HCY and D-galactose (both Sigma-Aldrich; Merck KGaA) were dissolved in cell culture media [complete medium containing Dulbecco's modified Eagle's Medium/F-12 (HyClone; Cytiva), 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% endothelial cell growth supplement (ScienCell Research Laboratories, Inc.), 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate (both Hyclone; Cytiva)]. 3-Methyladenine (3-MA) and rapamycin (both Gene Chem Co., Ltd., China) were dissolved in dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology Co., Ltd.). All of the reagents were diluted with cell culture media to experimental concentrations and were newly prepared for each experiment.

Primary HUVECs were immediately isolated from fresh umbilical cords of healthy pregnant individuals, as previously described (20). The present study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Urumchi, China; no. 20190225-13). Informed written consent was obtained from all participants (age, 20-25 years) from whom HUVECs were obtained. Human umbilical cords were collected into a sterile centrifuge tube containing PBS (Shanghai Sangon Biotech Co, Ltd.), 100 U/ml penicillin G and 100 ug/ml streptomycin sulfate and were isolated immediately. All procedures were performed out on a cleaning bench. Following washing with PBS, the umbilical vein was digested with 0.25% pancreatic enzyme at 37˚C for 10 min, centrifuged at 1,000 x g at 28˚C for 5 min, resuspended and cultured in Dulbecco's modified Eagle's medium/F-12 (Hyclone; Cytiva) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% endothelial cell growth supplement (ScienCell Research Laboratories, Inc.), 100 U/ml penicillin G and 100 ug/ml streptomycin sulfate (both Hyclone; Cytiva) in a humidified atmosphere with 5% CO₂ at 37˚C. The medium was changed and the cells were passaged every 2 days. Cells obtained between three and six passages were used to ensure cell line stability.

HUVECs were treated with various concentrations (0, 25, 100 and 500 µM) of HCY at 37˚C for 24 h, followed by incubation at 37˚C for 24 h. Cells treated with 5 g/l D-galactose at 37˚C for 24 h were used as a classic senescence model control. Then, cells were treated with 100 µM 3-MA or 5.5 nM rapamycin for 1 h at 37˚C before culture with 500 µM HCY for 24 h at 37˚C to test changes in autophagy and its role in HCY-induced senescence. The final concentration of DMSO used for dilution did not exceed 0.2%.

CCK-8 (Dojindo Laboratories, Inc.) assay was used to assess cell proliferation. Untreated HUVECs were plated at a density of 1x10³/well in 96-well plates and incubated as aforementioned. Following culture period, cells were washed with PBS three times. The cells were then cultured with 100 µl fresh cell culture media and 10 µl CCK-8 solution. After 3 h incubation at 37˚C with 5% CO₂, the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.). The cell proliferation was calculated using the following formula: Proliferation rate=[optical density (OD)_{experimental group}-OD_{blank group}]/(OD_{control group}-OD_{blank group}).

Propidium iodide (PI)/ribonuclease (RNase) staining buffer (BD Pharmingen; BD Biosciences) was used to measure cell cycle arrest. Cells in 100-mm petri dishes were cultured to 50-60% confluence and treated with HCY, D-galactose, 3-MA and rapamycin as aforementioned. Following treatment, cells were collected in 5 ml Eppendorf (EP) tubes, fixed at -4˚C for 24 h with precooled 70% ethanol and incubated at -4˚C overnight. Cells were centrifuged at 1,000 x g for 5 min at 28˚C, the supernatant was discarded and the cells were resuspended in PBS. The procedure was repeated three times. The cells were stained with PI/RNase staining buffer for 15 min at 28˚C in the dark. Each sample was analyzed using the BD LSRII Analyzer (BD Pharmingen; BD Biosciences) and Modfit version 5 (Becton, Dickinson and Company).

Cells in 6-well plates were cultured to 50-60% confluence. The cells were washed with PBS once after treatment with HCY, D-galactose, 3-MA and rapamycin as aforementioned, and stained with SA-β-Gal staining reagent (Beyotime Institute of Biotechnology) according to the manufacturer's instruction. The cells were still mounted. Blue-stained cells were manually counted in three randomly selected fields of view using light microscopy (x100 magnification).

Cells in 6-well plates were cultured to 50-60% confluence. The cells were washed with PBS. Cells were cultured for 20 min with DCFH-DA (Beyotime Institute of Biotechnology), which was diluted 1:1,000 in cell culture media without FBS, at 37˚C and 5% CO₂ in the dark. After washing the cells three times with Dulbecco's modified Eagle's Medium/F-12, images were obtained using a Leica DMI6000B fluorescence microscope (Leica Microsystems GmbH; x100 magnification) and the mean fluorescence intensity/cell was measured using ImageJ 1.53e (National Institutes of Health).

