H9c2 embryonic rat heart-derived cells were obtained from Academia Sinica (Shanghai, China). Picroside II (purity>99%) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and dissolved in sterile distilled water. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were provided from Gibco RBL (Grand Island, NY, USA). Trizol reagent was a product of Invitrogen Life Technologies (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit was purchased from MBI Fermentas, Inc. (Vilnius, Lithuania). Cytochrome c and β-actin antibodies were from Abcam (Cambridge, UK). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33342, caspase-3 activity assay kits and reactive oxygen species (ROS) detection kit were from Beyotime Biotechnology (Jiangsu, China). Lactate dehydrogenase (LDH) and creatine kinase (CK) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

H9c2 cells were cultured in DMEM supplemented with 15% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂, with medium changed every 2–3 days. The cells were passaged regularly. When they reached 80% confluence, cells were made quiescent by serum starvation (0.5% FBS) for 12 h, and subsequently were treated with picroside II (100 μg/ml) for 48 h prior to H/R. For the H/R experiment, a hypoxic incubator was used to produce an in vitro hypoxia challenge. Cells in the hypoxic incubator were subjected to hypoxia (<0.5% O₂) using pre-conditioned hypoxic medium [8 g NaCl, 0.4 g KCl, 0.14 g CaCl₂, 0.2 g MgSO₄·7H₂O, 1.2 g K₂HPO₄·12H₂O, 0.08 g KH₂PO₄ (pH 7.4)] at 37°C. Hypoxic medium was changed to fresh DMEM medium upon initiation of reoxygenation. The cells were subsequently cultured in the incubator under an atmosphere of 5% (v/v) CO₂ at 37°C. The selected period of hypoxia and reoxygenation time was based on the preliminary time course studies. The most suitable period of H/R was found to be 3 h hypoxia followed by 12 h reoxygenation. Control cells were cultured in normoxic conditions.

Cardiomyocyte viability was measured by the MTT quantitative colorimetric assay. After picroside II treatment and H/R, the medium was removed and the cardiomyocytes were washed twice with phosphate-buffered saline (PBS), subsequently 10 μl MTT solution was added to each well and incubated for an additional 4 h at 37°C. Following this, 100 μl dimethysulfoxide was added to dissolve the formazan product. The absorbance at 490 nm was read on a microplate reader and the data were expressed as a percentage of the control, which was considered 100% viable. CK and LDH, the indicators of cardiomyocyte injury, were detected by the commercially available colorimetric assay kits according to the manufacturer’s instructions.

Apoptotic cells were determined by fluorescent microscopy using the fluorescent dye Hoechst 33342. Briefly, H9c2 cells were seeded at a density of 1×10⁵ cells/well in 12-well plates and cultured as described above. Following picroside II treatment and H/R, the medium was aspirated and the cells were washed twice with PBS. Subsequently, Hoeshst 33342 solution (0.1 mg/ml) was added into each well of the 12-well plate for 20 min at 37°C in dark, followed by another three washes with PBS. Nuclear DNA staining was observed by a fluoresence microscope at 521 nm emission wavelength. The percent of apoptosis was expressed as a ratio of apoptotic to total cells. Caspase-3 is a key enzyme in the apoptosis cell-signaling cascade. The activity of caspase-3 was measured by the commercially available colorimetric assay kit according to the manufacturer’s instructions. The data were expressed as a percentage of the control.

Following picroside II treatment and H/R, total RNA was isolated from the H9c2 cells by the Trizol reagent and quantified by measuring the absorbance at 260 nm. A 1 μg RNA aliquot from each sample was reverse-transcripted to cDNA using the M-MLV Reverse transcriptase kit. The cDNA was used for qPCR. Quantitative analysis of mRNA expression was performed by the ABI 7300 real-time PCR system with the Power SYBR Green PCR Master Mix kit. PCR primers were as follows: caspase-3 primers (sense, 5′-CAAGTCGATGGACTCTGGAA-3′ and anti-sense, 5′-GTACCATTGCGAGCTGACAT-3′); GAPDH primers (sense, 5′-TGGCCTCCAAGGAGTAAGAAAC-3′ and anti-sense, 5′-GGCCTCTCTCTTGCTCTCAGTATC-3′). The PCR amplification profiles consisted of denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 60 sec. All the amplification reactions for each sample were carried out 4 times, and the relative expression values were normalized to the expression value of GAPDH.

H9c2 cells were cultured as described above. Following picroside II treatment and H/R, the cells were collected and the preparation of subcellular fractions was isolated using the Cell Mitochondria Isolation kit. The method was carried out according to the manufacturer’s instructions. Briefly, cells were washed in PBS and centrifuged at 600 × g for 10 min at 4°C. The supernatant was removed and the cells were resuspended in the mitochondria isolation reagent. Following a 15 min incubation on ice, lysates were homogenized and centrifuged at 600 × g for 10 min at 4°C. The supernatant was subsequently centrifuged at 11,000 × g for 10 min to isolate the mitochondria fraction in the pellet, while the supernatant was used to isolate the cytosol. The supernatant was subjected to centrifugation at 12,000 × g for 10 min. The resulting supernatant was designated as the cytosolic fraction, which was used for the detection of cytochrome c.

The levels of ROS were measured by oxidative conversion of cell permeable 2′,7′-dichlo-rofluorescein diacetate to fluorescent dichlorofluorescein. 2′,7′-dichlorofluorescein was added at a final concentration of 10 μmol/l and incubated for 20 min at 37°C. Fluorescence intensity was detected using a fluorescence plate reader at an excitation wavelength at 488 nm and an emission wavelength at 525 nm. The results were expressed as a percentage of the control.

