear factor (NF)-κB, phosphorylated (p)-NF-κB, NF-κB inhibitor α (IκBα), p-IκBα, extracellular signal-regulated kinase (ERK)1/2, p-ERK1/2 and GAPDH antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA). and were incubated with goat anti-mouse or anti-rabbit secondary antibodies (Beyotime Institute of Biotechnology). Enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) was used to visualize the blots.

Data are presented as the means ± standard deviation of at least three independent replicates. The results were analyzed using analysis of variance followed by the Tukey multiple comparisons test. GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform data analysis. P<0.05 was considered to indicate a statistically significant difference.

H&E and TUNEL staining were performed to evaluate the effects of I/R on control and TP-treated cardiac tissues. As shown in Fig. 1A, cells in the I/R group exhibited swelling, necrosis and degeneration, alongside marked neutrophil infiltration. Conversely, less severe cell necrosis was determined, and stained cells exhibited normal architecture in the PDTC- and TP-treated groups, thus indicating that I/R induced cell damage, whereas TP exerted protective effects against I/R. TUNEL staining demonstrated that I/R induced cell apoptosis (Fig. 1B); apoptotic cells were stained brown in the I/R group. The number of positively stained cells was reduced in the TP-treated groups, thus indicating the inhibitory effects of TP against I/R-induced cell apoptosis. In addition, Bcl2 was increased and Bax was decreased, as determined by western blot analysis, indicating that cell apoptosis was inhibited by TP compared with in the I/R group (Fig. 2A and B).

TNF-α, IL-1β and IL-6 were evaluated using ELISA, in order to investigate the inflammatory response following I/R in rat myocardial cells. Significant increases in the expression levels of TNF-α, IL-1β and IL-6 were detected in the I/R group, whereas these increased levels were attenuated in the TP-treated groups in a dose-dependent manner (Fig. 2C). Similarly, the relative nuclear and plasma protein expression levels of NF-κB were decreased in the TP-treated groups compared with in the I/R group (Fig. 2A and B).

The levels of cardiac enzymes, SOD, MDA and CAT, were measured to evaluate the oxidative damage caused by I/R (Fig. 2D and E). Although higher levels of SOD and MDA were detected in the I/R and TP-treated groups compared with in the control group, the enzymatic activities of SOD and MDA were reduced in the TP-treated groups compared with in the I/R group in a dose-dependent manner. In addition, CAT activity exhibited almost no alteration between the I/R and TP-treated groups. Furthermore, LOX-1 was measured by western blot analysis; the protein expression levels of LOX-1 were attenuated compared with in the I/R group (Fig. 2A and B), thus suggesting that TP exerts protective effects against I/R-induced peroxidation.

In order to investigate the possible mechanisms underlying the protective effects of TP against I/R injury in cardiac cells, an in vitro study was performed using H9C2 cells. The CCK-8 assay was performed to evaluate the viability of H9C2 cells after 2 h of ischemia and 6 h of reperfusion. Compared with in the I/R group, cell proliferation in the TP-treated groups was increased in a dose-dependent manner (Fig. 3), thus indicating the improved viability of H9C2 cells following TP treatment.

ELISA was used to measure the expression levels of TNF-α, IL-1β and IL-6 in H9C2 cells after I/R. Similar to the expression levels of TNF-α, IL-1β and IL-6 detected in cardiac tissues, the expression levels of these proteins were significantly increased in the I/R group compared with in the control group, and were decreased in the TP-treated groups compared with in the I/R group in a dose-dependent manner (Fig. 4).

Flow cytometry was used to evaluate apoptosis in the control and TP-treated H9C2 cells. I/R-induced cell apoptosis was detected using Annexin V-FITC/propidium iodide (PI) double staining (Fig. 5A). The apoptotic rate was calculated from the percentage of early apoptotic cells presented in the lower right quadrant of the histograms. The apoptotic rate was dose-dependently reduced in the TP-treated H9C2 cells compared with in the I/R group, thus revealing the inhibitory effect of TP against I/R. Hoechst 33258 staining was also used to morphologically detect apoptosis of H9C2 cells through fluorescence staining (Fig. 5B). Nuclear fragmentation and chromosomal condensation were improved in the cells treated with various concentrations of TP compared with in the I/R group. Furthermore, apoptosis of H9C2 cells was measured, in order to evaluate the protective effects of TP against H₂O₂-induced peroxidation. As shown in Fig. 6A and B, the apoptotic rate was increased in the H₂O₂ and I/R groups, whereas the apoptotic rate was significantly reduced in the NAC-, PDTC- and TP-treated groups.

ROS are produced by the mitochondrial electron transport chain in cells, and are involved in cell apoptosis via the regulation of caspases. ROS generation may be detected using dihydroethidium, which has free access into live cells through the cell membrane. As shown in Fig. 7A, compared with in the control group, the fluorescence intensity of ROS was increased in H9C2 cells in the I/R group. However, there was a significant dose-dependent decrease in ROS generation in the TP-treated groups compared with in the I/R group. In addition, reductions in SOD, CAT and MDA expression in the TP-treated groups indicated the antioxidative effects of TP (Fig. 7B and C). As presented in Fig. 8, TP exerted protective effects against H₂O₂-induced peroxidation, according to the decreased fluorescence intensity of ROS in NAC-, PDTA- and TP-treated groups.

The expression levels of inflammatory-associated proteins, NF-κB and IκBα, were measured by western blot analysis (Fig. 9A and B), as was the expression of iNOS (Fig. 9A and C). IκBα was downregulated in the I/R group, whereas NF-κB was upregulated. Conversely, the expression levels of NF-κB were decreased and IκBα was increased in the TP-treated groups in a dose-dependent manner; similar findings were detected in the NAC and PDTC groups. The phosphorylation of IκBα and NF-κB was increased in the I/R group, but decreased in the TP-treated groups (Fig. 10). In addition, upregulation of iNOS was detected following I/R, whereas in the TP-treated groups iNOS was decreased in a dose-dependent manner, thus indicating the inhibitory effects of TP during I/R.

COX2 and LOX-1 were measured using western blot analysis, in order to evaluate the peroxidative damage caused by I/R (Fig. 9A and C). COX2 and LOX-1 expression levels were increased following I/R, whereas in the TP-treated groups COX2 and LOX-1 were decreased in a dose-dependent manner.

ERK1/2 phosphorylation was also detected by western blot analysis (Fig. 9A and B). The phosphorylation of ERK1/2 was increased in the I/R group, but decreased in the TP-treated groups. Furthermore, the expression levels of caspase-3 and Bax were increased in the I/R group compared with in the control group, whereas Bcl2 was decreased (Fig. 9A and C). Conversely, caspase-3 and Bax expression were decreased by TP in a dose-dependent manner, whereas Bcl2 was increased in the TP-treated groups, thus indicating the inhibitory effects of TP against cell apoptosis.