Adult bone marrow-derived hMSCs were purchased from Cambrex (Walkersville, MD, USA). hMSCs (passages 4–10) were cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose containing 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA) at 37°C with 5% CO₂.

EGCG and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). To define the optimal concentrations for use in subsequent experiments, hMSCs were pre-incubated with different amounts of EGCG (50 and 100 μM) for 6 h and then the cells were exposed to 200 μM H₂O₂ (diluted in DMEM supplemented with 10% FBS) for 2 h. The cells were washed twice with DMEM to remove excess H₂O₂ and re-incubated in fresh complete medium for 24 h to prevent cell death and allow for the observation of senescent characteristics

The activity of senescence-associated β-galactosidase (SAβ-gal), a marker of senescence, was analyzed in hMSCs using a cellular senescence assay kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, the medium was aspirated and the cells were washed once with phosphate-buffered saline (PBS; pH 6.0). After fixing the cells with 1X fixing solution at room temperature for 10 min, the cells were washed again with PBS and incubated without light for at least 4 h with prepared SAβ-gal detection solution at 37°C without CO₂. The percentage of senescence-stained cells was obtained by counting the number of blue-stained cells and the total number of cells per field under the microscope (CKX41; Olympus, Tokyo, Japan; 100–200 cells in four random fields).

Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Briefly, hMSCs were cultured in 24-well tissue culture plates and exposed to 200 μM H₂O₂ for 2 h. After 24 h, the cells were stained with 1 mg/ml MTT (Sigma-Aldrich). The media were then carefully aspirated and 150 μl dimethyl sulfoxide (DMSO) was added to solubilize the colored formazan product. The optical density was read at 554 nm using a microplate reader (Floustar Optima; BMG Labtech, Ortenberg, Germany).

The cells were washed twice with cold PBS and lysed with RIPA buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.2 mg/ml leupeptin, 0.2 mg/ml aprotinin, 0.1 M phenylmethylsulfonylfluoride (PMSF), 1 mM Na₃VO₄ and 0.5 M NaF]. The lysates were centrifuged at 13,500 × g for 15 min at 4°C and the supernatants were loaded on to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The following primary antibodies were used: rabbit anti-p53 (1:1,000; sc-6243; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-acetyl p53 (1:1,000; 06-758; Upstate Biotechnology, Lake Placid, NY, USA); mouse anti-p21 (1:2,000; sc-6246) and rabbit anti-Nrf2 (1:1,000; SC-722) (both from Santa Cruz Biotechnology, Inc.); mouse anti-α-tubulin (1:5,000; T5168; Sigma-Aldrich) and goat anti-lamin B (1:2,000; sc-6216; Santa Cruz Biotechnology, Inc.). Primary antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse (A2554), -rabbit (A0545) (Sigma-Aldrich), or donkey anti-goat secondary antibodies (sc-2020; Santa Cruz Biotechnology, Inc.) and visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Rockford, IL, USA).

To obtain nuclear and cytoplasmic fractions, the cells were harvested and suspended in ice-cold cytoplasmic lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT and 0.2 mM PMSF) on ice for 15 min. The suspensions were then centrifuged at 13,500 × g for 10 min at 4°C and the supernatants were saved as the cytoplasmic fractions. The pellets were resuspended in nuclear lysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 0.4 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT and 0.2 mM PMSF) and incubated on ice for 40 min with occasional gentle shaking. The suspensions were then centrifuged at 13,500 × g for 15 min and the supernatants were used as nuclear fractions. Quantification of the results of western blot analysis was performed using ImageJ software (NIH, Bethesda, MD, USA).

The hMSCs were pre-incubated with 100 μM EGCG for 6 h, fixed in PBS containing 4% PFA and incubated overnight at 4°C with rabbit anti-Nrf2 (1:100). Alexa Fluor 546 anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) was used as a secondary antibody. The cells were counterstained with 100 ng/ml 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology, Inc.) for nuclear staining and visualized using a confocal laser scanning microscope (FV300; Olympus).

Human Nrf2-specific siRNA oligo nucleotides (SMARTpool) were purchased from Dharmacon (Lafayette, CO, USA). The following target specific siRNA sequences were used: 5′-UAAAGUGGCUGCUCAGAAU-3′; 5′-GAGUUACAGUGUCUUAAUA-3′; 5′-UGGAGUAAGUCGAGAAGUA-3′; and 5′-CACCUUAUAUCUCGAAGUU-3′. Non-targeting scrambled 20–25 nt siRNA oligonucleotides (Santa Cruz Biotechnology, Inc.) were used as a control. Transient transfections were performed using DharmaFECT 3 transfection reagent (Dharmacon) according to the manufacturer's instructions. Briefly, siRNA/lipid complexes were added to the wells at a final concentration of 100 nM siRNA and 1 μl/well of DharmaFECT 3. Nrf2 gene expression was determined at 48 h after transfection.

Statistical analysis was performed by analysis of variance (ANOVA) followed by a post hoc Newman-Keuls test. P-values <0.05 were considered to indicate a statistically significant difference. All data are presented as the means ± standard error of mean (SEM).

