MRTECs were purchased from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) with high glucose and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The EGCG was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Nrf2 (cat no. bs-1074R) and γ-GCS (cat no. YSRIBIO-068461) rabbit anti-rat immunoglobulin (Ig)G primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The streptavidin peroxidase immunocytochemical staining kit and 3,3′-diaminobenzidine(DAB) staining reagent were purchased from ZSGB-Bio (Beijing, China). The TRIzol total RNA extracting solution was purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd (Shanghai, China). The PrimeScript™ RT Reagent kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The RT-qPCR SYBR Premix Ex Taq™ kit was purchased from Takara Biotechnology Co., Ltd. The RT-qPCR primers were designed and synthesized by Takara Biotechnology Co., Ltd.

MRTECs were digested with 0.25% trypsin to form a single cell suspension and cultured in DMEM containing 100 ml/l fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin dual anti-solution, at 37°C in an atmosphere containing 5% CO₂. The medium was replaced every 24 h and 0.25% trypsin was used for digestion every 3 days; passaging was performed at a ratio of 1:3. The passaged cells were inoculated uniformly and, when the cells had reached the sub-integration state, serum-free medium was added for a 12-h synchronization, followed by the addition of 1% fetal bovine serum-containing medium for the experiments. The cells were seeded on coverslips, 6-well plates or flasks. When the cells had achieved ~80% fusion, the serum-free medium was replaced and, according to the experimental group, different dosages of drugs were added. Each experiment was performed with ≥3 different generations of cells.

The cultured cells were divided into the untreated normal group (N); the control group incubated with 250 µmol/l H₂O₂ for 6 h (C); and the EGCG groups incubated with 250 µmol/l H₂O₂, and co-cultured with EGCG at concentrations of 5 mg/l (T1), 10 mg/l (T2) and 20 mg/l (T3).

The MTT chromogenic assay was used to assess the cell viability of each group. The cells were transfected into 96-well plates, and cultured for 72 h. A total of 50 µl MTT (5 g/l) was added to each well and incubated for 4 h. Subsequently, the supernatant was discarded and 100 µl dimethyl sulfoxide was added for the detection of the absorbance at 570 nm (A570) using an automatic microplate reader. The number of viable cells was proportional to the value of A570: Live cell rate=A570 of the experimental group/A570 of the control group. Trypan blue staining was performed on the EGCG groups prior to the assay and the living cell rates were observed to be >90%, indicating that EGCG was non-toxic to the cells.

TRIzol was used to extract the total RNA and reverse transcription was performed to synthesize cDNA. The primer sequences for the RT-qPCR were as follows: The upstream and downstream primers of Nrf2 were 5′-TTGATTGACATCCTTTGG-3′ and 5′-GTTCCTTCTGGAGTTGCT-3′, respectively, with an amplified product of 142 bp; the upstream and downstream primers of γ-GCS were 5′-ATCTACCACGCAGTCAAG-3′ and 5′-CCGCCATTCAGTAACAAC-3′, respectively, with an amplified product of 119 bp; and the upstream and downstream primers of GAPDH-F were 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-ATGGTGGTGAAGACGCCAGT3′, respectively, with an amplified product of 142 bp. The PCR composition, reaction conditions (94°C for 2 min for 1 cycle, 94°C for 40 sec, 50–65°C for 40 sec and 72°C for 1 min for 25–35 cycles, and 72°C for 5 min for 1 cycle) and dissolution profile conditions were in accordance with the manufacturer's protocol.

The SYBR-Green 1 fluorescent dye embedment method was used to prepare the standard curves of the target genes, Nrf2 and γ-GCS, and the housekeeping gene GAPDH. The standard curves were used to quantifying the target gene and housekeeping gene levels in the samples. Through the correction of the housekeeping gene, the relative gene expression of Nrf2 and γ-GCS was measured in each group (11).

The coverslips containing cultured cells were prepared according to the streptavidin-biotin complex test kit protocol. The primary antibodies were rabbit anti-Nrf2 and γ-GCS polyclonal antibodies (both 1:200), which was incubated at 4°C overnight. After washing with PBS for 5 min 3 times, the secondary antibody was added, which was a biotinylated goat anti-rabbit IgG (1:200; Agrisera AB, Vännäs, Sweden; cat no. AS09608), which was incubated at 37°C for 30 min prior to washing with PBS as before. DAB staining was subsequently performed, and the DAB developer was added and incubated for 3 min at room temperature and then the reaction was stopped by subjecting cells to running water for 1 min. Cells were then stained with hematoxylin for 15 sec. In group N, PBS was used to replace the primary antibody and all subsequent steps were the same. The existence of black/dark grey particles inside the nucleus and/or cytoplasm was considered to be positive. The optical density values were calculated using MetaMorph image analysis software.

