APS was purchased from Nanjing Jingzhu Pharmaceutical Technology Co., Ltd. (Nanjing, China). H₂O₂ was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Dihydroethidium (DHE) was obtained from Shanghai Haoran Bio-Technology Co., Ltd. (Shanghai, China). MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). DAPI, Rhodamine 123 and the Cell Cycle and Apoptosis analysis kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). The cGMP assay kit was supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit anti-Copper-zinc superoxide dismutase (Cu/Zn-SOD; bs-10216R) and anti-endothelial nitric oxide synthase (eNOS; bs-20609R) antibodies were purchased from Beijing Boosen Biological Technology Co., Ltd (Beijing, China), and anti-β-actin (HRP-600008) was from Wuhan Sanying Biotechnology (Wuhan, China). All other chemicals and reagents used were of analytical grade.

Human umbilical vein endothelial cells (HUVECs) were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China) and were maintained in high-glucose Dulbecco's Modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 2 mmol/l L-glutamine, 5 µg/ml endothelial growth factor (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in a humidified incubator with 5% CO₂ at 37°C with media replenishment every 2 days and were passaged at 80–90% confluence. The cells were treated with various concentrations of APS or H₂O₂ for 24 h incubated in serum-free medium, prior to an MTT assay. Additional groups were pretreated with various concentrations of APS for 1 h prior to H₂O₂ exposure in fresh medium, following which they were harvested for further analysis. The cells were assigned into the following five groups; A, untreated control group; B, H₂O₂ (400 µM) model group; C, H₂O₂+APS (0.1 µg/ml) group; D, H₂O₂+APS (1 µg/ml) group; E, H₂O₂+APS (10 µg/ml) group.

The MTT assay was used to evaluate cell viability (22). In brief, HUVECs were cultured in a 96-well plate (1×104 cells/well). A total of 24 h following plating, cells were pretreated with 0.1–100 µg/ml APS for 1 h, following which 400 µM H₂O₂ was added into the plate and cultured at 37°C for a further 24 h. The viability of cells treated with APS (0.1–100 µg/ml) or H₂O₂ alone (0–500 µM) was also assessed. MTT was dissolved in PBS, added to each well and incubated at 37°C for 4 h, for a final concentration of 0.5 mg/ml. Subsequently, the culture medium was carefully removed and replaced with 150 µl DMSO. The plates were incubated for 10 min and measured at a wavelength of 490 nm on a microplate reader (DNM-9602G; Beijing Perlong New Technology Co., Ltd., Beijing, China). The viability of HUVECs was expressed as a percentage of the control group.

Apoptosis was evaluated using the Cell Cycle and Apoptosis analysis kit. Briefly, following treatment, floating and attached cells were harvested by trypsin and washed with PBS (concentration, 1×106 cells/ml). Cells were fixed with ice-cold 70% alcohol at 4°C overnight, and staining was performed with propidium (PI) according to the manufacturer's protocol. Samples were analyzed on a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest software v6.1x (BD Biosciences). Cells in the sub-G1 peak were considered apoptotic.

The morphological characteristics of nuclei were evaluated by DAPI staining as previously described (23). HUVECs were seeded onto a 6-well plate at a density of 1×105 cells/well. Following treatment, cells were fixed with 4% paraformaldehyde for 10 min. Cells were washed once with PBS and stained with 2 µg/ml DAPI for 15 min at 37°C in the dark. The stained cells were visualized under an inverted fluorescence microscope.

The mitochondrial membrane potential of HUVECs was assessed using the fluorescent dye Rhodamine 123 (24–26). Rhodamine 123, a selective fluorescent dye, permeates cell membranes and is sequestered by active mitochondria, therefore staining the mitochondria of living cells, and is widely used for the detection of mitochondrial membrane potential. Flavovirens fluorescence represents normal cells. HUVECs were cultured in a 6-well plate at a density of 1×105 cells/well. Following treatment, the culture medium was carefully removed and cells were washed twice with PBS. The cells were subsequently stained with 2 µM Rhodamine 123 for 20 min at 37°C in the dark. Cells were analyzed under an inverted fluorescence microscope. The fluorescence intensity was calculated with LAS Software (V4.3) (Leica Microsystems GmbH, Wetzlar, Germany).

HUVECs were pretreated with 0.1, 1 or 10 µg/ml APS for 1 h, followed by exposure to 400 µM H₂O₂ for 24 h. The cells were subsequently harvested with a Falcon scraper, centrifuged at 4°C for 5 min at 800 × g and sonicated. The homogenate was prepared in PBS and centrifuged at 1,000 × g for 5 min at 4°C. The cGMP levels in the supernatants were determined with a kit according to the manufacturer's protocol. cGMP levels were expressed as nmol/g protein. Protein concentration was calculated with a Bicinchoninic Acid (BCA) Protein assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). cGMP is a second messenger of NO, and its level reflects the level of nitric oxide (NO) (27).

The production of ROS was measured using DHE. DHE, an oxidative fluorescent dye, may be oxidized to ethidium bromide and intercalated into DNA in the presence of superoxide anions. HUVECs were seeded on a 6-well plate (1×105 cells/well). Following treatment, cells were washed with PBS and incubated with 2 mM DHE for 30 min at 37°C. Cells were washed with PBS and analyzed under an inverted fluorescence microscope. The fluorescence intensity was calculated using LAS Software V4.3.

Following treatment, cells were harvested and lysed with ice-cold radio immunoprecipitation assay buffer (Wuhan Boster Biotechnology Co., Ltd., Wuhan, China). Protein concentration was determined using a BCA Protein assay kit. Protein extracts (30 µg) were electrophoretically separated by 12 or 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk powder and incubated overnight at 4°C with the following primary antibodies: Anti-eNOS (1:1,500), anti-Cu/Zn-SOD (1:500) and β-actin (1:500). Following washing with TBS containing Tween-20 (10 mM Tris, 100 mM NaCl, and 0.1% Tween-20), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG; 1706515 and 1706516; 1:1,500; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 2 h at room temperature. Membranes were washed with TBS, and immunocomplexes were visualized using the Amersham ECL start Western Blotting Detection Reagent enhanced chemiluminescence system (GE Healthcare Life Sciences). β-actin served as an internal control for the experiments. The results were quantified by the Quantity One software v4.62 (Bio-Rad Laboratories, Inc.).

All data are expressed as the mean ± standard deviation. Each experiment was performed at least three times. Statistical analysis was performed using Student's t-test or one-way analysis of variance with SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA). The post hoc least significant difference test was used to evaluate differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

Initially, the cytotoxicity of APS on HUVECs was measured using the MTT assay. As presented in Fig. 1A, incubation of HUVECs with 0.1–100 µg/ml APS for 24 h did not significantly affect the cell viability (P>0.05). However, treatment of HUVECs with 100 to 500 µM H₂O₂ for 24 h resulted in a concentration-dependent decrease in cell viability, when compared with the control group (P<0.05; Fig. 1B). Pretreatment of HUVECs with 0.1, 1 or 10 µg/ml APS for 1 h prior to exposure to H₂O₂ significantly increased viability in a dose-dependent manner (Fig. 1C). These results indicated that APS may protect HUVECs from oxidative stress-induced injury. Based on these results, further studies employed a H₂O₂ concentration of 400 µM, and APS concentrations of 0.1, 1.0 and 10 µg/ml.

To further evaluate the protective effect of APS against H₂O₂-induced damage in HUVECs, apoptosis rates were measured by flow cytometry. As presented in Fig. 2, the percentage of apoptotic cells in the control group was 5.44±0.57%. Following exposure to 400 µM H₂O₂ for 24 h, the percentage of apoptosis increased to 43.14±4.25%. However, pretreatment of the cells with 0.1, 1 and 10 µg/ml APS for 1 h prior to H₂O₂ exposure reduced the percentage of apoptotic cells to 38.58±4.13, 27.60±2.25 and 24.10±3.26%, respectively. In addition, the morphology of HUVEC nuclei was evaluated by DAPI staining. Compared with the control group (Fig. 3A), nuclei of the H₂O₂ group (Fig. 3B) were shrunken, irregular and fragmented, typical features of apoptosis. However, APS (1 µg/ml) pretreatment (Fig. 3C) reduced these alterations.

The disruption of mitochondria has been reported to be involved in programmed cell death. The maintenance of the mitochondrial membrane potential is essential for mitochondrial integrity and bioenergetic function. To determine whether the anti-apoptotic effect of APS is associated with the inhibition of mitochondrial disruption, mitochondrial membrane potential variation in HUVECs was assessed by Rhodamine 123 staining. Rhodamine 123 is sequestered by active mitochondria of normal HUVECs and revealed yellow-green flavovirens fluorescence. Compared with control cells (Fig. 4A), H₂O₂-treated cells presented a weak green fluorescence intensity, which reflected the loss of mitochondrial membrane potential (Fig. 4B); this decrease in fluorescence was partially reversed by APS (1 µg/ml) pretreatment (Fig. 4C). Quantification of fluorescence intensity revealed that these alterations were significant (Fig. 4D). This may be a possible underlying mechanism by which APS protects against injury.

To further investigate the protective effects of APS, the influence of H₂O₂ on cGMP accumulation was measured. As presented in Fig. 5, basal production of cGMP decreased from 9.8±0.8 nmol/g protein in control cells to 4.2±0.4 nmol/g protein in H₂O₂-treated cells. However, pretreatment of cells with 0.1–10 µg/ml APS for 1 h prior to H₂O₂ exposure increased the levels of cGMP compared with H₂O₂ treatment alone, in a dose-dependent manner.

The secondary generation of ROS is another potential mechanism underlying cell damage induced by H₂O₂. Therefore, intracellular ROS levels were measured by DHE staining. Compared with the control group (Fig. 6A), DHE fluorescence in HUVECs exposed to H₂O₂ was increased (Fig. 6B). This increase was attenuated by pretreatment with APS (Fig. 6C). Quantification of fluorescence revealed significantly increased ROS production in H₂O₂-treated cells (P<0.01), and a significant and dose-dependent effect of APS (P<0.01; Fig. 6D).

The protein expression levels of eNOS and Cu/Zn-SOD were examined to investigate the potential underlying molecular mechanisms contributing to APS cytoprotection. Protein expression levels of Cu/Zn-SOD (Fig. 7A) and eNOS (Fig. 7B) in HUVECs exposed to H₂O₂ were significantly decreased (P<0.01). However, pretreatment with 10, 1 or 0.1 µg/ml APS significantly attenuated the H₂O₂-mediated decrease of eNOS and Cu/Zn-SOD expression (P<0.01), which may therefore contribute to the APS-mediated protective effects of HUVECs.