Human chondrocytes (cat. no. 4650; ScienCell Research Laboratories, Inc., San Diego, CA, USA) were cultured in a 6-well plate (1×10⁵/well) at 37°C in a 5% CO₂ incubator with Dulbecco's modified Eagle medium containing 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin and streptomycin (Sangon Biotech Co., Ltd., Shanghai, China). Cells were fixed with 95% ethanol for 15 sec at room temperature and then stained with 1% toluidine blue (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 5 min at room temperature and observed under a light microscope (magnification, ×100). Theaflavin-3-3′-digallate (purity >90%) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). At ~80% confluence, the cells were serum-starved overnight and were then divided into three treatment groups for comparison: i) Control group; ii) model group, in which cells were treated with 0.3 mM H₂O₂ for 6 h; and iii) TF pretreatment groups, in which cells were pretreated with various doses of TFs (10 and 20 µg/ml) for 12 h, followed by 6 h H₂O₂ incubation. The TF concentrations employed were based on previous literature (18,19). For the activation of AKT, cells were pretreated with 50 ng/ml insulin-like growth factor I (IGF-I; R&D Systems, Inc., Minneapolis, MN, USA) for 10 min prior to H₂O₂ or TF treatment, according to previous studies (20,21).

The cells were serum starved overnight in a 96-well plate (1×10⁵ cells/well), and were then treated with H₂O₂ (0.1–0.5 µM) in serum-free medium for various durations (6, 12, 24 and 48 h). A CKK-8 kit was used to detect cell viability, according to the manufacturer's protocol (Beijing Kangwei Century Biotechnology Co., Ltd., Beijing, China). Briefly, the CCK-8 solution was added to each well and the cells were incubated for 4 h at 37°C, after which, absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate and were treated as aforementioned. Matrix metalloproteinase (MMP)-13 (cat. no. DY511) and interleukin (IL)-1β (cat. no. DLB50) activities were detected using ELISA kits (R&D Systems, Inc.), according to the manufacturer's protocols. The ELISA kit used to measure the expression of cartilage glycoprotein 39 (Cgp-39; cat. no. HC021) was purchased from Shanghai GeFan Biotechnology, Co., Ltd. (Shanghai, China).

The cells (1×10⁴ cells/well) were treated as aforementioned. Subsequently, the cells were stained with H2DCHF-DA (Invitrogen; Thermo Fisher Scientific, Inc.) for ROS measurement, as previously described (22). BD FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA) running BD CellQuest^™ software version 3.3 (BD Biosciences) was used to perform flow cytometric analysis. All data are representatives of at least three independent experiments.

The cells (1×10⁵/well) were cultured in 6-well plates. An Annexin V/propidium iodide (PI) apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect apoptosis. According to the manufacturer's protocol, Annexin-V and PI staining was analyzed using analyzed using a flow cytometer (BD FACSCanto II) with FACSDiva software version 6.1 (both BD Biosciences).

According to a previous study (23), DNA damage was estimated using flow cytometry-based detection of γ-H2A histone family, member X (γ-H2AX). Briefly, the collected cells were suspended in BD Cytofix/Cytoperm fixation and permeabilization solution (BD Biosciences). After a 15-min incubation at 37°C, the cells were washed using PBS and blocked with 10% normal goat serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Cells were incubated with anti-γ-H2AX (pS139) antibody (cat. no. ab26350; 1:100; Abcam, Cambridge, UK) at 4°C overnight and then incubated with fluorescein isothiocyanate-conjugated secondary antibodies (cat. no. ab7064; 1:1,000; Abcam) for 45 min at 37°C. Subsequently, the fluorescent signals were measured using a BD flow cytometer (BD Biosciences).

Total RNA was isolated using RNAiso reagent (Takara Bio, Inc., Otsu, Japan). Subsequently, cDNA was reverse transcribed from total RNA using ReverTra Ace (Toyobo Life Science, Osaka, Japan) and oligo-dT (Takara Bio, Inc.), according to manufacturer's protocol. The mRNA expression levels were quantified using an ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using AceQ qPCR SYBR Green Master Mix (Vazyme, Piscataway, NJ, USA). The thermocycling conditions were as follows: 95°C for 5 min, followed by 30 cycles of 94°C for 15 sec and 60°C for 30 sec, and final extension at 72°C for 5 min. Relative expression levels were calculated using the 2^−ΔΔCq method (24). Primer sequences for RT-qPCR were as follows: ATR serine/threonine kinase (ATR) forward, 5′-GGAATCACGACTCGCTGA; AC-3′ reverse, 5′-AAATCGGCCCACTAGTAGCA-3′; ATM serine/threonine kinase (ATM) forward, 5′-CGAGGCGTACAATGGTGAAG-3′ and reverse, 5′-CCTCCGGCTAAGCGAAATTC-3′; B-cell lymphoma 2-associated X protein (Bax) forward, 5′-GAGCGGCGGTGATGGA-3′ and reverse, 5′-TGGATGAAACCCTGAAGCAAA-3′; and β-actin forward, 5′-CTCTTCCAGCCTTCCTTCC-3′; and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′.