Total Nrf2 nucleoprotein and γ-GCS cytoplasmic protein was extracted using radioimmunoprecipiation assay lysis buffer (BioTeke Corporation, Beijing, China), and the assay was performed according to the manufacturer's protocol. 3% SDS-PAGE 3% gel and 10% separation gel was used, and 10 µl protein was loaded per lane. The protein was then transfered to nitrocellulose membrane and blocked with 5% skimmed milk powder at 4°C overnight. The primary antibodies (rabbit anti-Nrf2 and γ-GCS polyclonal antibody) were diluted 1:1,000, incubated at 4°C overnight and the reaction was stopped with washing solution, then the goat anti-rabbit IgG secondary antibody was added (1:2,000) at room temperature for 1 h and stopped with washing solution as before. Staining was performed using the illuminating kit (BestBio, ShangHai; http://bestbio.biogo.net). Quantitative analysis was performed using Fluor Chem software (version 2.0; ProteinSimple; Bio-Techne, Minneapolis, MN, USA) and β-actin (1:200; cat no. A5441; Sigma-Aldrich; Merck KGaA) was used as the internal reference for the analysis, and was incubated at room temperature for 1 h.

All of the experiments were repeated ≥3 times and the results are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used for the comparisons of overall and intergroup differences. P<0.05 was considered to indicate a statistically significant difference.

Subsequent to 6 h treatment, in the control group, the cytoactivity of MRTECs was reduced. Among the treatment groups, at an EGCG concentration of 5 mg/l, the cytoactivity was significantly increased compared with the control group (P<0.01); at an EGCG concentration of 10 or 20 mg/l, cytoactivity was significantly increased compared with the control group (P<0.01). EGCG protected against the H₂O₂-mediated decrease in cytoactivity in a dose-dependent manner (Table I).

The standard curves of Nrf2, γ-GCS and GAPDH were prepared, and the correlation coefficients were 0.998, 0.990 and 0.995, respectively. The linearities were good, and the fusion curves demonstrated that the specificity was good.

The standard curve was used to quantify the expression of Nrf2, γ-GCS and GAPDH in the samples, and it was observed that, compared with the N group, the Nrf2 and γ-GCS mRNA in the C group was increased; compared with the C group, the expression of Nrf2 and γ-GCS mRNA was increased in the T1 group (P<0.05), and in the T1 and T3 groups (P<0.01), indicating that EGCG was able to upregulate the gene expression of Nrf2 and γ-GCS in MRTECs in a dose-dependent manner (Table II; Fig. 1).

Nrf2-positive expression is exhibited as brown particles in the MRTECs. When stimulated with 250 µmol/l H₂O₂ for 6 h, the Nrf2-positive expression increased. At increasing concentrations of EGCG, the expression of Nrf2 in the nucleus increased markedly, exhibited by deeper staining, most notably in the T3 group (Fig. 2). The γ-GCS-positive expression is exhibited as brown particles, and also located in the cytoplasm of normal MRTECs. When stimulated with H₂O₂ for 6 h, the γ-GCS-positive expression increased. At increasing concentrations of EGCG, the expression of γ-GCS in the nucleus increased markedly, exhibited by deeper staining, most notably in the T3 group (Fig. 3). Compared with the normal control group, the average optical density values in the control group and each treatment group were significantly increased (P<0.01); compared with the H₂O₂ control group, the average optical density values in the T1 (P<0.05), and the T2 and T3 groups (P<0.01) significantly increased (Table III).

The western blotting analysis results demonstrated that, compared with the normal group, Nrf2 and γ-GCS protein expression in the H₂O₂ control group significantly increased (P<0.01). Compared with the H₂O₂ control group, Nrf2 and γ-GCS protein expression increased in the T1 (P<0.05), and the T2 and T3 groups (P<0.01), suggesting that EGCG was able to increase Nrf2 and γ-GCS protein expression in MRTECs in a dose-dependent manner (Table IV; Fig. 4).