HUVECs were provided by the Basic Medical College of Jilin University (Changchun, China). In brief, HUVECs were cultured in RPMI-1640 medium (P004-1, Nanjing Jiancheng Bioengineering Institute) containing 10% fetal bovine serum (F8318, Gibco) and 1% penicillin in a 37°C, 5% CO₂ incubator. The treatment conditions of H₂O₂ and GEN (cat. no. HY-14596; MedChemExpress) were determined according to previous studies (10,12). Subsequent experiments were divided into four groups: control group, GEN treatment group, H₂O₂ treatment group and a combined treatment group of GEN and H₂O₂.

H₂O₂ (130 µM) was used to induce oxidation injury in HUVECs (1×10⁵ cells per well was seeded) according to pre-experiments and previous studies (10,12). GEN with different concentrations (100 nM, 1 µM, 10 µM) were pretreated to reduce the oxidant injury of H₂O₂. The CCK-8 kit (ck04, Dojindo, the absorbance values at 450 nm were recorded) was used to detect cell viability referring to operating instructions. A total of 10 µl CCK-8 solution was added to each well and incubated at 37°C with 5% CO₂ in incubator for 2 h. An optimal dose of GEN was utilized for its antioxidant effect and for observing its antioxidant mechanism.

HUVECs were seeded in six-well plates at a density of 1.2×10⁵/ml per well for 24 h. Then, Dichloro-dihydro-fluorescein diacetate (DCFH-DA) Assay (cat. no. S0033S; Beyotime Institute of Biotechnology) was implemented. A total of 10 µmol/l GEN was added to the GEN and GEN + H₂O₂ group for 1 h; 130 µmol/l H₂O₂ was added to the GEN + H₂O₂ group and the H₂O₂ group for 1 h. After the treatment was completed, the six-well plates were taken out from the incubator and washed by adding 1 ml of PBS to each well once, then the PBS was discarded. The DCFH-DA probes were diluted with the serum-free RPMI-1640 culture medium at the ratio of 1:1,000. The discarded culture medium from the six-well plates was aspirated, and the cells were washed three times with 1 ml of serum-free RPMI-1640 culture medium per well to avoid eluting the cells from the wall. After aspirating the culture medium used for washing, 1 ml of prepared DCFH-DA probe was added to each well and the six-well plate loaded with the probe was incubated in the incubator for 40 min to prevent degradation and inactivation of the probe, and the HUVECs were washed with serum-free cell RPMI-1640 culture medium for 3 times after 40 min to wash away the DCFH-DA that had not entered into the cells. Then, 200 µl of 0.25% trypsin was added to each well, the digestion was terminated, centrifugation followed and the supernatant was discarded. A total of 200 µl of PBS was added to each tube and was blown several times to make a single-cell suspension, which was transferred to a flow tube protected from light, and put on a flow cytometer (C6, BD, USA, 488 nm excitation light and 525 nm emission used) for ROS detection.

Apoptosis was detected using Annexin V-FITC kit (cat. no. C1062L; Beyotime Institute of Biotechnology) (13). In brief, HUVECs were seeded in six-well plates at a density of 1.2×10⁵/ml per well for 24 h. Then, the desired concentrations of GEN and H₂O₂ were added and washed once with PBS according to previous description. After that, cells were collected and supplemented with Annexin V-FITC conjugate. The flow tubes were incubated for 20 min in a dark place at room temperature and analyzed immediately by a flow cytometer (C6 and BD Accuri™ C6 Software, version 227.4).

Cells in logarithmic growth phase were cultured in 5-mm cell culture dishes, and cell samples were collected after adding GEN and H₂O₂. The cells were removed from the incubator, placed in an ice bath, and the adherent cells were collected in 1.5 ml EP tubes by scraping with a cell scraper. A total of 100 µl of cell lysate was added to each tube and placed in liquid nitrogen for 3 min, 37°C water bath for 3 min, and repeated three times. The cells were pre-cooled at 4°C in advance in a centrifuge and centrifuged at 12,000 × g for 10 min, and the supernatant was taken for the assay of enzyme activity. The intracellular enzyme activity was detected according to the instructions of GPx kits (cat. no. S0058; Beyotime Institute of Biotechnology) (14), SOD kits (cat. no. BC0175; Beijing Solarbio Science & Technology Co., Ltd.) (15), and reduced glutathione (GSH) kits (cat. no. BC1175; Beijing Solarbio Science & Technology Co., Ltd.), respectively (16).

RNA [An RNA extraction kit was used (12183018A, PureLink™ RNA; Thermo Fisher)] was extracted from HUVECs and qPCR [The TBGreen^® Premix Ex Taq (Takara) was used] was conducted after reverse transcription according to the reagent manufacturer's instructions (T2210, Solarbio, China). GAPDH was used as a reference gene. Primer sequences are listed in Table I. Thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles including denaturation at 95°C for 10 sec, annealing at 55°C and extension at 72°C for 30 sec. A standard measure of mRNA expression (2-ΔΔ cycle threshold) was calculated (17).

