HUVECs were purchased from the American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cultures were maintained in a 37°C incubator under a humidified atmosphere of 5% CO₂/95% air.

Cultured HUVECs were pre-incubated with MDG-1 (5, 10 or 50 mM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 12, 24 and 48 h prior to H₂O₂ treatment (100, 300 or 500 µM). Following the indicated time, the medium was removed, and the cells were subjected to the subsequent experiments.

The Cell Counting Kit-8 (CCK-8; Dojindo Molecular technologies, Inc., Rockville, MD, USA) was used to assess cell viability in HUVECs. In brief, HUVECs in the logarithmic growth-phase were collected, and 5×10⁴ cells/well were dispensed into 96-well culture plates with 100 µl culture medium. After 24 h of culture, different concentrations of MDG-1 (5, 10 or 50 mM) were added to each well prior to H₂O₂ treatment (100, 300 or 500 µM). Each of the concentrations above was regarded as one treatment group. Culture plates were then incubated for 4, 6 and 12 h, and cell viability was evaluated by CCK-8, following the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The optical density of indicated groups was used as a surrogate measurement for cell viability.

HUVECs were treated with different concentrations of MDG-1 (5, 10 or 50 mM) prior to H₂O₂ treatment (100, 300 or 500 µM) for 12 h. Cell apoptosis was then analyzed using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. In brief, HUVECs following treatment were washed three times with PBS, trypsinized, centrifuged (400 × g at room temperature) for 10 min, and resuspended to a final concentration of 5×10⁴ cells/ml in binding buffer containing Annexin V-FITC and PI. Apoptotic cells were then analyzed using a BD Accuri C6 flow cytometer equipped with BD Accuri C6 software (version 1.0.264) (both from BD Biosciences).

HUVECs (3×10³ cells/well) were cultured on a slide in DMEM and the indicated treatments were performed. Following treatment, the cells were washed three times with PBS, trypsinized, centrifuged (400 × g at room temperature) for 10 min and resuspended to a final concentration of 5×10⁴ cells/ml. The dihydroethidium (DHE; 50 µM; Beyotime Institute of Biotechnology) probe was then added to the cells at 37°C for 30 min, following which the cells were analyzed by flow cytometry (BD Biosciences).

HUVECs (5×10⁴ cells/well) were seeded in 6-well culture plates and the indicated treatments were performed. The concentration of each secreted inflammatory factor in the cell supernatant was measured by ELISA, according to the manufacturer's protocol. ELISA kits were purchased as following: Human tumor necrosis factor (TNF-α; 070133h; Shanghai WuHao Trading Co., Ltd., Shanghai, China); human interleukin 1β (IL-1β; DL-IL1b-Hu; Wuxi DonglinSci & Tech Development Co., Ltd., Wuxi, China); human IL-6 (ab46042; Abcam, Cambridge, MA, USA); human cyclooxygenase-2 (Cox-2; ESK5229-48T; Sangon Biotech Co., Ltd., Shanghai, China). ELISA kits were used according to the manufacturer's protocol.

HUVECs were harvested, washed twice with PBS, and lyzed with radio immuno precipitation assay buffer (Beyotime Institute of Biotechnology) with freshly added 0.01% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min. Cell lysates were centrifuged at 1,000 × g for 10 min at 4°C. The supernatant (20–30 µg of protein) was separated on 10% SDS-PAGE and transferred electrophoretically to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The blots were blocked with 5% skim milk overnight at 4°C, followed by incubation with primary antibodies overnight at 4°C. Rabbit polyclonal antibodies against Bcl-2 (cat. no. sc-492; 1:150) and Bax (cat. no. sc-493; 1:100) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A rabbit polyclonal antibody against caspase-3 (cat. no. ab2302; 1:500) was purchased from Abcam. A rabbit monoclonal antibody against GAPDH (cat. no. 5174; 1:1,500) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Blots were then incubated with goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology) for 1 h at 37°C and visualized using enhanced chemiluminescence reagents (EMD Millipore). Quantity One 4.62 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantitatively analyze protein expression levels.

Data were expressed as the mean ± standard deviation of triplicate experiments. One-way analysis of variance, followed by Tukey's post hoc test, was used to analyze the significance of differences between groups with SPSS 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To identify whether H₂O₂ may cause cytotoxicity in vitro, the effect of H₂O₂ on HUVEC viability was determined using the CCK-8 assay. HUVECs were treated with H₂O₂ at concentrations of 100, 300 or 500 µM, and exhibited significantly decreased cell viability in a dose-dependent manner at 6 and 12 h (Fig. 1A). A flow cytometry assay was then used in order to assess the effect of H₂O₂ on HUVEC apoptosis. HUVECs were treated with increasing concentrations of H₂O₂ for 12 h and exhibited a significant dose-dependent increase in the % of apoptotic cells over total, compared with untreated cells (Fig. 1B and C).

To investigate the effect of MDG-1 on H₂O₂-induced cytotoxicity, HUVECs were pretreated with MDG-1 at different concentrations prior to exposure to 300 µM H₂O₂ for 12 h. As presented in Fig. 2A, pretreatment of HUVECs with 5, 10 or 50 µM MDG-1 for 12, 24 or 48 h significantly increased cell viability in a dose-dependent manner, compared with cells treated with H₂O₂ alone. In addition, pretreatment of HUVECs with MDG-1 at concentrations of 5, 10 or 50 mM for 24 h significantly decreased cell apoptosis in a dose-dependent manner, compared with cells treated with H₂O₂ alone (Fig. 2B and C). These results indicate that MDG-1 pretreatment protected HUVECs against H₂O₂-induced toxicity.

To elucidate the possible mechanisms by which MDG-1 prevented H₂O₂-induced HUVEC apoptosis, ROS generation was measured in HUVECs that were treated with H₂O₂ and/or MDG-1. Exposure of HUVECs to 300 µM H₂O₂ for 12 h significantly enhanced ROS generation compared with untreated cells (Fig. 3). However, pretreatment with MDG-1 for 24 h (at concentrations of 5, 10 or 50 mM) significantly attenuated the H₂O₂-induced increase in ROS generation in a dose-dependent manner (Fig. 3). These findings demonstrated that the protective function of MDG-1 on HUVEC H₂O₂-induced toxicity may be associated through its antioxidant effect.

To determine the effect of MDG-1 in the inflammatory response of endothelial cells, secretion of the inflammatory factors TNF-α, IL-1β, IL-6 and Cox-2 was measured by ELISA in HUVECs that were treated with H₂O₂ and/or MDG-1. The concentration of TNF-α, IL-1β, IL-6 and Cox-2 was significantly increased in H₂O₂-induced HUVECs, compared with untreated cells (Fig. 4). However, pretreatment with MDG-1 for 24 h resulted in a significant and dose-dependent decrease in TNF-α, IL-1β, IL-6 and Cox-2 secretion, compared with cells induced with H₂O₂ alone (Fig. 4). These results indicated that MDG-1 pretreatment protected against H₂O₂-induced inflammatory responses in HUVECs.

Western blot analysis was performed to detect the protein expression levels of Bax, Bcl-2 and caspase-3 in HUVECs treated with 300 µM H₂O₂ for 12 h. H₂O₂ treatment decreased the expression of the antiapoptotic protein Bcl-2, while it increased the expression of the proapoptotic proteins caspase-3 and Bax, compared with untreated cells (Fig. 5). Notably, MDG-1 pretreatment at concentrations of 5, 10 and 50 mM markedly reversed the effects induced by H₂O₂ treatment on apoptosis-related protein expression, compared with cells treated with H₂O₂ alone (Fig. 5).