Chemicals and reagents.
Ginsenoside Rg2 (purity of 98.18%) was obtained from Manster Biotechnology (Chengdu, China). The rat myocardial cell line H9c2 was acquired from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. Enhanced Cell Counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) one-step apoptosis assay kit, immune staining fix solution, enhanced immunostaining permeabilization solution, reactive oxygen species (ROS) assay kit, RIPA lysis buffer, bicinchoninic acid (BCA) protein concentration determination kit, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit, SDS-PAGE loading buffer, SDS-PAGE electrophoresis buffer with Tris-Gly, western transfer liquid, BeyoECL Star detection kit, and LY294002 (LY294) were all purchased from Beyotime Technology (Shanghai, China). Multicolor prestained protein ladder and protein sample loading buffer were obtained from Epizyme Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’ s medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA, penicillin-streptomycin (P-S), and phosphate buffered saline (PBS) were purchased from Gibco (Waltham, MA, USA), whereas doxorubicin (DOX) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (San Luis, MO, USA). Polyvinylidene difluoride (PVDF) was obtained from Merck Chemical Technology (Shanghai, China). Antibodies against Akt, p-Akt, p53, p-p53, and GAPDH were acquired from Cell Signaling Technology (Danvers, MA, USA).
Cell culture.
H9c2 cells were cultured in DMEM containing 10% FBS and 1% P-S at 37 ℃ in a 5% CO2 incubator. H9c2 cells were treated with 0.25% trypsin at 80% confluency and passaged at a 1:4 ratio approximately every three days. The cells used in subsequent experiments were in the logarithmic growth phase.
Cell cytotoxicity assay.
H9c2 cells were seeded in 96-well plates at a density of 1×104 cells/well and incubated at 37°C for 24 h. The cells were then treated with different concentrations of DOX (2.5, 5, 10, 15, and 20 µM) and ginsenoside Rg2 (200, 250, 300, 350, and 400 µM) for another 24 h. After that, the cell supernatant was replaced with 10% CCK-8 diluent and incubated at 37°C for 1 h before the CCK-8 assay was performed. The absorbance value was measured at 450 nm using a multifunctional enzyme standard instrument (SYNERGY H1, BioTek).
To investigate the effect of ginsenoside Rg2 on DOX-induced cardiomyocyte apoptosis, H9c2 cells were first treated with ginsenoside Rg2 for 24 h followed by the addition of DOX for another 24 h. Thereafter, the CCK-8 assay was performed. To determine the underlying mechanism, H9c2 cells were pre-treated with LY294 (1 µM), a PI3K inhibitor, for 30 min before ginsenoside Rg2 intervention.
Apoptosis assay.
TUNEL staining was used to detect morphological features of apoptosis. After treatment with different concentrations (100, 200, and 250 µM) of ginsenoside Rg2 for 24 h, H9c2 cells were treated with DOX (5 µM) for another 24 h. Next, the cell supernatant was replaced with immune staining fix solution for 30 min. After washing with PBS, the cells were mixed with enhanced immunostaining permeabilization solution for 5 min. The TUNEL solution was then added to the cells and incubated for 60 min at 37 ℃, followed by detection using a 200-fold fluorescence microscope (Olympus BX50, Tokyo, Japan).
ROS assay.
The ROS assay kit was used to analyze ROS levels in cardiomyocytes. H9c2 cells were seeded in 96-well plates at a density of 1×104 cells/well. After intervention with ginsenoside Rg2, DOX, and LY294, DCFH-DA (10 µM) was added to the cells, followed by incubation for 20 min at 37 ℃. The cells were then washed three times with DMEM and ROS absorbance was measured at 488 nm using a multifunctional enzyme standard instrument.
Western blot analysis.
Briefly, the total proteins of H9c2 cells were extracted according to the manufacturer's instructions. The protein concentration was calculated using the BCA kit. After denaturation at 95°C for 5 min, proteins were separated via SDS-PAGE (10% separating gel and 5% stacking gel, 60 v for 30 min, 100 V for 1 h). The separated proteins were transferred to PVDF membranes at 350 mA for 1 h. Protein ladder markers were used to observe the molecular weight positions. The membranes were pre-incubated in blocking buffer-TBST containing 5% skim milk (w/v) for 1 h at room temperature (nearly 20°C) and then incubated overnight at 4°C with primary antibodies at a dilution of 1:1000. After washing with TBST, the membranes were incubated with a secondary antibody (1:2000 dilution) for 1 h at room temperature. Protein bands were detected using the ECL chemiluminescence system and a gel imaging system (Chemiscope 6300). ImageJ software was used for the gray value statistics. GAPDH was used as the loading control.
Statistical analysis.
All data are presented as mean ± standard deviation and were analyzed using SPSS Statistics 25. An independent sample t-test was used to compare the two groups. The comparison among the three groups (above) was performed via one-way analysis of variance and the least significant difference test was performed between groups. Non-normal distribution was represented by the median, and the rank sum test was adopted. Statistical significance was assumed if P reached a value < 0.05. Graphs were created using GraphPad Prism 7.0.