A total of 50 µg of cell protein were collected from four groups [Negative control (NC), GEN, GEN + H₂O₂, H₂O₂] with a cell scraper and lysed with RIPA (cat. no. P0013B; Beyotime Institute of Biotechnology) and 1% PMSF (cat. no. ST506; Beyotime Institute of Biotechnology). Electrophoresis was carried out, placing 50 µg of protein in each well, using 10% preformed gel. After transferring the proteins to the membrane (PVDF), it was blocked with 5% skimmed milk powder at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies (all diluted at 1:1,000): Nuclear factor erythroid2-related factor 2 (Nrf2; cat. no. 12721S), heme oxygenase (HO-1; cat. no. 43966), SOD1 (37385S), Bax (cat. no. 5023S; all from Cell Signaling Technology, Inc.), Caspase-3 (cat. no. 14220S; Abcam) and Bcl-2 (cat. no. orb10173; Biorbyt, Ltd). Following the primary incubation, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3,000; cat. no. 7074S; Cell Signaling Technology, Inc.) for 1 h at room temperature. As the same grouping (NC, GEN, GEN + H₂O₂, H₂O₂) in western blot detection, the same GAPDH (cat. no. AC001; ABclonal Biotech Co., Ltd.) was used as control in each experiment which was repeated at least three times. ImageJ software (1.52a, National Institutes of Health) was used to analyze the grey value of each resulting image, and the control protein was used to normalize the grey values of the target protein for statistical analysis.

Statistical analysis was performed using GraphPad Prism 8 (Dotmatics). One way ANOVA was used for multiple group comparisons, corrected by Bonferroni's method. Two independent samples unpaired t-test was used for comparison between two groups. All experiments were repeated more than 3 times. P<0.05 was considered to indicate a statistically significant difference.

To determine whether GEN could play a protective role against H₂O₂-induced cell damage, in combination with H₂O₂ stress for 1 h, HUVECs were pretreated with GEN (100 nM, 1 and 10 µM) for 1 h. As demonstrated in Fig. 1, the cell viability increased significantly after pretreatment of GEN compared with control group (****P<0.0001). Thus, 10 µM GEN pretreatment for 1 h was used as the treatment condition for the following experiments.

To investigate whether GEN could reduce the level of intracellular ROS, DCFH-DA fluorescent probe was used to detect the intracellular ROS in the labeled cells. As revealed in Fig. 2, intracellular ROS levels increased significantly after H₂O₂ stress (*P<0.05). By contrast, after GEN pretreatment, the intracellular ROS levels reduced significantly (*P<0.05).

To verify whether GEN could further attenuate the occurrence of apoptosis by attenuating the aggregation of ROS, Annexin-V/FITC Assay was applied to stain cells pretreated with the GEN. The apoptotic rates were counted separately for different periods. As shown in Fig. 3, GEN decreased the early apoptotic rate and the total apoptotic rate significantly (P<0.05), while no effect was detected for the late apoptotic rate.

To further verify the mitigating effect of GEN on the intracellular ROS, intracellular redox-responsive enzymes were measured. As demonstrated in Fig. 4A, the enzyme activity of intracellular GPx was significantly reduced in the H₂O₂ group (*P<0.05) and increased after GEN pretreatment (*P<0.05). The intracellular SOD activity was also significantly reduced (**P<0.01) and increased after GEN pretreatment (*P<0.05; Fig. 4B). In addition, the activity of intracellular GSH was significantly reduced in the H₂O₂ group (***P<0.001) and increased after GEN pretreatment (***P<0.001; Fig. 4C).

To verify whether the protective effect of GEN on oxidative damage after H₂O₂ stress was mediated through the Nrf2 pathway, the expression of Nrf2 pathway-related molecules (Nrf2, HO-1 and SOD1) was detected. As revealed in Fig. 5A, GEN pretreatment increased the mRNA expression of Nrf2 (**P<0.01), HO-1 (P<0.001) and SOD1 (**P<0.01) simultaneously under H₂O₂ stress. As shown in Fig. 5B and C, compared with the control group, the protein expression of Nrf2 (***P<0.001), HO-1 (***P<0.001) and SOD1 (****P<0.0001) significantly decreased after H₂O₂ stress. Compared with the H₂O₂ group, the protein expression of Nrf2 (*P<0.05), HO-1 (**P<0.01) and SOD1 (*P<0.05) increased significantly after GEN pretreatment.

To verify the expression of apoptosis-related genes after GEN treatment, the expression of Bax, Bcl-2, and Caspase-3 were detected by RT-qPCR and western blotting, respectively. As shown in Fig. 6A, compared with the control group, the ratio of Bax/Bcl-2 was significantly higher after H₂O₂ stress (*P<0.05), which was decreased after GEN pretreatment (**P<0.01). The expression of Caspase-3 was also significantly higher in the H₂O₂ group (***P<0.001), and lower in the GEN pretreatment group (***P<0.001; Fig. 6B and C. Compared with the control group, the ratio of Bax/Bcl-2 (***P<0.001) and the protein expression of Caspase-3 were significantly higher (*P<0.05) in the H₂O₂ group. However, the ratio of Bax/Bcl-2 was decreased (P<0.01) and the expression of Caspase-3 was also decreased (**P<0.01) after GEN pretreatment.