NIH 3T3 fibroblasts were used for all experiments. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies CA, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) in a humidified incubator at 37°C with 5% CO2. All experiments were performed in triplicate.
Cell viability assay
Cell survival was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega, WI, USA). Fibroblasts were plated at a density of 5,000 cells per well on 96-well plates and incubated for 24 h in 100 μl of DMEM containing 10% FBS. After incubation with serum-free medium for 24 h, cells were treated for 30 min with various concentrations of (+)-catechin (0–400 μM; Sigma Aldrich, PA, USA), and then subjected to oxidative stress induction with 0.1 mM hydrogen peroxide (H2O2). After 24 h, 20 μl of One Solution Reagent was added into each well and incubated at 37°C for 2 h in a humidified, 5% CO2 atmosphere. The production of formazan by viable cells was detected by measuring the absorbance at 490 nm using a 96-well plate reader.
Another series of experiments were conducted to compare cytotoxicity between (+)-catechin and EGCG. Fibroblasts were treated with various concentrations of (+)-catechin or EGCG (0–400 μM; Sigma Aldrich) without H2O2 for 24 h and the cells were then subjected to MTT assay.
Apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) using the In Situ Cell Death Detection Kit TMR Red (Roche, Mannheim, Germany), according to the manufacturer’s instructions. In brief, fibroblasts were maintained in DMEM containing 10% FBS for 2 days and then cultured in serum-free DMEM. Oxidative stress was induced by addition of 0.1 mM H2O2 prior to treatment with 10 μM (+)-catechin or vehicle. After 24 h of incubation with H2O2 and (+)-catechin or vehicle, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 60 min at room temperature, followed by five washes with PBS. Next, permeabilization was performed by incubation with 0.1% Triton X-100 in PBS for 10 min, and cells were mixed with TUNEL reaction mixtures containing TdT and tetramethylrhodamine (TMR) red-labeled nucleotides for 1 h. Coverslips were mounted onto slides using VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole dihydrochloride (Vector Laboratories, Peterborough, England). Fluorescence images were taken using a microscope (IX-70; Olympus) equipped with a charge-coupled device camera (CoolSNAP HQ; Nippon Roper, Chiba, Japan). For each experiment, 100 cells were randomly selected, and the percentage of TUNEL-positive cells was measured.
Western blot analysis
Cultured fibroblasts were serum-starved for 24 h in serum-free DMEM and then incubated with 10 μM (+)-catechin for 30 min prior to oxidative stress induction by 0.1 mM H2O2. After H2O2 challenge for 1 h, cells were harvested and lysed in radioimmunoprecipitation assay buffer containing 1 mM Na3VO4, 1 mM NaF, and Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland) for 20 min at 4°C. After centrifugation at 15,000 × g for 15 min at 4°C, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon-P Transfer Membranes (Millipore Japan, Tokyo, Japan). Membranes were incubated for 60 min in Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 and then blotted with the following primary antibodies at 4°C overnight: anti-phospho-JNK (1:1,000), anti-JNK (1:1,000), anti-phospho-p38 (1:1,000), anti-p38 (1:1,000), anti-cleaved caspase-3 (1:200), and anti-caspase-3 antibodies (1:200). All antibodies were purchased from Cell Signaling Technology, MA, USA. Next, membranes were incubated for 1 h with an anti-mouse or anti-rabbit HRP-linked secondary antibody (1:2,000; Cell Signaling Technology). Reaction products were visualized by chemiluminescence detection using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA). Quantification of relative band densities was performed by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA).
All data shown are expressed as the mean ± SE of three independent experiments. Data from each experiment were normalized to the respective control sample. Differences between conditions were analyzed by Student’s t test. Multiple-group comparisons were performed using a one-way analysis of variance, followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant.