The animal use in this study was performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). The use of animals was also reviewed and approved by the China Medical University Animal Care Review Committee. Neonatal Sprague-Dawley 1-to 3-day-old rats [Grade II, Certificate Number SCXK-(Liao) 2009-0001] were purchased from China Medical University (Shenyang, China).

Picroside II (purity >99%), was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological products (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), DMEM glucose-free, new-born calf serum and trypsin were purchased from Gibco-BRL (Carlsbad, CA, USA). Collagenase II was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). ECL reagent kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). Antibodies against Akt, phospho-Akt (Ser-473), CREB and phospho-CREB, were obtained from Bioworld Technology, Inc. (Minneapolis, MN, USA). anti-Bcl-2 antibody, anti-Bax antibody and anti-β-actin antibody was purchased from Boster Bilogical Technology, Ltd. (Wuhan, China). The detection kits of lactate dehydrogenase (LDH), were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). bicinchoninic acid (BCA) protein assay kit, 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin were supplied by Byeotime (Jianshu, China). Annexin V-FITC apoptosis detection kits were obtained from Nanjing KeyGen Biotech, Co, Ltd. (Nanjing, China). All other chemicals were purchased from Sigma-Aldrich. The purity of all reagents was at least analytical grade.

The primary culture of neonatal rat cardiac ventricular myocytes was prepared from Sprague-Dawley rats (1- to 3-day-old) based on a previously published protocol with some modifications (15). Briefly, hearts were harvested and placed in ice-cold D-Hank’s buffer. Ventricles were separated and cut into 1-mm³ pieces. The tissue fragments were digested by treatment with 0.06% trypsin once (37°C) and subsequently with 0.08% collagenase II 4–5 times (37°C). The cell suspension was then filtered and centrifuged for 8 min (120 × g, 4°C), and finally resuspended in Dulbecco’s modifed Eagle’s media supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 U/ml). Resuspended cells were plated onto 6-well plates in a humidified incubator (5% CO₂, 95% air, 37°C) for 2 h to exclude fibroblasts cells based on the fact that the fibroblasts cells attach to the surface more rapidly. To prevent proliferation of fibroblasts, 0.1 mmol/l 5-bromo-2′-deoxyuridine (BrdU) was added to the culture medium. Non-adherent cells were then replated onto fresh 96- and 6-well plates (1.2×10⁵ and 2×10⁶ cells/well, respectively) and incubated for 3–4 days before the experiment.

In the present study, we used the model of hypoxia-reoxygenation in vitro which was similar to that described by Zhu et al (16). Briefly, before starting the experiments, the cultured cardiomyocytes were carefully washed 3 times with Hank’s solution (5 mM HEPES, 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.5 mM CaCl₂; pH 7.2). The cells were incubated in a glucose-free DMEM base medium and then subjected to hypoxia to mimic the in vivo condition of myocardial ischemia. The cells were placed in an incubator at 37°C. N₂ (95%) and CO₂ (5%) were flushed into the incubator to bring the oxygen content down to 1% monitored by an oxygen probe. After 3 h of hypoxia, the cells were reoxygenated by immediately replacing a glucose-free DMEM base medium with a DMEM base medium with 5.5 mM glucose (pH 7.4) followed by incubation under normoxia for 2 h.

After cardiomyocytes had been cultured for ∼72 h, they were in the state of confluence and beat synchronously. The cultured cardiomyocytes were randomly divided into 7 groups: i) control group: cardiomyocytes were incubated under normoxic conditions at 37°C during the entire experimental period; ii) hypoxia/reoxygenation group: cardiomyocytes were incubated with a glucose-free DMEM base medium for 3 h of hypoxia followed by reoxygenation for 2 h as described above; iii) picroside II-50 group: picroside II (50 μg/ml) was applied 48 h prior to hypoxia/reoxygenation; iv) picroside II-100 group: picroside II (100 μg/ml) was applied 48 h prior to hypoxia/reoxygenation; v) picroside II (200 μg/ml) group: picroside II (200 μg/ml) was applied 48 h prior to hypoxia/reoxygenation; vi–vii) cells co-treated with hypoxia/reoxygenation and picroside II (200 μg/ml) were challenged with the alternative PI3K inhibitor wortmannin (wortmannin, 50 nM: picroside II + wortmannin group) or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, 15 μM: picroside II + LY294002 group).

Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cardiomyocytes were seeded on 96-well plates at 1.2×10⁵ cells/well. Following exposure to hypoxia-reoxygenation, cardiomyocytes were treated by the addition of 20 μl MTT solution (5 mg/ml phosphate buffer) for 4 h and the media were removed. The formazan blue crystals, formed by oxidation of the MTT dye, were dissolved in 150 μl DMSO for 10 min at the condition of vibration. The absorbance at 490 nm was measured using a microplate reader (Sunrise RC; Tecan Group, Ltd., Mannedorf, Switzerland) and cell viability was expressed as a percentage of the control culture value.

LDH activity was measured as the LDH content was released in the culture medium. After hypoxia/reoxygenation, 0.2 ml of culture medium was taken and assayed for LDH activity using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with a microplate reader (Sunrise RC; Tecan Group, Ltd.), following the manufacturer’s instructions.

Caspase-3 activity was measured using the caspase-3 assay kit/colorimetric according to the manufacturer’s instructions. Briefly, at the end of hypoxia/reoxygenation, 2×10⁶ cells were collected, centrifuged 1,000 × g for 10 min at 4°C. The cells were resuspended in the lysis buffer, incubated for 5 min on ice bath and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was then reacted with Ac-DEVD-pNA (2 mM), which were the substrates of caspase-3, at 37°C for 2 h. The protein concentration in the supernatant was determined by the BCA method. The absorbance at 405 nm was measured using a microplate reader (Sunrise RC; Tecan Group, Ltd.).

The nuclei of cardiomyocytes were stained with the chromatin dye Hoechst 33258. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After three washes with PBS, the cells were incubated with Hoechst 33258 (5 μg/ml in PBS) at room temperature for 15 min. After rinsing with PBS again, the cells were examined under a fluorescence microscope.

The apoptotic cells were measured by Annexin V-FITC/PI staining. At the end of hypoxia/reoxygenation, cells were harvested, washed with PBS, resuspended in binding buffer (10 mM Hepes/NaOH pH 7.4, 140 mM NaCl and 2.5 mM CaCl₂) and incubated with Annexin-V at room temperature in dark for 15 min. Then the cells were centrifuged, resuspended in the binding buffer and incubated with Propidium iodide (PI). After incubation, 400 μl of binding buffer was added and the cells were analyzed by flow cytometry (FACScalibur; BD Biosciences, USA).

Cardiomyocytes with various treatments were lysed in an ice-cold RIPA buffer [1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mmol/l EDTA, 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 10 mmol/NaF and 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF)]. The lysates were centrifuged at 12,000 rpm for 15 min at 4°C to remove debris. The protein concentration was determined with BCA protein assay. Equal amounts of denatured protein samples (50–100 μg protein/lane) were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were then washed with TTBS 3 times and incubated further with horseradish peroxidase-conjugated secondary antibody (1:2,000) for 2 h at room temperature. The blots were processed using an electrochemiluminescence (ECL) kit and and light emission was captured on X-ray film. The blots were visualized with ECL-plus reagent and then subjected to densitometric analysis. β-actin was used as the internal loading control.

The data are expressed as the mean ± SD. One-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. Differences were considered to be statistically significant at P<0.05.

The results show that pre-incubation of cardiomyocytes with different concentrations of picroside II (0, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg/ml) prevented the loss of viability that resulted from hypoxia/reoxygenation in a dose-dependent manner (data not shown). Maximum viability was apparent at a concentration of 200 μg/ml. However, higher concentrations (400 and 800 μg/ml) of picroside II did not cause any enhancement of this preventive effect. We analyzed the effective half-maximal concentration for protection (EC50) was 50 μg/ml. Therefore, we used concentrations of picroside II (50, 100 or 200 μg/ml) for our subsequent experiments. As shown in Fig. 2, hypoxia 3 h/reoxygenation for 30-150 min significantly decreased the percentage of survival cells in a time-dependent manner. Subjected to hypoxia 3 h/reoxygenation for 120 min, there were only 54.29±3.4% viable cells as compared to the control cells. Thus, hypoxia 3 h/reoxygenation 120 min was used as a standard apoptosis induction in the subsequent experiments.

