Human umbilical vein endothelial cells (HUVECs; China Center for Type Culture Collection, Wuhan, China) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l D-glucose supplemented with 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. The HUVECs were subcultured at a 1:2 ratio interval of 2 days. Vascular injury associated with DM in vitro was mimicked by incubation with H₂O₂ 100 μmol/l for 18 h to induce damage to the HUVECs, as previously described (24). The cells were treated with each agent in the medium for 1 h prior to exposure to H₂O₂.

The cells were divided into 3 groups as follows: i) the control group: cells were left untreated; ii) the model group: cells were treated with H₂O₂ 100 μmol/l for 18 h; and iii) the AST IV group: cells were treated with AST IV for 1 h and then treated with H₂O₂. In addition, some cells were treated with diphenyliodonium (DPI, a specific inhibitor of Nox4; from Sigma, St. Louis, MO, USA) or LY2109761 (a selective inhibitor of TGF-β1/Smad2; from MedChem Express, LLC, Princeton, NJ, USA).

The half maximal effective concentration (EC₅₀) of AST IV in preventing the H₂O₂-induced damage to HUVECs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). In brief, the cells were seeded at a density of 1×10⁴ cells/well in 96-well plates. They were then exposed to serial dilutions of AST IV in DMEM-high glucose medium and allowed to grow for the indicated periods of time. Following treatment, the cells were incubated with 100 μl DMEM-high glucose medium containing 5 mg/ml MTT. Following incubation for 4 h at 37°C, the supernatants were discarded, MTT crystals were dissolved in 100 μl dimethyl sulfoxide (DMSO) and the optical density (OD) was measured at 570 nm using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA, USA).

Intracellular ROS production was detected using a probe, the redox-sensitive fluorophore carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Sigma) as the following steps. After delivery, the cells were washed with phosphate-buffered saline (PBS) and incubated with 20 μmol/l H₂DCFDA in the dark for 30 min. The cells were then briefly exposed to 0.5 g/l trypsin. The deactivation of trypsin was accomplished by the addition of PBS supplemented with 3% FBS. All cells were examined using a FACScan flow cytometer (Beckman Coulter, Miami, FL, USA) and the data were processed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA) (25).

Following treatment, apoptosis was determined according to the following steps: the cells were collected by 0.25% ethylenediaminetetraacetic acid (EDTA)-free trypsin to digest the cells followed by centrifugation (at 1,500 rpm) to collect the cells; the cells then were suspended with 500 μl binding buffer, and the concentration was then adjusted to 1×10⁶ followed by the addition of 5 μl Annexin V-FITC staining fluid, gentle blending and incubation at 4°C in the dark for 15 min; the cells were then treated with PI dyeing liquid at 4°C in the dark for 5 min. Finally the cells were immediately examined using a FACScan flow cytometer (Beckman Coulter). Annexin V-FITC detection was carried out using the FL-1 and PI detection using the FL-2 channel.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using the First-Strand cDNA Synthesis kit (Life Technology, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA were used as the template for the qPCR amplification using oligo primers, and the internal control for the qPCR reaction was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences of the Nox4, TGF-β1, Smad2, Bax, Bcl-2 and caspase-3 genes (Shanghai Sangon Biotech, Shanghai, China) are presented in Table I. qPCR was performed using an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). qPCR was performed with a total volume of 12 μl in each well, containing 5 μl of SYBR-Green^® PCR Master Mix (Applied Biosystems), 5 μl of cDNA, and 1 μmol forward primers and 1 μmol reverse primers. Each sample was run in triplicate in a separate tube. The qPCR conditions were conducted as follows: 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Initial heating at 95°C for 10 min and a final extension at 72°C for 7 min was performed for all qPCR reactions. The cycle threshold (CT) values from all the qPCR experiments were calculated using the 2^−ΔΔCT method.

Following treatment, the cells were lysed using RIPA buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (PMSF; Sigma) on ice. The total protein concentrations were measured using the BCA Protein Assay kit (Beyotime). Proteins were separated by 8 or 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA). Subsequently, the membranes were blocked with 5% non-fat milk at room temperature (RT) for 1 h, and incubated with primary antibodies as follows: anti-Nox4 (ab109225; Abcam, Cambridge, UK), anti-TGF-β1 (3712S; Cell Signaling Technology Inc., Beverly, MA, USA), anti-Smad2 (ab33875; Abcam), anti-Bax (sc-6236), anti-Bcl-2 (sc-783) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-caspase 3 (9665S; Cell Signaling Technology Inc.) and anti-β-actin (A1978; Sigma) antibodies overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse, anti-rabbit antibodies (1:2,000 dilution) for 1 h at RT. Moreover, the proteins were visualized using an ECL advanced western blot detection kit (Pierce, Thermo, Rockford, IL, USA). Densitometric measurements of band intensity in the western blot analysis were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

