Lysophosphatidylcholine, baicalein, fura-2/AM, β-actin antibody and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Aldrich Chemical Company (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR). Antibodies to Bcl-2, Bax, ERK1/2, JNK and phosphorylated JNK were obtained from Upstate Biotechnology (Lake Placid, NY, USA) while antibodies of p38, phosphorylated p38 and cytochrome c were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Antibodies to cleaved caspase-3, cleaved caspase-9, and phosphorylated ERK1/2 were obtained from Cell Signaling Technology (Beverly MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO BRL Life Technologies (Grand Island, NY, USA). In the experiments, baicalein was prepared by dissolving with DMSO. Dilutions were made in phosphate-buffered saline (PBS) and filtered through a 0.22 μM syringe filter.
The rat ventricular myocardial cell line H9c2 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO BRL Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (Kibbutz Haemek, Israel) containing 100 U/ml of penicillin G, 100 μg/ml streptomycin and 0.25 mg/ml amphotericin B in a humidified atmosphere containing 5% CO2 at 37°C. Before experimental intervention, confluent-cultured cells were serum-starved for 24 h in DMEM supplemented with 0.1% fetal bovine serum.
Determination of cells viability
To assess H9c2 cell viability, MTT assay was performed according to the manufacturer’s instructions. After the experiments, MTT (0.5 mg/ml) was added in the medium for 4 h. The culture medium was removed and the cells were dissolved in isopropanol and shaken for 10 min. The amount of MTT formazan was quantified at absorbance of 540 nm and 630 nm using an ELISA reader (DYNEX Technologies, Germany).
Measurement of intracellular free calcium concentration
Intracellular calcium concentration ([Ca2+]i) was measured as we previously described . Trypsinized cells (1 × 106 cells/ml) were loaded with 2 μM of the ester form of fura-2 (fura-2/acetoxy methyl) for 30 min at 25°C in DMEM. After loading, the cells were kept in a balanced salt solution (BSS, mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 5 glucose, pH 7.4). Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring. The cuvette contained 1 ml of BSS and 5 × 105 cells. The H9c2 cells were incubated in Ca2+-containing buffer with various concentrations of baicalein for 5 min. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan) by recording excitation signals at 340 and 380 nm, and emission signals at 510 nm in two-second intervals.
Measurement of intracellular ROS production
Levels of intracellular O2
− and H2O2 were assessed spectrofluorometrically by oxidation of specific probes: DCFH-DA. Dye loading was performed by incubating the cardiomyocytes with 10 μM DCFH-DA for 30 min at 37°C, and the fluorescence intensity of the cells was determined using fluorescent microscope and fluorescence-activated cell sorting (FACS) analysis.
Hoechst 33342 staining
Apoptotic cells were confirmed by Hoechst 33342 staining. The cells showing nuclear fragmentation and chromatin condensation in Hoechst staining were categorized as apoptotic cells . After drug pretreatment for 24 h, the cultured H9c2 cells were exposed to 10 μM lysoPC. Then, cells were washed with 1X Hank’s balance salt solution (HBSS) and stained with 10 μg/ml of Hoechst 33342 for 60 min. The nuclear morphology of the cells was visualized using a fluorescence microscope (Zeiss Axioskop 2 plus, Japan).
Annexin V/propidium iodide staining
Quantitative assessment of apoptotic cells was determined by flow cytometry using the Annexin V-conjugated Alexa Fluor 488 Apoptosis Detection Kit following the manufacturer’s instructions. The apoptotic and necrotic cells from the same samples were quantified using quantitative FACS analysis. This method utilizes the binding of FITC-labeled Annexin V to phosphatidylserine in the cell membrane that surfaces only during the early phase of apoptosis, indicating the loss of cell membrane phospholipid asymmetry. However, the apoptotic cells with intact cell membranes do not stain with the propidium iodide. By utilizing the morphological changes that occur in both apoptotic and necrotic cells, the samples were stained simultaneously with Annexin V-FITC and propidium iodide. The samples were then subjected to flow cytometric analyses to detect the percentage of apoptotic (FITC-stained cells) and necrotic cells (PI-stained cells) in a given population. A minimum of 10,000 cells were maintained for all the samples. The samples were analyzed by a Coulter Epics XL-MCL (Beckman Coulter, USA).
After treatment with the indicated agents, cells were washed twice with cold PBS and then cells were harvested. Total cell extracts were prepared in lysis buffer [20 mM Tris–HCl (pH 7.5), 1 mM dithiothreitol (DTT), 5 mM EGTA, 2 mM EDTA, 0.5 mM PMSF, 20 μM leupeptin, and 20 μM aprotinin]. The cell lysate was centrifuged at 15,000 g for 30 min, and the supernatant fraction was collected for Western blot. An equivalent amount of protein was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (10-14%) and transferred to polyvinylidene difluoride membranes. After blocking for 1 h in 5% non-fat dry milk in Tris-buffered saline, the membrane was incubated with the desired primary antibody for 2 h. The membrane was then treated with appropriate horseradish peroxidase-conjugated secondary antibody (diluted 1:1000), and the immunoreactive bands were detected with enhanced chemiluminescence reagents (PerkinElmer Life and Analytical Sciences).
Measuring MAPK activity
H9c2 cells were pretreated with baicalein (0.1 to 10 μM) for 1 h followed by incubation with lysoPC (10 μM) for 10 min. Phosphorylated protein levels of ERK1/2, JNK, and p38 MAPK were determined using cell based ELISA kit (RayBio® Cell-Based ERK1/2, JNK, p38 MAPK phosphorylation ELISA Sampler Kit, Ray Biotech Inc., Norcross, GA, USA) as per manufacturer’s instructions.
Data are expressed as means ± SEM. Statistical differences were estimated by one-way analysis of variance (ANOVA) followed by Dunnett’s test. A value of P < 0.05 was considered significant.