Paeonol (2-hydroxy 4-methoxy acetophenone) (purity, >99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: Anti-ERK1/2 (rabbit polyclonal; V1141; 1:1,000; Promega Corporation, Madison, WI, USA), phosphorylated (p)-ERK1/2 (rabbit polyclonal; ab50011; 1:800; Abcam, Cambridge, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; rabbit polyclonal; sc-25778; 1:1,200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), VEGF₁₆₅ (rabbit polyclonal; sc-13083; 1:1,000). The ERK1/2 inhibitor, PD98059 was obtained from EMD Millipore (Billerica, MA, USA). The human umbilical vein endothelial cell (HUVEC) line was purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in Medium 199 supplemented with 20% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM L-glutamine (Sigma-Aldrich), 5 U/ml heparin (Sigma-Aldrich), 100 IU/ml penicillin (Sigma-Aldrich), 10 μg/ml streptomycin (Sigma-Aldrich) and 50 μg/ml endothelial cell growth supplement (American Type Culture Collection). Cells were cultured in a humidified 5% CO₂ incubator at 37°C and used for further experiments between passage 3 and 6.

The current study was approved by the Ethics Committee of Nanyang Institute of Technology (Nanyang, China). Male SD rats (weight, 350–400 g; age, 18 weeks) were provided by the Laboratory Animal Center of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) as rat models of thrombosis and housed in cages (10 rats/cage) at room temperature with food and water available ad libitum, and were maintained under at 12 h light/dark cycle. The rats (n=30) were randomly grouped into three equal groups (10 in each group), as follows: i) Control group, ii) paeonol group, and iii) aspirin group. In the control group, SD rats received 2 ml/kg normal saline (Sigma-Aldrich) by gavage every two days for two weeks; in the paeonol group, rats received 1.25 mg/kg paeonol by gavage every two days for two weeks; and in the aspirin group, rats received 50 mg/kg aspirin by gavage every two days for two weeks. All the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (17).

The rats were anesthetized at the end of the two weeks with 10% chloral hydrate (0.3 ml/100 g; Sigma-Aldrich) via intraperitoneal injection. Blood samples were collected from the abdominal aorta by opening the abdominal cavity, and immediately centrifuged at 2,200 × g at 4°C for 20 min to separate the serum, which was stored at −80°C for further use. The levels of FN, FIB, D-D, 6-keto-PGF_1α, and TXB₂, which are associated with PTS, were measured by ELISA. The level of VEGF₁₆₅ was also detected. All ELISAs were conducted using the FN (H140), FIB (F010), D-D (E029), 6-Keto-PGF_1α (H214), TXB₂ (R001) and VEGF₁₆₅ (H044) kits according to the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute, Nanjing, China): Briefly, plates were coated and incubated overnight at 4°C with 3.4 mg/ml nonbiotinylated 3D5 primary antibody (100 μl/well) in 200 mM NaHCO₃ (Sigma-Aldrich) at pH 9.6, and then washed 4 times with phosphate-buffered saline with 0.05% Tween 20 (PBST; Sigma-Aldrich). Following incubation with 150 μl/well blocking buffer (PBST containing 2.5% gelatin) for 2 h at 37°C, the plates were washed 4 times with PBST, and 100 μl serum samples (diluted 1:1 with PBS) were added to each well. The plates were incubated at 37°C for 2 h. Following washing 4 times with PBST, 100 μl 1 μg/ml biotinylated 3D5 secondary antibody in blocking buffer was added to each well, and incubated at 37°C for a further 2 h. The wells were washed 4 times with PBST and incubated with 100 μl/well ExtrAvidin-Alkaline phosphatase (Sigma-Aldrich) in blocking buffer (dilution, 1:5,000) and incubated for 1 h at 37°C. Following another 4 washes with PBST, the enzyme substrate alkaline phosphatase yellow (Sigma-Aldrich) was added to each well (100 μl/well) and incubated for 30 min at 37°C for color development. Optical density (OD) values were recorded using a microplate reader at 450 nm (Multiskan MK3; Thermo Fisher Scientific, Inc.). The content was estimated using a standard curve from serial dilutions.

The MTT assay was performed to determine the cytotoxicity of paeonol on HUVECs. Exponentially growing HUVECs from three to six passages (50 μl; 2×10⁵ cells/ml) were seeded into a 96-well plate in triplicate. The cells were treated with increasing concentrations of paeonol [0 (control), 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, and 50 μmol/l] for 24 h and each concentration was repeated in triplicate. Following paeonol treatment, 5 mg/ml MTT was added to each well and incubated for 4 h at 37°C. MTT is converted into purple-colored formazan in living cells, which was then solubilized with dimethylsulfoxide (Invitrogen; Thermo Fisher Scientific, Inc.), and the OD values in the wells were recorded using the microplate reader at 450 nm. The survival rates (%) of different treatments were calculated as: (OD value in treatment group - OD value in blank control group) / (OD value in negative control group - OD value in blank control group) ×100%.

The highest concentration with the greatest number of cells surviving was 0.5 μmol/l, thus, the cell proliferation assay was conducted at this concentration. HUVECs treated with 5 μmol/l paeonol were incubated for different times (0, 24, 48, 72 and 96 h). Normal HUVECs served as a control to determine the effect of paeonol on the viability of HUVECs over time. The detection of cell proliferation was conducted as described above and the OD values in different wells were recorded using the microplate reader at 450 nm.

HUVECs were separated into three groups, as follows: Normal HUVECs; HUVECs incubated with 0.5 μmol/l paeonol; and HUVECs incubated with 0.5 μmol/l paeonol + 50 μmol/l PD98059. For each treatment group, the protein expression levels of VEGF₁₆₅, ERK1/2, and pERK1/2 were detected in cell samples after 24 h incubation. GAPDH served as a loading control for western blot analysis. Total proteins were extracted using sodium dodecyl sulfate (SDS) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 30 min and the protein concentration was determined using the Bicinchoninic Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). All the extracts were boiled with loading buffer for 5 min prior to separation with SDS-polyacrylamide gel electrophoresis on 10% gels at 160 V for 60 min. Proteins were transferred onto polyvinylidene difluoride membranes. The membranes were washed with Tris-buffered saline with Tween 20 (TBST; Sigma-Aldrich) for 20 min, and the procedure was repeated three times. The membranes were incubated with primary antibodies overnight at room temperature. Following three additional washes with TBST, the horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibodies (Beyotime Institute of Biotechnology) were added and the membrane was incubated for 4 h at 37°C. Following three final washes with TBST, the blots were developed using BeyoECL Plus reagent (Beyotime Institute of Biotechnology) and the results were detected in the Gel Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

All the data were expressed as the mean ± standard deviation. Multiple comparisons were conducted using Fisher's Least Significant Difference method. All the statistical analyses were conducted using SPSS version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Using an ELISA, the levels of 6-keto-PGF_1α, FN, and VEGF₁₆₅ in serum were determined to be significantly upregulated by treatment with paeonol (P<0.05), however, the effect was smaller than that of aspirin treatment (Fig. 1). The levels of FIB, D-D, and TXB₂ were significantly downregulated by paeonol (P<0.05), however, the effect was also smaller than that of aspirin treatment (Fig. 2).

With increased concentration of paeonol, the cell viability of HUVECs decreased gradually (Fig. 3). No significant difference was detected for paeonol concentration <0.5 μmol/l. However, at concentrations >0.5 μmol/l, a significant difference in cell viability was observed (P<0.05; Fig. 3). At a concentration of 50 μmol/l paeonol, the cell viability was >70%, which indicates a safe result.

Based on the results of the MTT assay, 0.5 μmol/l was determined as the suitable concentration for further experiments. Paeonol at this concentration significantly improved the cell proliferation ability in a time-dependent manner compared with normal HUVECs (P<0.05; Fig. 4).

To elucidate the signaling pathway involved in the anti-thrombotic effect exerted by paeonol, the effect of paeonol on the expression levels of ERK1/2, p-ERK1/2, and VEGF₁₆₅ was investigated. As presented in Fig. 5, the expression levels of p-ERK1/2 and VEGF₁₆₅ were upregulated in HUVECs. Treatment with the ERK1/2 signaling pathway inhibitor, PD98059 markedly blocked the effect of paeonol on p-ERK1/2 and VEGF₁₆₅.