The mPTP opening of cardiomyocytes was measured with calcein-acetoxymethylester (calcein-AM) in the presence of cobalt chloride using the mPTP fluorescence assay kit according to the manufacturer’s instructions. In brief, cardiomyocytes were washed with Reagent A, and subsequently the cells were incubated with Reagents B and C for 20 min at 37°C, prior to washing twice with Reagent A again. Fluorescence intensity was determined by a SprctraMax M5 microplate reader at an excitation wavelength at 488 nm and an emission wavelength at 505 nm. Fluorescence intensity was normalized to total protein concentration in the corresponding cell and the data were expressed as normalized relative fluorescence units (U/mg protein).

Mitochondrial membrane potential was determined by the tetramethylrhodamine ethyl ester (TMRE) fluorescent dye. TMRE is positively charged and highly permeable across the mitochondrial membrane. Mitochondrial depolarization is able to result in the spread of TMRE from mitochondria to cytoplasm, and enhance the whole-cell fluorescence intensity. TMRE was added at a final concentration of 1×10⁻⁷ mol/l and incubated in the dark for 20 min at room temperature. Fluorescence intensity was determined (excitation wavelength at 514 nm/emission wavelength at 590 nm). The data were expressed as a percentage of the control.

Protein concentration was determined by a Bradford protein assay. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and transferred to a nitrocellulose membrane. After being blocked with 5% milk powder in TBST for 1 h at room temperature, the membranes were incubated with the primary antibody for cytochrome c (diluted 1 μg/ml, Cat. no. ab90529) and β-actin (diluted 1:1,000, Cat. no. ab1801) (both rabbit polyclonal antibodies; Abcam) at 4°C overnight, and followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit; (diluted 1:5,000, Cat. no. ab175773; Abcam) at room temperature for 1 h. Detection was performed using an ECL kit according to the manufacturer’s instructions. The results were normalized to β-actin expression.

Data are expressed as the means ± standard error of the mean. All the values were analyzed using analysis of variance and the Newman-Keuls Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay showed that the cell viability was significantly decreased after hypoxia for 3 h and reoxygenation for 12 h. Pretreatment with picroside II for 48 h prior to H/R was able to markedly inhibit the decrease in cell viability (Fig. 1A). In line with the MTT assay, the levels of CK and LDH, which are the indicators of cardiomyocyte injury, were significantly increased in the H/R group, which was also inhibited by pretreatment of picroside II (Fig. 1B). However, picroside II alone had no effect on cell viability and the production of CK and LDH.

The Hoechst 33342 staining assay showed that the percentage of apoptotic cells was significantly increased after hypoxia for 3 h and reoxygenation for 12 h compared to the control group. Consistent with the results of Hoechst 33342 staining, the mRNA expression and activity of caspase-3, the effector caspase of the mitochondrial apoptosis pathway, were also markedly increased in the H/R group. These effects induced by H/R were significantly inhibited by pretreatment of picroside II (Figs. 2 and 3). However, picroside II alone had no effect on cardiomyocyte apoptosis and the mRNA expression and activity of caspase-3.

Compared to the control group, the levels of intracellular ROS were clearly increased after hypoxia for 3 h and reoxygenation for 12 h, which was indicated by the increased fluorescence intensity. However, pretreatment of picroside II significantly inhibited the production of intracellular ROS induced by H/R (Fig. 4). Picroside II alone had no effect on the production of intracellular ROS.

The opening of mPTP was determined with calcein-AM in the presence of cobalt chloride. Calcein-AM fluorescent dye is able to move freely between mitochondrial and cytosol via the opening of mPTP. Cobalt chloride can quench calcein-AM fluorescence, but it cannot eliminate calcein-AM fluorescence in mitochondria when the mPTP closed. Due to this property, the fluorescence intensity of calcein-AM following treatment with cobalt chloride can reflect the extent of mPTP opening. The results showed that the fluorescence intensity was significantly decreased in the H/R group, indicating that the extent of mPTP opening is enhanced following H/R. However, pretreatment of picroside II was able to markedly increase the fluorescence intensity compared to the H/R group (Fig. 5). Picroside II alone had no effect on the fluorescence intensity. These results indicated that picroside II decreased the degree of mPTP opening in response of H/R-induced injury.

The results showed that mitochondrial membrane potential was depolarized after hypoxia for 3 h and reoxygenation for 12 h, which was indicated by the increased fluorescence intensity of TMRE. Pretreatment of picroside II was able to markedly decrease the fluorescence intensity of TMRE compared to the H/R group (Fig. 6). However, picroside II alone had no effect on the fluorescence intensity.

In mitochondria, there was a high level of cytochrome c expression in the control group. However, the protein expression of cytochrome c in mitochondria was notably decreased following H/R. This H/R-induced effect was significantly inhibited by pretreatment of picroside II. By contrast, the cytochrome c expression in the cytosol was lower in the control group compared to the H/R group. Pretreatment of picroside II was able to significantly reduce the increase in cytosolic cytochrome c expression induced by H/R (Fig. 7). Picroside II alone had no effect on cytochrome c expression in the mitochondria and cytosol. Taken together, these results suggested that picroside II has the ability to inhibit cytochrome c release from the mitochondria to cytosol.