All experiments were carried out 3 times. All quantified results are expressed as the means ± SEM in graphical representation. Data analysis of all results was carried out using one-way analysis of variance (ANOVA) followed by Fisher’s LSD-based post-hoc analysis. All P-values were two sided and considered significant when P<0.05. Statistical analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

MTT (Sigma) assay was performed to determine the EC₅₀ of AST IV in inhibiting H₂O₂-induced damage to HUVECs. The cells were treated with AST IV at 5, 10, 25, 50, 100, 200, 400 and 800 μmol/l for 1 h prior to treatment with H₂O₂ and cell viability was then examined by MTT assay. The results revealed that the EC₅₀ of AST IV in inhibiting H₂O₂-induced injury to HUVECs was 100 μmol/l [calculated by regression equation (x, drug concentration; y, cell survival rate)].

To assess the protective effects of AST IV against H₂O₂-induced HUVEC apoptosis, the mRNA and protein expression levels of Nox4, TGF-β1, Smad2, Bcl-2, Bax and caspase-3 were determined; the intercellular ROS level and the apoptotic rate were also determined. Nox4 expression in the model group was significantly increased in comparison to the control group (P<0.01; Fig. 2B and C). The geometric mean fluorescence intensity indicating ROS production in the control group was lower, whereas that in the model group was markedly increased (P<0.01; Fig. 2A). In comparison with the control group, the expression of TGF-β1/Smad2 was markedly elevated in the model group (P<0.01). In addition, the expression of Bax (pro-apoptotic gene) was lower and that of Bcl-2 (anti-apoptotic gene) was higher in the control group compared to the model group (P<0.01; Fig. 2B and C). The expression of caspase-3, a main terminal shear enzyme involved in apoptosis, was barely detectable in the control group; by contrast, its expression was upregulated in the model group (P<0.01; Fig. 2B and C). In comparison with the control group, the apoptotic rate was significantly increased in the model group (P<0.01; Fig. 2D). However, treatment with AST IV100 μmol/l reversed these effects (P<0.01).

To explore the molecular mechanisms responsible for H₂O₂-induced HUVEC apoptosis, the HUVECs were treated with diphenyliodonium (DPI; 10 μmol, a Nox4 inhibitor) prior to treatment with H₂O₂. We found that treatment with DPI or AST IV decreased the expression of Nox4 (Fig. 3B and C), as well as the intercellular ROS levels in the HUVECs treated with H₂O₂ (Fig. 3A). Our results revealed that DPI or AST decreased TGF-β1/Smad2 expression in the HUVECs damaged by H₂O₂ (Fig. 3B and C). Our results also revealed that treatment with DPI or AST IV decreased Bax and caspase-3 expression, and increased Bcl-2 expression (Fig. 3B and C). In addition, treatment with DPI or AST IV decreased the HUVEC apoptotic rate (Fig. 3D). The exposure of the cells to AST IV at 100 μmol/l had a similar effect to that of treament with DPI (Fig. 3).

To further investigate the role of the TGF-β1/Smad2 signaling pathway in H₂O₂-induced HUVEC apoptosis, LY2109761 (0.1 μmol/l), a selective inhibitor of TGF-β1/Smad2, was used to suppress the activation of the TGF-β1/Smad2 pathway. The results revealed that TGF-β1 and Smad2 mRNA and protein expression was detected at extremely low levels in the control group, whereas the overexpression of TGF-β1 and Smad2 was observed in the model group; there was a statistically significant difference between the control group and the model group (P<0.01; Fig. 4B and C). However, treatment with LY2109761 decreased TGF-β1 and Smad2 expression, as well as Bax and caspase-3 expression, and increased Bcl-2 expression (P<0.01; Fig. 4B and C). In addition, treatment with LY2109761 decreased HUVEC apoptosis (P<0.01; Fig. 4D), but had no effect on Nox4 expression and the ROS levels (P>0.05; Fig. 4A–C). Treatment with AST IV at 100 μmol/l significantly ameliorated these risk factors; it downregulated Nox4 expression (Fig. 4B and C), decreased ROS levels (Fig. 4A), decreased TGF-β1, Smad2, Bax and caspase-3 expression (Fig. 4B and C) and upregulated Bcl-2 expression (Fig. 4B and C) (P<0.05 and P<0.01 compared to model group), further decreasing HUVEC apoptosis (Fig. 4D).