Tannic acid (TA) (catalog number T0801) was purchased from Topscience (Shanghai, China). N-Acetylcysteine (NAC) (catalog number HY-B0215) was purchased from Med Chem Express (Monmouth Junction, NJ,USA). LPS (Lipopolysaccharide from Escherichia coli O55:B5) was purchased from Sigma Aldrich (St. Louis, MO, USA), and the rat heart myoblast cell line H9C2(2-1) (catalog number GNR 5) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). High-glucose DMEM (catalog number 12800017) was purchased from GIBCO BRL (Grand Island, NY, USA). Phosphate-buffered saline (PBS) (catalog number SH30256.01) and trypsin (catalog number SH30042.01) were purchased from GE Healthcare (Logan, UT, USA). Pierce™ BCA Protein Assay Kit (catalog number 23227) and fetal bovine serum (FBS) (catalog number 10270106) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). ROS assay kit was purchased from Nan jing Jian cheng Bioengineering Institute (Nanjing, China). Cell Counting Kit-8 assay (CCK-8) was purchased from 7sea biotech (Shanghai, China). Culture dishes (catalog numbers 430166 and 430167) were purchased from Corning Costar Company (Cambridge, MA, USA).
Primary antibody directed against GAPDH (catalog number ab9485) was purchased from Abcam (Cambridge, MA, USA); primary antibodies against Cleaved Caspase 3 (catalog number BF0711), Cleaved Caspase 9 (catalog number AF5240), Bax (catalog number AF0120), Bcl-2 (catalog number AF6139), ATF6 (catalog number DF6009), CHOP (catalog number DF6025), Phospho-PERK (catalog number DF7576), PERK (catalog number AF4799), Phospho-IRE1 (catalog number AF7150), IRE1 (catalog number DF7709), Phospho-JNK (catalog number AF3318) and JNK (catalog number AF6318) were obtained from Affinity (Cincinnati, OH, USA). HRP-conjugated secondary antibodies (catalog numbers ab6721 and ab6885) and a FITC-conjugated secondary antibody (catalog number ab6717) were purchased from Abcam (Cambridge, MA, USA).
Cell culture and treatment
The H9C2 cells were cultured in high-glucose DMEM supplemented containing 10% (v/v) FBS, 1.5 g/L NaHCO3 and 1% penicillin-streptomycin (Life Technologies Corporation, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere with 5% CO2, and maintained in a logarithmic growth phase for all experiments. H9C2 cells were treated with LPS (15 μg/mL) for 12 h, and different concentrations of TA (0, 5, 10, 20, 30, and 40 µM) for 24 h. H9C2 cells were pretreated with TA 2 h prior to treatment with LPS. For ROS inhibition experiments, inhibitor N-Acetylcysteine (NAC; 2 mM) was added prior to TA. Then 8 h later, and cells were harvested for further analysis.
The H9C2 cells were seeded in 96-well plate at a density of 5000 cells/well, and treated with the following conditions: fresh culture medium alone (control), fresh culture medium with different concentrations (0–50 µM) of TA, and/or fresh culture medium with 15 μg/mL LPS for the indicated times. Cell viability was assessed using the cell counting kit, CCK-8, according to manufacturer’s instructions. Briefly, after treatment, the CCK-8 solution (10 µL) was added to each well and the cultures were incubated at 37 °C in humidified 95% air and 5% CO2 for 1 h. Absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA). Values were expressed as the percentage of viable cells as follows: Relative viability (%) = [OD450 (treated) - OD450 (blank)] / [OD450 (control) - OD450 (blank)] × 100%.
Measurement of cellular ROS by laser confocal microscopy
The cellular ROS Detection Assay Kit contains the fluorogenic dye, 2,7-dichlorofluorescin-diacetate (DCF-DA), that measures ROS activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’–dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm. The H9C2 cells were incubated with 2,7-dichlorofluorescein-diacetate (DCFH-DA, 10µM, Jian cheng, China) for 30 min at 37 °C in the dark. The cells were subsequently washed twice with PBS and observed under a confocal laser scanning microscope (Leica), and the images were captured. The mean fluorescence intensity of DCF was analyzed.
Flow cytometric analysis of apoptosis
Fluorescein-conjugated annexin V (annexin V-FITC) was used to measure the apoptosis in H9C2 cells as described previously . The necrotic cells were counter stained with propidium iodide (PI). Cells were plated in 60-mm dishes and exposed to different treatments once reaching 80-85% confluence. Detached and adherent cells were collected, washed twice with cold PBS, and then resuspended in the 1X binding buffer at a concentration of 1×106 cells/mL. The resuspended solution containing 1×105 cells (100 µL) was transferred to a 5 mL culture tube, to which 5 µL of FITC Annexin V and 5 µL PI were added, and gently vortexed, prior to 15 min incubation at 25 °C in the dark. Binding buffer 1X (400 µL) was added to each tube, and the percentages of apoptotic cells were analyzed by flow cytometry within 1 h.
Real-time quantitative polymerase chain reaction (RT-PCR) analysis was performed as described previously . The primer sequences are described in Table 1. Briefly, total RNA was extracted using Trizol (catalog number 15596026, Life technologies, California, USA), and then DNase was used to remove internal DNA contamination. Random primers were used in reverse transcription reactions to obtain first-strand cDNA, and SYBR Green qPCR Master Mix (catalog number FP202, Tiangen, Beijing, China) was used to amplify the target fragments. GAPDH cDNA amplification was used as an internal control. The general reaction procedure included an initial denaturing step at 95 °C for 15 minutes followed by 37 cycles of denaturing at 95 °C for 10 sec, annealing at 57 °C for 30 sec, and extension at 72 °C for 30 sec, with a plate read at 95 °C for 5 sec. Each experimental group was performed in triplicate. IBM SPSS (version 19) software was used to analyze differences in expression.
Western blotting was performed as describing previously . Briefly, cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, and 1% NP-40 with a protease inhibitor cocktail and phosphatase inhibitor cocktail, The insoluble components were removed by centrifugation at 12,000 × g for 15 minutes at 4 °C. Subsequently, protein concentrations were measured using the BCA method. After denaturation, the total proteins were separated by 10% SDS-PAGE and transferred onto 0.2 µm or 0.45 µm polyvinylidene fluoride membranes. After blocking with 5% (w/v) non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The membranes were subsequently washed three times with 1 × TBST, and further incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After subsequent washes of the membrane, three times with 1 × TBST, antibody detection was performed using the Immobilon Western Chemiluminescent HRP Substrate (ECL, catalog number WBKLS0500, Merck Millipore, MA, USA) and the detection kit using a ChemiDoc™ XRS+ system (Bio-Rad, Hercules, California, USA). ImageJ (version 1.46) was used to compare the gray values of the target bands; GAPDH served as an internal control.
Data is presented as the mean ± standard error of the mean (SEM) from at three independent experiments. GraphPad Prism 8.0.2 (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. A P value <0.05 was considered statistically significant. The number of independent experiments performed is indicated in the figure legends.