Fig. 3 shows the results of the time-response investigation during which cells were exposed to 200 μg/ml picroside II for ≤72 h. The magnitude of cell survival peaked at 48 h, when cell viability was 92.3±3.02%. Fig. 4 shows that after exposure to hypoxia 3 h/reoxygenation for 2 h the survival rate of cardiomyocytes was 48.25±4.31%. Pre-incubation with picroside II (50, 100 and 200 μg/ml) prevented cardiomyocytes from hypoxia/reoxygenation-induced damage, and caused a dose-dependent attenuation in cell survival to 74.17± 3.51, 83 ±4.5 and 92.02±3.6%, respectively (all P<0.01 vs. hypoxia/reoxygenation).

The release of LDH in the medium is widely known as an indicator of cardiomyocyte injury. As shown in Fig. 5, LDH concentrations in medium from control group cells were minimal (5.63±0.39 U/l), while LDH activity induced by hypoxia/reoxygenation increased up to (41.5±2.71 U/l). Treatment with picroside II (50, 100 and 200 μg/ml) significantly attenuated LDH activity in a dose-dependent manner (concentrations were 24.02±2.0 U/l, 16.69±1.92 U/l and 10.09±1.38 U/l, respectively).

Hoechest 33258 staining assay showed that cardiomyocytes exposed to hypoxia/reoxygenation featured typical characteristics of apoptosis, including the condensed chromatin and the fragmented apoptotic nuclei. However, the development of these apoptotic features were significantly suppressed when cells were treated with picroside II (200 μg/ml) (Fig. 6).

Flow cytometry revealed that hypoxia-reoxygenation caused a significant increase in the percentage of apoptotic cells from 3.3±0.95% in control cells to 27.09±3.42% (P<0.01) (Fig. 7). However, when picroside II (200 μg/ml) was applied 48 h prior to hypoxia-reoxygenation, it caused a reduction in the percentage of apoptotic cells to 9.05±2.0% (P<0.01) (Fig. 8).

Cardiomyocytes exposed to hypoxia/reoygenation insults had an increase in caspase-3 activation (368.3±32.12) comparing to the control group (100±12.01). Treatment with picroside II (200 μg/ml) for 48 h significantly decreased this hypoxia/reoygenation-induced caspase-3 activation (Fig. 9).

However, the protection of picroside II was partially blocked by pretreating cells with wortmannin or LY294002. These results provide direct evidence that picroside II can significantly inhibit apoptosis in cardiomyocytes induced by hypoxia/reoygenation, and that this effect is partially inhibited by concurrent wortmannin or LY294002 treatment.

The kinetics of Akt activation were examined, to evaluate a possible mechanism for the anti-apoptotic effect of picroside II. As shown in Fig. 10A, hypoxia/reoxygenation significantly reduced the phosphorylation of Akt without alteration of the total Akt expression. Pretreatment of cardiomyocytes with picroside II, prior to hypoxia/reoxygenation exposure, resulted in a significant increase in the phosphorylation of Akt. In addition, CREB, a downstream target of Akt, was also markedly phosphorylated (Fig. 10B). Both 50 nM wortmannin and 15 μM LY294002 markedly inhibited picroside II-induced Akt and CREB phosphorylation.

Akt phosphorylates the transcription factor CREB, implicated in the transcription of the anti-apoptotic Bcl-2 gene (17). We investigated the protein expression of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax. As shown in Fig. 10C, the expression of Bcl-2 protein decreased in the hypoxia/reoxygenation group compared to the control group. However, cells treated with picroside II showed an increase in Bcl-2 protein expression as detected by western blot analysis. Picroside II pretreatment downregulated Bax levels in cells exposed to hypoxia/reoxygenation comparing to untreated cells (Fig. 10D). This effect was partially inhibited by concurrent wortmannin or LY294002 treatment. Picroside II treatment mediated phosphorylation of Akt and CREB, increased Bcl-2 protein expression, and decreased Bax protein expression and caspase-3 activity in cardiomyocytes hypoxia/reoxygenation, which was abolished by LY294002 or wortmannin treatment.