A Total Extraction Sample kit (Sigma-Aldrich; Merck KGaA) was used to extract total proteins. Protein concentration was determined using the Pierce^™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The proteins (20 µg/lane) were separated by 10% SDS-PAGE and were then transferred onto a polyvinylidene fluoride membrane. To block non-specific proteins, non-fat milk (3%) was used to incubate the membrane for 2 h at room temperature. Following incubation with primary antibodies overnight at 4°C, the membrane was incubated with secondary antibodies for 2 h at room temperature, and the bands were developed using an enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Little Chalfont, UK). Blot density was determined using Quantity One software version 4.6.9 (Bio-Rad Laboratories, Inc.). The primary antibodies used were as follows: Anti-ATR (cat. no. ab2905; 1:1,000), anti-Bax (cat. no. ab53154; 1:1,000; both Abcam, Cambridge, UK), anti-cleaved caspase-3 (cat. no. 9664; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ATM (cat. no. ab78; 1:2,000), anti-γH2AX (cat. no. ab11175; 1:8,000; both Abcam), anti-phosphorylated (p)-AKT (cat. no. 13038; 1:1,000; Cell Signaling Technology, Inc.), anti-AKT1/2 (cat. no. ab182729; 1:5,000), anti-p-FOXO3a (cat. no. ab53287; 1:1,000; both Abcam), anti-FOXO3a (cat. no. 2497; 1:1,000; CST), anti-Gpx1 (cat. no. ab22604; 1:1,000), anti-CAT (cat. no. ab16731; 1:2,000; both Abcam) and anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit; cat. no. ab205718; 1:2,000 and goat anti-mouse; cat. no. ab205719; 1:5,000) were obtained from Abcam.

All experiments were independently performed ≥3 times. Data are presented as the means ± standard deviation. GraphPad software version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to compare differences between groups by one-way analysis of variance followed by Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The cartilage cells presented with spindle morphology, and toluidine blue staining was conducted to identify the cells. It was identified that the cytoplasm was stained light blue and the nucleus was stained dark blue, indicating that these cells were chondrocytes (Fig. 1A and B). Subsequently, CCK-8 assay was conducted to evaluate the effects of H₂O₂ on the viability of cartilage cells. It was demonstrated that cell viability was suppressed by H₂O₂ in a dose-dependent manner. A significant difference emerged in the group that was treated with 0.3 mM H₂O₂ for 6 h, in which cell viability was deceased by 14% (Fig. 1B). Therefore, 0.3 mM H₂O₂ was subsequently used to treat cartilage cells for 6 h, in order to mimic the progression of cartilage degeneration. To measure cartilage degeneration following H₂O₂ treatment, the expression levels of catabolic factors, MMP-13, IL-1β and Cgp-39, were detected. It was demonstrated that the expression levels of MMP-13, IL-1β and Cgp-39 were increased by H₂O₂, but were decreased by TF pretreatment (Fig. 2A). These findings suggested that TFs may inhibit cartilage degeneration. Furthermore, according to flow cytometric analysis, it was demonstrated that ROS levels were markedly decreased in the TF pretreatment groups (Fig. 2B and C).

The results of flow cytometric analysis revealed that H₂O₂-induced apoptosis was suppressed by pretreatment with TFs (Fig. 3A and B). DNA double-strand breaks (DSBs) are a type of detrimental DNA damage, for which γH2AX is considered a surrogate marker. In response to DSBs, H2AX is phosphorylated at Ser139 (γH2AX) (25). The present results demonstrated that TFs could decrease γH2AX expression compared with in the model group (Fig. 4A and B). In addition, western blotting confirmed that TFs inhibited the expression levels of γH2AX (Fig. 4C). The expression levels of apoptosis-associated factors, including cleaved caspase-3 and Bax, were decreased in the TF pretreatment groups compared with in the model group. The expression levels of DNA damage-response genes, ATM and ATR, were also decreased following TF pretreatment (Fig. 5A-C). However, the protein expression levels of ATR were slightly, but not significantly, increased in the T1 + H₂O₂ group.

Emerging evidences have demonstrated that FOXO proteins are important mediators of oxidative stress (15,16). Compared with in the model group, the protein expression levels of p-AKT and p-FOXO3a were mitigated by TF pretreatment, whereas the expression levels of Gpx1 and CAT were enhanced (Fig. 6A and B).

To further confirm the role of AKT in the present study, apoptosis and DNA damage were detected following treatment with the AKT activator, IGF-I. The results demonstrated that TF pretreatment did not significantly reverse H₂O₂-induced apoptosis following persistent activation of AKT (Fig. 7A and B). In addition, TF-induced inhibition of DNA damage was reversed following persistent activation of AKT (Fig. 8A-C).