The stimulation of cells with exogenous ROS activates various signaling pathways that result in DNA damage, cellular senescence and apoptosis (3). In order to examine the effects of H₂O₂ exposure on cellular senescence, the hMSCs were exposed to 200 μM H₂O₂ diluted in DMEM supplemented with 10% FBS for 2 h, in order to allow the observation of senescent characteristics without significant cell death. In the present study, the activity of SAβ-gal was measured by SAβ-gal staining at 24 h after H₂O₂ exposure. Approximately 75% of H₂O₂-exposed hMSCs were positive for SAβ-gal (blue cytoplasmic stain) (74.6±3.6%), whereas only 20% of the control cells without H₂O₂ exposure were SAβ-gal-positive (P<0.01) (Fig. 1A, B and E). However, the pre-treatment of hMSCs with 50 or 100 μM EGCG for 6 h reduced the percentage of SAβ-gal-positive cells following H₂O₂ exposure to 50.7±4.8 and 30.4±1.9%, respectively (P<0.01) (Fig. 1C–E). Taken together, these results suggest that cellular senescence in hMSCs is accelerated by H₂O₂ exposure and EGCG pre-treatment reduces this acceleration in a dose-dependent manner. Furthermore, there were no significant differences in cell death among the experimental groups, indicating that H₂O₂ exposure induced cellular senescence without causing significant cell death (Fig. 1F).

To further evaluate H₂O₂-induced changes in senescent cells, we next examined the protein levels of acetyl-p53, p53 and p21 in hMSCs at different times following 200 μM H₂O₂ exposure. The expression of p53 and p21 is known to correlate with senescence in human primary cells and p53 acetylation has been shown to strongly promote cellular senescence (8,12). Consistent with the findings of previous studies, there were senescence-associated increases in the protein levels of acetyl-p53, p21 and p53 following 200 μM H₂O₂ exposure (Fig. 2A–D). Particularly after 24 h of H₂O₂ exposure, the protein levels of acetyl-p53 and p21 were significantly increased by up to 4.4- and 5.9-fold, respectively, compared with the controls, (P<0.01) (Fig. 2B and C). Despite an increasing trend in total p53 protein levels following H₂O₂ exposure, the increase did not reach statistical significance (Fig. 2D).

Thus, we decided to examine the effect of EGCG pretreatment on acetyl-p53 and p21 protein levels in hMSCs after 24 h of H₂O₂ exposure. As previously shown, there were significant increases in acetyl-p53 and p21 protein levels at 24 h after 200 μM H₂O₂ exposure (P<0.01) (Fig. 2E–G). However, EGCG pre-treatment (100 μM) significantly decreased the protein levels of acetyl-p53 and p21 in the H₂O₂-exposed hMSCs by 46.3±8.1 and 35.1±6.5% (P<0.01), respectively, compared with the cells given no EGCG pretreatment (Fig. 2E–G).

To determine whether the suppression of cellular senescence by EGCG in H₂O₂-exposed hMSCs is associated with Nrf2 activation, we performed double-labeling experiments with anti-Nrf2 antibody and DAPI after 6 h of EGCG treatment (100 μM). Nrf2 was mostly found to be localized in the cytoplasm in the untreated cells (Fig. 3A, left panel). However, marked translocation of Nrf2 to the nuclei was observed after 6 h of EGCG treatment, although some Nrf2 remained in the cytoplasm (Fig. 3A, right panel). In addition, nuclear fractions were subjected to western blot analysis, showing that pre-treatment with EGCG increased nuclear Nrf2 protein levels 2.5-fold compared with the untreated cells (P<0.05) (Fig. 3B).

We hypothesized that Nrf2 activation may play an important role in the anti-senescence effects of EGCG. To test this hypothesis, we performed SAβ-gal staining at 48 h after siRNA-mediated Nrf2 knockdown or control siRNA transfection (Fig. 4A). As previously shown in Fig. 1, the percentage of SAβ-gal-positive cells in the 100 μM EGCG-pretreated/H₂O₂-exposed group was significantly reduced (35.1±2.5%) compared with the H₂O₂-exposed cells without pre-treatment (78.4±3.7%) (P<0.01) (Fig. 4A, panels b and c and 4B). However, EGCG-pre-treated and H₂O₂-exposed/Nrf2-siRNA-transfected cells exhibited increased positive staining for SAβ-gal (65.6±3.9%) (P<0.01), which is similar to that of the H₂O₂-exposed cells (Fig. 4A, panels b and d and 4B). By contrast, EGCG-pre-treated/H₂O₂-exposed/control-siRNA-transfected cells stained positive at a significantly lower rate of 36±4.2%, which is similar to that of the EGCG-pre-treated/H₂O₂-treated cells (Fig. 4A, panels c and e and 4B). We confirmed that Nrf2 protein levels were reduced to 30±5.4% at 48 h after Nrf2 siRNA transfection compared with the control siRNA (P<0.05) (Fig. 4C). These results suggest that Nrf2 may play an important role in the anti-senescence activity of EGCG.

We next examined acetyl-p53 and p21 protein levels in Nrf2-knockdown hMSCs. As previously shown (Fig. 2E–G), acetyl-p53 and p21 protein levels were significantly reduced by 44.6±3.7 and 39.7±5.4%, respectively, in the EGCG-pretreated/H₂O₂-exposed cells compared with the H₂O₂-exposed cells (P<0.01) (Fig. 4D–F). However, at 48 h after Nrf2-siRNA transfection, acetyl-p53 and p21 protein levels were significantly increased in the EGCG-pre-treated/H₂O₂-exposed cells. The protein levels of acetyl-p53 and p21 were similar to those in the H₂O₂-exposed cells (Fig. 4D–F). By contrast, control siRNA transfection did not change the acetyl-p53 and p21 protein levels in the EGCG-pre-treated/H₂O₂-exposed cells. Taken together, these results indicate that Nrf2 activation by EGCG pre-treatment suppresses H₂O₂-induced cellular senescence and the expression of acetyl-p53 and p21 in hMSCs (Fig. 5).