Cells in 96-well plates were cultured to 10-20% confluence. The cells were transfected with 100 µl mixed liquid containing 10 µl autophagy-related lentivirus, 4 µl HitransG A (both Shanghai GeneChem Co., Ltd) and 86 µl cell culture media, followed by incubation for 12-16 h at 37˚C. The Stub-RFP-Sens-GFP-LC3 autophagy-related double fluorescence lentivirus is a lentivirus expressing red fluorescent and green fluorescent protein and autophagy marker protein LC3 that was used according to the manufacturer's instructions. The Sens-GFP is an acid-sensitive protein and the Stub-RFP is a stable fluorescent protein. When the autophagic flux increases, the expression of GFP and RFP increases simultaneously. Then autophagosomes fuse with lysosomes, changing the environment to acid, which quenches green fluorescence. The fluorescence color change from yellow to red indicates autophagy flux change (21). The transfected cells were cultured in a 100-mm Petri dish for 36-72 h at 37˚C. Successfully transfected cells were cultured for 24 h at 37˚C in confocal dishes and treated with HCY, D-galactose, 3-MA and rapamycin as aforementioned. Images were captured using a confocal laser microscope (Leica Microsystems GmbH; x400 magnification). The number of autophagy-related fluorescent dots/cell was manually counted in three randomly selected fields of view.

All data are presented as the mean ± standard deviation of three independent experimental repeats. SPSS version 20.0 (IBM Corp.) was used to perform statistical analysis. Comparisons between two independent samples were performed using an unpaired t-test. One-way analysis of variance followed by Dunnett's post hoc test was used for comparisons between >2 groups. P<0.05 was considered to indicate a statistically significant difference.

D-galactose is a classical senescence inducer (4). D-galactose was used to establish the classical senescence model. The cell proliferation rate in each group was tested by CCK-8 and examined via assessing cell viability. Compared with that in the control group, the cell proliferation rate of D-galactose group was reduced by 40.87%, and that of 500 µM HCY group was decreased by 49.46%. HCY decreased cell proliferation activity significantly in a dose-dependent manner (Fig. 1A). The cell cycle arrest was tested by flow cytometry. Compared with that in the control group, the number of G₁/G₀ stage cells increased by 15.46% in the D-galactose intervention group and 11.40% in the 500µM HCY group. Cell cycle in the HCY-treated group was arrested in a dose-dependent manner and HCY significantly arrested HUVECs in the G₁/G₀ stage (Fig. 1B and C).

The activity of SA-β-Gal is upregulated during cell senescence and the test of SA-β-Gal activation is used as a senescence marker (9). Blue-stained cells represent senescent cells. The number of senescent cells increased significantly in HUVECs subjected to HCY treatment (Fig. 1D and E). Higher HCY concentration resulted in more senescent cells. Compared with that in the control group, the number of senescent cells increased 6.04 times in the D-galactose intervention group and 5.39 times in the 500 µM HCY group.

LC3 is an autophagy marker protein (22). To observe intracellular autophagic flux, the Stub-RFP-Sens-GFP-LC3 autophagy-related double fluorescence lentivirus was used. Autophagy-associated yellow dots increased significantly in HUVECs treated with HCY (Fig. 2A and B). The autophagy dots in the 500 µM HCY group increased by 2.53 times compared with those of the control group.

To investigate the effect of autophagy on HCY-induced HUVEC senescence, 3-MA, a commonly used autophagy inhibitor, and rapamycin, an autophagy inducer, were used to inhibit and induce autophagy, respectively (22). Compared with the 500 µM HCY group, 3-MA decreased the number of autophagic dots in HUVECs treated with HCY by 30.10%, while rapamycin increased these by 82.94% (Fig. 2C and D). The HVUECs treated with 3-MA exhibited decreased cell viability and cell cycle progression (Fig. 2E-G). Treatment with rapamycin increased cell viability and alleviated cell cycle arrest. Compared with the 500 µM HCY group, 3-MA reduced the cell viability of HUVECs treated with HCY by 29.13%, and increased the number of G₁/G₀ stage cells by 5.66%, while rapamycin increased the cell viability of HUVECs treated with HCY by 35.56%, and reduced the number of G₁/G₀ stage cells by 6.94%. Furthermore, compared with the 500 µM HCY group, 3-MA treatment increased the number of senescent cells in the HCY-treated group by 57.81%, while rapamycin decreased the number of senescent cells by 42.74% (Fig. 2H and I).

ROS are increased during cell senescence and participate in the mechanisms of senescence (1). To test ROS in HCY-treated HUVECs, an inverted fluorescence microscope was used. The mean fluorescence intensity, indicative of the ROS concentration, was significantly higher in HUVECs treated with HCY and D-galactose than in the control group (Fig. 3A and B). Compared with that in the control group, the mean fluorescence intensity in the 500 µM HCY intervention group increased by 1.61 times, and in the D-galactose intervention group, by 1.76 times.

Previous studies have shown that ROS induces autophagy, while autophagy can delay cell senescence by decreasing intracellular ROS (4,23). To investigate the role of ROS in autophagy and HCY-induced endothelial cell senescence, 3-MA and rapamycin were used. Compared with the 500 µM HCY group, exposure to 3-MA increased ROS levels in HUVECs treated with HCY by 13.46%, while exposure to rapamycin decreased ROS levels in HUVECs treated with HCY by 18.59% (Fig. 3C and D).